前列腺胰蛋白酶样丝氨酸内肽酶称为起始蛋白(BmIni)是触发家蚕精子成熟反应的重要因素,家蚕.已预测BmIni会特异性切割某些精浆和精子蛋白中存在的两个连续精氨酸残基的羧基。但是实际的底物仍然未知。试图阐明精子成熟信号通路的分子机制,在这项研究中,我们检查了BmIni是否通过特异性降解激活精浆羧肽酶B(BmCPB)蛋白。首先,我们在体外证实,未交配的雄性囊泡中存在无活性的BmCPB(v。)半精索通过用BmIni或胰蛋白酶处理而被激活。编码精浆BmCPB蛋白的基因的分子克隆表明,BmCPB作为分泌的酶原产生,并且可以在胰蛋白酶样蛋白酶切割前结构域和酶位点之间的边界后被激活。为了支持这些发现,胰蛋白酶和BmIni均显着激活重组Pro-BmCPB,在大肠杆菌中成功表达和纯化为酶原;此外,在BmIni的激活中出现了两种特定的裂解形式,而在胰蛋白酶中没有出现。因此,具有突变二精氨酸基序(Arg109-Arg110)的重组蛋白,基于其与牛CPB的高度同源性,推测为BmCPB的预切割位点,制备并用BmIni处理。因此,不再观察到两种特定的降解肽,同时激活被抑制。一起来看,这些发现得出的结论是,酶原BmCPB,在男性生殖器官中合成和分泌,在射精过程中和雌性生殖器官中被BmIni的序列依赖性蛋白水解激活,为BmIni在B.mori的精子成熟级联中精浆和/或精子蛋白降解的潜在机制提供了线索。
A prostate trypsin-like serine endopeptidase called initiatorin (BmIni) is an essential factor in triggering the sperm maturation response of the silkworm, Bombyx mori. BmIni has been predicted to specifically cleave the carboxyl side of two consecutive arginine residues present in certain seminal plasma and sperm proteins, but the actual substrates are still unknown. In an attempt to elucidate the molecular mechanism underlying the sperm maturation signaling pathway, in this study, we examined whether BmIni activates the seminal carboxypeptidase B (BmCPB) protein through specific degradation. First, we confirmed in vitro that the inactive BmCPB present in unmated male vesicula (v.) seminalis is activated by treatment with BmIni or trypsin. Molecular cloning of the gene encoding the seminal BmCPB protein has shown that BmCPB is produced as a secreted proenzyme and may be activated after a trypsin-like protease cleaves the boundary between the prodomain and the enzyme site. In support of these findings, both trypsin and BmIni significantly activated recombinant Pro-BmCPB, which was successfully expressed and purified as a proenzyme in Escherichia coli; moreover, two specific cleavage forms appeared in the activation by BmIni that did not appear in that by trypsin. Therefore, a recombinant protein with a mutated diarginine motif (Arg109-Arg110), which is presumed to be a pre-cleavage site of BmCPB based on its high homology with bovine CPB, was prepared and treated with BmIni. As a result, the two specific degraded peptides were no longer observed, and simultaneously the activation was suppressed. Taken together, these findings lead to the conclusion that zymogen BmCPB, which is synthesized and secreted in male reproductive organs, is activated by sequence-dependent proteolysis by BmIni during ejaculation and in the female reproductive organs, providing a clue to the mechanism underlying seminal plasma and/or sperm protein degradation by BmIni in the sperm maturation cascade of B. mori.