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(GPI)-Anchored Glycoprotein ACA ISOLATION, PURIFICATION, PRIMARY STRUCTURE DETERMINATION, and MOLECULAR PARAMETERS of ITS LIPID STRUCTURE*

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(GPI)-Anchored Glycoprotein ACA ISOLATION, PURIFICATION, PRIMARY STRUCTURE DETERMINATION, and MOLECULAR PARAMETERS of ITS LIPID STRUCTURE* THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 43, Issue of October 25, pp. 40472–40478, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. A Novel Human Erythrocyte Glycosylphosphatidylinositol (GPI)-anchored Glycoprotein ACA ISOLATION, PURIFICATION, PRIMARY STRUCTURE DETERMINATION, AND MOLECULAR PARAMETERS OF ITS LIPID STRUCTURE* Received for publication, March 12, 2002, and in revised form, August 2, 2002 Published, JBC Papers in Press, August 7, 2002, DOI 10.1074/jbc.M202416200 Zorica A. Becker Kojic´‡ and Peter Terness From the Institute of Immunology, Department of Transplantation Immunology, University of Heidelberg, Im Neuenheimer Feld 305, 69120 Heidelberg, Germany A method has been elaborated to isolate and purify up no cytoplasmic tail, and are therefore located exclusively on the to homogeneity a novel membrane glycoprotein contain- extracellular side of the plasma membrane. Glycosylated phos- ing a glycosyl-phosphatidylinositol (GPI) anchor by phoinositol-anchored molecules are a structurally diverse fam- means of salting out with ammonium sulfate (40–80% ily of biomolecules (1–3) that includes: protozoan coat compo- saturation), followed by preparative SDS-PAGE, chro- nents, activation antigens, complement regulatory proteins, matography and acetone precipitation. The preparation adhesion molecules, membrane-associated enzymes, and many obtained was homogeneous upon electrophoresis in the others glycoproteins. This type of cell membrane attachment Downloaded from presence of 0.1% SDS after reduction with 2-mercapto- implies that the protein moieties of these antigens are located ethanol. It is protein-soluble at its isoelectrical point outside the membrane, have potentially high lateral mobility, (pH 5.5) with molecular mass of 65,000 daltons. The iso- and may be released from the cell by the action of GPI-specific lated protein is linked to the membrane via glycosyl- phospholipase C. GPI anchors may be considered as a reason- phosphatidylinositol susceptible to cleavage by purified ably well defined class of the structures because important phospholipase C. The hydrophobic portion of the glyco- http://www.jbc.org/ lipid membrane anchor of the protein was radiolabeled elements are conserved over a wide range of phylogeny, from with the photoactivated reagent 3-(trifluoromethyl)-3- protozoan parasites to mammals. This anchor contains a linear 125 ␣ ␣ (m-[ I]iodophenyl)diazirine and hydrolyzed with gly- core glycan sequence of ethanolamine-PO4-6 Man 1–2Man 1– ␣ ␣ cosyl-phosphatidylinositol-specific phospholipase C, 6Man 1–4GlcNH2- 1–6myo-inositol-1-PO4-lipid. The hetero- followed by enzymatic deacetylation of the remaining geneity among different GPI-anchored proteins occurs mainly lipid. Thin-layer chromatography showed that the gen- in the lipid composition. The variant surface glycoprotein by guest on May 16, 2018 erated radiolabeled fragment migrates with the same (VSG) anchors contain only dimyristoyl phosphatidylinositol mobility as that of variant surface glycoprotein (VSG), (4), whereas GPIs of Leishmania promastigote surface protein- obtained in the same manner. In this study we describe ase and many mammalian GPI-anchored proteins including a novel erythrocyte membrane GPI-linked protein with those on the erythrocyte such as acetylcholinesterase (AchE), the structural feature of an anchor that, in contrast to decay-accelerating factor (DAF), and membrane inhibitor of other GPI-linked erythrocyte proteins, has a non-acety- reactive lysis (MIRL) (5), contain almost exclusively 1-alkyl-2- lated inositol ring and diacylglycerol rather than alkyl- acylinositol phospholipid, which appear to be characteristic of acyl glycerol as a lipid tail of the anchor. human anchors in general (6). In addition, some GPI anchors contain an additional fatty acid (palmitate) in a hydroxyester linkage to the inositol ring, which renders them resistant to the Many proteins of eukaryotic cells are anchored to the mem- action of bacterial phosphatidylinositol-specific phospholipase brane by covalent linkage to glycosyl-phosphatidylinositol C (PI-PLC) and the GPI-specific phospholipase C (GPI-PLC) (GPI)1 (1). Those proteins lack a transmembrane domain, have from Trypanosoma brucei. The basis of this phospholipase re- sistance was first described for human erythrocyte acetylcho- * The costs of publication of this article were defrayed in part by the linesterase (7), and subsequently for human erythrocyte decay- payment of page charges. This article must therefore be hereby marked accelerating factor (5). The presence of a substitution on the “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 2-position of the inositol ring would explain the PI-PLC resist- The nucleotide sequence reported in this paper has been submitted to ance of the palmitoylated anchors since the bacterial PLC the SWISS-PROT/TrEMBL Database under the accession no. P83408. enzyme operates via nucleophilic attack of the phosphorus ‡ Supported by the University of Heidelberg, Hochschulsonderpro- atom by the hydroxyl group at the 2-position of the inositol gramm II (HSP II). Present address: Otto Meyerhof Centre, Dr. Terness Laboratory, Im Neuenheimer Feld 350, 69120 Heidelberg, Germany. To ring. This finding led to the suggestion that occupation of the whom correspondence and reprint requests should be addressed. Tel.: myo-inositol 2-position hydroxy group by palmitate would au- 49-6221-380986; Fax: 49-6221-382292; E-mail: ZBecker-Kojic@ tomatically preclude the action of this enzyme (8). As shown for t-online.de. DAF and some other GPI-linked proteins they can exhibit 1 The abbreviations used are: GPI, glycosylphosphatidylinositol; differential inositol acetylation, and this structural variation is VSG, variant surface glycoprotein; AchE, acetylcholinesterase; DAF, decay-accelerating factor; MIRL, membrane inhibitor of reactive lysis; regulated in a cell-specific fashion, but can also be protein-de- PI-PLC, phosphatidylinositol specific phospholipase C; GPI-PLC, gly- pendent (9). The biological relevance of GPI anchor structural cosylphosphatidylinositol specific phospholipase C; PI-PLD, phosphati- variability among different blood cell types remains incom- dylinositol phospholipase D; CRD, cross-reacting determinant; GK, pletely understood. The presence of additional fatty acid chain glycerokinase; LDH, lactate dehydrogenase; [125I]TID, 3-(trifluoro- methyl)-3-(m[125I]iodophenyl) diazirine; TLC, thin-layer chromatogra- (acylation) in the hydrophobic domain probably improves the phy; s/mVSG, soluble/membrane VSG; IEF, isoelectrical focusing. attachment of the molecule to the external leaflet of the mem- 40472 This paper is available on line at http://www.jbc.org Novel Human Membrane Glycoprotein 40473 brane. The presence of this acyl-chain on the inositol ring in gas phase sequencer. Subsequently, a search in the human sequence erythrocyte-associated anchors renders these structures resist- data bank was performed (www.ncbi.nlm.nih.gov./entrez/query. ϭ ant to cleavage by PI-PLC and less sensitive to PI-PLD cleav- Fcgi?db protein). Cell-free Extracts—Preparation of ghosts was done according to pro- age when purified. The resistance to be released by phospho- tocol approved by the Institutional Review Board of the New York Blood lipases, either by blocking the lytic mechanism of a Center. Briefly, about 2.3 ϫ 106/ml packed red cells and erythroleuke- phosphatidyl-specific phospholipase C or by retaining mem- mia cells (K-562) were washed once with phosphate-buffered saline brane association following cleavage of phosphatidic acid by buffer at 5000 rpm for 5 min. After the supernatant was decanted, the anchor-specific phospholipase D, may enhance the membrane cells were lysed with 1:20.5 dilution of phosphate-buffered saline in distilled water and centrifuge at 15,000 rpm for 10 min. Supernatant stability of these proteins over time, consistent with the longer was decanted, and the discrete pellet of the white cells adhering to the (120 day) life span in the circulation of erythrocytes as com- centrifuge tube was removed, too (12). Resuspended ghosts were pared with leukocytes. We report here on a novel GPI-linked washed with 10 mM Tris-HCl (pH 7.8) until they became white and erythrocyte membrane protein with an anchor sensitive to fluffy. Equal volume of simple buffer was added and boiled for 2 min phospholipases C and unusual structure of the lipid portion of before subjected to SDS-PAGE using 4.5 stacking gel and 4–15% sep- protein, distinct from that of other erythrocyte (DAF, LFA, arating gel. Immunoblotting—Membrane proteins (10–30 ␮g) prepared from the MIRL, and acetylcholinesterase) proteins. erythrocyte ghosts and homogenized tissue of erythroleukemia (K-652) cells were mixed with reducing sample buffer and subjected to SDS- EXPERIMENTAL PROCEDURES PAGE and then transferred to nitrocellulose membrane. The filters Purification and Isolation of Protein—Human blood withdrawn from were blocked with 3% bovine serum albumin in phosphate-buffered healthy donor was centrifuged at 1500 ϫ g at 4 °C for 10 min. Plasma saline and used for testing antibody reactivity. Polyclonal mouse serum and buffy coat were removed by aspiration, and packed cells were against a 65-kDa GPI-protein was used at a dilution of 1:5000. Visual- washed twice in 150 mM/15 mM sodium phosphate (pH 7.6) and lysed in ization was performed using anti-IgG secondary antibody and conju- ϫ CH3COOH/distilled
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