Introduction to glycobiology

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

Introduction to glycobiology

bull Learn the basic language of glycobiologybull Monosaccharides (vertebrate)bull Glycans (oligosaccharide amp glycoconjugate basic concepts)

bull Learn basic glycan functionsbull Glycans in glycoprotein activity folding traffickingbull O-GlcNAc-mediated regulationbull Glycolipids in membrane recognition amp regulationbull Proteoglycans in the extracellular matrixbull Glycan binding proteins

bull Learn the basic concepts of glycan biosynthesisbull Learn the basic tools of glycomics (analysis)

Course Objectives

Introduction to glycobiologyDate Day Lecture Faculty

Mar 16 Mon Saccharides amp The Glyco World Dr R Schnaar

Mar 18 Wed Glycoproteins 1 Dr N Zachara

Mar 20 Fri Glycoproteins 2 Dr N Zachara

Mar 23 Mon Hyaluronan and Proteoglycans Dr N Zachara

Mar 25 Wed Glycolipids and GPI anchors Dr R Schnaar

Mar 27 Fri O-GlcNAc Dr G Hart

Mar 30 Mon Carbohydrate Engineering Dr K Yarema

Apr 1 Wed Protein-Glycan Recognition Dr R Schnaar

Apr 3 Fri Glycan Binding Protein Functions Dr R Schnaar

Apr 6 Mon Glycomics (Analytical Glycobiology) Dr G Hart

Apr 8 Wed Glycans and Disease Dr G Hart

Introduction to glycobiology

Course co-directors Ronald Schnaar (schnaarjhuedu) Natasha Zachara (nzacharajhmiedu)Lecturers

Gerald Hart (gwhartjhmiedu)Kevin Yarema (kyarema1jhuedu)

Introduction to glycobiology

Oxford University Press New York Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Content freely available at httpwwwncbinlmnihgovbooksNBK1908

What are

GlycobiologyGlycoscience

Glycomics

GlycobiologyThe study of the biological functions structures recognition and biosynthesis of glycans (sugar chains saccharides) in the context of the biological scaffolds to which they are attached (eg glycolipids amp glycoproteins)

GlycoscienceGlycobiology amp Glycochemistry includinghellip

- Chemical synthesis - Chemoenzymatic synthesis - Metabolic engineering - Glycomimetics - Glycan conformation and structure - Material Sciences - Plant and bacterial polysaccharides

Glycomics - Analytical glycosciences - Glycan bioinformatics

ldquoGlycomerdquo ndash The total complement of glycans in a cell or organism

Glycobiology is hellip NOT Carbohydrates as food

Berg Tymoczko amp Stryer (2006) Biochemistry Sixth Edition WH Freeman New York

ldquoCarbohydrates in food are important sources of energy Starch found in plant-derived food such as pasta rdquo

Glycobiology is NOT ldquoardquo post-translational modification it is thousands

Modified from httpwww-chesyredu facultyhouglandhtml

bull Combinatorial linkage position orientation and branching provide the potential for millions of different glycan structures ndash functional diversity

bull Glycans may be larger and are more diverse than their (protein) carriers

bull A glycanrsquos function can supersede that of its (protein) carrier

bull 1-2 of the human genome is devoted to glycosylation

Cummings RD (2009) Molecular BioSystems 5 1087

Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical petasaccharide sequeces in glycosaminoglycans

Glycobiology is hellip NOT a ldquodecorationrdquo

ldquoStructurerdquo of HIV gp120Zolla-Pazner (2004)Nature Reviews Immunology 4 199

N-linked carbohydrate can form both an immunologically silent facemdashwith carbohydrate masquerading as selfmdashand also can protect neighboring epitopes through an evolving glycan shield

httpwwwniaidnihgovlabsandresourceslabsaboutlabsvrcstructuralbiologylaboratoryPageskwongaspx

CD59 a complement defense glycoprotein

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchor

Proteins typically fold inward whereas glycans spread out in space

ldquonakedrdquo CD59 Huang et al (2007)Acta Crystallographica 63 714

Rudd et al (1997) J Biol Chem 272 7229

Scanning electron microscopy of C neoformans yeast cellsVan Duin et al (2004) Antimicrobial Agents and Chemotherapy 482014

Light microscopy micrograph of Cryptococcus neoformans

Steenbergen et al (2003) Microbes and Infection 5667

What if a MAJOR cell component was invisible by standard microscopy

Light microscopy micrograph of Cryptococcus neoformans capsule delineated by India ink The inner circle represents the fungal cell with the wide outer circle being the capsuleSteenbergen et al (2003) Microbes and Infection 5667

Electron microscopic thin section ofEscherichia coli K1Amako et al (1988) J Bacteriol 1704960

Unique to yeast ndash NO

Electron microscopic thin section ofKlebsiella pneumoniaeAmako et al (1988) J Bacteriol 1704960

The Eukaryotic Cell Surface

Tiny sugar ldquodecorationsrdquoadded as an afterthought

Lodish et al (1995) Molecular Cell Biology 3rd ed

The ldquoglycocalyxrdquo surrounding a fibroblast Cell surface carbohydrates are stained black

lipid bilayer

fibroblast

extracellular

intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface -- The Real Picture

Tropical Forest

Canopy

Cohen amp Varki (2010) OMICS 4455

ldquoEvolution has failed to generate a living cell devoid of surface glycosylationrdquo - A Varki

lipid bilayer

fibroblast intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface

Real image textbook image

bull Among their diverse functions glycans hellipndash encode intermolecular and cell-cell recognitionndash regulate the activities of proteins (enzymes ion channels

receptors)ndash Define biophysical space (large hydrated)

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

Introduction to glycobiology

bull Learn the basic language of glycobiologybull Monosaccharides (vertebrate)bull Glycans (oligosaccharide amp glycoconjugate basic concepts)

bull Learn basic glycan functionsbull Glycans in glycoprotein activity folding traffickingbull O-GlcNAc-mediated regulationbull Glycolipids in membrane recognition amp regulationbull Proteoglycans in the extracellular matrixbull Glycan binding proteins

bull Learn the basic concepts of glycan biosynthesisbull Learn the basic tools of glycomics (analysis)

Course Objectives

Introduction to glycobiologyDate Day Lecture Faculty

Mar 16 Mon Saccharides amp The Glyco World Dr R Schnaar

Mar 18 Wed Glycoproteins 1 Dr N Zachara

Mar 20 Fri Glycoproteins 2 Dr N Zachara

Mar 23 Mon Hyaluronan and Proteoglycans Dr N Zachara

Mar 25 Wed Glycolipids and GPI anchors Dr R Schnaar

Mar 27 Fri O-GlcNAc Dr G Hart

Mar 30 Mon Carbohydrate Engineering Dr K Yarema

Apr 1 Wed Protein-Glycan Recognition Dr R Schnaar

Apr 3 Fri Glycan Binding Protein Functions Dr R Schnaar

Apr 6 Mon Glycomics (Analytical Glycobiology) Dr G Hart

Apr 8 Wed Glycans and Disease Dr G Hart

Introduction to glycobiology

Course co-directors Ronald Schnaar (schnaarjhuedu) Natasha Zachara (nzacharajhmiedu)Lecturers

Gerald Hart (gwhartjhmiedu)Kevin Yarema (kyarema1jhuedu)

Introduction to glycobiology

Oxford University Press New York Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Content freely available at httpwwwncbinlmnihgovbooksNBK1908

What are

GlycobiologyGlycoscience

Glycomics

GlycobiologyThe study of the biological functions structures recognition and biosynthesis of glycans (sugar chains saccharides) in the context of the biological scaffolds to which they are attached (eg glycolipids amp glycoproteins)

GlycoscienceGlycobiology amp Glycochemistry includinghellip

- Chemical synthesis - Chemoenzymatic synthesis - Metabolic engineering - Glycomimetics - Glycan conformation and structure - Material Sciences - Plant and bacterial polysaccharides

Glycomics - Analytical glycosciences - Glycan bioinformatics

ldquoGlycomerdquo ndash The total complement of glycans in a cell or organism

Glycobiology is hellip NOT Carbohydrates as food

Berg Tymoczko amp Stryer (2006) Biochemistry Sixth Edition WH Freeman New York

ldquoCarbohydrates in food are important sources of energy Starch found in plant-derived food such as pasta rdquo

Glycobiology is NOT ldquoardquo post-translational modification it is thousands

Modified from httpwww-chesyredu facultyhouglandhtml

bull Combinatorial linkage position orientation and branching provide the potential for millions of different glycan structures ndash functional diversity

bull Glycans may be larger and are more diverse than their (protein) carriers

bull A glycanrsquos function can supersede that of its (protein) carrier

bull 1-2 of the human genome is devoted to glycosylation

Cummings RD (2009) Molecular BioSystems 5 1087

Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical petasaccharide sequeces in glycosaminoglycans

Glycobiology is hellip NOT a ldquodecorationrdquo

ldquoStructurerdquo of HIV gp120Zolla-Pazner (2004)Nature Reviews Immunology 4 199

N-linked carbohydrate can form both an immunologically silent facemdashwith carbohydrate masquerading as selfmdashand also can protect neighboring epitopes through an evolving glycan shield

httpwwwniaidnihgovlabsandresourceslabsaboutlabsvrcstructuralbiologylaboratoryPageskwongaspx

CD59 a complement defense glycoprotein

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchor

Proteins typically fold inward whereas glycans spread out in space

ldquonakedrdquo CD59 Huang et al (2007)Acta Crystallographica 63 714

Rudd et al (1997) J Biol Chem 272 7229

Scanning electron microscopy of C neoformans yeast cellsVan Duin et al (2004) Antimicrobial Agents and Chemotherapy 482014

Light microscopy micrograph of Cryptococcus neoformans

Steenbergen et al (2003) Microbes and Infection 5667

What if a MAJOR cell component was invisible by standard microscopy

Light microscopy micrograph of Cryptococcus neoformans capsule delineated by India ink The inner circle represents the fungal cell with the wide outer circle being the capsuleSteenbergen et al (2003) Microbes and Infection 5667

Electron microscopic thin section ofEscherichia coli K1Amako et al (1988) J Bacteriol 1704960

Unique to yeast ndash NO

Electron microscopic thin section ofKlebsiella pneumoniaeAmako et al (1988) J Bacteriol 1704960

The Eukaryotic Cell Surface

Tiny sugar ldquodecorationsrdquoadded as an afterthought

Lodish et al (1995) Molecular Cell Biology 3rd ed

The ldquoglycocalyxrdquo surrounding a fibroblast Cell surface carbohydrates are stained black

lipid bilayer

fibroblast

extracellular

intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface -- The Real Picture

Tropical Forest

Canopy

Cohen amp Varki (2010) OMICS 4455

ldquoEvolution has failed to generate a living cell devoid of surface glycosylationrdquo - A Varki

lipid bilayer

fibroblast intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface

Real image textbook image

bull Among their diverse functions glycans hellipndash encode intermolecular and cell-cell recognitionndash regulate the activities of proteins (enzymes ion channels

receptors)ndash Define biophysical space (large hydrated)

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

Introduction to glycobiology

bull Learn the basic language of glycobiologybull Monosaccharides (vertebrate)bull Glycans (oligosaccharide amp glycoconjugate basic concepts)

bull Learn basic glycan functionsbull Glycans in glycoprotein activity folding traffickingbull O-GlcNAc-mediated regulationbull Glycolipids in membrane recognition amp regulationbull Proteoglycans in the extracellular matrixbull Glycan binding proteins

bull Learn the basic concepts of glycan biosynthesisbull Learn the basic tools of glycomics (analysis)

Course Objectives

Introduction to glycobiologyDate Day Lecture Faculty

Mar 16 Mon Saccharides amp The Glyco World Dr R Schnaar

Mar 18 Wed Glycoproteins 1 Dr N Zachara

Mar 20 Fri Glycoproteins 2 Dr N Zachara

Mar 23 Mon Hyaluronan and Proteoglycans Dr N Zachara

Mar 25 Wed Glycolipids and GPI anchors Dr R Schnaar

Mar 27 Fri O-GlcNAc Dr G Hart

Mar 30 Mon Carbohydrate Engineering Dr K Yarema

Apr 1 Wed Protein-Glycan Recognition Dr R Schnaar

Apr 3 Fri Glycan Binding Protein Functions Dr R Schnaar

Apr 6 Mon Glycomics (Analytical Glycobiology) Dr G Hart

Apr 8 Wed Glycans and Disease Dr G Hart

Introduction to glycobiology

Course co-directors Ronald Schnaar (schnaarjhuedu) Natasha Zachara (nzacharajhmiedu)Lecturers

Gerald Hart (gwhartjhmiedu)Kevin Yarema (kyarema1jhuedu)

Introduction to glycobiology

Oxford University Press New York Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Content freely available at httpwwwncbinlmnihgovbooksNBK1908

What are

GlycobiologyGlycoscience

Glycomics

GlycobiologyThe study of the biological functions structures recognition and biosynthesis of glycans (sugar chains saccharides) in the context of the biological scaffolds to which they are attached (eg glycolipids amp glycoproteins)

GlycoscienceGlycobiology amp Glycochemistry includinghellip

- Chemical synthesis - Chemoenzymatic synthesis - Metabolic engineering - Glycomimetics - Glycan conformation and structure - Material Sciences - Plant and bacterial polysaccharides

Glycomics - Analytical glycosciences - Glycan bioinformatics

ldquoGlycomerdquo ndash The total complement of glycans in a cell or organism

Glycobiology is hellip NOT Carbohydrates as food

Berg Tymoczko amp Stryer (2006) Biochemistry Sixth Edition WH Freeman New York

ldquoCarbohydrates in food are important sources of energy Starch found in plant-derived food such as pasta rdquo

Glycobiology is NOT ldquoardquo post-translational modification it is thousands

Modified from httpwww-chesyredu facultyhouglandhtml

bull Combinatorial linkage position orientation and branching provide the potential for millions of different glycan structures ndash functional diversity

bull Glycans may be larger and are more diverse than their (protein) carriers

bull A glycanrsquos function can supersede that of its (protein) carrier

bull 1-2 of the human genome is devoted to glycosylation

Cummings RD (2009) Molecular BioSystems 5 1087

Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical petasaccharide sequeces in glycosaminoglycans

Glycobiology is hellip NOT a ldquodecorationrdquo

ldquoStructurerdquo of HIV gp120Zolla-Pazner (2004)Nature Reviews Immunology 4 199

N-linked carbohydrate can form both an immunologically silent facemdashwith carbohydrate masquerading as selfmdashand also can protect neighboring epitopes through an evolving glycan shield

httpwwwniaidnihgovlabsandresourceslabsaboutlabsvrcstructuralbiologylaboratoryPageskwongaspx

CD59 a complement defense glycoprotein

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchor

Proteins typically fold inward whereas glycans spread out in space

ldquonakedrdquo CD59 Huang et al (2007)Acta Crystallographica 63 714

Rudd et al (1997) J Biol Chem 272 7229

Scanning electron microscopy of C neoformans yeast cellsVan Duin et al (2004) Antimicrobial Agents and Chemotherapy 482014

Light microscopy micrograph of Cryptococcus neoformans

Steenbergen et al (2003) Microbes and Infection 5667

What if a MAJOR cell component was invisible by standard microscopy

Light microscopy micrograph of Cryptococcus neoformans capsule delineated by India ink The inner circle represents the fungal cell with the wide outer circle being the capsuleSteenbergen et al (2003) Microbes and Infection 5667

Electron microscopic thin section ofEscherichia coli K1Amako et al (1988) J Bacteriol 1704960

Unique to yeast ndash NO

Electron microscopic thin section ofKlebsiella pneumoniaeAmako et al (1988) J Bacteriol 1704960

The Eukaryotic Cell Surface

Tiny sugar ldquodecorationsrdquoadded as an afterthought

Lodish et al (1995) Molecular Cell Biology 3rd ed

The ldquoglycocalyxrdquo surrounding a fibroblast Cell surface carbohydrates are stained black

lipid bilayer

fibroblast

extracellular

intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface -- The Real Picture

Tropical Forest

Canopy

Cohen amp Varki (2010) OMICS 4455

ldquoEvolution has failed to generate a living cell devoid of surface glycosylationrdquo - A Varki

lipid bilayer

fibroblast intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface

Real image textbook image

bull Among their diverse functions glycans hellipndash encode intermolecular and cell-cell recognitionndash regulate the activities of proteins (enzymes ion channels

receptors)ndash Define biophysical space (large hydrated)

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

Introduction to glycobiologyDate Day Lecture Faculty

Mar 16 Mon Saccharides amp The Glyco World Dr R Schnaar

Mar 18 Wed Glycoproteins 1 Dr N Zachara

Mar 20 Fri Glycoproteins 2 Dr N Zachara

Mar 23 Mon Hyaluronan and Proteoglycans Dr N Zachara

Mar 25 Wed Glycolipids and GPI anchors Dr R Schnaar

Mar 27 Fri O-GlcNAc Dr G Hart

Mar 30 Mon Carbohydrate Engineering Dr K Yarema

Apr 1 Wed Protein-Glycan Recognition Dr R Schnaar

Apr 3 Fri Glycan Binding Protein Functions Dr R Schnaar

Apr 6 Mon Glycomics (Analytical Glycobiology) Dr G Hart

Apr 8 Wed Glycans and Disease Dr G Hart

Introduction to glycobiology

Course co-directors Ronald Schnaar (schnaarjhuedu) Natasha Zachara (nzacharajhmiedu)Lecturers

Gerald Hart (gwhartjhmiedu)Kevin Yarema (kyarema1jhuedu)

Introduction to glycobiology

Oxford University Press New York Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Content freely available at httpwwwncbinlmnihgovbooksNBK1908

What are

GlycobiologyGlycoscience

Glycomics

GlycobiologyThe study of the biological functions structures recognition and biosynthesis of glycans (sugar chains saccharides) in the context of the biological scaffolds to which they are attached (eg glycolipids amp glycoproteins)

GlycoscienceGlycobiology amp Glycochemistry includinghellip

- Chemical synthesis - Chemoenzymatic synthesis - Metabolic engineering - Glycomimetics - Glycan conformation and structure - Material Sciences - Plant and bacterial polysaccharides

Glycomics - Analytical glycosciences - Glycan bioinformatics

ldquoGlycomerdquo ndash The total complement of glycans in a cell or organism

Glycobiology is hellip NOT Carbohydrates as food

Berg Tymoczko amp Stryer (2006) Biochemistry Sixth Edition WH Freeman New York

ldquoCarbohydrates in food are important sources of energy Starch found in plant-derived food such as pasta rdquo

Glycobiology is NOT ldquoardquo post-translational modification it is thousands

Modified from httpwww-chesyredu facultyhouglandhtml

bull Combinatorial linkage position orientation and branching provide the potential for millions of different glycan structures ndash functional diversity

bull Glycans may be larger and are more diverse than their (protein) carriers

bull A glycanrsquos function can supersede that of its (protein) carrier

bull 1-2 of the human genome is devoted to glycosylation

Cummings RD (2009) Molecular BioSystems 5 1087

Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical petasaccharide sequeces in glycosaminoglycans

Glycobiology is hellip NOT a ldquodecorationrdquo

ldquoStructurerdquo of HIV gp120Zolla-Pazner (2004)Nature Reviews Immunology 4 199

N-linked carbohydrate can form both an immunologically silent facemdashwith carbohydrate masquerading as selfmdashand also can protect neighboring epitopes through an evolving glycan shield

httpwwwniaidnihgovlabsandresourceslabsaboutlabsvrcstructuralbiologylaboratoryPageskwongaspx

CD59 a complement defense glycoprotein

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchor

Proteins typically fold inward whereas glycans spread out in space

ldquonakedrdquo CD59 Huang et al (2007)Acta Crystallographica 63 714

Rudd et al (1997) J Biol Chem 272 7229

Scanning electron microscopy of C neoformans yeast cellsVan Duin et al (2004) Antimicrobial Agents and Chemotherapy 482014

Light microscopy micrograph of Cryptococcus neoformans

Steenbergen et al (2003) Microbes and Infection 5667

What if a MAJOR cell component was invisible by standard microscopy

Light microscopy micrograph of Cryptococcus neoformans capsule delineated by India ink The inner circle represents the fungal cell with the wide outer circle being the capsuleSteenbergen et al (2003) Microbes and Infection 5667

Electron microscopic thin section ofEscherichia coli K1Amako et al (1988) J Bacteriol 1704960

Unique to yeast ndash NO

Electron microscopic thin section ofKlebsiella pneumoniaeAmako et al (1988) J Bacteriol 1704960

The Eukaryotic Cell Surface

Tiny sugar ldquodecorationsrdquoadded as an afterthought

Lodish et al (1995) Molecular Cell Biology 3rd ed

The ldquoglycocalyxrdquo surrounding a fibroblast Cell surface carbohydrates are stained black

lipid bilayer

fibroblast

extracellular

intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface -- The Real Picture

Tropical Forest

Canopy

Cohen amp Varki (2010) OMICS 4455

ldquoEvolution has failed to generate a living cell devoid of surface glycosylationrdquo - A Varki

lipid bilayer

fibroblast intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface

Real image textbook image

bull Among their diverse functions glycans hellipndash encode intermolecular and cell-cell recognitionndash regulate the activities of proteins (enzymes ion channels

receptors)ndash Define biophysical space (large hydrated)

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

Introduction to glycobiology

Course co-directors Ronald Schnaar (schnaarjhuedu) Natasha Zachara (nzacharajhmiedu)Lecturers

Gerald Hart (gwhartjhmiedu)Kevin Yarema (kyarema1jhuedu)

Introduction to glycobiology

Oxford University Press New York Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Content freely available at httpwwwncbinlmnihgovbooksNBK1908

What are

GlycobiologyGlycoscience

Glycomics

GlycobiologyThe study of the biological functions structures recognition and biosynthesis of glycans (sugar chains saccharides) in the context of the biological scaffolds to which they are attached (eg glycolipids amp glycoproteins)

GlycoscienceGlycobiology amp Glycochemistry includinghellip

- Chemical synthesis - Chemoenzymatic synthesis - Metabolic engineering - Glycomimetics - Glycan conformation and structure - Material Sciences - Plant and bacterial polysaccharides

Glycomics - Analytical glycosciences - Glycan bioinformatics

ldquoGlycomerdquo ndash The total complement of glycans in a cell or organism

Glycobiology is hellip NOT Carbohydrates as food

Berg Tymoczko amp Stryer (2006) Biochemistry Sixth Edition WH Freeman New York

ldquoCarbohydrates in food are important sources of energy Starch found in plant-derived food such as pasta rdquo

Glycobiology is NOT ldquoardquo post-translational modification it is thousands

Modified from httpwww-chesyredu facultyhouglandhtml

bull Combinatorial linkage position orientation and branching provide the potential for millions of different glycan structures ndash functional diversity

bull Glycans may be larger and are more diverse than their (protein) carriers

bull A glycanrsquos function can supersede that of its (protein) carrier

bull 1-2 of the human genome is devoted to glycosylation

Cummings RD (2009) Molecular BioSystems 5 1087

Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical petasaccharide sequeces in glycosaminoglycans

Glycobiology is hellip NOT a ldquodecorationrdquo

ldquoStructurerdquo of HIV gp120Zolla-Pazner (2004)Nature Reviews Immunology 4 199

N-linked carbohydrate can form both an immunologically silent facemdashwith carbohydrate masquerading as selfmdashand also can protect neighboring epitopes through an evolving glycan shield

httpwwwniaidnihgovlabsandresourceslabsaboutlabsvrcstructuralbiologylaboratoryPageskwongaspx

CD59 a complement defense glycoprotein

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchor

Proteins typically fold inward whereas glycans spread out in space

ldquonakedrdquo CD59 Huang et al (2007)Acta Crystallographica 63 714

Rudd et al (1997) J Biol Chem 272 7229

Scanning electron microscopy of C neoformans yeast cellsVan Duin et al (2004) Antimicrobial Agents and Chemotherapy 482014

Light microscopy micrograph of Cryptococcus neoformans

Steenbergen et al (2003) Microbes and Infection 5667

What if a MAJOR cell component was invisible by standard microscopy

Light microscopy micrograph of Cryptococcus neoformans capsule delineated by India ink The inner circle represents the fungal cell with the wide outer circle being the capsuleSteenbergen et al (2003) Microbes and Infection 5667

Electron microscopic thin section ofEscherichia coli K1Amako et al (1988) J Bacteriol 1704960

Unique to yeast ndash NO

Electron microscopic thin section ofKlebsiella pneumoniaeAmako et al (1988) J Bacteriol 1704960

The Eukaryotic Cell Surface

Tiny sugar ldquodecorationsrdquoadded as an afterthought

Lodish et al (1995) Molecular Cell Biology 3rd ed

The ldquoglycocalyxrdquo surrounding a fibroblast Cell surface carbohydrates are stained black

lipid bilayer

fibroblast

extracellular

intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface -- The Real Picture

Tropical Forest

Canopy

Cohen amp Varki (2010) OMICS 4455

ldquoEvolution has failed to generate a living cell devoid of surface glycosylationrdquo - A Varki

lipid bilayer

fibroblast intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface

Real image textbook image

bull Among their diverse functions glycans hellipndash encode intermolecular and cell-cell recognitionndash regulate the activities of proteins (enzymes ion channels

receptors)ndash Define biophysical space (large hydrated)

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

Introduction to glycobiology

Oxford University Press New York Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Content freely available at httpwwwncbinlmnihgovbooksNBK1908

What are

GlycobiologyGlycoscience

Glycomics

GlycobiologyThe study of the biological functions structures recognition and biosynthesis of glycans (sugar chains saccharides) in the context of the biological scaffolds to which they are attached (eg glycolipids amp glycoproteins)

GlycoscienceGlycobiology amp Glycochemistry includinghellip

- Chemical synthesis - Chemoenzymatic synthesis - Metabolic engineering - Glycomimetics - Glycan conformation and structure - Material Sciences - Plant and bacterial polysaccharides

Glycomics - Analytical glycosciences - Glycan bioinformatics

ldquoGlycomerdquo ndash The total complement of glycans in a cell or organism

Glycobiology is hellip NOT Carbohydrates as food

Berg Tymoczko amp Stryer (2006) Biochemistry Sixth Edition WH Freeman New York

ldquoCarbohydrates in food are important sources of energy Starch found in plant-derived food such as pasta rdquo

Glycobiology is NOT ldquoardquo post-translational modification it is thousands

Modified from httpwww-chesyredu facultyhouglandhtml

bull Combinatorial linkage position orientation and branching provide the potential for millions of different glycan structures ndash functional diversity

bull Glycans may be larger and are more diverse than their (protein) carriers

bull A glycanrsquos function can supersede that of its (protein) carrier

bull 1-2 of the human genome is devoted to glycosylation

Cummings RD (2009) Molecular BioSystems 5 1087

Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical petasaccharide sequeces in glycosaminoglycans

Glycobiology is hellip NOT a ldquodecorationrdquo

ldquoStructurerdquo of HIV gp120Zolla-Pazner (2004)Nature Reviews Immunology 4 199

N-linked carbohydrate can form both an immunologically silent facemdashwith carbohydrate masquerading as selfmdashand also can protect neighboring epitopes through an evolving glycan shield

httpwwwniaidnihgovlabsandresourceslabsaboutlabsvrcstructuralbiologylaboratoryPageskwongaspx

CD59 a complement defense glycoprotein

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchor

Proteins typically fold inward whereas glycans spread out in space

ldquonakedrdquo CD59 Huang et al (2007)Acta Crystallographica 63 714

Rudd et al (1997) J Biol Chem 272 7229

Scanning electron microscopy of C neoformans yeast cellsVan Duin et al (2004) Antimicrobial Agents and Chemotherapy 482014

Light microscopy micrograph of Cryptococcus neoformans

Steenbergen et al (2003) Microbes and Infection 5667

What if a MAJOR cell component was invisible by standard microscopy

Light microscopy micrograph of Cryptococcus neoformans capsule delineated by India ink The inner circle represents the fungal cell with the wide outer circle being the capsuleSteenbergen et al (2003) Microbes and Infection 5667

Electron microscopic thin section ofEscherichia coli K1Amako et al (1988) J Bacteriol 1704960

Unique to yeast ndash NO

Electron microscopic thin section ofKlebsiella pneumoniaeAmako et al (1988) J Bacteriol 1704960

The Eukaryotic Cell Surface

Tiny sugar ldquodecorationsrdquoadded as an afterthought

Lodish et al (1995) Molecular Cell Biology 3rd ed

The ldquoglycocalyxrdquo surrounding a fibroblast Cell surface carbohydrates are stained black

lipid bilayer

fibroblast

extracellular

intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface -- The Real Picture

Tropical Forest

Canopy

Cohen amp Varki (2010) OMICS 4455

ldquoEvolution has failed to generate a living cell devoid of surface glycosylationrdquo - A Varki

lipid bilayer

fibroblast intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface

Real image textbook image

bull Among their diverse functions glycans hellipndash encode intermolecular and cell-cell recognitionndash regulate the activities of proteins (enzymes ion channels

receptors)ndash Define biophysical space (large hydrated)

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

What are

GlycobiologyGlycoscience

Glycomics

GlycobiologyThe study of the biological functions structures recognition and biosynthesis of glycans (sugar chains saccharides) in the context of the biological scaffolds to which they are attached (eg glycolipids amp glycoproteins)

GlycoscienceGlycobiology amp Glycochemistry includinghellip

- Chemical synthesis - Chemoenzymatic synthesis - Metabolic engineering - Glycomimetics - Glycan conformation and structure - Material Sciences - Plant and bacterial polysaccharides

Glycomics - Analytical glycosciences - Glycan bioinformatics

ldquoGlycomerdquo ndash The total complement of glycans in a cell or organism

Glycobiology is hellip NOT Carbohydrates as food

Berg Tymoczko amp Stryer (2006) Biochemistry Sixth Edition WH Freeman New York

ldquoCarbohydrates in food are important sources of energy Starch found in plant-derived food such as pasta rdquo

Glycobiology is NOT ldquoardquo post-translational modification it is thousands

Modified from httpwww-chesyredu facultyhouglandhtml

bull Combinatorial linkage position orientation and branching provide the potential for millions of different glycan structures ndash functional diversity

bull Glycans may be larger and are more diverse than their (protein) carriers

bull A glycanrsquos function can supersede that of its (protein) carrier

bull 1-2 of the human genome is devoted to glycosylation

Cummings RD (2009) Molecular BioSystems 5 1087

Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical petasaccharide sequeces in glycosaminoglycans

Glycobiology is hellip NOT a ldquodecorationrdquo

ldquoStructurerdquo of HIV gp120Zolla-Pazner (2004)Nature Reviews Immunology 4 199

N-linked carbohydrate can form both an immunologically silent facemdashwith carbohydrate masquerading as selfmdashand also can protect neighboring epitopes through an evolving glycan shield

httpwwwniaidnihgovlabsandresourceslabsaboutlabsvrcstructuralbiologylaboratoryPageskwongaspx

CD59 a complement defense glycoprotein

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchor

Proteins typically fold inward whereas glycans spread out in space

ldquonakedrdquo CD59 Huang et al (2007)Acta Crystallographica 63 714

Rudd et al (1997) J Biol Chem 272 7229

Scanning electron microscopy of C neoformans yeast cellsVan Duin et al (2004) Antimicrobial Agents and Chemotherapy 482014

Light microscopy micrograph of Cryptococcus neoformans

Steenbergen et al (2003) Microbes and Infection 5667

What if a MAJOR cell component was invisible by standard microscopy

Light microscopy micrograph of Cryptococcus neoformans capsule delineated by India ink The inner circle represents the fungal cell with the wide outer circle being the capsuleSteenbergen et al (2003) Microbes and Infection 5667

Electron microscopic thin section ofEscherichia coli K1Amako et al (1988) J Bacteriol 1704960

Unique to yeast ndash NO

Electron microscopic thin section ofKlebsiella pneumoniaeAmako et al (1988) J Bacteriol 1704960

The Eukaryotic Cell Surface

Tiny sugar ldquodecorationsrdquoadded as an afterthought

Lodish et al (1995) Molecular Cell Biology 3rd ed

The ldquoglycocalyxrdquo surrounding a fibroblast Cell surface carbohydrates are stained black

lipid bilayer

fibroblast

extracellular

intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface -- The Real Picture

Tropical Forest

Canopy

Cohen amp Varki (2010) OMICS 4455

ldquoEvolution has failed to generate a living cell devoid of surface glycosylationrdquo - A Varki

lipid bilayer

fibroblast intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface

Real image textbook image

bull Among their diverse functions glycans hellipndash encode intermolecular and cell-cell recognitionndash regulate the activities of proteins (enzymes ion channels

receptors)ndash Define biophysical space (large hydrated)

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

GlycobiologyThe study of the biological functions structures recognition and biosynthesis of glycans (sugar chains saccharides) in the context of the biological scaffolds to which they are attached (eg glycolipids amp glycoproteins)

GlycoscienceGlycobiology amp Glycochemistry includinghellip

- Chemical synthesis - Chemoenzymatic synthesis - Metabolic engineering - Glycomimetics - Glycan conformation and structure - Material Sciences - Plant and bacterial polysaccharides

Glycomics - Analytical glycosciences - Glycan bioinformatics

ldquoGlycomerdquo ndash The total complement of glycans in a cell or organism

Glycobiology is hellip NOT Carbohydrates as food

Berg Tymoczko amp Stryer (2006) Biochemistry Sixth Edition WH Freeman New York

ldquoCarbohydrates in food are important sources of energy Starch found in plant-derived food such as pasta rdquo

Glycobiology is NOT ldquoardquo post-translational modification it is thousands

Modified from httpwww-chesyredu facultyhouglandhtml

bull Combinatorial linkage position orientation and branching provide the potential for millions of different glycan structures ndash functional diversity

bull Glycans may be larger and are more diverse than their (protein) carriers

bull A glycanrsquos function can supersede that of its (protein) carrier

bull 1-2 of the human genome is devoted to glycosylation

Cummings RD (2009) Molecular BioSystems 5 1087

Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical petasaccharide sequeces in glycosaminoglycans

Glycobiology is hellip NOT a ldquodecorationrdquo

ldquoStructurerdquo of HIV gp120Zolla-Pazner (2004)Nature Reviews Immunology 4 199

N-linked carbohydrate can form both an immunologically silent facemdashwith carbohydrate masquerading as selfmdashand also can protect neighboring epitopes through an evolving glycan shield

httpwwwniaidnihgovlabsandresourceslabsaboutlabsvrcstructuralbiologylaboratoryPageskwongaspx

CD59 a complement defense glycoprotein

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchor

Proteins typically fold inward whereas glycans spread out in space

ldquonakedrdquo CD59 Huang et al (2007)Acta Crystallographica 63 714

Rudd et al (1997) J Biol Chem 272 7229

Scanning electron microscopy of C neoformans yeast cellsVan Duin et al (2004) Antimicrobial Agents and Chemotherapy 482014

Light microscopy micrograph of Cryptococcus neoformans

Steenbergen et al (2003) Microbes and Infection 5667

What if a MAJOR cell component was invisible by standard microscopy

Light microscopy micrograph of Cryptococcus neoformans capsule delineated by India ink The inner circle represents the fungal cell with the wide outer circle being the capsuleSteenbergen et al (2003) Microbes and Infection 5667

Electron microscopic thin section ofEscherichia coli K1Amako et al (1988) J Bacteriol 1704960

Unique to yeast ndash NO

Electron microscopic thin section ofKlebsiella pneumoniaeAmako et al (1988) J Bacteriol 1704960

The Eukaryotic Cell Surface

Tiny sugar ldquodecorationsrdquoadded as an afterthought

Lodish et al (1995) Molecular Cell Biology 3rd ed

The ldquoglycocalyxrdquo surrounding a fibroblast Cell surface carbohydrates are stained black

lipid bilayer

fibroblast

extracellular

intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface -- The Real Picture

Tropical Forest

Canopy

Cohen amp Varki (2010) OMICS 4455

ldquoEvolution has failed to generate a living cell devoid of surface glycosylationrdquo - A Varki

lipid bilayer

fibroblast intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface

Real image textbook image

bull Among their diverse functions glycans hellipndash encode intermolecular and cell-cell recognitionndash regulate the activities of proteins (enzymes ion channels

receptors)ndash Define biophysical space (large hydrated)

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

GlycoscienceGlycobiology amp Glycochemistry includinghellip

- Chemical synthesis - Chemoenzymatic synthesis - Metabolic engineering - Glycomimetics - Glycan conformation and structure - Material Sciences - Plant and bacterial polysaccharides

Glycomics - Analytical glycosciences - Glycan bioinformatics

ldquoGlycomerdquo ndash The total complement of glycans in a cell or organism

Glycobiology is hellip NOT Carbohydrates as food

Berg Tymoczko amp Stryer (2006) Biochemistry Sixth Edition WH Freeman New York

ldquoCarbohydrates in food are important sources of energy Starch found in plant-derived food such as pasta rdquo

Glycobiology is NOT ldquoardquo post-translational modification it is thousands

Modified from httpwww-chesyredu facultyhouglandhtml

bull Combinatorial linkage position orientation and branching provide the potential for millions of different glycan structures ndash functional diversity

bull Glycans may be larger and are more diverse than their (protein) carriers

bull A glycanrsquos function can supersede that of its (protein) carrier

bull 1-2 of the human genome is devoted to glycosylation

Cummings RD (2009) Molecular BioSystems 5 1087

Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical petasaccharide sequeces in glycosaminoglycans

Glycobiology is hellip NOT a ldquodecorationrdquo

ldquoStructurerdquo of HIV gp120Zolla-Pazner (2004)Nature Reviews Immunology 4 199

N-linked carbohydrate can form both an immunologically silent facemdashwith carbohydrate masquerading as selfmdashand also can protect neighboring epitopes through an evolving glycan shield

httpwwwniaidnihgovlabsandresourceslabsaboutlabsvrcstructuralbiologylaboratoryPageskwongaspx

CD59 a complement defense glycoprotein

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchor

Proteins typically fold inward whereas glycans spread out in space

ldquonakedrdquo CD59 Huang et al (2007)Acta Crystallographica 63 714

Rudd et al (1997) J Biol Chem 272 7229

Scanning electron microscopy of C neoformans yeast cellsVan Duin et al (2004) Antimicrobial Agents and Chemotherapy 482014

Light microscopy micrograph of Cryptococcus neoformans

Steenbergen et al (2003) Microbes and Infection 5667

What if a MAJOR cell component was invisible by standard microscopy

Light microscopy micrograph of Cryptococcus neoformans capsule delineated by India ink The inner circle represents the fungal cell with the wide outer circle being the capsuleSteenbergen et al (2003) Microbes and Infection 5667

Electron microscopic thin section ofEscherichia coli K1Amako et al (1988) J Bacteriol 1704960

Unique to yeast ndash NO

Electron microscopic thin section ofKlebsiella pneumoniaeAmako et al (1988) J Bacteriol 1704960

The Eukaryotic Cell Surface

Tiny sugar ldquodecorationsrdquoadded as an afterthought

Lodish et al (1995) Molecular Cell Biology 3rd ed

The ldquoglycocalyxrdquo surrounding a fibroblast Cell surface carbohydrates are stained black

lipid bilayer

fibroblast

extracellular

intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface -- The Real Picture

Tropical Forest

Canopy

Cohen amp Varki (2010) OMICS 4455

ldquoEvolution has failed to generate a living cell devoid of surface glycosylationrdquo - A Varki

lipid bilayer

fibroblast intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface

Real image textbook image

bull Among their diverse functions glycans hellipndash encode intermolecular and cell-cell recognitionndash regulate the activities of proteins (enzymes ion channels

receptors)ndash Define biophysical space (large hydrated)

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

Glycomics - Analytical glycosciences - Glycan bioinformatics

ldquoGlycomerdquo ndash The total complement of glycans in a cell or organism

Glycobiology is hellip NOT Carbohydrates as food

Berg Tymoczko amp Stryer (2006) Biochemistry Sixth Edition WH Freeman New York

ldquoCarbohydrates in food are important sources of energy Starch found in plant-derived food such as pasta rdquo

Glycobiology is NOT ldquoardquo post-translational modification it is thousands

Modified from httpwww-chesyredu facultyhouglandhtml

bull Combinatorial linkage position orientation and branching provide the potential for millions of different glycan structures ndash functional diversity

bull Glycans may be larger and are more diverse than their (protein) carriers

bull A glycanrsquos function can supersede that of its (protein) carrier

bull 1-2 of the human genome is devoted to glycosylation

Cummings RD (2009) Molecular BioSystems 5 1087

Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical petasaccharide sequeces in glycosaminoglycans

Glycobiology is hellip NOT a ldquodecorationrdquo

ldquoStructurerdquo of HIV gp120Zolla-Pazner (2004)Nature Reviews Immunology 4 199

N-linked carbohydrate can form both an immunologically silent facemdashwith carbohydrate masquerading as selfmdashand also can protect neighboring epitopes through an evolving glycan shield

httpwwwniaidnihgovlabsandresourceslabsaboutlabsvrcstructuralbiologylaboratoryPageskwongaspx

CD59 a complement defense glycoprotein

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchor

Proteins typically fold inward whereas glycans spread out in space

ldquonakedrdquo CD59 Huang et al (2007)Acta Crystallographica 63 714

Rudd et al (1997) J Biol Chem 272 7229

Scanning electron microscopy of C neoformans yeast cellsVan Duin et al (2004) Antimicrobial Agents and Chemotherapy 482014

Light microscopy micrograph of Cryptococcus neoformans

Steenbergen et al (2003) Microbes and Infection 5667

What if a MAJOR cell component was invisible by standard microscopy

Light microscopy micrograph of Cryptococcus neoformans capsule delineated by India ink The inner circle represents the fungal cell with the wide outer circle being the capsuleSteenbergen et al (2003) Microbes and Infection 5667

Electron microscopic thin section ofEscherichia coli K1Amako et al (1988) J Bacteriol 1704960

Unique to yeast ndash NO

Electron microscopic thin section ofKlebsiella pneumoniaeAmako et al (1988) J Bacteriol 1704960

The Eukaryotic Cell Surface

Tiny sugar ldquodecorationsrdquoadded as an afterthought

Lodish et al (1995) Molecular Cell Biology 3rd ed

The ldquoglycocalyxrdquo surrounding a fibroblast Cell surface carbohydrates are stained black

lipid bilayer

fibroblast

extracellular

intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface -- The Real Picture

Tropical Forest

Canopy

Cohen amp Varki (2010) OMICS 4455

ldquoEvolution has failed to generate a living cell devoid of surface glycosylationrdquo - A Varki

lipid bilayer

fibroblast intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface

Real image textbook image

bull Among their diverse functions glycans hellipndash encode intermolecular and cell-cell recognitionndash regulate the activities of proteins (enzymes ion channels

receptors)ndash Define biophysical space (large hydrated)

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

Glycobiology is hellip NOT Carbohydrates as food

Berg Tymoczko amp Stryer (2006) Biochemistry Sixth Edition WH Freeman New York

ldquoCarbohydrates in food are important sources of energy Starch found in plant-derived food such as pasta rdquo

Glycobiology is NOT ldquoardquo post-translational modification it is thousands

Modified from httpwww-chesyredu facultyhouglandhtml

bull Combinatorial linkage position orientation and branching provide the potential for millions of different glycan structures ndash functional diversity

bull Glycans may be larger and are more diverse than their (protein) carriers

bull A glycanrsquos function can supersede that of its (protein) carrier

bull 1-2 of the human genome is devoted to glycosylation

Cummings RD (2009) Molecular BioSystems 5 1087

Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical petasaccharide sequeces in glycosaminoglycans

Glycobiology is hellip NOT a ldquodecorationrdquo

ldquoStructurerdquo of HIV gp120Zolla-Pazner (2004)Nature Reviews Immunology 4 199

N-linked carbohydrate can form both an immunologically silent facemdashwith carbohydrate masquerading as selfmdashand also can protect neighboring epitopes through an evolving glycan shield

httpwwwniaidnihgovlabsandresourceslabsaboutlabsvrcstructuralbiologylaboratoryPageskwongaspx

CD59 a complement defense glycoprotein

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchor

Proteins typically fold inward whereas glycans spread out in space

ldquonakedrdquo CD59 Huang et al (2007)Acta Crystallographica 63 714

Rudd et al (1997) J Biol Chem 272 7229

Scanning electron microscopy of C neoformans yeast cellsVan Duin et al (2004) Antimicrobial Agents and Chemotherapy 482014

Light microscopy micrograph of Cryptococcus neoformans

Steenbergen et al (2003) Microbes and Infection 5667

What if a MAJOR cell component was invisible by standard microscopy

Light microscopy micrograph of Cryptococcus neoformans capsule delineated by India ink The inner circle represents the fungal cell with the wide outer circle being the capsuleSteenbergen et al (2003) Microbes and Infection 5667

Electron microscopic thin section ofEscherichia coli K1Amako et al (1988) J Bacteriol 1704960

Unique to yeast ndash NO

Electron microscopic thin section ofKlebsiella pneumoniaeAmako et al (1988) J Bacteriol 1704960

The Eukaryotic Cell Surface

Tiny sugar ldquodecorationsrdquoadded as an afterthought

Lodish et al (1995) Molecular Cell Biology 3rd ed

The ldquoglycocalyxrdquo surrounding a fibroblast Cell surface carbohydrates are stained black

lipid bilayer

fibroblast

extracellular

intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface -- The Real Picture

Tropical Forest

Canopy

Cohen amp Varki (2010) OMICS 4455

ldquoEvolution has failed to generate a living cell devoid of surface glycosylationrdquo - A Varki

lipid bilayer

fibroblast intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface

Real image textbook image

bull Among their diverse functions glycans hellipndash encode intermolecular and cell-cell recognitionndash regulate the activities of proteins (enzymes ion channels

receptors)ndash Define biophysical space (large hydrated)

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

Glycobiology is NOT ldquoardquo post-translational modification it is thousands

Modified from httpwww-chesyredu facultyhouglandhtml

bull Combinatorial linkage position orientation and branching provide the potential for millions of different glycan structures ndash functional diversity

bull Glycans may be larger and are more diverse than their (protein) carriers

bull A glycanrsquos function can supersede that of its (protein) carrier

bull 1-2 of the human genome is devoted to glycosylation

Cummings RD (2009) Molecular BioSystems 5 1087

Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical petasaccharide sequeces in glycosaminoglycans

Glycobiology is hellip NOT a ldquodecorationrdquo

ldquoStructurerdquo of HIV gp120Zolla-Pazner (2004)Nature Reviews Immunology 4 199

N-linked carbohydrate can form both an immunologically silent facemdashwith carbohydrate masquerading as selfmdashand also can protect neighboring epitopes through an evolving glycan shield

httpwwwniaidnihgovlabsandresourceslabsaboutlabsvrcstructuralbiologylaboratoryPageskwongaspx

CD59 a complement defense glycoprotein

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchor

Proteins typically fold inward whereas glycans spread out in space

ldquonakedrdquo CD59 Huang et al (2007)Acta Crystallographica 63 714

Rudd et al (1997) J Biol Chem 272 7229

Scanning electron microscopy of C neoformans yeast cellsVan Duin et al (2004) Antimicrobial Agents and Chemotherapy 482014

Light microscopy micrograph of Cryptococcus neoformans

Steenbergen et al (2003) Microbes and Infection 5667

What if a MAJOR cell component was invisible by standard microscopy

Light microscopy micrograph of Cryptococcus neoformans capsule delineated by India ink The inner circle represents the fungal cell with the wide outer circle being the capsuleSteenbergen et al (2003) Microbes and Infection 5667

Electron microscopic thin section ofEscherichia coli K1Amako et al (1988) J Bacteriol 1704960

Unique to yeast ndash NO

Electron microscopic thin section ofKlebsiella pneumoniaeAmako et al (1988) J Bacteriol 1704960

The Eukaryotic Cell Surface

Tiny sugar ldquodecorationsrdquoadded as an afterthought

Lodish et al (1995) Molecular Cell Biology 3rd ed

The ldquoglycocalyxrdquo surrounding a fibroblast Cell surface carbohydrates are stained black

lipid bilayer

fibroblast

extracellular

intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface -- The Real Picture

Tropical Forest

Canopy

Cohen amp Varki (2010) OMICS 4455

ldquoEvolution has failed to generate a living cell devoid of surface glycosylationrdquo - A Varki

lipid bilayer

fibroblast intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface

Real image textbook image

bull Among their diverse functions glycans hellipndash encode intermolecular and cell-cell recognitionndash regulate the activities of proteins (enzymes ion channels

receptors)ndash Define biophysical space (large hydrated)

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

Glycobiology is hellip NOT a ldquodecorationrdquo

ldquoStructurerdquo of HIV gp120Zolla-Pazner (2004)Nature Reviews Immunology 4 199

N-linked carbohydrate can form both an immunologically silent facemdashwith carbohydrate masquerading as selfmdashand also can protect neighboring epitopes through an evolving glycan shield

httpwwwniaidnihgovlabsandresourceslabsaboutlabsvrcstructuralbiologylaboratoryPageskwongaspx

CD59 a complement defense glycoprotein

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchor

Proteins typically fold inward whereas glycans spread out in space

ldquonakedrdquo CD59 Huang et al (2007)Acta Crystallographica 63 714

Rudd et al (1997) J Biol Chem 272 7229

Scanning electron microscopy of C neoformans yeast cellsVan Duin et al (2004) Antimicrobial Agents and Chemotherapy 482014

Light microscopy micrograph of Cryptococcus neoformans

Steenbergen et al (2003) Microbes and Infection 5667

What if a MAJOR cell component was invisible by standard microscopy

Light microscopy micrograph of Cryptococcus neoformans capsule delineated by India ink The inner circle represents the fungal cell with the wide outer circle being the capsuleSteenbergen et al (2003) Microbes and Infection 5667

Electron microscopic thin section ofEscherichia coli K1Amako et al (1988) J Bacteriol 1704960

Unique to yeast ndash NO

Electron microscopic thin section ofKlebsiella pneumoniaeAmako et al (1988) J Bacteriol 1704960

The Eukaryotic Cell Surface

Tiny sugar ldquodecorationsrdquoadded as an afterthought

Lodish et al (1995) Molecular Cell Biology 3rd ed

The ldquoglycocalyxrdquo surrounding a fibroblast Cell surface carbohydrates are stained black

lipid bilayer

fibroblast

extracellular

intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface -- The Real Picture

Tropical Forest

Canopy

Cohen amp Varki (2010) OMICS 4455

ldquoEvolution has failed to generate a living cell devoid of surface glycosylationrdquo - A Varki

lipid bilayer

fibroblast intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface

Real image textbook image

bull Among their diverse functions glycans hellipndash encode intermolecular and cell-cell recognitionndash regulate the activities of proteins (enzymes ion channels

receptors)ndash Define biophysical space (large hydrated)

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

CD59 a complement defense glycoprotein

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchor

Proteins typically fold inward whereas glycans spread out in space

ldquonakedrdquo CD59 Huang et al (2007)Acta Crystallographica 63 714

Rudd et al (1997) J Biol Chem 272 7229

Scanning electron microscopy of C neoformans yeast cellsVan Duin et al (2004) Antimicrobial Agents and Chemotherapy 482014

Light microscopy micrograph of Cryptococcus neoformans

Steenbergen et al (2003) Microbes and Infection 5667

What if a MAJOR cell component was invisible by standard microscopy

Light microscopy micrograph of Cryptococcus neoformans capsule delineated by India ink The inner circle represents the fungal cell with the wide outer circle being the capsuleSteenbergen et al (2003) Microbes and Infection 5667

Electron microscopic thin section ofEscherichia coli K1Amako et al (1988) J Bacteriol 1704960

Unique to yeast ndash NO

Electron microscopic thin section ofKlebsiella pneumoniaeAmako et al (1988) J Bacteriol 1704960

The Eukaryotic Cell Surface

Tiny sugar ldquodecorationsrdquoadded as an afterthought

Lodish et al (1995) Molecular Cell Biology 3rd ed

The ldquoglycocalyxrdquo surrounding a fibroblast Cell surface carbohydrates are stained black

lipid bilayer

fibroblast

extracellular

intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface -- The Real Picture

Tropical Forest

Canopy

Cohen amp Varki (2010) OMICS 4455

ldquoEvolution has failed to generate a living cell devoid of surface glycosylationrdquo - A Varki

lipid bilayer

fibroblast intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface

Real image textbook image

bull Among their diverse functions glycans hellipndash encode intermolecular and cell-cell recognitionndash regulate the activities of proteins (enzymes ion channels

receptors)ndash Define biophysical space (large hydrated)

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

Scanning electron microscopy of C neoformans yeast cellsVan Duin et al (2004) Antimicrobial Agents and Chemotherapy 482014

Light microscopy micrograph of Cryptococcus neoformans

Steenbergen et al (2003) Microbes and Infection 5667

What if a MAJOR cell component was invisible by standard microscopy

Light microscopy micrograph of Cryptococcus neoformans capsule delineated by India ink The inner circle represents the fungal cell with the wide outer circle being the capsuleSteenbergen et al (2003) Microbes and Infection 5667

Electron microscopic thin section ofEscherichia coli K1Amako et al (1988) J Bacteriol 1704960

Unique to yeast ndash NO

Electron microscopic thin section ofKlebsiella pneumoniaeAmako et al (1988) J Bacteriol 1704960

The Eukaryotic Cell Surface

Tiny sugar ldquodecorationsrdquoadded as an afterthought

Lodish et al (1995) Molecular Cell Biology 3rd ed

The ldquoglycocalyxrdquo surrounding a fibroblast Cell surface carbohydrates are stained black

lipid bilayer

fibroblast

extracellular

intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface -- The Real Picture

Tropical Forest

Canopy

Cohen amp Varki (2010) OMICS 4455

ldquoEvolution has failed to generate a living cell devoid of surface glycosylationrdquo - A Varki

lipid bilayer

fibroblast intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface

Real image textbook image

bull Among their diverse functions glycans hellipndash encode intermolecular and cell-cell recognitionndash regulate the activities of proteins (enzymes ion channels

receptors)ndash Define biophysical space (large hydrated)

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

Electron microscopic thin section ofEscherichia coli K1Amako et al (1988) J Bacteriol 1704960

Unique to yeast ndash NO

Electron microscopic thin section ofKlebsiella pneumoniaeAmako et al (1988) J Bacteriol 1704960

The Eukaryotic Cell Surface

Tiny sugar ldquodecorationsrdquoadded as an afterthought

Lodish et al (1995) Molecular Cell Biology 3rd ed

The ldquoglycocalyxrdquo surrounding a fibroblast Cell surface carbohydrates are stained black

lipid bilayer

fibroblast

extracellular

intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface -- The Real Picture

Tropical Forest

Canopy

Cohen amp Varki (2010) OMICS 4455

ldquoEvolution has failed to generate a living cell devoid of surface glycosylationrdquo - A Varki

lipid bilayer

fibroblast intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface

Real image textbook image

bull Among their diverse functions glycans hellipndash encode intermolecular and cell-cell recognitionndash regulate the activities of proteins (enzymes ion channels

receptors)ndash Define biophysical space (large hydrated)

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

The Eukaryotic Cell Surface

Tiny sugar ldquodecorationsrdquoadded as an afterthought

Lodish et al (1995) Molecular Cell Biology 3rd ed

The ldquoglycocalyxrdquo surrounding a fibroblast Cell surface carbohydrates are stained black

lipid bilayer

fibroblast

extracellular

intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface -- The Real Picture

Tropical Forest

Canopy

Cohen amp Varki (2010) OMICS 4455

ldquoEvolution has failed to generate a living cell devoid of surface glycosylationrdquo - A Varki

lipid bilayer

fibroblast intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface

Real image textbook image

bull Among their diverse functions glycans hellipndash encode intermolecular and cell-cell recognitionndash regulate the activities of proteins (enzymes ion channels

receptors)ndash Define biophysical space (large hydrated)

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

The ldquoglycocalyxrdquo surrounding a fibroblast Cell surface carbohydrates are stained black

lipid bilayer

fibroblast

extracellular

intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface -- The Real Picture

Tropical Forest

Canopy

Cohen amp Varki (2010) OMICS 4455

ldquoEvolution has failed to generate a living cell devoid of surface glycosylationrdquo - A Varki

lipid bilayer

fibroblast intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface

Real image textbook image

bull Among their diverse functions glycans hellipndash encode intermolecular and cell-cell recognitionndash regulate the activities of proteins (enzymes ion channels

receptors)ndash Define biophysical space (large hydrated)

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

Tropical Forest

Canopy

Cohen amp Varki (2010) OMICS 4455

ldquoEvolution has failed to generate a living cell devoid of surface glycosylationrdquo - A Varki

lipid bilayer

fibroblast intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface

Real image textbook image

bull Among their diverse functions glycans hellipndash encode intermolecular and cell-cell recognitionndash regulate the activities of proteins (enzymes ion channels

receptors)ndash Define biophysical space (large hydrated)

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

lipid bilayer

fibroblast intracellular

Martinez-Palomo A et al Cancer Res 29 925-937 1969

The Cell Surface

Real image textbook image

bull Among their diverse functions glycans hellipndash encode intermolecular and cell-cell recognitionndash regulate the activities of proteins (enzymes ion channels

receptors)ndash Define biophysical space (large hydrated)

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

Rosetta Stone British Museum

Glycobiology = Language

semiotics the theory of signs and symbols

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

Carbohydrates ndash the building blocks of Glycobiology

(CH2O)n = ldquocarbordquo ldquohydraterdquo

Generalized Ketose

Generalized Aldose

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

glucose fructose

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

Enantiomers mirror images of each other that are not superimposable

Diastereomers stereoisomers that are not enantiomers

Generalized Aldose

Monosaccharide structural identity is all about stereochemistry

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

Fischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

L-glyceraldehydeD-glyceraldehyde

Enantiomers

Highest numbered asymmetric carbon = reference carbon

Diasteriomers

Highest numbered asymmetric carbon = reference carbon

Enantiomers amp Diasteriomers

Highest numbered asymmetric carbon = reference carbon

carbons asymmetric carbons

diaster-

iomers

diasteriomers amp

enantiomers

3 1 0 2

4 2 2 4

5 3 4 8

6 4 8 16

Essentials of Glycobiology Second Edition

epimers differ at a single stereogenic center

Monosaccharides equilibrate between open chain and ring formsTwo ring forms (anomeric configurations) are possible α and β

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

D-glucose

α-D-glucose

β-D-glucose

Monosaccharide conformation ndash Chair configuations

Taylor amp Drickamer (2006) Introduction to Glycobiology 2nd edition

Nearly all vertebrate glycans are built from only 9 sugars

3 hexosesbull Glucosebull Mannosebull Galactose

2 N-acetylhexosaminesbull N-acetylglucosaminebull N-acetylgalactosamine

bull xylosebull glucuronic acidbull sialic acidbull L-fucose

all D configuation except fucose

Seven eukaryotic sugars and their relationship to glucose

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

Two important terminal eukaryotic sugars

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

Glycan properties for molecular recognition and binding energy

Glucose Galactose

Sialic Acid

Mannose

Enantiomers amp Diasteriomers

Highest numbered asymmetric carbon = reference carbon

carbons asymmetric carbons

diaster-

iomers

diasteriomers amp

enantiomers

3 1 0 2

4 2 2 4

5 3 4 8

6 4 8 16

Essentials of Glycobiology Second Edition

epimers differ at a single stereogenic center

Monosaccharides equilibrate between open chain and ring formsTwo ring forms (anomeric configurations) are possible α and β

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

D-glucose

α-D-glucose

β-D-glucose

Monosaccharide conformation ndash Chair configuations

Taylor amp Drickamer (2006) Introduction to Glycobiology 2nd edition

Nearly all vertebrate glycans are built from only 9 sugars

3 hexosesbull Glucosebull Mannosebull Galactose

2 N-acetylhexosaminesbull N-acetylglucosaminebull N-acetylgalactosamine

bull xylosebull glucuronic acidbull sialic acidbull L-fucose

all D configuation except fucose

Seven eukaryotic sugars and their relationship to glucose

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

Two important terminal eukaryotic sugars

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

Glycan properties for molecular recognition and binding energy

Glucose Galactose

Sialic Acid

Mannose

Essentials of Glycobiology Second Edition

epimers differ at a single stereogenic center

Monosaccharides equilibrate between open chain and ring formsTwo ring forms (anomeric configurations) are possible α and β

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

D-glucose

α-D-glucose

β-D-glucose

Monosaccharide conformation ndash Chair configuations

Taylor amp Drickamer (2006) Introduction to Glycobiology 2nd edition

Nearly all vertebrate glycans are built from only 9 sugars

3 hexosesbull Glucosebull Mannosebull Galactose

2 N-acetylhexosaminesbull N-acetylglucosaminebull N-acetylgalactosamine

bull xylosebull glucuronic acidbull sialic acidbull L-fucose

all D configuation except fucose

Seven eukaryotic sugars and their relationship to glucose

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

Two important terminal eukaryotic sugars

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

Glycan properties for molecular recognition and binding energy

Glucose Galactose

Sialic Acid

Mannose

Monosaccharides equilibrate between open chain and ring formsTwo ring forms (anomeric configurations) are possible α and β

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

D-glucose

α-D-glucose

β-D-glucose

Monosaccharide conformation ndash Chair configuations

Taylor amp Drickamer (2006) Introduction to Glycobiology 2nd edition

Nearly all vertebrate glycans are built from only 9 sugars

3 hexosesbull Glucosebull Mannosebull Galactose

2 N-acetylhexosaminesbull N-acetylglucosaminebull N-acetylgalactosamine

bull xylosebull glucuronic acidbull sialic acidbull L-fucose

all D configuation except fucose

Seven eukaryotic sugars and their relationship to glucose

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

Two important terminal eukaryotic sugars

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

Glycan properties for molecular recognition and binding energy

Glucose Galactose

Sialic Acid

Mannose

Monosaccharide conformation ndash Chair configuations

Taylor amp Drickamer (2006) Introduction to Glycobiology 2nd edition

Nearly all vertebrate glycans are built from only 9 sugars

3 hexosesbull Glucosebull Mannosebull Galactose

2 N-acetylhexosaminesbull N-acetylglucosaminebull N-acetylgalactosamine

bull xylosebull glucuronic acidbull sialic acidbull L-fucose

all D configuation except fucose

Seven eukaryotic sugars and their relationship to glucose

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

Two important terminal eukaryotic sugars

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

Glycan properties for molecular recognition and binding energy

Glucose Galactose

Sialic Acid

Mannose

Nearly all vertebrate glycans are built from only 9 sugars

3 hexosesbull Glucosebull Mannosebull Galactose

2 N-acetylhexosaminesbull N-acetylglucosaminebull N-acetylgalactosamine

bull xylosebull glucuronic acidbull sialic acidbull L-fucose

all D configuation except fucose

Seven eukaryotic sugars and their relationship to glucose

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

Two important terminal eukaryotic sugars

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

Glycan properties for molecular recognition and binding energy

Glucose Galactose

Sialic Acid

Mannose

Seven eukaryotic sugars and their relationship to glucose

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

Two important terminal eukaryotic sugars

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

Glycan properties for molecular recognition and binding energy

Glucose Galactose

Sialic Acid

Mannose

Two important terminal eukaryotic sugars

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

Glycan properties for molecular recognition and binding energy

Glucose Galactose

Sialic Acid

Mannose

Glycan properties for molecular recognition and binding energy

Glucose Galactose

Sialic Acid

Mannose

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

When a glycosidic bond is formed the anomeric configuration is ldquolockedrdquo

Glycosidic bonds link sugars together

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

(lactose) CCRC

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

When a glycosidic bond is formed the anomeric configuration is ldquolockedrdquo

Glycosidic bonds link sugars together

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

(lactose) CCRC

When a glycosidic bond is formed the anomeric configuration is ldquolockedrdquo

Glycosidic bonds link sugars together

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

(lactose) CCRC

Nomenclature

-Name the non-reducing (left-most) sugar (Gal) -Name the anomeric configuration (β) -Name the anomeric carbon number (1) -Name the substituted carbon number (4) -Name the substituted sugar (Glc)

RESULT Gal β1-4 Glc

Taylor amp Drickamer (2011) Introduction to Glycobiology 3rd edition

NeuAc α2-3 Gal β1-4 (Fuc α1-3) GlcNAc

Nomenclature Branches are placed in parentheses

Varki et al (2009) Proteomics 95398

Oligosaccharides Molecular diversity of glycans

Polypeptides Glycans

Building blocks amino acids monosaccharides

Number of different monomers 20 common 9 common

Linkage sites per monomer 1 3-4

Possible linkage configurations 1 2

Possible homodimer structures 1 6-8

Linkage modes linear linear or branched

HO

HO

O

HO

O

GalHO

OHO

HO

O

HO

O

GalHO

O

H2N C

Ala

OH

OH2N C

Ala

OH

O

Three different amino acids (Ala Ser Tyr) ndash 6 structures

Ala-Ser-TyrAla-Tyr-SerSer-Ala-TyrSer-Tyr-AlaTyr-Ala-SerTyr-Ser-Ala

Three different sugars (Glc Gal Man) ndash 1056 structures hellip

Gal a1-2 Glc a1-2 Man a1-Gal a1-3 Glc a1-2 Man a1-Gal a1-4 Glc a1-2 Man a1-Gal a1-6 Glc a1-2 Man a1-Gal b1-2 Glc a1-2 Man a1-Gal b1-3 Glc a1-2 Man a1-Gal b1-4 Glc a1-2 Man a1-Gal b1-6 Glc a1-2 Man a1-Gal a1-2 Glc a1-3 Man a1-Gal a1-3 Glc a1-3 Man a1-Gal a1-4 Glc a1-3 Man a1-Gal a1-6 Glc a1-3 Man a1-Gal b1-2 Glc a1-3 Man a1-Gal b1-3 Glc a1-3 Man a1-Gal b1-4 Glc a1-3 Man a1-Gal b1-6 Glc a1-3 Man a1-Gal a1-2 Glc a1-4 Man a1-Gal a1-3 Glc a1-4 Man a1-Gal a1-4 Glc a1-4 Man a1-Gal a1-6 Glc a1-4 Man a1-Gal b1-2 Glc a1-4 Man a1-Gal b1-3 Glc a1-4 Man a1-Gal b1-4 Glc a1-4 Man a1-Gal b1-6 Glc a1-4 Man a1-Gal a1-2 Glc a1-6 Man a1-Gal a1-3 Glc a1-6 Man a1-Gal a1-4 Glc a1-6 Man a1-Gal a1-6 Glc a1-6 Man a1-Gal b1-2 Glc a1-6 Man a1-Gal b1-3 Glc a1-6 Man a1-Gal b1-4 Glc a1-6 Man a1-Gal b1-6 Glc a1-6 Man a1-Gal a1-2 Glc b1-2 Man a1-Gal a1-3 Glc b1-2 Man a1-Gal a1-4 Glc b1-2 Man a1-Gal a1-6 Glc b1-2 Man a1-Gal b1-2 Glc b1-2 Man a1-Gal b1-3 Glc b1-2 Man a1-Gal b1-4 Glc b1-2 Man a1-Gal b1-6 Glc b1-2 Man a1-Gal a1-2 Glc b1-3 Man a1-Gal a1-3 Glc b1-3 Man a1-Gal a1-4 Glc b1-3 Man a1-Gal a1-6 Glc b1-3 Man a1-Gal b1-2 Glc b1-3 Man a1-Gal b1-3 Glc b1-3 Man a1-Gal b1-4 Glc b1-3 Man a1-Gal b1-6 Glc b1-3 Man a1-Gal a1-2 Glc b1-4 Man a1-Gal a1-3 Glc b1-4 Man a1-Gal a1-4 Glc b1-4 Man a1-Gal a1-6 Glc b1-4 Man a1-Gal b1-2 Glc b1-4 Man a1-Gal b1-3 Glc b1-4 Man a1-Gal b1-4 Glc b1-4 Man a1-Gal b1-6 Glc b1-4 Man a1-Gal a1-2 Glc b1-6 Man a1-Gal a1-3 Glc b1-6 Man a1-Gal a1-4 Glc b1-6 Man a1-Gal a1-6 Glc b1-6 Man a1-Gal b1-2 Glc b1-6 Man a1-Gal b1-3 Glc b1-6 Man a1-Gal b1-4 Glc b1-6 Man a1-Gal b1-6 Glc b1-6 Man a1-Gal a1-2 Glc a1-2 Man b1-Gal a1-3 Glc a1-2 Man b1-Gal a1-4 Glc a1-2 Man b1-Gal a1-6 Glc a1-2 Man b1-Gal b1-2 Glc a1-2 Man b1-Gal b1-3 Glc a1-2 Man b1-Gal b1-4 Glc a1-2 Man b1-Gal b1-6 Glc a1-2 Man b1-Gal a1-2 Glc a1-3 Man b1-Gal a1-3 Glc a1-3 Man b1-Gal a1-4 Glc a1-3 Man b1-Gal a1-6 Glc a1-3 Man b1-Gal b1-2 Glc a1-3 Man b1-Gal b1-3 Glc a1-3 Man b1-Gal b1-4 Glc a1-3 Man b1-Gal b1-6 Glc a1-3 Man b1-Gal a1-2 Glc a1-4 Man b1-Gal a1-3 Glc a1-4 Man b1-Gal a1-4 Glc a1-4 Man b1-Gal a1-6 Glc a1-4 Man b1-Gal b1-2 Glc a1-4 Man b1-Gal b1-3 Glc a1-4 Man b1-Gal b1-4 Glc a1-4 Man b1-Gal b1-6 Glc a1-4 Man b1-Gal a1-2 Glc a1-6 Man b1-Gal a1-3 Glc a1-6 Man b1-Gal a1-4 Glc a1-6 Man b1-Gal a1-6 Glc a1-6 Man b1-Gal b1-2 Glc a1-6 Man b1-

Gal b1-3 Glc a1-6 Man b1-Gal b1-4 Glc a1-6 Man b1-Gal b1-6 Glc a1-6 Man b1-Gal a1-2 Glc b1-2 Man b1-Gal a1-3 Glc b1-2 Man b1-Gal a1-4 Glc b1-2 Man b1-Gal a1-6 Glc b1-2 Man b1-Gal b1-2 Glc b1-2 Man b1-Gal b1-3 Glc b1-2 Man b1-Gal b1-4 Glc b1-2 Man b1-Gal b1-6 Glc b1-2 Man b1-Gal a1-2 Glc b1-3 Man b1-Gal a1-3 Glc b1-3 Man b1-Gal a1-4 Glc b1-3 Man b1-Gal a1-6 Glc b1-3 Man b1-Gal b1-2 Glc b1-3 Man b1-Gal b1-3 Glc b1-3 Man b1-Gal b1-4 Glc b1-3 Man b1-Gal b1-6 Glc b1-3 Man b1-Gal a1-2 Glc b1-4 Man b1-Gal a1-3 Glc b1-4 Man b1-Gal a1-4 Glc b1-4 Man b1-Gal a1-6 Glc b1-4 Man b1-Gal b1-2 Glc b1-4 Man b1-Gal b1-3 Glc b1-4 Man b1-Gal b1-4 Glc b1-4 Man b1-Gal b1-6 Glc b1-4 Man b1-Gal a1-2 Glc b1-6 Man b1-Gal a1-3 Glc b1-6 Man b1-Gal a1-4 Glc b1-6 Man b1-Gal a1-6 Glc b1-6 Man b1-Gal b1-2 Glc b1-6 Man b1-Gal b1-3 Glc b1-6 Man b1-Gal b1-4 Glc b1-6 Man b1-Gal b1-6 Glc b1-6 Man b1-Gal a1-2 Man a1-2 Glc a1-Gal a1-3 Man a1-2 Glc a1-Gal a1-4 Man a1-2 Glc a1-Gal a1-6 Man a1-2 Glc a1-Gal b1-2 Man a1-2 Glc a1-Gal b1-3 Man a1-2 Glc a1-Gal b1-4 Man a1-2 Glc a1-Gal b1-6 Man a1-2 Glc a1-Gal a1-2 Man a1-3 Glc a1-Gal a1-3 Man a1-3 Glc a1-Gal a1-4 Man a1-3 Glc a1-Gal a1-6 Man a1-3 Glc a1-Gal b1-2 Man a1-3 Glc a1-Gal b1-3 Man a1-3 Glc a1-Gal b1-4 Man a1-3 Glc a1-Gal b1-6 Man a1-3 Glc a1-Gal a1-2 Man a1-4 Glc a1-Gal a1-3 Man a1-4 Glc a1-Gal a1-4 Man a1-4 Glc a1-Gal a1-6 Man a1-4 Glc a1-Gal b1-2 Man a1-4 Glc a1-Gal b1-3 Man a1-4 Glc a1-Gal b1-4 Man a1-4 Glc a1-Gal b1-6 Man a1-4 Glc a1-Gal a1-2 Man a1-6 Glc a1-Gal a1-3 Man a1-6 Glc a1-Gal a1-4 Man a1-6 Glc a1-Gal a1-6 Man a1-6 Glc a1-Gal b1-2 Man a1-6 Glc a1-Gal b1-3 Man a1-6 Glc a1-Gal b1-4 Man a1-6 Glc a1-Gal b1-6 Man a1-6 Glc a1-Gal a1-2 Man b1-2 Glc a1-Gal a1-3 Man b1-2 Glc a1-Gal a1-4 Man b1-2 Glc a1-Gal a1-6 Man b1-2 Glc a1-Gal b1-2 Man b1-2 Glc a1-Gal b1-3 Man b1-2 Glc a1-Gal b1-4 Man b1-2 Glc a1-Gal b1-6 Man b1-2 Glc a1-Gal a1-2 Man b1-3 Glc a1-Gal a1-3 Man b1-3 Glc a1-Gal a1-4 Man b1-3 Glc a1-Gal a1-6 Man b1-3 Glc a1-Gal b1-2 Man b1-3 Glc a1-Gal b1-3 Man b1-3 Glc a1-Gal b1-4 Man b1-3 Glc a1-Gal b1-6 Man b1-3 Glc a1-Gal a1-2 Man b1-4 Glc a1-Gal a1-3 Man b1-4 Glc a1-Gal a1-4 Man b1-4 Glc a1-Gal a1-6 Man b1-4 Glc a1-Gal b1-2 Man b1-4 Glc a1-Gal b1-3 Man b1-4 Glc a1-Gal b1-4 Man b1-4 Glc a1-Gal b1-6 Man b1-4 Glc a1-Gal a1-2 Man b1-6 Glc a1-Gal a1-3 Man b1-6 Glc a1-

Gal a1-4 Man b1-6 Glc a1-Gal a1-6 Man b1-6 Glc a1-Gal b1-2 Man b1-6 Glc a1-Gal b1-3 Man b1-6 Glc a1-Gal b1-4 Man b1-6 Glc a1-Gal b1-6 Man b1-6 Glc a1-Gal a1-2 Man a1-2 Glc b1-Gal a1-3 Man a1-2 Glc b1-Gal a1-4 Man a1-2 Glc b1-Gal a1-6 Man a1-2 Glc b1-Gal b1-2 Man a1-2 Glc b1-Gal b1-3 Man a1-2 Glc b1-Gal b1-4 Man a1-2 Glc b1-Gal b1-6 Man a1-2 Glc b1-Gal a1-2 Man a1-3 Glc b1-Gal a1-3 Man a1-3 Glc b1-Gal a1-4 Man a1-3 Glc b1-Gal a1-6 Man a1-3 Glc b1-Gal b1-2 Man a1-3 Glc b1-Gal b1-3 Man a1-3 Glc b1-Gal b1-4 Man a1-3 Glc b1-Gal b1-6 Man a1-3 Glc b1-Gal a1-2 Man a1-4 Glc b1-Gal a1-3 Man a1-4 Glc b1-Gal a1-4 Man a1-4 Glc b1-Gal a1-6 Man a1-4 Glc b1-Gal b1-2 Man a1-4 Glc b1-Gal b1-3 Man a1-4 Glc b1-Gal b1-4 Man a1-4 Glc b1-Gal b1-6 Man a1-4 Glc b1-Gal a1-2 Man a1-6 Glc b1-Gal a1-3 Man a1-6 Glc b1-Gal a1-4 Man a1-6 Glc b1-Gal a1-6 Man a1-6 Glc b1-Gal b1-2 Man a1-6 Glc b1-Gal b1-3 Man a1-6 Glc b1-Gal b1-4 Man a1-6 Glc b1-Gal b1-6 Man a1-6 Glc b1-Gal a1-2 Man b1-2 Glc b1-Gal a1-3 Man b1-2 Glc b1-Gal a1-4 Man b1-2 Glc b1-Gal a1-6 Man b1-2 Glc b1-Gal b1-2 Man b1-2 Glc b1-Gal b1-3 Man b1-2 Glc b1-Gal b1-4 Man b1-2 Glc b1-Gal b1-6 Man b1-2 Glc b1-Gal a1-2 Man b1-3 Glc b1-Gal a1-3 Man b1-3 Glc b1-Gal a1-4 Man b1-3 Glc b1-Gal a1-6 Man b1-3 Glc b1-Gal b1-2 Man b1-3 Glc b1-Gal b1-3 Man b1-3 Glc b1-Gal b1-4 Man b1-3 Glc b1-Gal b1-6 Man b1-3 Glc b1-Gal a1-2 Man b1-4 Glc b1-Gal a1-3 Man b1-4 Glc b1-Gal a1-4 Man b1-4 Glc b1-Gal a1-6 Man b1-4 Glc b1-Gal b1-2 Man b1-4 Glc b1-Gal b1-3 Man b1-4 Glc b1-Gal b1-4 Man b1-4 Glc b1-Gal b1-6 Man b1-4 Glc b1-Gal a1-2 Man b1-6 Glc b1-Gal a1-3 Man b1-6 Glc b1-Gal a1-4 Man b1-6 Glc b1-Gal a1-6 Man b1-6 Glc b1-Gal b1-2 Man b1-6 Glc b1-Gal b1-3 Man b1-6 Glc b1-Gal b1-4 Man b1-6 Glc b1-Gal b1-6 Man b1-6 Glc b1-Man a1-2 Glc a1-2 Gal a1-Man a1-3 Glc a1-2 Gal a1-Man a1-4 Glc a1-2 Gal a1-Man a1-6 Glc a1-2 Gal a1-Man b1-2 Glc a1-2 Gal a1-Man b1-3 Glc a1-2 Gal a1-Man b1-4 Glc a1-2 Gal a1-Man b1-6 Glc a1-2 Gal a1-Man a1-2 Glc a1-3 Gal a1-Man a1-3 Glc a1-3 Gal a1-Man a1-4 Glc a1-3 Gal a1-Man a1-6 Glc a1-3 Gal a1-Man b1-2 Glc a1-3 Gal a1-Man b1-3 Glc a1-3 Gal a1-Man b1-4 Glc a1-3 Gal a1-Man b1-6 Glc a1-3 Gal a1-Man a1-2 Glc a1-4 Gal a1-Man a1-3 Glc a1-4 Gal a1-Man a1-4 Glc a1-4 Gal a1-Man a1-6 Glc a1-4 Gal a1-Man b1-2 Glc a1-4 Gal a1-Man b1-3 Glc a1-4 Gal a1-Man b1-4 Glc a1-4 Gal a1-

Man b1-6 Glc a1-4 Gal a1-Man a1-2 Glc a1-6 Gal a1-Man a1-3 Glc a1-6 Gal a1-Man a1-4 Glc a1-6 Gal a1-Man a1-6 Glc a1-6 Gal a1-Man b1-2 Glc a1-6 Gal a1-Man b1-3 Glc a1-6 Gal a1-Man b1-4 Glc a1-6 Gal a1-Man b1-6 Glc a1-6 Gal a1-Man a1-2 Glc b1-2 Gal a1-Man a1-3 Glc b1-2 Gal a1-Man a1-4 Glc b1-2 Gal a1-Man a1-6 Glc b1-2 Gal a1-Man b1-2 Glc b1-2 Gal a1-Man b1-3 Glc b1-2 Gal a1-Man b1-4 Glc b1-2 Gal a1-Man b1-6 Glc b1-2 Gal a1-Man a1-2 Glc b1-3 Gal a1-Man a1-3 Glc b1-3 Gal a1-Man a1-4 Glc b1-3 Gal a1-Man a1-6 Glc b1-3 Gal a1-Man b1-2 Glc b1-3 Gal a1-Man b1-3 Glc b1-3 Gal a1-Man b1-4 Glc b1-3 Gal a1-Man b1-6 Glc b1-3 Gal a1-Man a1-2 Glc b1-4 Gal a1-Man a1-3 Glc b1-4 Gal a1-Man a1-4 Glc b1-4 Gal a1-Man a1-6 Glc b1-4 Gal a1-Man b1-2 Glc b1-4 Gal a1-Man b1-3 Glc b1-4 Gal a1-Man b1-4 Glc b1-4 Gal a1-Man b1-6 Glc b1-4 Gal a1-Man a1-2 Glc b1-6 Gal a1-Man a1-3 Glc b1-6 Gal a1-Man a1-4 Glc b1-6 Gal a1-Man a1-6 Glc b1-6 Gal a1-Man b1-2 Glc b1-6 Gal a1-Man b1-3 Glc b1-6 Gal a1-Man b1-4 Glc b1-6 Gal a1-Man b1-6 Glc b1-6 Gal a1-Man a1-2 Glc a1-2 Gal b1-Man a1-3 Glc a1-2 Gal b1-Man a1-4 Glc a1-2 Gal b1-Man a1-6 Glc a1-2 Gal b1-Man b1-2 Glc a1-2 Gal b1-Man b1-3 Glc a1-2 Gal b1-Man b1-4 Glc a1-2 Gal b1-Man b1-6 Glc a1-2 Gal b1-Man a1-2 Glc a1-3 Gal b1-Man a1-3 Glc a1-3 Gal b1-Man a1-4 Glc a1-3 Gal b1-Man a1-6 Glc a1-3 Gal b1-Man b1-2 Glc a1-3 Gal b1-Man b1-3 Glc a1-3 Gal b1-Man b1-4 Glc a1-3 Gal b1-Man b1-6 Glc a1-3 Gal b1-Man a1-2 Glc a1-4 Gal b1-Man a1-3 Glc a1-4 Gal b1-Man a1-4 Glc a1-4 Gal b1-Man a1-6 Glc a1-4 Gal b1-Man b1-2 Glc a1-4 Gal b1-Man b1-3 Glc a1-4 Gal b1-Man b1-4 Glc a1-4 Gal b1-Man b1-6 Glc a1-4 Gal b1-Man a1-2 Glc a1-6 Gal b1-Man a1-3 Glc a1-6 Gal b1-Man a1-4 Glc a1-6 Gal b1-Man a1-6 Glc a1-6 Gal b1-Man b1-2 Glc a1-6 Gal b1-Man b1-3 Glc a1-6 Gal b1-Man b1-4 Glc a1-6 Gal b1-Man b1-6 Glc a1-6 Gal b1-Man a1-2 Glc b1-2 Gal b1-Man a1-3 Glc b1-2 Gal b1-Man a1-4 Glc b1-2 Gal b1-Man a1-6 Glc b1-2 Gal b1-Man b1-2 Glc b1-2 Gal b1-Man b1-3 Glc b1-2 Gal b1-Man b1-4 Glc b1-2 Gal b1-Man b1-6 Glc b1-2 Gal b1-Man a1-2 Glc b1-3 Gal b1-Man a1-3 Glc b1-3 Gal b1-Man a1-4 Glc b1-3 Gal b1-Man a1-6 Glc b1-3 Gal b1-Man b1-2 Glc b1-3 Gal b1-Man b1-3 Glc b1-3 Gal b1-Man b1-4 Glc b1-3 Gal b1-Man b1-6 Glc b1-3 Gal b1-Man a1-2 Glc b1-4 Gal b1-Man a1-3 Glc b1-4 Gal b1-Man a1-4 Glc b1-4 Gal b1-Man a1-6 Glc b1-4 Gal b1-

Man b1-2 Glc b1-4 Gal b1-Man b1-3 Glc b1-4 Gal b1-Man b1-4 Glc b1-4 Gal b1-Man b1-6 Glc b1-4 Gal b1-Man a1-2 Glc b1-6 Gal b1-Man a1-3 Glc b1-6 Gal b1-Man a1-4 Glc b1-6 Gal b1-Man a1-6 Glc b1-6 Gal b1-Man b1-2 Glc b1-6 Gal b1-Man b1-3 Glc b1-6 Gal b1-Man b1-4 Glc b1-6 Gal b1-Man b1-6 Glc b1-6 Gal b1-Man a1-2 Gal a1-2 Glc a1-Man a1-3 Gal a1-2 Glc a1-Man a1-4 Gal a1-2 Glc a1-Man a1-6 Gal a1-2 Glc a1-Man b1-2 Gal a1-2 Glc a1-Man b1-3 Gal a1-2 Glc a1-Man b1-4 Gal a1-2 Glc a1-Man b1-6 Gal a1-2 Glc a1-Man a1-2 Gal a1-3 Glc a1-Man a1-3 Gal a1-3 Glc a1-Man a1-4 Gal a1-3 Glc a1-Man a1-6 Gal a1-3 Glc a1-Man b1-2 Gal a1-3 Glc a1-Man b1-3 Gal a1-3 Glc a1-Man b1-4 Gal a1-3 Glc a1-Man b1-6 Gal a1-3 Glc a1-Man a1-2 Gal a1-4 Glc a1-Man a1-3 Gal a1-4 Glc a1-Man a1-4 Gal a1-4 Glc a1-Man a1-6 Gal a1-4 Glc a1-Man b1-2 Gal a1-4 Glc a1-Man b1-3 Gal a1-4 Glc a1-Man b1-4 Gal a1-4 Glc a1-Man b1-6 Gal a1-4 Glc a1-Man a1-2 Gal a1-6 Glc a1-Man a1-3 Gal a1-6 Glc a1-Man a1-4 Gal a1-6 Glc a1-Man a1-6 Gal a1-6 Glc a1-Man b1-2 Gal a1-6 Glc a1-Man b1-3 Gal a1-6 Glc a1-Man b1-4 Gal a1-6 Glc a1-Man b1-6 Gal a1-6 Glc a1-Man a1-2 Gal b1-2 Glc a1-Man a1-3 Gal b1-2 Glc a1-Man a1-4 Gal b1-2 Glc a1-Man a1-6 Gal b1-2 Glc a1-Man b1-2 Gal b1-2 Glc a1-Man b1-3 Gal b1-2 Glc a1-Man b1-4 Gal b1-2 Glc a1-Man b1-6 Gal b1-2 Glc a1-Man a1-2 Gal b1-3 Glc a1-Man a1-3 Gal b1-3 Glc a1-Man a1-4 Gal b1-3 Glc a1-Man a1-6 Gal b1-3 Glc a1-Man b1-2 Gal b1-3 Glc a1-Man b1-3 Gal b1-3 Glc a1-Man b1-4 Gal b1-3 Glc a1-Man b1-6 Gal b1-3 Glc a1-Man a1-2 Gal b1-4 Glc a1-Man a1-3 Gal b1-4 Glc a1-Man a1-4 Gal b1-4 Glc a1-Man a1-6 Gal b1-4 Glc a1-Man b1-2 Gal b1-4 Glc a1-Man b1-3 Gal b1-4 Glc a1-Man b1-4 Gal b1-4 Glc a1-Man b1-6 Gal b1-4 Glc a1-Man a1-2 Gal b1-6 Glc a1-Man a1-3 Gal b1-6 Glc a1-Man a1-4 Gal b1-6 Glc a1-Man a1-6 Gal b1-6 Glc a1-Man b1-2 Gal b1-6 Glc a1-Man b1-3 Gal b1-6 Glc a1-Man b1-4 Gal b1-6 Glc a1-Man b1-6 Gal b1-6 Glc a1-Man a1-2 Gal a1-2 Glc b1Man a1-3 Gal a1-2 Glc b1Man a1-4 Gal a1-2 Glc b1Man a1-6 Gal a1-2 Glc b1Man b1-2 Gal a1-2 Glc b1Man b1-3 Gal a1-2 Glc b1Man b1-4 Gal a1-2 Glc b1Man b1-6 Gal a1-2 Glc b1Man a1-2 Gal a1-3 Glc b1Man a1-3 Gal a1-3 Glc b1Man a1-4 Gal a1-3 Glc b1Man a1-6 Gal a1-3 Glc b1Man b1-2 Gal a1-3 Glc b1Man b1-3 Gal a1-3 Glc b1Man b1-4 Gal a1-3 Glc b1Man b1-6 Gal a1-3 Glc b1Man a1-2 Gal a1-4 Glc b1

Man a1-3 Gal a1-4 Glc b1Man a1-4 Gal a1-4 Glc b1Man a1-6 Gal a1-4 Glc b1Man b1-2 Gal a1-4 Glc b1Man b1-3 Gal a1-4 Glc b1Man b1-4 Gal a1-4 Glc b1Man b1-6 Gal a1-4 Glc b1Man a1-2 Gal a1-6 Glc b1Man a1-3 Gal a1-6 Glc b1Man a1-4 Gal a1-6 Glc b1Man a1-6 Gal a1-6 Glc b1Man b1-2 Gal a1-6 Glc b1Man b1-3 Gal a1-6 Glc b1Man b1-4 Gal a1-6 Glc b1Man b1-6 Gal a1-6 Glc b1Man a1-2 Gal b1-2 Glc b1Man a1-3 Gal b1-2 Glc b1Man a1-4 Gal b1-2 Glc b1Man a1-6 Gal b1-2 Glc b1Man b1-2 Gal b1-2 Glc b1Man b1-3 Gal b1-2 Glc b1Man b1-4 Gal b1-2 Glc b1Man b1-6 Gal b1-2 Glc b1Man a1-2 Gal b1-3 Glc b1Man a1-3 Gal b1-3 Glc b1Man a1-4 Gal b1-3 Glc b1Man a1-6 Gal b1-3 Glc b1Man b1-2 Gal b1-3 Glc b1Man b1-3 Gal b1-3 Glc b1Man b1-4 Gal b1-3 Glc b1Man b1-6 Gal b1-3 Glc b1Man a1-2 Gal b1-4 Glc b1Man a1-3 Gal b1-4 Glc b1Man a1-4 Gal b1-4 Glc b1Man a1-6 Gal b1-4 Glc b1Man b1-2 Gal b1-4 Glc b1Man b1-3 Gal b1-4 Glc b1Man b1-4 Gal b1-4 Glc b1Man b1-6 Gal b1-4 Glc b1Man a1-2 Gal b1-6 Glc b1Man a1-3 Gal b1-6 Glc b1Man a1-4 Gal b1-6 Glc b1Man a1-6 Gal b1-6 Glc b1Man b1-2 Gal b1-6 Glc b1Man b1-3 Gal b1-6 Glc b1Man b1-4 Gal b1-6 Glc b1Man b1-6 Gal b1-6 Glc b1Glc a1-2 Man a1-2 Gal a1-Glc a1-3 Man a1-2 Gal a1-Glc a1-4 Man a1-2 Gal a1-Glc a1-6 Man a1-2 Gal a1-Glc b1-2 Man a1-2 Gal a1-Glc b1-3 Man a1-2 Gal a1-Glc b1-4 Man a1-2 Gal a1-Glc b1-6 Man a1-2 Gal a1-Glc a1-2 Man a1-3 Gal a1-Glc a1-3 Man a1-3 Gal a1-Glc a1-4 Man a1-3 Gal a1-Glc a1-6 Man a1-3 Gal a1-Glc b1-2 Man a1-3 Gal a1-Glc b1-3 Man a1-3 Gal a1-Glc b1-4 Man a1-3 Gal a1-Glc b1-6 Man a1-3 Gal a1-Glc a1-2 Man a1-4 Gal a1-Glc a1-3 Man a1-4 Gal a1-Glc a1-4 Man a1-4 Gal a1-Glc a1-6 Man a1-4 Gal a1-Glc b1-2 Man a1-4 Gal a1-Glc b1-3 Man a1-4 Gal a1-Glc b1-4 Man a1-4 Gal a1-Glc b1-6 Man a1-4 Gal a1-Glc a1-2 Man a1-6 Gal a1-Glc a1-3 Man a1-6 Gal a1-Glc a1-4 Man a1-6 Gal a1-Glc a1-6 Man a1-6 Gal a1-Glc b1-2 Man a1-6 Gal a1-Glc b1-3 Man a1-6 Gal a1-Glc b1-4 Man a1-6 Gal a1-Glc b1-6 Man a1-6 Gal a1-Glc a1-2 Man b1-2 Gal a1-Glc a1-3 Man b1-2 Gal a1-Glc a1-4 Man b1-2 Gal a1-Glc a1-6 Man b1-2 Gal a1-Glc b1-2 Man b1-2 Gal a1-Glc b1-3 Man b1-2 Gal a1-Glc b1-4 Man b1-2 Gal a1-Glc b1-6 Man b1-2 Gal a1-Glc a1-2 Man b1-3 Gal a1-Glc a1-3 Man b1-3 Gal a1-Glc a1-4 Man b1-3 Gal a1-Glc a1-6 Man b1-3 Gal a1-Glc b1-2 Man b1-3 Gal a1-Glc b1-3 Man b1-3 Gal a1-

Glc b1-4 Man b1-3 Gal a1-Glc b1-6 Man b1-3 Gal a1-Glc a1-2 Man b1-4 Gal a1-Glc a1-3 Man b1-4 Gal a1-Glc a1-4 Man b1-4 Gal a1-Glc a1-6 Man b1-4 Gal a1-Glc b1-2 Man b1-4 Gal a1-Glc b1-3 Man b1-4 Gal a1-Glc b1-4 Man b1-4 Gal a1-Glc b1-6 Man b1-4 Gal a1-Glc a1-2 Man b1-6 Gal a1-Glc a1-3 Man b1-6 Gal a1-Glc a1-4 Man b1-6 Gal a1-Glc a1-6 Man b1-6 Gal a1-Glc b1-2 Man b1-6 Gal a1-Glc b1-3 Man b1-6 Gal a1-Glc b1-4 Man b1-6 Gal a1-Glc b1-6 Man b1-6 Gal a1-Glc a1-2 Man a1-2 Gal b1-Glc a1-3 Man a1-2 Gal b1-Glc a1-4 Man a1-2 Gal b1-Glc a1-6 Man a1-2 Gal b1-Glc b1-2 Man a1-2 Gal b1-Glc b1-3 Man a1-2 Gal b1-Glc b1-4 Man a1-2 Gal b1-Glc b1-6 Man a1-2 Gal b1-Glc a1-2 Man a1-3 Gal b1-Glc a1-3 Man a1-3 Gal b1-Glc a1-4 Man a1-3 Gal b1-Glc a1-6 Man a1-3 Gal b1-Glc b1-2 Man a1-3 Gal b1-Glc b1-3 Man a1-3 Gal b1-Glc b1-4 Man a1-3 Gal b1-Glc b1-6 Man a1-3 Gal b1-Glc a1-2 Man a1-4 Gal b1-Glc a1-3 Man a1-4 Gal b1-Glc a1-4 Man a1-4 Gal b1-Glc a1-6 Man a1-4 Gal b1-Glc b1-2 Man a1-4 Gal b1-Glc b1-3 Man a1-4 Gal b1-Glc b1-4 Man a1-4 Gal b1-Glc b1-6 Man a1-4 Gal b1-Glc a1-2 Man a1-6 Gal b1-Glc a1-3 Man a1-6 Gal b1-Glc a1-4 Man a1-6 Gal b1-Glc a1-6 Man a1-6 Gal b1-Glc b1-2 Man a1-6 Gal b1-Glc b1-3 Man a1-6 Gal b1-Glc b1-4 Man a1-6 Gal b1-Glc b1-6 Man a1-6 Gal b1-Glc a1-2 Man b1-2 Gal b1-Glc a1-3 Man b1-2 Gal b1-Glc a1-4 Man b1-2 Gal b1-Glc a1-6 Man b1-2 Gal b1-Glc b1-2 Man b1-2 Gal b1-Glc b1-3 Man b1-2 Gal b1-Glc b1-4 Man b1-2 Gal b1-Glc b1-6 Man b1-2 Gal b1-Glc a1-2 Man b1-3 Gal b1-Glc a1-3 Man b1-3 Gal b1-Glc a1-4 Man b1-3 Gal b1-Glc a1-6 Man b1-3 Gal b1-Glc b1-2 Man b1-3 Gal b1-Glc b1-3 Man b1-3 Gal b1-Glc b1-4 Man b1-3 Gal b1-Glc b1-6 Man b1-3 Gal b1-Glc a1-2 Man b1-4 Gal b1-Glc a1-3 Man b1-4 Gal b1-Glc a1-4 Man b1-4 Gal b1-Glc a1-6 Man b1-4 Gal b1-Glc b1-2 Man b1-4 Gal b1-Glc b1-3 Man b1-4 Gal b1-Glc b1-4 Man b1-4 Gal b1-Glc b1-6 Man b1-4 Gal b1-Glc a1-2 Man b1-6 Gal b1-Glc a1-3 Man b1-6 Gal b1-Glc a1-4 Man b1-6 Gal b1-Glc a1-6 Man b1-6 Gal b1-Glc b1-2 Man b1-6 Gal b1-Glc b1-3 Man b1-6 Gal b1-Glc b1-4 Man b1-6 Gal b1-Glc b1-6 Man b1-6 Gal b1-Glc a1-2 Gal a1-2 Man a1-Glc a1-3 Gal a1-2 Man a1-Glc a1-4 Gal a1-2 Man a1-Glc a1-6 Gal a1-2 Man a1-Glc b1-2 Gal a1-2 Man a1-Glc b1-3 Gal a1-2 Man a1-Glc b1-4 Gal a1-2 Man a1-Glc b1-6 Gal a1-2 Man a1-Glc a1-2 Gal a1-3 Man a1-Glc a1-3 Gal a1-3 Man a1-Glc a1-4 Gal a1-3 Man a1-

Glc a1-6 Gal a1-3 Man a1-Glc b1-2 Gal a1-3 Man a1-Glc b1-3 Gal a1-3 Man a1-Glc b1-4 Gal a1-3 Man a1-Glc b1-6 Gal a1-3 Man a1-Glc a1-2 Gal a1-4 Man a1-Glc a1-3 Gal a1-4 Man a1-Glc a1-4 Gal a1-4 Man a1-Glc a1-6 Gal a1-4 Man a1-Glc b1-2 Gal a1-4 Man a1-Glc b1-3 Gal a1-4 Man a1-Glc b1-4 Gal a1-4 Man a1-Glc b1-6 Gal a1-4 Man a1-Glc a1-2 Gal a1-6 Man a1-Glc a1-3 Gal a1-6 Man a1-Glc a1-4 Gal a1-6 Man a1-Glc a1-6 Gal a1-6 Man a1-Glc b1-2 Gal a1-6 Man a1-Glc b1-3 Gal a1-6 Man a1-Glc b1-4 Gal a1-6 Man a1-Glc b1-6 Gal a1-6 Man a1-Glc a1-2 Gal b1-2 Man a1-Glc a1-3 Gal b1-2 Man a1-Glc a1-4 Gal b1-2 Man a1-Glc a1-6 Gal b1-2 Man a1-Glc b1-2 Gal b1-2 Man a1-Glc b1-3 Gal b1-2 Man a1-Glc b1-4 Gal b1-2 Man a1-Glc b1-6 Gal b1-2 Man a1-Glc a1-2 Gal b1-3 Man a1-Glc a1-3 Gal b1-3 Man a1-Glc a1-4 Gal b1-3 Man a1-Glc a1-6 Gal b1-3 Man a1-Glc b1-2 Gal b1-3 Man a1-Glc b1-3 Gal b1-3 Man a1-Glc b1-4 Gal b1-3 Man a1-Glc b1-6 Gal b1-3 Man a1-Glc a1-2 Gal b1-4 Man a1-Glc a1-3 Gal b1-4 Man a1-Glc a1-4 Gal b1-4 Man a1-Glc a1-6 Gal b1-4 Man a1-Glc b1-2 Gal b1-4 Man a1-Glc b1-3 Gal b1-4 Man a1-Glc b1-4 Gal b1-4 Man a1-Glc b1-6 Gal b1-4 Man a1-Glc a1-2 Gal b1-6 Man a1-Glc a1-3 Gal b1-6 Man a1-Glc a1-4 Gal b1-6 Man a1-Glc a1-6 Gal b1-6 Man a1-Glc b1-2 Gal b1-6 Man a1-Glc b1-3 Gal b1-6 Man a1-Glc b1-4 Gal b1-6 Man a1-Glc b1-6 Gal b1-6 Man a1-Glc a1-2 Gal a1-2 Man b1-Glc a1-3 Gal a1-2 Man b1-Glc a1-4 Gal a1-2 Man b1-Glc a1-6 Gal a1-2 Man b1-Glc b1-2 Gal a1-2 Man b1-Glc b1-3 Gal a1-2 Man b1-Glc b1-4 Gal a1-2 Man b1-Glc b1-6 Gal a1-2 Man b1-Glc a1-2 Gal a1-3 Man b1-Glc a1-3 Gal a1-3 Man b1-Glc a1-4 Gal a1-3 Man b1-Glc a1-6 Gal a1-3 Man b1-Glc b1-2 Gal a1-3 Man b1-Glc b1-3 Gal a1-3 Man b1-Glc b1-4 Gal a1-3 Man b1-Glc b1-6 Gal a1-3 Man b1-Glc a1-2 Gal a1-4 Man b1-Glc a1-3 Gal a1-4 Man b1-Glc a1-4 Gal a1-4 Man b1-Glc a1-6 Gal a1-4 Man b1-Glc b1-2 Gal a1-4 Man b1-Glc b1-3 Gal a1-4 Man b1-Glc b1-4 Gal a1-4 Man b1-Glc b1-6 Gal a1-4 Man b1-Glc a1-2 Gal a1-6 Man b1-Glc a1-3 Gal a1-6 Man b1-Glc a1-4 Gal a1-6 Man b1-Glc a1-6 Gal a1-6 Man b1-Glc b1-2 Gal a1-6 Man b1-Glc b1-3 Gal a1-6 Man b1-Glc b1-4 Gal a1-6 Man b1-Glc b1-6 Gal a1-6 Man b1-Glc a1-2 Gal b1-2 Man b1-Glc a1-3 Gal b1-2 Man b1-Glc a1-4 Gal b1-2 Man b1-Glc a1-6 Gal b1-2 Man b1-Glc b1-2 Gal b1-2 Man b1-Glc b1-3 Gal b1-2 Man b1-Glc b1-4 Gal b1-2 Man b1-Glc b1-6 Gal b1-2 Man b1-

Glc a1-2 Gal b1-3 Man b1-Glc a1-3 Gal b1-3 Man b1-Glc a1-4 Gal b1-3 Man b1-Glc a1-6 Gal b1-3 Man b1-Glc b1-2 Gal b1-3 Man b1-Glc b1-3 Gal b1-3 Man b1-Glc b1-4 Gal b1-3 Man b1-Glc b1-6 Gal b1-3 Man b1-Glc a1-2 Gal b1-4 Man b1-Glc a1-3 Gal b1-4 Man b1-Glc a1-4 Gal b1-4 Man b1-Glc a1-6 Gal b1-4 Man b1-Glc b1-2 Gal b1-4 Man b1-Glc b1-3 Gal b1-4 Man b1-Glc b1-4 Gal b1-4 Man b1-Glc b1-6 Gal b1-4 Man b1-Glc a1-2 Gal b1-6 Man b1-Glc a1-3 Gal b1-6 Man b1-Glc a1-4 Gal b1-6 Man b1-Glc a1-6 Gal b1-6 Man b1-Glc b1-2 Gal b1-6 Man b1-Glc b1-3 Gal b1-6 Man b1-Glc b1-4 Gal b1-6 Man b1-Glc b1-6 Gal b1-6 Man b1-Gal a1-2 (Glc a1-3) Man a1-Gal a1-2 (Glc a1-4) Man a1-Gal a1-2 (Glc a1-6) Man a1-Gal a1-3 (Glc a1-2) Man a1-Gal a1-3 (Glc a1-4) Man a1-Gal a1-3 (Glc a1-6) Man a1-Gal a1-4 (Glc a1-2) Man a1-Gal a1-4 (Glc a1-3) Man a1-Gal a1-4 (Glc a1-6) Man a1-Gal a1-6 (Glc a1-2) Man a1-Gal a1-6 (Glc a1-3) Man a1-Gal a1-6 (Glc a1-4) Man a1-Gal b1-2 (Glc a1-3) Man a1-Gal b1-2 (Glc a1-4) Man a1-Gal b1-2 (Glc a1-6) Man a1-Gal b1-3 (Glc a1-2) Man a1-Gal b1-3 (Glc a1-4) Man a1-Gal b1-3 (Glc a1-6) Man a1-Gal b1-4 (Glc a1-2) Man a1-Gal b1-4 (Glc a1-3) Man a1-Gal b1-4 (Glc a1-6) Man a1-Gal b1-6 (Glc a1-2) Man a1-Gal b1-6 (Glc a1-3) Man a1-Gal b1-6 (Glc a1-4) Man a1-Gal a1-2 (Glc b1-3) Man a1-Gal a1-2 (Glc b1-4) Man a1-Gal a1-2 (Glc b1-6) Man a1-Gal a1-3 (Glc b1-2) Man a1-Gal a1-3 (Glc b1-4) Man a1-Gal a1-3 (Glc b1-6) Man a1-Gal a1-4 (Glc b1-2) Man a1-Gal a1-4 (Glc b1-3) Man a1-Gal a1-4 (Glc b1-6) Man a1-Gal a1-6 (Glc b1-2) Man a1-Gal a1-6 (Glc b1-3) Man a1-Gal a1-6 (Glc b1-4) Man a1-Gal b1-2 (Glc b1-3) Man a1-Gal b1-2 (Glc b1-4) Man a1-Gal b1-2 (Glc b1-6) Man a1-Gal b1-3 (Glc b1-2) Man a1-Gal b1-3 (Glc b1-4) Man a1-Gal b1-3 (Glc b1-6) Man a1-Gal b1-4 (Glc b1-2) Man a1-Gal b1-4 (Glc b1-3) Man a1-Gal b1-4 (Glc b1-6) Man a1-Gal b1-6 (Glc b1-2) Man a1-Gal b1-6 (Glc b1-3) Man a1-Gal b1-6 (Glc b1-4) Man a1-Gal a1-2 (Glc a1-3) Man b1-Gal a1-2 (Glc a1-4) Man b1-Gal a1-2 (Glc a1-6) Man b1-Gal a1-3 (Glc a1-2) Man b1-Gal a1-3 (Glc a1-4) Man b1-Gal a1-3 (Glc a1-6) Man b1-Gal a1-4 (Glc a1-2) Man b1-Gal a1-4 (Glc a1-3) Man b1-Gal a1-4 (Glc a1-6) Man b1-Gal a1-6 (Glc a1-2) Man b1-Gal a1-6 (Glc a1-3) Man b1-Gal a1-6 (Glc a1-4) Man b1-Gal b1-2 (Glc a1-3) Man b1-Gal b1-2 (Glc a1-4) Man b1-Gal b1-2 (Glc a1-6) Man b1-Gal b1-3 (Glc a1-2) Man b1-Gal b1-3 (Glc a1-4) Man b1-Gal b1-3 (Glc a1-6) Man b1-Gal b1-4 (Glc a1-2) Man b1-Gal b1-4 (Glc a1-3) Man b1-Gal b1-4 (Glc a1-6) Man b1-

Gal b1-6 (Glc a1-2) Man b1-Gal b1-6 (Glc a1-3) Man b1-Gal b1-6 (Glc a1-4) Man b1-Gal a1-2 (Glc b1-3) Man b1-Gal a1-2 (Glc b1-4) Man b1-Gal a1-2 (Glc b1-6) Man b1-Gal a1-3 (Glc b1-2) Man b1-Gal a1-3 (Glc b1-4) Man b1-Gal a1-3 (Glc b1-6) Man b1-Gal a1-4 (Glc b1-2) Man b1-Gal a1-4 (Glc b1-3) Man b1-Gal a1-4 (Glc b1-6) Man b1-Gal a1-6 (Glc b1-2) Man b1-Gal a1-6 (Glc b1-3) Man b1-Gal a1-6 (Glc b1-4) Man b1-Gal b1-2 (Glc b1-3) Man b1-Gal b1-2 (Glc b1-4) Man b1-Gal b1-2 (Glc b1-6) Man b1-Gal b1-3 (Glc b1-2) Man b1-Gal b1-3 (Glc b1-4) Man b1-Gal b1-3 (Glc b1-6) Man b1-Gal b1-4 (Glc b1-2) Man b1-Gal b1-4 (Glc b1-3) Man b1-Gal b1-4 (Glc b1-6) Man b1-Gal b1-6 (Glc b1-2) Man b1-Gal b1-6 (Glc b1-3) Man b1-Gal b1-6 (Glc b1-4) Man b1-Gal a1-2 (Man a1-3) Glc a1-Gal a1-2 (Man a1-4) Glc a1-Gal a1-2 (Man a1-6) Glc a1-Gal a1-3 (Man a1-2) Glc a1-Gal a1-3 (Man a1-4) Glc a1-Gal a1-3 (Man a1-6) Glc a1-Gal a1-4 (Man a1-2) Glc a1-Gal a1-4 (Man a1-3) Glc a1-Gal a1-4 (Man a1-6) Glc a1-Gal a1-6 (Man a1-2) Glc a1-Gal a1-6 (Man a1-3) Glc a1-Gal a1-6 (Man a1-4) Glc a1-Gal b1-2 (Man a1-3) Glc a1-Gal b1-2 (Man a1-4) Glc a1-Gal b1-2 (Man a1-6) Glc a1-Gal b1-3 (Man a1-2) Glc a1-Gal b1-3 (Man a1-4) Glc a1-Gal b1-3 (Man a1-6) Glc a1-Gal b1-4 (Man a1-2) Glc a1-Gal b1-4 (Man a1-3) Glc a1-Gal b1-4 (Man a1-6) Glc a1-Gal b1-6 (Man a1-2) Glc a1-Gal b1-6 (Man a1-3) Glc a1-Gal b1-6 (Man a1-4) Glc a1-Gal a1-2 (Man b1-3) Glc a1-Gal a1-2 (Man b1-4) Glc a1-Gal a1-2 (Man b1-6) Glc a1-Gal a1-3 (Man b1-2) Glc a1-Gal a1-3 (Man b1-4) Glc a1-Gal a1-3 (Man b1-6) Glc a1-Gal a1-4 (Man b1-2) Glc a1-Gal a1-4 (Man b1-3) Glc a1-Gal a1-4 (Man b1-6) Glc a1-Gal a1-6 (Man b1-2) Glc a1-Gal a1-6 (Man b1-3) Glc a1-Gal a1-6 (Man b1-4) Glc a1-Gal b1-2 (Man b1-3) Glc a1-Gal b1-2 (Man b1-4) Glc a1-Gal b1-2 (Man b1-6) Glc a1-Gal b1-3 (Man b1-2) Glc a1-Gal b1-3 (Man b1-4) Glc a1-Gal b1-3 (Man b1-6) Glc a1-Gal b1-4 (Man b1-2) Glc a1-Gal b1-4 (Man b1-3) Glc a1-Gal b1-4 (Man b1-6) Glc a1-Gal b1-6 (Man b1-2) Glc a1-Gal b1-6 (Man b1-3) Glc a1-Gal b1-6 (Man b1-4) Glc a1-Gal a1-2 (Man a1-3) Glc b1-Gal a1-2 (Man a1-4) Glc b1-Gal a1-2 (Man a1-6) Glc b1-Gal a1-3 (Man a1-2) Glc b1-Gal a1-3 (Man a1-4) Glc b1-Gal a1-3 (Man a1-6) Glc b1-

Gal a1-4 (Man a1-2) Glc b1-Gal a1-4 (Man a1-3) Glc b1-Gal a1-4 (Man a1-6) Glc b1-Gal a1-6 (Man a1-2) Glc b1-Gal a1-6 (Man a1-3) Glc b1-Gal a1-6 (Man a1-4) Glc b1-Gal b1-2 (Man a1-3) Glc b1-Gal b1-2 (Man a1-4) Glc b1-Gal b1-2 (Man a1-6) Glc b1-Gal b1-3 (Man a1-2) Glc b1-Gal b1-3 (Man a1-4) Glc b1-Gal b1-3 (Man a1-6) Glc b1-Gal b1-4 (Man a1-2) Glc b1-Gal b1-4 (Man a1-3) Glc b1-Gal b1-4 (Man a1-6) Glc b1-Gal b1-6 (Man a1-2) Glc b1-Gal b1-6 (Man a1-3) Glc b1-Gal b1-6 (Man a1-4) Glc b1-Gal a1-2 (Man b1-3) Glc b1-Gal a1-2 (Man b1-4) Glc b1-Gal a1-2 (Man b1-6) Glc b1-Gal a1-3 (Man b1-2) Glc b1-Gal a1-3 (Man b1-4) Glc b1-Gal a1-3 (Man b1-6) Glc b1-Gal a1-4 (Man b1-2) Glc b1-Gal a1-4 (Man b1-3) Glc b1-Gal a1-4 (Man b1-6) Glc b1-Gal a1-6 (Man b1-2) Glc b1-Gal a1-6 (Man b1-3) Glc b1-Gal a1-6 (Man b1-4) Glc b1-Gal b1-2 (Man b1-3) Glc b1-Gal b1-2 (Man b1-4) Glc b1-Gal b1-2 (Man b1-6) Glc b1-Gal b1-3 (Man b1-2) Glc b1-Gal b1-3 (Man b1-4) Glc b1-Gal b1-3 (Man b1-6) Glc b1-Gal b1-4 (Man b1-2) Glc b1-Gal b1-4 (Man b1-3) Glc b1-Gal b1-4 (Man b1-6) Glc b1-Gal b1-6 (Man b1-2) Glc b1-Gal b1-6 (Man b1-3) Glc b1-Gal b1-6 (Man b1-4) Glc b1-Glc a1-2 (Man a1-3) Gal a1-Glc a1-2 (Man a1-4) Gal a1-Glc a1-2 (Man a1-6) Gal a1-Glc a1-3 (Man a1-2) Gal a1-Glc a1-3 (Man a1-4) Gal a1-Glc a1-3 (Man a1-6) Gal a1-Glc a1-4 (Man a1-2) Gal a1-Glc a1-4 (Man a1-3) Gal a1-Glc a1-4 (Man a1-6) Gal a1-Glc a1-6 (Man a1-2) Gal a1-Glc a1-6 (Man a1-3) Gal a1-Glc a1-6 (Man a1-4) Gal a1-Glc b1-2 (Man a1-3) Gal a1-Glc b1-2 (Man a1-4) Gal a1-Glc b1-2 (Man a1-6) Gal a1-Glc b1-3 (Man a1-2) Gal a1-Glc b1-3 (Man a1-4) Gal a1-Glc b1-3 (Man a1-6) Gal a1-Glc b1-4 (Man a1-2) Gal a1-Glc b1-4 (Man a1-3) Gal a1-Glc b1-4 (Man a1-6) Gal a1-Glc b1-6 (Man a1-2) Gal a1-Glc b1-6 (Man a1-3) Gal a1-Glc b1-6 (Man a1-4) Gal a1-Glc a1-2 (Man b1-3) Gal a1-Glc a1-2 (Man b1-4) Gal a1-Glc a1-2 (Man b1-6) Gal a1-Glc a1-3 (Man b1-2) Gal a1-Glc a1-3 (Man b1-4) Gal a1-Glc a1-3 (Man b1-6) Gal a1-Glc a1-4 (Man b1-2) Gal a1-Glc a1-4 (Man b1-3) Gal a1-Glc a1-4 (Man b1-6) Gal a1-Glc a1-6 (Man b1-2) Gal a1-Glc a1-6 (Man b1-3) Gal a1-Glc a1-6 (Man b1-4) Gal a1-Glc b1-2 (Man b1-3) Gal a1-Glc b1-2 (Man b1-4) Gal a1-Glc b1-2 (Man b1-6) Gal a1-

Glc b1-3 (Man b1-2) Gal a1-Glc b1-3 (Man b1-4) Gal a1-Glc b1-3 (Man b1-6) Gal a1-Glc b1-4 (Man b1-2) Gal a1-Glc b1-4 (Man b1-3) Gal a1-Glc b1-4 (Man b1-6) Gal a1-Glc b1-6 (Man b1-2) Gal a1-Glc b1-6 (Man b1-3) Gal a1-Glc b1-6 (Man b1-4) Gal a1-Glc a1-2 (Man a1-3) Gal b1-Glc a1-2 (Man a1-4) Gal b1-Glc a1-2 (Man a1-6) Gal b1-Glc a1-3 (Man a1-2) Gal b1-Glc a1-3 (Man a1-4) Gal b1-Glc a1-3 (Man a1-6) Gal b1-Glc a1-4 (Man a1-2) Gal b1-Glc a1-4 (Man a1-3) Gal b1-Glc a1-4 (Man a1-6) Gal b1-Glc a1-6 (Man a1-2) Gal b1-Glc a1-6 (Man a1-3) Gal b1-Glc a1-6 (Man a1-4) Gal b1-Glc b1-2 (Man a1-3) Gal b1-Glc b1-2 (Man a1-4) Gal b1-Glc b1-2 (Man a1-6) Gal b1-Glc b1-3 (Man a1-2) Gal b1-Glc b1-3 (Man a1-4) Gal b1-Glc b1-3 (Man a1-6) Gal b1-Glc b1-4 (Man a1-2) Gal b1-Glc b1-4 (Man a1-3) Gal b1-Glc b1-4 (Man a1-6) Gal b1-Glc b1-6 (Man a1-2) Gal b1-Glc b1-6 (Man a1-3) Gal b1-Glc b1-6 (Man a1-4) Gal b1-Glc a1-2 (Man b1-3) Gal b1-Glc a1-2 (Man b1-4) Gal b1-Glc a1-2 (Man b1-6) Gal b1-Glc a1-3 (Man b1-2) Gal b1-Glc a1-3 (Man b1-4) Gal b1-Glc a1-3 (Man b1-6) Gal b1-Glc a1-4 (Man b1-2) Gal b1-Glc a1-4 (Man b1-3) Gal b1-Glc a1-4 (Man b1-6) Gal b1-Glc a1-6 (Man b1-2) Gal b1-Glc a1-6 (Man b1-3) Gal b1-Glc a1-6 (Man b1-4) Gal b1-Glc b1-2 (Man b1-3) Gal b1-Glc b1-2 (Man b1-4) Gal b1-Glc b1-2 (Man b1-6) Gal b1-Glc b1-3 (Man b1-2) Gal b1-Glc b1-3 (Man b1-4) Gal b1-Glc b1-3 (Man b1-6) Gal b1-Glc b1-4 (Man b1-2) Gal b1-Glc b1-4 (Man b1-3) Gal b1-Glc b1-4 (Man b1-6) Gal b1-Glc b1-6 (Man b1-2) Gal b1-Glc b1-6 (Man b1-3) Gal b1-Glc b1-6 (Man b1-4) Gal b1-

Man a1-4 Gal a1-3 Glc b1Man a1-6 Gal a1-3 Glc b1Man b1-2 Gal a1-3 Glc b1Man b1-3 Gal a1-3 Glc b1Man b1-4 Gal a1-3 Glc b1Man b1-6 Gal a1-3 Glc b1Man a1-2 Gal a1-4 Glc b1Man a1-3 Gal a1-4 Glc b1Man a1-4 Gal a1-4 Glc b1Man a1-6 Gal a1-4 Glc b1Man b1-2 Gal a1-4 Glc b1Man b1-3 Gal a1-4 Glc b1Man b1-4 Gal a1-4 Glc b1Man b1-6 Gal a1-4 Glc b1Man a1-2 Gal a1-6 Glc b1Man a1-3 Gal a1-6 Glc b1

Cummings RD (2009) Molecular BioSystems 5 1087

Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical petasaccharide sequeces in glycosaminoglycans

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoN-linkedrdquo glycan linkage to protein asparagine residues

Families of N-linked glycan structures

Man

4GlcNAc1

2Man

Man

Man

Man

2Man

2Man

2Man

63

Man63 4GlcNAc1 Asn

4GlcNAc1

Man62

Man63 4GlcNAc1 Asn

Man42

4GlcNAc1

4GlcNAc1

4GlcNAc1

4GlcNAc1

6Gal

6Gal

6Gal

6Gal

NeuAc

NeuAc

NeuAcNeuAc

4GlcNAc1

Man

Man63 4GlcNAc1 Asn

2Man4GlcNAc13GalNeuAc

Man

Man

63

core

core

core

High Mannose

Complex

Hybrid

Examples of structures found as termini on the branches of complex N-linked glycoprotein glycanshellip

Terminal Saccharide Diversity --- A key to recognition

NeuAc 2-6 Gal 1-4 GlcNAc---NeuAc 2-3 Gal 1-4 GlcNAc---

Gal 1-3 Gal 1-4 GlcNAc---4-SO3-GalNAc 1-4 GlcNAc---

(-Gal 1-4 GlcNAc 1-3-)n = polylactosamine repeats (n may be gt6)

(-NeuAc 2-8-) n = polysialic acid (n may be gt50)

Fuc residues (eg Fuc 1-2 Gal Fuc 1-3 GlcNAc Fuc 1-4 GlcNAc

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoO-linkedrdquo glycan linkage to protein serine residues

Examples of variations of O-linked glycans

GalNAc Serine

GalNAc Serine

NeuAc 26

GalNAc Serine

Gal 1

NeuAc 263

GalNAc SerineNeuAc 2-3 Gal 1

NeuAc 263

Gal 1-4 GlcNAc 1

Fuc 1

GalNAc Serine

GlcNAc 1

63

4NeuAc 2-3 Gal 1

3

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Varki et al (2009) Proteomics 95398

Oligosaccharides Molecular diversity of glycans

Polypeptides Glycans

Building blocks amino acids monosaccharides

Number of different monomers 20 common 9 common

Linkage sites per monomer 1 3-4

Possible linkage configurations 1 2

Possible homodimer structures 1 6-8

Linkage modes linear linear or branched

HO

HO

O

HO

O

GalHO

OHO

HO

O

HO

O

GalHO

O

H2N C

Ala

OH

OH2N C

Ala

OH

O

Three different amino acids (Ala Ser Tyr) ndash 6 structures

Ala-Ser-TyrAla-Tyr-SerSer-Ala-TyrSer-Tyr-AlaTyr-Ala-SerTyr-Ser-Ala

Three different sugars (Glc Gal Man) ndash 1056 structures hellip

Gal a1-2 Glc a1-2 Man a1-Gal a1-3 Glc a1-2 Man a1-Gal a1-4 Glc a1-2 Man a1-Gal a1-6 Glc a1-2 Man a1-Gal b1-2 Glc a1-2 Man a1-Gal b1-3 Glc a1-2 Man a1-Gal b1-4 Glc a1-2 Man a1-Gal b1-6 Glc a1-2 Man a1-Gal a1-2 Glc a1-3 Man a1-Gal a1-3 Glc a1-3 Man a1-Gal a1-4 Glc a1-3 Man a1-Gal a1-6 Glc a1-3 Man a1-Gal b1-2 Glc a1-3 Man a1-Gal b1-3 Glc a1-3 Man a1-Gal b1-4 Glc a1-3 Man a1-Gal b1-6 Glc a1-3 Man a1-Gal a1-2 Glc a1-4 Man a1-Gal a1-3 Glc a1-4 Man a1-Gal a1-4 Glc a1-4 Man a1-Gal a1-6 Glc a1-4 Man a1-Gal b1-2 Glc a1-4 Man a1-Gal b1-3 Glc a1-4 Man a1-Gal b1-4 Glc a1-4 Man a1-Gal b1-6 Glc a1-4 Man a1-Gal a1-2 Glc a1-6 Man a1-Gal a1-3 Glc a1-6 Man a1-Gal a1-4 Glc a1-6 Man a1-Gal a1-6 Glc a1-6 Man a1-Gal b1-2 Glc a1-6 Man a1-Gal b1-3 Glc a1-6 Man a1-Gal b1-4 Glc a1-6 Man a1-Gal b1-6 Glc a1-6 Man a1-Gal a1-2 Glc b1-2 Man a1-Gal a1-3 Glc b1-2 Man a1-Gal a1-4 Glc b1-2 Man a1-Gal a1-6 Glc b1-2 Man a1-Gal b1-2 Glc b1-2 Man a1-Gal b1-3 Glc b1-2 Man a1-Gal b1-4 Glc b1-2 Man a1-Gal b1-6 Glc b1-2 Man a1-Gal a1-2 Glc b1-3 Man a1-Gal a1-3 Glc b1-3 Man a1-Gal a1-4 Glc b1-3 Man a1-Gal a1-6 Glc b1-3 Man a1-Gal b1-2 Glc b1-3 Man a1-Gal b1-3 Glc b1-3 Man a1-Gal b1-4 Glc b1-3 Man a1-Gal b1-6 Glc b1-3 Man a1-Gal a1-2 Glc b1-4 Man a1-Gal a1-3 Glc b1-4 Man a1-Gal a1-4 Glc b1-4 Man a1-Gal a1-6 Glc b1-4 Man a1-Gal b1-2 Glc b1-4 Man a1-Gal b1-3 Glc b1-4 Man a1-Gal b1-4 Glc b1-4 Man a1-Gal b1-6 Glc b1-4 Man a1-Gal a1-2 Glc b1-6 Man a1-Gal a1-3 Glc b1-6 Man a1-Gal a1-4 Glc b1-6 Man a1-Gal a1-6 Glc b1-6 Man a1-Gal b1-2 Glc b1-6 Man a1-Gal b1-3 Glc b1-6 Man a1-Gal b1-4 Glc b1-6 Man a1-Gal b1-6 Glc b1-6 Man a1-Gal a1-2 Glc a1-2 Man b1-Gal a1-3 Glc a1-2 Man b1-Gal a1-4 Glc a1-2 Man b1-Gal a1-6 Glc a1-2 Man b1-Gal b1-2 Glc a1-2 Man b1-Gal b1-3 Glc a1-2 Man b1-Gal b1-4 Glc a1-2 Man b1-Gal b1-6 Glc a1-2 Man b1-Gal a1-2 Glc a1-3 Man b1-Gal a1-3 Glc a1-3 Man b1-Gal a1-4 Glc a1-3 Man b1-Gal a1-6 Glc a1-3 Man b1-Gal b1-2 Glc a1-3 Man b1-Gal b1-3 Glc a1-3 Man b1-Gal b1-4 Glc a1-3 Man b1-Gal b1-6 Glc a1-3 Man b1-Gal a1-2 Glc a1-4 Man b1-Gal a1-3 Glc a1-4 Man b1-Gal a1-4 Glc a1-4 Man b1-Gal a1-6 Glc a1-4 Man b1-Gal b1-2 Glc a1-4 Man b1-Gal b1-3 Glc a1-4 Man b1-Gal b1-4 Glc a1-4 Man b1-Gal b1-6 Glc a1-4 Man b1-Gal a1-2 Glc a1-6 Man b1-Gal a1-3 Glc a1-6 Man b1-Gal a1-4 Glc a1-6 Man b1-Gal a1-6 Glc a1-6 Man b1-Gal b1-2 Glc a1-6 Man b1-

Gal b1-3 Glc a1-6 Man b1-Gal b1-4 Glc a1-6 Man b1-Gal b1-6 Glc a1-6 Man b1-Gal a1-2 Glc b1-2 Man b1-Gal a1-3 Glc b1-2 Man b1-Gal a1-4 Glc b1-2 Man b1-Gal a1-6 Glc b1-2 Man b1-Gal b1-2 Glc b1-2 Man b1-Gal b1-3 Glc b1-2 Man b1-Gal b1-4 Glc b1-2 Man b1-Gal b1-6 Glc b1-2 Man b1-Gal a1-2 Glc b1-3 Man b1-Gal a1-3 Glc b1-3 Man b1-Gal a1-4 Glc b1-3 Man b1-Gal a1-6 Glc b1-3 Man b1-Gal b1-2 Glc b1-3 Man b1-Gal b1-3 Glc b1-3 Man b1-Gal b1-4 Glc b1-3 Man b1-Gal b1-6 Glc b1-3 Man b1-Gal a1-2 Glc b1-4 Man b1-Gal a1-3 Glc b1-4 Man b1-Gal a1-4 Glc b1-4 Man b1-Gal a1-6 Glc b1-4 Man b1-Gal b1-2 Glc b1-4 Man b1-Gal b1-3 Glc b1-4 Man b1-Gal b1-4 Glc b1-4 Man b1-Gal b1-6 Glc b1-4 Man b1-Gal a1-2 Glc b1-6 Man b1-Gal a1-3 Glc b1-6 Man b1-Gal a1-4 Glc b1-6 Man b1-Gal a1-6 Glc b1-6 Man b1-Gal b1-2 Glc b1-6 Man b1-Gal b1-3 Glc b1-6 Man b1-Gal b1-4 Glc b1-6 Man b1-Gal b1-6 Glc b1-6 Man b1-Gal a1-2 Man a1-2 Glc a1-Gal a1-3 Man a1-2 Glc a1-Gal a1-4 Man a1-2 Glc a1-Gal a1-6 Man a1-2 Glc a1-Gal b1-2 Man a1-2 Glc a1-Gal b1-3 Man a1-2 Glc a1-Gal b1-4 Man a1-2 Glc a1-Gal b1-6 Man a1-2 Glc a1-Gal a1-2 Man a1-3 Glc a1-Gal a1-3 Man a1-3 Glc a1-Gal a1-4 Man a1-3 Glc a1-Gal a1-6 Man a1-3 Glc a1-Gal b1-2 Man a1-3 Glc a1-Gal b1-3 Man a1-3 Glc a1-Gal b1-4 Man a1-3 Glc a1-Gal b1-6 Man a1-3 Glc a1-Gal a1-2 Man a1-4 Glc a1-Gal a1-3 Man a1-4 Glc a1-Gal a1-4 Man a1-4 Glc a1-Gal a1-6 Man a1-4 Glc a1-Gal b1-2 Man a1-4 Glc a1-Gal b1-3 Man a1-4 Glc a1-Gal b1-4 Man a1-4 Glc a1-Gal b1-6 Man a1-4 Glc a1-Gal a1-2 Man a1-6 Glc a1-Gal a1-3 Man a1-6 Glc a1-Gal a1-4 Man a1-6 Glc a1-Gal a1-6 Man a1-6 Glc a1-Gal b1-2 Man a1-6 Glc a1-Gal b1-3 Man a1-6 Glc a1-Gal b1-4 Man a1-6 Glc a1-Gal b1-6 Man a1-6 Glc a1-Gal a1-2 Man b1-2 Glc a1-Gal a1-3 Man b1-2 Glc a1-Gal a1-4 Man b1-2 Glc a1-Gal a1-6 Man b1-2 Glc a1-Gal b1-2 Man b1-2 Glc a1-Gal b1-3 Man b1-2 Glc a1-Gal b1-4 Man b1-2 Glc a1-Gal b1-6 Man b1-2 Glc a1-Gal a1-2 Man b1-3 Glc a1-Gal a1-3 Man b1-3 Glc a1-Gal a1-4 Man b1-3 Glc a1-Gal a1-6 Man b1-3 Glc a1-Gal b1-2 Man b1-3 Glc a1-Gal b1-3 Man b1-3 Glc a1-Gal b1-4 Man b1-3 Glc a1-Gal b1-6 Man b1-3 Glc a1-Gal a1-2 Man b1-4 Glc a1-Gal a1-3 Man b1-4 Glc a1-Gal a1-4 Man b1-4 Glc a1-Gal a1-6 Man b1-4 Glc a1-Gal b1-2 Man b1-4 Glc a1-Gal b1-3 Man b1-4 Glc a1-Gal b1-4 Man b1-4 Glc a1-Gal b1-6 Man b1-4 Glc a1-Gal a1-2 Man b1-6 Glc a1-Gal a1-3 Man b1-6 Glc a1-

Gal a1-4 Man b1-6 Glc a1-Gal a1-6 Man b1-6 Glc a1-Gal b1-2 Man b1-6 Glc a1-Gal b1-3 Man b1-6 Glc a1-Gal b1-4 Man b1-6 Glc a1-Gal b1-6 Man b1-6 Glc a1-Gal a1-2 Man a1-2 Glc b1-Gal a1-3 Man a1-2 Glc b1-Gal a1-4 Man a1-2 Glc b1-Gal a1-6 Man a1-2 Glc b1-Gal b1-2 Man a1-2 Glc b1-Gal b1-3 Man a1-2 Glc b1-Gal b1-4 Man a1-2 Glc b1-Gal b1-6 Man a1-2 Glc b1-Gal a1-2 Man a1-3 Glc b1-Gal a1-3 Man a1-3 Glc b1-Gal a1-4 Man a1-3 Glc b1-Gal a1-6 Man a1-3 Glc b1-Gal b1-2 Man a1-3 Glc b1-Gal b1-3 Man a1-3 Glc b1-Gal b1-4 Man a1-3 Glc b1-Gal b1-6 Man a1-3 Glc b1-Gal a1-2 Man a1-4 Glc b1-Gal a1-3 Man a1-4 Glc b1-Gal a1-4 Man a1-4 Glc b1-Gal a1-6 Man a1-4 Glc b1-Gal b1-2 Man a1-4 Glc b1-Gal b1-3 Man a1-4 Glc b1-Gal b1-4 Man a1-4 Glc b1-Gal b1-6 Man a1-4 Glc b1-Gal a1-2 Man a1-6 Glc b1-Gal a1-3 Man a1-6 Glc b1-Gal a1-4 Man a1-6 Glc b1-Gal a1-6 Man a1-6 Glc b1-Gal b1-2 Man a1-6 Glc b1-Gal b1-3 Man a1-6 Glc b1-Gal b1-4 Man a1-6 Glc b1-Gal b1-6 Man a1-6 Glc b1-Gal a1-2 Man b1-2 Glc b1-Gal a1-3 Man b1-2 Glc b1-Gal a1-4 Man b1-2 Glc b1-Gal a1-6 Man b1-2 Glc b1-Gal b1-2 Man b1-2 Glc b1-Gal b1-3 Man b1-2 Glc b1-Gal b1-4 Man b1-2 Glc b1-Gal b1-6 Man b1-2 Glc b1-Gal a1-2 Man b1-3 Glc b1-Gal a1-3 Man b1-3 Glc b1-Gal a1-4 Man b1-3 Glc b1-Gal a1-6 Man b1-3 Glc b1-Gal b1-2 Man b1-3 Glc b1-Gal b1-3 Man b1-3 Glc b1-Gal b1-4 Man b1-3 Glc b1-Gal b1-6 Man b1-3 Glc b1-Gal a1-2 Man b1-4 Glc b1-Gal a1-3 Man b1-4 Glc b1-Gal a1-4 Man b1-4 Glc b1-Gal a1-6 Man b1-4 Glc b1-Gal b1-2 Man b1-4 Glc b1-Gal b1-3 Man b1-4 Glc b1-Gal b1-4 Man b1-4 Glc b1-Gal b1-6 Man b1-4 Glc b1-Gal a1-2 Man b1-6 Glc b1-Gal a1-3 Man b1-6 Glc b1-Gal a1-4 Man b1-6 Glc b1-Gal a1-6 Man b1-6 Glc b1-Gal b1-2 Man b1-6 Glc b1-Gal b1-3 Man b1-6 Glc b1-Gal b1-4 Man b1-6 Glc b1-Gal b1-6 Man b1-6 Glc b1-Man a1-2 Glc a1-2 Gal a1-Man a1-3 Glc a1-2 Gal a1-Man a1-4 Glc a1-2 Gal a1-Man a1-6 Glc a1-2 Gal a1-Man b1-2 Glc a1-2 Gal a1-Man b1-3 Glc a1-2 Gal a1-Man b1-4 Glc a1-2 Gal a1-Man b1-6 Glc a1-2 Gal a1-Man a1-2 Glc a1-3 Gal a1-Man a1-3 Glc a1-3 Gal a1-Man a1-4 Glc a1-3 Gal a1-Man a1-6 Glc a1-3 Gal a1-Man b1-2 Glc a1-3 Gal a1-Man b1-3 Glc a1-3 Gal a1-Man b1-4 Glc a1-3 Gal a1-Man b1-6 Glc a1-3 Gal a1-Man a1-2 Glc a1-4 Gal a1-Man a1-3 Glc a1-4 Gal a1-Man a1-4 Glc a1-4 Gal a1-Man a1-6 Glc a1-4 Gal a1-Man b1-2 Glc a1-4 Gal a1-Man b1-3 Glc a1-4 Gal a1-Man b1-4 Glc a1-4 Gal a1-

Man b1-6 Glc a1-4 Gal a1-Man a1-2 Glc a1-6 Gal a1-Man a1-3 Glc a1-6 Gal a1-Man a1-4 Glc a1-6 Gal a1-Man a1-6 Glc a1-6 Gal a1-Man b1-2 Glc a1-6 Gal a1-Man b1-3 Glc a1-6 Gal a1-Man b1-4 Glc a1-6 Gal a1-Man b1-6 Glc a1-6 Gal a1-Man a1-2 Glc b1-2 Gal a1-Man a1-3 Glc b1-2 Gal a1-Man a1-4 Glc b1-2 Gal a1-Man a1-6 Glc b1-2 Gal a1-Man b1-2 Glc b1-2 Gal a1-Man b1-3 Glc b1-2 Gal a1-Man b1-4 Glc b1-2 Gal a1-Man b1-6 Glc b1-2 Gal a1-Man a1-2 Glc b1-3 Gal a1-Man a1-3 Glc b1-3 Gal a1-Man a1-4 Glc b1-3 Gal a1-Man a1-6 Glc b1-3 Gal a1-Man b1-2 Glc b1-3 Gal a1-Man b1-3 Glc b1-3 Gal a1-Man b1-4 Glc b1-3 Gal a1-Man b1-6 Glc b1-3 Gal a1-Man a1-2 Glc b1-4 Gal a1-Man a1-3 Glc b1-4 Gal a1-Man a1-4 Glc b1-4 Gal a1-Man a1-6 Glc b1-4 Gal a1-Man b1-2 Glc b1-4 Gal a1-Man b1-3 Glc b1-4 Gal a1-Man b1-4 Glc b1-4 Gal a1-Man b1-6 Glc b1-4 Gal a1-Man a1-2 Glc b1-6 Gal a1-Man a1-3 Glc b1-6 Gal a1-Man a1-4 Glc b1-6 Gal a1-Man a1-6 Glc b1-6 Gal a1-Man b1-2 Glc b1-6 Gal a1-Man b1-3 Glc b1-6 Gal a1-Man b1-4 Glc b1-6 Gal a1-Man b1-6 Glc b1-6 Gal a1-Man a1-2 Glc a1-2 Gal b1-Man a1-3 Glc a1-2 Gal b1-Man a1-4 Glc a1-2 Gal b1-Man a1-6 Glc a1-2 Gal b1-Man b1-2 Glc a1-2 Gal b1-Man b1-3 Glc a1-2 Gal b1-Man b1-4 Glc a1-2 Gal b1-Man b1-6 Glc a1-2 Gal b1-Man a1-2 Glc a1-3 Gal b1-Man a1-3 Glc a1-3 Gal b1-Man a1-4 Glc a1-3 Gal b1-Man a1-6 Glc a1-3 Gal b1-Man b1-2 Glc a1-3 Gal b1-Man b1-3 Glc a1-3 Gal b1-Man b1-4 Glc a1-3 Gal b1-Man b1-6 Glc a1-3 Gal b1-Man a1-2 Glc a1-4 Gal b1-Man a1-3 Glc a1-4 Gal b1-Man a1-4 Glc a1-4 Gal b1-Man a1-6 Glc a1-4 Gal b1-Man b1-2 Glc a1-4 Gal b1-Man b1-3 Glc a1-4 Gal b1-Man b1-4 Glc a1-4 Gal b1-Man b1-6 Glc a1-4 Gal b1-Man a1-2 Glc a1-6 Gal b1-Man a1-3 Glc a1-6 Gal b1-Man a1-4 Glc a1-6 Gal b1-Man a1-6 Glc a1-6 Gal b1-Man b1-2 Glc a1-6 Gal b1-Man b1-3 Glc a1-6 Gal b1-Man b1-4 Glc a1-6 Gal b1-Man b1-6 Glc a1-6 Gal b1-Man a1-2 Glc b1-2 Gal b1-Man a1-3 Glc b1-2 Gal b1-Man a1-4 Glc b1-2 Gal b1-Man a1-6 Glc b1-2 Gal b1-Man b1-2 Glc b1-2 Gal b1-Man b1-3 Glc b1-2 Gal b1-Man b1-4 Glc b1-2 Gal b1-Man b1-6 Glc b1-2 Gal b1-Man a1-2 Glc b1-3 Gal b1-Man a1-3 Glc b1-3 Gal b1-Man a1-4 Glc b1-3 Gal b1-Man a1-6 Glc b1-3 Gal b1-Man b1-2 Glc b1-3 Gal b1-Man b1-3 Glc b1-3 Gal b1-Man b1-4 Glc b1-3 Gal b1-Man b1-6 Glc b1-3 Gal b1-Man a1-2 Glc b1-4 Gal b1-Man a1-3 Glc b1-4 Gal b1-Man a1-4 Glc b1-4 Gal b1-Man a1-6 Glc b1-4 Gal b1-

Man b1-2 Glc b1-4 Gal b1-Man b1-3 Glc b1-4 Gal b1-Man b1-4 Glc b1-4 Gal b1-Man b1-6 Glc b1-4 Gal b1-Man a1-2 Glc b1-6 Gal b1-Man a1-3 Glc b1-6 Gal b1-Man a1-4 Glc b1-6 Gal b1-Man a1-6 Glc b1-6 Gal b1-Man b1-2 Glc b1-6 Gal b1-Man b1-3 Glc b1-6 Gal b1-Man b1-4 Glc b1-6 Gal b1-Man b1-6 Glc b1-6 Gal b1-Man a1-2 Gal a1-2 Glc a1-Man a1-3 Gal a1-2 Glc a1-Man a1-4 Gal a1-2 Glc a1-Man a1-6 Gal a1-2 Glc a1-Man b1-2 Gal a1-2 Glc a1-Man b1-3 Gal a1-2 Glc a1-Man b1-4 Gal a1-2 Glc a1-Man b1-6 Gal a1-2 Glc a1-Man a1-2 Gal a1-3 Glc a1-Man a1-3 Gal a1-3 Glc a1-Man a1-4 Gal a1-3 Glc a1-Man a1-6 Gal a1-3 Glc a1-Man b1-2 Gal a1-3 Glc a1-Man b1-3 Gal a1-3 Glc a1-Man b1-4 Gal a1-3 Glc a1-Man b1-6 Gal a1-3 Glc a1-Man a1-2 Gal a1-4 Glc a1-Man a1-3 Gal a1-4 Glc a1-Man a1-4 Gal a1-4 Glc a1-Man a1-6 Gal a1-4 Glc a1-Man b1-2 Gal a1-4 Glc a1-Man b1-3 Gal a1-4 Glc a1-Man b1-4 Gal a1-4 Glc a1-Man b1-6 Gal a1-4 Glc a1-Man a1-2 Gal a1-6 Glc a1-Man a1-3 Gal a1-6 Glc a1-Man a1-4 Gal a1-6 Glc a1-Man a1-6 Gal a1-6 Glc a1-Man b1-2 Gal a1-6 Glc a1-Man b1-3 Gal a1-6 Glc a1-Man b1-4 Gal a1-6 Glc a1-Man b1-6 Gal a1-6 Glc a1-Man a1-2 Gal b1-2 Glc a1-Man a1-3 Gal b1-2 Glc a1-Man a1-4 Gal b1-2 Glc a1-Man a1-6 Gal b1-2 Glc a1-Man b1-2 Gal b1-2 Glc a1-Man b1-3 Gal b1-2 Glc a1-Man b1-4 Gal b1-2 Glc a1-Man b1-6 Gal b1-2 Glc a1-Man a1-2 Gal b1-3 Glc a1-Man a1-3 Gal b1-3 Glc a1-Man a1-4 Gal b1-3 Glc a1-Man a1-6 Gal b1-3 Glc a1-Man b1-2 Gal b1-3 Glc a1-Man b1-3 Gal b1-3 Glc a1-Man b1-4 Gal b1-3 Glc a1-Man b1-6 Gal b1-3 Glc a1-Man a1-2 Gal b1-4 Glc a1-Man a1-3 Gal b1-4 Glc a1-Man a1-4 Gal b1-4 Glc a1-Man a1-6 Gal b1-4 Glc a1-Man b1-2 Gal b1-4 Glc a1-Man b1-3 Gal b1-4 Glc a1-Man b1-4 Gal b1-4 Glc a1-Man b1-6 Gal b1-4 Glc a1-Man a1-2 Gal b1-6 Glc a1-Man a1-3 Gal b1-6 Glc a1-Man a1-4 Gal b1-6 Glc a1-Man a1-6 Gal b1-6 Glc a1-Man b1-2 Gal b1-6 Glc a1-Man b1-3 Gal b1-6 Glc a1-Man b1-4 Gal b1-6 Glc a1-Man b1-6 Gal b1-6 Glc a1-Man a1-2 Gal a1-2 Glc b1Man a1-3 Gal a1-2 Glc b1Man a1-4 Gal a1-2 Glc b1Man a1-6 Gal a1-2 Glc b1Man b1-2 Gal a1-2 Glc b1Man b1-3 Gal a1-2 Glc b1Man b1-4 Gal a1-2 Glc b1Man b1-6 Gal a1-2 Glc b1Man a1-2 Gal a1-3 Glc b1Man a1-3 Gal a1-3 Glc b1Man a1-4 Gal a1-3 Glc b1Man a1-6 Gal a1-3 Glc b1Man b1-2 Gal a1-3 Glc b1Man b1-3 Gal a1-3 Glc b1Man b1-4 Gal a1-3 Glc b1Man b1-6 Gal a1-3 Glc b1Man a1-2 Gal a1-4 Glc b1

Man a1-3 Gal a1-4 Glc b1Man a1-4 Gal a1-4 Glc b1Man a1-6 Gal a1-4 Glc b1Man b1-2 Gal a1-4 Glc b1Man b1-3 Gal a1-4 Glc b1Man b1-4 Gal a1-4 Glc b1Man b1-6 Gal a1-4 Glc b1Man a1-2 Gal a1-6 Glc b1Man a1-3 Gal a1-6 Glc b1Man a1-4 Gal a1-6 Glc b1Man a1-6 Gal a1-6 Glc b1Man b1-2 Gal a1-6 Glc b1Man b1-3 Gal a1-6 Glc b1Man b1-4 Gal a1-6 Glc b1Man b1-6 Gal a1-6 Glc b1Man a1-2 Gal b1-2 Glc b1Man a1-3 Gal b1-2 Glc b1Man a1-4 Gal b1-2 Glc b1Man a1-6 Gal b1-2 Glc b1Man b1-2 Gal b1-2 Glc b1Man b1-3 Gal b1-2 Glc b1Man b1-4 Gal b1-2 Glc b1Man b1-6 Gal b1-2 Glc b1Man a1-2 Gal b1-3 Glc b1Man a1-3 Gal b1-3 Glc b1Man a1-4 Gal b1-3 Glc b1Man a1-6 Gal b1-3 Glc b1Man b1-2 Gal b1-3 Glc b1Man b1-3 Gal b1-3 Glc b1Man b1-4 Gal b1-3 Glc b1Man b1-6 Gal b1-3 Glc b1Man a1-2 Gal b1-4 Glc b1Man a1-3 Gal b1-4 Glc b1Man a1-4 Gal b1-4 Glc b1Man a1-6 Gal b1-4 Glc b1Man b1-2 Gal b1-4 Glc b1Man b1-3 Gal b1-4 Glc b1Man b1-4 Gal b1-4 Glc b1Man b1-6 Gal b1-4 Glc b1Man a1-2 Gal b1-6 Glc b1Man a1-3 Gal b1-6 Glc b1Man a1-4 Gal b1-6 Glc b1Man a1-6 Gal b1-6 Glc b1Man b1-2 Gal b1-6 Glc b1Man b1-3 Gal b1-6 Glc b1Man b1-4 Gal b1-6 Glc b1Man b1-6 Gal b1-6 Glc b1Glc a1-2 Man a1-2 Gal a1-Glc a1-3 Man a1-2 Gal a1-Glc a1-4 Man a1-2 Gal a1-Glc a1-6 Man a1-2 Gal a1-Glc b1-2 Man a1-2 Gal a1-Glc b1-3 Man a1-2 Gal a1-Glc b1-4 Man a1-2 Gal a1-Glc b1-6 Man a1-2 Gal a1-Glc a1-2 Man a1-3 Gal a1-Glc a1-3 Man a1-3 Gal a1-Glc a1-4 Man a1-3 Gal a1-Glc a1-6 Man a1-3 Gal a1-Glc b1-2 Man a1-3 Gal a1-Glc b1-3 Man a1-3 Gal a1-Glc b1-4 Man a1-3 Gal a1-Glc b1-6 Man a1-3 Gal a1-Glc a1-2 Man a1-4 Gal a1-Glc a1-3 Man a1-4 Gal a1-Glc a1-4 Man a1-4 Gal a1-Glc a1-6 Man a1-4 Gal a1-Glc b1-2 Man a1-4 Gal a1-Glc b1-3 Man a1-4 Gal a1-Glc b1-4 Man a1-4 Gal a1-Glc b1-6 Man a1-4 Gal a1-Glc a1-2 Man a1-6 Gal a1-Glc a1-3 Man a1-6 Gal a1-Glc a1-4 Man a1-6 Gal a1-Glc a1-6 Man a1-6 Gal a1-Glc b1-2 Man a1-6 Gal a1-Glc b1-3 Man a1-6 Gal a1-Glc b1-4 Man a1-6 Gal a1-Glc b1-6 Man a1-6 Gal a1-Glc a1-2 Man b1-2 Gal a1-Glc a1-3 Man b1-2 Gal a1-Glc a1-4 Man b1-2 Gal a1-Glc a1-6 Man b1-2 Gal a1-Glc b1-2 Man b1-2 Gal a1-Glc b1-3 Man b1-2 Gal a1-Glc b1-4 Man b1-2 Gal a1-Glc b1-6 Man b1-2 Gal a1-Glc a1-2 Man b1-3 Gal a1-Glc a1-3 Man b1-3 Gal a1-Glc a1-4 Man b1-3 Gal a1-Glc a1-6 Man b1-3 Gal a1-Glc b1-2 Man b1-3 Gal a1-Glc b1-3 Man b1-3 Gal a1-

Glc b1-4 Man b1-3 Gal a1-Glc b1-6 Man b1-3 Gal a1-Glc a1-2 Man b1-4 Gal a1-Glc a1-3 Man b1-4 Gal a1-Glc a1-4 Man b1-4 Gal a1-Glc a1-6 Man b1-4 Gal a1-Glc b1-2 Man b1-4 Gal a1-Glc b1-3 Man b1-4 Gal a1-Glc b1-4 Man b1-4 Gal a1-Glc b1-6 Man b1-4 Gal a1-Glc a1-2 Man b1-6 Gal a1-Glc a1-3 Man b1-6 Gal a1-Glc a1-4 Man b1-6 Gal a1-Glc a1-6 Man b1-6 Gal a1-Glc b1-2 Man b1-6 Gal a1-Glc b1-3 Man b1-6 Gal a1-Glc b1-4 Man b1-6 Gal a1-Glc b1-6 Man b1-6 Gal a1-Glc a1-2 Man a1-2 Gal b1-Glc a1-3 Man a1-2 Gal b1-Glc a1-4 Man a1-2 Gal b1-Glc a1-6 Man a1-2 Gal b1-Glc b1-2 Man a1-2 Gal b1-Glc b1-3 Man a1-2 Gal b1-Glc b1-4 Man a1-2 Gal b1-Glc b1-6 Man a1-2 Gal b1-Glc a1-2 Man a1-3 Gal b1-Glc a1-3 Man a1-3 Gal b1-Glc a1-4 Man a1-3 Gal b1-Glc a1-6 Man a1-3 Gal b1-Glc b1-2 Man a1-3 Gal b1-Glc b1-3 Man a1-3 Gal b1-Glc b1-4 Man a1-3 Gal b1-Glc b1-6 Man a1-3 Gal b1-Glc a1-2 Man a1-4 Gal b1-Glc a1-3 Man a1-4 Gal b1-Glc a1-4 Man a1-4 Gal b1-Glc a1-6 Man a1-4 Gal b1-Glc b1-2 Man a1-4 Gal b1-Glc b1-3 Man a1-4 Gal b1-Glc b1-4 Man a1-4 Gal b1-Glc b1-6 Man a1-4 Gal b1-Glc a1-2 Man a1-6 Gal b1-Glc a1-3 Man a1-6 Gal b1-Glc a1-4 Man a1-6 Gal b1-Glc a1-6 Man a1-6 Gal b1-Glc b1-2 Man a1-6 Gal b1-Glc b1-3 Man a1-6 Gal b1-Glc b1-4 Man a1-6 Gal b1-Glc b1-6 Man a1-6 Gal b1-Glc a1-2 Man b1-2 Gal b1-Glc a1-3 Man b1-2 Gal b1-Glc a1-4 Man b1-2 Gal b1-Glc a1-6 Man b1-2 Gal b1-Glc b1-2 Man b1-2 Gal b1-Glc b1-3 Man b1-2 Gal b1-Glc b1-4 Man b1-2 Gal b1-Glc b1-6 Man b1-2 Gal b1-Glc a1-2 Man b1-3 Gal b1-Glc a1-3 Man b1-3 Gal b1-Glc a1-4 Man b1-3 Gal b1-Glc a1-6 Man b1-3 Gal b1-Glc b1-2 Man b1-3 Gal b1-Glc b1-3 Man b1-3 Gal b1-Glc b1-4 Man b1-3 Gal b1-Glc b1-6 Man b1-3 Gal b1-Glc a1-2 Man b1-4 Gal b1-Glc a1-3 Man b1-4 Gal b1-Glc a1-4 Man b1-4 Gal b1-Glc a1-6 Man b1-4 Gal b1-Glc b1-2 Man b1-4 Gal b1-Glc b1-3 Man b1-4 Gal b1-Glc b1-4 Man b1-4 Gal b1-Glc b1-6 Man b1-4 Gal b1-Glc a1-2 Man b1-6 Gal b1-Glc a1-3 Man b1-6 Gal b1-Glc a1-4 Man b1-6 Gal b1-Glc a1-6 Man b1-6 Gal b1-Glc b1-2 Man b1-6 Gal b1-Glc b1-3 Man b1-6 Gal b1-Glc b1-4 Man b1-6 Gal b1-Glc b1-6 Man b1-6 Gal b1-Glc a1-2 Gal a1-2 Man a1-Glc a1-3 Gal a1-2 Man a1-Glc a1-4 Gal a1-2 Man a1-Glc a1-6 Gal a1-2 Man a1-Glc b1-2 Gal a1-2 Man a1-Glc b1-3 Gal a1-2 Man a1-Glc b1-4 Gal a1-2 Man a1-Glc b1-6 Gal a1-2 Man a1-Glc a1-2 Gal a1-3 Man a1-Glc a1-3 Gal a1-3 Man a1-Glc a1-4 Gal a1-3 Man a1-

Glc a1-6 Gal a1-3 Man a1-Glc b1-2 Gal a1-3 Man a1-Glc b1-3 Gal a1-3 Man a1-Glc b1-4 Gal a1-3 Man a1-Glc b1-6 Gal a1-3 Man a1-Glc a1-2 Gal a1-4 Man a1-Glc a1-3 Gal a1-4 Man a1-Glc a1-4 Gal a1-4 Man a1-Glc a1-6 Gal a1-4 Man a1-Glc b1-2 Gal a1-4 Man a1-Glc b1-3 Gal a1-4 Man a1-Glc b1-4 Gal a1-4 Man a1-Glc b1-6 Gal a1-4 Man a1-Glc a1-2 Gal a1-6 Man a1-Glc a1-3 Gal a1-6 Man a1-Glc a1-4 Gal a1-6 Man a1-Glc a1-6 Gal a1-6 Man a1-Glc b1-2 Gal a1-6 Man a1-Glc b1-3 Gal a1-6 Man a1-Glc b1-4 Gal a1-6 Man a1-Glc b1-6 Gal a1-6 Man a1-Glc a1-2 Gal b1-2 Man a1-Glc a1-3 Gal b1-2 Man a1-Glc a1-4 Gal b1-2 Man a1-Glc a1-6 Gal b1-2 Man a1-Glc b1-2 Gal b1-2 Man a1-Glc b1-3 Gal b1-2 Man a1-Glc b1-4 Gal b1-2 Man a1-Glc b1-6 Gal b1-2 Man a1-Glc a1-2 Gal b1-3 Man a1-Glc a1-3 Gal b1-3 Man a1-Glc a1-4 Gal b1-3 Man a1-Glc a1-6 Gal b1-3 Man a1-Glc b1-2 Gal b1-3 Man a1-Glc b1-3 Gal b1-3 Man a1-Glc b1-4 Gal b1-3 Man a1-Glc b1-6 Gal b1-3 Man a1-Glc a1-2 Gal b1-4 Man a1-Glc a1-3 Gal b1-4 Man a1-Glc a1-4 Gal b1-4 Man a1-Glc a1-6 Gal b1-4 Man a1-Glc b1-2 Gal b1-4 Man a1-Glc b1-3 Gal b1-4 Man a1-Glc b1-4 Gal b1-4 Man a1-Glc b1-6 Gal b1-4 Man a1-Glc a1-2 Gal b1-6 Man a1-Glc a1-3 Gal b1-6 Man a1-Glc a1-4 Gal b1-6 Man a1-Glc a1-6 Gal b1-6 Man a1-Glc b1-2 Gal b1-6 Man a1-Glc b1-3 Gal b1-6 Man a1-Glc b1-4 Gal b1-6 Man a1-Glc b1-6 Gal b1-6 Man a1-Glc a1-2 Gal a1-2 Man b1-Glc a1-3 Gal a1-2 Man b1-Glc a1-4 Gal a1-2 Man b1-Glc a1-6 Gal a1-2 Man b1-Glc b1-2 Gal a1-2 Man b1-Glc b1-3 Gal a1-2 Man b1-Glc b1-4 Gal a1-2 Man b1-Glc b1-6 Gal a1-2 Man b1-Glc a1-2 Gal a1-3 Man b1-Glc a1-3 Gal a1-3 Man b1-Glc a1-4 Gal a1-3 Man b1-Glc a1-6 Gal a1-3 Man b1-Glc b1-2 Gal a1-3 Man b1-Glc b1-3 Gal a1-3 Man b1-Glc b1-4 Gal a1-3 Man b1-Glc b1-6 Gal a1-3 Man b1-Glc a1-2 Gal a1-4 Man b1-Glc a1-3 Gal a1-4 Man b1-Glc a1-4 Gal a1-4 Man b1-Glc a1-6 Gal a1-4 Man b1-Glc b1-2 Gal a1-4 Man b1-Glc b1-3 Gal a1-4 Man b1-Glc b1-4 Gal a1-4 Man b1-Glc b1-6 Gal a1-4 Man b1-Glc a1-2 Gal a1-6 Man b1-Glc a1-3 Gal a1-6 Man b1-Glc a1-4 Gal a1-6 Man b1-Glc a1-6 Gal a1-6 Man b1-Glc b1-2 Gal a1-6 Man b1-Glc b1-3 Gal a1-6 Man b1-Glc b1-4 Gal a1-6 Man b1-Glc b1-6 Gal a1-6 Man b1-Glc a1-2 Gal b1-2 Man b1-Glc a1-3 Gal b1-2 Man b1-Glc a1-4 Gal b1-2 Man b1-Glc a1-6 Gal b1-2 Man b1-Glc b1-2 Gal b1-2 Man b1-Glc b1-3 Gal b1-2 Man b1-Glc b1-4 Gal b1-2 Man b1-Glc b1-6 Gal b1-2 Man b1-

Glc a1-2 Gal b1-3 Man b1-Glc a1-3 Gal b1-3 Man b1-Glc a1-4 Gal b1-3 Man b1-Glc a1-6 Gal b1-3 Man b1-Glc b1-2 Gal b1-3 Man b1-Glc b1-3 Gal b1-3 Man b1-Glc b1-4 Gal b1-3 Man b1-Glc b1-6 Gal b1-3 Man b1-Glc a1-2 Gal b1-4 Man b1-Glc a1-3 Gal b1-4 Man b1-Glc a1-4 Gal b1-4 Man b1-Glc a1-6 Gal b1-4 Man b1-Glc b1-2 Gal b1-4 Man b1-Glc b1-3 Gal b1-4 Man b1-Glc b1-4 Gal b1-4 Man b1-Glc b1-6 Gal b1-4 Man b1-Glc a1-2 Gal b1-6 Man b1-Glc a1-3 Gal b1-6 Man b1-Glc a1-4 Gal b1-6 Man b1-Glc a1-6 Gal b1-6 Man b1-Glc b1-2 Gal b1-6 Man b1-Glc b1-3 Gal b1-6 Man b1-Glc b1-4 Gal b1-6 Man b1-Glc b1-6 Gal b1-6 Man b1-Gal a1-2 (Glc a1-3) Man a1-Gal a1-2 (Glc a1-4) Man a1-Gal a1-2 (Glc a1-6) Man a1-Gal a1-3 (Glc a1-2) Man a1-Gal a1-3 (Glc a1-4) Man a1-Gal a1-3 (Glc a1-6) Man a1-Gal a1-4 (Glc a1-2) Man a1-Gal a1-4 (Glc a1-3) Man a1-Gal a1-4 (Glc a1-6) Man a1-Gal a1-6 (Glc a1-2) Man a1-Gal a1-6 (Glc a1-3) Man a1-Gal a1-6 (Glc a1-4) Man a1-Gal b1-2 (Glc a1-3) Man a1-Gal b1-2 (Glc a1-4) Man a1-Gal b1-2 (Glc a1-6) Man a1-Gal b1-3 (Glc a1-2) Man a1-Gal b1-3 (Glc a1-4) Man a1-Gal b1-3 (Glc a1-6) Man a1-Gal b1-4 (Glc a1-2) Man a1-Gal b1-4 (Glc a1-3) Man a1-Gal b1-4 (Glc a1-6) Man a1-Gal b1-6 (Glc a1-2) Man a1-Gal b1-6 (Glc a1-3) Man a1-Gal b1-6 (Glc a1-4) Man a1-Gal a1-2 (Glc b1-3) Man a1-Gal a1-2 (Glc b1-4) Man a1-Gal a1-2 (Glc b1-6) Man a1-Gal a1-3 (Glc b1-2) Man a1-Gal a1-3 (Glc b1-4) Man a1-Gal a1-3 (Glc b1-6) Man a1-Gal a1-4 (Glc b1-2) Man a1-Gal a1-4 (Glc b1-3) Man a1-Gal a1-4 (Glc b1-6) Man a1-Gal a1-6 (Glc b1-2) Man a1-Gal a1-6 (Glc b1-3) Man a1-Gal a1-6 (Glc b1-4) Man a1-Gal b1-2 (Glc b1-3) Man a1-Gal b1-2 (Glc b1-4) Man a1-Gal b1-2 (Glc b1-6) Man a1-Gal b1-3 (Glc b1-2) Man a1-Gal b1-3 (Glc b1-4) Man a1-Gal b1-3 (Glc b1-6) Man a1-Gal b1-4 (Glc b1-2) Man a1-Gal b1-4 (Glc b1-3) Man a1-Gal b1-4 (Glc b1-6) Man a1-Gal b1-6 (Glc b1-2) Man a1-Gal b1-6 (Glc b1-3) Man a1-Gal b1-6 (Glc b1-4) Man a1-Gal a1-2 (Glc a1-3) Man b1-Gal a1-2 (Glc a1-4) Man b1-Gal a1-2 (Glc a1-6) Man b1-Gal a1-3 (Glc a1-2) Man b1-Gal a1-3 (Glc a1-4) Man b1-Gal a1-3 (Glc a1-6) Man b1-Gal a1-4 (Glc a1-2) Man b1-Gal a1-4 (Glc a1-3) Man b1-Gal a1-4 (Glc a1-6) Man b1-Gal a1-6 (Glc a1-2) Man b1-Gal a1-6 (Glc a1-3) Man b1-Gal a1-6 (Glc a1-4) Man b1-Gal b1-2 (Glc a1-3) Man b1-Gal b1-2 (Glc a1-4) Man b1-Gal b1-2 (Glc a1-6) Man b1-Gal b1-3 (Glc a1-2) Man b1-Gal b1-3 (Glc a1-4) Man b1-Gal b1-3 (Glc a1-6) Man b1-Gal b1-4 (Glc a1-2) Man b1-Gal b1-4 (Glc a1-3) Man b1-Gal b1-4 (Glc a1-6) Man b1-

Gal b1-6 (Glc a1-2) Man b1-Gal b1-6 (Glc a1-3) Man b1-Gal b1-6 (Glc a1-4) Man b1-Gal a1-2 (Glc b1-3) Man b1-Gal a1-2 (Glc b1-4) Man b1-Gal a1-2 (Glc b1-6) Man b1-Gal a1-3 (Glc b1-2) Man b1-Gal a1-3 (Glc b1-4) Man b1-Gal a1-3 (Glc b1-6) Man b1-Gal a1-4 (Glc b1-2) Man b1-Gal a1-4 (Glc b1-3) Man b1-Gal a1-4 (Glc b1-6) Man b1-Gal a1-6 (Glc b1-2) Man b1-Gal a1-6 (Glc b1-3) Man b1-Gal a1-6 (Glc b1-4) Man b1-Gal b1-2 (Glc b1-3) Man b1-Gal b1-2 (Glc b1-4) Man b1-Gal b1-2 (Glc b1-6) Man b1-Gal b1-3 (Glc b1-2) Man b1-Gal b1-3 (Glc b1-4) Man b1-Gal b1-3 (Glc b1-6) Man b1-Gal b1-4 (Glc b1-2) Man b1-Gal b1-4 (Glc b1-3) Man b1-Gal b1-4 (Glc b1-6) Man b1-Gal b1-6 (Glc b1-2) Man b1-Gal b1-6 (Glc b1-3) Man b1-Gal b1-6 (Glc b1-4) Man b1-Gal a1-2 (Man a1-3) Glc a1-Gal a1-2 (Man a1-4) Glc a1-Gal a1-2 (Man a1-6) Glc a1-Gal a1-3 (Man a1-2) Glc a1-Gal a1-3 (Man a1-4) Glc a1-Gal a1-3 (Man a1-6) Glc a1-Gal a1-4 (Man a1-2) Glc a1-Gal a1-4 (Man a1-3) Glc a1-Gal a1-4 (Man a1-6) Glc a1-Gal a1-6 (Man a1-2) Glc a1-Gal a1-6 (Man a1-3) Glc a1-Gal a1-6 (Man a1-4) Glc a1-Gal b1-2 (Man a1-3) Glc a1-Gal b1-2 (Man a1-4) Glc a1-Gal b1-2 (Man a1-6) Glc a1-Gal b1-3 (Man a1-2) Glc a1-Gal b1-3 (Man a1-4) Glc a1-Gal b1-3 (Man a1-6) Glc a1-Gal b1-4 (Man a1-2) Glc a1-Gal b1-4 (Man a1-3) Glc a1-Gal b1-4 (Man a1-6) Glc a1-Gal b1-6 (Man a1-2) Glc a1-Gal b1-6 (Man a1-3) Glc a1-Gal b1-6 (Man a1-4) Glc a1-Gal a1-2 (Man b1-3) Glc a1-Gal a1-2 (Man b1-4) Glc a1-Gal a1-2 (Man b1-6) Glc a1-Gal a1-3 (Man b1-2) Glc a1-Gal a1-3 (Man b1-4) Glc a1-Gal a1-3 (Man b1-6) Glc a1-Gal a1-4 (Man b1-2) Glc a1-Gal a1-4 (Man b1-3) Glc a1-Gal a1-4 (Man b1-6) Glc a1-Gal a1-6 (Man b1-2) Glc a1-Gal a1-6 (Man b1-3) Glc a1-Gal a1-6 (Man b1-4) Glc a1-Gal b1-2 (Man b1-3) Glc a1-Gal b1-2 (Man b1-4) Glc a1-Gal b1-2 (Man b1-6) Glc a1-Gal b1-3 (Man b1-2) Glc a1-Gal b1-3 (Man b1-4) Glc a1-Gal b1-3 (Man b1-6) Glc a1-Gal b1-4 (Man b1-2) Glc a1-Gal b1-4 (Man b1-3) Glc a1-Gal b1-4 (Man b1-6) Glc a1-Gal b1-6 (Man b1-2) Glc a1-Gal b1-6 (Man b1-3) Glc a1-Gal b1-6 (Man b1-4) Glc a1-Gal a1-2 (Man a1-3) Glc b1-Gal a1-2 (Man a1-4) Glc b1-Gal a1-2 (Man a1-6) Glc b1-Gal a1-3 (Man a1-2) Glc b1-Gal a1-3 (Man a1-4) Glc b1-Gal a1-3 (Man a1-6) Glc b1-

Gal a1-4 (Man a1-2) Glc b1-Gal a1-4 (Man a1-3) Glc b1-Gal a1-4 (Man a1-6) Glc b1-Gal a1-6 (Man a1-2) Glc b1-Gal a1-6 (Man a1-3) Glc b1-Gal a1-6 (Man a1-4) Glc b1-Gal b1-2 (Man a1-3) Glc b1-Gal b1-2 (Man a1-4) Glc b1-Gal b1-2 (Man a1-6) Glc b1-Gal b1-3 (Man a1-2) Glc b1-Gal b1-3 (Man a1-4) Glc b1-Gal b1-3 (Man a1-6) Glc b1-Gal b1-4 (Man a1-2) Glc b1-Gal b1-4 (Man a1-3) Glc b1-Gal b1-4 (Man a1-6) Glc b1-Gal b1-6 (Man a1-2) Glc b1-Gal b1-6 (Man a1-3) Glc b1-Gal b1-6 (Man a1-4) Glc b1-Gal a1-2 (Man b1-3) Glc b1-Gal a1-2 (Man b1-4) Glc b1-Gal a1-2 (Man b1-6) Glc b1-Gal a1-3 (Man b1-2) Glc b1-Gal a1-3 (Man b1-4) Glc b1-Gal a1-3 (Man b1-6) Glc b1-Gal a1-4 (Man b1-2) Glc b1-Gal a1-4 (Man b1-3) Glc b1-Gal a1-4 (Man b1-6) Glc b1-Gal a1-6 (Man b1-2) Glc b1-Gal a1-6 (Man b1-3) Glc b1-Gal a1-6 (Man b1-4) Glc b1-Gal b1-2 (Man b1-3) Glc b1-Gal b1-2 (Man b1-4) Glc b1-Gal b1-2 (Man b1-6) Glc b1-Gal b1-3 (Man b1-2) Glc b1-Gal b1-3 (Man b1-4) Glc b1-Gal b1-3 (Man b1-6) Glc b1-Gal b1-4 (Man b1-2) Glc b1-Gal b1-4 (Man b1-3) Glc b1-Gal b1-4 (Man b1-6) Glc b1-Gal b1-6 (Man b1-2) Glc b1-Gal b1-6 (Man b1-3) Glc b1-Gal b1-6 (Man b1-4) Glc b1-Glc a1-2 (Man a1-3) Gal a1-Glc a1-2 (Man a1-4) Gal a1-Glc a1-2 (Man a1-6) Gal a1-Glc a1-3 (Man a1-2) Gal a1-Glc a1-3 (Man a1-4) Gal a1-Glc a1-3 (Man a1-6) Gal a1-Glc a1-4 (Man a1-2) Gal a1-Glc a1-4 (Man a1-3) Gal a1-Glc a1-4 (Man a1-6) Gal a1-Glc a1-6 (Man a1-2) Gal a1-Glc a1-6 (Man a1-3) Gal a1-Glc a1-6 (Man a1-4) Gal a1-Glc b1-2 (Man a1-3) Gal a1-Glc b1-2 (Man a1-4) Gal a1-Glc b1-2 (Man a1-6) Gal a1-Glc b1-3 (Man a1-2) Gal a1-Glc b1-3 (Man a1-4) Gal a1-Glc b1-3 (Man a1-6) Gal a1-Glc b1-4 (Man a1-2) Gal a1-Glc b1-4 (Man a1-3) Gal a1-Glc b1-4 (Man a1-6) Gal a1-Glc b1-6 (Man a1-2) Gal a1-Glc b1-6 (Man a1-3) Gal a1-Glc b1-6 (Man a1-4) Gal a1-Glc a1-2 (Man b1-3) Gal a1-Glc a1-2 (Man b1-4) Gal a1-Glc a1-2 (Man b1-6) Gal a1-Glc a1-3 (Man b1-2) Gal a1-Glc a1-3 (Man b1-4) Gal a1-Glc a1-3 (Man b1-6) Gal a1-Glc a1-4 (Man b1-2) Gal a1-Glc a1-4 (Man b1-3) Gal a1-Glc a1-4 (Man b1-6) Gal a1-Glc a1-6 (Man b1-2) Gal a1-Glc a1-6 (Man b1-3) Gal a1-Glc a1-6 (Man b1-4) Gal a1-Glc b1-2 (Man b1-3) Gal a1-Glc b1-2 (Man b1-4) Gal a1-Glc b1-2 (Man b1-6) Gal a1-

Glc b1-3 (Man b1-2) Gal a1-Glc b1-3 (Man b1-4) Gal a1-Glc b1-3 (Man b1-6) Gal a1-Glc b1-4 (Man b1-2) Gal a1-Glc b1-4 (Man b1-3) Gal a1-Glc b1-4 (Man b1-6) Gal a1-Glc b1-6 (Man b1-2) Gal a1-Glc b1-6 (Man b1-3) Gal a1-Glc b1-6 (Man b1-4) Gal a1-Glc a1-2 (Man a1-3) Gal b1-Glc a1-2 (Man a1-4) Gal b1-Glc a1-2 (Man a1-6) Gal b1-Glc a1-3 (Man a1-2) Gal b1-Glc a1-3 (Man a1-4) Gal b1-Glc a1-3 (Man a1-6) Gal b1-Glc a1-4 (Man a1-2) Gal b1-Glc a1-4 (Man a1-3) Gal b1-Glc a1-4 (Man a1-6) Gal b1-Glc a1-6 (Man a1-2) Gal b1-Glc a1-6 (Man a1-3) Gal b1-Glc a1-6 (Man a1-4) Gal b1-Glc b1-2 (Man a1-3) Gal b1-Glc b1-2 (Man a1-4) Gal b1-Glc b1-2 (Man a1-6) Gal b1-Glc b1-3 (Man a1-2) Gal b1-Glc b1-3 (Man a1-4) Gal b1-Glc b1-3 (Man a1-6) Gal b1-Glc b1-4 (Man a1-2) Gal b1-Glc b1-4 (Man a1-3) Gal b1-Glc b1-4 (Man a1-6) Gal b1-Glc b1-6 (Man a1-2) Gal b1-Glc b1-6 (Man a1-3) Gal b1-Glc b1-6 (Man a1-4) Gal b1-Glc a1-2 (Man b1-3) Gal b1-Glc a1-2 (Man b1-4) Gal b1-Glc a1-2 (Man b1-6) Gal b1-Glc a1-3 (Man b1-2) Gal b1-Glc a1-3 (Man b1-4) Gal b1-Glc a1-3 (Man b1-6) Gal b1-Glc a1-4 (Man b1-2) Gal b1-Glc a1-4 (Man b1-3) Gal b1-Glc a1-4 (Man b1-6) Gal b1-Glc a1-6 (Man b1-2) Gal b1-Glc a1-6 (Man b1-3) Gal b1-Glc a1-6 (Man b1-4) Gal b1-Glc b1-2 (Man b1-3) Gal b1-Glc b1-2 (Man b1-4) Gal b1-Glc b1-2 (Man b1-6) Gal b1-Glc b1-3 (Man b1-2) Gal b1-Glc b1-3 (Man b1-4) Gal b1-Glc b1-3 (Man b1-6) Gal b1-Glc b1-4 (Man b1-2) Gal b1-Glc b1-4 (Man b1-3) Gal b1-Glc b1-4 (Man b1-6) Gal b1-Glc b1-6 (Man b1-2) Gal b1-Glc b1-6 (Man b1-3) Gal b1-Glc b1-6 (Man b1-4) Gal b1-

Man a1-4 Gal a1-3 Glc b1Man a1-6 Gal a1-3 Glc b1Man b1-2 Gal a1-3 Glc b1Man b1-3 Gal a1-3 Glc b1Man b1-4 Gal a1-3 Glc b1Man b1-6 Gal a1-3 Glc b1Man a1-2 Gal a1-4 Glc b1Man a1-3 Gal a1-4 Glc b1Man a1-4 Gal a1-4 Glc b1Man a1-6 Gal a1-4 Glc b1Man b1-2 Gal a1-4 Glc b1Man b1-3 Gal a1-4 Glc b1Man b1-4 Gal a1-4 Glc b1Man b1-6 Gal a1-4 Glc b1Man a1-2 Gal a1-6 Glc b1Man a1-3 Gal a1-6 Glc b1

Cummings RD (2009) Molecular BioSystems 5 1087

Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical petasaccharide sequeces in glycosaminoglycans

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoN-linkedrdquo glycan linkage to protein asparagine residues

Families of N-linked glycan structures

Man

4GlcNAc1

2Man

Man

Man

Man

2Man

2Man

2Man

63

Man63 4GlcNAc1 Asn

4GlcNAc1

Man62

Man63 4GlcNAc1 Asn

Man42

4GlcNAc1

4GlcNAc1

4GlcNAc1

4GlcNAc1

6Gal

6Gal

6Gal

6Gal

NeuAc

NeuAc

NeuAcNeuAc

4GlcNAc1

Man

Man63 4GlcNAc1 Asn

2Man4GlcNAc13GalNeuAc

Man

Man

63

core

core

core

High Mannose

Complex

Hybrid

Examples of structures found as termini on the branches of complex N-linked glycoprotein glycanshellip

Terminal Saccharide Diversity --- A key to recognition

NeuAc 2-6 Gal 1-4 GlcNAc---NeuAc 2-3 Gal 1-4 GlcNAc---

Gal 1-3 Gal 1-4 GlcNAc---4-SO3-GalNAc 1-4 GlcNAc---

(-Gal 1-4 GlcNAc 1-3-)n = polylactosamine repeats (n may be gt6)

(-NeuAc 2-8-) n = polysialic acid (n may be gt50)

Fuc residues (eg Fuc 1-2 Gal Fuc 1-3 GlcNAc Fuc 1-4 GlcNAc

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoO-linkedrdquo glycan linkage to protein serine residues

Examples of variations of O-linked glycans

GalNAc Serine

GalNAc Serine

NeuAc 26

GalNAc Serine

Gal 1

NeuAc 263

GalNAc SerineNeuAc 2-3 Gal 1

NeuAc 263

Gal 1-4 GlcNAc 1

Fuc 1

GalNAc Serine

GlcNAc 1

63

4NeuAc 2-3 Gal 1

3

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Oligosaccharides Molecular diversity of glycans

Polypeptides Glycans

Building blocks amino acids monosaccharides

Number of different monomers 20 common 9 common

Linkage sites per monomer 1 3-4

Possible linkage configurations 1 2

Possible homodimer structures 1 6-8

Linkage modes linear linear or branched

HO

HO

O

HO

O

GalHO

OHO

HO

O

HO

O

GalHO

O

H2N C

Ala

OH

OH2N C

Ala

OH

O

Three different amino acids (Ala Ser Tyr) ndash 6 structures

Ala-Ser-TyrAla-Tyr-SerSer-Ala-TyrSer-Tyr-AlaTyr-Ala-SerTyr-Ser-Ala

Three different sugars (Glc Gal Man) ndash 1056 structures hellip

Gal a1-2 Glc a1-2 Man a1-Gal a1-3 Glc a1-2 Man a1-Gal a1-4 Glc a1-2 Man a1-Gal a1-6 Glc a1-2 Man a1-Gal b1-2 Glc a1-2 Man a1-Gal b1-3 Glc a1-2 Man a1-Gal b1-4 Glc a1-2 Man a1-Gal b1-6 Glc a1-2 Man a1-Gal a1-2 Glc a1-3 Man a1-Gal a1-3 Glc a1-3 Man a1-Gal a1-4 Glc a1-3 Man a1-Gal a1-6 Glc a1-3 Man a1-Gal b1-2 Glc a1-3 Man a1-Gal b1-3 Glc a1-3 Man a1-Gal b1-4 Glc a1-3 Man a1-Gal b1-6 Glc a1-3 Man a1-Gal a1-2 Glc a1-4 Man a1-Gal a1-3 Glc a1-4 Man a1-Gal a1-4 Glc a1-4 Man a1-Gal a1-6 Glc a1-4 Man a1-Gal b1-2 Glc a1-4 Man a1-Gal b1-3 Glc a1-4 Man a1-Gal b1-4 Glc a1-4 Man a1-Gal b1-6 Glc a1-4 Man a1-Gal a1-2 Glc a1-6 Man a1-Gal a1-3 Glc a1-6 Man a1-Gal a1-4 Glc a1-6 Man a1-Gal a1-6 Glc a1-6 Man a1-Gal b1-2 Glc a1-6 Man a1-Gal b1-3 Glc a1-6 Man a1-Gal b1-4 Glc a1-6 Man a1-Gal b1-6 Glc a1-6 Man a1-Gal a1-2 Glc b1-2 Man a1-Gal a1-3 Glc b1-2 Man a1-Gal a1-4 Glc b1-2 Man a1-Gal a1-6 Glc b1-2 Man a1-Gal b1-2 Glc b1-2 Man a1-Gal b1-3 Glc b1-2 Man a1-Gal b1-4 Glc b1-2 Man a1-Gal b1-6 Glc b1-2 Man a1-Gal a1-2 Glc b1-3 Man a1-Gal a1-3 Glc b1-3 Man a1-Gal a1-4 Glc b1-3 Man a1-Gal a1-6 Glc b1-3 Man a1-Gal b1-2 Glc b1-3 Man a1-Gal b1-3 Glc b1-3 Man a1-Gal b1-4 Glc b1-3 Man a1-Gal b1-6 Glc b1-3 Man a1-Gal a1-2 Glc b1-4 Man a1-Gal a1-3 Glc b1-4 Man a1-Gal a1-4 Glc b1-4 Man a1-Gal a1-6 Glc b1-4 Man a1-Gal b1-2 Glc b1-4 Man a1-Gal b1-3 Glc b1-4 Man a1-Gal b1-4 Glc b1-4 Man a1-Gal b1-6 Glc b1-4 Man a1-Gal a1-2 Glc b1-6 Man a1-Gal a1-3 Glc b1-6 Man a1-Gal a1-4 Glc b1-6 Man a1-Gal a1-6 Glc b1-6 Man a1-Gal b1-2 Glc b1-6 Man a1-Gal b1-3 Glc b1-6 Man a1-Gal b1-4 Glc b1-6 Man a1-Gal b1-6 Glc b1-6 Man a1-Gal a1-2 Glc a1-2 Man b1-Gal a1-3 Glc a1-2 Man b1-Gal a1-4 Glc a1-2 Man b1-Gal a1-6 Glc a1-2 Man b1-Gal b1-2 Glc a1-2 Man b1-Gal b1-3 Glc a1-2 Man b1-Gal b1-4 Glc a1-2 Man b1-Gal b1-6 Glc a1-2 Man b1-Gal a1-2 Glc a1-3 Man b1-Gal a1-3 Glc a1-3 Man b1-Gal a1-4 Glc a1-3 Man b1-Gal a1-6 Glc a1-3 Man b1-Gal b1-2 Glc a1-3 Man b1-Gal b1-3 Glc a1-3 Man b1-Gal b1-4 Glc a1-3 Man b1-Gal b1-6 Glc a1-3 Man b1-Gal a1-2 Glc a1-4 Man b1-Gal a1-3 Glc a1-4 Man b1-Gal a1-4 Glc a1-4 Man b1-Gal a1-6 Glc a1-4 Man b1-Gal b1-2 Glc a1-4 Man b1-Gal b1-3 Glc a1-4 Man b1-Gal b1-4 Glc a1-4 Man b1-Gal b1-6 Glc a1-4 Man b1-Gal a1-2 Glc a1-6 Man b1-Gal a1-3 Glc a1-6 Man b1-Gal a1-4 Glc a1-6 Man b1-Gal a1-6 Glc a1-6 Man b1-Gal b1-2 Glc a1-6 Man b1-

Gal b1-3 Glc a1-6 Man b1-Gal b1-4 Glc a1-6 Man b1-Gal b1-6 Glc a1-6 Man b1-Gal a1-2 Glc b1-2 Man b1-Gal a1-3 Glc b1-2 Man b1-Gal a1-4 Glc b1-2 Man b1-Gal a1-6 Glc b1-2 Man b1-Gal b1-2 Glc b1-2 Man b1-Gal b1-3 Glc b1-2 Man b1-Gal b1-4 Glc b1-2 Man b1-Gal b1-6 Glc b1-2 Man b1-Gal a1-2 Glc b1-3 Man b1-Gal a1-3 Glc b1-3 Man b1-Gal a1-4 Glc b1-3 Man b1-Gal a1-6 Glc b1-3 Man b1-Gal b1-2 Glc b1-3 Man b1-Gal b1-3 Glc b1-3 Man b1-Gal b1-4 Glc b1-3 Man b1-Gal b1-6 Glc b1-3 Man b1-Gal a1-2 Glc b1-4 Man b1-Gal a1-3 Glc b1-4 Man b1-Gal a1-4 Glc b1-4 Man b1-Gal a1-6 Glc b1-4 Man b1-Gal b1-2 Glc b1-4 Man b1-Gal b1-3 Glc b1-4 Man b1-Gal b1-4 Glc b1-4 Man b1-Gal b1-6 Glc b1-4 Man b1-Gal a1-2 Glc b1-6 Man b1-Gal a1-3 Glc b1-6 Man b1-Gal a1-4 Glc b1-6 Man b1-Gal a1-6 Glc b1-6 Man b1-Gal b1-2 Glc b1-6 Man b1-Gal b1-3 Glc b1-6 Man b1-Gal b1-4 Glc b1-6 Man b1-Gal b1-6 Glc b1-6 Man b1-Gal a1-2 Man a1-2 Glc a1-Gal a1-3 Man a1-2 Glc a1-Gal a1-4 Man a1-2 Glc a1-Gal a1-6 Man a1-2 Glc a1-Gal b1-2 Man a1-2 Glc a1-Gal b1-3 Man a1-2 Glc a1-Gal b1-4 Man a1-2 Glc a1-Gal b1-6 Man a1-2 Glc a1-Gal a1-2 Man a1-3 Glc a1-Gal a1-3 Man a1-3 Glc a1-Gal a1-4 Man a1-3 Glc a1-Gal a1-6 Man a1-3 Glc a1-Gal b1-2 Man a1-3 Glc a1-Gal b1-3 Man a1-3 Glc a1-Gal b1-4 Man a1-3 Glc a1-Gal b1-6 Man a1-3 Glc a1-Gal a1-2 Man a1-4 Glc a1-Gal a1-3 Man a1-4 Glc a1-Gal a1-4 Man a1-4 Glc a1-Gal a1-6 Man a1-4 Glc a1-Gal b1-2 Man a1-4 Glc a1-Gal b1-3 Man a1-4 Glc a1-Gal b1-4 Man a1-4 Glc a1-Gal b1-6 Man a1-4 Glc a1-Gal a1-2 Man a1-6 Glc a1-Gal a1-3 Man a1-6 Glc a1-Gal a1-4 Man a1-6 Glc a1-Gal a1-6 Man a1-6 Glc a1-Gal b1-2 Man a1-6 Glc a1-Gal b1-3 Man a1-6 Glc a1-Gal b1-4 Man a1-6 Glc a1-Gal b1-6 Man a1-6 Glc a1-Gal a1-2 Man b1-2 Glc a1-Gal a1-3 Man b1-2 Glc a1-Gal a1-4 Man b1-2 Glc a1-Gal a1-6 Man b1-2 Glc a1-Gal b1-2 Man b1-2 Glc a1-Gal b1-3 Man b1-2 Glc a1-Gal b1-4 Man b1-2 Glc a1-Gal b1-6 Man b1-2 Glc a1-Gal a1-2 Man b1-3 Glc a1-Gal a1-3 Man b1-3 Glc a1-Gal a1-4 Man b1-3 Glc a1-Gal a1-6 Man b1-3 Glc a1-Gal b1-2 Man b1-3 Glc a1-Gal b1-3 Man b1-3 Glc a1-Gal b1-4 Man b1-3 Glc a1-Gal b1-6 Man b1-3 Glc a1-Gal a1-2 Man b1-4 Glc a1-Gal a1-3 Man b1-4 Glc a1-Gal a1-4 Man b1-4 Glc a1-Gal a1-6 Man b1-4 Glc a1-Gal b1-2 Man b1-4 Glc a1-Gal b1-3 Man b1-4 Glc a1-Gal b1-4 Man b1-4 Glc a1-Gal b1-6 Man b1-4 Glc a1-Gal a1-2 Man b1-6 Glc a1-Gal a1-3 Man b1-6 Glc a1-

Gal a1-4 Man b1-6 Glc a1-Gal a1-6 Man b1-6 Glc a1-Gal b1-2 Man b1-6 Glc a1-Gal b1-3 Man b1-6 Glc a1-Gal b1-4 Man b1-6 Glc a1-Gal b1-6 Man b1-6 Glc a1-Gal a1-2 Man a1-2 Glc b1-Gal a1-3 Man a1-2 Glc b1-Gal a1-4 Man a1-2 Glc b1-Gal a1-6 Man a1-2 Glc b1-Gal b1-2 Man a1-2 Glc b1-Gal b1-3 Man a1-2 Glc b1-Gal b1-4 Man a1-2 Glc b1-Gal b1-6 Man a1-2 Glc b1-Gal a1-2 Man a1-3 Glc b1-Gal a1-3 Man a1-3 Glc b1-Gal a1-4 Man a1-3 Glc b1-Gal a1-6 Man a1-3 Glc b1-Gal b1-2 Man a1-3 Glc b1-Gal b1-3 Man a1-3 Glc b1-Gal b1-4 Man a1-3 Glc b1-Gal b1-6 Man a1-3 Glc b1-Gal a1-2 Man a1-4 Glc b1-Gal a1-3 Man a1-4 Glc b1-Gal a1-4 Man a1-4 Glc b1-Gal a1-6 Man a1-4 Glc b1-Gal b1-2 Man a1-4 Glc b1-Gal b1-3 Man a1-4 Glc b1-Gal b1-4 Man a1-4 Glc b1-Gal b1-6 Man a1-4 Glc b1-Gal a1-2 Man a1-6 Glc b1-Gal a1-3 Man a1-6 Glc b1-Gal a1-4 Man a1-6 Glc b1-Gal a1-6 Man a1-6 Glc b1-Gal b1-2 Man a1-6 Glc b1-Gal b1-3 Man a1-6 Glc b1-Gal b1-4 Man a1-6 Glc b1-Gal b1-6 Man a1-6 Glc b1-Gal a1-2 Man b1-2 Glc b1-Gal a1-3 Man b1-2 Glc b1-Gal a1-4 Man b1-2 Glc b1-Gal a1-6 Man b1-2 Glc b1-Gal b1-2 Man b1-2 Glc b1-Gal b1-3 Man b1-2 Glc b1-Gal b1-4 Man b1-2 Glc b1-Gal b1-6 Man b1-2 Glc b1-Gal a1-2 Man b1-3 Glc b1-Gal a1-3 Man b1-3 Glc b1-Gal a1-4 Man b1-3 Glc b1-Gal a1-6 Man b1-3 Glc b1-Gal b1-2 Man b1-3 Glc b1-Gal b1-3 Man b1-3 Glc b1-Gal b1-4 Man b1-3 Glc b1-Gal b1-6 Man b1-3 Glc b1-Gal a1-2 Man b1-4 Glc b1-Gal a1-3 Man b1-4 Glc b1-Gal a1-4 Man b1-4 Glc b1-Gal a1-6 Man b1-4 Glc b1-Gal b1-2 Man b1-4 Glc b1-Gal b1-3 Man b1-4 Glc b1-Gal b1-4 Man b1-4 Glc b1-Gal b1-6 Man b1-4 Glc b1-Gal a1-2 Man b1-6 Glc b1-Gal a1-3 Man b1-6 Glc b1-Gal a1-4 Man b1-6 Glc b1-Gal a1-6 Man b1-6 Glc b1-Gal b1-2 Man b1-6 Glc b1-Gal b1-3 Man b1-6 Glc b1-Gal b1-4 Man b1-6 Glc b1-Gal b1-6 Man b1-6 Glc b1-Man a1-2 Glc a1-2 Gal a1-Man a1-3 Glc a1-2 Gal a1-Man a1-4 Glc a1-2 Gal a1-Man a1-6 Glc a1-2 Gal a1-Man b1-2 Glc a1-2 Gal a1-Man b1-3 Glc a1-2 Gal a1-Man b1-4 Glc a1-2 Gal a1-Man b1-6 Glc a1-2 Gal a1-Man a1-2 Glc a1-3 Gal a1-Man a1-3 Glc a1-3 Gal a1-Man a1-4 Glc a1-3 Gal a1-Man a1-6 Glc a1-3 Gal a1-Man b1-2 Glc a1-3 Gal a1-Man b1-3 Glc a1-3 Gal a1-Man b1-4 Glc a1-3 Gal a1-Man b1-6 Glc a1-3 Gal a1-Man a1-2 Glc a1-4 Gal a1-Man a1-3 Glc a1-4 Gal a1-Man a1-4 Glc a1-4 Gal a1-Man a1-6 Glc a1-4 Gal a1-Man b1-2 Glc a1-4 Gal a1-Man b1-3 Glc a1-4 Gal a1-Man b1-4 Glc a1-4 Gal a1-

Man b1-6 Glc a1-4 Gal a1-Man a1-2 Glc a1-6 Gal a1-Man a1-3 Glc a1-6 Gal a1-Man a1-4 Glc a1-6 Gal a1-Man a1-6 Glc a1-6 Gal a1-Man b1-2 Glc a1-6 Gal a1-Man b1-3 Glc a1-6 Gal a1-Man b1-4 Glc a1-6 Gal a1-Man b1-6 Glc a1-6 Gal a1-Man a1-2 Glc b1-2 Gal a1-Man a1-3 Glc b1-2 Gal a1-Man a1-4 Glc b1-2 Gal a1-Man a1-6 Glc b1-2 Gal a1-Man b1-2 Glc b1-2 Gal a1-Man b1-3 Glc b1-2 Gal a1-Man b1-4 Glc b1-2 Gal a1-Man b1-6 Glc b1-2 Gal a1-Man a1-2 Glc b1-3 Gal a1-Man a1-3 Glc b1-3 Gal a1-Man a1-4 Glc b1-3 Gal a1-Man a1-6 Glc b1-3 Gal a1-Man b1-2 Glc b1-3 Gal a1-Man b1-3 Glc b1-3 Gal a1-Man b1-4 Glc b1-3 Gal a1-Man b1-6 Glc b1-3 Gal a1-Man a1-2 Glc b1-4 Gal a1-Man a1-3 Glc b1-4 Gal a1-Man a1-4 Glc b1-4 Gal a1-Man a1-6 Glc b1-4 Gal a1-Man b1-2 Glc b1-4 Gal a1-Man b1-3 Glc b1-4 Gal a1-Man b1-4 Glc b1-4 Gal a1-Man b1-6 Glc b1-4 Gal a1-Man a1-2 Glc b1-6 Gal a1-Man a1-3 Glc b1-6 Gal a1-Man a1-4 Glc b1-6 Gal a1-Man a1-6 Glc b1-6 Gal a1-Man b1-2 Glc b1-6 Gal a1-Man b1-3 Glc b1-6 Gal a1-Man b1-4 Glc b1-6 Gal a1-Man b1-6 Glc b1-6 Gal a1-Man a1-2 Glc a1-2 Gal b1-Man a1-3 Glc a1-2 Gal b1-Man a1-4 Glc a1-2 Gal b1-Man a1-6 Glc a1-2 Gal b1-Man b1-2 Glc a1-2 Gal b1-Man b1-3 Glc a1-2 Gal b1-Man b1-4 Glc a1-2 Gal b1-Man b1-6 Glc a1-2 Gal b1-Man a1-2 Glc a1-3 Gal b1-Man a1-3 Glc a1-3 Gal b1-Man a1-4 Glc a1-3 Gal b1-Man a1-6 Glc a1-3 Gal b1-Man b1-2 Glc a1-3 Gal b1-Man b1-3 Glc a1-3 Gal b1-Man b1-4 Glc a1-3 Gal b1-Man b1-6 Glc a1-3 Gal b1-Man a1-2 Glc a1-4 Gal b1-Man a1-3 Glc a1-4 Gal b1-Man a1-4 Glc a1-4 Gal b1-Man a1-6 Glc a1-4 Gal b1-Man b1-2 Glc a1-4 Gal b1-Man b1-3 Glc a1-4 Gal b1-Man b1-4 Glc a1-4 Gal b1-Man b1-6 Glc a1-4 Gal b1-Man a1-2 Glc a1-6 Gal b1-Man a1-3 Glc a1-6 Gal b1-Man a1-4 Glc a1-6 Gal b1-Man a1-6 Glc a1-6 Gal b1-Man b1-2 Glc a1-6 Gal b1-Man b1-3 Glc a1-6 Gal b1-Man b1-4 Glc a1-6 Gal b1-Man b1-6 Glc a1-6 Gal b1-Man a1-2 Glc b1-2 Gal b1-Man a1-3 Glc b1-2 Gal b1-Man a1-4 Glc b1-2 Gal b1-Man a1-6 Glc b1-2 Gal b1-Man b1-2 Glc b1-2 Gal b1-Man b1-3 Glc b1-2 Gal b1-Man b1-4 Glc b1-2 Gal b1-Man b1-6 Glc b1-2 Gal b1-Man a1-2 Glc b1-3 Gal b1-Man a1-3 Glc b1-3 Gal b1-Man a1-4 Glc b1-3 Gal b1-Man a1-6 Glc b1-3 Gal b1-Man b1-2 Glc b1-3 Gal b1-Man b1-3 Glc b1-3 Gal b1-Man b1-4 Glc b1-3 Gal b1-Man b1-6 Glc b1-3 Gal b1-Man a1-2 Glc b1-4 Gal b1-Man a1-3 Glc b1-4 Gal b1-Man a1-4 Glc b1-4 Gal b1-Man a1-6 Glc b1-4 Gal b1-

Man b1-2 Glc b1-4 Gal b1-Man b1-3 Glc b1-4 Gal b1-Man b1-4 Glc b1-4 Gal b1-Man b1-6 Glc b1-4 Gal b1-Man a1-2 Glc b1-6 Gal b1-Man a1-3 Glc b1-6 Gal b1-Man a1-4 Glc b1-6 Gal b1-Man a1-6 Glc b1-6 Gal b1-Man b1-2 Glc b1-6 Gal b1-Man b1-3 Glc b1-6 Gal b1-Man b1-4 Glc b1-6 Gal b1-Man b1-6 Glc b1-6 Gal b1-Man a1-2 Gal a1-2 Glc a1-Man a1-3 Gal a1-2 Glc a1-Man a1-4 Gal a1-2 Glc a1-Man a1-6 Gal a1-2 Glc a1-Man b1-2 Gal a1-2 Glc a1-Man b1-3 Gal a1-2 Glc a1-Man b1-4 Gal a1-2 Glc a1-Man b1-6 Gal a1-2 Glc a1-Man a1-2 Gal a1-3 Glc a1-Man a1-3 Gal a1-3 Glc a1-Man a1-4 Gal a1-3 Glc a1-Man a1-6 Gal a1-3 Glc a1-Man b1-2 Gal a1-3 Glc a1-Man b1-3 Gal a1-3 Glc a1-Man b1-4 Gal a1-3 Glc a1-Man b1-6 Gal a1-3 Glc a1-Man a1-2 Gal a1-4 Glc a1-Man a1-3 Gal a1-4 Glc a1-Man a1-4 Gal a1-4 Glc a1-Man a1-6 Gal a1-4 Glc a1-Man b1-2 Gal a1-4 Glc a1-Man b1-3 Gal a1-4 Glc a1-Man b1-4 Gal a1-4 Glc a1-Man b1-6 Gal a1-4 Glc a1-Man a1-2 Gal a1-6 Glc a1-Man a1-3 Gal a1-6 Glc a1-Man a1-4 Gal a1-6 Glc a1-Man a1-6 Gal a1-6 Glc a1-Man b1-2 Gal a1-6 Glc a1-Man b1-3 Gal a1-6 Glc a1-Man b1-4 Gal a1-6 Glc a1-Man b1-6 Gal a1-6 Glc a1-Man a1-2 Gal b1-2 Glc a1-Man a1-3 Gal b1-2 Glc a1-Man a1-4 Gal b1-2 Glc a1-Man a1-6 Gal b1-2 Glc a1-Man b1-2 Gal b1-2 Glc a1-Man b1-3 Gal b1-2 Glc a1-Man b1-4 Gal b1-2 Glc a1-Man b1-6 Gal b1-2 Glc a1-Man a1-2 Gal b1-3 Glc a1-Man a1-3 Gal b1-3 Glc a1-Man a1-4 Gal b1-3 Glc a1-Man a1-6 Gal b1-3 Glc a1-Man b1-2 Gal b1-3 Glc a1-Man b1-3 Gal b1-3 Glc a1-Man b1-4 Gal b1-3 Glc a1-Man b1-6 Gal b1-3 Glc a1-Man a1-2 Gal b1-4 Glc a1-Man a1-3 Gal b1-4 Glc a1-Man a1-4 Gal b1-4 Glc a1-Man a1-6 Gal b1-4 Glc a1-Man b1-2 Gal b1-4 Glc a1-Man b1-3 Gal b1-4 Glc a1-Man b1-4 Gal b1-4 Glc a1-Man b1-6 Gal b1-4 Glc a1-Man a1-2 Gal b1-6 Glc a1-Man a1-3 Gal b1-6 Glc a1-Man a1-4 Gal b1-6 Glc a1-Man a1-6 Gal b1-6 Glc a1-Man b1-2 Gal b1-6 Glc a1-Man b1-3 Gal b1-6 Glc a1-Man b1-4 Gal b1-6 Glc a1-Man b1-6 Gal b1-6 Glc a1-Man a1-2 Gal a1-2 Glc b1Man a1-3 Gal a1-2 Glc b1Man a1-4 Gal a1-2 Glc b1Man a1-6 Gal a1-2 Glc b1Man b1-2 Gal a1-2 Glc b1Man b1-3 Gal a1-2 Glc b1Man b1-4 Gal a1-2 Glc b1Man b1-6 Gal a1-2 Glc b1Man a1-2 Gal a1-3 Glc b1Man a1-3 Gal a1-3 Glc b1Man a1-4 Gal a1-3 Glc b1Man a1-6 Gal a1-3 Glc b1Man b1-2 Gal a1-3 Glc b1Man b1-3 Gal a1-3 Glc b1Man b1-4 Gal a1-3 Glc b1Man b1-6 Gal a1-3 Glc b1Man a1-2 Gal a1-4 Glc b1

Man a1-3 Gal a1-4 Glc b1Man a1-4 Gal a1-4 Glc b1Man a1-6 Gal a1-4 Glc b1Man b1-2 Gal a1-4 Glc b1Man b1-3 Gal a1-4 Glc b1Man b1-4 Gal a1-4 Glc b1Man b1-6 Gal a1-4 Glc b1Man a1-2 Gal a1-6 Glc b1Man a1-3 Gal a1-6 Glc b1Man a1-4 Gal a1-6 Glc b1Man a1-6 Gal a1-6 Glc b1Man b1-2 Gal a1-6 Glc b1Man b1-3 Gal a1-6 Glc b1Man b1-4 Gal a1-6 Glc b1Man b1-6 Gal a1-6 Glc b1Man a1-2 Gal b1-2 Glc b1Man a1-3 Gal b1-2 Glc b1Man a1-4 Gal b1-2 Glc b1Man a1-6 Gal b1-2 Glc b1Man b1-2 Gal b1-2 Glc b1Man b1-3 Gal b1-2 Glc b1Man b1-4 Gal b1-2 Glc b1Man b1-6 Gal b1-2 Glc b1Man a1-2 Gal b1-3 Glc b1Man a1-3 Gal b1-3 Glc b1Man a1-4 Gal b1-3 Glc b1Man a1-6 Gal b1-3 Glc b1Man b1-2 Gal b1-3 Glc b1Man b1-3 Gal b1-3 Glc b1Man b1-4 Gal b1-3 Glc b1Man b1-6 Gal b1-3 Glc b1Man a1-2 Gal b1-4 Glc b1Man a1-3 Gal b1-4 Glc b1Man a1-4 Gal b1-4 Glc b1Man a1-6 Gal b1-4 Glc b1Man b1-2 Gal b1-4 Glc b1Man b1-3 Gal b1-4 Glc b1Man b1-4 Gal b1-4 Glc b1Man b1-6 Gal b1-4 Glc b1Man a1-2 Gal b1-6 Glc b1Man a1-3 Gal b1-6 Glc b1Man a1-4 Gal b1-6 Glc b1Man a1-6 Gal b1-6 Glc b1Man b1-2 Gal b1-6 Glc b1Man b1-3 Gal b1-6 Glc b1Man b1-4 Gal b1-6 Glc b1Man b1-6 Gal b1-6 Glc b1Glc a1-2 Man a1-2 Gal a1-Glc a1-3 Man a1-2 Gal a1-Glc a1-4 Man a1-2 Gal a1-Glc a1-6 Man a1-2 Gal a1-Glc b1-2 Man a1-2 Gal a1-Glc b1-3 Man a1-2 Gal a1-Glc b1-4 Man a1-2 Gal a1-Glc b1-6 Man a1-2 Gal a1-Glc a1-2 Man a1-3 Gal a1-Glc a1-3 Man a1-3 Gal a1-Glc a1-4 Man a1-3 Gal a1-Glc a1-6 Man a1-3 Gal a1-Glc b1-2 Man a1-3 Gal a1-Glc b1-3 Man a1-3 Gal a1-Glc b1-4 Man a1-3 Gal a1-Glc b1-6 Man a1-3 Gal a1-Glc a1-2 Man a1-4 Gal a1-Glc a1-3 Man a1-4 Gal a1-Glc a1-4 Man a1-4 Gal a1-Glc a1-6 Man a1-4 Gal a1-Glc b1-2 Man a1-4 Gal a1-Glc b1-3 Man a1-4 Gal a1-Glc b1-4 Man a1-4 Gal a1-Glc b1-6 Man a1-4 Gal a1-Glc a1-2 Man a1-6 Gal a1-Glc a1-3 Man a1-6 Gal a1-Glc a1-4 Man a1-6 Gal a1-Glc a1-6 Man a1-6 Gal a1-Glc b1-2 Man a1-6 Gal a1-Glc b1-3 Man a1-6 Gal a1-Glc b1-4 Man a1-6 Gal a1-Glc b1-6 Man a1-6 Gal a1-Glc a1-2 Man b1-2 Gal a1-Glc a1-3 Man b1-2 Gal a1-Glc a1-4 Man b1-2 Gal a1-Glc a1-6 Man b1-2 Gal a1-Glc b1-2 Man b1-2 Gal a1-Glc b1-3 Man b1-2 Gal a1-Glc b1-4 Man b1-2 Gal a1-Glc b1-6 Man b1-2 Gal a1-Glc a1-2 Man b1-3 Gal a1-Glc a1-3 Man b1-3 Gal a1-Glc a1-4 Man b1-3 Gal a1-Glc a1-6 Man b1-3 Gal a1-Glc b1-2 Man b1-3 Gal a1-Glc b1-3 Man b1-3 Gal a1-

Glc b1-4 Man b1-3 Gal a1-Glc b1-6 Man b1-3 Gal a1-Glc a1-2 Man b1-4 Gal a1-Glc a1-3 Man b1-4 Gal a1-Glc a1-4 Man b1-4 Gal a1-Glc a1-6 Man b1-4 Gal a1-Glc b1-2 Man b1-4 Gal a1-Glc b1-3 Man b1-4 Gal a1-Glc b1-4 Man b1-4 Gal a1-Glc b1-6 Man b1-4 Gal a1-Glc a1-2 Man b1-6 Gal a1-Glc a1-3 Man b1-6 Gal a1-Glc a1-4 Man b1-6 Gal a1-Glc a1-6 Man b1-6 Gal a1-Glc b1-2 Man b1-6 Gal a1-Glc b1-3 Man b1-6 Gal a1-Glc b1-4 Man b1-6 Gal a1-Glc b1-6 Man b1-6 Gal a1-Glc a1-2 Man a1-2 Gal b1-Glc a1-3 Man a1-2 Gal b1-Glc a1-4 Man a1-2 Gal b1-Glc a1-6 Man a1-2 Gal b1-Glc b1-2 Man a1-2 Gal b1-Glc b1-3 Man a1-2 Gal b1-Glc b1-4 Man a1-2 Gal b1-Glc b1-6 Man a1-2 Gal b1-Glc a1-2 Man a1-3 Gal b1-Glc a1-3 Man a1-3 Gal b1-Glc a1-4 Man a1-3 Gal b1-Glc a1-6 Man a1-3 Gal b1-Glc b1-2 Man a1-3 Gal b1-Glc b1-3 Man a1-3 Gal b1-Glc b1-4 Man a1-3 Gal b1-Glc b1-6 Man a1-3 Gal b1-Glc a1-2 Man a1-4 Gal b1-Glc a1-3 Man a1-4 Gal b1-Glc a1-4 Man a1-4 Gal b1-Glc a1-6 Man a1-4 Gal b1-Glc b1-2 Man a1-4 Gal b1-Glc b1-3 Man a1-4 Gal b1-Glc b1-4 Man a1-4 Gal b1-Glc b1-6 Man a1-4 Gal b1-Glc a1-2 Man a1-6 Gal b1-Glc a1-3 Man a1-6 Gal b1-Glc a1-4 Man a1-6 Gal b1-Glc a1-6 Man a1-6 Gal b1-Glc b1-2 Man a1-6 Gal b1-Glc b1-3 Man a1-6 Gal b1-Glc b1-4 Man a1-6 Gal b1-Glc b1-6 Man a1-6 Gal b1-Glc a1-2 Man b1-2 Gal b1-Glc a1-3 Man b1-2 Gal b1-Glc a1-4 Man b1-2 Gal b1-Glc a1-6 Man b1-2 Gal b1-Glc b1-2 Man b1-2 Gal b1-Glc b1-3 Man b1-2 Gal b1-Glc b1-4 Man b1-2 Gal b1-Glc b1-6 Man b1-2 Gal b1-Glc a1-2 Man b1-3 Gal b1-Glc a1-3 Man b1-3 Gal b1-Glc a1-4 Man b1-3 Gal b1-Glc a1-6 Man b1-3 Gal b1-Glc b1-2 Man b1-3 Gal b1-Glc b1-3 Man b1-3 Gal b1-Glc b1-4 Man b1-3 Gal b1-Glc b1-6 Man b1-3 Gal b1-Glc a1-2 Man b1-4 Gal b1-Glc a1-3 Man b1-4 Gal b1-Glc a1-4 Man b1-4 Gal b1-Glc a1-6 Man b1-4 Gal b1-Glc b1-2 Man b1-4 Gal b1-Glc b1-3 Man b1-4 Gal b1-Glc b1-4 Man b1-4 Gal b1-Glc b1-6 Man b1-4 Gal b1-Glc a1-2 Man b1-6 Gal b1-Glc a1-3 Man b1-6 Gal b1-Glc a1-4 Man b1-6 Gal b1-Glc a1-6 Man b1-6 Gal b1-Glc b1-2 Man b1-6 Gal b1-Glc b1-3 Man b1-6 Gal b1-Glc b1-4 Man b1-6 Gal b1-Glc b1-6 Man b1-6 Gal b1-Glc a1-2 Gal a1-2 Man a1-Glc a1-3 Gal a1-2 Man a1-Glc a1-4 Gal a1-2 Man a1-Glc a1-6 Gal a1-2 Man a1-Glc b1-2 Gal a1-2 Man a1-Glc b1-3 Gal a1-2 Man a1-Glc b1-4 Gal a1-2 Man a1-Glc b1-6 Gal a1-2 Man a1-Glc a1-2 Gal a1-3 Man a1-Glc a1-3 Gal a1-3 Man a1-Glc a1-4 Gal a1-3 Man a1-

Glc a1-6 Gal a1-3 Man a1-Glc b1-2 Gal a1-3 Man a1-Glc b1-3 Gal a1-3 Man a1-Glc b1-4 Gal a1-3 Man a1-Glc b1-6 Gal a1-3 Man a1-Glc a1-2 Gal a1-4 Man a1-Glc a1-3 Gal a1-4 Man a1-Glc a1-4 Gal a1-4 Man a1-Glc a1-6 Gal a1-4 Man a1-Glc b1-2 Gal a1-4 Man a1-Glc b1-3 Gal a1-4 Man a1-Glc b1-4 Gal a1-4 Man a1-Glc b1-6 Gal a1-4 Man a1-Glc a1-2 Gal a1-6 Man a1-Glc a1-3 Gal a1-6 Man a1-Glc a1-4 Gal a1-6 Man a1-Glc a1-6 Gal a1-6 Man a1-Glc b1-2 Gal a1-6 Man a1-Glc b1-3 Gal a1-6 Man a1-Glc b1-4 Gal a1-6 Man a1-Glc b1-6 Gal a1-6 Man a1-Glc a1-2 Gal b1-2 Man a1-Glc a1-3 Gal b1-2 Man a1-Glc a1-4 Gal b1-2 Man a1-Glc a1-6 Gal b1-2 Man a1-Glc b1-2 Gal b1-2 Man a1-Glc b1-3 Gal b1-2 Man a1-Glc b1-4 Gal b1-2 Man a1-Glc b1-6 Gal b1-2 Man a1-Glc a1-2 Gal b1-3 Man a1-Glc a1-3 Gal b1-3 Man a1-Glc a1-4 Gal b1-3 Man a1-Glc a1-6 Gal b1-3 Man a1-Glc b1-2 Gal b1-3 Man a1-Glc b1-3 Gal b1-3 Man a1-Glc b1-4 Gal b1-3 Man a1-Glc b1-6 Gal b1-3 Man a1-Glc a1-2 Gal b1-4 Man a1-Glc a1-3 Gal b1-4 Man a1-Glc a1-4 Gal b1-4 Man a1-Glc a1-6 Gal b1-4 Man a1-Glc b1-2 Gal b1-4 Man a1-Glc b1-3 Gal b1-4 Man a1-Glc b1-4 Gal b1-4 Man a1-Glc b1-6 Gal b1-4 Man a1-Glc a1-2 Gal b1-6 Man a1-Glc a1-3 Gal b1-6 Man a1-Glc a1-4 Gal b1-6 Man a1-Glc a1-6 Gal b1-6 Man a1-Glc b1-2 Gal b1-6 Man a1-Glc b1-3 Gal b1-6 Man a1-Glc b1-4 Gal b1-6 Man a1-Glc b1-6 Gal b1-6 Man a1-Glc a1-2 Gal a1-2 Man b1-Glc a1-3 Gal a1-2 Man b1-Glc a1-4 Gal a1-2 Man b1-Glc a1-6 Gal a1-2 Man b1-Glc b1-2 Gal a1-2 Man b1-Glc b1-3 Gal a1-2 Man b1-Glc b1-4 Gal a1-2 Man b1-Glc b1-6 Gal a1-2 Man b1-Glc a1-2 Gal a1-3 Man b1-Glc a1-3 Gal a1-3 Man b1-Glc a1-4 Gal a1-3 Man b1-Glc a1-6 Gal a1-3 Man b1-Glc b1-2 Gal a1-3 Man b1-Glc b1-3 Gal a1-3 Man b1-Glc b1-4 Gal a1-3 Man b1-Glc b1-6 Gal a1-3 Man b1-Glc a1-2 Gal a1-4 Man b1-Glc a1-3 Gal a1-4 Man b1-Glc a1-4 Gal a1-4 Man b1-Glc a1-6 Gal a1-4 Man b1-Glc b1-2 Gal a1-4 Man b1-Glc b1-3 Gal a1-4 Man b1-Glc b1-4 Gal a1-4 Man b1-Glc b1-6 Gal a1-4 Man b1-Glc a1-2 Gal a1-6 Man b1-Glc a1-3 Gal a1-6 Man b1-Glc a1-4 Gal a1-6 Man b1-Glc a1-6 Gal a1-6 Man b1-Glc b1-2 Gal a1-6 Man b1-Glc b1-3 Gal a1-6 Man b1-Glc b1-4 Gal a1-6 Man b1-Glc b1-6 Gal a1-6 Man b1-Glc a1-2 Gal b1-2 Man b1-Glc a1-3 Gal b1-2 Man b1-Glc a1-4 Gal b1-2 Man b1-Glc a1-6 Gal b1-2 Man b1-Glc b1-2 Gal b1-2 Man b1-Glc b1-3 Gal b1-2 Man b1-Glc b1-4 Gal b1-2 Man b1-Glc b1-6 Gal b1-2 Man b1-

Glc a1-2 Gal b1-3 Man b1-Glc a1-3 Gal b1-3 Man b1-Glc a1-4 Gal b1-3 Man b1-Glc a1-6 Gal b1-3 Man b1-Glc b1-2 Gal b1-3 Man b1-Glc b1-3 Gal b1-3 Man b1-Glc b1-4 Gal b1-3 Man b1-Glc b1-6 Gal b1-3 Man b1-Glc a1-2 Gal b1-4 Man b1-Glc a1-3 Gal b1-4 Man b1-Glc a1-4 Gal b1-4 Man b1-Glc a1-6 Gal b1-4 Man b1-Glc b1-2 Gal b1-4 Man b1-Glc b1-3 Gal b1-4 Man b1-Glc b1-4 Gal b1-4 Man b1-Glc b1-6 Gal b1-4 Man b1-Glc a1-2 Gal b1-6 Man b1-Glc a1-3 Gal b1-6 Man b1-Glc a1-4 Gal b1-6 Man b1-Glc a1-6 Gal b1-6 Man b1-Glc b1-2 Gal b1-6 Man b1-Glc b1-3 Gal b1-6 Man b1-Glc b1-4 Gal b1-6 Man b1-Glc b1-6 Gal b1-6 Man b1-Gal a1-2 (Glc a1-3) Man a1-Gal a1-2 (Glc a1-4) Man a1-Gal a1-2 (Glc a1-6) Man a1-Gal a1-3 (Glc a1-2) Man a1-Gal a1-3 (Glc a1-4) Man a1-Gal a1-3 (Glc a1-6) Man a1-Gal a1-4 (Glc a1-2) Man a1-Gal a1-4 (Glc a1-3) Man a1-Gal a1-4 (Glc a1-6) Man a1-Gal a1-6 (Glc a1-2) Man a1-Gal a1-6 (Glc a1-3) Man a1-Gal a1-6 (Glc a1-4) Man a1-Gal b1-2 (Glc a1-3) Man a1-Gal b1-2 (Glc a1-4) Man a1-Gal b1-2 (Glc a1-6) Man a1-Gal b1-3 (Glc a1-2) Man a1-Gal b1-3 (Glc a1-4) Man a1-Gal b1-3 (Glc a1-6) Man a1-Gal b1-4 (Glc a1-2) Man a1-Gal b1-4 (Glc a1-3) Man a1-Gal b1-4 (Glc a1-6) Man a1-Gal b1-6 (Glc a1-2) Man a1-Gal b1-6 (Glc a1-3) Man a1-Gal b1-6 (Glc a1-4) Man a1-Gal a1-2 (Glc b1-3) Man a1-Gal a1-2 (Glc b1-4) Man a1-Gal a1-2 (Glc b1-6) Man a1-Gal a1-3 (Glc b1-2) Man a1-Gal a1-3 (Glc b1-4) Man a1-Gal a1-3 (Glc b1-6) Man a1-Gal a1-4 (Glc b1-2) Man a1-Gal a1-4 (Glc b1-3) Man a1-Gal a1-4 (Glc b1-6) Man a1-Gal a1-6 (Glc b1-2) Man a1-Gal a1-6 (Glc b1-3) Man a1-Gal a1-6 (Glc b1-4) Man a1-Gal b1-2 (Glc b1-3) Man a1-Gal b1-2 (Glc b1-4) Man a1-Gal b1-2 (Glc b1-6) Man a1-Gal b1-3 (Glc b1-2) Man a1-Gal b1-3 (Glc b1-4) Man a1-Gal b1-3 (Glc b1-6) Man a1-Gal b1-4 (Glc b1-2) Man a1-Gal b1-4 (Glc b1-3) Man a1-Gal b1-4 (Glc b1-6) Man a1-Gal b1-6 (Glc b1-2) Man a1-Gal b1-6 (Glc b1-3) Man a1-Gal b1-6 (Glc b1-4) Man a1-Gal a1-2 (Glc a1-3) Man b1-Gal a1-2 (Glc a1-4) Man b1-Gal a1-2 (Glc a1-6) Man b1-Gal a1-3 (Glc a1-2) Man b1-Gal a1-3 (Glc a1-4) Man b1-Gal a1-3 (Glc a1-6) Man b1-Gal a1-4 (Glc a1-2) Man b1-Gal a1-4 (Glc a1-3) Man b1-Gal a1-4 (Glc a1-6) Man b1-Gal a1-6 (Glc a1-2) Man b1-Gal a1-6 (Glc a1-3) Man b1-Gal a1-6 (Glc a1-4) Man b1-Gal b1-2 (Glc a1-3) Man b1-Gal b1-2 (Glc a1-4) Man b1-Gal b1-2 (Glc a1-6) Man b1-Gal b1-3 (Glc a1-2) Man b1-Gal b1-3 (Glc a1-4) Man b1-Gal b1-3 (Glc a1-6) Man b1-Gal b1-4 (Glc a1-2) Man b1-Gal b1-4 (Glc a1-3) Man b1-Gal b1-4 (Glc a1-6) Man b1-

Gal b1-6 (Glc a1-2) Man b1-Gal b1-6 (Glc a1-3) Man b1-Gal b1-6 (Glc a1-4) Man b1-Gal a1-2 (Glc b1-3) Man b1-Gal a1-2 (Glc b1-4) Man b1-Gal a1-2 (Glc b1-6) Man b1-Gal a1-3 (Glc b1-2) Man b1-Gal a1-3 (Glc b1-4) Man b1-Gal a1-3 (Glc b1-6) Man b1-Gal a1-4 (Glc b1-2) Man b1-Gal a1-4 (Glc b1-3) Man b1-Gal a1-4 (Glc b1-6) Man b1-Gal a1-6 (Glc b1-2) Man b1-Gal a1-6 (Glc b1-3) Man b1-Gal a1-6 (Glc b1-4) Man b1-Gal b1-2 (Glc b1-3) Man b1-Gal b1-2 (Glc b1-4) Man b1-Gal b1-2 (Glc b1-6) Man b1-Gal b1-3 (Glc b1-2) Man b1-Gal b1-3 (Glc b1-4) Man b1-Gal b1-3 (Glc b1-6) Man b1-Gal b1-4 (Glc b1-2) Man b1-Gal b1-4 (Glc b1-3) Man b1-Gal b1-4 (Glc b1-6) Man b1-Gal b1-6 (Glc b1-2) Man b1-Gal b1-6 (Glc b1-3) Man b1-Gal b1-6 (Glc b1-4) Man b1-Gal a1-2 (Man a1-3) Glc a1-Gal a1-2 (Man a1-4) Glc a1-Gal a1-2 (Man a1-6) Glc a1-Gal a1-3 (Man a1-2) Glc a1-Gal a1-3 (Man a1-4) Glc a1-Gal a1-3 (Man a1-6) Glc a1-Gal a1-4 (Man a1-2) Glc a1-Gal a1-4 (Man a1-3) Glc a1-Gal a1-4 (Man a1-6) Glc a1-Gal a1-6 (Man a1-2) Glc a1-Gal a1-6 (Man a1-3) Glc a1-Gal a1-6 (Man a1-4) Glc a1-Gal b1-2 (Man a1-3) Glc a1-Gal b1-2 (Man a1-4) Glc a1-Gal b1-2 (Man a1-6) Glc a1-Gal b1-3 (Man a1-2) Glc a1-Gal b1-3 (Man a1-4) Glc a1-Gal b1-3 (Man a1-6) Glc a1-Gal b1-4 (Man a1-2) Glc a1-Gal b1-4 (Man a1-3) Glc a1-Gal b1-4 (Man a1-6) Glc a1-Gal b1-6 (Man a1-2) Glc a1-Gal b1-6 (Man a1-3) Glc a1-Gal b1-6 (Man a1-4) Glc a1-Gal a1-2 (Man b1-3) Glc a1-Gal a1-2 (Man b1-4) Glc a1-Gal a1-2 (Man b1-6) Glc a1-Gal a1-3 (Man b1-2) Glc a1-Gal a1-3 (Man b1-4) Glc a1-Gal a1-3 (Man b1-6) Glc a1-Gal a1-4 (Man b1-2) Glc a1-Gal a1-4 (Man b1-3) Glc a1-Gal a1-4 (Man b1-6) Glc a1-Gal a1-6 (Man b1-2) Glc a1-Gal a1-6 (Man b1-3) Glc a1-Gal a1-6 (Man b1-4) Glc a1-Gal b1-2 (Man b1-3) Glc a1-Gal b1-2 (Man b1-4) Glc a1-Gal b1-2 (Man b1-6) Glc a1-Gal b1-3 (Man b1-2) Glc a1-Gal b1-3 (Man b1-4) Glc a1-Gal b1-3 (Man b1-6) Glc a1-Gal b1-4 (Man b1-2) Glc a1-Gal b1-4 (Man b1-3) Glc a1-Gal b1-4 (Man b1-6) Glc a1-Gal b1-6 (Man b1-2) Glc a1-Gal b1-6 (Man b1-3) Glc a1-Gal b1-6 (Man b1-4) Glc a1-Gal a1-2 (Man a1-3) Glc b1-Gal a1-2 (Man a1-4) Glc b1-Gal a1-2 (Man a1-6) Glc b1-Gal a1-3 (Man a1-2) Glc b1-Gal a1-3 (Man a1-4) Glc b1-Gal a1-3 (Man a1-6) Glc b1-

Gal a1-4 (Man a1-2) Glc b1-Gal a1-4 (Man a1-3) Glc b1-Gal a1-4 (Man a1-6) Glc b1-Gal a1-6 (Man a1-2) Glc b1-Gal a1-6 (Man a1-3) Glc b1-Gal a1-6 (Man a1-4) Glc b1-Gal b1-2 (Man a1-3) Glc b1-Gal b1-2 (Man a1-4) Glc b1-Gal b1-2 (Man a1-6) Glc b1-Gal b1-3 (Man a1-2) Glc b1-Gal b1-3 (Man a1-4) Glc b1-Gal b1-3 (Man a1-6) Glc b1-Gal b1-4 (Man a1-2) Glc b1-Gal b1-4 (Man a1-3) Glc b1-Gal b1-4 (Man a1-6) Glc b1-Gal b1-6 (Man a1-2) Glc b1-Gal b1-6 (Man a1-3) Glc b1-Gal b1-6 (Man a1-4) Glc b1-Gal a1-2 (Man b1-3) Glc b1-Gal a1-2 (Man b1-4) Glc b1-Gal a1-2 (Man b1-6) Glc b1-Gal a1-3 (Man b1-2) Glc b1-Gal a1-3 (Man b1-4) Glc b1-Gal a1-3 (Man b1-6) Glc b1-Gal a1-4 (Man b1-2) Glc b1-Gal a1-4 (Man b1-3) Glc b1-Gal a1-4 (Man b1-6) Glc b1-Gal a1-6 (Man b1-2) Glc b1-Gal a1-6 (Man b1-3) Glc b1-Gal a1-6 (Man b1-4) Glc b1-Gal b1-2 (Man b1-3) Glc b1-Gal b1-2 (Man b1-4) Glc b1-Gal b1-2 (Man b1-6) Glc b1-Gal b1-3 (Man b1-2) Glc b1-Gal b1-3 (Man b1-4) Glc b1-Gal b1-3 (Man b1-6) Glc b1-Gal b1-4 (Man b1-2) Glc b1-Gal b1-4 (Man b1-3) Glc b1-Gal b1-4 (Man b1-6) Glc b1-Gal b1-6 (Man b1-2) Glc b1-Gal b1-6 (Man b1-3) Glc b1-Gal b1-6 (Man b1-4) Glc b1-Glc a1-2 (Man a1-3) Gal a1-Glc a1-2 (Man a1-4) Gal a1-Glc a1-2 (Man a1-6) Gal a1-Glc a1-3 (Man a1-2) Gal a1-Glc a1-3 (Man a1-4) Gal a1-Glc a1-3 (Man a1-6) Gal a1-Glc a1-4 (Man a1-2) Gal a1-Glc a1-4 (Man a1-3) Gal a1-Glc a1-4 (Man a1-6) Gal a1-Glc a1-6 (Man a1-2) Gal a1-Glc a1-6 (Man a1-3) Gal a1-Glc a1-6 (Man a1-4) Gal a1-Glc b1-2 (Man a1-3) Gal a1-Glc b1-2 (Man a1-4) Gal a1-Glc b1-2 (Man a1-6) Gal a1-Glc b1-3 (Man a1-2) Gal a1-Glc b1-3 (Man a1-4) Gal a1-Glc b1-3 (Man a1-6) Gal a1-Glc b1-4 (Man a1-2) Gal a1-Glc b1-4 (Man a1-3) Gal a1-Glc b1-4 (Man a1-6) Gal a1-Glc b1-6 (Man a1-2) Gal a1-Glc b1-6 (Man a1-3) Gal a1-Glc b1-6 (Man a1-4) Gal a1-Glc a1-2 (Man b1-3) Gal a1-Glc a1-2 (Man b1-4) Gal a1-Glc a1-2 (Man b1-6) Gal a1-Glc a1-3 (Man b1-2) Gal a1-Glc a1-3 (Man b1-4) Gal a1-Glc a1-3 (Man b1-6) Gal a1-Glc a1-4 (Man b1-2) Gal a1-Glc a1-4 (Man b1-3) Gal a1-Glc a1-4 (Man b1-6) Gal a1-Glc a1-6 (Man b1-2) Gal a1-Glc a1-6 (Man b1-3) Gal a1-Glc a1-6 (Man b1-4) Gal a1-Glc b1-2 (Man b1-3) Gal a1-Glc b1-2 (Man b1-4) Gal a1-Glc b1-2 (Man b1-6) Gal a1-

Glc b1-3 (Man b1-2) Gal a1-Glc b1-3 (Man b1-4) Gal a1-Glc b1-3 (Man b1-6) Gal a1-Glc b1-4 (Man b1-2) Gal a1-Glc b1-4 (Man b1-3) Gal a1-Glc b1-4 (Man b1-6) Gal a1-Glc b1-6 (Man b1-2) Gal a1-Glc b1-6 (Man b1-3) Gal a1-Glc b1-6 (Man b1-4) Gal a1-Glc a1-2 (Man a1-3) Gal b1-Glc a1-2 (Man a1-4) Gal b1-Glc a1-2 (Man a1-6) Gal b1-Glc a1-3 (Man a1-2) Gal b1-Glc a1-3 (Man a1-4) Gal b1-Glc a1-3 (Man a1-6) Gal b1-Glc a1-4 (Man a1-2) Gal b1-Glc a1-4 (Man a1-3) Gal b1-Glc a1-4 (Man a1-6) Gal b1-Glc a1-6 (Man a1-2) Gal b1-Glc a1-6 (Man a1-3) Gal b1-Glc a1-6 (Man a1-4) Gal b1-Glc b1-2 (Man a1-3) Gal b1-Glc b1-2 (Man a1-4) Gal b1-Glc b1-2 (Man a1-6) Gal b1-Glc b1-3 (Man a1-2) Gal b1-Glc b1-3 (Man a1-4) Gal b1-Glc b1-3 (Man a1-6) Gal b1-Glc b1-4 (Man a1-2) Gal b1-Glc b1-4 (Man a1-3) Gal b1-Glc b1-4 (Man a1-6) Gal b1-Glc b1-6 (Man a1-2) Gal b1-Glc b1-6 (Man a1-3) Gal b1-Glc b1-6 (Man a1-4) Gal b1-Glc a1-2 (Man b1-3) Gal b1-Glc a1-2 (Man b1-4) Gal b1-Glc a1-2 (Man b1-6) Gal b1-Glc a1-3 (Man b1-2) Gal b1-Glc a1-3 (Man b1-4) Gal b1-Glc a1-3 (Man b1-6) Gal b1-Glc a1-4 (Man b1-2) Gal b1-Glc a1-4 (Man b1-3) Gal b1-Glc a1-4 (Man b1-6) Gal b1-Glc a1-6 (Man b1-2) Gal b1-Glc a1-6 (Man b1-3) Gal b1-Glc a1-6 (Man b1-4) Gal b1-Glc b1-2 (Man b1-3) Gal b1-Glc b1-2 (Man b1-4) Gal b1-Glc b1-2 (Man b1-6) Gal b1-Glc b1-3 (Man b1-2) Gal b1-Glc b1-3 (Man b1-4) Gal b1-Glc b1-3 (Man b1-6) Gal b1-Glc b1-4 (Man b1-2) Gal b1-Glc b1-4 (Man b1-3) Gal b1-Glc b1-4 (Man b1-6) Gal b1-Glc b1-6 (Man b1-2) Gal b1-Glc b1-6 (Man b1-3) Gal b1-Glc b1-6 (Man b1-4) Gal b1-

Man a1-4 Gal a1-3 Glc b1Man a1-6 Gal a1-3 Glc b1Man b1-2 Gal a1-3 Glc b1Man b1-3 Gal a1-3 Glc b1Man b1-4 Gal a1-3 Glc b1Man b1-6 Gal a1-3 Glc b1Man a1-2 Gal a1-4 Glc b1Man a1-3 Gal a1-4 Glc b1Man a1-4 Gal a1-4 Glc b1Man a1-6 Gal a1-4 Glc b1Man b1-2 Gal a1-4 Glc b1Man b1-3 Gal a1-4 Glc b1Man b1-4 Gal a1-4 Glc b1Man b1-6 Gal a1-4 Glc b1Man a1-2 Gal a1-6 Glc b1Man a1-3 Gal a1-6 Glc b1

Cummings RD (2009) Molecular BioSystems 5 1087

Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical petasaccharide sequeces in glycosaminoglycans

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoN-linkedrdquo glycan linkage to protein asparagine residues

Families of N-linked glycan structures

Man

4GlcNAc1

2Man

Man

Man

Man

2Man

2Man

2Man

63

Man63 4GlcNAc1 Asn

4GlcNAc1

Man62

Man63 4GlcNAc1 Asn

Man42

4GlcNAc1

4GlcNAc1

4GlcNAc1

4GlcNAc1

6Gal

6Gal

6Gal

6Gal

NeuAc

NeuAc

NeuAcNeuAc

4GlcNAc1

Man

Man63 4GlcNAc1 Asn

2Man4GlcNAc13GalNeuAc

Man

Man

63

core

core

core

High Mannose

Complex

Hybrid

Examples of structures found as termini on the branches of complex N-linked glycoprotein glycanshellip

Terminal Saccharide Diversity --- A key to recognition

NeuAc 2-6 Gal 1-4 GlcNAc---NeuAc 2-3 Gal 1-4 GlcNAc---

Gal 1-3 Gal 1-4 GlcNAc---4-SO3-GalNAc 1-4 GlcNAc---

(-Gal 1-4 GlcNAc 1-3-)n = polylactosamine repeats (n may be gt6)

(-NeuAc 2-8-) n = polysialic acid (n may be gt50)

Fuc residues (eg Fuc 1-2 Gal Fuc 1-3 GlcNAc Fuc 1-4 GlcNAc

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoO-linkedrdquo glycan linkage to protein serine residues

Examples of variations of O-linked glycans

GalNAc Serine

GalNAc Serine

NeuAc 26

GalNAc Serine

Gal 1

NeuAc 263

GalNAc SerineNeuAc 2-3 Gal 1

NeuAc 263

Gal 1-4 GlcNAc 1

Fuc 1

GalNAc Serine

GlcNAc 1

63

4NeuAc 2-3 Gal 1

3

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Three different amino acids (Ala Ser Tyr) ndash 6 structures

Ala-Ser-TyrAla-Tyr-SerSer-Ala-TyrSer-Tyr-AlaTyr-Ala-SerTyr-Ser-Ala

Three different sugars (Glc Gal Man) ndash 1056 structures hellip

Gal a1-2 Glc a1-2 Man a1-Gal a1-3 Glc a1-2 Man a1-Gal a1-4 Glc a1-2 Man a1-Gal a1-6 Glc a1-2 Man a1-Gal b1-2 Glc a1-2 Man a1-Gal b1-3 Glc a1-2 Man a1-Gal b1-4 Glc a1-2 Man a1-Gal b1-6 Glc a1-2 Man a1-Gal a1-2 Glc a1-3 Man a1-Gal a1-3 Glc a1-3 Man a1-Gal a1-4 Glc a1-3 Man a1-Gal a1-6 Glc a1-3 Man a1-Gal b1-2 Glc a1-3 Man a1-Gal b1-3 Glc a1-3 Man a1-Gal b1-4 Glc a1-3 Man a1-Gal b1-6 Glc a1-3 Man a1-Gal a1-2 Glc a1-4 Man a1-Gal a1-3 Glc a1-4 Man a1-Gal a1-4 Glc a1-4 Man a1-Gal a1-6 Glc a1-4 Man a1-Gal b1-2 Glc a1-4 Man a1-Gal b1-3 Glc a1-4 Man a1-Gal b1-4 Glc a1-4 Man a1-Gal b1-6 Glc a1-4 Man a1-Gal a1-2 Glc a1-6 Man a1-Gal a1-3 Glc a1-6 Man a1-Gal a1-4 Glc a1-6 Man a1-Gal a1-6 Glc a1-6 Man a1-Gal b1-2 Glc a1-6 Man a1-Gal b1-3 Glc a1-6 Man a1-Gal b1-4 Glc a1-6 Man a1-Gal b1-6 Glc a1-6 Man a1-Gal a1-2 Glc b1-2 Man a1-Gal a1-3 Glc b1-2 Man a1-Gal a1-4 Glc b1-2 Man a1-Gal a1-6 Glc b1-2 Man a1-Gal b1-2 Glc b1-2 Man a1-Gal b1-3 Glc b1-2 Man a1-Gal b1-4 Glc b1-2 Man a1-Gal b1-6 Glc b1-2 Man a1-Gal a1-2 Glc b1-3 Man a1-Gal a1-3 Glc b1-3 Man a1-Gal a1-4 Glc b1-3 Man a1-Gal a1-6 Glc b1-3 Man a1-Gal b1-2 Glc b1-3 Man a1-Gal b1-3 Glc b1-3 Man a1-Gal b1-4 Glc b1-3 Man a1-Gal b1-6 Glc b1-3 Man a1-Gal a1-2 Glc b1-4 Man a1-Gal a1-3 Glc b1-4 Man a1-Gal a1-4 Glc b1-4 Man a1-Gal a1-6 Glc b1-4 Man a1-Gal b1-2 Glc b1-4 Man a1-Gal b1-3 Glc b1-4 Man a1-Gal b1-4 Glc b1-4 Man a1-Gal b1-6 Glc b1-4 Man a1-Gal a1-2 Glc b1-6 Man a1-Gal a1-3 Glc b1-6 Man a1-Gal a1-4 Glc b1-6 Man a1-Gal a1-6 Glc b1-6 Man a1-Gal b1-2 Glc b1-6 Man a1-Gal b1-3 Glc b1-6 Man a1-Gal b1-4 Glc b1-6 Man a1-Gal b1-6 Glc b1-6 Man a1-Gal a1-2 Glc a1-2 Man b1-Gal a1-3 Glc a1-2 Man b1-Gal a1-4 Glc a1-2 Man b1-Gal a1-6 Glc a1-2 Man b1-Gal b1-2 Glc a1-2 Man b1-Gal b1-3 Glc a1-2 Man b1-Gal b1-4 Glc a1-2 Man b1-Gal b1-6 Glc a1-2 Man b1-Gal a1-2 Glc a1-3 Man b1-Gal a1-3 Glc a1-3 Man b1-Gal a1-4 Glc a1-3 Man b1-Gal a1-6 Glc a1-3 Man b1-Gal b1-2 Glc a1-3 Man b1-Gal b1-3 Glc a1-3 Man b1-Gal b1-4 Glc a1-3 Man b1-Gal b1-6 Glc a1-3 Man b1-Gal a1-2 Glc a1-4 Man b1-Gal a1-3 Glc a1-4 Man b1-Gal a1-4 Glc a1-4 Man b1-Gal a1-6 Glc a1-4 Man b1-Gal b1-2 Glc a1-4 Man b1-Gal b1-3 Glc a1-4 Man b1-Gal b1-4 Glc a1-4 Man b1-Gal b1-6 Glc a1-4 Man b1-Gal a1-2 Glc a1-6 Man b1-Gal a1-3 Glc a1-6 Man b1-Gal a1-4 Glc a1-6 Man b1-Gal a1-6 Glc a1-6 Man b1-Gal b1-2 Glc a1-6 Man b1-

Gal b1-3 Glc a1-6 Man b1-Gal b1-4 Glc a1-6 Man b1-Gal b1-6 Glc a1-6 Man b1-Gal a1-2 Glc b1-2 Man b1-Gal a1-3 Glc b1-2 Man b1-Gal a1-4 Glc b1-2 Man b1-Gal a1-6 Glc b1-2 Man b1-Gal b1-2 Glc b1-2 Man b1-Gal b1-3 Glc b1-2 Man b1-Gal b1-4 Glc b1-2 Man b1-Gal b1-6 Glc b1-2 Man b1-Gal a1-2 Glc b1-3 Man b1-Gal a1-3 Glc b1-3 Man b1-Gal a1-4 Glc b1-3 Man b1-Gal a1-6 Glc b1-3 Man b1-Gal b1-2 Glc b1-3 Man b1-Gal b1-3 Glc b1-3 Man b1-Gal b1-4 Glc b1-3 Man b1-Gal b1-6 Glc b1-3 Man b1-Gal a1-2 Glc b1-4 Man b1-Gal a1-3 Glc b1-4 Man b1-Gal a1-4 Glc b1-4 Man b1-Gal a1-6 Glc b1-4 Man b1-Gal b1-2 Glc b1-4 Man b1-Gal b1-3 Glc b1-4 Man b1-Gal b1-4 Glc b1-4 Man b1-Gal b1-6 Glc b1-4 Man b1-Gal a1-2 Glc b1-6 Man b1-Gal a1-3 Glc b1-6 Man b1-Gal a1-4 Glc b1-6 Man b1-Gal a1-6 Glc b1-6 Man b1-Gal b1-2 Glc b1-6 Man b1-Gal b1-3 Glc b1-6 Man b1-Gal b1-4 Glc b1-6 Man b1-Gal b1-6 Glc b1-6 Man b1-Gal a1-2 Man a1-2 Glc a1-Gal a1-3 Man a1-2 Glc a1-Gal a1-4 Man a1-2 Glc a1-Gal a1-6 Man a1-2 Glc a1-Gal b1-2 Man a1-2 Glc a1-Gal b1-3 Man a1-2 Glc a1-Gal b1-4 Man a1-2 Glc a1-Gal b1-6 Man a1-2 Glc a1-Gal a1-2 Man a1-3 Glc a1-Gal a1-3 Man a1-3 Glc a1-Gal a1-4 Man a1-3 Glc a1-Gal a1-6 Man a1-3 Glc a1-Gal b1-2 Man a1-3 Glc a1-Gal b1-3 Man a1-3 Glc a1-Gal b1-4 Man a1-3 Glc a1-Gal b1-6 Man a1-3 Glc a1-Gal a1-2 Man a1-4 Glc a1-Gal a1-3 Man a1-4 Glc a1-Gal a1-4 Man a1-4 Glc a1-Gal a1-6 Man a1-4 Glc a1-Gal b1-2 Man a1-4 Glc a1-Gal b1-3 Man a1-4 Glc a1-Gal b1-4 Man a1-4 Glc a1-Gal b1-6 Man a1-4 Glc a1-Gal a1-2 Man a1-6 Glc a1-Gal a1-3 Man a1-6 Glc a1-Gal a1-4 Man a1-6 Glc a1-Gal a1-6 Man a1-6 Glc a1-Gal b1-2 Man a1-6 Glc a1-Gal b1-3 Man a1-6 Glc a1-Gal b1-4 Man a1-6 Glc a1-Gal b1-6 Man a1-6 Glc a1-Gal a1-2 Man b1-2 Glc a1-Gal a1-3 Man b1-2 Glc a1-Gal a1-4 Man b1-2 Glc a1-Gal a1-6 Man b1-2 Glc a1-Gal b1-2 Man b1-2 Glc a1-Gal b1-3 Man b1-2 Glc a1-Gal b1-4 Man b1-2 Glc a1-Gal b1-6 Man b1-2 Glc a1-Gal a1-2 Man b1-3 Glc a1-Gal a1-3 Man b1-3 Glc a1-Gal a1-4 Man b1-3 Glc a1-Gal a1-6 Man b1-3 Glc a1-Gal b1-2 Man b1-3 Glc a1-Gal b1-3 Man b1-3 Glc a1-Gal b1-4 Man b1-3 Glc a1-Gal b1-6 Man b1-3 Glc a1-Gal a1-2 Man b1-4 Glc a1-Gal a1-3 Man b1-4 Glc a1-Gal a1-4 Man b1-4 Glc a1-Gal a1-6 Man b1-4 Glc a1-Gal b1-2 Man b1-4 Glc a1-Gal b1-3 Man b1-4 Glc a1-Gal b1-4 Man b1-4 Glc a1-Gal b1-6 Man b1-4 Glc a1-Gal a1-2 Man b1-6 Glc a1-Gal a1-3 Man b1-6 Glc a1-

Gal a1-4 Man b1-6 Glc a1-Gal a1-6 Man b1-6 Glc a1-Gal b1-2 Man b1-6 Glc a1-Gal b1-3 Man b1-6 Glc a1-Gal b1-4 Man b1-6 Glc a1-Gal b1-6 Man b1-6 Glc a1-Gal a1-2 Man a1-2 Glc b1-Gal a1-3 Man a1-2 Glc b1-Gal a1-4 Man a1-2 Glc b1-Gal a1-6 Man a1-2 Glc b1-Gal b1-2 Man a1-2 Glc b1-Gal b1-3 Man a1-2 Glc b1-Gal b1-4 Man a1-2 Glc b1-Gal b1-6 Man a1-2 Glc b1-Gal a1-2 Man a1-3 Glc b1-Gal a1-3 Man a1-3 Glc b1-Gal a1-4 Man a1-3 Glc b1-Gal a1-6 Man a1-3 Glc b1-Gal b1-2 Man a1-3 Glc b1-Gal b1-3 Man a1-3 Glc b1-Gal b1-4 Man a1-3 Glc b1-Gal b1-6 Man a1-3 Glc b1-Gal a1-2 Man a1-4 Glc b1-Gal a1-3 Man a1-4 Glc b1-Gal a1-4 Man a1-4 Glc b1-Gal a1-6 Man a1-4 Glc b1-Gal b1-2 Man a1-4 Glc b1-Gal b1-3 Man a1-4 Glc b1-Gal b1-4 Man a1-4 Glc b1-Gal b1-6 Man a1-4 Glc b1-Gal a1-2 Man a1-6 Glc b1-Gal a1-3 Man a1-6 Glc b1-Gal a1-4 Man a1-6 Glc b1-Gal a1-6 Man a1-6 Glc b1-Gal b1-2 Man a1-6 Glc b1-Gal b1-3 Man a1-6 Glc b1-Gal b1-4 Man a1-6 Glc b1-Gal b1-6 Man a1-6 Glc b1-Gal a1-2 Man b1-2 Glc b1-Gal a1-3 Man b1-2 Glc b1-Gal a1-4 Man b1-2 Glc b1-Gal a1-6 Man b1-2 Glc b1-Gal b1-2 Man b1-2 Glc b1-Gal b1-3 Man b1-2 Glc b1-Gal b1-4 Man b1-2 Glc b1-Gal b1-6 Man b1-2 Glc b1-Gal a1-2 Man b1-3 Glc b1-Gal a1-3 Man b1-3 Glc b1-Gal a1-4 Man b1-3 Glc b1-Gal a1-6 Man b1-3 Glc b1-Gal b1-2 Man b1-3 Glc b1-Gal b1-3 Man b1-3 Glc b1-Gal b1-4 Man b1-3 Glc b1-Gal b1-6 Man b1-3 Glc b1-Gal a1-2 Man b1-4 Glc b1-Gal a1-3 Man b1-4 Glc b1-Gal a1-4 Man b1-4 Glc b1-Gal a1-6 Man b1-4 Glc b1-Gal b1-2 Man b1-4 Glc b1-Gal b1-3 Man b1-4 Glc b1-Gal b1-4 Man b1-4 Glc b1-Gal b1-6 Man b1-4 Glc b1-Gal a1-2 Man b1-6 Glc b1-Gal a1-3 Man b1-6 Glc b1-Gal a1-4 Man b1-6 Glc b1-Gal a1-6 Man b1-6 Glc b1-Gal b1-2 Man b1-6 Glc b1-Gal b1-3 Man b1-6 Glc b1-Gal b1-4 Man b1-6 Glc b1-Gal b1-6 Man b1-6 Glc b1-Man a1-2 Glc a1-2 Gal a1-Man a1-3 Glc a1-2 Gal a1-Man a1-4 Glc a1-2 Gal a1-Man a1-6 Glc a1-2 Gal a1-Man b1-2 Glc a1-2 Gal a1-Man b1-3 Glc a1-2 Gal a1-Man b1-4 Glc a1-2 Gal a1-Man b1-6 Glc a1-2 Gal a1-Man a1-2 Glc a1-3 Gal a1-Man a1-3 Glc a1-3 Gal a1-Man a1-4 Glc a1-3 Gal a1-Man a1-6 Glc a1-3 Gal a1-Man b1-2 Glc a1-3 Gal a1-Man b1-3 Glc a1-3 Gal a1-Man b1-4 Glc a1-3 Gal a1-Man b1-6 Glc a1-3 Gal a1-Man a1-2 Glc a1-4 Gal a1-Man a1-3 Glc a1-4 Gal a1-Man a1-4 Glc a1-4 Gal a1-Man a1-6 Glc a1-4 Gal a1-Man b1-2 Glc a1-4 Gal a1-Man b1-3 Glc a1-4 Gal a1-Man b1-4 Glc a1-4 Gal a1-

Man b1-6 Glc a1-4 Gal a1-Man a1-2 Glc a1-6 Gal a1-Man a1-3 Glc a1-6 Gal a1-Man a1-4 Glc a1-6 Gal a1-Man a1-6 Glc a1-6 Gal a1-Man b1-2 Glc a1-6 Gal a1-Man b1-3 Glc a1-6 Gal a1-Man b1-4 Glc a1-6 Gal a1-Man b1-6 Glc a1-6 Gal a1-Man a1-2 Glc b1-2 Gal a1-Man a1-3 Glc b1-2 Gal a1-Man a1-4 Glc b1-2 Gal a1-Man a1-6 Glc b1-2 Gal a1-Man b1-2 Glc b1-2 Gal a1-Man b1-3 Glc b1-2 Gal a1-Man b1-4 Glc b1-2 Gal a1-Man b1-6 Glc b1-2 Gal a1-Man a1-2 Glc b1-3 Gal a1-Man a1-3 Glc b1-3 Gal a1-Man a1-4 Glc b1-3 Gal a1-Man a1-6 Glc b1-3 Gal a1-Man b1-2 Glc b1-3 Gal a1-Man b1-3 Glc b1-3 Gal a1-Man b1-4 Glc b1-3 Gal a1-Man b1-6 Glc b1-3 Gal a1-Man a1-2 Glc b1-4 Gal a1-Man a1-3 Glc b1-4 Gal a1-Man a1-4 Glc b1-4 Gal a1-Man a1-6 Glc b1-4 Gal a1-Man b1-2 Glc b1-4 Gal a1-Man b1-3 Glc b1-4 Gal a1-Man b1-4 Glc b1-4 Gal a1-Man b1-6 Glc b1-4 Gal a1-Man a1-2 Glc b1-6 Gal a1-Man a1-3 Glc b1-6 Gal a1-Man a1-4 Glc b1-6 Gal a1-Man a1-6 Glc b1-6 Gal a1-Man b1-2 Glc b1-6 Gal a1-Man b1-3 Glc b1-6 Gal a1-Man b1-4 Glc b1-6 Gal a1-Man b1-6 Glc b1-6 Gal a1-Man a1-2 Glc a1-2 Gal b1-Man a1-3 Glc a1-2 Gal b1-Man a1-4 Glc a1-2 Gal b1-Man a1-6 Glc a1-2 Gal b1-Man b1-2 Glc a1-2 Gal b1-Man b1-3 Glc a1-2 Gal b1-Man b1-4 Glc a1-2 Gal b1-Man b1-6 Glc a1-2 Gal b1-Man a1-2 Glc a1-3 Gal b1-Man a1-3 Glc a1-3 Gal b1-Man a1-4 Glc a1-3 Gal b1-Man a1-6 Glc a1-3 Gal b1-Man b1-2 Glc a1-3 Gal b1-Man b1-3 Glc a1-3 Gal b1-Man b1-4 Glc a1-3 Gal b1-Man b1-6 Glc a1-3 Gal b1-Man a1-2 Glc a1-4 Gal b1-Man a1-3 Glc a1-4 Gal b1-Man a1-4 Glc a1-4 Gal b1-Man a1-6 Glc a1-4 Gal b1-Man b1-2 Glc a1-4 Gal b1-Man b1-3 Glc a1-4 Gal b1-Man b1-4 Glc a1-4 Gal b1-Man b1-6 Glc a1-4 Gal b1-Man a1-2 Glc a1-6 Gal b1-Man a1-3 Glc a1-6 Gal b1-Man a1-4 Glc a1-6 Gal b1-Man a1-6 Glc a1-6 Gal b1-Man b1-2 Glc a1-6 Gal b1-Man b1-3 Glc a1-6 Gal b1-Man b1-4 Glc a1-6 Gal b1-Man b1-6 Glc a1-6 Gal b1-Man a1-2 Glc b1-2 Gal b1-Man a1-3 Glc b1-2 Gal b1-Man a1-4 Glc b1-2 Gal b1-Man a1-6 Glc b1-2 Gal b1-Man b1-2 Glc b1-2 Gal b1-Man b1-3 Glc b1-2 Gal b1-Man b1-4 Glc b1-2 Gal b1-Man b1-6 Glc b1-2 Gal b1-Man a1-2 Glc b1-3 Gal b1-Man a1-3 Glc b1-3 Gal b1-Man a1-4 Glc b1-3 Gal b1-Man a1-6 Glc b1-3 Gal b1-Man b1-2 Glc b1-3 Gal b1-Man b1-3 Glc b1-3 Gal b1-Man b1-4 Glc b1-3 Gal b1-Man b1-6 Glc b1-3 Gal b1-Man a1-2 Glc b1-4 Gal b1-Man a1-3 Glc b1-4 Gal b1-Man a1-4 Glc b1-4 Gal b1-Man a1-6 Glc b1-4 Gal b1-

Man b1-2 Glc b1-4 Gal b1-Man b1-3 Glc b1-4 Gal b1-Man b1-4 Glc b1-4 Gal b1-Man b1-6 Glc b1-4 Gal b1-Man a1-2 Glc b1-6 Gal b1-Man a1-3 Glc b1-6 Gal b1-Man a1-4 Glc b1-6 Gal b1-Man a1-6 Glc b1-6 Gal b1-Man b1-2 Glc b1-6 Gal b1-Man b1-3 Glc b1-6 Gal b1-Man b1-4 Glc b1-6 Gal b1-Man b1-6 Glc b1-6 Gal b1-Man a1-2 Gal a1-2 Glc a1-Man a1-3 Gal a1-2 Glc a1-Man a1-4 Gal a1-2 Glc a1-Man a1-6 Gal a1-2 Glc a1-Man b1-2 Gal a1-2 Glc a1-Man b1-3 Gal a1-2 Glc a1-Man b1-4 Gal a1-2 Glc a1-Man b1-6 Gal a1-2 Glc a1-Man a1-2 Gal a1-3 Glc a1-Man a1-3 Gal a1-3 Glc a1-Man a1-4 Gal a1-3 Glc a1-Man a1-6 Gal a1-3 Glc a1-Man b1-2 Gal a1-3 Glc a1-Man b1-3 Gal a1-3 Glc a1-Man b1-4 Gal a1-3 Glc a1-Man b1-6 Gal a1-3 Glc a1-Man a1-2 Gal a1-4 Glc a1-Man a1-3 Gal a1-4 Glc a1-Man a1-4 Gal a1-4 Glc a1-Man a1-6 Gal a1-4 Glc a1-Man b1-2 Gal a1-4 Glc a1-Man b1-3 Gal a1-4 Glc a1-Man b1-4 Gal a1-4 Glc a1-Man b1-6 Gal a1-4 Glc a1-Man a1-2 Gal a1-6 Glc a1-Man a1-3 Gal a1-6 Glc a1-Man a1-4 Gal a1-6 Glc a1-Man a1-6 Gal a1-6 Glc a1-Man b1-2 Gal a1-6 Glc a1-Man b1-3 Gal a1-6 Glc a1-Man b1-4 Gal a1-6 Glc a1-Man b1-6 Gal a1-6 Glc a1-Man a1-2 Gal b1-2 Glc a1-Man a1-3 Gal b1-2 Glc a1-Man a1-4 Gal b1-2 Glc a1-Man a1-6 Gal b1-2 Glc a1-Man b1-2 Gal b1-2 Glc a1-Man b1-3 Gal b1-2 Glc a1-Man b1-4 Gal b1-2 Glc a1-Man b1-6 Gal b1-2 Glc a1-Man a1-2 Gal b1-3 Glc a1-Man a1-3 Gal b1-3 Glc a1-Man a1-4 Gal b1-3 Glc a1-Man a1-6 Gal b1-3 Glc a1-Man b1-2 Gal b1-3 Glc a1-Man b1-3 Gal b1-3 Glc a1-Man b1-4 Gal b1-3 Glc a1-Man b1-6 Gal b1-3 Glc a1-Man a1-2 Gal b1-4 Glc a1-Man a1-3 Gal b1-4 Glc a1-Man a1-4 Gal b1-4 Glc a1-Man a1-6 Gal b1-4 Glc a1-Man b1-2 Gal b1-4 Glc a1-Man b1-3 Gal b1-4 Glc a1-Man b1-4 Gal b1-4 Glc a1-Man b1-6 Gal b1-4 Glc a1-Man a1-2 Gal b1-6 Glc a1-Man a1-3 Gal b1-6 Glc a1-Man a1-4 Gal b1-6 Glc a1-Man a1-6 Gal b1-6 Glc a1-Man b1-2 Gal b1-6 Glc a1-Man b1-3 Gal b1-6 Glc a1-Man b1-4 Gal b1-6 Glc a1-Man b1-6 Gal b1-6 Glc a1-Man a1-2 Gal a1-2 Glc b1Man a1-3 Gal a1-2 Glc b1Man a1-4 Gal a1-2 Glc b1Man a1-6 Gal a1-2 Glc b1Man b1-2 Gal a1-2 Glc b1Man b1-3 Gal a1-2 Glc b1Man b1-4 Gal a1-2 Glc b1Man b1-6 Gal a1-2 Glc b1Man a1-2 Gal a1-3 Glc b1Man a1-3 Gal a1-3 Glc b1Man a1-4 Gal a1-3 Glc b1Man a1-6 Gal a1-3 Glc b1Man b1-2 Gal a1-3 Glc b1Man b1-3 Gal a1-3 Glc b1Man b1-4 Gal a1-3 Glc b1Man b1-6 Gal a1-3 Glc b1Man a1-2 Gal a1-4 Glc b1

Man a1-3 Gal a1-4 Glc b1Man a1-4 Gal a1-4 Glc b1Man a1-6 Gal a1-4 Glc b1Man b1-2 Gal a1-4 Glc b1Man b1-3 Gal a1-4 Glc b1Man b1-4 Gal a1-4 Glc b1Man b1-6 Gal a1-4 Glc b1Man a1-2 Gal a1-6 Glc b1Man a1-3 Gal a1-6 Glc b1Man a1-4 Gal a1-6 Glc b1Man a1-6 Gal a1-6 Glc b1Man b1-2 Gal a1-6 Glc b1Man b1-3 Gal a1-6 Glc b1Man b1-4 Gal a1-6 Glc b1Man b1-6 Gal a1-6 Glc b1Man a1-2 Gal b1-2 Glc b1Man a1-3 Gal b1-2 Glc b1Man a1-4 Gal b1-2 Glc b1Man a1-6 Gal b1-2 Glc b1Man b1-2 Gal b1-2 Glc b1Man b1-3 Gal b1-2 Glc b1Man b1-4 Gal b1-2 Glc b1Man b1-6 Gal b1-2 Glc b1Man a1-2 Gal b1-3 Glc b1Man a1-3 Gal b1-3 Glc b1Man a1-4 Gal b1-3 Glc b1Man a1-6 Gal b1-3 Glc b1Man b1-2 Gal b1-3 Glc b1Man b1-3 Gal b1-3 Glc b1Man b1-4 Gal b1-3 Glc b1Man b1-6 Gal b1-3 Glc b1Man a1-2 Gal b1-4 Glc b1Man a1-3 Gal b1-4 Glc b1Man a1-4 Gal b1-4 Glc b1Man a1-6 Gal b1-4 Glc b1Man b1-2 Gal b1-4 Glc b1Man b1-3 Gal b1-4 Glc b1Man b1-4 Gal b1-4 Glc b1Man b1-6 Gal b1-4 Glc b1Man a1-2 Gal b1-6 Glc b1Man a1-3 Gal b1-6 Glc b1Man a1-4 Gal b1-6 Glc b1Man a1-6 Gal b1-6 Glc b1Man b1-2 Gal b1-6 Glc b1Man b1-3 Gal b1-6 Glc b1Man b1-4 Gal b1-6 Glc b1Man b1-6 Gal b1-6 Glc b1Glc a1-2 Man a1-2 Gal a1-Glc a1-3 Man a1-2 Gal a1-Glc a1-4 Man a1-2 Gal a1-Glc a1-6 Man a1-2 Gal a1-Glc b1-2 Man a1-2 Gal a1-Glc b1-3 Man a1-2 Gal a1-Glc b1-4 Man a1-2 Gal a1-Glc b1-6 Man a1-2 Gal a1-Glc a1-2 Man a1-3 Gal a1-Glc a1-3 Man a1-3 Gal a1-Glc a1-4 Man a1-3 Gal a1-Glc a1-6 Man a1-3 Gal a1-Glc b1-2 Man a1-3 Gal a1-Glc b1-3 Man a1-3 Gal a1-Glc b1-4 Man a1-3 Gal a1-Glc b1-6 Man a1-3 Gal a1-Glc a1-2 Man a1-4 Gal a1-Glc a1-3 Man a1-4 Gal a1-Glc a1-4 Man a1-4 Gal a1-Glc a1-6 Man a1-4 Gal a1-Glc b1-2 Man a1-4 Gal a1-Glc b1-3 Man a1-4 Gal a1-Glc b1-4 Man a1-4 Gal a1-Glc b1-6 Man a1-4 Gal a1-Glc a1-2 Man a1-6 Gal a1-Glc a1-3 Man a1-6 Gal a1-Glc a1-4 Man a1-6 Gal a1-Glc a1-6 Man a1-6 Gal a1-Glc b1-2 Man a1-6 Gal a1-Glc b1-3 Man a1-6 Gal a1-Glc b1-4 Man a1-6 Gal a1-Glc b1-6 Man a1-6 Gal a1-Glc a1-2 Man b1-2 Gal a1-Glc a1-3 Man b1-2 Gal a1-Glc a1-4 Man b1-2 Gal a1-Glc a1-6 Man b1-2 Gal a1-Glc b1-2 Man b1-2 Gal a1-Glc b1-3 Man b1-2 Gal a1-Glc b1-4 Man b1-2 Gal a1-Glc b1-6 Man b1-2 Gal a1-Glc a1-2 Man b1-3 Gal a1-Glc a1-3 Man b1-3 Gal a1-Glc a1-4 Man b1-3 Gal a1-Glc a1-6 Man b1-3 Gal a1-Glc b1-2 Man b1-3 Gal a1-Glc b1-3 Man b1-3 Gal a1-

Glc b1-4 Man b1-3 Gal a1-Glc b1-6 Man b1-3 Gal a1-Glc a1-2 Man b1-4 Gal a1-Glc a1-3 Man b1-4 Gal a1-Glc a1-4 Man b1-4 Gal a1-Glc a1-6 Man b1-4 Gal a1-Glc b1-2 Man b1-4 Gal a1-Glc b1-3 Man b1-4 Gal a1-Glc b1-4 Man b1-4 Gal a1-Glc b1-6 Man b1-4 Gal a1-Glc a1-2 Man b1-6 Gal a1-Glc a1-3 Man b1-6 Gal a1-Glc a1-4 Man b1-6 Gal a1-Glc a1-6 Man b1-6 Gal a1-Glc b1-2 Man b1-6 Gal a1-Glc b1-3 Man b1-6 Gal a1-Glc b1-4 Man b1-6 Gal a1-Glc b1-6 Man b1-6 Gal a1-Glc a1-2 Man a1-2 Gal b1-Glc a1-3 Man a1-2 Gal b1-Glc a1-4 Man a1-2 Gal b1-Glc a1-6 Man a1-2 Gal b1-Glc b1-2 Man a1-2 Gal b1-Glc b1-3 Man a1-2 Gal b1-Glc b1-4 Man a1-2 Gal b1-Glc b1-6 Man a1-2 Gal b1-Glc a1-2 Man a1-3 Gal b1-Glc a1-3 Man a1-3 Gal b1-Glc a1-4 Man a1-3 Gal b1-Glc a1-6 Man a1-3 Gal b1-Glc b1-2 Man a1-3 Gal b1-Glc b1-3 Man a1-3 Gal b1-Glc b1-4 Man a1-3 Gal b1-Glc b1-6 Man a1-3 Gal b1-Glc a1-2 Man a1-4 Gal b1-Glc a1-3 Man a1-4 Gal b1-Glc a1-4 Man a1-4 Gal b1-Glc a1-6 Man a1-4 Gal b1-Glc b1-2 Man a1-4 Gal b1-Glc b1-3 Man a1-4 Gal b1-Glc b1-4 Man a1-4 Gal b1-Glc b1-6 Man a1-4 Gal b1-Glc a1-2 Man a1-6 Gal b1-Glc a1-3 Man a1-6 Gal b1-Glc a1-4 Man a1-6 Gal b1-Glc a1-6 Man a1-6 Gal b1-Glc b1-2 Man a1-6 Gal b1-Glc b1-3 Man a1-6 Gal b1-Glc b1-4 Man a1-6 Gal b1-Glc b1-6 Man a1-6 Gal b1-Glc a1-2 Man b1-2 Gal b1-Glc a1-3 Man b1-2 Gal b1-Glc a1-4 Man b1-2 Gal b1-Glc a1-6 Man b1-2 Gal b1-Glc b1-2 Man b1-2 Gal b1-Glc b1-3 Man b1-2 Gal b1-Glc b1-4 Man b1-2 Gal b1-Glc b1-6 Man b1-2 Gal b1-Glc a1-2 Man b1-3 Gal b1-Glc a1-3 Man b1-3 Gal b1-Glc a1-4 Man b1-3 Gal b1-Glc a1-6 Man b1-3 Gal b1-Glc b1-2 Man b1-3 Gal b1-Glc b1-3 Man b1-3 Gal b1-Glc b1-4 Man b1-3 Gal b1-Glc b1-6 Man b1-3 Gal b1-Glc a1-2 Man b1-4 Gal b1-Glc a1-3 Man b1-4 Gal b1-Glc a1-4 Man b1-4 Gal b1-Glc a1-6 Man b1-4 Gal b1-Glc b1-2 Man b1-4 Gal b1-Glc b1-3 Man b1-4 Gal b1-Glc b1-4 Man b1-4 Gal b1-Glc b1-6 Man b1-4 Gal b1-Glc a1-2 Man b1-6 Gal b1-Glc a1-3 Man b1-6 Gal b1-Glc a1-4 Man b1-6 Gal b1-Glc a1-6 Man b1-6 Gal b1-Glc b1-2 Man b1-6 Gal b1-Glc b1-3 Man b1-6 Gal b1-Glc b1-4 Man b1-6 Gal b1-Glc b1-6 Man b1-6 Gal b1-Glc a1-2 Gal a1-2 Man a1-Glc a1-3 Gal a1-2 Man a1-Glc a1-4 Gal a1-2 Man a1-Glc a1-6 Gal a1-2 Man a1-Glc b1-2 Gal a1-2 Man a1-Glc b1-3 Gal a1-2 Man a1-Glc b1-4 Gal a1-2 Man a1-Glc b1-6 Gal a1-2 Man a1-Glc a1-2 Gal a1-3 Man a1-Glc a1-3 Gal a1-3 Man a1-Glc a1-4 Gal a1-3 Man a1-

Glc a1-6 Gal a1-3 Man a1-Glc b1-2 Gal a1-3 Man a1-Glc b1-3 Gal a1-3 Man a1-Glc b1-4 Gal a1-3 Man a1-Glc b1-6 Gal a1-3 Man a1-Glc a1-2 Gal a1-4 Man a1-Glc a1-3 Gal a1-4 Man a1-Glc a1-4 Gal a1-4 Man a1-Glc a1-6 Gal a1-4 Man a1-Glc b1-2 Gal a1-4 Man a1-Glc b1-3 Gal a1-4 Man a1-Glc b1-4 Gal a1-4 Man a1-Glc b1-6 Gal a1-4 Man a1-Glc a1-2 Gal a1-6 Man a1-Glc a1-3 Gal a1-6 Man a1-Glc a1-4 Gal a1-6 Man a1-Glc a1-6 Gal a1-6 Man a1-Glc b1-2 Gal a1-6 Man a1-Glc b1-3 Gal a1-6 Man a1-Glc b1-4 Gal a1-6 Man a1-Glc b1-6 Gal a1-6 Man a1-Glc a1-2 Gal b1-2 Man a1-Glc a1-3 Gal b1-2 Man a1-Glc a1-4 Gal b1-2 Man a1-Glc a1-6 Gal b1-2 Man a1-Glc b1-2 Gal b1-2 Man a1-Glc b1-3 Gal b1-2 Man a1-Glc b1-4 Gal b1-2 Man a1-Glc b1-6 Gal b1-2 Man a1-Glc a1-2 Gal b1-3 Man a1-Glc a1-3 Gal b1-3 Man a1-Glc a1-4 Gal b1-3 Man a1-Glc a1-6 Gal b1-3 Man a1-Glc b1-2 Gal b1-3 Man a1-Glc b1-3 Gal b1-3 Man a1-Glc b1-4 Gal b1-3 Man a1-Glc b1-6 Gal b1-3 Man a1-Glc a1-2 Gal b1-4 Man a1-Glc a1-3 Gal b1-4 Man a1-Glc a1-4 Gal b1-4 Man a1-Glc a1-6 Gal b1-4 Man a1-Glc b1-2 Gal b1-4 Man a1-Glc b1-3 Gal b1-4 Man a1-Glc b1-4 Gal b1-4 Man a1-Glc b1-6 Gal b1-4 Man a1-Glc a1-2 Gal b1-6 Man a1-Glc a1-3 Gal b1-6 Man a1-Glc a1-4 Gal b1-6 Man a1-Glc a1-6 Gal b1-6 Man a1-Glc b1-2 Gal b1-6 Man a1-Glc b1-3 Gal b1-6 Man a1-Glc b1-4 Gal b1-6 Man a1-Glc b1-6 Gal b1-6 Man a1-Glc a1-2 Gal a1-2 Man b1-Glc a1-3 Gal a1-2 Man b1-Glc a1-4 Gal a1-2 Man b1-Glc a1-6 Gal a1-2 Man b1-Glc b1-2 Gal a1-2 Man b1-Glc b1-3 Gal a1-2 Man b1-Glc b1-4 Gal a1-2 Man b1-Glc b1-6 Gal a1-2 Man b1-Glc a1-2 Gal a1-3 Man b1-Glc a1-3 Gal a1-3 Man b1-Glc a1-4 Gal a1-3 Man b1-Glc a1-6 Gal a1-3 Man b1-Glc b1-2 Gal a1-3 Man b1-Glc b1-3 Gal a1-3 Man b1-Glc b1-4 Gal a1-3 Man b1-Glc b1-6 Gal a1-3 Man b1-Glc a1-2 Gal a1-4 Man b1-Glc a1-3 Gal a1-4 Man b1-Glc a1-4 Gal a1-4 Man b1-Glc a1-6 Gal a1-4 Man b1-Glc b1-2 Gal a1-4 Man b1-Glc b1-3 Gal a1-4 Man b1-Glc b1-4 Gal a1-4 Man b1-Glc b1-6 Gal a1-4 Man b1-Glc a1-2 Gal a1-6 Man b1-Glc a1-3 Gal a1-6 Man b1-Glc a1-4 Gal a1-6 Man b1-Glc a1-6 Gal a1-6 Man b1-Glc b1-2 Gal a1-6 Man b1-Glc b1-3 Gal a1-6 Man b1-Glc b1-4 Gal a1-6 Man b1-Glc b1-6 Gal a1-6 Man b1-Glc a1-2 Gal b1-2 Man b1-Glc a1-3 Gal b1-2 Man b1-Glc a1-4 Gal b1-2 Man b1-Glc a1-6 Gal b1-2 Man b1-Glc b1-2 Gal b1-2 Man b1-Glc b1-3 Gal b1-2 Man b1-Glc b1-4 Gal b1-2 Man b1-Glc b1-6 Gal b1-2 Man b1-

Glc a1-2 Gal b1-3 Man b1-Glc a1-3 Gal b1-3 Man b1-Glc a1-4 Gal b1-3 Man b1-Glc a1-6 Gal b1-3 Man b1-Glc b1-2 Gal b1-3 Man b1-Glc b1-3 Gal b1-3 Man b1-Glc b1-4 Gal b1-3 Man b1-Glc b1-6 Gal b1-3 Man b1-Glc a1-2 Gal b1-4 Man b1-Glc a1-3 Gal b1-4 Man b1-Glc a1-4 Gal b1-4 Man b1-Glc a1-6 Gal b1-4 Man b1-Glc b1-2 Gal b1-4 Man b1-Glc b1-3 Gal b1-4 Man b1-Glc b1-4 Gal b1-4 Man b1-Glc b1-6 Gal b1-4 Man b1-Glc a1-2 Gal b1-6 Man b1-Glc a1-3 Gal b1-6 Man b1-Glc a1-4 Gal b1-6 Man b1-Glc a1-6 Gal b1-6 Man b1-Glc b1-2 Gal b1-6 Man b1-Glc b1-3 Gal b1-6 Man b1-Glc b1-4 Gal b1-6 Man b1-Glc b1-6 Gal b1-6 Man b1-Gal a1-2 (Glc a1-3) Man a1-Gal a1-2 (Glc a1-4) Man a1-Gal a1-2 (Glc a1-6) Man a1-Gal a1-3 (Glc a1-2) Man a1-Gal a1-3 (Glc a1-4) Man a1-Gal a1-3 (Glc a1-6) Man a1-Gal a1-4 (Glc a1-2) Man a1-Gal a1-4 (Glc a1-3) Man a1-Gal a1-4 (Glc a1-6) Man a1-Gal a1-6 (Glc a1-2) Man a1-Gal a1-6 (Glc a1-3) Man a1-Gal a1-6 (Glc a1-4) Man a1-Gal b1-2 (Glc a1-3) Man a1-Gal b1-2 (Glc a1-4) Man a1-Gal b1-2 (Glc a1-6) Man a1-Gal b1-3 (Glc a1-2) Man a1-Gal b1-3 (Glc a1-4) Man a1-Gal b1-3 (Glc a1-6) Man a1-Gal b1-4 (Glc a1-2) Man a1-Gal b1-4 (Glc a1-3) Man a1-Gal b1-4 (Glc a1-6) Man a1-Gal b1-6 (Glc a1-2) Man a1-Gal b1-6 (Glc a1-3) Man a1-Gal b1-6 (Glc a1-4) Man a1-Gal a1-2 (Glc b1-3) Man a1-Gal a1-2 (Glc b1-4) Man a1-Gal a1-2 (Glc b1-6) Man a1-Gal a1-3 (Glc b1-2) Man a1-Gal a1-3 (Glc b1-4) Man a1-Gal a1-3 (Glc b1-6) Man a1-Gal a1-4 (Glc b1-2) Man a1-Gal a1-4 (Glc b1-3) Man a1-Gal a1-4 (Glc b1-6) Man a1-Gal a1-6 (Glc b1-2) Man a1-Gal a1-6 (Glc b1-3) Man a1-Gal a1-6 (Glc b1-4) Man a1-Gal b1-2 (Glc b1-3) Man a1-Gal b1-2 (Glc b1-4) Man a1-Gal b1-2 (Glc b1-6) Man a1-Gal b1-3 (Glc b1-2) Man a1-Gal b1-3 (Glc b1-4) Man a1-Gal b1-3 (Glc b1-6) Man a1-Gal b1-4 (Glc b1-2) Man a1-Gal b1-4 (Glc b1-3) Man a1-Gal b1-4 (Glc b1-6) Man a1-Gal b1-6 (Glc b1-2) Man a1-Gal b1-6 (Glc b1-3) Man a1-Gal b1-6 (Glc b1-4) Man a1-Gal a1-2 (Glc a1-3) Man b1-Gal a1-2 (Glc a1-4) Man b1-Gal a1-2 (Glc a1-6) Man b1-Gal a1-3 (Glc a1-2) Man b1-Gal a1-3 (Glc a1-4) Man b1-Gal a1-3 (Glc a1-6) Man b1-Gal a1-4 (Glc a1-2) Man b1-Gal a1-4 (Glc a1-3) Man b1-Gal a1-4 (Glc a1-6) Man b1-Gal a1-6 (Glc a1-2) Man b1-Gal a1-6 (Glc a1-3) Man b1-Gal a1-6 (Glc a1-4) Man b1-Gal b1-2 (Glc a1-3) Man b1-Gal b1-2 (Glc a1-4) Man b1-Gal b1-2 (Glc a1-6) Man b1-Gal b1-3 (Glc a1-2) Man b1-Gal b1-3 (Glc a1-4) Man b1-Gal b1-3 (Glc a1-6) Man b1-Gal b1-4 (Glc a1-2) Man b1-Gal b1-4 (Glc a1-3) Man b1-Gal b1-4 (Glc a1-6) Man b1-

Gal b1-6 (Glc a1-2) Man b1-Gal b1-6 (Glc a1-3) Man b1-Gal b1-6 (Glc a1-4) Man b1-Gal a1-2 (Glc b1-3) Man b1-Gal a1-2 (Glc b1-4) Man b1-Gal a1-2 (Glc b1-6) Man b1-Gal a1-3 (Glc b1-2) Man b1-Gal a1-3 (Glc b1-4) Man b1-Gal a1-3 (Glc b1-6) Man b1-Gal a1-4 (Glc b1-2) Man b1-Gal a1-4 (Glc b1-3) Man b1-Gal a1-4 (Glc b1-6) Man b1-Gal a1-6 (Glc b1-2) Man b1-Gal a1-6 (Glc b1-3) Man b1-Gal a1-6 (Glc b1-4) Man b1-Gal b1-2 (Glc b1-3) Man b1-Gal b1-2 (Glc b1-4) Man b1-Gal b1-2 (Glc b1-6) Man b1-Gal b1-3 (Glc b1-2) Man b1-Gal b1-3 (Glc b1-4) Man b1-Gal b1-3 (Glc b1-6) Man b1-Gal b1-4 (Glc b1-2) Man b1-Gal b1-4 (Glc b1-3) Man b1-Gal b1-4 (Glc b1-6) Man b1-Gal b1-6 (Glc b1-2) Man b1-Gal b1-6 (Glc b1-3) Man b1-Gal b1-6 (Glc b1-4) Man b1-Gal a1-2 (Man a1-3) Glc a1-Gal a1-2 (Man a1-4) Glc a1-Gal a1-2 (Man a1-6) Glc a1-Gal a1-3 (Man a1-2) Glc a1-Gal a1-3 (Man a1-4) Glc a1-Gal a1-3 (Man a1-6) Glc a1-Gal a1-4 (Man a1-2) Glc a1-Gal a1-4 (Man a1-3) Glc a1-Gal a1-4 (Man a1-6) Glc a1-Gal a1-6 (Man a1-2) Glc a1-Gal a1-6 (Man a1-3) Glc a1-Gal a1-6 (Man a1-4) Glc a1-Gal b1-2 (Man a1-3) Glc a1-Gal b1-2 (Man a1-4) Glc a1-Gal b1-2 (Man a1-6) Glc a1-Gal b1-3 (Man a1-2) Glc a1-Gal b1-3 (Man a1-4) Glc a1-Gal b1-3 (Man a1-6) Glc a1-Gal b1-4 (Man a1-2) Glc a1-Gal b1-4 (Man a1-3) Glc a1-Gal b1-4 (Man a1-6) Glc a1-Gal b1-6 (Man a1-2) Glc a1-Gal b1-6 (Man a1-3) Glc a1-Gal b1-6 (Man a1-4) Glc a1-Gal a1-2 (Man b1-3) Glc a1-Gal a1-2 (Man b1-4) Glc a1-Gal a1-2 (Man b1-6) Glc a1-Gal a1-3 (Man b1-2) Glc a1-Gal a1-3 (Man b1-4) Glc a1-Gal a1-3 (Man b1-6) Glc a1-Gal a1-4 (Man b1-2) Glc a1-Gal a1-4 (Man b1-3) Glc a1-Gal a1-4 (Man b1-6) Glc a1-Gal a1-6 (Man b1-2) Glc a1-Gal a1-6 (Man b1-3) Glc a1-Gal a1-6 (Man b1-4) Glc a1-Gal b1-2 (Man b1-3) Glc a1-Gal b1-2 (Man b1-4) Glc a1-Gal b1-2 (Man b1-6) Glc a1-Gal b1-3 (Man b1-2) Glc a1-Gal b1-3 (Man b1-4) Glc a1-Gal b1-3 (Man b1-6) Glc a1-Gal b1-4 (Man b1-2) Glc a1-Gal b1-4 (Man b1-3) Glc a1-Gal b1-4 (Man b1-6) Glc a1-Gal b1-6 (Man b1-2) Glc a1-Gal b1-6 (Man b1-3) Glc a1-Gal b1-6 (Man b1-4) Glc a1-Gal a1-2 (Man a1-3) Glc b1-Gal a1-2 (Man a1-4) Glc b1-Gal a1-2 (Man a1-6) Glc b1-Gal a1-3 (Man a1-2) Glc b1-Gal a1-3 (Man a1-4) Glc b1-Gal a1-3 (Man a1-6) Glc b1-

Gal a1-4 (Man a1-2) Glc b1-Gal a1-4 (Man a1-3) Glc b1-Gal a1-4 (Man a1-6) Glc b1-Gal a1-6 (Man a1-2) Glc b1-Gal a1-6 (Man a1-3) Glc b1-Gal a1-6 (Man a1-4) Glc b1-Gal b1-2 (Man a1-3) Glc b1-Gal b1-2 (Man a1-4) Glc b1-Gal b1-2 (Man a1-6) Glc b1-Gal b1-3 (Man a1-2) Glc b1-Gal b1-3 (Man a1-4) Glc b1-Gal b1-3 (Man a1-6) Glc b1-Gal b1-4 (Man a1-2) Glc b1-Gal b1-4 (Man a1-3) Glc b1-Gal b1-4 (Man a1-6) Glc b1-Gal b1-6 (Man a1-2) Glc b1-Gal b1-6 (Man a1-3) Glc b1-Gal b1-6 (Man a1-4) Glc b1-Gal a1-2 (Man b1-3) Glc b1-Gal a1-2 (Man b1-4) Glc b1-Gal a1-2 (Man b1-6) Glc b1-Gal a1-3 (Man b1-2) Glc b1-Gal a1-3 (Man b1-4) Glc b1-Gal a1-3 (Man b1-6) Glc b1-Gal a1-4 (Man b1-2) Glc b1-Gal a1-4 (Man b1-3) Glc b1-Gal a1-4 (Man b1-6) Glc b1-Gal a1-6 (Man b1-2) Glc b1-Gal a1-6 (Man b1-3) Glc b1-Gal a1-6 (Man b1-4) Glc b1-Gal b1-2 (Man b1-3) Glc b1-Gal b1-2 (Man b1-4) Glc b1-Gal b1-2 (Man b1-6) Glc b1-Gal b1-3 (Man b1-2) Glc b1-Gal b1-3 (Man b1-4) Glc b1-Gal b1-3 (Man b1-6) Glc b1-Gal b1-4 (Man b1-2) Glc b1-Gal b1-4 (Man b1-3) Glc b1-Gal b1-4 (Man b1-6) Glc b1-Gal b1-6 (Man b1-2) Glc b1-Gal b1-6 (Man b1-3) Glc b1-Gal b1-6 (Man b1-4) Glc b1-Glc a1-2 (Man a1-3) Gal a1-Glc a1-2 (Man a1-4) Gal a1-Glc a1-2 (Man a1-6) Gal a1-Glc a1-3 (Man a1-2) Gal a1-Glc a1-3 (Man a1-4) Gal a1-Glc a1-3 (Man a1-6) Gal a1-Glc a1-4 (Man a1-2) Gal a1-Glc a1-4 (Man a1-3) Gal a1-Glc a1-4 (Man a1-6) Gal a1-Glc a1-6 (Man a1-2) Gal a1-Glc a1-6 (Man a1-3) Gal a1-Glc a1-6 (Man a1-4) Gal a1-Glc b1-2 (Man a1-3) Gal a1-Glc b1-2 (Man a1-4) Gal a1-Glc b1-2 (Man a1-6) Gal a1-Glc b1-3 (Man a1-2) Gal a1-Glc b1-3 (Man a1-4) Gal a1-Glc b1-3 (Man a1-6) Gal a1-Glc b1-4 (Man a1-2) Gal a1-Glc b1-4 (Man a1-3) Gal a1-Glc b1-4 (Man a1-6) Gal a1-Glc b1-6 (Man a1-2) Gal a1-Glc b1-6 (Man a1-3) Gal a1-Glc b1-6 (Man a1-4) Gal a1-Glc a1-2 (Man b1-3) Gal a1-Glc a1-2 (Man b1-4) Gal a1-Glc a1-2 (Man b1-6) Gal a1-Glc a1-3 (Man b1-2) Gal a1-Glc a1-3 (Man b1-4) Gal a1-Glc a1-3 (Man b1-6) Gal a1-Glc a1-4 (Man b1-2) Gal a1-Glc a1-4 (Man b1-3) Gal a1-Glc a1-4 (Man b1-6) Gal a1-Glc a1-6 (Man b1-2) Gal a1-Glc a1-6 (Man b1-3) Gal a1-Glc a1-6 (Man b1-4) Gal a1-Glc b1-2 (Man b1-3) Gal a1-Glc b1-2 (Man b1-4) Gal a1-Glc b1-2 (Man b1-6) Gal a1-

Glc b1-3 (Man b1-2) Gal a1-Glc b1-3 (Man b1-4) Gal a1-Glc b1-3 (Man b1-6) Gal a1-Glc b1-4 (Man b1-2) Gal a1-Glc b1-4 (Man b1-3) Gal a1-Glc b1-4 (Man b1-6) Gal a1-Glc b1-6 (Man b1-2) Gal a1-Glc b1-6 (Man b1-3) Gal a1-Glc b1-6 (Man b1-4) Gal a1-Glc a1-2 (Man a1-3) Gal b1-Glc a1-2 (Man a1-4) Gal b1-Glc a1-2 (Man a1-6) Gal b1-Glc a1-3 (Man a1-2) Gal b1-Glc a1-3 (Man a1-4) Gal b1-Glc a1-3 (Man a1-6) Gal b1-Glc a1-4 (Man a1-2) Gal b1-Glc a1-4 (Man a1-3) Gal b1-Glc a1-4 (Man a1-6) Gal b1-Glc a1-6 (Man a1-2) Gal b1-Glc a1-6 (Man a1-3) Gal b1-Glc a1-6 (Man a1-4) Gal b1-Glc b1-2 (Man a1-3) Gal b1-Glc b1-2 (Man a1-4) Gal b1-Glc b1-2 (Man a1-6) Gal b1-Glc b1-3 (Man a1-2) Gal b1-Glc b1-3 (Man a1-4) Gal b1-Glc b1-3 (Man a1-6) Gal b1-Glc b1-4 (Man a1-2) Gal b1-Glc b1-4 (Man a1-3) Gal b1-Glc b1-4 (Man a1-6) Gal b1-Glc b1-6 (Man a1-2) Gal b1-Glc b1-6 (Man a1-3) Gal b1-Glc b1-6 (Man a1-4) Gal b1-Glc a1-2 (Man b1-3) Gal b1-Glc a1-2 (Man b1-4) Gal b1-Glc a1-2 (Man b1-6) Gal b1-Glc a1-3 (Man b1-2) Gal b1-Glc a1-3 (Man b1-4) Gal b1-Glc a1-3 (Man b1-6) Gal b1-Glc a1-4 (Man b1-2) Gal b1-Glc a1-4 (Man b1-3) Gal b1-Glc a1-4 (Man b1-6) Gal b1-Glc a1-6 (Man b1-2) Gal b1-Glc a1-6 (Man b1-3) Gal b1-Glc a1-6 (Man b1-4) Gal b1-Glc b1-2 (Man b1-3) Gal b1-Glc b1-2 (Man b1-4) Gal b1-Glc b1-2 (Man b1-6) Gal b1-Glc b1-3 (Man b1-2) Gal b1-Glc b1-3 (Man b1-4) Gal b1-Glc b1-3 (Man b1-6) Gal b1-Glc b1-4 (Man b1-2) Gal b1-Glc b1-4 (Man b1-3) Gal b1-Glc b1-4 (Man b1-6) Gal b1-Glc b1-6 (Man b1-2) Gal b1-Glc b1-6 (Man b1-3) Gal b1-Glc b1-6 (Man b1-4) Gal b1-

Man a1-4 Gal a1-3 Glc b1Man a1-6 Gal a1-3 Glc b1Man b1-2 Gal a1-3 Glc b1Man b1-3 Gal a1-3 Glc b1Man b1-4 Gal a1-3 Glc b1Man b1-6 Gal a1-3 Glc b1Man a1-2 Gal a1-4 Glc b1Man a1-3 Gal a1-4 Glc b1Man a1-4 Gal a1-4 Glc b1Man a1-6 Gal a1-4 Glc b1Man b1-2 Gal a1-4 Glc b1Man b1-3 Gal a1-4 Glc b1Man b1-4 Gal a1-4 Glc b1Man b1-6 Gal a1-4 Glc b1Man a1-2 Gal a1-6 Glc b1Man a1-3 Gal a1-6 Glc b1

Cummings RD (2009) Molecular BioSystems 5 1087

Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical petasaccharide sequeces in glycosaminoglycans

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoN-linkedrdquo glycan linkage to protein asparagine residues

Families of N-linked glycan structures

Man

4GlcNAc1

2Man

Man

Man

Man

2Man

2Man

2Man

63

Man63 4GlcNAc1 Asn

4GlcNAc1

Man62

Man63 4GlcNAc1 Asn

Man42

4GlcNAc1

4GlcNAc1

4GlcNAc1

4GlcNAc1

6Gal

6Gal

6Gal

6Gal

NeuAc

NeuAc

NeuAcNeuAc

4GlcNAc1

Man

Man63 4GlcNAc1 Asn

2Man4GlcNAc13GalNeuAc

Man

Man

63

core

core

core

High Mannose

Complex

Hybrid

Examples of structures found as termini on the branches of complex N-linked glycoprotein glycanshellip

Terminal Saccharide Diversity --- A key to recognition

NeuAc 2-6 Gal 1-4 GlcNAc---NeuAc 2-3 Gal 1-4 GlcNAc---

Gal 1-3 Gal 1-4 GlcNAc---4-SO3-GalNAc 1-4 GlcNAc---

(-Gal 1-4 GlcNAc 1-3-)n = polylactosamine repeats (n may be gt6)

(-NeuAc 2-8-) n = polysialic acid (n may be gt50)

Fuc residues (eg Fuc 1-2 Gal Fuc 1-3 GlcNAc Fuc 1-4 GlcNAc

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoO-linkedrdquo glycan linkage to protein serine residues

Examples of variations of O-linked glycans

GalNAc Serine

GalNAc Serine

NeuAc 26

GalNAc Serine

Gal 1

NeuAc 263

GalNAc SerineNeuAc 2-3 Gal 1

NeuAc 263

Gal 1-4 GlcNAc 1

Fuc 1

GalNAc Serine

GlcNAc 1

63

4NeuAc 2-3 Gal 1

3

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Gal a1-2 Glc a1-2 Man a1-Gal a1-3 Glc a1-2 Man a1-Gal a1-4 Glc a1-2 Man a1-Gal a1-6 Glc a1-2 Man a1-Gal b1-2 Glc a1-2 Man a1-Gal b1-3 Glc a1-2 Man a1-Gal b1-4 Glc a1-2 Man a1-Gal b1-6 Glc a1-2 Man a1-Gal a1-2 Glc a1-3 Man a1-Gal a1-3 Glc a1-3 Man a1-Gal a1-4 Glc a1-3 Man a1-Gal a1-6 Glc a1-3 Man a1-Gal b1-2 Glc a1-3 Man a1-Gal b1-3 Glc a1-3 Man a1-Gal b1-4 Glc a1-3 Man a1-Gal b1-6 Glc a1-3 Man a1-Gal a1-2 Glc a1-4 Man a1-Gal a1-3 Glc a1-4 Man a1-Gal a1-4 Glc a1-4 Man a1-Gal a1-6 Glc a1-4 Man a1-Gal b1-2 Glc a1-4 Man a1-Gal b1-3 Glc a1-4 Man a1-Gal b1-4 Glc a1-4 Man a1-Gal b1-6 Glc a1-4 Man a1-Gal a1-2 Glc a1-6 Man a1-Gal a1-3 Glc a1-6 Man a1-Gal a1-4 Glc a1-6 Man a1-Gal a1-6 Glc a1-6 Man a1-Gal b1-2 Glc a1-6 Man a1-Gal b1-3 Glc a1-6 Man a1-Gal b1-4 Glc a1-6 Man a1-Gal b1-6 Glc a1-6 Man a1-Gal a1-2 Glc b1-2 Man a1-Gal a1-3 Glc b1-2 Man a1-Gal a1-4 Glc b1-2 Man a1-Gal a1-6 Glc b1-2 Man a1-Gal b1-2 Glc b1-2 Man a1-Gal b1-3 Glc b1-2 Man a1-Gal b1-4 Glc b1-2 Man a1-Gal b1-6 Glc b1-2 Man a1-Gal a1-2 Glc b1-3 Man a1-Gal a1-3 Glc b1-3 Man a1-Gal a1-4 Glc b1-3 Man a1-Gal a1-6 Glc b1-3 Man a1-Gal b1-2 Glc b1-3 Man a1-Gal b1-3 Glc b1-3 Man a1-Gal b1-4 Glc b1-3 Man a1-Gal b1-6 Glc b1-3 Man a1-Gal a1-2 Glc b1-4 Man a1-Gal a1-3 Glc b1-4 Man a1-Gal a1-4 Glc b1-4 Man a1-Gal a1-6 Glc b1-4 Man a1-Gal b1-2 Glc b1-4 Man a1-Gal b1-3 Glc b1-4 Man a1-Gal b1-4 Glc b1-4 Man a1-Gal b1-6 Glc b1-4 Man a1-Gal a1-2 Glc b1-6 Man a1-Gal a1-3 Glc b1-6 Man a1-Gal a1-4 Glc b1-6 Man a1-Gal a1-6 Glc b1-6 Man a1-Gal b1-2 Glc b1-6 Man a1-Gal b1-3 Glc b1-6 Man a1-Gal b1-4 Glc b1-6 Man a1-Gal b1-6 Glc b1-6 Man a1-Gal a1-2 Glc a1-2 Man b1-Gal a1-3 Glc a1-2 Man b1-Gal a1-4 Glc a1-2 Man b1-Gal a1-6 Glc a1-2 Man b1-Gal b1-2 Glc a1-2 Man b1-Gal b1-3 Glc a1-2 Man b1-Gal b1-4 Glc a1-2 Man b1-Gal b1-6 Glc a1-2 Man b1-Gal a1-2 Glc a1-3 Man b1-Gal a1-3 Glc a1-3 Man b1-Gal a1-4 Glc a1-3 Man b1-Gal a1-6 Glc a1-3 Man b1-Gal b1-2 Glc a1-3 Man b1-Gal b1-3 Glc a1-3 Man b1-Gal b1-4 Glc a1-3 Man b1-Gal b1-6 Glc a1-3 Man b1-Gal a1-2 Glc a1-4 Man b1-Gal a1-3 Glc a1-4 Man b1-Gal a1-4 Glc a1-4 Man b1-Gal a1-6 Glc a1-4 Man b1-Gal b1-2 Glc a1-4 Man b1-Gal b1-3 Glc a1-4 Man b1-Gal b1-4 Glc a1-4 Man b1-Gal b1-6 Glc a1-4 Man b1-Gal a1-2 Glc a1-6 Man b1-Gal a1-3 Glc a1-6 Man b1-Gal a1-4 Glc a1-6 Man b1-Gal a1-6 Glc a1-6 Man b1-Gal b1-2 Glc a1-6 Man b1-

Gal b1-3 Glc a1-6 Man b1-Gal b1-4 Glc a1-6 Man b1-Gal b1-6 Glc a1-6 Man b1-Gal a1-2 Glc b1-2 Man b1-Gal a1-3 Glc b1-2 Man b1-Gal a1-4 Glc b1-2 Man b1-Gal a1-6 Glc b1-2 Man b1-Gal b1-2 Glc b1-2 Man b1-Gal b1-3 Glc b1-2 Man b1-Gal b1-4 Glc b1-2 Man b1-Gal b1-6 Glc b1-2 Man b1-Gal a1-2 Glc b1-3 Man b1-Gal a1-3 Glc b1-3 Man b1-Gal a1-4 Glc b1-3 Man b1-Gal a1-6 Glc b1-3 Man b1-Gal b1-2 Glc b1-3 Man b1-Gal b1-3 Glc b1-3 Man b1-Gal b1-4 Glc b1-3 Man b1-Gal b1-6 Glc b1-3 Man b1-Gal a1-2 Glc b1-4 Man b1-Gal a1-3 Glc b1-4 Man b1-Gal a1-4 Glc b1-4 Man b1-Gal a1-6 Glc b1-4 Man b1-Gal b1-2 Glc b1-4 Man b1-Gal b1-3 Glc b1-4 Man b1-Gal b1-4 Glc b1-4 Man b1-Gal b1-6 Glc b1-4 Man b1-Gal a1-2 Glc b1-6 Man b1-Gal a1-3 Glc b1-6 Man b1-Gal a1-4 Glc b1-6 Man b1-Gal a1-6 Glc b1-6 Man b1-Gal b1-2 Glc b1-6 Man b1-Gal b1-3 Glc b1-6 Man b1-Gal b1-4 Glc b1-6 Man b1-Gal b1-6 Glc b1-6 Man b1-Gal a1-2 Man a1-2 Glc a1-Gal a1-3 Man a1-2 Glc a1-Gal a1-4 Man a1-2 Glc a1-Gal a1-6 Man a1-2 Glc a1-Gal b1-2 Man a1-2 Glc a1-Gal b1-3 Man a1-2 Glc a1-Gal b1-4 Man a1-2 Glc a1-Gal b1-6 Man a1-2 Glc a1-Gal a1-2 Man a1-3 Glc a1-Gal a1-3 Man a1-3 Glc a1-Gal a1-4 Man a1-3 Glc a1-Gal a1-6 Man a1-3 Glc a1-Gal b1-2 Man a1-3 Glc a1-Gal b1-3 Man a1-3 Glc a1-Gal b1-4 Man a1-3 Glc a1-Gal b1-6 Man a1-3 Glc a1-Gal a1-2 Man a1-4 Glc a1-Gal a1-3 Man a1-4 Glc a1-Gal a1-4 Man a1-4 Glc a1-Gal a1-6 Man a1-4 Glc a1-Gal b1-2 Man a1-4 Glc a1-Gal b1-3 Man a1-4 Glc a1-Gal b1-4 Man a1-4 Glc a1-Gal b1-6 Man a1-4 Glc a1-Gal a1-2 Man a1-6 Glc a1-Gal a1-3 Man a1-6 Glc a1-Gal a1-4 Man a1-6 Glc a1-Gal a1-6 Man a1-6 Glc a1-Gal b1-2 Man a1-6 Glc a1-Gal b1-3 Man a1-6 Glc a1-Gal b1-4 Man a1-6 Glc a1-Gal b1-6 Man a1-6 Glc a1-Gal a1-2 Man b1-2 Glc a1-Gal a1-3 Man b1-2 Glc a1-Gal a1-4 Man b1-2 Glc a1-Gal a1-6 Man b1-2 Glc a1-Gal b1-2 Man b1-2 Glc a1-Gal b1-3 Man b1-2 Glc a1-Gal b1-4 Man b1-2 Glc a1-Gal b1-6 Man b1-2 Glc a1-Gal a1-2 Man b1-3 Glc a1-Gal a1-3 Man b1-3 Glc a1-Gal a1-4 Man b1-3 Glc a1-Gal a1-6 Man b1-3 Glc a1-Gal b1-2 Man b1-3 Glc a1-Gal b1-3 Man b1-3 Glc a1-Gal b1-4 Man b1-3 Glc a1-Gal b1-6 Man b1-3 Glc a1-Gal a1-2 Man b1-4 Glc a1-Gal a1-3 Man b1-4 Glc a1-Gal a1-4 Man b1-4 Glc a1-Gal a1-6 Man b1-4 Glc a1-Gal b1-2 Man b1-4 Glc a1-Gal b1-3 Man b1-4 Glc a1-Gal b1-4 Man b1-4 Glc a1-Gal b1-6 Man b1-4 Glc a1-Gal a1-2 Man b1-6 Glc a1-Gal a1-3 Man b1-6 Glc a1-

Gal a1-4 Man b1-6 Glc a1-Gal a1-6 Man b1-6 Glc a1-Gal b1-2 Man b1-6 Glc a1-Gal b1-3 Man b1-6 Glc a1-Gal b1-4 Man b1-6 Glc a1-Gal b1-6 Man b1-6 Glc a1-Gal a1-2 Man a1-2 Glc b1-Gal a1-3 Man a1-2 Glc b1-Gal a1-4 Man a1-2 Glc b1-Gal a1-6 Man a1-2 Glc b1-Gal b1-2 Man a1-2 Glc b1-Gal b1-3 Man a1-2 Glc b1-Gal b1-4 Man a1-2 Glc b1-Gal b1-6 Man a1-2 Glc b1-Gal a1-2 Man a1-3 Glc b1-Gal a1-3 Man a1-3 Glc b1-Gal a1-4 Man a1-3 Glc b1-Gal a1-6 Man a1-3 Glc b1-Gal b1-2 Man a1-3 Glc b1-Gal b1-3 Man a1-3 Glc b1-Gal b1-4 Man a1-3 Glc b1-Gal b1-6 Man a1-3 Glc b1-Gal a1-2 Man a1-4 Glc b1-Gal a1-3 Man a1-4 Glc b1-Gal a1-4 Man a1-4 Glc b1-Gal a1-6 Man a1-4 Glc b1-Gal b1-2 Man a1-4 Glc b1-Gal b1-3 Man a1-4 Glc b1-Gal b1-4 Man a1-4 Glc b1-Gal b1-6 Man a1-4 Glc b1-Gal a1-2 Man a1-6 Glc b1-Gal a1-3 Man a1-6 Glc b1-Gal a1-4 Man a1-6 Glc b1-Gal a1-6 Man a1-6 Glc b1-Gal b1-2 Man a1-6 Glc b1-Gal b1-3 Man a1-6 Glc b1-Gal b1-4 Man a1-6 Glc b1-Gal b1-6 Man a1-6 Glc b1-Gal a1-2 Man b1-2 Glc b1-Gal a1-3 Man b1-2 Glc b1-Gal a1-4 Man b1-2 Glc b1-Gal a1-6 Man b1-2 Glc b1-Gal b1-2 Man b1-2 Glc b1-Gal b1-3 Man b1-2 Glc b1-Gal b1-4 Man b1-2 Glc b1-Gal b1-6 Man b1-2 Glc b1-Gal a1-2 Man b1-3 Glc b1-Gal a1-3 Man b1-3 Glc b1-Gal a1-4 Man b1-3 Glc b1-Gal a1-6 Man b1-3 Glc b1-Gal b1-2 Man b1-3 Glc b1-Gal b1-3 Man b1-3 Glc b1-Gal b1-4 Man b1-3 Glc b1-Gal b1-6 Man b1-3 Glc b1-Gal a1-2 Man b1-4 Glc b1-Gal a1-3 Man b1-4 Glc b1-Gal a1-4 Man b1-4 Glc b1-Gal a1-6 Man b1-4 Glc b1-Gal b1-2 Man b1-4 Glc b1-Gal b1-3 Man b1-4 Glc b1-Gal b1-4 Man b1-4 Glc b1-Gal b1-6 Man b1-4 Glc b1-Gal a1-2 Man b1-6 Glc b1-Gal a1-3 Man b1-6 Glc b1-Gal a1-4 Man b1-6 Glc b1-Gal a1-6 Man b1-6 Glc b1-Gal b1-2 Man b1-6 Glc b1-Gal b1-3 Man b1-6 Glc b1-Gal b1-4 Man b1-6 Glc b1-Gal b1-6 Man b1-6 Glc b1-Man a1-2 Glc a1-2 Gal a1-Man a1-3 Glc a1-2 Gal a1-Man a1-4 Glc a1-2 Gal a1-Man a1-6 Glc a1-2 Gal a1-Man b1-2 Glc a1-2 Gal a1-Man b1-3 Glc a1-2 Gal a1-Man b1-4 Glc a1-2 Gal a1-Man b1-6 Glc a1-2 Gal a1-Man a1-2 Glc a1-3 Gal a1-Man a1-3 Glc a1-3 Gal a1-Man a1-4 Glc a1-3 Gal a1-Man a1-6 Glc a1-3 Gal a1-Man b1-2 Glc a1-3 Gal a1-Man b1-3 Glc a1-3 Gal a1-Man b1-4 Glc a1-3 Gal a1-Man b1-6 Glc a1-3 Gal a1-Man a1-2 Glc a1-4 Gal a1-Man a1-3 Glc a1-4 Gal a1-Man a1-4 Glc a1-4 Gal a1-Man a1-6 Glc a1-4 Gal a1-Man b1-2 Glc a1-4 Gal a1-Man b1-3 Glc a1-4 Gal a1-Man b1-4 Glc a1-4 Gal a1-

Man b1-6 Glc a1-4 Gal a1-Man a1-2 Glc a1-6 Gal a1-Man a1-3 Glc a1-6 Gal a1-Man a1-4 Glc a1-6 Gal a1-Man a1-6 Glc a1-6 Gal a1-Man b1-2 Glc a1-6 Gal a1-Man b1-3 Glc a1-6 Gal a1-Man b1-4 Glc a1-6 Gal a1-Man b1-6 Glc a1-6 Gal a1-Man a1-2 Glc b1-2 Gal a1-Man a1-3 Glc b1-2 Gal a1-Man a1-4 Glc b1-2 Gal a1-Man a1-6 Glc b1-2 Gal a1-Man b1-2 Glc b1-2 Gal a1-Man b1-3 Glc b1-2 Gal a1-Man b1-4 Glc b1-2 Gal a1-Man b1-6 Glc b1-2 Gal a1-Man a1-2 Glc b1-3 Gal a1-Man a1-3 Glc b1-3 Gal a1-Man a1-4 Glc b1-3 Gal a1-Man a1-6 Glc b1-3 Gal a1-Man b1-2 Glc b1-3 Gal a1-Man b1-3 Glc b1-3 Gal a1-Man b1-4 Glc b1-3 Gal a1-Man b1-6 Glc b1-3 Gal a1-Man a1-2 Glc b1-4 Gal a1-Man a1-3 Glc b1-4 Gal a1-Man a1-4 Glc b1-4 Gal a1-Man a1-6 Glc b1-4 Gal a1-Man b1-2 Glc b1-4 Gal a1-Man b1-3 Glc b1-4 Gal a1-Man b1-4 Glc b1-4 Gal a1-Man b1-6 Glc b1-4 Gal a1-Man a1-2 Glc b1-6 Gal a1-Man a1-3 Glc b1-6 Gal a1-Man a1-4 Glc b1-6 Gal a1-Man a1-6 Glc b1-6 Gal a1-Man b1-2 Glc b1-6 Gal a1-Man b1-3 Glc b1-6 Gal a1-Man b1-4 Glc b1-6 Gal a1-Man b1-6 Glc b1-6 Gal a1-Man a1-2 Glc a1-2 Gal b1-Man a1-3 Glc a1-2 Gal b1-Man a1-4 Glc a1-2 Gal b1-Man a1-6 Glc a1-2 Gal b1-Man b1-2 Glc a1-2 Gal b1-Man b1-3 Glc a1-2 Gal b1-Man b1-4 Glc a1-2 Gal b1-Man b1-6 Glc a1-2 Gal b1-Man a1-2 Glc a1-3 Gal b1-Man a1-3 Glc a1-3 Gal b1-Man a1-4 Glc a1-3 Gal b1-Man a1-6 Glc a1-3 Gal b1-Man b1-2 Glc a1-3 Gal b1-Man b1-3 Glc a1-3 Gal b1-Man b1-4 Glc a1-3 Gal b1-Man b1-6 Glc a1-3 Gal b1-Man a1-2 Glc a1-4 Gal b1-Man a1-3 Glc a1-4 Gal b1-Man a1-4 Glc a1-4 Gal b1-Man a1-6 Glc a1-4 Gal b1-Man b1-2 Glc a1-4 Gal b1-Man b1-3 Glc a1-4 Gal b1-Man b1-4 Glc a1-4 Gal b1-Man b1-6 Glc a1-4 Gal b1-Man a1-2 Glc a1-6 Gal b1-Man a1-3 Glc a1-6 Gal b1-Man a1-4 Glc a1-6 Gal b1-Man a1-6 Glc a1-6 Gal b1-Man b1-2 Glc a1-6 Gal b1-Man b1-3 Glc a1-6 Gal b1-Man b1-4 Glc a1-6 Gal b1-Man b1-6 Glc a1-6 Gal b1-Man a1-2 Glc b1-2 Gal b1-Man a1-3 Glc b1-2 Gal b1-Man a1-4 Glc b1-2 Gal b1-Man a1-6 Glc b1-2 Gal b1-Man b1-2 Glc b1-2 Gal b1-Man b1-3 Glc b1-2 Gal b1-Man b1-4 Glc b1-2 Gal b1-Man b1-6 Glc b1-2 Gal b1-Man a1-2 Glc b1-3 Gal b1-Man a1-3 Glc b1-3 Gal b1-Man a1-4 Glc b1-3 Gal b1-Man a1-6 Glc b1-3 Gal b1-Man b1-2 Glc b1-3 Gal b1-Man b1-3 Glc b1-3 Gal b1-Man b1-4 Glc b1-3 Gal b1-Man b1-6 Glc b1-3 Gal b1-Man a1-2 Glc b1-4 Gal b1-Man a1-3 Glc b1-4 Gal b1-Man a1-4 Glc b1-4 Gal b1-Man a1-6 Glc b1-4 Gal b1-

Man b1-2 Glc b1-4 Gal b1-Man b1-3 Glc b1-4 Gal b1-Man b1-4 Glc b1-4 Gal b1-Man b1-6 Glc b1-4 Gal b1-Man a1-2 Glc b1-6 Gal b1-Man a1-3 Glc b1-6 Gal b1-Man a1-4 Glc b1-6 Gal b1-Man a1-6 Glc b1-6 Gal b1-Man b1-2 Glc b1-6 Gal b1-Man b1-3 Glc b1-6 Gal b1-Man b1-4 Glc b1-6 Gal b1-Man b1-6 Glc b1-6 Gal b1-Man a1-2 Gal a1-2 Glc a1-Man a1-3 Gal a1-2 Glc a1-Man a1-4 Gal a1-2 Glc a1-Man a1-6 Gal a1-2 Glc a1-Man b1-2 Gal a1-2 Glc a1-Man b1-3 Gal a1-2 Glc a1-Man b1-4 Gal a1-2 Glc a1-Man b1-6 Gal a1-2 Glc a1-Man a1-2 Gal a1-3 Glc a1-Man a1-3 Gal a1-3 Glc a1-Man a1-4 Gal a1-3 Glc a1-Man a1-6 Gal a1-3 Glc a1-Man b1-2 Gal a1-3 Glc a1-Man b1-3 Gal a1-3 Glc a1-Man b1-4 Gal a1-3 Glc a1-Man b1-6 Gal a1-3 Glc a1-Man a1-2 Gal a1-4 Glc a1-Man a1-3 Gal a1-4 Glc a1-Man a1-4 Gal a1-4 Glc a1-Man a1-6 Gal a1-4 Glc a1-Man b1-2 Gal a1-4 Glc a1-Man b1-3 Gal a1-4 Glc a1-Man b1-4 Gal a1-4 Glc a1-Man b1-6 Gal a1-4 Glc a1-Man a1-2 Gal a1-6 Glc a1-Man a1-3 Gal a1-6 Glc a1-Man a1-4 Gal a1-6 Glc a1-Man a1-6 Gal a1-6 Glc a1-Man b1-2 Gal a1-6 Glc a1-Man b1-3 Gal a1-6 Glc a1-Man b1-4 Gal a1-6 Glc a1-Man b1-6 Gal a1-6 Glc a1-Man a1-2 Gal b1-2 Glc a1-Man a1-3 Gal b1-2 Glc a1-Man a1-4 Gal b1-2 Glc a1-Man a1-6 Gal b1-2 Glc a1-Man b1-2 Gal b1-2 Glc a1-Man b1-3 Gal b1-2 Glc a1-Man b1-4 Gal b1-2 Glc a1-Man b1-6 Gal b1-2 Glc a1-Man a1-2 Gal b1-3 Glc a1-Man a1-3 Gal b1-3 Glc a1-Man a1-4 Gal b1-3 Glc a1-Man a1-6 Gal b1-3 Glc a1-Man b1-2 Gal b1-3 Glc a1-Man b1-3 Gal b1-3 Glc a1-Man b1-4 Gal b1-3 Glc a1-Man b1-6 Gal b1-3 Glc a1-Man a1-2 Gal b1-4 Glc a1-Man a1-3 Gal b1-4 Glc a1-Man a1-4 Gal b1-4 Glc a1-Man a1-6 Gal b1-4 Glc a1-Man b1-2 Gal b1-4 Glc a1-Man b1-3 Gal b1-4 Glc a1-Man b1-4 Gal b1-4 Glc a1-Man b1-6 Gal b1-4 Glc a1-Man a1-2 Gal b1-6 Glc a1-Man a1-3 Gal b1-6 Glc a1-Man a1-4 Gal b1-6 Glc a1-Man a1-6 Gal b1-6 Glc a1-Man b1-2 Gal b1-6 Glc a1-Man b1-3 Gal b1-6 Glc a1-Man b1-4 Gal b1-6 Glc a1-Man b1-6 Gal b1-6 Glc a1-Man a1-2 Gal a1-2 Glc b1Man a1-3 Gal a1-2 Glc b1Man a1-4 Gal a1-2 Glc b1Man a1-6 Gal a1-2 Glc b1Man b1-2 Gal a1-2 Glc b1Man b1-3 Gal a1-2 Glc b1Man b1-4 Gal a1-2 Glc b1Man b1-6 Gal a1-2 Glc b1Man a1-2 Gal a1-3 Glc b1Man a1-3 Gal a1-3 Glc b1Man a1-4 Gal a1-3 Glc b1Man a1-6 Gal a1-3 Glc b1Man b1-2 Gal a1-3 Glc b1Man b1-3 Gal a1-3 Glc b1Man b1-4 Gal a1-3 Glc b1Man b1-6 Gal a1-3 Glc b1Man a1-2 Gal a1-4 Glc b1

Man a1-3 Gal a1-4 Glc b1Man a1-4 Gal a1-4 Glc b1Man a1-6 Gal a1-4 Glc b1Man b1-2 Gal a1-4 Glc b1Man b1-3 Gal a1-4 Glc b1Man b1-4 Gal a1-4 Glc b1Man b1-6 Gal a1-4 Glc b1Man a1-2 Gal a1-6 Glc b1Man a1-3 Gal a1-6 Glc b1Man a1-4 Gal a1-6 Glc b1Man a1-6 Gal a1-6 Glc b1Man b1-2 Gal a1-6 Glc b1Man b1-3 Gal a1-6 Glc b1Man b1-4 Gal a1-6 Glc b1Man b1-6 Gal a1-6 Glc b1Man a1-2 Gal b1-2 Glc b1Man a1-3 Gal b1-2 Glc b1Man a1-4 Gal b1-2 Glc b1Man a1-6 Gal b1-2 Glc b1Man b1-2 Gal b1-2 Glc b1Man b1-3 Gal b1-2 Glc b1Man b1-4 Gal b1-2 Glc b1Man b1-6 Gal b1-2 Glc b1Man a1-2 Gal b1-3 Glc b1Man a1-3 Gal b1-3 Glc b1Man a1-4 Gal b1-3 Glc b1Man a1-6 Gal b1-3 Glc b1Man b1-2 Gal b1-3 Glc b1Man b1-3 Gal b1-3 Glc b1Man b1-4 Gal b1-3 Glc b1Man b1-6 Gal b1-3 Glc b1Man a1-2 Gal b1-4 Glc b1Man a1-3 Gal b1-4 Glc b1Man a1-4 Gal b1-4 Glc b1Man a1-6 Gal b1-4 Glc b1Man b1-2 Gal b1-4 Glc b1Man b1-3 Gal b1-4 Glc b1Man b1-4 Gal b1-4 Glc b1Man b1-6 Gal b1-4 Glc b1Man a1-2 Gal b1-6 Glc b1Man a1-3 Gal b1-6 Glc b1Man a1-4 Gal b1-6 Glc b1Man a1-6 Gal b1-6 Glc b1Man b1-2 Gal b1-6 Glc b1Man b1-3 Gal b1-6 Glc b1Man b1-4 Gal b1-6 Glc b1Man b1-6 Gal b1-6 Glc b1Glc a1-2 Man a1-2 Gal a1-Glc a1-3 Man a1-2 Gal a1-Glc a1-4 Man a1-2 Gal a1-Glc a1-6 Man a1-2 Gal a1-Glc b1-2 Man a1-2 Gal a1-Glc b1-3 Man a1-2 Gal a1-Glc b1-4 Man a1-2 Gal a1-Glc b1-6 Man a1-2 Gal a1-Glc a1-2 Man a1-3 Gal a1-Glc a1-3 Man a1-3 Gal a1-Glc a1-4 Man a1-3 Gal a1-Glc a1-6 Man a1-3 Gal a1-Glc b1-2 Man a1-3 Gal a1-Glc b1-3 Man a1-3 Gal a1-Glc b1-4 Man a1-3 Gal a1-Glc b1-6 Man a1-3 Gal a1-Glc a1-2 Man a1-4 Gal a1-Glc a1-3 Man a1-4 Gal a1-Glc a1-4 Man a1-4 Gal a1-Glc a1-6 Man a1-4 Gal a1-Glc b1-2 Man a1-4 Gal a1-Glc b1-3 Man a1-4 Gal a1-Glc b1-4 Man a1-4 Gal a1-Glc b1-6 Man a1-4 Gal a1-Glc a1-2 Man a1-6 Gal a1-Glc a1-3 Man a1-6 Gal a1-Glc a1-4 Man a1-6 Gal a1-Glc a1-6 Man a1-6 Gal a1-Glc b1-2 Man a1-6 Gal a1-Glc b1-3 Man a1-6 Gal a1-Glc b1-4 Man a1-6 Gal a1-Glc b1-6 Man a1-6 Gal a1-Glc a1-2 Man b1-2 Gal a1-Glc a1-3 Man b1-2 Gal a1-Glc a1-4 Man b1-2 Gal a1-Glc a1-6 Man b1-2 Gal a1-Glc b1-2 Man b1-2 Gal a1-Glc b1-3 Man b1-2 Gal a1-Glc b1-4 Man b1-2 Gal a1-Glc b1-6 Man b1-2 Gal a1-Glc a1-2 Man b1-3 Gal a1-Glc a1-3 Man b1-3 Gal a1-Glc a1-4 Man b1-3 Gal a1-Glc a1-6 Man b1-3 Gal a1-Glc b1-2 Man b1-3 Gal a1-Glc b1-3 Man b1-3 Gal a1-

Glc b1-4 Man b1-3 Gal a1-Glc b1-6 Man b1-3 Gal a1-Glc a1-2 Man b1-4 Gal a1-Glc a1-3 Man b1-4 Gal a1-Glc a1-4 Man b1-4 Gal a1-Glc a1-6 Man b1-4 Gal a1-Glc b1-2 Man b1-4 Gal a1-Glc b1-3 Man b1-4 Gal a1-Glc b1-4 Man b1-4 Gal a1-Glc b1-6 Man b1-4 Gal a1-Glc a1-2 Man b1-6 Gal a1-Glc a1-3 Man b1-6 Gal a1-Glc a1-4 Man b1-6 Gal a1-Glc a1-6 Man b1-6 Gal a1-Glc b1-2 Man b1-6 Gal a1-Glc b1-3 Man b1-6 Gal a1-Glc b1-4 Man b1-6 Gal a1-Glc b1-6 Man b1-6 Gal a1-Glc a1-2 Man a1-2 Gal b1-Glc a1-3 Man a1-2 Gal b1-Glc a1-4 Man a1-2 Gal b1-Glc a1-6 Man a1-2 Gal b1-Glc b1-2 Man a1-2 Gal b1-Glc b1-3 Man a1-2 Gal b1-Glc b1-4 Man a1-2 Gal b1-Glc b1-6 Man a1-2 Gal b1-Glc a1-2 Man a1-3 Gal b1-Glc a1-3 Man a1-3 Gal b1-Glc a1-4 Man a1-3 Gal b1-Glc a1-6 Man a1-3 Gal b1-Glc b1-2 Man a1-3 Gal b1-Glc b1-3 Man a1-3 Gal b1-Glc b1-4 Man a1-3 Gal b1-Glc b1-6 Man a1-3 Gal b1-Glc a1-2 Man a1-4 Gal b1-Glc a1-3 Man a1-4 Gal b1-Glc a1-4 Man a1-4 Gal b1-Glc a1-6 Man a1-4 Gal b1-Glc b1-2 Man a1-4 Gal b1-Glc b1-3 Man a1-4 Gal b1-Glc b1-4 Man a1-4 Gal b1-Glc b1-6 Man a1-4 Gal b1-Glc a1-2 Man a1-6 Gal b1-Glc a1-3 Man a1-6 Gal b1-Glc a1-4 Man a1-6 Gal b1-Glc a1-6 Man a1-6 Gal b1-Glc b1-2 Man a1-6 Gal b1-Glc b1-3 Man a1-6 Gal b1-Glc b1-4 Man a1-6 Gal b1-Glc b1-6 Man a1-6 Gal b1-Glc a1-2 Man b1-2 Gal b1-Glc a1-3 Man b1-2 Gal b1-Glc a1-4 Man b1-2 Gal b1-Glc a1-6 Man b1-2 Gal b1-Glc b1-2 Man b1-2 Gal b1-Glc b1-3 Man b1-2 Gal b1-Glc b1-4 Man b1-2 Gal b1-Glc b1-6 Man b1-2 Gal b1-Glc a1-2 Man b1-3 Gal b1-Glc a1-3 Man b1-3 Gal b1-Glc a1-4 Man b1-3 Gal b1-Glc a1-6 Man b1-3 Gal b1-Glc b1-2 Man b1-3 Gal b1-Glc b1-3 Man b1-3 Gal b1-Glc b1-4 Man b1-3 Gal b1-Glc b1-6 Man b1-3 Gal b1-Glc a1-2 Man b1-4 Gal b1-Glc a1-3 Man b1-4 Gal b1-Glc a1-4 Man b1-4 Gal b1-Glc a1-6 Man b1-4 Gal b1-Glc b1-2 Man b1-4 Gal b1-Glc b1-3 Man b1-4 Gal b1-Glc b1-4 Man b1-4 Gal b1-Glc b1-6 Man b1-4 Gal b1-Glc a1-2 Man b1-6 Gal b1-Glc a1-3 Man b1-6 Gal b1-Glc a1-4 Man b1-6 Gal b1-Glc a1-6 Man b1-6 Gal b1-Glc b1-2 Man b1-6 Gal b1-Glc b1-3 Man b1-6 Gal b1-Glc b1-4 Man b1-6 Gal b1-Glc b1-6 Man b1-6 Gal b1-Glc a1-2 Gal a1-2 Man a1-Glc a1-3 Gal a1-2 Man a1-Glc a1-4 Gal a1-2 Man a1-Glc a1-6 Gal a1-2 Man a1-Glc b1-2 Gal a1-2 Man a1-Glc b1-3 Gal a1-2 Man a1-Glc b1-4 Gal a1-2 Man a1-Glc b1-6 Gal a1-2 Man a1-Glc a1-2 Gal a1-3 Man a1-Glc a1-3 Gal a1-3 Man a1-Glc a1-4 Gal a1-3 Man a1-

Glc a1-6 Gal a1-3 Man a1-Glc b1-2 Gal a1-3 Man a1-Glc b1-3 Gal a1-3 Man a1-Glc b1-4 Gal a1-3 Man a1-Glc b1-6 Gal a1-3 Man a1-Glc a1-2 Gal a1-4 Man a1-Glc a1-3 Gal a1-4 Man a1-Glc a1-4 Gal a1-4 Man a1-Glc a1-6 Gal a1-4 Man a1-Glc b1-2 Gal a1-4 Man a1-Glc b1-3 Gal a1-4 Man a1-Glc b1-4 Gal a1-4 Man a1-Glc b1-6 Gal a1-4 Man a1-Glc a1-2 Gal a1-6 Man a1-Glc a1-3 Gal a1-6 Man a1-Glc a1-4 Gal a1-6 Man a1-Glc a1-6 Gal a1-6 Man a1-Glc b1-2 Gal a1-6 Man a1-Glc b1-3 Gal a1-6 Man a1-Glc b1-4 Gal a1-6 Man a1-Glc b1-6 Gal a1-6 Man a1-Glc a1-2 Gal b1-2 Man a1-Glc a1-3 Gal b1-2 Man a1-Glc a1-4 Gal b1-2 Man a1-Glc a1-6 Gal b1-2 Man a1-Glc b1-2 Gal b1-2 Man a1-Glc b1-3 Gal b1-2 Man a1-Glc b1-4 Gal b1-2 Man a1-Glc b1-6 Gal b1-2 Man a1-Glc a1-2 Gal b1-3 Man a1-Glc a1-3 Gal b1-3 Man a1-Glc a1-4 Gal b1-3 Man a1-Glc a1-6 Gal b1-3 Man a1-Glc b1-2 Gal b1-3 Man a1-Glc b1-3 Gal b1-3 Man a1-Glc b1-4 Gal b1-3 Man a1-Glc b1-6 Gal b1-3 Man a1-Glc a1-2 Gal b1-4 Man a1-Glc a1-3 Gal b1-4 Man a1-Glc a1-4 Gal b1-4 Man a1-Glc a1-6 Gal b1-4 Man a1-Glc b1-2 Gal b1-4 Man a1-Glc b1-3 Gal b1-4 Man a1-Glc b1-4 Gal b1-4 Man a1-Glc b1-6 Gal b1-4 Man a1-Glc a1-2 Gal b1-6 Man a1-Glc a1-3 Gal b1-6 Man a1-Glc a1-4 Gal b1-6 Man a1-Glc a1-6 Gal b1-6 Man a1-Glc b1-2 Gal b1-6 Man a1-Glc b1-3 Gal b1-6 Man a1-Glc b1-4 Gal b1-6 Man a1-Glc b1-6 Gal b1-6 Man a1-Glc a1-2 Gal a1-2 Man b1-Glc a1-3 Gal a1-2 Man b1-Glc a1-4 Gal a1-2 Man b1-Glc a1-6 Gal a1-2 Man b1-Glc b1-2 Gal a1-2 Man b1-Glc b1-3 Gal a1-2 Man b1-Glc b1-4 Gal a1-2 Man b1-Glc b1-6 Gal a1-2 Man b1-Glc a1-2 Gal a1-3 Man b1-Glc a1-3 Gal a1-3 Man b1-Glc a1-4 Gal a1-3 Man b1-Glc a1-6 Gal a1-3 Man b1-Glc b1-2 Gal a1-3 Man b1-Glc b1-3 Gal a1-3 Man b1-Glc b1-4 Gal a1-3 Man b1-Glc b1-6 Gal a1-3 Man b1-Glc a1-2 Gal a1-4 Man b1-Glc a1-3 Gal a1-4 Man b1-Glc a1-4 Gal a1-4 Man b1-Glc a1-6 Gal a1-4 Man b1-Glc b1-2 Gal a1-4 Man b1-Glc b1-3 Gal a1-4 Man b1-Glc b1-4 Gal a1-4 Man b1-Glc b1-6 Gal a1-4 Man b1-Glc a1-2 Gal a1-6 Man b1-Glc a1-3 Gal a1-6 Man b1-Glc a1-4 Gal a1-6 Man b1-Glc a1-6 Gal a1-6 Man b1-Glc b1-2 Gal a1-6 Man b1-Glc b1-3 Gal a1-6 Man b1-Glc b1-4 Gal a1-6 Man b1-Glc b1-6 Gal a1-6 Man b1-Glc a1-2 Gal b1-2 Man b1-Glc a1-3 Gal b1-2 Man b1-Glc a1-4 Gal b1-2 Man b1-Glc a1-6 Gal b1-2 Man b1-Glc b1-2 Gal b1-2 Man b1-Glc b1-3 Gal b1-2 Man b1-Glc b1-4 Gal b1-2 Man b1-Glc b1-6 Gal b1-2 Man b1-

Glc a1-2 Gal b1-3 Man b1-Glc a1-3 Gal b1-3 Man b1-Glc a1-4 Gal b1-3 Man b1-Glc a1-6 Gal b1-3 Man b1-Glc b1-2 Gal b1-3 Man b1-Glc b1-3 Gal b1-3 Man b1-Glc b1-4 Gal b1-3 Man b1-Glc b1-6 Gal b1-3 Man b1-Glc a1-2 Gal b1-4 Man b1-Glc a1-3 Gal b1-4 Man b1-Glc a1-4 Gal b1-4 Man b1-Glc a1-6 Gal b1-4 Man b1-Glc b1-2 Gal b1-4 Man b1-Glc b1-3 Gal b1-4 Man b1-Glc b1-4 Gal b1-4 Man b1-Glc b1-6 Gal b1-4 Man b1-Glc a1-2 Gal b1-6 Man b1-Glc a1-3 Gal b1-6 Man b1-Glc a1-4 Gal b1-6 Man b1-Glc a1-6 Gal b1-6 Man b1-Glc b1-2 Gal b1-6 Man b1-Glc b1-3 Gal b1-6 Man b1-Glc b1-4 Gal b1-6 Man b1-Glc b1-6 Gal b1-6 Man b1-Gal a1-2 (Glc a1-3) Man a1-Gal a1-2 (Glc a1-4) Man a1-Gal a1-2 (Glc a1-6) Man a1-Gal a1-3 (Glc a1-2) Man a1-Gal a1-3 (Glc a1-4) Man a1-Gal a1-3 (Glc a1-6) Man a1-Gal a1-4 (Glc a1-2) Man a1-Gal a1-4 (Glc a1-3) Man a1-Gal a1-4 (Glc a1-6) Man a1-Gal a1-6 (Glc a1-2) Man a1-Gal a1-6 (Glc a1-3) Man a1-Gal a1-6 (Glc a1-4) Man a1-Gal b1-2 (Glc a1-3) Man a1-Gal b1-2 (Glc a1-4) Man a1-Gal b1-2 (Glc a1-6) Man a1-Gal b1-3 (Glc a1-2) Man a1-Gal b1-3 (Glc a1-4) Man a1-Gal b1-3 (Glc a1-6) Man a1-Gal b1-4 (Glc a1-2) Man a1-Gal b1-4 (Glc a1-3) Man a1-Gal b1-4 (Glc a1-6) Man a1-Gal b1-6 (Glc a1-2) Man a1-Gal b1-6 (Glc a1-3) Man a1-Gal b1-6 (Glc a1-4) Man a1-Gal a1-2 (Glc b1-3) Man a1-Gal a1-2 (Glc b1-4) Man a1-Gal a1-2 (Glc b1-6) Man a1-Gal a1-3 (Glc b1-2) Man a1-Gal a1-3 (Glc b1-4) Man a1-Gal a1-3 (Glc b1-6) Man a1-Gal a1-4 (Glc b1-2) Man a1-Gal a1-4 (Glc b1-3) Man a1-Gal a1-4 (Glc b1-6) Man a1-Gal a1-6 (Glc b1-2) Man a1-Gal a1-6 (Glc b1-3) Man a1-Gal a1-6 (Glc b1-4) Man a1-Gal b1-2 (Glc b1-3) Man a1-Gal b1-2 (Glc b1-4) Man a1-Gal b1-2 (Glc b1-6) Man a1-Gal b1-3 (Glc b1-2) Man a1-Gal b1-3 (Glc b1-4) Man a1-Gal b1-3 (Glc b1-6) Man a1-Gal b1-4 (Glc b1-2) Man a1-Gal b1-4 (Glc b1-3) Man a1-Gal b1-4 (Glc b1-6) Man a1-Gal b1-6 (Glc b1-2) Man a1-Gal b1-6 (Glc b1-3) Man a1-Gal b1-6 (Glc b1-4) Man a1-Gal a1-2 (Glc a1-3) Man b1-Gal a1-2 (Glc a1-4) Man b1-Gal a1-2 (Glc a1-6) Man b1-Gal a1-3 (Glc a1-2) Man b1-Gal a1-3 (Glc a1-4) Man b1-Gal a1-3 (Glc a1-6) Man b1-Gal a1-4 (Glc a1-2) Man b1-Gal a1-4 (Glc a1-3) Man b1-Gal a1-4 (Glc a1-6) Man b1-Gal a1-6 (Glc a1-2) Man b1-Gal a1-6 (Glc a1-3) Man b1-Gal a1-6 (Glc a1-4) Man b1-Gal b1-2 (Glc a1-3) Man b1-Gal b1-2 (Glc a1-4) Man b1-Gal b1-2 (Glc a1-6) Man b1-Gal b1-3 (Glc a1-2) Man b1-Gal b1-3 (Glc a1-4) Man b1-Gal b1-3 (Glc a1-6) Man b1-Gal b1-4 (Glc a1-2) Man b1-Gal b1-4 (Glc a1-3) Man b1-Gal b1-4 (Glc a1-6) Man b1-

Gal b1-6 (Glc a1-2) Man b1-Gal b1-6 (Glc a1-3) Man b1-Gal b1-6 (Glc a1-4) Man b1-Gal a1-2 (Glc b1-3) Man b1-Gal a1-2 (Glc b1-4) Man b1-Gal a1-2 (Glc b1-6) Man b1-Gal a1-3 (Glc b1-2) Man b1-Gal a1-3 (Glc b1-4) Man b1-Gal a1-3 (Glc b1-6) Man b1-Gal a1-4 (Glc b1-2) Man b1-Gal a1-4 (Glc b1-3) Man b1-Gal a1-4 (Glc b1-6) Man b1-Gal a1-6 (Glc b1-2) Man b1-Gal a1-6 (Glc b1-3) Man b1-Gal a1-6 (Glc b1-4) Man b1-Gal b1-2 (Glc b1-3) Man b1-Gal b1-2 (Glc b1-4) Man b1-Gal b1-2 (Glc b1-6) Man b1-Gal b1-3 (Glc b1-2) Man b1-Gal b1-3 (Glc b1-4) Man b1-Gal b1-3 (Glc b1-6) Man b1-Gal b1-4 (Glc b1-2) Man b1-Gal b1-4 (Glc b1-3) Man b1-Gal b1-4 (Glc b1-6) Man b1-Gal b1-6 (Glc b1-2) Man b1-Gal b1-6 (Glc b1-3) Man b1-Gal b1-6 (Glc b1-4) Man b1-Gal a1-2 (Man a1-3) Glc a1-Gal a1-2 (Man a1-4) Glc a1-Gal a1-2 (Man a1-6) Glc a1-Gal a1-3 (Man a1-2) Glc a1-Gal a1-3 (Man a1-4) Glc a1-Gal a1-3 (Man a1-6) Glc a1-Gal a1-4 (Man a1-2) Glc a1-Gal a1-4 (Man a1-3) Glc a1-Gal a1-4 (Man a1-6) Glc a1-Gal a1-6 (Man a1-2) Glc a1-Gal a1-6 (Man a1-3) Glc a1-Gal a1-6 (Man a1-4) Glc a1-Gal b1-2 (Man a1-3) Glc a1-Gal b1-2 (Man a1-4) Glc a1-Gal b1-2 (Man a1-6) Glc a1-Gal b1-3 (Man a1-2) Glc a1-Gal b1-3 (Man a1-4) Glc a1-Gal b1-3 (Man a1-6) Glc a1-Gal b1-4 (Man a1-2) Glc a1-Gal b1-4 (Man a1-3) Glc a1-Gal b1-4 (Man a1-6) Glc a1-Gal b1-6 (Man a1-2) Glc a1-Gal b1-6 (Man a1-3) Glc a1-Gal b1-6 (Man a1-4) Glc a1-Gal a1-2 (Man b1-3) Glc a1-Gal a1-2 (Man b1-4) Glc a1-Gal a1-2 (Man b1-6) Glc a1-Gal a1-3 (Man b1-2) Glc a1-Gal a1-3 (Man b1-4) Glc a1-Gal a1-3 (Man b1-6) Glc a1-Gal a1-4 (Man b1-2) Glc a1-Gal a1-4 (Man b1-3) Glc a1-Gal a1-4 (Man b1-6) Glc a1-Gal a1-6 (Man b1-2) Glc a1-Gal a1-6 (Man b1-3) Glc a1-Gal a1-6 (Man b1-4) Glc a1-Gal b1-2 (Man b1-3) Glc a1-Gal b1-2 (Man b1-4) Glc a1-Gal b1-2 (Man b1-6) Glc a1-Gal b1-3 (Man b1-2) Glc a1-Gal b1-3 (Man b1-4) Glc a1-Gal b1-3 (Man b1-6) Glc a1-Gal b1-4 (Man b1-2) Glc a1-Gal b1-4 (Man b1-3) Glc a1-Gal b1-4 (Man b1-6) Glc a1-Gal b1-6 (Man b1-2) Glc a1-Gal b1-6 (Man b1-3) Glc a1-Gal b1-6 (Man b1-4) Glc a1-Gal a1-2 (Man a1-3) Glc b1-Gal a1-2 (Man a1-4) Glc b1-Gal a1-2 (Man a1-6) Glc b1-Gal a1-3 (Man a1-2) Glc b1-Gal a1-3 (Man a1-4) Glc b1-Gal a1-3 (Man a1-6) Glc b1-

Gal a1-4 (Man a1-2) Glc b1-Gal a1-4 (Man a1-3) Glc b1-Gal a1-4 (Man a1-6) Glc b1-Gal a1-6 (Man a1-2) Glc b1-Gal a1-6 (Man a1-3) Glc b1-Gal a1-6 (Man a1-4) Glc b1-Gal b1-2 (Man a1-3) Glc b1-Gal b1-2 (Man a1-4) Glc b1-Gal b1-2 (Man a1-6) Glc b1-Gal b1-3 (Man a1-2) Glc b1-Gal b1-3 (Man a1-4) Glc b1-Gal b1-3 (Man a1-6) Glc b1-Gal b1-4 (Man a1-2) Glc b1-Gal b1-4 (Man a1-3) Glc b1-Gal b1-4 (Man a1-6) Glc b1-Gal b1-6 (Man a1-2) Glc b1-Gal b1-6 (Man a1-3) Glc b1-Gal b1-6 (Man a1-4) Glc b1-Gal a1-2 (Man b1-3) Glc b1-Gal a1-2 (Man b1-4) Glc b1-Gal a1-2 (Man b1-6) Glc b1-Gal a1-3 (Man b1-2) Glc b1-Gal a1-3 (Man b1-4) Glc b1-Gal a1-3 (Man b1-6) Glc b1-Gal a1-4 (Man b1-2) Glc b1-Gal a1-4 (Man b1-3) Glc b1-Gal a1-4 (Man b1-6) Glc b1-Gal a1-6 (Man b1-2) Glc b1-Gal a1-6 (Man b1-3) Glc b1-Gal a1-6 (Man b1-4) Glc b1-Gal b1-2 (Man b1-3) Glc b1-Gal b1-2 (Man b1-4) Glc b1-Gal b1-2 (Man b1-6) Glc b1-Gal b1-3 (Man b1-2) Glc b1-Gal b1-3 (Man b1-4) Glc b1-Gal b1-3 (Man b1-6) Glc b1-Gal b1-4 (Man b1-2) Glc b1-Gal b1-4 (Man b1-3) Glc b1-Gal b1-4 (Man b1-6) Glc b1-Gal b1-6 (Man b1-2) Glc b1-Gal b1-6 (Man b1-3) Glc b1-Gal b1-6 (Man b1-4) Glc b1-Glc a1-2 (Man a1-3) Gal a1-Glc a1-2 (Man a1-4) Gal a1-Glc a1-2 (Man a1-6) Gal a1-Glc a1-3 (Man a1-2) Gal a1-Glc a1-3 (Man a1-4) Gal a1-Glc a1-3 (Man a1-6) Gal a1-Glc a1-4 (Man a1-2) Gal a1-Glc a1-4 (Man a1-3) Gal a1-Glc a1-4 (Man a1-6) Gal a1-Glc a1-6 (Man a1-2) Gal a1-Glc a1-6 (Man a1-3) Gal a1-Glc a1-6 (Man a1-4) Gal a1-Glc b1-2 (Man a1-3) Gal a1-Glc b1-2 (Man a1-4) Gal a1-Glc b1-2 (Man a1-6) Gal a1-Glc b1-3 (Man a1-2) Gal a1-Glc b1-3 (Man a1-4) Gal a1-Glc b1-3 (Man a1-6) Gal a1-Glc b1-4 (Man a1-2) Gal a1-Glc b1-4 (Man a1-3) Gal a1-Glc b1-4 (Man a1-6) Gal a1-Glc b1-6 (Man a1-2) Gal a1-Glc b1-6 (Man a1-3) Gal a1-Glc b1-6 (Man a1-4) Gal a1-Glc a1-2 (Man b1-3) Gal a1-Glc a1-2 (Man b1-4) Gal a1-Glc a1-2 (Man b1-6) Gal a1-Glc a1-3 (Man b1-2) Gal a1-Glc a1-3 (Man b1-4) Gal a1-Glc a1-3 (Man b1-6) Gal a1-Glc a1-4 (Man b1-2) Gal a1-Glc a1-4 (Man b1-3) Gal a1-Glc a1-4 (Man b1-6) Gal a1-Glc a1-6 (Man b1-2) Gal a1-Glc a1-6 (Man b1-3) Gal a1-Glc a1-6 (Man b1-4) Gal a1-Glc b1-2 (Man b1-3) Gal a1-Glc b1-2 (Man b1-4) Gal a1-Glc b1-2 (Man b1-6) Gal a1-

Glc b1-3 (Man b1-2) Gal a1-Glc b1-3 (Man b1-4) Gal a1-Glc b1-3 (Man b1-6) Gal a1-Glc b1-4 (Man b1-2) Gal a1-Glc b1-4 (Man b1-3) Gal a1-Glc b1-4 (Man b1-6) Gal a1-Glc b1-6 (Man b1-2) Gal a1-Glc b1-6 (Man b1-3) Gal a1-Glc b1-6 (Man b1-4) Gal a1-Glc a1-2 (Man a1-3) Gal b1-Glc a1-2 (Man a1-4) Gal b1-Glc a1-2 (Man a1-6) Gal b1-Glc a1-3 (Man a1-2) Gal b1-Glc a1-3 (Man a1-4) Gal b1-Glc a1-3 (Man a1-6) Gal b1-Glc a1-4 (Man a1-2) Gal b1-Glc a1-4 (Man a1-3) Gal b1-Glc a1-4 (Man a1-6) Gal b1-Glc a1-6 (Man a1-2) Gal b1-Glc a1-6 (Man a1-3) Gal b1-Glc a1-6 (Man a1-4) Gal b1-Glc b1-2 (Man a1-3) Gal b1-Glc b1-2 (Man a1-4) Gal b1-Glc b1-2 (Man a1-6) Gal b1-Glc b1-3 (Man a1-2) Gal b1-Glc b1-3 (Man a1-4) Gal b1-Glc b1-3 (Man a1-6) Gal b1-Glc b1-4 (Man a1-2) Gal b1-Glc b1-4 (Man a1-3) Gal b1-Glc b1-4 (Man a1-6) Gal b1-Glc b1-6 (Man a1-2) Gal b1-Glc b1-6 (Man a1-3) Gal b1-Glc b1-6 (Man a1-4) Gal b1-Glc a1-2 (Man b1-3) Gal b1-Glc a1-2 (Man b1-4) Gal b1-Glc a1-2 (Man b1-6) Gal b1-Glc a1-3 (Man b1-2) Gal b1-Glc a1-3 (Man b1-4) Gal b1-Glc a1-3 (Man b1-6) Gal b1-Glc a1-4 (Man b1-2) Gal b1-Glc a1-4 (Man b1-3) Gal b1-Glc a1-4 (Man b1-6) Gal b1-Glc a1-6 (Man b1-2) Gal b1-Glc a1-6 (Man b1-3) Gal b1-Glc a1-6 (Man b1-4) Gal b1-Glc b1-2 (Man b1-3) Gal b1-Glc b1-2 (Man b1-4) Gal b1-Glc b1-2 (Man b1-6) Gal b1-Glc b1-3 (Man b1-2) Gal b1-Glc b1-3 (Man b1-4) Gal b1-Glc b1-3 (Man b1-6) Gal b1-Glc b1-4 (Man b1-2) Gal b1-Glc b1-4 (Man b1-3) Gal b1-Glc b1-4 (Man b1-6) Gal b1-Glc b1-6 (Man b1-2) Gal b1-Glc b1-6 (Man b1-3) Gal b1-Glc b1-6 (Man b1-4) Gal b1-

Man a1-4 Gal a1-3 Glc b1Man a1-6 Gal a1-3 Glc b1Man b1-2 Gal a1-3 Glc b1Man b1-3 Gal a1-3 Glc b1Man b1-4 Gal a1-3 Glc b1Man b1-6 Gal a1-3 Glc b1Man a1-2 Gal a1-4 Glc b1Man a1-3 Gal a1-4 Glc b1Man a1-4 Gal a1-4 Glc b1Man a1-6 Gal a1-4 Glc b1Man b1-2 Gal a1-4 Glc b1Man b1-3 Gal a1-4 Glc b1Man b1-4 Gal a1-4 Glc b1Man b1-6 Gal a1-4 Glc b1Man a1-2 Gal a1-6 Glc b1Man a1-3 Gal a1-6 Glc b1

Cummings RD (2009) Molecular BioSystems 5 1087

Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical petasaccharide sequeces in glycosaminoglycans

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoN-linkedrdquo glycan linkage to protein asparagine residues

Families of N-linked glycan structures

Man

4GlcNAc1

2Man

Man

Man

Man

2Man

2Man

2Man

63

Man63 4GlcNAc1 Asn

4GlcNAc1

Man62

Man63 4GlcNAc1 Asn

Man42

4GlcNAc1

4GlcNAc1

4GlcNAc1

4GlcNAc1

6Gal

6Gal

6Gal

6Gal

NeuAc

NeuAc

NeuAcNeuAc

4GlcNAc1

Man

Man63 4GlcNAc1 Asn

2Man4GlcNAc13GalNeuAc

Man

Man

63

core

core

core

High Mannose

Complex

Hybrid

Examples of structures found as termini on the branches of complex N-linked glycoprotein glycanshellip

Terminal Saccharide Diversity --- A key to recognition

NeuAc 2-6 Gal 1-4 GlcNAc---NeuAc 2-3 Gal 1-4 GlcNAc---

Gal 1-3 Gal 1-4 GlcNAc---4-SO3-GalNAc 1-4 GlcNAc---

(-Gal 1-4 GlcNAc 1-3-)n = polylactosamine repeats (n may be gt6)

(-NeuAc 2-8-) n = polysialic acid (n may be gt50)

Fuc residues (eg Fuc 1-2 Gal Fuc 1-3 GlcNAc Fuc 1-4 GlcNAc

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoO-linkedrdquo glycan linkage to protein serine residues

Examples of variations of O-linked glycans

GalNAc Serine

GalNAc Serine

NeuAc 26

GalNAc Serine

Gal 1

NeuAc 263

GalNAc SerineNeuAc 2-3 Gal 1

NeuAc 263

Gal 1-4 GlcNAc 1

Fuc 1

GalNAc Serine

GlcNAc 1

63

4NeuAc 2-3 Gal 1

3

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoN-linkedrdquo glycan linkage to protein asparagine residues

Families of N-linked glycan structures

Man

4GlcNAc1

2Man

Man

Man

Man

2Man

2Man

2Man

63

Man63 4GlcNAc1 Asn

4GlcNAc1

Man62

Man63 4GlcNAc1 Asn

Man42

4GlcNAc1

4GlcNAc1

4GlcNAc1

4GlcNAc1

6Gal

6Gal

6Gal

6Gal

NeuAc

NeuAc

NeuAcNeuAc

4GlcNAc1

Man

Man63 4GlcNAc1 Asn

2Man4GlcNAc13GalNeuAc

Man

Man

63

core

core

core

High Mannose

Complex

Hybrid

Examples of structures found as termini on the branches of complex N-linked glycoprotein glycanshellip

Terminal Saccharide Diversity --- A key to recognition

NeuAc 2-6 Gal 1-4 GlcNAc---NeuAc 2-3 Gal 1-4 GlcNAc---

Gal 1-3 Gal 1-4 GlcNAc---4-SO3-GalNAc 1-4 GlcNAc---

(-Gal 1-4 GlcNAc 1-3-)n = polylactosamine repeats (n may be gt6)

(-NeuAc 2-8-) n = polysialic acid (n may be gt50)

Fuc residues (eg Fuc 1-2 Gal Fuc 1-3 GlcNAc Fuc 1-4 GlcNAc

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoO-linkedrdquo glycan linkage to protein serine residues

Examples of variations of O-linked glycans

GalNAc Serine

GalNAc Serine

NeuAc 26

GalNAc Serine

Gal 1

NeuAc 263

GalNAc SerineNeuAc 2-3 Gal 1

NeuAc 263

Gal 1-4 GlcNAc 1

Fuc 1

GalNAc Serine

GlcNAc 1

63

4NeuAc 2-3 Gal 1

3

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Saccharides

bull Carbohydrates The language of glycobiologybull The glycosidic bond and glycan nomenclaturebull Major vertebrate glycan classes

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoN-linkedrdquo glycan linkage to protein asparagine residues

Families of N-linked glycan structures

Man

4GlcNAc1

2Man

Man

Man

Man

2Man

2Man

2Man

63

Man63 4GlcNAc1 Asn

4GlcNAc1

Man62

Man63 4GlcNAc1 Asn

Man42

4GlcNAc1

4GlcNAc1

4GlcNAc1

4GlcNAc1

6Gal

6Gal

6Gal

6Gal

NeuAc

NeuAc

NeuAcNeuAc

4GlcNAc1

Man

Man63 4GlcNAc1 Asn

2Man4GlcNAc13GalNeuAc

Man

Man

63

core

core

core

High Mannose

Complex

Hybrid

Examples of structures found as termini on the branches of complex N-linked glycoprotein glycanshellip

Terminal Saccharide Diversity --- A key to recognition

NeuAc 2-6 Gal 1-4 GlcNAc---NeuAc 2-3 Gal 1-4 GlcNAc---

Gal 1-3 Gal 1-4 GlcNAc---4-SO3-GalNAc 1-4 GlcNAc---

(-Gal 1-4 GlcNAc 1-3-)n = polylactosamine repeats (n may be gt6)

(-NeuAc 2-8-) n = polysialic acid (n may be gt50)

Fuc residues (eg Fuc 1-2 Gal Fuc 1-3 GlcNAc Fuc 1-4 GlcNAc

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoO-linkedrdquo glycan linkage to protein serine residues

Examples of variations of O-linked glycans

GalNAc Serine

GalNAc Serine

NeuAc 26

GalNAc Serine

Gal 1

NeuAc 263

GalNAc SerineNeuAc 2-3 Gal 1

NeuAc 263

Gal 1-4 GlcNAc 1

Fuc 1

GalNAc Serine

GlcNAc 1

63

4NeuAc 2-3 Gal 1

3

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoN-linkedrdquo glycan linkage to protein asparagine residues

Families of N-linked glycan structures

Man

4GlcNAc1

2Man

Man

Man

Man

2Man

2Man

2Man

63

Man63 4GlcNAc1 Asn

4GlcNAc1

Man62

Man63 4GlcNAc1 Asn

Man42

4GlcNAc1

4GlcNAc1

4GlcNAc1

4GlcNAc1

6Gal

6Gal

6Gal

6Gal

NeuAc

NeuAc

NeuAcNeuAc

4GlcNAc1

Man

Man63 4GlcNAc1 Asn

2Man4GlcNAc13GalNeuAc

Man

Man

63

core

core

core

High Mannose

Complex

Hybrid

Examples of structures found as termini on the branches of complex N-linked glycoprotein glycanshellip

Terminal Saccharide Diversity --- A key to recognition

NeuAc 2-6 Gal 1-4 GlcNAc---NeuAc 2-3 Gal 1-4 GlcNAc---

Gal 1-3 Gal 1-4 GlcNAc---4-SO3-GalNAc 1-4 GlcNAc---

(-Gal 1-4 GlcNAc 1-3-)n = polylactosamine repeats (n may be gt6)

(-NeuAc 2-8-) n = polysialic acid (n may be gt50)

Fuc residues (eg Fuc 1-2 Gal Fuc 1-3 GlcNAc Fuc 1-4 GlcNAc

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoO-linkedrdquo glycan linkage to protein serine residues

Examples of variations of O-linked glycans

GalNAc Serine

GalNAc Serine

NeuAc 26

GalNAc Serine

Gal 1

NeuAc 263

GalNAc SerineNeuAc 2-3 Gal 1

NeuAc 263

Gal 1-4 GlcNAc 1

Fuc 1

GalNAc Serine

GlcNAc 1

63

4NeuAc 2-3 Gal 1

3

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoN-linkedrdquo glycan linkage to protein asparagine residues

Families of N-linked glycan structures

Man

4GlcNAc1

2Man

Man

Man

Man

2Man

2Man

2Man

63

Man63 4GlcNAc1 Asn

4GlcNAc1

Man62

Man63 4GlcNAc1 Asn

Man42

4GlcNAc1

4GlcNAc1

4GlcNAc1

4GlcNAc1

6Gal

6Gal

6Gal

6Gal

NeuAc

NeuAc

NeuAcNeuAc

4GlcNAc1

Man

Man63 4GlcNAc1 Asn

2Man4GlcNAc13GalNeuAc

Man

Man

63

core

core

core

High Mannose

Complex

Hybrid

Examples of structures found as termini on the branches of complex N-linked glycoprotein glycanshellip

Terminal Saccharide Diversity --- A key to recognition

NeuAc 2-6 Gal 1-4 GlcNAc---NeuAc 2-3 Gal 1-4 GlcNAc---

Gal 1-3 Gal 1-4 GlcNAc---4-SO3-GalNAc 1-4 GlcNAc---

(-Gal 1-4 GlcNAc 1-3-)n = polylactosamine repeats (n may be gt6)

(-NeuAc 2-8-) n = polysialic acid (n may be gt50)

Fuc residues (eg Fuc 1-2 Gal Fuc 1-3 GlcNAc Fuc 1-4 GlcNAc

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoO-linkedrdquo glycan linkage to protein serine residues

Examples of variations of O-linked glycans

GalNAc Serine

GalNAc Serine

NeuAc 26

GalNAc Serine

Gal 1

NeuAc 263

GalNAc SerineNeuAc 2-3 Gal 1

NeuAc 263

Gal 1-4 GlcNAc 1

Fuc 1

GalNAc Serine

GlcNAc 1

63

4NeuAc 2-3 Gal 1

3

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoN-linkedrdquo glycan linkage to protein asparagine residues

Families of N-linked glycan structures

Man

4GlcNAc1

2Man

Man

Man

Man

2Man

2Man

2Man

63

Man63 4GlcNAc1 Asn

4GlcNAc1

Man62

Man63 4GlcNAc1 Asn

Man42

4GlcNAc1

4GlcNAc1

4GlcNAc1

4GlcNAc1

6Gal

6Gal

6Gal

6Gal

NeuAc

NeuAc

NeuAcNeuAc

4GlcNAc1

Man

Man63 4GlcNAc1 Asn

2Man4GlcNAc13GalNeuAc

Man

Man

63

core

core

core

High Mannose

Complex

Hybrid

Examples of structures found as termini on the branches of complex N-linked glycoprotein glycanshellip

Terminal Saccharide Diversity --- A key to recognition

NeuAc 2-6 Gal 1-4 GlcNAc---NeuAc 2-3 Gal 1-4 GlcNAc---

Gal 1-3 Gal 1-4 GlcNAc---4-SO3-GalNAc 1-4 GlcNAc---

(-Gal 1-4 GlcNAc 1-3-)n = polylactosamine repeats (n may be gt6)

(-NeuAc 2-8-) n = polysialic acid (n may be gt50)

Fuc residues (eg Fuc 1-2 Gal Fuc 1-3 GlcNAc Fuc 1-4 GlcNAc

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoO-linkedrdquo glycan linkage to protein serine residues

Examples of variations of O-linked glycans

GalNAc Serine

GalNAc Serine

NeuAc 26

GalNAc Serine

Gal 1

NeuAc 263

GalNAc SerineNeuAc 2-3 Gal 1

NeuAc 263

Gal 1-4 GlcNAc 1

Fuc 1

GalNAc Serine

GlcNAc 1

63

4NeuAc 2-3 Gal 1

3

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoN-linkedrdquo glycan linkage to protein asparagine residues

Families of N-linked glycan structures

Man

4GlcNAc1

2Man

Man

Man

Man

2Man

2Man

2Man

63

Man63 4GlcNAc1 Asn

4GlcNAc1

Man62

Man63 4GlcNAc1 Asn

Man42

4GlcNAc1

4GlcNAc1

4GlcNAc1

4GlcNAc1

6Gal

6Gal

6Gal

6Gal

NeuAc

NeuAc

NeuAcNeuAc

4GlcNAc1

Man

Man63 4GlcNAc1 Asn

2Man4GlcNAc13GalNeuAc

Man

Man

63

core

core

core

High Mannose

Complex

Hybrid

Examples of structures found as termini on the branches of complex N-linked glycoprotein glycanshellip

Terminal Saccharide Diversity --- A key to recognition

NeuAc 2-6 Gal 1-4 GlcNAc---NeuAc 2-3 Gal 1-4 GlcNAc---

Gal 1-3 Gal 1-4 GlcNAc---4-SO3-GalNAc 1-4 GlcNAc---

(-Gal 1-4 GlcNAc 1-3-)n = polylactosamine repeats (n may be gt6)

(-NeuAc 2-8-) n = polysialic acid (n may be gt50)

Fuc residues (eg Fuc 1-2 Gal Fuc 1-3 GlcNAc Fuc 1-4 GlcNAc

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoO-linkedrdquo glycan linkage to protein serine residues

Examples of variations of O-linked glycans

GalNAc Serine

GalNAc Serine

NeuAc 26

GalNAc Serine

Gal 1

NeuAc 263

GalNAc SerineNeuAc 2-3 Gal 1

NeuAc 263

Gal 1-4 GlcNAc 1

Fuc 1

GalNAc Serine

GlcNAc 1

63

4NeuAc 2-3 Gal 1

3

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

ldquoN-linkedrdquo glycan linkage to protein asparagine residues

Families of N-linked glycan structures

Man

4GlcNAc1

2Man

Man

Man

Man

2Man

2Man

2Man

63

Man63 4GlcNAc1 Asn

4GlcNAc1

Man62

Man63 4GlcNAc1 Asn

Man42

4GlcNAc1

4GlcNAc1

4GlcNAc1

4GlcNAc1

6Gal

6Gal

6Gal

6Gal

NeuAc

NeuAc

NeuAcNeuAc

4GlcNAc1

Man

Man63 4GlcNAc1 Asn

2Man4GlcNAc13GalNeuAc

Man

Man

63

core

core

core

High Mannose

Complex

Hybrid

Examples of structures found as termini on the branches of complex N-linked glycoprotein glycanshellip

Terminal Saccharide Diversity --- A key to recognition

NeuAc 2-6 Gal 1-4 GlcNAc---NeuAc 2-3 Gal 1-4 GlcNAc---

Gal 1-3 Gal 1-4 GlcNAc---4-SO3-GalNAc 1-4 GlcNAc---

(-Gal 1-4 GlcNAc 1-3-)n = polylactosamine repeats (n may be gt6)

(-NeuAc 2-8-) n = polysialic acid (n may be gt50)

Fuc residues (eg Fuc 1-2 Gal Fuc 1-3 GlcNAc Fuc 1-4 GlcNAc

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoO-linkedrdquo glycan linkage to protein serine residues

Examples of variations of O-linked glycans

GalNAc Serine

GalNAc Serine

NeuAc 26

GalNAc Serine

Gal 1

NeuAc 263

GalNAc SerineNeuAc 2-3 Gal 1

NeuAc 263

Gal 1-4 GlcNAc 1

Fuc 1

GalNAc Serine

GlcNAc 1

63

4NeuAc 2-3 Gal 1

3

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Families of N-linked glycan structures

Man

4GlcNAc1

2Man

Man

Man

Man

2Man

2Man

2Man

63

Man63 4GlcNAc1 Asn

4GlcNAc1

Man62

Man63 4GlcNAc1 Asn

Man42

4GlcNAc1

4GlcNAc1

4GlcNAc1

4GlcNAc1

6Gal

6Gal

6Gal

6Gal

NeuAc

NeuAc

NeuAcNeuAc

4GlcNAc1

Man

Man63 4GlcNAc1 Asn

2Man4GlcNAc13GalNeuAc

Man

Man

63

core

core

core

High Mannose

Complex

Hybrid

Examples of structures found as termini on the branches of complex N-linked glycoprotein glycanshellip

Terminal Saccharide Diversity --- A key to recognition

NeuAc 2-6 Gal 1-4 GlcNAc---NeuAc 2-3 Gal 1-4 GlcNAc---

Gal 1-3 Gal 1-4 GlcNAc---4-SO3-GalNAc 1-4 GlcNAc---

(-Gal 1-4 GlcNAc 1-3-)n = polylactosamine repeats (n may be gt6)

(-NeuAc 2-8-) n = polysialic acid (n may be gt50)

Fuc residues (eg Fuc 1-2 Gal Fuc 1-3 GlcNAc Fuc 1-4 GlcNAc

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoO-linkedrdquo glycan linkage to protein serine residues

Examples of variations of O-linked glycans

GalNAc Serine

GalNAc Serine

NeuAc 26

GalNAc Serine

Gal 1

NeuAc 263

GalNAc SerineNeuAc 2-3 Gal 1

NeuAc 263

Gal 1-4 GlcNAc 1

Fuc 1

GalNAc Serine

GlcNAc 1

63

4NeuAc 2-3 Gal 1

3

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Examples of structures found as termini on the branches of complex N-linked glycoprotein glycanshellip

Terminal Saccharide Diversity --- A key to recognition

NeuAc 2-6 Gal 1-4 GlcNAc---NeuAc 2-3 Gal 1-4 GlcNAc---

Gal 1-3 Gal 1-4 GlcNAc---4-SO3-GalNAc 1-4 GlcNAc---

(-Gal 1-4 GlcNAc 1-3-)n = polylactosamine repeats (n may be gt6)

(-NeuAc 2-8-) n = polysialic acid (n may be gt50)

Fuc residues (eg Fuc 1-2 Gal Fuc 1-3 GlcNAc Fuc 1-4 GlcNAc

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoO-linkedrdquo glycan linkage to protein serine residues

Examples of variations of O-linked glycans

GalNAc Serine

GalNAc Serine

NeuAc 26

GalNAc Serine

Gal 1

NeuAc 263

GalNAc SerineNeuAc 2-3 Gal 1

NeuAc 263

Gal 1-4 GlcNAc 1

Fuc 1

GalNAc Serine

GlcNAc 1

63

4NeuAc 2-3 Gal 1

3

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoO-linkedrdquo glycan linkage to protein serine residues

Examples of variations of O-linked glycans

GalNAc Serine

GalNAc Serine

NeuAc 26

GalNAc Serine

Gal 1

NeuAc 263

GalNAc SerineNeuAc 2-3 Gal 1

NeuAc 263

Gal 1-4 GlcNAc 1

Fuc 1

GalNAc Serine

GlcNAc 1

63

4NeuAc 2-3 Gal 1

3

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

ldquoO-linkedrdquo glycan linkage to protein serine residues

Examples of variations of O-linked glycans

GalNAc Serine

GalNAc Serine

NeuAc 26

GalNAc Serine

Gal 1

NeuAc 263

GalNAc SerineNeuAc 2-3 Gal 1

NeuAc 263

Gal 1-4 GlcNAc 1

Fuc 1

GalNAc Serine

GlcNAc 1

63

4NeuAc 2-3 Gal 1

3

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

ldquoO-linkedrdquo glycan linkage to protein serine residues

Examples of variations of O-linked glycans

GalNAc Serine

GalNAc Serine

NeuAc 26

GalNAc Serine

Gal 1

NeuAc 263

GalNAc SerineNeuAc 2-3 Gal 1

NeuAc 263

Gal 1-4 GlcNAc 1

Fuc 1

GalNAc Serine

GlcNAc 1

63

4NeuAc 2-3 Gal 1

3

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Examples of variations of O-linked glycans

GalNAc Serine

GalNAc Serine

NeuAc 26

GalNAc Serine

Gal 1

NeuAc 263

GalNAc SerineNeuAc 2-3 Gal 1

NeuAc 263

Gal 1-4 GlcNAc 1

Fuc 1

GalNAc Serine

GlcNAc 1

63

4NeuAc 2-3 Gal 1

3

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

GPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

15 kDa GPI

anchorplasma membrane

CD59 a complement defense glycoprotein

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Glycoproteinsbull N-Linkedbull O-Linkedbull GPI Anchorbull O-GlcNAc

A non-inclusive list

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

O-GlcNAc

Note No further sugar substitutions have been confirmedHighly dynamic post-translational modification

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

A Glycosphingolipid

Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

A Ganglioside

ldquoGD1ardquo

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Major Glycans of Eukaryotes

bull Glycoproteinsbull Glycolipidsbull Proteoglycans

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Glycosaminoglycans (GAGrsquos) and Proteoglycans

bull GLYCOSAMINOGLYCANS (GAGrsquos)ndash long linear glycans made of repeating

disaccharidesndash Hyaluronic acid is the only ldquostand-alonerdquo

GAG other GAGrsquos are constituents of hellipbull PROTEOGLYCANS

ndash GAGrsquos on proteinsndash Defined by their repeating disaccharide unitsndash GAGrsquos on proteoglycans are sulfated

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Hyaluronic acid -- a ldquosimplerdquo space filling molecule

(GlcU β4 GlcNAc)n

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Cartilage proteoglycan

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Glycan Biosynthesis

Glycosyltransferase reaction (UDP-Galglucose β4 galactosyltransferase)

UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAc

UDP-GlcAUDP-XylGDP-FucCMP-NeuAc

Activated Sugars

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Glycoprotein biosynthesis

bull N-Linked ndash en-bloc preconstructed core trimming terminal elaboration

bull O-Linked ndash stepwise sugar by sugar additionbull O-GlcNAc - dynamic transferaseglycosidasebull GPI Anchor ndash en-bloc preconstructed core elaboration

Glycolipid biosynthesis

bull Stepwise sugar-by-sugar addition

Proteoglycan biosynthesis

bull Stepwise sugar-by-sugar addition (core and repeating disaccharide)

bull Post-polymerization modifications

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Introduction to glycobiology

ldquoWhat the matter Sal not up on your glycobiology

H Ford (2010) Extraordinary Measures CBS Films Los Angeles CA USA

>

Harrison Ford Asks Not up on your glycobiology - YouTube

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Hurler(alpha-L

iduronidase)

Hunter(iduronate-2-

sulfatase)

I-cell(GlcNAc

phosphotransferase)

Lysosomal storage diseasesare often due to faulty glycan catabolism

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Lysosomal storage diseases can be studied using patient cells in culture

I-cell lyphocytesvan der Meer et al (2001) J Clin Pathol 54724

I-cell fibroblastsHanai et al (1971) Am J Dis Child 12234

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Fibroblasts from Hunter amp Hurler patients build up glycosaminoglycans

Co-incubation with normal fibroblasts or each other corrects the builduphellip

Correction is via a soluble factor (direct contact not required)

Fratantoni et al (1968) Science 162170

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Corrective factor properties

bull Purified lysosomal hydrolases are taken up by fibroblasts ndash different enzymes correct storage of different diseases

bull Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake

bull Enzyme uptake was selectively blocked by mannosebull Enzyme uptake was blocked better mannose-6-phosphatebull Phosphatase pre-treatment of the enzyme blocked its uptakebull Fibroblasts do not take up I-cell lysosomal hydrolasesbull Normal lysosomal hydrolases have mannose-6-phosphate

residues (unusual) on their N-linked glycansbull I-cell patients are deficient in addition of 6-linked phosphate

to mannose on their N-linked glycans

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
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• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Lysosomal enzyme targeting

Dahms et al (1989) J Biol Chem 26412115

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
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• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Discovery A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes

Bohnsack et al (2009) J Biol Chem 28435215

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
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• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Sugar-modifying enzyme lacking in I-cell diseaseN-acetylglucosamine-1-phosphate transferase

Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
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• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

H Ford and B Fraser (2010) Extraordinary Measures CBS Films Los Angeles CA USA

William Canfield and John Crowley Novazyme (httpblognewokcom)

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
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• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90

Introduction to glycobiology

• Slide 1
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
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• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Nearly all vertebrate glycans are built from only 9 sugars
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Major Glycans of Eukaryotes
• Major Glycans of Eukaryotes (2)
• Glycoproteins
• Glycoproteins (2)
• Slide 53
• Slide 54
• Slide 55
• Glycoproteins (3)
• Glycoproteins (4)
• Slide 58
• Slide 59
• Glycoproteins (5)
• Glycoproteins (6)
• Slide 62
• Slide 63
• Glycoproteins (7)
• Glycoproteins (8)
• O-GlcNAc
• Major Glycans of Eukaryotes (3)
• Major Glycans of Eukaryotes (4)
• Slide 69
• Slide 70
• Major Glycans of Eukaryotes (5)
• Major Glycans of Eukaryotes (6)
• Glycosaminoglycans (GAGrsquos) and Proteoglycans
• Slide 74
• Slide 75
• Slide 76
• Slide 77
• Glycan Biosynthesis
• Glycoprotein biosynthesis
• Slide 80
• Slide 81
• Slide 82
• Slide 83
• Slide 84
• Slide 85
• Slide 86
• Slide 87
• Slide 88
• Slide 89
• Slide 90