Identification and characterization of lipocalin-type prostaglandin D2 synthase homologs in the urine of male rockfish
Identification and characterization of lipocalin-type prostaglandin D2 synthase homologs in the urine of male rockfish
复制标题
雄性石斑鱼尿液中脂质运载蛋白型前列腺素 D2 合酶同系物的鉴定和表征
DOI：
10.1016/j.gene.2022.147093
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发表时间：
2023
期刊：
影响因子：
3.5
通讯作者：
Hiramatsu Naoshi
中科院分区：
文献类型：
--
作者：
Yamaguchi Yo;Namgung Jin;Nagata Jun;Kawasaki Takuma;Hara Akihiko;Todo Takashi;Hiramatsu Naoshi
黑岩鱼（学名：Acropites schlegelii）是一种胎生海鱼。雄性在交配季节会产生尿蛋白;然而，这些蛋白的身份是未知的。在这项研究中，我们专注于高分子量尿蛋白（HMWups）在男性黑岩鱼。采用液相色谱-串联质谱（LC-MS/MS）技术对HMWup进行鉴定，并通过计算机模拟分析RNA-seq数据预测候选HMWup转录本的组织分布及其基因结构。克隆候选cDNA并制备主要候选物的重组蛋白。使用针对重组蛋白的抗血清进行尿蛋白质印迹，以再次确认LC-MS/MS结果。采用实时定量聚合酶链反应（qRT-PCR）和免疫组织化学分别验证RNA-seq的预测和鉴定产生HMWups的细胞。LC-MS/MS结合蛋白质印迹和cDNA克隆，鉴定了HMWups为脂质运载蛋白型前列腺素D2合酶（l-PGDS）同源物。RNA-seq分析和qRT-PCR显示，thel-PGDS同源物转录本主要在睾丸和雄性肾脏中表达;支持细胞和肾小管上皮细胞呈免疫反应性。这些结果表明，在尿中的主要蛋白质成分的雄性黑岩鱼是l-PGDS同系物，可能产生的肾小管在肾脏。雄性岩鱼被认为在交配行为中释放未知的信息素物质。在这项研究中获得的知识和工具授权研究的作用（S）的HMWups在信息素系统的岩鱼繁殖。迄今尚未发现蛋白质型硬骨鱼信息素。
Black rockfish (Sebastes schlegelii) and its relatives are viviparous marine fish. Males produce urinary proteins during the copulation season; however, the identity of these proteins was unknown. In this study, we focused on high-molecular-weight urinary proteins (HMWups) in male black rockfish. The HMWups were identified by liquid chromatography and tandem mass spectrometry (LC-MS/MS) of urine.In silicoanalyses of RNA-seq data predicted the tissue distribution of candidate HMWup transcripts and their gene structures. Candidate cDNAs were cloned and a recombinant protein of a major candidate was prepared. Western blotting of urine using an antiserum against the recombinant protein was performed to reconfirm the LC-MS/MS results. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry were employed to validate the prediction by RNA-seq and identify the cells producing HMWups, respectively. LC-MS/MS, in conjunction with Western blotting and cDNA cloning, identified the HMWups as lipocalin-type prostaglandin D2synthase (l-PGDS) homologs. RNA-seq analyses and qRT-PCR revealed that thel-PGDS homolog transcripts were dominantly expressed in the testis and male kidney; Sertoli cells and epithelial cells in the renal tubules were immunoreactive. These results indicated that major protein components in the urine of male black rockfish arel-PGDS homologs, potentially produced by the renal tubules in the kidney. Male rockfish (genusSebastes) are thought to release unknown pheromone substances during mating behavior. The knowledge and tools obtained in this study empower research into the role(s) of HMWups in pheromone systems underlying rockfish reproduction. No protein-type teleost pheromone has heretofore been discovered.