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

Major Glycan Classes in Animal Cells 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 ProteoglycanGLYCOPHOSPHO-LIPIDANCHOR N-LINKED CHAINS O-LINKEDCHAIN HYALURONAN GLYCOSAMINO-GLYCANS HEPARAN SULFATE CHONDROITIN SULFATE SULFATE Sialic Acids GLYCOSPHINGOLIPID O-LINKED GlcNAc

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

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

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  2-3Gal  1-3GalNAc(  1- Sia  2-8Sia  2-3Gal  1-3GalNAc  1- Sia  2-3Gal  1-3GlcNAc  Sia  Gal  1-(3)4GlcNAc  1- Gal  1-3GalNAc(  1- Sia  2-3Gal  1-3GalNAc  Sia  Gal  1-3GalNAc  Sia  Sia  2-6GalNAc  1- GalNAc(  1- Sia  2-3Gal  1-(3)4GlcNAc  1- (4)3 1 Fuc  Sialyl-Lewis X(A)

-6P UDP- Degradation and Recycling of Glycans Cytosol Golgi Lysosome ER Glucose Galactose1= Transporter 2= Transferase 3 = Acceptor 3 1 UTP PPi -1PUDP- UTP PPi -1P UDP- UDP UMP

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

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

Major Glycan Classes in Animal Cells 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 ProteoglycanGLYCOPHOSPHO-LIPIDANCHOR N-LINKED CHAINS O-LINKEDCHAIN HYALURONAN GLYCOSAMINO-GLYCANS HEPARAN SULFATE CHONDROITIN SULFATE SULFATE Sialic Acids GLYCOSPHINGOLIPID O-LINKED GlcNAc

FUNCTIONAL EFFECTS OF MODIFYING OR ELIMINATING N-LINKED CHAINS ON GLYCOPROTEINS 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

GENERAL PRINCIPLES REGARDING THE BIOLOGICAL 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

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

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

Elimination or Alteration of Major Glycan Classes in vivo causes Embryonic Lethality O Se r GLYCOPHOSPHO-LIPIDANCHOR O Ser/Thr N Asn Ser-O- N-LINKED CHAINS O-LINKEDCHAINGLYCOSPHINGOLIPID OUTSIDE INSIDE N Asn S SS -O-Ser S S S SS GLYCOSAMINO-GLYCANS Etn P INOSITOL P NH Ac P NS O-LINKED GlcNAc Ac S 2 P Glycoprotein Proteoglycan HYALURONAN HEPARAN SULFATE CHONDROITIN SULFATE SULFATE Sialic Acids

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

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

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

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

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

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

"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

Relatively Little is Known about Glycan Diversity in Nature and its Evolutionary Origins 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?

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

The Red Queen Effect: One Possible Explanation for the Dominance of Sexual Reproduction during Evolution 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?

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

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

Exogenous oligosaccharide recognition may be much commoner than endogenous recognition 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”?

Do more Gene disruption studies in mice 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

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

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

SOME APPROACHES TO EXPLORING SPECIFIC BIOLOGICAL ROLES OF OLIGOSACCHARIDES IN MULTICELLULAR ANIMALS 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 OLIGOSACCHARIDE RECEPTOR

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

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

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

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

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

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

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

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

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