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Essentials of Glycobiology Lecture 2 March 30, 2004 Ajit Varki General pathways for Biosynthesis Biological roles Evolutionary considerations Major Glycan Classes in Animal Cells CHONDROITIN SULFATE HYALURONAN P GLYCOSAMINOGLYCANS HEPARAN SULFATE S S S S S S Ser-O- S S -O-Ser NS NS Proteoglycan N-LINKED CHAINS Ac O-LINKED CHAIN GLYCOPHOSPHOLIPID Etn ANCHOR P S P O Ser/Thr GLYCOSPHINGOLIPID N Asn N Asn NH 2 INOSITOL Glycoprotein Ac OUTSIDE Sialic Acids O-LINKED GlcNAc INSIDE O Ser P Biosynthesis of different classes of glycans within the ER-Golgi Pathway N-glycans O-glycans ROUGH ER N-GlcNAc LINKED O-GalNAc LINKED GSLs GAGs GPIs O-Xyl LINKED GPILINKED Glc-Cer LINKED G ? G G *G GOLGI APPARATUS * * * ** ** **** SECRETORY GRANULE * * **** ** ** ** G * ** ENDOSOME *G ** G * * ** * G * LYSOSOME Common Outer Chains Shared by Different Classes of Glycans = Sialic acid S N-LINKED CHAIN O Ser/Thr N Asn O-LINKED CHAIN GLYCOSPHINGOLIPID Secreted Protein S O Ser/Thr N Asn Membrane Protein OUTSIDE CELL MEMBRANE INSIDE Some Sialic Acid (Sia) Terminated Sequences Recognized by Siglec-2 Gal1-(3)4GlcNAc1- Sia2-6Gal1-4GlcNAc1- Sia2-3Gal1-(3)4GlcNAc1Recognized by Selectins Sia2-8Sia2-3Gal1-4Glc1Sia2-3Gal1-3GlcNAc16 2 Sia Sia2-3Gal1-(3)4GlcNAc1(4)3 1 Sialyl-Lewis X(A) Fuc Sia2-3Gal1-3GalNAc16 2 Sia Sia2-8Sia2-3Gal1-3GalNAc1- Gal1-3GalNAc16 2 Sia Sia2-3Gal1-3GalNAc(1- Gal1-3GalNAc(1- Sia2-6GalNAc1- GalNAc(1- Degradation and Recycling of Glycans Lysosome Golgi 1 1 UMP UTP 1 1 PPi -1P UDP 3 UDPUDP- -6P UTP 1 Glucose 2 UDP- -1P Cytosol PPi 3 ER Galactose 1= Transporter 2= Transferase 3 = Acceptor 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 CHONDROITIN SULFATE HYALURONAN P GLYCOSAMINOGLYCANS HEPARAN SULFATE S S S S S S Ser-O- S S -O-Ser NS NS Proteoglycan N-LINKED CHAINS Ac O-LINKED CHAIN GLYCOPHOSPHOLIPID Etn ANCHOR P S P O Ser/Thr GLYCOSPHINGOLIPID N Asn N Asn NH 2 INOSITOL Glycoprotein Ac OUTSIDE Sialic Acids O-LINKED GlcNAc INSIDE O Ser P 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 Extrinsic Recognition = Non-self Intrinsic Recognition = Self EXTRINSIC RECEPTOR INTRINSIC RECEPTOR SELF M SELF SIALYLATED OLIGOSACCHARIDE = M = Microorganism or Toxin Molecular Mimicry Elimination of many Major Glycan Classes still permits Cell Viability in vitro CHONDROITIN SULFATE HYALURONAN P GLYCOSAMINOGLYCANS HEPARAN SULFATE S S S S S Ser-O- S S -O-Ser NS NS Proteoglycan N-LINKED CHAINS Ac O-LINKED CHAIN GLYCOPHOSPHOLIPID Etn ANCHOR P P S O Ser/Thr GLYCOSPHINGOLIPID N Asn N Asn NH 2 INOSITOL Glycoprotein Ac OUTSIDE Sialic Acids INSIDE O-LINKED GlcNAc LETHAL O Se r P S Elimination or Alteration of Major Glycan Classes in vivo causes Embryonic Lethality CHONDROITIN SULFATE HYALURONAN P GLYCOSAMINOGLYCANS HEPARAN SULFATE S S S S -O-Ser NS NS Proteoglycan N-LINKED CHAINS Ac O-LINKED CHAIN GLYCOPHOSPHOLIPID ANCHOR P S O Ser/Thr GLYCOSPHINGOLIPID S N Asn N Asn INOSITOL Glycoprotein Ac Sialic Acids INSIDE O Se r Etn P NH 2 OUTSIDE O-LINKED GlcNAc S S Ser-O- P S 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 ANALYSIS OF MUTANTS METABOLIC LABELLING EXPERIMENTS MONOCLONAL ANTIBODIES DISCOVERY OF A NEW OLIGOSACCHARIDE SEQUENCE PHYSICAL METHODS OF STRUCTURAL ANALYSIS PROOF OF THE STRUCTURE / DETAILS & VARIATIONS TISSUE DISTRIBUTION 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 FIND RECEPTOR FUNCTIONAL CONSEQUENCES OF REMOVAL, ALTERATION OR COMPETITION TISSUE DISTRIBUTION WHAT ARE ITS FUNCTIONS? FUNCTIONAL CONSEQUENCES IN MUTANTS NATURALLY OCURRING MUTANTS (RARE) EXPERIMENTALLY DERIVED MUTANTS IN INTACT MULTICELLULAR SYSTEMS FUNCTIONAL EFFECTS OF ALTERED SYNTHESIS IN TISSUE CULTURE 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 “Intelligent Design” “Optimal Design” EVOLUTIONISTS "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 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 Glycan recognition is more common? Structural Structural OR Intrinsic Recognition Extrinsic Recognition Extrinsic Intrinsic Recognition Recognition The two classes of Glycan recognition are under different types and rates of evolutionary selection pressures 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 interspecies 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 INTRINSIC RECEPTOR EXTRINSIC RECEPTOR M SELF OLIGOSACCHARIDE = M = Micro-organism Pathogen Toxin Symbiont How to Evade Microbial Recognition without loosing Endogenous Function? Evading Microbial Recognition without loosing Endogenous Function M SELF Ac SELF Change linkage M Add modification M Mask with new residue M Add branch Ac SELF SELF SELF M Substitute residue Extrinsic Glycan recognition may be much commoner than endogenous recognition Structural Extrinsic Recognition Intrinsic Recognition If intrinsic recognition is responsible for only a small fraction of glycan 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 Comparative Glycomics - an approach to uncovering the endogenous roles of oligosaccharide structures Species 1 Species 2 Species 3 Species 4 = in situ localization of a specific oligosaccharide structure Species 5 How Much more Complex is the Glycome of an organism in Comparison with its Genome? Glycome Proteome Genome DNA RNA Transcriptome PROTEINS GLYCANS (SUGAR CHAINS) ENZYMES Zymome? LIPIDS Lipome Variations in structure, time and space. Changes in response to environment SOME APPROACHES TO EXPLORING SPECIFIC BIOLOGICAL ROLES OF OLIGOSACCHARIDES IN MULTICELLULAR ANIMALS OLIGOSACCHARIDE RECEPTOR • 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 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 OLIGOSACCHARIDE RECEPTOR • • • Need pure oligosaccharides in large quantities May require multivalency to block effectively May cross-react with other receptors 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 AFFINITY PURIFICATION SCREEN EXPRESSION LIBRARIES 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 LECTIN RECEPTOR GLYCOSIDASE Normal Overexpress transferase Overexpress "competing" transferase Ablate receptor CORE OLIGOSACCHARIDE OUTER MONOSACCHARIDES Ablate core transferase Ablate outer transferase Express "masking" transferase Express membranebound glycosidase Example: Stepwise production of mSiglec-F R114A “Knock-in” And mSiglec-F Null Mice : lox neo : exon : point-mutation Targeting Construct tk Wild-type Locus in ES cells Transfection, Neo Selection neo ES Cell with targeted allele tk Transient Cre expression Gancyclovir Selection Knocked-in allele - Produce Mice Mate with mice expressing ZP3-Cre Knockout allele Essentials of Glycobiology Lecture 2 March 30, 2004 Ajit Varki General pathways for Biosynthesis Biological roles Evolutionary considerations
