GPI-anchored glycoproteins Mike Ferguson

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1 GPI-Anchored Glycoproteins Prof. College of Life Sciences University of Dundee UK 1 What are GPI membrane anchors? GPI stands for glycosylphosphatidylinositol In other words, a phosphatidylinositol (PI) phospholipid linked via a glycosyl, or sugar chain, component to the C-terminus of a protein 2 How are proteins bound to membranes? GPI-anchored proteins 3 1

2 Examples of GPI-anchored proteins 4 GPI-anchored Protein Protozoa Trypanosoma brucei variant surface glycoprotein (VSG) Leishmania major promastigote surface protease (PSP) Trypanosoma cruzi GPI-anchored mucins Plasmodium falciparum merozoite surface protein 1 (MSP-1) Toxoplasma gondii surface antigen 1 (SAG-1) Entamoeba histolytica GPI proteophosphoglycans Function Protective coat Bound complement degradation Host cell invasion Erythrocyte invasion Host cell invasion Virulence factor Examples of GPI-anchored proteins (2) 5 GPI-anchored Protein Yeast, fungi, slime mold Saccharomyces cerevisiae a-agglutinin Saccharomyces cerevisiae GAS1p Aspergillus fumigatus GEL1p Candida albicans HWP1 Dictyostelium discoideum prespore antigen (PsA) Dictyostelium discoideum contact-site A (CsA) Plants Pyrus arabinogalactan proteins (AGP) Arabidopsis thaliana metallo and aspartyl proteases Arabidopsis thaliana b1-3 glucanase Function Adhesion molecule Cell-wall biogenesis Cell-wall biogenesis Adhesion molecule Adhesion molecule Adhesion molecule Cell-wall biogenesis Pollen tube development Cell-wall biogenesis Examples of GPI-anchored proteins (3) 6 GPI-anchored Protein Mammals Erythrocyte CD59 and decay acceleration factor (DAF) Alkaline phosphatase 5 -Nucleotidase Renal dipeptidase Trehalase a Neural cell adhesion molecule 120 (NCAM-120) Neural cell adhesion molecule TAG-1 a CD58 a FcgIII receptor Ciliary neurotrophic factor receptor (CNTFR) a subunit Glial-cell-derived neurotrophic factor receptor (GDNFR) a CD14 Prion protein (PrP) Glypican family of GPI-anchored proteoglycans Function Complement regulation Cell-surface hydrolase Cell-surface hydrolase Cell-surface hydrolase Cell-surface hydrolase Adhesion molecule Adhesion molecule Adhesion molecule Fc receptor Neural receptor Neural receptor LPS receptor Unknown Extracellular matrix 2

3 Features of GPI-anchored proteins and their processing by GPI transamidase 7 GPI transamidase 8 N = Nucleophile (e.g. Thiol) Ohishi Furukawa et al., et al., The Biocimica EMBO Journal et Biophysica (2001)20: Acta Biomembranes (1997) 1328 (2): Maeda Y, Kinoshita T (2011) Structural remodeling, trafficking and functions 9of glycosylphosphatidylinositol-anchored proteins; Progress in Lipid Research 50:

4 10 Maeda Y, Kinoshita T (2011) Structural remodeling, trafficking and functions of glycosylphosphatidylinositol-anchored proteins; Progress in Lipid Research 50: Maeda Y, Kinoshita T (2011) Structural remodeling, trafficking and functions of glycosylphosphatidylinositol-anchored proteins; Progress in Lipid Research 50: Maeda Y, Kinoshita T (2011) Structural remodeling, trafficking and functions of glycosylphosphatidylinositol-anchored proteins; Progress in Lipid Research 50:

5 13 Maeda Y, Kinoshita T (2011) Structural remodeling, trafficking and functions of glycosylphosphatidylinositol-anchored proteins; Progress in Lipid Research 50: Maeda Y, Kinoshita T (2011) Structural remodeling, trafficking and functions of glycosylphosphatidylinositol-anchored proteins; Progress in Lipid Research 50: Maeda Y, Kinoshita T (2011) Structural remodeling, trafficking and functions of glycosylphosphatidylinositol-anchored proteins; Progress in Lipid Research 50:

6 General structure of GPI anchors Human African Sleeping Sickness Trypanosoma brucei 18 6

7 Transmission EM Scanning EM 19 The surface coat is made of a dense monolayer of variant surface glycoprotein (VSG) (Prof. George Cross FRS) Uniform thickness 20 Many protein variants Common GPI anchor 21 7

8 First GPI structure solved using about 1 g of VSG (could only have been done with trypanosome VSG 10 million copies per cell) Break into component parts 24 8

9 Remove contaminants Myristate Galactose Glucosamine Glycerol Mannose Inositol Phosphate Ethanolamine 25 Find some associations Dimyrisoylglycerol 26 Find some more associations 27 Glucosamine-(dimyristoyl)phosphatidylinositol 9

10 Complete the associations with NMR and MS 28 Chemical and enzymatic reactions of GPI anchors 29 Essentials of Glycobiology, Second Edition, Chapter 11, Figure 3 30 Ferguson MAJ, Kinoshita T, Hart GW (2009) Glycosylphosphatidylinositol Anchors; In: Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P et al., editors; Essentials of Glycobiology, 2nd ed. Cold Spring Harbor (NY) 10

11 31 Differences in parasite and host GPI pathways T. brucei Human 32 Güther and Ferguson EMBO J. (1995) The cell-free system 33 11

12 The modified cell-free system 34 Substrate retrosynthetic strategy 1 2 D 6 1 D 6 1 NHEt 3 NHEt 3 35 An example of testing for (a) substrates and (b) inhibitors of the T. brucei GPI pathway using the cell-free system GPIs 36 Lane 2: +ve control, add synthetic substrate (GPI bands formed) 12

13 Both a and b forms recognised by T. brucei but only a by HeLa 2-OH not needed by T. brucei but essential for HeLa Any lipid will do ( can simplify lipid stereochemistry) 37 This allows an opportunity to make selective inhibitors Proposed mechanism of action for the GlcNAc-PI de-n-acetylase 38 Urbaniak et al., JBC, 280, p2831 Postulated transglycosylation reaction in which a GPI-cell wall protein becomes cross-linked to cell wall b1,6-glucan via its GPI glycan 39 Marlyn Gonzalez, Peter N. Lipke, Rafael Ovalle; Chapter 15 GPI Proteins in Biogenesis and Structure of Yeast Cell Walls The Enzymes, Volume 26, 2009, Pages

14 General functions of GPI anchors Stable association of proteins with the plasma membrane, but with measurable off-rates from the membrane and the potential to be shed by phosholipases The potential for very high lateral mobility on the plane of the lipid bilayer The potential to insulate the protein domain from the cell interior (may be important in protozoa) The potential to participate in signaling through association with other membrane-spanning components in lipid rafts 40 Lipid rafts 41 Some membrane components are excluded from the rafts See: Leslie, M. (2011) Do Lipid Rafts Exist? Science, 334: for a recent discussion on lipid rafts Thank you for listening! The research on GPI anchors is supported by The Wellcome Trust 42 14

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