Essentials of Glycobiology Lecture 2 April 2, 2002 Ajit Varki

1. Essentials of Glycobiology Lecture 2April 2, 2002Ajit Varki General pathways for Biosynthesis Biological roles Evolutionary considerations

2. CHONDROITIN SULFATE HYALURONAN GLYCOSAMINO- GLYCANS HEPARAN SULFATE N-LINKED CHAINS O-LINKED CHAIN GLYCOPHOSPHO- LIPID ANCHOR GLYCOSPHINGOLIPID O-LINKED GlcNAc Major Glycan Classes in Animal Cells P S S S Ser-O- S S S S S -O-Ser NS NS Proteoglycan Ac P Etn S P N O N NH Asn Ser/Thr 2 Asn INOSITOL Glycoprotein Ac OUTSIDE P Sialic Acids INSIDE O Ser

3. G G * G * * G * * * G * * * * * G * * * * * * * * * * * * * * * G * * * * * * * * * * O-glycans GSLs GAGs N-glycans GPIs GPI- Glc-Cer O-Xyl N-GlcNAc O-GalNAc LINKED LINKED LINKED LINKED LINKED ROUGH ER G ? Biosynthesis of different classes of glycans within the ER-Golgi Pathway GOLGI APPARATUS SECRETORY LYSOSOME GRANULE ENDOSOME

4. = Sialic acid Common Outer Chains Shared by Different Classes of Glycans S N-LINKED CHAIN O N Ser/Thr Asn O-LINKED CHAIN Secreted Protein GLYCOSPHINGOLIPID S O N Membrane Protein Ser/Thr Asn OUTSIDE CELL MEMBRANE INSIDE

5. Gal1-(3)4GlcNAc1- Sia2-3Gal1-(3)4GlcNAc1- (4)3 1 Fuc Sialyl-Lewis X(A) Sia2-3Gal1-3GlcNAc1- 6 2 Sia Sia2-3Gal1-3GalNAc1- 6 2 Sia Gal1-3GalNAc1- 6 2 Sia GalNAc(1- Some Sialic Acid (Sia) Terminated Sequences Sia2-6Gal1-4GlcNAc1- Sia2-3Gal1-(3)4GlcNAc1- Sia2-8Sia2-3Gal1-4Glc1- Sia2-8Sia2-3Gal1-3GalNAc1- Sia2-3Gal1-3GalNAc(1- Gal1-3GalNAc(1- Sia2-6GalNAc1-

6. 1 1 UMP UDP PPi U TP -1P UDP- UDP- UDP- -6P -1P U TP PPi 1 3 Lysosome Golgi Degradation and Recycling of Glycans 1 1 3 2 Cytosol ER Glucose Galactose 1= Transporter 2= Transferase 3 = Acceptor

7. Essentials of Glycobiology Lecture 2April 2, 2002Ajit Varki General pathways for Biosynthesis Biological roles Evolutionary considerations

8. “...while the functions of DNA and proteins are generally known.....it is much less clear what carbohydrates do...” Ciba Foundation Symposium 1988

9. CHONDROITIN SULFATE HYALURONAN GLYCOSAMINO- GLYCANS HEPARAN SULFATE N-LINKED CHAINS O-LINKED CHAIN GLYCOPHOSPHO- LIPID ANCHOR GLYCOSPHINGOLIPID O-LINKED GlcNAc Major Glycan Classes in Animal Cells P S S S Ser-O- S S S S S -O-Ser NS NS Proteoglycan Ac P Etn S P N O N NH Asn Ser/Thr 2 Asn INOSITOL Glycoprotein Ac OUTSIDE P Sialic Acids INSIDE O Ser

10. Biosynthesis and folding Stability in the ER Secretion rate Intracellular trafficking Cell surface expression Intracellular stability and turnover rate Range or specificity of function Activity of enzymes, hormones & cytokines Signal transduction function of receptors Susceptibility to proteases or denaturants Recognition by antibodies Circulatory half-life Targetting to specific cell types or organs FUNCTIONAL EFFECTS OF MODIFYING OR ELIMINATING N-LINKED CHAINS ON GLYCOPROTEINS

11. GENERAL PRINCIPLES REGARDING THEBIOLOGICAL ROLES OF OLIGOSACCHARIDES (GLYCANS) The biological roles of glycans appear to span the spectrum from those that are trivial, to the those that are crucial for the development, function and survival of an organism While all of the theories regarding the biological roles of glycans appear to be correct, exceptions to each can also be found It is difficult to predict a priori the functions a given glycan on a given glycoconjugate might be mediating, or its relative importance to the organism The only common features of the varied functions of glycans are that they mediate: Structural and modulatory roles or Specific recognition events

12. Exogenous Recognition = Non-self Endogenous Recognition = Self Molecular Mimicry EXOGENOUS RECEPTOR ENDOGENOUS RECEPTOR SELF M SELF M = Micro- organism or Toxin SIALYLATED OLIGOSACCHARIDE = Biological Roles of Glycans Structural/Physical

13. GLYCOSPHINGOLIPID LETHAL CHONDROITIN SULFATE HYALURONAN Elimination of many Major Glycan Classes still permits Cell Viability in vitro GLYCOSAMINO- GLYCANS P S S S Ser-O- HEPARAN SULFATE S S S S S -O-Ser NS NS Proteoglycan N-LINKED CHAINS Ac O-LINKED CHAIN GLYCOPHOSPHO- LIPID ANCHOR P Etn S P N O N NH Asn Ser/Thr 2 Asn INOSITOL Glycoprotein Ac OUTSIDE P Sialic Acids INSIDE O-LINKED GlcNAc O Ser

14. GLYCOSPHINGOLIPID CHONDROITIN SULFATE HYALURONAN Elimination or Alteration of Major Glycan Classes in vivo causes Embryonic Lethality GLYCOSAMINO- GLYCANS P S S S Ser-O- HEPARAN SULFATE S S S S S -O-Ser NS NS Proteoglycan N-LINKED CHAINS Ac O-LINKED CHAIN GLYCOPHOSPHO- LIPID ANCHOR P Etn S P N O N NH Asn Ser/Thr 2 Asn INOSITOL Glycoprotein Ac OUTSIDE P Sialic Acids INSIDE O-LINKED GlcNAc O Ser

15. ACTIVITY ASSAY FOR ACTIVITY PURIFICATION OF THE PROTEIN ANTIBODIES PEPTIDE SEQUENCES cDNA CLONING / GENOMIC CLONING / GENE REGULATION

16. STEPS IN THE STUDY OF A NEW OLIGOSACCHARIDE SEQUENCE METABOLIC LABELLING EXPERIMENTS MONOCLONAL ANTIBODIES ANALYSIS OF MUTANTS DISCOVERY OF A NEW OLIGOSACCHARIDE SEQUENCE PHYSICAL METHODS OF STRUCTURAL ANALYSIS PROOF OF THE STRUCTURE / DETAILS & VARIATIONS BIOSYNTHETIC PATHWAYS & ENZYMOLOGY TISSUE DISTRIBUTION CHANGES IN DEVELOPMENT & MALIGNANCY

17. COMPLETE STRUCTURE AND BIOSYNTHESIS OF A NEW OLIGOSACCHARIDE SEQUENCE IS WORKED OUT FIND RECEPTOR FUNCTIONAL CONSEQUENCES OF REMOVAL, ALTERATION OR COMPETITION TISSUE DISTRIBUTION FUNCTIONAL CONSEQUENCES IN MUTANTS IN TISSUE CULTURE IN INTACT MULTICELLULAR SYSTEMS WHAT ARE THE FUNCTIONS OF THE OLIGOSACCHARIDE? WHAT ARE ITS FUNCTIONS? FUNCTIONAL EFFECTS OF ALTERED SYNTHESIS NATURALLY OCURRING MUTANTS (RARE) EXPERIMENTALLY DERIVED MUTANTS

18. Essentials of Glycobiology Lecture 2April 2, 2002Ajit Varki General pathways for Biosynthesis Biological roles Evolutionary considerations

19. “Nothing in biology makes sense, except in the light of evolution”. Theodosius Dobzhnasky

20. “Intelligent Design” “Optimal Design” EVOLUTIONISTS MANY BIOLOGISTS ASSUME THAT EVOLUTION USUALLY RESULTS IN OPTIMAL DESIGN CREATIONISTS

21. "Although no biological explanation makes sense except in the light of evolution, it does not follow that all evolutionary explanations make sense." John M. Coffin In “The Evolution of HIV” Keith A. Crandall Ed The John Hopkins University press Baltimore and London 1999 ISBN 0-8018-6151-9

22. Questions about oligosaccharide (glycan) diversification in evolution What is the rate of glycan diversification? Is there a “molecular clock” for glycan diversification? What are selective forces driving glycan diversification? What are the relative roles of the different selective forces? What is the functional significance of glycan diversification during evolution? Can exploration of evolutionary diversification educate us about glycan function? Relatively Little is Known about Glycan Diversity in Nature and its Evolutionary Origins

23. Which class of oligosaccharide recognition is more common? Structural Structural OR Exogenous Recognition Exogenous Recognition Endogenous Recognition Endogenous Recognition The two classes of oligosaccharide recognition are under different types and rates of evolutionary selection pressures

24. Large multi-cellular organisms with long life cycles must constantly change, in order to survive the onslaught of potentially lethal microorganisms and parasites which, having much shorter life cycles, can evolve much faster. Sexual reproduction provides a mechanism to generate and maintain diversity at many genetic loci What is the Relevance to the Evolution of Glycan Diversity? Most pathogenic organisms must first bind to their target cells via recognition of specific glycans. It is very likely that at least some of the intra- and inter-species variation in glycosylation is the consequence of such ongoing host-pathogen interactions during evolution. Question: how much of the diversity in glycan structure seen among vertebrates can be attributed to this selection mechanism? The Red Queen Effect: One Possible Explanation for the Dominance of Sexual Reproduction during Evolution

25. Glycans have probably been involved in an Ongoing Arms Race during Evolution EXOGENOUS RECEPTOR ENDOGENOUS RECEPTOR M SELF M = Micro-organism Pathogen Toxin Symbiont OLIGOSACCHARIDE = How to Evade Microbial Recognition without loosing Endogenous Function?

26. Change linkage Add modification Mask with new residue Add branch Substitute residue M SELF M Ac SELF Ac M SELF M SELF M SELF Evading Microbial Recognition without loosing Endogenous Function

27. Endogenous Recognition Exogenous oligosaccharide recognition may be much commoner than endogenous recognition Structural Exogenous Recognition If endogenous recognition is responsible for only a small fraction of oligosaccharide diversity, how can we find this “needle in a haystack”?

28. Define the phenotypic consequences of eliminating each gene Define the number of genes involved in producing each linkage Define the phenotypic consequences of eliminating each linkage Do more Systematic Comparative Glycomics Define the rate of oligosaccharide diversification during evolution Find out if there a “molecular clock” for diversification Define the relative roles of exogenous and endogenous selection Better understand functional significance of glycan diversification Make predictions about endogenous glycan function Do more Gene disruption studies in mice

29. Glycome GLYCANS (SUGAR CHAINS) Proteome Genome Zymome? ENZYMES Transcriptome LIPIDS Lipome How Much more Complex is the Glycome of an organism in Comparison with its Genome? DNA RNA PROTEINS Variations in structure, time and space. Changes in response to environment

30. Comparative Glycomics - an approach to uncovering the endogenous roles of oligosaccharide structures Species 5 Species 3 Species 4 Species 2 Species 1 = in situ localization of a specific oligosaccharide structure

31. Localize specific oligosaccharides by lectins or antibodies Interfere with specific oligosaccharides by lectins or antibodies Metabolic inhibition or alteration of glycosylation Find natural oligosaccharide ligands for specific receptors Find receptors recognizing specific oligosaccharides Eliminate specific receptors by gene targetting Eliminate specific oligosaccharides by glycosidases Study natural glycosylation mutants in intact animals Construct glycosylation mutants in intact animals SOME APPROACHES TO EXPLORING SPECIFIC BIOLOGICAL ROLES OF OLIGOSACCHARIDES IN MULTICELLULAR ANIMALS OLIGOSACCHARIDE RECEPTOR

32. Localization or interference by lectins or antibodies recognizing specific oligosaccharides OLIGOSACCHARIDE RECEPTOR LECTIN OR ANTIBODY • Plant lectins not very specific for animal oligosaccharides • Multivalency can cause non-specific adhesion • Need pure oligosaccharides for immunization • IgM antibodies common - have weak affinity and show cross-reactivity • High-affinity IgG antibodies preferred, but hard to get

33. Interference by soluble oligosaccharides or mimics OLIGOSACCHARIDE RECEPTOR • Need pure oligosaccharides in large quantities • May require multivalency to block effectively • May cross-react with other receptors

34. Finding natural oligosaccharide ligands for cell surface receptors OLIGOSACCHARIDE RECEPTOR • Where to look? • Monovalent affinity may not be high • Is it biologically relevant?

35. RECEPTOR DETECTION AFFINITY PURIFICATION SCREEN EXPRESSION LIBRARIES Finding receptors recognizing specific oligosaccharides OLIGOSACCHARIDE RECEPTOR ? • Need pure defined glycans • Probably need multivalency • Where to look for receptor?

36. Studying natural glycosylation mutants in cultured cells OLIGOSACCHARIDE RECEPTOR • Very common • Phenotypes often minor or undetectable • Receptor may not be in the same cell

37. Studying natural glycosylation mutants in intact animals OLIGOSACCHARIDE RECEPTOR • Relatively rare • Phenotypes unpredictable and variable • Pleiotropic effects on multiple systems

38. LECTIN RECEPTOR CORE OLIGOSACCHARIDE APPROACHES TO GENETIC MANIPULATION OF GLYCOSYLATION OUTER MONOSACCHARIDES GLYCOSIDASE Normal Ablate core transferase Overexpress transferase Ablate outer transferase Express "masking" transferase Overexpress "competing" transferase Express membrane- bound glycosidase Ablate receptor

39. Stepwise production of mSiglec-F R114A “Knock-in” And mSiglec-F Null Mice tk tk neo neo Targeting Construct Wild-type Locus in ES cells ES Cell with targeted allele : point-mutation : lox : exon Transient Cre expression Gancyclovir Selection Knocked-in allele - Produce Mice Mate with mice expressing ZP3-Cre Knockout allele Takashi Angata Transfection, Neo Selection

40. Essentials of Glycobiology Lecture 2April 2, 2002Ajit Varki General pathways for Biosynthesis Biological roles Evolutionary considerations