Isolation and characterization of the proteins from dioscorea species capable of up-regulating estradiol secretion from ovarian granulosa cells

Abstract

 Estradiol-stimulating protein, DOI, was previously isolated from the rhizomes of Dioscorea opposite Thunb., which is one of classical Chinese herbal medicines for treating menopausal symptoms. In this study, the DOI-like proteins have been isolated from four Dioscorea species, including Dioscorea alata rhizomes, Dioscorea hypoglancae rhizomes, Dioscorea opposite rhizomes and Dioscorea zingiberensis rhizomes, by a newly-developed DOI-antibody affinity column. The proteins isolated from four Dioscorea species shared a similar denatured molecular weight compared with DOI. The estradiol-stimulating properties and underlying mechanism in ovarian granulosa cell model were studied. The cell plating density and the incubation time of ovarian granulosa cell model have been optimized based on the highest level of estradiol secretion response to forskolin. Results of estradiol concentration in the cell culture medium demonstrated that the estradiol-stimulating effect of DOI protein from Dioscorea opposite rhizomes is the highest. Moreover, DOI isolated from Dioscorea opposite rhizome sup-regulated the protein expression of estradiol-metabolizing enzymes (CYP19 and StAR) and estrogen receptors (ERαand ERβ), while DOI-like proteins from other Dioscorea spp did not. In order to further biological charactering DOI isolated from Dioscorea opposite rhizomes, the estradiol-stimulating effect of DOI on FSHR antibody induced FSHR-attenuated ovarian granulosa cell model has been measured. Result showed estradiol-stimulating effect of DOI was abolished in FSHR-attenuated ovarian granulosa cell model, so it may regulate estradiol biosynthesis through FSHR pathway. Then, the estradiol-stimulating effect of trypsin-digested DOI and proteinase K-digested DOI has been evaluated. Results demonstrated that trypsin-digested DOI could up-regulate estradiol concentration in ovarian granulosa cells. For the molecular characterization of DOI, the nucleic acid sequence and partial amino acid sequence of DOI were determined by DNA sequence and mass spectrometry, respectively. Full nucleic acid sequence and 128 partial amino acid sequences were obtained. According to the nucleic acid sequence of DOI, 286 amino acid residues in DOI was deducted, and the N-terminal sequence has low similarity between the deducted DOI amino acid sequence and DOI aminoacid sequence detected by mass spectrometry. Based on the sequences, we found DOI without chitinase activity had high sequence similarity with chitinase, which is a kind of enzyme, at least with reported antifungal activity, that exists in plants. Thus, the activity of antifungal of DOI was determined and it shows the antifungal effect at 0.5 mg/ml concentration. In conclusion, DOI protein isolated from Dioscorea opposite Thunb. had the highest estradiol-stimulating effect compared with proteins isolated from other Dioscorea spp. DOI may regulate the estradiol biosynthesis through FSHR, the fragment of DOI also had estradiol-stimulating effect in ovarian granulosa cell model, the amino acid sequence of DOI showed high similarity with chitinase and it showed antifungal property.