Folate binding protein from kidney brush border membranes contains components characteristic of a glycoinositol phospholipid anchor.
A number of cell surface proteins have been shown to be anchored to the plasma membrane by a covalently attached glycoinositol phospholipid (GPL) in amide linkage to the C-terminus of the mature protein. We applied several criteria to establish that folate binding protein (FBP) in brush border membranes of rat kidney contains a GPL anchor. Brush border membranes were isolated and labeled with [3H]folate, and the complex of FBP and [3H]folate was shown to be released to the supernatant by incubation with purified bacterial phosphatidylinositol-specific phospholipase C (PIPLC) but not by incubation with a purified bacterial phosphatidylcholine-specific phospholipase C. The FBP-[3H]folate complex both in crude extracts and after FBP purification by ligand-directed affinity chromatography interacted with Triton X-114 micelles, and prior incubation with PIPLC prevented this detergent interaction. Individual residues characteristic of GPL anchors were found to be covalently associated with FBP following polyacrylamide gel electrophoresis in sodium dodecyl sulfate. These included glucosamine and ethanolamine, which were radiolabeled by reductive methylation and identified by chromatography on an amino acid analyzer, and inositol phosphate, which was inferred by Western blotting with an anti-CRD antisera. This antisera gave positive immunostaining only after FBP had been cleaved by PIPLC, a reliable diagnostic of a GPL anchor. The relationship between GPL-anchored FBP in biological membranes and soluble FBP in biological fluids also is discussed.
United States Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts 02111.
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Lee
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Shoda
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Krall
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Foster
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Selhub
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Rosenberry
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D002417:Cattle; D003429:Cross Reactions; D003902:Detergents; D000939:Epitopes / Q000276:immunology; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000737:chemistry*; D006017:Glycolipids / Q000032:analysis* / Q000276:immunology; D017261:Glycosylphosphatidylinositols; D006801:Humans; D006868:Hydrolysis; D007106:Immune Sera; D007668:Kidney / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure*; D008745:Methylation; D008871:Microvilli / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology; D017830:Octoxynol; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000032:analysis* / Q000276:immunology; D054801:Phosphoinositide Phospholipase C; D010740:Phospholipases; D010727:Phosphoric Diester Hydrolases; D011092:Polyethylene Glycols; D051381:Rats; D011956:Receptors, Cell Surface
D002352:Carrier Proteins; D003902:Detergents; D000939:Epitopes; D058973:Folate Receptors, GPI-Anchored; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D007106:Immune Sera; D010716:Phosphatidylinositols; D011956:Receptors, Cell Surface; D011092:Polyethylene Glycols; D017830:Octoxynol; D005492:Folic Acid; D010740:Phospholipases; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase