essentials of glycobiology lecture 2 april 2, 2002 ajit varki general pathways for biosynthesis...

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


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


Lecture 2

April 2, 2002

Ajit Varki


Biological roles


“...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




ANCHORANCHOR




GLYCOSAMINO-GLYCOSAMINO-GLYCANSGLYCANS HEPARAN SULFATEHEPARAN SULFATE





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


ANCHORANCHOR

OSer/Thr

NAsn

Ser-O-



GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPIDOUTSIDE

INSIDE

NAsn

S S S

-O-SerS SSS S

GLYCOSAMINO-GLYCOSAMINO-GLYCANSGLYCANS

EtnP

INOSITOL

P

NH

Ac

P

NS NS


Ac

S

2

P




HEPARAN SULFATEHEPARAN SULFATE



LETHAL

Elimination or

Alteration of Major Glycan

Classes in vivo causes

Embryonic Lethality

OSer


ANCHORANCHOR

OSer/Thr

NAsn

Ser-O-



GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPIDOUTSIDE

INSIDE

NAsn

S S S

-O-SerS SSS S

GLYCOSAMINO-GLYCOSAMINO-GLYCANSGLYCANS

EtnP

INOSITOL

P

NH

Ac

P

NS NS


Ac

S

2

P




HEPARAN SULFATEHEPARAN SULFATE



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


Lecture 2

April 2, 2002

Ajit Varki


Biological roles


“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?


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


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


Lecture 2

April 2, 2002

Ajit Varki


Biological roles


essentials of glycobiology lecture 2 april 2, 2002 ajit varki general pathways for biosynthesis...

Documents

sia sia

sia gal

outside inside n asn

g g g g g g

sequences sia

sialic acid sia

biological roles of

p udp udp ump slide