Folate binding protein from kidney brush border membranes contains components characteristic of a glycoinositol phospholipid anchor

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-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.

Folate binding protein: molecular characterization and transcript distribution in pig liver, kidney and jejunum

“…Conceptually, the folate-FBP complex might be internalized in caveolae by a process termed potocytosis, following which folate is dissociated by low pH and crosses into the cytoplasm by an anion carrier and is polyglutamylated while the FBP recycles back to the cell surface [29]. Although this concept was developed from studies with isolated monkey kidney MA104 cells [30], it is also applicable to the proposed mechanism for folate uptake by Caco-2 cells [8] and the rat kidney proximal tubule [31] in which uptake was prevented by the cleavage of membrane-bound [$H]folic acid from transfected cells and isolated rat kidney brush border membranes by phosphatidylinositol phospholipase C [8,31]. The sequence analysis of pig liver FBP might be consistent with the glycosylphosphatidylinositol anchoring system, because 9 of the 14 C-terminal residues were identical with those described in isolated human Caco-2 and KB cells ( Figure 2) [2,7,8].…”

Folate and homocysteine metabolisms and their roles in the biochemical basis of neuropsychiatry

“…There are 3 physiological mechanisms for transportation from the cell membrane. The first is transport by the folate-binding proteins/folate receptors (FRs) family, which moves folate into the cell unidirectionally (8). FRs operate by receptor-mediated endocytosis.…”

Compositional analysis of Glucosaminyl(acyl)phosphatidylinositol Accumulated in HeLa S3 Cells

“…6 . The quantity of GlcN was calculated from the previously calibrated specific radioactivity of stock [ 14C]HCH0 (69 cpdpmol) (Haas and Rosenberry, 1985;Lee et al, 1992). Samples were run on TLC, and the radioactive band corresponding to GlcN(acy1)PtdIns or GlcN(acy1)inositol were scraped, subjected to acid methanolysis, and analyzed for fatty acid methyl esters.…”

“…These data indicate that both lipids a and b contain a hexosamine with a free amine group. This hexosamine was identified as GlcN by reductive radiomethylation, a technique used previously to detect and quantitate GlcN and Etn in glycosyl-PtdIns-anchored proteins Medof et al, 1986;Fatemi et al, 1987;Lee et a]., 1992) and in free glycosyl-Ptdlns species (Deeg et al, 1992b). The main peaks detected by cation-exchange chromatography of acid hydrolyzates from [ "Clmethylated lipid a had retention times corresponding to GlcN(Me)2 and Gl~N(Me)~1ns (Fig.…”