Essentials of Glycobiology Lecture 10 April 13th. 2004 Ajit Varki The Sialic Acids.

Essentials of Glycobiology

Lecture 10

April 13th. 2004

Ajit Varki

The Sialic Acids

GLYCOPHOSPHO-GLYCOPHOSPHO-LIPIDLIPID

ANCHORANCHOR

Sialic Acids on

Vetebrate Glycans

OSer

OSer/Thr

NAsn

Ser-O-

OUTSIDE

INSIDE

NAsn

S S S

-O-SerS SSS S

EtnP

INOSITOL

P

NH

Ac

P

NS NS

Ac

S

2

P

GlycoproteinGlycoprotein

ProteoglycanProteoglycanN-LINKED CHAINSN-LINKED CHAINS

O-LINKED O-LINKED CHAINCHAIN

HYALURONANHYALURONAN

GLYCOSAMINO-GLYCOSAMINO-GLYCANSGLYCANS HEPARAN SULFATEHEPARAN SULFATE

CHONDROITINCHONDROITIN SULFATESULFATE

Sialic AcidsSialic Acids

GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPID

O-LINKED GlcNAcO-LINKED GlcNAc

The "Primary" Sialic Acids

H-C-OH

H-C-OH

H-C-OH

C OH-C-C-N

H

H

C C

C C

H

OH

H

OH

1

2

4 3

6

5

C-O

9

8

7

O

H O H

H

H

-C C

H-C-OH

H-C-OH

H-C-OH

C OHO

HH

H

C C

H

OH

H

OH

1

2

4 3

6

5

C-O

9

8

7

O

-

-D-KDN-D-Neu5Ac

Thought to be the metabolic precursors of all other sialic acids

N-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-

D-glycero-D-galacto-nonulosonic acid)“Neu5Ac” “NANA”, “NeuAc”

KDN (2-keto-3-deoxy-D-glycero-

D-galacto-nonulosonic acid)

NUCLEUS

Pathways of N-acetylneuraminic Acid (Neu5Ac) metabolism

PLASMA MEMBRANESECRETORY PATHWAYS

LYOSOSOMES

ER

Newly synthesized glycoconjugate

GOLGI

Neu5AcManNAc

CYTOSOL

CELL MEMBRANE

CMP-Neu5Ac

1

1 CMP-Sialic Acid Synthase

5

5 Sialic Acid Exporter

4 Lysosomal Sialidase

Neu5Ac

4-Neu5Ac

ENDO/PINOCYTOSIS

-Neu5Ac

-Neu5Ac

UDP-GlcNAc

2 CMP-Sialic acid transporter3 Sialyltransferases

-Neu5Ac2

3

ManNAc

Neu5Ac

Feedback Inhibition

Sialyltransferases in the Mammalian Genome

Enzyme

Acceptor

Linkage

ST3Gal-IST3Gal-IIST3Gal-IIIST3Gal-IVST3Gal-VST3Gal-VI

ST6Gal-IST6GalNAc-IIST6GalNAc-III

ST6GalNAc-VST6GalNAc-IV

ST8Sia-IST8Sia-IIST8Sia-IIIST8Sia-IVST8Sia-V

Sialic acidGalactose N-AcetylgalactosamineGalactose

2-32-6 2-6 2-8

ST6GalNAc-VI

ST6GalNAc-I

ST8Sia-VI

ST6Gal-II

Jamey Marth

Biological Roles of Sialic Acids

Structural/Physical Roles

INTRINSIC RECEPTOR

SELF

IntrinsicRecognition

“Self”

SiglecsFactor HSelectins

Uterine AgglutininLaminins

SIALYLATED OLIGOSACCHARIDE =

M

EXTRINSIC RECEPTOR

M = Micro-organism or Toxin

ExtrinsicRecognition“Non-self”

InfluenzaMalariaCholera

HelicobacterMycoplasma

RotavirusPolyoma virusCoronavirusPertussis

Tetanus etc.

SELF

MolecularMimicry

E.ColiGonococcus

MeningococcusCampylobacterTrypanosoma

StreptococcusEtc.

DID THE SIALIC ACIDS APPEAR LATE IN EVOLUTION?

Adapted from : Schauer (1982)

Exceptions

• Certain bacteria (mostly animal pathogens)

• Certain protozoa (mostly animal pathogens)

• Certain fungi (animal pathogens)

• Rare cultured insect cell lines?

• Rare insect embryo stages?

• Octopus and Squid brain?

MammaliaAvesReptiliaAmphibiaDipnoi?TeleosteiChondrosteiElasmobranchiAgnathaCephalochordataUrochordataHemichordataEchinodermata

InsectaCrustaceaArachinidaAnnelidiaMolluscaBrachiopodaBryozoa

DETECTED

NOTDETECTED

De

ute

ros

tom

esP

roto

stom

es

Microbial genes in the human genome: lateral transfer or gene loss?

Salzberg et al. Science 2001, 292, 1903-1906

• 40 Candidate Genes fulfilling criteria suggesting Lateral transfer from Vertebrates to Bacteria

OUR ANALYSIS OF THE LIST:

• 7 of the 40 are involved in sialic acid biosynthesis, turnover or degradation

• Thus, pathway involving ~0.1% of the human genome represents almost 20% of the potential examples of lateral gene transfer between vertebrates and bacteria!

Phylogenetic relationships of enzymes

involved in the

metabolism of sialic acids

Neu5Ac/bacteria

GlcNAc-6-phosphate

ManNAc

Neu5Ac

CMP-Neu5Ac

ManNAc-6 -phosphate

Pi

PEP

Pi

CTP

PPi

HOMOLOGYHOMOLOGY

PhosphatasePhosphatase

GlcNAc-6-GlcNAc-6-phosphatephosphate

2-epimerase2-epimerase

Neu5AcNeu5Acsynthetasesynthetase

CMP-Neu5AcCMP-Neu5Acsynthetasesynthetase

Neu5Ac/Vertebrates

UDP-GlcNAc

ManNAc

Neu5Ac-9-phosphate

Neu5Ac

CMP-Neu5Ac

ATP

ADP

UDP

PEP

Pi

Pi

CTP

PPi

ManNAc-6 -phosphate

ManNAc kinaseManNAc kinase

UDP-GlcNAcUDP-GlcNAc2-epimerase2-epimerase

Neu5Ac-9-Neu5Ac-9-phosphatephosphatesynthetasesynthetase


Neu5Ac-9-phosphateNeu5Ac-9-phosphatephosphatasephosphatase

Angata and Varki Chemical

Reviews 102, 439-469, 2002.

PlantsFungiEukaryaArchaeaCommon ancestor of cellular life

EuryarchaeotaCrenarchaeotaProtozoaProtostomesDeuterostomesSpirochetesChlamydiaThermus/DeinococcusCyanobacteriaAquifexGram-positiveHigh G+CGram-positiveLow G+C Gram-negativeBacteria"Universal tree" of cellular

organisms and occurrence of sialic acids

BiochemicalEvidence

GeneticEvidence

Phylogenetic relationships of enzymes

involved in the

metabolism of sialic acids

and KDO

Neu5Ac/bacteria

GlcNAc-6-phosphate

ManNAc

Neu5Ac

CMP-Neu5Ac

ManNAc-6 -phosphate

Pi

PEP

Pi

CTP

PPi

KDO/bacteria

Ara-5-phosphate

KDO-8-phosphate

KDO

CMP-KDO

PEP

Pi

Pi

CTP

PPi

PhosphatasePhosphataseHOMOLOGYHOMOLOGY

PhosphatasePhosphatase

GlcNAc-6-GlcNAc-6-phosphatephosphate

2-epimerase2-epimerase

Neu5AcNeu5Acsynthetasesynthetase


Neu5Ac/Vertebrates

UDP-GlcNAc

ManNAc

Neu5Ac-9-phosphate

Neu5Ac

CMP-Neu5Ac

ATP

ADP

UDP

PEP

Pi

Pi

CTP

PPi

ManNAc-6 -phosphate

ManNAc kinaseManNAc kinase

UDP-GlcNAcUDP-GlcNAc2-epimerase2-epimerase

Neu5Ac-9-Neu5Ac-9-phosphatephosphatesynthetasesynthetase


Neu5Ac-9-phosphateNeu5Ac-9-phosphatephosphatasephosphatase

CMP-KDOCMP-KDOsynthetasesynthetase

KDO-8-phosphateKDO-8-phosphatesynthetasesynthetase

Angata and Varki Chemical

Reviews 102, 439-469, 2002.

Possible Scenarios for the Phylogenetic Origins of Sialic Acids

Scenario 1: Ancient inventionBacteriaArchaeaEukaryaInvention of SiaDeuterostome Lineage

Loss or partial loss in many lineages

BiochemicalEvidence

GeneticEvidence

Lateral TransferAmongst

Prokaryotes


Scenario 2: Invention in bacteriaInvention of SiaBacteriaArchaeaEukaryaDeuterostome Lineage


BiochemicalEvidence

GeneticEvidence


Prokaryotes

Lateral Transfer

toEarly

Animal Ancestor


Scenario 3: Invention in early eukaryoteInvention of SiaBacteriaArchaeaEukaryaDeuterostome Lineage


BiochemicalEvidence

GeneticEvidence


Prokaryotes

Lateral Transfer

ToProkaryotes

fromEarly

Animal Ancestor



INTRINSIC RECEPTOR

SELF

IntrinsicRecognition

“Self”



SIALYLATED OLIGOSACCHARIDE =

M

EXTRINSIC RECEPTOR

M = Micro-organism or Toxin

ExtrinsicRecognition“Non-self”



RotavirusPolyoma virusCoronavirusPertussis

Tetanus etc.

SELF

MolecularMimicry

E.ColiGonococcus

MeningococcusCampylobacterTrypanosoma

StreptococcusEtc.

Terminal Sialic acids, Oligosialic acids, Polysialic acidsand the Enzymes that can degrade them

SIALIDASE (NEURAMINIDASE)

Gangliosideor

N-Glycanor

O-glycanOr

BacterialGlycan

Sia2-3(6)Gal

Polysialic Acid

Oligosialic Acid

Terminal Sialic Acid

Sia2-8Sia2-3(6)Gal

Sia2-8Sia2-8Sia2-8Sia2-8Sia2-3(6)Gal

ENDOSIALIDASE (ENDO-NEURAMINIDASE)

The Neural Cell Adhesion

Molecule(N-CAM)

Expression of Polysialic Acid on N-CAM also

affects Cell-Cell Interactions involving

Other Adhesion Molecules

WHY DID THE NCAM-PSA MECHANISM EVOLVE?

MOLECULAR MECHANISM FOR GLOBAL REGULATION:

PSA evolved to allow regulation of an expanded array of different CAMs without the requirement that all of these CAMs be affected by the same signaling pathways

EXPANDED NEED FOR PLASTICITY:Migration of precursor cellsPathfinding by large groups of axonsRetention of plasticity in certain adult brain tissues

Evolutionary History of N-CAMNCAM/apCAM/FasII

Progenitor

MouseNCAM

ChimpanzeeNCAM

AplysiaapCAM

DrosophilaFasII

VERTEBRATES+PSA, +NCAM-2

INVERTEBRATES(no PSA)

More adult PSA(brain)

More “hardwired”More “plastic”

HumanNCAM

Fly: “most of my genes are like his”

Albert: “vive la différence”

SELF

Ligands forIntrinsic

Receptors



INTRINSIC RECEPTORINTRINSIC RECEPTOR

SIALYLATED GLYCAN = M = Micro-organism/Toxin

SELF

M

EXTRINSIC RECEPTOREXTRINSIC RECEPTOR



RotavirusPolyoma virusCoronavirus

Pertussis Tetanus etc.

Ligands for ExtrinsicReceptors



MolecularMimicry

E.ColiGonococcus

MeningococcusCampylobacterTrypanosomaStreptococcus

Etc.

?

INFLUENZA A & B VIRUSES BIND TO CELL SURFACES VIAA HEMAGGLUTININ THAT RECOGNIZES SIALIC ACIDS

THE "RECEPTOR-DESTROYING ENZYME"

IS A NEURAMINIDASE (SIALIDASE)

LINKAGE TOUNDERLYINGSUGAR CHAIN

HEMAGGLUTININ

NEURAMINIDASE

INFLUENZAA OR B VIRUS

99

Sialic Acids Restrict Complement Activation on Cell Surfaces

FLUIDPHASE

Requires side chain

of Sia

C3 Bb C3b

P

Factor H

C3b iC3b

Bb

I

C3bBb C3b

C5

C5b

P

MembraneAttackComplex

ACTIVATING SURFACE

Sia

Factor H

C3b iC3b

I

Bb

NON- ACTIVATINGSURFACE

Sialic Acid-binding Lectins are Widespread in NatureGROUP SUB-GROUP EXAMPLEVertebrate C - type lectins L-Selectin

I - type lectins CD22 (Siglec-2)Unclassified Complement Factor H

Bacterial Adhesin E.coli S-adhesins Toxin Vibrio cholerae toxin

Viral Hemagglutinin Influenza A virusHemagglutinin-esterase Influenza C virusHemagglutinin-neuraminidase Sendai virus

Protozoal Plasmodium P. falciparum erythrocyte-binding antigen

Tritrichomonas Tritrichomonas mobilensis Lectin.Crustacean Crab Limulin

Lobster L-Agglutinin IPrawn Monodin

Arachnid Scorpion Vaejovis spinigerus lectinBeetle Allomyrina dichotoma lectinsSpider Aphonopelma lectin

Mollusc Slug Limax flavus agglutininSnail AchatininH

Plant Seed Maackia Amurensis lectinBark Sambucus Nigra lectinRoot Tricosanthes japonicum lectin

Fungal Mushroom Hericium erinaceum lectin

Natural substitutions can change the mass and shape of the Sialic Acid molecule

Carbon Oxygen Nitrogen Hydrogen

8-O-METHYL-7,9 DI-O-ACETYL-7,9 DI-O-ACETYL-N-GLYCOLYLN-GLYCOLYLNEURAMINIC

ACID

N -ACETYLNEURAMINIC ACID

8877

11

44

22

33

55

66

99

8877

11

44

22

33

55

66

99

THE SIALIC ACIDS

R2

R2

R5

R5N-acetyl

Hydrogen or:-linkage to: -Gal (-3 -4 -6) or -GalNAc (-6) or -GlcNAc(-4 -6) or -Sialic Acid (-8 -9) or-linkage to CMP orAbsent in: - 2,3dehydro or - 2,7anhydro (double-bond when R2 absent)

R5

R5

R5

N-glycolyl

Hydroxyl

Amino

*

-at physio-logical pH,ionized or

lactonized orlactamized

R11122

3344

55

6677

88

99

*

CARBON OXYGEN HYDROGEN

R1

= hydrogen or:

R8

R9

R7

R4

R8R9 R7 R4

Acetyl

R8R9 R7 R4

Phosphate R9

Methyl

R8

Sulfate R8

LactylR9

Nomenclature and Abbreviations

Combinations of:

Neu = neuraminic acid

KDN = 2-keto-3-deoxy-nonulosonic acid.

Ac = acetyl, Gc = glycolyl, Me = methyl, Lt = lactyl, S=sulfate

Examples:

N-glycolyl-neuraminic acid = Neu5Gc

9-O-acetyl-8-O-methyl-N-acetyl-neuraminic acid = Neu5,9Ac 8Me

7,8,9-tri-O-acetyl-N-glycolyl-neuraminic acid = Neu5Gc7,8,9Ac

Uncertain of the type of the sialic acid ? Use generic abbreviation Sia

Sialic acid of unknown type with O- acetyl at 9-position = Sia9Ac

3

2

O-ACETYL ESTERS AT THE 7- AND 9-POSITIONS

OF SIALIC ACIDS

7

9

8

7

9

8

7

9

8

7-O-ACETYL-N-ACETYL-NEURAMINIC ACID

O-acetyltransferases

9-O-acetylesterases

O-acetyl MigrationMigrase Enzyme?

INFLUENZA A VIRUSINFLUENZA A VIRUS

N-ACETYL-NEURAMINIC ACID

INFLUENZA C INFLUENZA C CORONAVIRUSESCORONAVIRUSES

9-O-ACETYL-N-ACETYL-NEURAMINIC ACID

INFLUENZA C AND CORONAVIRUS HEMAGGLUTININ-ESTERASES SPECIFICALLY RECOGNIZE

9-O-ACETYLATED SIALIC ACIDS

LINKAGE TOUNDERLYING

SUGAR

THE "RECEPTOR-DESTROYING ENZYME" IS A SIALIC ACID-SPECIFIC 9-O-ACETYL-ESTERASE

AT 37OC, THE ESTERASE ACTIVITY IS DOMINANT

INFLUENZAC VIRUS

HEMAGGLUTININ

ESTERASE

9

Ser

Sialic Acid-binding Proteins recognize specific features of Sialic Acids in natural ligands: some examples

Underlying Importance for Recognition Lectins Linkage Saccharide Carbo- N-Acyl Side 9-O-Acetylation

xylate Group Chain of Side chain

Selectins Sia2-3 Gal1-(3)4GlcNAc1- yes No No No? 1 Fuc(3)4

CD22 (Siglec-2) Sia2-6 Gal1-4GlcNAc1 yes yes yes Blocks

Sialoadhesin (Siglec-1) Sia2-3 Gal1-4GlcNAc1 yes yes yes Blocks

Complement Factor H Sia2-? ? yes no? yes Blocks

P. falciparum Sia2-3 Gal1-4GlcNAc1- ? no? ? Blocks? merozoite lectin

Influenza A Sia2-3 ? yes variable yes Blocks Hemagglutinin Sia2-6

Influenza C Hemagglutinin-esterase Sia2-X - yes no? yes Required

Carbon Oxygen Nitrogen Hydrogen

Two Major Kinds of Sialic Acidsin Mammalian Cells

N-ACETYLNEURAMINIC ACID

(Neu5Ac)88

77

1122

33

55

66

99


SUGAR CHAIN

44

N-GLYCOLYLNEURAMINIC ACID

(Neu5Gc)

8877

11

44

22

33

55

66

99


SUGAR CHAIN

“Great Apes”

Evolutionary Relationships

amongst Humans and the

Great Apes

10

5

Mil

lio

ns

of

Yea

rs B

efo

re P

rese

nt* 0

*Precise Timing Uncertain

Gorilla gorilla

GorillaPan paniscusBonobo

Pan troglodytes Chimpanzee

MEAN Amino AcidDifference

~0.5%<1.0%

Homo sapiens Human

Pongo pygmaeusOrangutan

Neu5Ac Neu5Gc

Genetic Mutation

Causing lossOf Neu5Gc

R3

R2

The Sialic AcidsCARBON

NITROGEN

OXYGEN

HYDROGEN

11

22

33

44

55

66

77

88

99

= H, ACETYL (4,7,8,9), LACTYL (9), METHYL (8), SULFATE (8,9), PHOSPHATE (9),

ANHYDRO (4,8 or 2,7), SIALIC ACID (8,9), FUCOSE(4), GLUCOSE(8), OR GALACTOSE(4)

= N-ACETYL, N-GLYCOLYL, N-GLYCOLYL-O-ACETYL, AMINO, HYDROXYL

= Gal (3/4/6), GalNAc(6), GlcNAc(4/6), Sia (8/9) or 5-O-Neu5Gc, (absent in 2,6 / 2,7 ANHYDRO)

R1

R1

R1

R1

R1

R2

R3

Essentials of Glycobiology Lecture 10 April 13th. 2004 Ajit Varki The Sialic Acids.

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Transcript of Essentials of Glycobiology Lecture 10 April 13th. 2004 Ajit Varki The Sialic Acids.