Biochemical characterization of zebrafish Paqr5b

Previously, we established a gene knock-out strain of paqr5b in zebrafish and showed that the gene is essential for the formation of neurons in the zebrafish olfactory rosette. The results suggested that Paqr5b might play a role as a receptor for neurosteroids and contribute to the differentiation of olfactory neurons. In this study, we attempted to express the recombinant zebrafish Paqr5b protein and analyze its affinity for steroids. Full-length zebrafish Paqr5b (zPaqr5b) was expressed in Pichia pastris according to the method established for goldfish and human Paqr7. Solubilized zPaqr5b was purified by two column chromatography steps, nickel-nitrilotriacetic acid (Ni-NTA) column and gel column (Sephacryl S-300). The protein fraction showed a binding affinity of Kd = 4.6 nM and Bmax = 0.72 nM for progesterone. The result showed that zPaqr5b was successfully fractionated as the active form. The specificity of zPaqr5b against steroids was then analyzed by steroid binding assay. The zPaqr5b showed specific binding to progesterone as well as to the neurosteroid, allopregnanolone (ALLO). In addition, zPaqr5b showed high affinity for 17α,20β-dehydroxyprogesterone (DHP), a pheromone used to induce sexual behavior. In contrast, it was observed that other steroids, estradiol, testosterone and cortisol, showed no affinity, even when present at high doses. These results suggest that zPaqr5b is responsible for a receptor of progestogenic neurosteroids in the differentiation of neurons in the olfactory rosette (OR) and for a receptor for pheromones in developed neuronal cells in the OR.