Essentials of Glycobiology

Lecture 8

April 11, 2002

Ajit Varki

Structure, biosynthesis and general biology of

Glycosphosphlipid (GPI) Anchors

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

Lecture Overview

Historical Background of DiscoveryDefining the Core Structure Biosynthesis & Transfer of GPI AnchorsThe Signal for Addition of GPI AnchorsOccurrence and Variations in NaturePostulated Biological RolesGenetic DisordersPerspectives & Future Directions

Historical Background of the Discovery of GPI-Anchors.

First data suggesting existence of protein-lipid anchors in 1963 - crude bacterial phospholipase C releases alkaline phosphatase from mammalian cells.

Inositol-containing phospholipid protein anchors postulated by Hiro Ikezawa in Japan, and Martin Low in the U.S. in mid-1970’s. Predictions were based upon ability of highly-purified bacterial phosphatidylinositol phospholipase C to release certain enzymes, such as alkaline phosphatase, from cell surfaces.

Alan William’s in U.K. independently noted that antigen Thy-1 had attributes of glycolipid and glycoprotein.

However, without supporting structural data,

existence of GPI-anchorsnot widely accepted.

Historical Background of the Discovery of GPI-Anchors. The C-terminus of Thy-1 glycoprotein was subsequently

found to contain both fatty acids and ethanolamine. In 1981, Tony Holder and George Cross groups showed

that soluble form of the variant surface glycoprotein (sVSG) of African trypanosomes contains an immuno-crossreactive carbohydrate (CRD) attached to its C-terminus via an amide linkage involving ethanolamine.

Mervyn Turner’s group showed that trypanosomes contain an enzyme which rapidly releases the membrane-associated VSG (mfVSG) upon cellular damage. Conversion of mfVSG to water soluble

sVSG so rapid membrane form is only

detected by rapidly boiling trypanosomes in

(SDS) prior to electrophoresis.

Historical Background of the Discovery of GPI-Anchors. In 1985, Hart & Englund groups at Johns Hopkins

demonstrate that the lipid-anchor on VSG is added within one minute of the polypeptide’s synthesis in the endoplasmic reticulum (ER). They postulated that the membrane anchor might be first pre-assembled in the ER and attached en bloc.

Later in 1985, Michael Ferguson and colleagues at Oxford publish a tour de force structural elucidation of the glycolipid attached to the mfVSG of trypanosomes. These studies first to structurally define the term ‘glycosyl-phosphatidylinositol’ (GPI).

Basic Glycosylphosphatidylinositol (GPI) AnchorBasic Glycosylphosphatidylinositol (GPI) Anchor

Phospholipid

Structure of the Basic GPI Anchor

Structural Analysis of the GPI Anchor

Enzymatic and chemical

cleavage sites are useful in identifying

GPI anchored membrane proteins

Examples of GPI-Anchored ProteinsExamples of GPI-Anchored Proteins

Cell surface hydrolasesalkaline phosphataseacetylcholinesterase5’ nucleotidase

Adhesion molecules

neural cell adhesion molecule

heparan sulfate proteoglycan

Others

decay accelerating factor

scrapie prion proteinfolate receptor

Protozoal antigenstrypanosome VSGleishmanial proteaseplasmodium antigens

Mammalian antigens

Thy-1carcinoembryonic antigen

Studying GPI Biosynthesis in vitroStudying GPI Biosynthesis in vitro

cell membranessalts,buffersradiolabeled sugardonor

30 °C

add solventsspin

evaporate

F

O

thin layerchromatography

O F

Biosynthesis of GPI anchors

• The first step in biosynthesis of the GPI anchor requires at least four genes

• One of them, PIG-A is an X-linked gene

Examples of C-Terminal Sequences Signalling the Addition of GPI-Anchors

Bold AA is site of GPI attachment Sequence to right is cleaved by the transpeptidase upon Anchor addition

Rules for C-Terminal Sequences Signalling the Addition of GPI-Anchors

Residue to which anchor is attached (termed site) and residue two amino acids on carboxyl side ( + 2 site) always have small side-chains

+ 1 site can have large side-chains.

+ 2 site followed by 5 to 10 hydrophilic amino acids,

This is followed by fifteen to twenty

hydrophobic amino acids at or near

the carboxy-terminus

Proposed branched

pathway for biosynthesis of mammalian GPI

anchors

GPI Anchor FunctionsGPI Anchor FunctionsDense packing of Proteins on Cell SurfaceIncreased Protein mobility on Cell SurfaceTargeting of proteins to Apical DomainsSpecific release from Cell SurfaceControl of Exit from ER?Developmental regulation of protein

expression?Generation of Protein ComplexitySignal transduction?Toxin BindingParasite Cell structure

Possible Role of the GPI-Anchor in ER ExitPossible Role of the GPI-Anchor in ER Exit

Sialic AcidsSialic Acids

GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPID Ac

O-LINKED GlcNAcO-LINKED GlcNAcOSer

OSer/Thr

NAsn

Ser-O-

N-LINKED CHAINSN-LINKED CHAINS

O-LINKED O-LINKED CHAINCHAIN

NAsn

S S S

-O-SerS SSS S

GLYCOSAMINO-GLYCOSAMINO-GLYCANSGLYCANS

P

NS NS

Ac

S

P

GlycoproteinGlycoprotein

HYALURONANHYALURONAN

HEPARAN SULFATEHEPARAN SULFATE

CHONDROITINCHONDROITIN SULFATESULFATE

Paroxysmal Nocturnal Hemoglobinuria: Somatic Loss of Glycophospholipid Anchors

in Hematopoietic Stem Cells

INSIDE

OUTSIDE

INOSITOL

P

NH2

GLYCOPHOSPHO-GLYCOPHOSPHO-LIPIDLIPID

ANCHORANCHOR EtnP

Biosynthesis of GPI anchors

• The first step in biosynthesis of the GPI anchor requires at least four genes

• One of them, PIG-A is an X-linked gene

Mutation in PNH

Paroxysmal Nocturnal Hemoglobinuria

An acquired clonal hematopoietic stem cell disorder characterized by intravascular hemolytic anemia. Abnormal blood cells lack GPI-anchored proteins due to a mutation in the PIG-A gene.

Lack of GPI-anchored complement regulatory proteins, such as decay-accelerating factor (DAF) and CD59, results in complement-mediated hemolysis and hemoglobinuria.

Factors that determine why mutant clones

expand have not been determined.

Paroxysmal Nocturnal Hemoglobinuria

It has been suggested that existing PNH clones have a conditional growth advantage depending on some factor present in the marrow environment of PNH patients.

However, cells with the PNH phenotype have been found at a frequency of 22 per million in normal individuals. These rare cells were collected by flow sorting and had PIG-A mutations.

Thus, PIG-A gene mutations are

not sufficient for the development

of clinically evident PNH.

Basic Glycosylphosphatidylinositol (GPI) AnchorBasic Glycosylphosphatidylinositol (GPI) Anchor

Phospholipid