Integral membrane proteins maintain intimate contact with the hydrophobic phase of phospholipid bilayer membranes and require detergents for solubilization. These proteins generally are thought to retain one or more transmembrane polypeptide segments that result from cotranslational discharge into the endoplasmic reticulum lumen. Reports on acetylcholinesterase (AChE) and decay accelerating factor (mDAF) from human erythrocytes (Ehu), trypanosome variant surface glycoproteins (mfVSGs), and Thy-1 indicate a new class of integral membrane proteins: those with a C-terminal hydrophobic anchor composed of a covalently linked glycophospholipid. Data on Ehu AChE and mDAF suggest that this anchor is composed of an oligosaccharide with glucosamine at the reducing terminus in glycosidic linkage to a phospholipid similar to phosphatidylinositol (PI). To extend this characterization, anchor fragments will be produced by protease digestion, alkaline hydrolysis, digestion with PI-specific phospholipase C, acetolysis, deamination, and methanolysis; isolated by high pressure liquid chromatography (HPLC) or thin layer chromatography (TLC); and analyzed for neutral sugars, amines fatty acids, inositol, phosphate and glycerol, and characterized by fast atom bombardment mass spectroscopy (FAB-MS).
A second aim i s to initiate biosynthetic studies of these anchors. Thy-1 and DAF in cultured cells will be radiolabeled biosynthetically with anchor precursors. Successful labeling has already been demonstrated with (3H) ethanolamine. Control cell lines, mutant lymphomas that secrete Thy-1, and DAF-positive and -negative lymphocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) will be investigated. Incorporation of anchor precursor radiolabel into secreted forms of DAF and Thy-1 will be compared to the incorporation into membrane-bound forms of these proteins to determine whether anchor metabolism is involved in secretion.
A third aim i s to explore the possibility that an anchor glycolipid is synthesized as a discrete precursor and transferred en bloc to the polypeptide. Such a precursor will be pursued by systematic analyses of radiolabeled components that appear in chloroform-methanol and aqueous extracts during the biosynthetic labeling experiments.
A final aim i s to seek more abundant proteins with glycolipid anchors. These proteins will be isolated and monoclonal antibodies will be prepared, both to extend the studies of anchor biosynthesis and to identify and characterize the new proteins.
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