Presentation on theme: "Essentials of Glycobiology Lecture 8 April 8, 2004 Hud Freeze Structure, biosynthesis and general biology of Glycophospholipid (GPI) Anchors."— Presentation transcript:
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 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 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
Discovery of GPI-Anchors. 1963 1963-First data suggests protein-lipid anchors: crude bacterial phospholipase C releases alkaline phosphatase from mammalian cells. mid-1970’s mid-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-Anchors The 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!!!
Proposed branched pathway for biosynthesis of mammalian GPI anchors
Examples of C-Terminal Sequences Signaling 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 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 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
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 MUTATIONS IN DOL-P-MAN SYN AND 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.
PATIENT HAS MPDU1 DEFICIENCY Inefficient 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