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Glycobiology BCMB 8130 Clinical Correlations!-sign up for 1st and 2nd choice (1/2)!

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Glycosylation in Diabetes!

O-Mannosylation in Human Pathophysiology!

Glycoconjugate Trafficking and Disorders!

Evolution of Immunity and Sialic Acid/Siglec Biology!

Glycan Signaling and Development (Notch/Fringe)!

Glycosylation in Metastasis and as Biomarkers!

Glycans linked to lipids and lipid precursors

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 Large O-linked Glycosaminoglycans and poly-lactosamine structures

 Glycoprotein N-linked and O-linked oligosaccharides

 Glycolipid oligosaccharides

Glycan synthesis in a cellular context

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Overview

From ER

through

Trans-Golgi and points inbetween

On and into the ER

Dolichol-P-X Glycosyl phosphatidylinositol (GPI)

Glycosphingolipids (GSL)

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ER glycolipid synthesis

Dolichol-P-X Glycosyl phosphatidylinositol (GPI)

Glycosphingolipids (GSL)

Minimal Defining Structure of a Glycosphingolipid

Glycan-O-Ceramide Essentials of Glycobiology

Second Edition

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Biosynthesis of Ceramide and Glucosylceramide

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Golgi processing of Glycosphingolipids

cis

medial

trans

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Major Classes of Glycosphingolipids

Different Core structures generate unique shapes and are expressed in a cell-type specific manner

After Varki, A

Nomenclature Issues Glycosphingolipid (GSL) = Glycan + Sphingolipid

(named after the Egyptian Sphinx) Glycosphingolipids often just referred to as “Glycolipids”.

“Ganglioside": a GSL one or more sialic acid residues Example of nomenclature:

Galβ3GalNAcβ4(Neu5Acα3)Galβ4Glcβ1Cer = GM1 in the Svennerholm nomenclature

OR II3Neu5Ac-GgOSe4-Cer

in the official IUPAC-IUB designation

After Varki, A

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Pathways for Ganglio-series Glycosphingolipid biosynthesis

Essentials of Glycobiology Second Edition

ST-I

ST-II

ST-III

ST-IV ST-V GalT-II GalNAcT GalT-I

• Ceramide is utilized for Ganglioside synthesis and for GalCer synthesis • GM3 is a branching substrate for production of all complex gangliosides • Biosynthesis exhibits branch exclusivity (sialyltransferases cannot take a-series to b-series or b-series to c-series • Extension with neutral residues utilizes the same transferases regardless of branch

Turnover and Degradation of Glycosphingolipids •  Internalized from plasma membrane via endocytosis •  Pass through endosomes (some remodelling possible?) •  Terminal degradation in lysosomes - stepwise reactions

by specific enzymes. •  Some final steps involve cleavages close to the cell

membrane, and require facilitation by specific sphingolipid activator proteins (SAPs, also known as “liftases”).

•  Individual components, available for re-utilization in various pathways.

•  At least some of glucosylceramide may remain intact and be recycled

•  Human diseases in which specific enzymes or SAPs are genetically deficient (storage diseases)

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Biological Roles of Glycosphingolipids •  Thought to be critical components of the epidermal (skin)

permeability barrier (Glc-Cer delivers Cer to stratum corneum) •  Organizing role in cell membrane. Thought to associate with

GPI anchors in the trans-Golgi, forming “rafts” which target to apical domains of polarized epithelial cells

•  May also be in glycosphingolipid enriched domains (“GEMs”) which are associated with cytosolic oncogenes and signalling molecules

•  Physical protection against hostile environnments •  Binding sites for the adhesion of symbiont bacteria. •  Highly specific receptor targets for a variety of bacteria, toxins

and viruses.

Biological Roles of Glycosphingolipids •  Specific association of certain glycosphingolipids with

certain membrane receptors. •  Can mediate low-affinity but high specificity

carbohydrate-carbohydrate interactions between different cell types.

•  Targets for autoimmune antibodies in Guillian-Barre and Miller-Fisher syndromes following Campylobacter infections and in some patients with human myeloma

•  Shed in large amounts by certain cancers - these are found to have a strong immunosuppressive effects, via as yet unknown mechanisms

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Embryonic Lethal. Embryogenesis proceeded into gastrulation with differentiation into primitive germ layers and embryo patterning but abruptly halted by a major apoptotic process. Deficient embryonic stem cells able to form endodermal, mesodermal, and ectodermal derivatives but were strikingly deficient in ability to form well differentiated tissues. However, hematopoietic and neuronal differentiation could be induced.

Consequences of Glycosylceramide Synthase gene disruption

Early embryonic lethal, associated primarily

with extra-embryonic tissues (trophoblast,

extraembryonic membranes)

Consequences of Lactosylceramide Synthase gene disruption

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Enhanced sensitivity to insulin. Enhanced insulin receptor phosphorylation

in skeletal muscle. Protection from high-fat

diet-induced insulin resistance.

Is GM3 ganglioside a negative regulator of

insulin signaling?

Consequences of SialylTransferase I (GM3 synthase) gene disruption

Viable, fertile, normal life span, sensory deficits especially

related to pain sensation

Consequences of SialylTransferase II (GD3 synthase) gene disruption

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Male Sterility. Late Onset Peripheral Nerve De-myelination possibly

related to loss of ligands for Myelin Associated

Glycoprotein (Siglec-4). Reduction in neural

conduction velocity in some nerves.

Compensatory increase in GM3 and GD3 in the

brain

Consequences of GalNAc Transferase I gene disruption

Sheikh, KA, et al. (1999) PNAS 96, 7532

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Sudden death phenotype Extremely susceptible to induction of lethal

seizures by loud sounds Further characterization

in progress

Double KO : Mice Expressing only GM3 in the Brain

On and into the ER

Glycosyl phosphatidylinositol (GPI)

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Examples of GPI-Anchored Proteins

Cell surface hydrolases alkaline phosphatase acetylcholinesterase 5ʼ nucleotidase

Adhesion molecules

neural cell adhesion molecule

heparan sulfate proteoglycan

Others

decay accelerating factor

scrapie prion protein folate receptor

Protozoal antigens trypanosome VSG leishmanial protease plasmodium antigens

Mammalian antigens

Thy-1 carcinoembryonic antigen

Structure of the Basic GPI Anchor

E t nP

INOSITOL

P

= Mannose (Man) = GlucosamineEtn = EthanolamineP = Phosphate

N H2

N H2Pig-A

PNH Defect

GPI-Linked Protein

Cell SurfaceMembrane

After Hart, G

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Chemical and enzymatic reactions of GPI anchors

Essentials of Glycobiology Second Edition

Essentials of Glycobiology Second Edition

GPI-biosynthetic pathways of mammalian

cells and Trypanosoma

brucei

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After Freeze, H

After Freeze, H

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Essentials of Glycobiology Second Edition

Essentials of Glycobiology Second Edition

Examples of C-Terminal Sequences Signaling �the Addition of GPI-Anchors

5-10 hydrophilic, 15-20 hydrophobic

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After Freeze, H

• The first step in biosynthesis of the GPI anchor requires at least four genes

• One of them, PIG-A is an X-linked gene

PIG-A is mutated in PNH

Paroxysmal Nocturnal Hemoglobinuria

 A hematopoietic stem cell disorder characterized by intravascular hemolytic anemia (sudden onset, intermittent/episodic). Abnormal blood cells lack GPI-anchored proteins due to a mutation in the PIG-A gene.

 Lack of GPI-anchored complement regulatory proteins, such as decay-accelerating factor (DAF) and CD59, results in complement-mediated hemolysis and hemoglobinuria.

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Nocturnal hemoglobinuria, not clear why morning urine is enriched in hemoglobin breakdown products (variable course)

Morning Report, Toronto General Hospital; http://morningreporttgh.blogspot.com/2010/05/

GPI-linked proteins protect RBCs from complement-mediated lysis

Rother RP, et al. (2007) Nature Biotechnology 25, 1256

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Accumulation of hemoglobin breakdown products in kidney tubule eventually leads to tubule pathology

Tsai C-W, et al. (2007) Kidney International 71, 1187

On and into the ER

Dolichol-P-X

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Schenk, B. et al. Glycobiology 2001 11:61R-70R; doi:10.1093/glycob/11.5.61R

Topological model for the enzymatic reactions leading to Dol-P biosynthesis de novo on the cytoplasmic face of the ER

Biosynthesis of N-Glycans:�Production of GlcNAc-P-P-Dolichol

Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79

GlcNAc Man

Gal Sia Fuc

Glc

Dolichol

Tunicamycin Blocks - not very specific!

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Biosynthesis of the N-Glycan�Precursor on the Cytosolic Leaflet of the

Endoplasmic Reticulum (ER)

Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79

GlcNAc Man

Gal Sia Fuc

Glc

CDG = Congenital Disorder of Glycosylation in Humans

Biosynthesis of the N-Glycan�Precursor on Lumenal Leaflet of ER

Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79

GlcNAc Man

Gal Sia Fuc

Glc

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Completion of Biosynthesis of N-Glycan�Precursor on Lumenal Leaflet of ER�

- and Transfer to Protein

Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79

GlcNAc Man

Gal Sia Fuc

Glc

Schenk, B. et al. Glycobiology 2001 11:61R-70R; doi:10.1093/glycob/11.5.61R

Topological model for lipid intermediate synthesis, translocation and the role for Dol-P-P/Dol-P phosphatases in the recycling of Dol-P-P/Dol-P in the ER

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General Principles Regarding Lipid-Linked Glycans – Glycan synthesis is compartmentalized within cells – Precursors begin as lipid-linked species on the cytoplasmic

face of the ER, requiring that substrates be flipped for further processsing

– Donor substrates contribute to more than one class of glycoconjugate

– Nucleotide sugar donors are used for cytoplasmic face extension and for Golgi extension; Dolichol-linked donors are used for ER extension of N-linked glycan precursors

– The assembly-line model for glycan extension in the Golgi apparatus may not be as applicable to glycolipid synthesis as it is to glycoprotein glycosylation

– Some precursors and intermediates in glycolipid synthesis influence signaling pathways

– Glycosphingolipids and GPI-anchored proteins associate in membrane microdomains (rafts, GEMs)

Essentials of Glycobiology Second Edition

Processing and maturation of an N-glycan