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 O Ser O Ser/Thr N Asn Ser-O- OUTSIDE INSIDE N Asn S SS -O-Ser S S S SS Etn P INOSITOL P NH Ac P NS Ac S 2 P Glycoprotein ProteoglycanGLYCOPHOSPHO-LIPIDANCHOR N-LINKED CHAINS O-LINKEDCHAIN HYALURONAN GLYCOSAMINO-GLYCANS HEPARAN SULFATE CHONDROITIN SULFATE SULFATE Sialic Acids GLYCOSPHINGOLIPID O-LINKED GlcNAc

Lecture Overview Historical Background of Discovery Defining the Core Structure Biosynthesis & Transfer of GPI Anchors The Signal for Addition of GPI Anchors Occurrence and Variations in Nature Postulated Biological Roles Genetic Disorders Perspectives & Future Directions

Historical Background of the Discovery of GPI-Anchors. First data suggesting existence of protein-lipid anchors in 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) 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 Proteins Cell surface hydrolases alkaline phosphatase acetylcholinesterase 5’ nucleotidase Adhesion molecules neural cell adhesion molecule heparan sulfate proteoglycan Others decay accelerating factor scrapie prion protein folate receptor Protozoal antigens trypanosome VSG leishmanial protease plasmodium antigens Mammalian antigens Thy-1 carcinoembryonic antigen

Studying GPI Biosynthesis in vitro cell membranes salts,buffers radiolabeled sugardonor 30 °C add solvents spin evaporate F O thin layer chromatography OF

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 Functions Dense packing of Proteins on Cell Surface Increased Protein mobility on Cell Surface Targeting of proteins to Apical Domains Specific release from Cell Surface Control of Exit from ER? Developmental regulation of protein expression? Generation of Protein Complexity Signal transduction? Toxin Binding Parasite Cell structure

Possible Role of the GPI-Anchor in ER Exit

Sialic Acids GLYCOSPHINGOLIPID Ac O-LINKED GlcNAc O Ser O Ser/Thr N Asn Ser-O- N-LINKED CHAINS O-LINKEDCHAIN N Asn S SS -O-Ser S S S SS GLYCOSAMINO-GLYCANS P NS Ac S P Glycoprotein HYALURONAN HEPARAN SULFATE CHONDROITIN SULFATE SULFATE Paroxysmal Nocturnal Hemoglobinuria: Somatic Loss of Glycophospholipid Anchors in Hematopoietic Stem Cells INSIDE OUTSIDE INOSITOL P NH 2 GLYCOPHOSPHO-LIPIDANCHOR Etn P

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) Anchor Phospholipid