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Essentials of Glycobiology Lecture 8 April 8, 2004 Hud Freeze

Essentials of Glycobiology Lecture 8 April 8, 2004 Hud Freeze. Structure, biosynthesis and general biology of Glycophospholipid (GPI) Anchors. CHONDROITIN SULFATE. HYALURONAN. GLYCOSAMINO- GLYCANS. HEPARAN SULFATE. N-LINKED CHAINS. O-LINKED CHAIN. GLYCOPHOSPHO- LIPID ANCHOR.

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Essentials of Glycobiology Lecture 8 April 8, 2004 Hud Freeze

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  1. Essentials of Glycobiology Lecture 8April 8, 2004Hud Freeze Structure, biosynthesis and general biology of Glycophospholipid (GPI) Anchors

  2. CHONDROITIN SULFATE HYALURONAN GLYCOSAMINO- GLYCANS HEPARAN SULFATE N-LINKED CHAINS O-LINKED CHAIN GLYCOPHOSPHO- LIPID ANCHOR GLYCOSPHINGOLIPID O-LINKED GlcNAc Major Glycan Classes in Animal Cells P S S S Ser-O- S S S S S -O-Ser NS NS Proteoglycan Ac P Etn S P N O N NH Asn Ser/Thr 2 Asn INOSITOL Glycoprotein Ac OUTSIDE P Sialic Acids INSIDE O Ser

  3. Basic Glycosylphosphatidylinositol (GPI) Anchor Phospholipid

  4. 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 Lecture Overview

  5. 1963-First data suggests protein-lipid anchors: crude bacterial phospholipase C releases alkaline phosphatase from mammalian cells. 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.

  6. 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

  7. 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). Discovery of GPI-Anchors. • THE LESSON: SHOW ME THE STRUCTURE!!!

  8. Basic Glycosylphosphatidylinositol (GPI) Anchor Phospholipid

  9. Cell surface hydrolases Protozoal antigens alkaline phosphatase trypanosome VSG acetylcholinesterase leishmanial protease 5’ nucleotidase plasmodium antigens Adhesion molecules Mammalian antigens neural cell adhesion molecule carcinoembryonic antigen heparan sulfate proteoglycan Thy-1 Others scrapie prion protein folate receptor decay accelerating factor Examples of GPI-Anchored Proteins

  10. Structure of the Basic GPI Anchor

  11. PLANTS ALSO MAKE GPI-ANCHORS Casper Vroemen,http://www.dpw.wau.nl/molbi/mediacenter/images/embryo11.jpg

  12. Studying GPI Biosynthesis invitro thin layer chromatography F 30 °C O add solvents cell membranes spin salts, buffers radiolabeled sugardonor evaporate O F

  13. Structural Analysis of the GPI AnchorEnzymatic and chemical cleavage sites are useful in identifying GPI anchored membrane proteins

  14. Proposed branched pathway for biosynthesis of mammalian GPI anchors

  15. 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

  16. Rules for C-Terminal Sequences Signaling the Addition of GPI-Anchors • Residue to which anchor is attached (termed w site) and residue two amino acids on carboxyl side (w + 2 site) always have small side-chains • w + 1 site can have large side-chains. • w + 2 site followed by 5 to 10 hydrophilic amino acids, • Next, add fifteen to twenty hydrophobic amino acids at or near the carboxy-terminus

  17. 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

  18. Possible Role of the GPI-Anchor in ER Exit

  19. UPS AND DOWNS OF GPI-LINKED PLACENTAL ALKALINE PHOSPHATASE Frances J. Sharom www.chembio.uoguelph.ca/ sharom/

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

  21. GLYCOSPHINGOLIPID CHONDROITIN SULFATE HYALURONAN Paroxysmal Nocturnal Hemoglobinuria: Somatic Loss of Glycophospholipid Anchors in Hematopoietic StemCells GLYCOSAMINO- GLYCANS P S S S Ser-O- HEPARAN SULFATE S S S S S -O-Ser NS NS N-LINKED CHAINS Ac O-LINKED CHAIN GLYCOPHOSPHO- LIPID ANCHOR P Etn S P N O N NH Asn Ser/Thr 2 Asn INOSITOL Glycoprotein Ac OUTSIDE P Sialic Acids INSIDE O-LINKED GlcNAc O Ser

  22. Taroh Kinoshita

  23. Mutation in PNH MUTATIONS IN DOL-P-MAN SYN AND USE • The first step in biosynthesis of the GPI anchor requires at least four genes • One of them, PIG-A is an X-linked gene Biosynthesis of GPI anchors

  24. 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

  25. 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. Paroxysmal Nocturnal Hemoglobinuria

  26. NON-POLITICAL FLIP-FLOP: A GOOD THING

  27. Dol

  28. CDG-If

  29. PTPATIENT WITH DPM1 DEFICIENCY Decreased Dol-P-Man synthesis PATIENT HAS MPDU1 DEFICIENCY Inefficient use of Dol-P-Man and Dol-P-Glc Both patients are blind with severe developmental delay Pathology may result from impaired N-linked or GPI-anchor synthesis

  30. FUTURE PERSPECTIVES THE FUNCTION OF GPI-ANCHORS IS STILL UNRESOLVED LIKE THE FUNCTIONS OF GLYCOYSLATION MAYBE ALL THE THEORIES ARE CORRECT

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