Essentials of Glycobiology

Lecture 8

April 8, 2004

Hud Freeze

Structure, biosynthesis and general biology of

Glycophospholipid (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

Basic Glycosylphosphatidylinositol (GPI) AnchorBasic Glycosylphosphatidylinositol (GPI) Anchor

Protein

Glycan

Phospholipid

Lecture Overview

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

Discovery of GPI-AnchorsDiscovery of GPI-Anchors..

19631963-First data suggests protein-lipid anchors: crude bacterial phospholipase C releases alkaline phosphatase from mammalian cells.

mid-1970’smid-1970’s Hiro Ikezawa in Japan, and Martin Low in the U.S. show that purified bacterial phosphatidylinositol phospholipase C releases some enzymes, e.g.,alkaline phosphatase, from cell surfaces. Propose Inositol-containing phospholipid protein linkage

Alan Williams in U.K. notes that antigen Thy-1 properties of glycolipid and glycoprotein.

However: No structural data!

GPI-anchors? Really?

Discovery of GPI-AnchorsDiscovery of GPI-AnchorsThe C-terminus of Thy-1 glycoprotein found to have 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. mfVSG becomes water soluble.

sVSG so rapid membrane form is only detected by rapidly boiling trypanosomes in (SDS) prior to electrophoresis.

Discovery of GPI-Anchors. 1985: Hart & Englund groups at Johns Hopkins show

that the lipid-anchor on VSG is added within one minute of the polypeptide’s synthesis in the endoplasmic reticulum (ER). They postulate a pre-assembled membrane anchor is attached en bloc.

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

THE LESSON: SHOW ME THE STRUCTURE!!!THE LESSON: SHOW ME THE STRUCTURE!!!

Basic Glycosylphosphatidylinositol (GPI) AnchorBasic Glycosylphosphatidylinositol (GPI) Anchor

Protein

Glycan

Phospholipid

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

Structure of the Basic GPI Anchor

EtnP

INOSITOL

P

= Mannose (Man)

= Glucosamine

Etn = Ethanolamine

P = Phosphate

NH2

NH2Pig-A

PNH Defect

GPI-Linked Protein

Cell SurfaceMembrane

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PLANTS ALSO MAKE GPI-ANCHORS

Casper Vroemen,http://www.dpw.wau.nl/molbi/mediacenter/images/embryo11.jpg

Studying GPI Biosynthesis in vitroStudying GPI Biosynthesis in vitro

cell membranessalts, buffersradiolabeled sugardonor

30 °C

add solventsspin

evaporate

F

O

thin layerchromatography

O F

Structural Analysis Structural Analysis of the GPI Anchorof the GPI Anchor

Enzymatic and Enzymatic and chemical chemical

cleavage sites cleavage sites are useful in are useful in identifying identifying

GPI anchored GPI anchored membrane proteinsmembrane proteins

Proposed branched

pathway for biosynthesis of mammalian GPI

anchors

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

Protein GPI-Signal Sequence

Acetylcholinesterase (Torpedo) NQFLPKLLNATAC DGELSSSGTSSSKGIIFYVLFSILYLIFY

Alkaline Phosphatase (placenta) TACDLAPPAGTTD AAHPGRSVVPALLPLLAGTLLLLETATAP

Decay Accelerating Factor HETTPNKGSGTTS GTTRLLSGHTCFTLTGLLGTLVTMGLLT

PARP (T. Brucei) EPEPEPEPEPEPG AATLKSVALPFAIAAAALVAAF

Prion Protein (hamster) QKESQAYYDGRRS SAVLFSSPPVILLISFLIFLMVG

Thy-1 (rat) KTINVIRDKLVKC GGISLLVQNTSWLLLLLLSLSFLQATDFISI

Variant Surface Glycoprotein (T. Brucei) ESNCKWENNACKD SSILVTKKFALTVVSAAFVALLF

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

Rules for C-Terminal Sequences Signaling 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,

Next, add fifteen to twenty hydrophobic amino acids at or near the carboxy-terminus

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

- -

transport vesicles

+

+

transport vesicles

Positive Signal: Anchor is recognized by packaging machinery

Negative Signal: Retention mechanism is displaced by anchor

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UPS AND DOWNS OF GPI-LINKED PLACENTAL ALKALINE PHOSPHATASE

Frances J. Sharom www.chembio.uoguelph.ca/ sharom/

Sean Munro, Cell, 115, 377-388, Nov 2003

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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

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Taroh Kinoshita

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

MUTATIONS INDOL-P-MAN SYNAND USE

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

Pre 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.

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NON-POLITICAL FLIP-FLOP: A GOOD THING

Dol

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CDG-If

PATIENT HAS MPDU1 DEFICIENCYInefficient use of Dol-P-Man and Dol-P-Glc

PTPATIENT WITH DPM1 DEFICIENCY Decreased Dol-P-Man synthesis

Both patients are blind with severe developmental delay

Pathology may result from impaired N-linked or GPI-anchor synthesis

FUTURE PERSPECTIVES

THE FUNCTION OF GPI-ANCHORS IS STILL UNRESOLVED

LIKE THE FUNCTIONS OF GLYCOYSLATION

MAYBE ALL THE THEORIES ARE CORRECT