Cloning of a cysteine protease required for the molting of Onchocerca volvulus third stage larvae
Cloning of a cysteine protease required for the molting of Onchocerca volvulus third stage larvae
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DOI：
10.1074/jbc.271.47.30181
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发表时间：
1996-11-22
影响因子：
4.8
通讯作者：
Brotman, B
中科院分区：
文献类型：
--
作者：
Lustigman, S;McKerrow, JH;Brotman, B
我们研究了半胱氨酸蛋白酶抑制剂对盘尾丝虫第三期幼虫（L3）存活和体外蜕皮的影响，探讨了半胱氨酸蛋白酶在盘尾丝虫第四期幼虫（L4）发育中的作用。当幼虫在特异性抑制剂肽基单氟甲基酮存在下培养时，L3或L4的活力不受影响，然而，抑制剂以时间和剂量依赖的方式减少体外蜕皮为L4的L3的数量，在50-250 μ M抑制剂存在下，蜕皮被完全抑制，未蜕皮的L3幼虫超微结构观察表明，L4角质层有新的合成，但L3和L4角质层之间没有分离。蜕皮幼虫与标记的抑制剂结合后，检测到内源性半胱氨酸蛋白酶，和针对重组O.肠扭转L3半胱氨酸蛋白酶基因的克隆和表达。在蜕皮幼虫的角质层区域检测到该酶，这些研究表明，蜕皮和L4的成功发育取决于半胱氨酸蛋白酶的表达和释放。
We have investigated the involvement of a cysteine protease in the development of Onchocerca volvulus fourth stage larvae (L4) by testing the effect of cysteine protease inhibitors on the survival of third stage larvae (L3), and the molting of L3 to L4 in vitro. When larvae were cultured in the presence of specific inhibitors, the peptidyl monofluoromethylketones, viability of either L3 or L4 was not affected, However, the inhibitors reduced the number of L3 that molted to L4 in vitro in a time- and dose-dependent manner, Molting was completely inhibited in the presence of 50-250 mu M inhibitor, Ultrastructural examination of L3 that did not molt in the presence of inhibitors indicated that new L4 cuticle was synthesized, but there was no separation between the L3 and the L4 cuticles, The endogenous cysteine protease was detected in molting larvae after binding to labeled inhibitors, and by antibodies directed against a recombinant O. volvulus L3 cysteine protease that was cloned and expressed. The enzyme was detected in cuticle regions where the separation between the cuticles occurs in molting larvae, These studies suggest that molting and successful development of L4 depends on the expression and release of a cysteine protease.