title: The conversion of the human membrane-associated folate binding protein (folate receptor) to the soluble folate binding protein by a membrane-associated metalloprotease.
abstract: The membrane-associated (M-FBP) and soluble (S-FBP) forms of human folate binding proteins (FBP) have been well characterized. Although related in a precursor-product manner, the mechanism of conversion and the basis for differences between M-FBP and S-FBP are not known. The conversion of M-FBP to S-FBP in crude human nasopharyngeal carcinoma (KB) cell preparations is demonstrated based on characteristic gel filtration elution profiles of M-FBP and S-FBP (Ve/V0 = 1.3 and 1.7, respectively) in Triton X-100. M-FBP is stoichiometrically converted to S-FBP in a time- and temperature-dependent reaction by a metalloprotease which is: heat-labile; particulate; contained in human KB cell and placental membranes, and rat kidney homogenates; inhibited by EDTA, 1,10-phenanthroline, and parahydroxymercuribenzoate; requires divalent cations; is maximally active at neutral pH; and is active in the presence or absence of detergent. The purified soluble FBP product appears to be identical to S-FBP. Conversion of purified endogenously [3H]leucine-labeled M-FBP yields a soluble FBP characterized by a 45% decrease in specific activity (moles of 3H/mol folate bound) relative to M-FBP and a non-folate binding fragment which contains 45% of the [3H]leucine from M-FBP, requires detergent and/or urea to remain soluble, and migrates aberrantly on gel filtration in 1% (v/v) Triton X-100 and 8 M urea. Based on changes in the specific activity and the gel filtration elution profiles of purified labeled M-FBP associated with conversion to S-FBP, the endoproteolytic cleavage site is predicted between residues 226 and 229 of the cDNA predicted human FBP amino acid sequence. These results suggest that the cDNA predicted hydrophobic carboxyl terminus (residues 227-257) remains intact on the fully processed, membrane-anchored M-FBP, contains the Triton binding domain, and is involved in the formation of the membrane anchor of M-FBP.
authors.affiliation: Medicine Branch, National Cancer Institute, Bethesda, Maryland 20892.
authors.forename: P C
authors.initials: PC
authors.lastname: Elwood
authors.forename: J C
authors.initials: JC
authors.lastname: Deutsch
authors.forename: J F
authors.initials: JF
authors.lastname: Kolhouse
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007624:KB Cells; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism*; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012995:Solubility; D013696:Temperature
chemical_list: D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D005492:Folic Acid; D008666:Metalloendopeptidases