Identification of a Mg2+-dependent protease in human placenta which cleaves hydrophobic folate-binding proteins to hydrophilic forms

A. C. Antony, R. S. Verma, A. R. Unune, J. A. LaRosa

Research output: Contribution to journalArticle

28 Scopus citations

Abstract

Hydrophobic folate-binding proteins (FBPs), which are only 5-10 kDa larger than 40-kDa hydrophilic FBPs, bind significant qualities 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 (> 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 anti-serum, 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.

Original languageEnglish (US)
Pages (from-to)1911-1914
Number of pages4
JournalJournal of Biological Chemistry
Volume264
Issue number4
StatePublished - Jan 1 1989

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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