Identification of the sperm-activating factor initiatorin, a prostatic endopeptidase of the silkworm, Bombyx mori
Identification of the sperm-activating factor initiatorin, a prostatic endopeptidase of the silkworm, Bombyx mori
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DOI：
10.1016/j.ibmb.2012.04.004
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发表时间：
2012-08-01
影响因子：
3.8
通讯作者：
Kamei, Kaeko
中科院分区：
文献类型：
--
作者：
Nagaoka, Sumiharu;Kato, Kumiko;Kamei, Kaeko
雄性马立克次氏体在射精管后段分泌物中有一种胰蛋白酶类型的蛋白酶，称为起始蛋白，被认为与精子活力的获得有关，尽管这一推论仍有待证明。在此，我们修改了包括纯化在内的实验步骤，并明确鉴定了纯化的起始蛋白为B的激活因子。mod精子通过体外研究，我们处理分离的精子与这种酶。cDNA序列分析表明，initiatorin由281个氨基酸组成，序列与牛胰蛋白酶相似，与鳞翅目昆虫和直翅目昆虫的射精附腺蛋白高度同源。重组initiatorin，在大肠杆菌中表达和纯化，也表现出蛋白水解和精子激活活性。RT-PCR和Westernblot分析表明，initiatorin在腺（g.）南极洲它也表明，pro-initiatorin的合成和储存在g。射精后，精子会转化为成熟的精子。具有双碱基序列的荧光肽能被initiatorin有效地切割，其中一种底物BOC-Gly-Arg-Arg-MCA能抑制G.南极洲这些结果描述了这样的想法，即起始蛋白具有最合适的蛋白酶性质作为蛋白质降解级联的起始剂，因为它释放游离的精氨酸，其反过来成为精子运动的能量来源。(C)2012爱思唯尔有限公司保留所有权利。
Male Bombyx mod has a trypsin-type protease, called initiatorin, in the secretion from the posterior segment of the ejaculatory duct that is thought to be involved in the acquisition of sperm motility, although this inference remains to be demonstrated. Here, we revised the experimental procedures including that for purification and definitely identified the purified initiatorin protein as an activation factor of B. mod sperm by an in vitro study in which we treated isolated spermatozoa with this enzyme. Analysis of cDNA revealed that initiatorin consists of 281 amino acids with sequence similarity to bovine trypsin, and is highly homologous to the ejaculated accessory gland proteins not only of other Lepidoptera but also of Orthoptera. Recombinant initiatorin, expressed in Escherichia coli and purified, also showed proteolytic and sperm-activating activities. RT-PCR and Western blot analyses indicated that initiatorin is abundantly expressed in the glandula (g.) prostatica. It was also shown that pro-initiatorin is synthesized and stored in g. prostatica, and then converted to the mature form upon ejaculation. Fluorogenic peptides with a dibasic sequence were efficiently cleaved by initiatorin, and one such substrate, BOC-Gly-Arg-Arg-MCA, inhibited sperm activation by the extract of g. prostatica. These results delineate the idea that initiatorin has the most suitable protease property as an initiator of the protein degradation cascade in that it releases free arginines, which in turn become an energy resource for sperm motility. (C) 2012 Elsevier Ltd. All rights reserved.