essentials of glycobiology lecture 2 april 2, 2002 ajit varki general pathways for biosynthesis...
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Essentials of Glycobiology
Lecture 2
April 2, 2002
Ajit Varki
General pathways for Biosynthesis
Biological roles
Evolutionary considerations
Major Glycan
Classes in Animal Cells
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
ProteoglycanProteoglycan
GLYCOPHOSPHO-GLYCOPHOSPHO-LIPIDLIPID
ANCHORANCHOR
N-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
Biosynthesis of different classes of glycans within the ER-Golgi Pathway
SECRETORYGRANULE LYSOSOMEENDOSOME
GOLGIAPPARATUS
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
OSer/Thr
NAsn
N-LINKED CHAINN-LINKED CHAIN
O-LINKED CHAINO-LINKED CHAIN
GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPID
OUTSIDE
INSIDE
S
CELLMEMBRANE
Membrane ProteinMembrane Protein
OSer/Thr
NAsn
S
= Sialic acid
Secreted ProteinSecreted Protein
Some Sialic Acid (Sia) Terminated Sequences
Sia2-6Gal1-4GlcNAc1- Sia2-3Gal1-(3)4GlcNAc1-
Sia2-8Sia2-3Gal1-4Glc1-
Sia2-3Gal1-3GalNAc(1-
Sia2-8Sia2-3Gal1-3GalNAc1-
Sia2-3Gal1-3GlcNAc1- 6 2Sia
Gal1-(3)4GlcNAc1-
Gal1-3GalNAc(1-
Sia2-3Gal1-3GalNAc1- 6 2Sia
Gal1-3GalNAc1- 6 2Sia
Sia2-6GalNAc1-
GalNAc(1-
Sia2-3Gal1-(3)4GlcNAc1- (4)3 1Fuc
Sialyl-Lewis X(A)
-6P
UDP-
Degradation and Recycling of
Glycans
Cytosol
Golgi
Lysosome
ER
113
2
1
1
Glucose Galactose 1= Transporter 2= Transferase 3 = Acceptor
3
1
UTPPPi
-1P UDP-
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
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
ProteoglycanProteoglycan
GLYCOPHOSPHO-GLYCOPHOSPHO-LIPIDLIPID
ANCHORANCHOR
N-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
FUNCTIONAL EFFECTS OF MODIFYING OR ELIMINATING N-LINKED CHAINS ON GLYCOPROTEINS
Biosynthesis and foldingStability in the ERSecretion rateIntracellular trafficking Cell surface expressionIntracellular stability and turnover rateRange or specificity of functionActivity of enzymes, hormones & cytokinesSignal transduction function of receptorsSusceptibility to proteases or denaturantsRecognition by antibodiesCirculatory half-lifeTargetting to specific cell types or organs
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
Biological Roles of Glycans
Structural/Physical
M
M = Micro- organism or Toxin
EndogenousRecognition
= Self
ENDOGENOUS RECEPTOR
SELF
ExogenousRecognition= Non-self
EXOGENOUS RECEPTOR
SIALYLATED OLIGOSACCHARIDE =
SELF
MolecularMimicry
Elimination of many Major Glycan
Classes still permits Cell
Viability in vitro
OSer
GLYCOPHOSPHO-GLYCOPHOSPHO-LIPIDLIPID
ANCHORANCHOR
OSer/Thr
NAsn
Ser-O-
N-LINKED CHAINSN-LINKED CHAINS
O-LINKED O-LINKED CHAINCHAIN
GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPIDOUTSIDE
INSIDE
NAsn
S S S
-O-SerS SSS S
GLYCOSAMINO-GLYCOSAMINO-GLYCANSGLYCANS
EtnP
INOSITOL
P
NH
Ac
P
NS NS
O-LINKED GlcNAcO-LINKED GlcNAc
Ac
S
2
P
GlycoproteinGlycoprotein
ProteoglycanProteoglycan
HYALURONANHYALURONAN
HEPARAN SULFATEHEPARAN SULFATE
CHONDROITINCHONDROITIN SULFATESULFATE
Sialic AcidsSialic Acids
LETHAL
Elimination or
Alteration of Major Glycan
Classes in vivo causes
Embryonic Lethality
OSer
GLYCOPHOSPHO-GLYCOPHOSPHO-LIPIDLIPID
ANCHORANCHOR
OSer/Thr
NAsn
Ser-O-
N-LINKED CHAINSN-LINKED CHAINS
O-LINKED O-LINKED CHAINCHAIN
GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPIDOUTSIDE
INSIDE
NAsn
S S S
-O-SerS SSS S
GLYCOSAMINO-GLYCOSAMINO-GLYCANSGLYCANS
EtnP
INOSITOL
P
NH
Ac
P
NS NS
O-LINKED GlcNAcO-LINKED GlcNAc
Ac
S
2
P
GlycoproteinGlycoprotein
ProteoglycanProteoglycan
HYALURONANHYALURONAN
HEPARAN SULFATEHEPARAN SULFATE
CHONDROITINCHONDROITIN SULFATESULFATE
Sialic AcidsSialic 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 EdThe John Hopkins University pressBaltimore 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 oligosaccharide recognition is more common?
EndogenousRecognition
Structural
ExogenousRecognition Endogenous
Recognition
Structural
ExogenousRecognition
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
MSELF
MSELF
MSELF
Ac
Ac
MSELF
MSELF
Change linkage
Add modification
Mask with new residue
Add branch
Substitute residue
Exogenous oligosaccharide recognition may be much commoner than endogenous recognition
EndogenousRecognition
Structural
ExogenousRecognition
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 miceDefine 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
GlycomeGLYCANS
(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 3 Species 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 antibodiesInterfere with specific oligosaccharides by lectins or
antibodiesMetabolic inhibition or alteration of glycosylationFind natural oligosaccharide ligands for specific receptorsFind receptors recognizing specific oligosaccharidesEliminate specific receptors by gene targettingEliminate specific oligosaccharides by glycosidasesStudy natural glycosylation mutants in intact animalsConstruct 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 EXPRESSIONLIBRARIES
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 GLYCOSYLATIONCORE OLIGOSACCHARIDELECTIN RECEPTOR
GLYCOSIDASE OUTER MONOSACCHARIDES
Normal
Ablate outer transferase
Ablate receptor
Overexpress transferase
Express "masking"
transferase
Overexpress "competing"transferase
Express membrane-
boundglycosidase
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
tkneo ES Cell with targeted allele
tkneo 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