Although normal human chorionic villi-associated hydrophobic placental folate receptors (PFR) are converted to hydrophilic forms by an endogenous, EDTA-sensitive, Mg2+-dependent protease under serum-free conditions (Verma, R. S., and Antony, A. C. (1991) J. Biol. Chem. 266, 12522-12535), it is not known whether hydrophobic PFR are also susceptible to conversion by endogenous phospholipases. We isolated and characterized hydrophobic PFR, and tested the hypothesis that purified, in situ, and de novo-synthesized native PFR were covalently linked to glycosyl-phosphatidylinositol (GPI) anchors. 125I-hydrophobic PFR, but not 125I-hydrophilic PFR, (i) separated into the Triton X-114 micellar phase at 30 °C, (ii) efficiently incorporated into phosphatidylcholine-cholesterol liposomes, and (iii) were covalently labeled by the hydrophobic probe 3-(trifluoromethyl)-3-(meta[125I]iodophenyl)diazirine, [125I]TID. (iv) [125I]TID-labeled-and [phenyl-3H]Triton X-100-bound hydrophobic PFR, as well as native PFR in situ, were released as hydrophilic forms by recombinant (r) GPI-specific phospholipase(PL) C (GPI-PLC), and GPI-PLD (but not by PLC), in the absence and presence of a concentration of EDTA known to inhibit endogenous Mg2+-dependent protease. (v) Nitrous acid deamination of [125I]TID-labeled hydrophobic PFR as well as (r)GPI-PLC cleavage of [phenyl-3H]Triton-X-100- and [125I] TID-labeled hydrophobic PFR, released hydrophobic radiolabeled moieties which comigrated on thin layer chromatography distinct from free radiolabel. Finally, (vi) biosynthetic studies on chorionic villi cultured in vitro revealed incorporation of radiolabeled precursors into the GPI anchor of hydrophobic PFR. We conclude that native hydrophobic PFR are linked to GPI anchors and are therefore potential substrates for three distinct endogenous enzymes (GPI-PLC, GPI-PLD, and specific Mg2+-dependent metalloprotease) in maternal serum and placenta in vivo.
|Original language||English (US)|
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|State||Published - 1992|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology