Cathepsin A protein from the accessory sex gland of the Chinese mitten crab (Eriocheir sinensis) plays a key role in spermatophore digestion
Cathepsin A protein from the accessory sex gland of the Chinese mitten crab (Eriocheir sinensis) plays a key role in spermatophore digestion
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来自中华绒螯蟹 (Eriocheir sinensis) 副性腺的组织蛋白酶 A 蛋白在精囊消化中发挥关键作用
DOI：
10.1016/j.jinsphys.2013.07.003
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发表时间：
2013-09-01
影响因子：
2.2
通讯作者：
Wang，Qun
中科院分区：
文献类型：
--
作者：
Wang，Juan;Fang，Di-An;Wang，Qun
中华绒螯蟹（Eriocheir sinensis）的附性腺（ASG）分泌蛋白能有效地消化精荚壁。为了鉴定哪些蛋白参与精荚壁的消化，从ASG分泌的50-kDa蛋白通过一系列分离步骤纯化至均一，包括硫酸铵分级分离、Sephadex G-25 S凝胶过滤、DEAE-Sephacel柱上的离子交换层析和Sephacryl S-200凝胶过滤。纯化的蛋白质对精荚壁的破裂有明显的促进作用，HPLC-ESI-MS/MS鸟枪法分析表明其为组织蛋白酶A（cathA）。Western印迹分析和cathA抑制剂消化实验也证实了这一发现。ELISA分析表明，ASG分泌物中的cathA酶活性在纯化过程中不断增强。此外，酶活性在E. sinensis与腺体的最后发育阶段平行。此外，从cathA抑制剂，抑制精荚壁消化的分析表明，cathA是主要的酶参与。因此，我们首次报道了来自ASG的cathA的特性，它可能在消化E精荚壁中起关键作用。中国产。
Accessory sex gland (ASG) secretory proteins of the Chinese mitten crab (Eriocheir sinensis) can effectively digest the spermatophore wall. In order to identify which proteins participate in spermatophore wall digestion, a 50-kDa protein secreted from the ASG was purified to homogeneity by a series of isolation steps, including ammonium sulfate fractionation, Sephadex G-25 S gel-filtration, ion exchange chromatography on a DEAE-Sephacel column and Sephacryl S-200 gel-filtration. The purified protein was effective in spermatophore wall rupture, and the subsequent HPLC–ESI-MS/MS shotgun analysis showed the digestive protein to be cathepsin A (cathA). This finding was also confirmed by Western blot analysis and a cathA inhibitor digestion experiment. ELISA analysis showed that cathA enzymatic activity from ASG secretions increased during its purification process. Furthermore, enzymatic activity was significantly higher in the mating period ofE. sinensisparallel to the latest developmental stage of the gland. Moreover, analysis from a cathA inhibitor that inhibits spermatophore wall digestion showed that cathA is the main enzyme involved. Hence, we first report the characterization of cathA from the ASG, which might play a key role in digesting the spermatophore wall ofE. sinensis.