Retinol binding protein is a major carrier of progesterone in vitro and in human follicular fluid

Abstract

OBJECTIVE: The human retinol binding protein 4 (RBP) is a member of the lipocalin family of proteins and is the specific vitamin A carrier. It has diverse biological functions including intra- and extracellular transport of retinoids, regulation of reproduction and insulin resistance signaling. Our recent proteomic analysis of human follicular fluid (FF) demonstrate high level of RBP presence and nearly undetectable level of retinoids. This study was conducted to investigate the RBP function in human FF as a possible carrier of other than retinals hydrophobic ligands.DESIGN: Experimental study.MATERIALS AND METHODS: Recombinant human RBP (rhRBP) was isolated from bacteria as inclusion bodies and purified by HPLC. Renaturation was performed by dialysis in the presence of retinol, cholesterol, or progesterone as ligands. rhRBP complexation with the ligands was verified by HPLC, ELISA, and immunoprecipitation with RBP-specific antibody, A/G Sepharose, and Western blot. Presence of retinol and progesterone in the complexes was monitored through 3H-labeled ligands as a trace. Complexes were formed also with purified TTR. rhRBP-ligand and rhRBP-TTR-ligand complexes (0.1–0.4uM) were added to primary human ovarian fibroblast cell culture (FC). Ligand delivery to FC was monitored by measuring cellular 3H-ligand incorporation after 3h incubation. FF and FC were obtained at the time of vaginal oocyte retrieval from IVF patients (3 young donor patients, age <28 years) and cultured in DMEM medium. Fetal calf serum was omitted during the rhRBP-ligand and rhRBP-TTR-ligand incubation. HPLC and immunocapture were used to search for RBP-progesterone and RBP–cholesterol complexes in FF.RESULTS: We confirmed 3H-Progesterone-rhRBP and 3H-Progesterone-rhRBP-TTR complexation using purified (>99% purity) recombinant proteins. The binding affinity of progesterone-rhRBP complexation is similar to the binding affinity of the retinol-rhRBP binding. Cholesterol was found to bind rRBP and rhRBP-TTR with very low affinity (<3%). >40% of the progesterone from the progesterone-rhRBP complex was found to enter FC. The presence of TTR did not change dramatically progesterone uptake. More than 60% of the FF progesterone is present as a constituent of the RBP-TTR complex.CONCLUSIONS: rhRBP alone and in a complex with TTR is a progesterone but not cholesterol carrier. In FF, RBP-TTR is a major carrier of progesterone. In a complex with RBP or RBP-TTR progesterone is delivered into the cells. OBJECTIVE: The human retinol binding protein 4 (RBP) is a member of the lipocalin family of proteins and is the specific vitamin A carrier. It has diverse biological functions including intra- and extracellular transport of retinoids, regulation of reproduction and insulin resistance signaling. Our recent proteomic analysis of human follicular fluid (FF) demonstrate high level of RBP presence and nearly undetectable level of retinoids. This study was conducted to investigate the RBP function in human FF as a possible carrier of other than retinals hydrophobic ligands. DESIGN: Experimental study. MATERIALS AND METHODS: Recombinant human RBP (rhRBP) was isolated from bacteria as inclusion bodies and purified by HPLC. Renaturation was performed by dialysis in the presence of retinol, cholesterol, or progesterone as ligands. rhRBP complexation with the ligands was verified by HPLC, ELISA, and immunoprecipitation with RBP-specific antibody, A/G Sepharose, and Western blot. Presence of retinol and progesterone in the complexes was monitored through 3H-labeled ligands as a trace. Complexes were formed also with purified TTR. rhRBP-ligand and rhRBP-TTR-ligand complexes (0.1–0.4uM) were added to primary human ovarian fibroblast cell culture (FC). Ligand delivery to FC was monitored by measuring cellular 3H-ligand incorporation after 3h incubation. FF and FC were obtained at the time of vaginal oocyte retrieval from IVF patients (3 young donor patients, age <28 years) and cultured in DMEM medium. Fetal calf serum was omitted during the rhRBP-ligand and rhRBP-TTR-ligand incubation. HPLC and immunocapture were used to search for RBP-progesterone and RBP–cholesterol complexes in FF. RESULTS: We confirmed 3H-Progesterone-rhRBP and 3H-Progesterone-rhRBP-TTR complexation using purified (>99% purity) recombinant proteins. The binding affinity of progesterone-rhRBP complexation is similar to the binding affinity of the retinol-rhRBP binding. Cholesterol was found to bind rRBP and rhRBP-TTR with very low affinity (<3%). >40% of the progesterone from the progesterone-rhRBP complex was found to enter FC. The presence of TTR did not change dramatically progesterone uptake. More than 60% of the FF progesterone is present as a constituent of the RBP-TTR complex. CONCLUSIONS: rhRBP alone and in a complex with TTR is a progesterone but not cholesterol carrier. In FF, RBP-TTR is a major carrier of progesterone. In a complex with RBP or RBP-TTR progesterone is delivered into the cells.