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

2. GLYCOPHOSPHO-LIPIDANCHOR Sialic Acids on Vetebrate Glycans O Ser O Ser/Thr N Asn Ser-O- OUTSIDE INSIDE N Asn S SS -O-Ser S S S SS Etn P INOSITOL P NH Ac P NS Ac S 2 P Glycoprotein Proteoglycan N-LINKED CHAINS O-LINKEDCHAIN HYALURONAN GLYCOSAMINO-GLYCANS HEPARAN SULFATE CHONDROITIN SULFATE SULFATE Sialic Acids GLYCOSPHINGOLIPID O-LINKED GlcNAc

3. The "Primary" Sialic Acids H-C-OH C O H-C-C-N H H C H OH H 1 2 43 6 5 C-O 9 8 7 O H O H H H - C H-C-OH C O HO H H H C H OH H 1 2 43 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)

4. NUCLEUS Pathways of N-acetylneuraminic Acid (Neu5Ac) metabolism PLASMA MEMBRANE SECRETORY 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 UDP-GlcNAc 2 CMP-Sialic acid transporter 3 Sialyltransferases -Neu5Ac 2 3 ManNA c Neu5Ac Feedback Inhibition

5. Sialyltransferases in the Mammalian Genome Enzyme Acceptor Linkage ST3Gal-I ST3Gal-II ST3Gal-III ST3Gal-IV ST3Gal-V ST3Gal-VI ST6Gal-I ST6GalNAc-II ST6GalNAc-III ST6GalNAc-V ST6GalNAc-IV ST8Sia-I ST8Sia-II ST8Sia-III ST8Sia-IV ST8Sia-V Sialic acid Galactose N-AcetylgalactosamineGalactose  2-3  2-6  2-8 ST6GalNAc-VI ST6GalNAc-I ST8Sia-VI ST6Gal-II Jamey Marth

6. Biological Roles of Sialic Acids Structural/Physical Roles INTRINSIC RECEPTOR SELF Intrinsic Recognition “Self” Siglecs Factor H Selectins Uterine Agglutinin Laminins SIALYLATED OLIGOSACCHARIDE = M EXTRINSIC RECEPTOR M = Micro-organism or Toxin Extrinsic Recognition “Non-self” Influenza Malaria Cholera Helicobacter Mycoplasma Rotavirus Polyoma virus Coronavirus Pertussis Tetanus etc. SELF Molecular Mimicry E.Coli Gonococcus Meningococcus Campylobacter Trypanosoma Streptococcus Etc.

7. 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? Mammalia Aves Reptilia Amphibia Dipnoi? Teleostei Chondrostei Elasmobranchi Agnatha Cephalochordata Urochordata Hemichordata Echinodermata Insecta Crustacea Arachinida Annelidia Mollusca Brachiopoda Bryozoa DETECTED NOT DETECTED Deuterostomes Protostomes

8. 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!

9. 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 HOMOLOGY Phosphatase GlcNAc-6- phosphate 2-epimeraseNeu5Acsynthetase CMP-Neu5Acsynthetase Neu5Ac/Vertebrates UDP-GlcNAc ManNAc Neu5Ac-9-phosphate Neu5Ac CMP-Neu5Ac ATP ADP UDP PEP Pi CTP PPi ManNAc-6 -phosphate ManNAc kinase UDP-GlcNAc2-epimeraseNeu5Ac-9-phosphatesynthetase CMP-Neu5Acsynthetase Neu5Ac-9-phosphatephosphatase Angata and Varki Chemical Reviews 102, 439-469, 2002.

10. "Universal tree" of cellular organisms and occurrence of sialic acids Biochemical Evidence Genetic Evidence

11. 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 CTP PPi Phosphatase Phosphatase HOMOLOGY Phosphatase GlcNAc-6- phosphate 2-epimeraseNeu5Acsynthetase CMP-Neu5Acsynthetase Neu5Ac/Vertebrates UDP-GlcNAc ManNAc Neu5Ac-9-phosphate Neu5Ac CMP-Neu5Ac ATP ADP UDP PEP Pi CTP PPi ManNAc-6 -phosphate ManNAc kinase UDP-GlcNAc2-epimeraseNeu5Ac-9-phosphatesynthetase CMP-Neu5Acsynthetase Neu5Ac-9-phosphatephosphatase CMP-KDOsynthetaseKDO-8-phosphatesynthetase Angata and Varki Chemical Reviews 102, 439-469, 2002.

12. Possible Scenarios for the Phylogenetic Origins of Sialic Acids Loss or partial loss in many lineages Biochemical Evidence Genetic Evidence Lateral Transfer Amongst Prokaryotes

13. Possible Scenarios for the Phylogenetic Origins of Sialic Acids Loss or partial loss in many lineages Biochemical Evidence Genetic Evidence Lateral Transfer Amongst Prokaryotes Lateral Transfer to Early Animal Ancestor

14. Possible Scenarios for the Phylogenetic Origins of Sialic Acids Loss or partial loss in many lineages Biochemical Evidence Genetic Evidence Lateral Transfer Amongst Prokaryotes Lateral Transfer To Prokaryotes from Early Animal Ancestor

15. Biological Roles of Sialic Acids Structural/Physical Roles INTRINSIC RECEPTOR SELF Intrinsic Recognition “Self” Siglecs Factor H Selectins Uterine Agglutinin Laminins SIALYLATED OLIGOSACCHARIDE = M EXTRINSIC RECEPTOR M = Micro-organism or Toxin Extrinsic Recognition “Non-self” Influenza Malaria Cholera Helicobacter Mycoplasma Rotavirus Polyoma virus Coronavirus Pertussis Tetanus etc. SELF Molecular Mimicry E.Coli Gonococcus Meningococcus Campylobacter Trypanosoma Streptococcus Etc.

16. Terminal Sialic acids, Oligosialic acids, Polysialic acids and the Enzymes that can degrade them SIALIDASE (NEURAMINIDASE) Ganglioside or N-Glycan or O-glycan Or Bacterial Glycan Sia  2-3(6)Gal   Polysialic Acid Oligosialic Acid Terminal Sialic Acid Sia  2-8Sia  2-3(6)Gal   Sia  2-8Sia  2-8Sia  2-8Sia  2-8Sia  2-3(6)Gal   ENDOSIALIDASE (ENDO-NEURAMINIDASE)

17. The Neural Cell Adhesion Molecule (N-CAM)

18. Expression of Polysialic Acid on N-CAM also affects Cell-Cell Interactions involving Other Adhesion Molecules

19. 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 cells Pathfinding by large groups of axons Retention of plasticity in certain adult brain tissues

20. Evolutionary History of N-CAM NCAM/apCAM/FasII Progenitor Mouse NCAM Chimpanzee NCAM Aplysia apCAM Drosophila FasII VERTEBRATES +PSA, +NCAM-2 INVERTEBRATES (no PSA) More adult PSA (brain) More “hardwired” More “plastic” Human NCAM

21. Fly: “most of my genes are like his” Albert: “vive la différence”

22. SELF Ligands for Intrinsic Receptors Siglecs Factor H Selectins Uterine Agglutinin Laminins INTRINSIC RECEPTOR SIALYLATED GLYCAN = M = Micro-organism/Toxin SELF M EXTRINSIC RECEPTOR Influenza Malaria Cholera Helicobacter Mycoplasma Rotavirus Polyoma virus Coronavirus Pertussis Tetanus etc. Ligands for Extrinsic Receptors Biological Roles of Sialic Acids Structural/Physical Roles Molecular Mimicry E.Coli Gonococcus Meningococcus Campylobacter Trypanosoma Streptococcus Etc. ?

23. INFLUENZA A & B VIRUSES BIND TO CELL SURFACES VIA A HEMAGGLUTININ THAT RECOGNIZES SIALIC ACIDS THE "RECEPTOR-DESTROYING ENZYME" IS A NEURAMINIDASE (SIALIDASE) LINKAGE TO UNDERLYING SUGAR CHAIN HEMAGGLUTININ NEURAMINIDASE INFLUENZA A OR B VIRUS 9

24. Sialic Acids Restrict Complement Activation on Cell Surfaces FLUID PHASE Requires side chain of Sia C3 Bb C3b P Factor H C3b iC3b Bb I C3b Bb C3b C5 C5b P Membrane Attack Complex ACTIVATING SURFACE Sia Factor H C3b iC3b I Bb NON- ACTIVATING SURFACE

25. Sialic Acid-binding Lectins are Widespread in Nature GROUPSUB-GROUPEXAMPLE VertebrateC - type lectinsL-Selectin I - type lectinsCD22 (Siglec-2) Unclassified Complement Factor H BacterialAdhesinE.coli S-adhesins ToxinVibrio cholerae toxin Viral Hemagglutinin Influenza A virus Hemagglutinin-esterase Influenza C virus Hemagglutinin-neuraminidase Sendai virus Protozoal Plasmodium P. falciparum erythrocyte-binding antigen TritrichomonasTritrichomonas mobilensis Lectin. Crustacean CrabLimulin Lobster L-Agglutinin I Prawn Monodin ArachnidScorpionVaejovis spinigerus lectin Beetle Allomyrina dichotoma lectins SpiderAphonopelma lectin Mollusc SlugLimax flavus agglutinin SnailAchatininH PlantSeedMaackia Amurensis lectin BarkSambucus Nigra lectin RootTricosanthes japonicum lectin FungalMushroom Hericium erinaceum lectin

26. Natural substitutions can change the mass and shape of the Sialic Acid molecule Carbon Oxygen Nitrogen Hydrogen 7,9 DI-O-ACETYL- 8-O-METHYL-7,9 DI-O-ACETYL- N-GLYCOLYL N-GLYCOLYLNEURAMINIC ACID N -ACETYLNEURAMINIC ACID 8 7 1 4 2 3 5 6 9 8 7 1 4 2 3 5 6 9

27. THE SIALIC ACIDS R2 R5 N-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 or Absent in: - 2,3dehydro or - 2,7anhydro (double-bond when R2 absent) R5 N-glycolyl Hydroxyl Amino * -at physio- logical pH, ionized or lactonized or lactamized R1 1 2 3 4 5 6 7 89 * CARBON OXYGEN HYDROGEN R1 = hydrogen or: R8 R9 R7 R4 R8 R9 R7 R4 Acetyl R8 R9 R7 R4 Phosphate R9 Methyl R8 Sulfate R8 Lactyl R9

28. 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

29. 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 Migration Migrase Enzyme? INFLUENZA A VIRUS N-ACETYL-NEURAMINIC ACID INFLUENZA C CORONAVIRUSES 9-O-ACETYL-N-ACETYL-NEURAMINIC ACID

30. INFLUENZA C AND CORONAVIRUS HEMAGGLUTININ- ESTERASES SPECIFICALLY RECOGNIZE 9-O-ACETYLATED SIALIC ACIDS LINKAGE TO UNDERLYING SUGAR THE "RECEPTOR-DESTROYING ENZYME" IS A SIALIC ACID-SPECIFIC 9-O-ACETYL-ESTERASE AT 37 O C, THE ESTERASE ACTIVITY IS DOMINANT INFLUENZA C VIRUS HEMAGGLUTININ ESTERASE 9 Ser

31. Sialic Acid-binding Proteins recognize specific features of Sialic Acids in natural ligands: some examples Underlying Importance for Recognition LectinsLinkage Saccharide Carbo- N-Acyl Side 9-O-Acetylation xylate Group Chainof Side chain Selectins Sia  2-3 Gal  1-(3)4GlcNAc  1 - yesNoNoNo?  1 Fuc(3)4 CD22 (Siglec-2) Sia  2-6 Gal  1-4GlcNAc  1yesyesyesBlocks Sialoadhesin (Siglec-1) Sia  2-3 Gal  1-4GlcNAc  1yesyesyesBlocks Complement Factor H Sia  2-? ?yesno?yes Blocks P. falciparum Sia  2-3 Gal  1-4GlcNAc  1 - ? no? ? Blocks? merozoite lectin Influenza A Sia  2-3 ?yes variableyes Blocks Hemagglutinin Sia  2-6 Influenza C Hemagglutinin-esteraseSia  2-X -yesno?yes Required

32. Carbon Oxygen Nitrogen Hydrogen Two Major Kinds of Sialic Acids in Mammalian Cells N-ACETYLNEURAMINIC ACID (Neu5Ac) 8 7 1 2 3 5 6 9 LINKAGE TO UNDERLYING SUGAR CHAIN4 N-GLYCOLYLNEURAMINIC ACID (Neu5Gc) 8 7 1 4 2 3 5 6 9 LINKAGE TO UNDERLYING SUGAR CHAIN

33. “Great Apes” Evolutionary Relationships amongst Humans and the Great Apes 10 5 Millions of Years Before Present * 0 * Precise Timing Uncertain Gorilla gorilla Gorilla Pan paniscus Bonobo Pan troglodytes Chimpanzee MEAN Amino Acid Difference ~0.5% <1.0% H omo sapiens Human Pongo pygmaeus Orangutan Neu5Ac Neu5Gc Genetic Mutation Causing loss Of Neu5Gc

34. R3R2 The Sialic Acids CARBON NITROGEN OXYGEN HYDROGEN 1 2 3 4 5 6 7 8 9 = 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 R2R3