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

doi:10.1016/s0021-9258(18)94117-7

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.

Highlights

folate-binding proteins (FBPs) were the major species recovered on gel filtration in Triton X-100 (Fig. 1,left).To identify a putativehydrophobic incubated with radiolabeled folate in 3 mlof equilibration/elution FBP-directed placental protease, we reasoned that one apbuffer and applied to a 2.5 X 30-cm Sephacryl S-200 column which proach was to carry out placentalmembrane solubilization in was equilibrated and eluted with 10 mM potassium phosphate, pH the presence of known "broad-spectrum" protease inhibitors
Effect of EDTA on generation of 160-kDa and 40-kDa placental folate-binding proteins. 100 pl of crude solubilized placental membranes were incubated with 25 fmol of '%I-labeled pteroylglutamate and applied over a 2.5 X 30-cm Sephacryl S-200 column which was equilibrated in 10 mM potassium phosphate, pH 7.5, containing 1% Triton X-100
Membranes were solubilized with 10 mM potassium phosphate, pH 7.5, containing 1%Triton X-100 and either 20 mM EDTA (X), 10 mM EDTA (O),or no EDTA (0).Right, immunoprecipitation of '251-labeledpteroylglutamate-bound FBPs with anti-40-kDa FBP antiserum and crude rabbit preimmune serum

Summary

Introduction

FBPs were the major species recovered on gel filtration in Triton X-100 (Fig. 1,left).To identify a putativehydrophobic incubated with radiolabeled folate in 3 mlof equilibration/elution FBP-directed placental protease, we reasoned that one apbuffer and applied to a 2.5 X 30-cm Sephacryl S-200 column which proach was to carry out placentalmembrane solubilization in was equilibrated and eluted with 10 mM potassium phosphate, pH the presence of known "broad-spectrum" protease inhibitors. 100 pl of crude solubilized placental membranes were incubated with 25 fmol of '%I-labeled pteroylglutamate (histamine derivative) and applied over a 2.5 X 30-cm Sephacryl S-200 column which was equilibrated in 10 mM potassium phosphate, pH 7.5, containing 1% Triton X-100.

Results

Conclusion