title: Identification of a Mg2+-dependent protease in human placenta which cleaves hydrophobic folate-binding proteins to hydrophilic forms.
abstract: Hydrophobic folate-binding proteins (FBPs), which are only 5-10 kDa larger than 40-kDa hydrophilic FBPs, bind significant quantities of Triton X-100 micelles and elute as apparent 160-kDa species on Sephacryl S-200 gel filtration in Triton X-100. Detergent-solubilized placental membranes release a major (greater than 95%) 40-kDa hydrophilic FBP species as well as a minor apparent 160-kDa folate binding species when similarly analyzed. We tested the hypothesis that this recovery of predominantly hydrophilic FBPs was mediated by a putative hydrophobic FBP-specific placental protease. When placenta was solubilized in the presence of increasing concentrations of EDTA, there was a progressive increase in apparent 160-kDa folate binding moieties concomitant with a decrease in 40-kDa FBPs. At 20 mM EDTA, a single apparent 160-kDa folate binding species was recovered and the 40-kDa FBPs could not be detected by radioligand binding or specific radioimmunoassay. The apparent 160-kDa species specifically bound radiolabeled folates and were specifically immunoprecipitated by rabbit anti-40-kDa FBP antiserum. On 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose and probing with anti-40-kDa FBP antiserum, the apparent 160-kDa FBPs electrophoresed as 45-kDa species. Detergent binding studies indicated that apparent 160-kDa FBPs were hydrophobic, thus accounting for the molecular weight discrepancy in gel filtration in Triton X-100 versus sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The EDTA-mediated inhibition of conversion of hydrophobic FBPs to hydrophilic FBPs by protease was reversed in a dose-dependent manner by Mg2+. If this protease is physiologically relevant, it could play an important regulatory role in folate transport by influencing the net number of hydrophobic FBPs on the cell surface.
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis 46223.
authors.forename: A C
authors.initials: AC
authors.lastname: Antony
authors.forename: R S
authors.initials: RS
authors.lastname: Verma
authors.forename: A R
authors.initials: AR
authors.lastname: Unune
authors.forename: J A
authors.initials: JA
authors.lastname: LaRosa
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003902:Detergents; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D058973:Folate Receptors, GPI-Anchored; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011956:Receptors, Cell Surface / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D003902:Detergents; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D004492:Edetic Acid; D010450:Endopeptidases; D008274:Magnesium