A novel human erythrocyte glycosylphosphatidylinositol (GPI)-anchored glycoprotein ACA. Isolation, purification, primary structure determination, and molecular parameters of its lipid structure.
A method has been elaborated to isolate and purify up to homogeneity a novel membrane glycoprotein containing a glycosyl-phosphatidylinositol (GPI) anchor by means of salting out with ammonium sulfate (40-80% saturation), followed by preparative SDS-PAGE, chromatography and acetone precipitation. The preparation obtained was homogeneous upon electrophoresis in the presence of 0.1% SDS after reduction with 2-mercaptoethanol. It is protein-soluble at its isoelectrical point (pH 5.5) with molecular mass of 65,000 daltons. The isolated protein is linked to the membrane via glycosyl-phosphatidylinositol susceptible to cleavage by purified phospholipase C. The hydrophobic portion of the glycolipid membrane anchor of the protein was radiolabeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine and hydrolyzed with glycosyl-phosphatidylinositol-specific phospholipase C, followed by enzymatic deacetylation of the remaining lipid. Thin-layer chromatography showed that the generated radiolabeled fragment migrates with the same mobility as that of variant surface glycoprotein (VSG), obtained in the same manner. In this study we describe a novel erythrocyte membrane GPI-linked protein with the structural feature of an anchor that, in contrast to other GPI-linked erythrocyte proteins, has a non-acetylated inositol ring and diacylglycerol rather than alkyl-acyl glycerol as a lipid tail of the anchor.
Becker Kojić
Zorica A
ZA
Institute of Immunology, Department of Transplantation Immunology, University of Heidelberg, Otto Meyerhof Centre, Terness Laboratory, Im Neuenheimer Feld 305, 69120 Heidelberg, Germany. ZBecker-Kojic@t-online.de
Terness
Peter
P
D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification*; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010449:Peptide Mapping
C468767:ACA protein, human; D001798:Blood Proteins; D000939:Epitopes; D017261:Glycosylphosphatidylinositols; D008055:Lipids; D008562:Membrane Glycoproteins