Limited proteolysis by a prostatic endopeptidase, the sperm-activating factor initiatorin, regulates the activation of pro-carboxypeptidase B in the seminal fluid of the silkworm, Bombyx mori.
Limited proteolysis by a prostatic endopeptidase, the sperm-activating factor initiatorin, regulates the activation of pro-carboxypeptidase B in the seminal fluid of the silkworm, Bombyx mori.
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DOI：
10.1016/j.ibmb.2022.103819
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发表时间：
2022-08
影响因子：
3.8
通讯作者：
Miki Sakakura;Yuki Takata;Chikayo Kimura;Saki Matsuda;Tomoko Takamura;S. Nagaoka
中科院分区：
文献类型：
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作者：
Miki Sakakura;Yuki Takata;Chikayo Kimura;Saki Matsuda;Tomoko Takamura;S. Nagaoka
前列腺胰蛋白酶样丝氨酸内肽酶称为启动素（BmIni）是触发家蚕精子成熟反应的重要因素。据预测，BmIni可以特异性地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侧，但实际的底物尚不清楚。为了阐明精子成熟信号通路的分子机制，本研究考察了BmIni是否通过特异性降解激活精子羧基肽酶B （BmCPB）蛋白。首先，我们在体外证实，未交配雄性精囊虫（v.）中的失活BmCPB可以通过BmIni或胰蛋白酶激活。编码种子BmCPB蛋白的基因的分子克隆表明，BmCPB是作为分泌的前酶产生的，可能在胰蛋白酶样蛋白酶切割原结构体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都能显著激活重组Pro-BmCPB，该重组Pro-BmCPB在大肠杆菌中成功表达并纯化为原酶；此外，在BmIni激活过程中出现了两种特定的切割形式，而在胰蛋白酶激活过程中没有出现。因此，我们制备了一个含有突变的diarginine motif的重组蛋白（Arg109-Arg110），根据其与牛CPB的高度同源性，该蛋白被认为是BmCPB的预裂解位点。结果，两种特异性降解肽不再被观察到，同时激活被抑制。综上所述，这些研究结果表明，在雄性生殖器官中合成和分泌的酶原BmCPB在射精和雌性生殖器官中被序列依赖性蛋白水解激活，这为BmIni在家蚕精子成熟级联中降解精浆和/或精子蛋白的机制提供了线索。
A prostate trypsin-like serine endopeptidase called initiatorin (BmIni) is an essential factor in triggering the sperm maturation response of the silkworm,Bombyxmori. 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 confirmedin vitrothat 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 inEscherichia 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 ofB.mori.