Characterization of proteins involved in the regulation of sperm motility and their protein network

本研究旨在阐明精子细胞从信号分子接收到运动机制激活的分子网络或级联过程。结果发现了参与这一信号通路的部分蛋白质，并揭示了分子间的相互作用。在研究期间，通过二维凝胶电泳和肽质量指纹图谱，在海鞘的精子中检测到12种发生电点或量变化的蛋白质，其中可以鉴定出潜在重要的轴突蛋白：外臂动力蛋白中间链2(IC2)，称为IC116的内臂动力蛋白中间链，径向辐组分LRR37， camp依赖性蛋白激酶的调节亚基和一种94 kDa的新型犰狳重复蛋白，其他蛋白被认为参与信号接收或细胞内信号转导，如sp17样蛋白，TER ATPasae，TEFB和14-3-3蛋白。另一方面，通过轴突的选择性提取，一系列柱层析和免疫沉淀来检测蛋白质-蛋白质相互作用。因此，径向辐条的亚复结构可以被隔离。该亚复合物由akap样蛋白（RSP3）、HSP40、两个MORN重复蛋白、RSP4/6和一些新的蛋白组成，这些蛋白的同源物也存在于人体内，但功能尚未揭示。该亚复合体的分子相互作用将揭示径向辐条的分子功能，并最终揭示从海鞘到人类的鞭毛运动机制。这一结果也提示分子伴侣参与鞭毛运动的形成、维持或调节。

The research was carried out to elucidate the molecular network or cascade from the reception of signaling molecule to the activation of motile machinery in sperm cell. As a result a part of the proteins involved in this signaling pathway have been identified and the molecular interactions among the molecules have been revealed. In the research period twelve proteins that undergo changes in electric point or in the amount were detected in the sperm of the ascidian Ciona intestinalis on two-dimentional gel electrophoresis and peptide mass fingerprinting, Among them, potentially important axonemal proteins could be identified : outer arm dynein intermediate chain2(IC2), an intermediate chain of inner arm dynein termed IC116, radial spoke component LRR37, the regulatory subunit of cAMP-dependent protein kinase and a 94 kDa novel armadillo repeat protein, Another proteins are though to be involved in the reception of signals or intracellular signal transduction, such as SP17-like protein, TER ATPasae,TEFB,and 14-3-3 proteins. On the other hand, protein-protein interaction was examined by means of selective extraction of the axonemes, a series of columm chromatography and immunoprecipitation. As a result, a subcomplex constitution the radial spoke could be isolated. The subcomptex was composed of AKAP-like protein (RSP3), HSP40, two MORN repeat proteins, RSP4/6 and several novel proteins whose homologs are also present in human but the functions have been unrevealed. The molecular interaction in this subcomplex would shed light on the molecular function of radial spoke and ultimately on the mechanism of flagellar motility conserved from ascidian to human. The result also suggests that molecular chaperone is involved in the formation, maintenance or regulation of flagellar motility.