Biochemical characterization of zebrafish Paqr5b

IF 2.3 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochemistry and Biophysics Reports Pub Date : 2025-04-02 DOI:10.1016/j.bbrep.2025.101994

Md Sohanur Rahman Sohan , Md Forhad Hossain , Shakhawat Hossain , Yuki Omori , Mohammad Tohidul Amin , Md Ekramul Hasan , Toshinobu Tokumoto

{"title":"Biochemical characterization of zebrafish Paqr5b","authors":"Md Sohanur Rahman Sohan , Md Forhad Hossain , Shakhawat Hossain , Yuki Omori , Mohammad Tohidul Amin , Md Ekramul Hasan , Toshinobu Tokumoto","doi":"10.1016/j.bbrep.2025.101994","DOIUrl":null,"url":null,"abstract":"<div><div>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.</div><div>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 <em>Pichia pastris</em> 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.</div></div>","PeriodicalId":8771,"journal":{"name":"Biochemistry and Biophysics Reports","volume":"42 ","pages":"Article 101994"},"PeriodicalIF":2.3000,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochemistry and Biophysics Reports","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2405580825000810","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}

引用次数: 0

Abstract

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.

查看原文本刊更多论文

斑马鱼Paqr5b的生化特性

在此之前，我们在斑马鱼中建立了一个基因敲除菌株paqr5b，并表明该基因对斑马鱼嗅觉玫瑰花中神经元的形成至关重要。结果提示，Paqr5b可能作为神经甾体受体，参与嗅觉神经元的分化。在本研究中，我们试图表达重组斑马鱼Paqr5b蛋白，并分析其对类固醇的亲和力。根据建立的金鱼和人Paqr7的表达方法，在毕赤酵母中表达斑马鱼全长Paqr5b （zPaqr5b）。通过镍腈三乙酸（Ni-NTA）柱和凝胶柱（Sephacryl S-300）两层色谱纯化溶解后的zPaqr5b。该蛋白对黄体酮的结合亲和力Kd = 4.6 nM, Bmax = 0.72 nM。结果表明，zPaqr5b被成功分离为活性形式。然后通过类固醇结合试验分析zPaqr5b对类固醇的特异性。zPaqr5b显示出与孕酮以及神经类固醇异孕酮（ALLO）的特异性结合。此外，zPaqr5b对17α，20β-去羟孕酮（DHP）具有高亲和力，DHP是一种用于诱导性行为的信息素。相比之下，其他类固醇，雌二醇，睾酮和皮质醇，即使在高剂量下也没有表现出亲和力。这些结果表明，zPaqr5b在嗅蔷薇丛（OR）神经元分化过程中是孕激素受体，在嗅蔷薇丛发育的神经元细胞中是信息素受体。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Biochemistry and Biophysics Reports Biochemistry, Genetics and Molecular Biology-Biophysics

CiteScore

4.60

自引率

0.00%

发文量

191

审稿时长

59 days

期刊介绍： Open access, online only, peer-reviewed international journal in the Life Sciences, established in 2014 Biochemistry and Biophysics Reports (BB Reports) publishes original research in all aspects of Biochemistry, Biophysics and related areas like Molecular and Cell Biology. BB Reports welcomes solid though more preliminary, descriptive and small scale results if they have the potential to stimulate and/or contribute to future research, leading to new insights or hypothesis. Primary criteria for acceptance is that the work is original, scientifically and technically sound and provides valuable knowledge to life sciences research. We strongly believe all results deserve to be published and documented for the advancement of science. BB Reports specifically appreciates receiving reports on: Negative results, Replication studies, Reanalysis of previous datasets.