Abstract
Membrane-associated and soluble forms of folate binding protein (FBP) have been identified in mammalian tissues and biological fluids. Despite their solubility differences, these two forms are functionally similar, immunologically cross-reacting, and have the same apparent molecular weights. In this study we demonstrate, for the first time, that the membrane FBP of cultured human KB cells contains a glycosyl-phosphatidylinositol (GPI) tail which is responsible for its hydrophobic properties and distinguishes it from the soluble FBP released into the medium. Treatment of the purified membrane FBP with phospholipase C specific for phosphatidylinositol (PI-PLC) removed the GPI tail and converted it to the soluble form without a change in apparent Mr by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, virtually all of the folate binding sites on the plasma membrane of the intact cells were released as soluble, functional FBP following treatment with PI-PLC. The GPI tail contained 1-O-alkyl-2-O-acylglycerol as a mixture of fatty alcohols in ether linkage at C1 of the glycerol backbone and almost exclusively docosanoic acid (22:0) as the fatty acid on C2. The inositol also contained a mixture of fatty acids (16:0, 18:0, 18:1, 20:4, 22:0) located on a site other than the C2 position since the FBP was susceptible to PI-PLC cleavage. After nitrous acid deamination, the aqueous portion of the FBP contained covalently bound fatty acids, predominantly palmitate (16:0) and stearate (18:0), indicating the presence of additional acyl groups attached to the peptide in the form of amide, ester, or thioester linkage.
Highlights
Two forms of folate binding protein (FBP),' soluble and membrane-associated, have been described in mammalian tissues and biological fluids [1]
One model which has been used to study the relationship between these two forms is the human KB cell, an epidermoid carcinoma cell line, because it contains both a hydrophobic membrane-associated FBP, located predominantly on the plasma membrane [2], and a soluble FBP (s-FBP),which is released into theculture medium [3, 4]
Recent reports [5,6,7] suggest that the s-FBP is derived from the m-FBPby proteolytic cleavage of a hydrophobic sequence of amino acids which serves to anchor the protein inthe membrane. Another possibility is that the m-FBP is anchored to the plasma membrane by a lipid structure since covalently bound fatty acids have been identified in this form of FBP [8]
Summary
Two forms of folate binding protein (FBP),' soluble and membrane-associated, have been described in mammalian tissues and biological fluids [1]. The cell monolayers were washed with Hanks' BSS, solubilized in 2 ml of 0.05 M Tris-HC1, pH 8.5, 1% TritonX-100, 5 mM EDTA, 0.15 M NaCl containing Trayslol (200 units/ml) (lysis buffer) and centrifuged at 30,000 X g for 30 min at 4 "C.
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