Folate Binding Protein from Kidney Brush Border Membranes Contains Components Characteristic of a Glycoinositol Phospholipid Anchor

He Chung Lee, Ryosuke Shoda, Jacob Selhub, Jennifer A. Krall, John D. Foster, Terrone L. Rosenberry

Research output: Contribution to journalArticle

30 Scopus citations

Abstract

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-l 14 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 immu-nostaining 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.

Original languageEnglish (US)
Pages (from-to)3236-3243
Number of pages8
JournalBiochemistry
Volume31
Issue number12
DOIs
StatePublished - 1992

ASJC Scopus subject areas

  • Biochemistry

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