Characterization of an antibody to the cross-reacting determinant of the glycosyl-phosphatidylinositol anchor of human membrane dipeptidase.
A polyclonal antiserum raised to the phospholipase C-solubilized form of membrane dipeptidase (EC 3.4.13.11) purified from human kidney was found to cross-react with unrelated trypanosomal and porcine glycosyl-phosphatidylinositol anchored proteins. Those antibodies recognising the cross-reacting determinant (CRD) were isolated by chromatography on a column of immobilized phospholipase C-solubilized porcine aminopeptidase P (EC 3.4.11.9), and the epitopes involved in the recognition were then characterized by immunoelectrophoretic blot analysis and by a competitive ELISA. The phospholipase C-solubilized forms of human and porcine membrane dipeptidase, porcine aminopeptidase P and trypanosome variant surface glycoprotein were recognised by the anti-CRD antiserum, and this recognition was abolished by prior treatment of the proteins with either mild acid or nitrous acid. In contrast, the detergent-solubilized, membrane-forms of human and porcine membrane dipeptidase were not recognised. Of a range of components of the glycosyl-phosphatidylinositol anchor, only inositol 1,2-cyclic monophosphate and the insulin-mimetic disaccharide, glucosaminyl-1,6-inositol 1,2-cyclic monophosphate, inhibited in the micromolar range the binding of the anti-CRD antiserum to immobilized porcine aminopeptidase P. These results indicate that the major epitope recognised by this anti-CRD antiserum is the inositol 1,2-cyclic monophosphate formed on phospholipase C cleavage of the glycosyl-phosphatidylinositol anchor.
Broomfield
S J
SJ
Department of Biochemistry and Molecular Biology, University of Leeds, UK.
Hooper
N M
NM
D000626:Aminopeptidases / Q000737:chemistry / Q000276:immunology; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D002240:Carbohydrate Sequence; D003429:Cross Reactions; D004150:Dipeptidases / Q000737:chemistry* / Q000276:immunology; D004187:Disaccharides / Q000737:chemistry / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004800:Enzymes, Immobilized; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D006801:Humans; D007122:Immunoelectrophoresis; D007295:Inositol Phosphates / Q000737:chemistry / Q000276:immunology; D008565:Membrane Proteins / Q000737:chemistry* / Q000276:immunology; D008969:Molecular Sequence Data; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases; D013552:Swine
D000906:Antibodies; D004187:Disaccharides; D004800:Enzymes, Immobilized; D000939:Epitopes; D017261:Glycosylphosphatidylinositols; D007295:Inositol Phosphates; D008565:Membrane Proteins; C079713:glucosaminyl-1,6-inositol-1,2-cyclic monophosphate; C010199:inositol cyclic phosphate; D010727:Phosphoric Diester Hydrolases; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase; D004150:Dipeptidases; C038937:dipeptidase; D043265:Phosphatidylinositol Diacylglycerol-Lyase