Characterization of antibodies to the glycosyl-phosphatidylinositol membrane anchors of mammalian proteins.
Two polyclonal antisera were raised in rabbits to the phospholipase C-solubilized forms of pig renal dipeptidase (EC 3.4.13.11) and pig aminopeptidase P (EC 3.4.11.9). These antisera were purified and shown to cross-react with other glycosyl-phosphatidylinositol (G-PI)-anchored proteins isolated from pig, human and trypanosomes. The epitopes involved in this cross-reactivity were characterized by Western-blot analysis after mild acid or nitrous acid treatment of the G-PI-anchored proteins and by a competitive e.l.i.s.a. with other G-PI-anchored proteins and individual components of the anchor structure. These studies revealed that the primary epitope for both antisera is the inositol 1.2-(cyclic)monophosphate that is formed on phospholipase C cleavage of the intact G-PI anchor. Other minor epitopes, such as phosphoethanolamine, probably involve side-chain modifications to the core anchor structure that may be species-specific.
Hooper
N M
NM
Department of Biochemistry and Molecular Biology, University of Leeds, U.K.
Broomfield
S J
SJ
Turner
A J
AJ
D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification; D001667:Binding, Competitive; D015153:Blotting, Western; D002462:Cell Membrane / Q000276:immunology; D003429:Cross Reactions; D004150:Dipeptidases / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D004983:Ethanolamines / Q000276:immunology*; D006017:Glycolipids / Q000276:immunology*; D017261:Glycosylphosphatidylinositols; D006801:Humans; D006868:Hydrolysis; D007295:Inositol Phosphates / Q000276:immunology*; D007672:Kidney Cortex / Q000737:chemistry / Q000201:enzymology; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000276:immunology*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D013045:Species Specificity; D013552:Swine; D014345:Trypanosoma / Q000737:chemistry
D000906:Antibodies; D000939:Epitopes; D004983:Ethanolamines; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D007295:Inositol Phosphates; D010716:Phosphatidylinositols; C010199:inositol cyclic phosphate; C005448:phosphorylethanolamine; D010727:Phosphoric Diester Hydrolases; D004150:Dipeptidases; C038937:dipeptidase; D043265:Phosphatidylinositol Diacylglycerol-Lyase