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华支睾吸虫Rab亚家族成员Rab8的鉴定与特征分析

Identification and characterization of a member of Rab subfamily, Rab8, from Clonorchis sinensis.

作者信息

Liang Pei, He Lei, Yu Jinyun, Xie Zhizhi, Chen Xueqing, Mao Qiang, Liang Chi, Huang Yan, Lu Gang, Yu Xinbing

机构信息

Department of Pathogen Biology, Hainan Medical College, Haikou, Hainan, 571199, China.

出版信息

Parasitol Res. 2015 May;114(5):1857-64. doi: 10.1007/s00436-015-4372-8. Epub 2015 Mar 15.

Abstract

The Rabs act as a binary molecular switch that utilizes the conformational changes associated with the GTP/GDP cycle to elicit responses from target proteins. It regulates a broad spectrum of cellular processes including cell proliferation, cytoskeletal assembly, and intracellular membrane trafficking in eukaryotes. The Rab8 from Clonorchis sinensis (CsRab8) was composed of 199 amino acids. The deduced amino acid sequence shared above 50% identities with other species from trematode, tapeworm, mammal, insecta, nematode, and reptile, respectively. The homologous analysis of sequences showed the conservative domains: G1 box (GDSGVGKS), G2 box (T), G3 box (DTAG), G4 box (GNKCDL), and G5 box. In addition, the structure modeling had also shown other functional domains: GTP/Mg(2+) binding sites, switch I region, and switch II region. A phylogenic tree analysis indicated that the CsRab8 was clustered with the Rab from Schistosoma japonicum, and trematode and tapeworm came from the same branch, which was different from an evolutional branch built by other species, such as mammal animal, insecta, nematode, and reptile. The recombinant CsRab8 protein was expressed in Escherichia coli and the purified protein was a soluble molecule by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. CsRab8 was identified as a component of excretory/secretory products of C. sinensis by western blot analysis. The transcriptional level of CsRab8 at metacercaria stage was the highest at the four stages and higher by 56.49-folds than that at adult worm, 1.23-folds than that at excysted metacercaria, and 2.69-folds than that at egg stage. Immunohistochemical localization analysis showed that CsRab8 was specifically distributed in the tegument, vitellarium, eggs, and testicle of adult worms, and detected on the vitellarium and tegument of metacercaria. Combined with the results, CsRab8 is indispensable for survival and development of parasites, especially for regulating excretory/secretory products secretion.

摘要

Rabs作为一种二元分子开关,利用与GTP/GDP循环相关的构象变化来引发靶蛋白的反应。它调节着广泛的细胞过程,包括真核生物中的细胞增殖、细胞骨架组装和细胞内膜运输。华支睾吸虫的Rab8(CsRab8)由199个氨基酸组成。推导的氨基酸序列与吸虫、绦虫、哺乳动物、昆虫、线虫和爬行动物等其他物种的序列分别具有50%以上的同源性。序列同源性分析显示了保守结构域:G1框(GDSGVGKS)、G2框(T)、G3框(DTAG)、G4框(GNKCDL)和G5框。此外,结构建模还显示了其他功能结构域:GTP/Mg(2+)结合位点、开关I区域和开关II区域。系统发育树分析表明,CsRab8与日本血吸虫的Rab聚集在一起,吸虫和绦虫来自同一分支,这与由其他物种如哺乳动物、昆虫、线虫和爬行动物构建的进化分支不同。重组CsRab8蛋白在大肠杆菌中表达,通过12%十二烷基硫酸钠聚丙烯酰胺凝胶电泳分析,纯化后的蛋白是一种可溶性分子。通过蛋白质印迹分析,CsRab8被鉴定为华支睾吸虫排泄/分泌产物的一个组成部分。CsRab8在尾蚴阶段的转录水平在四个阶段中最高,比成虫阶段高56.49倍,比脱囊尾蚴阶段高1.23倍,比虫卵阶段高2.69倍。免疫组织化学定位分析表明,CsRab8特异性分布于成虫的体表、卵黄腺、虫卵和睾丸,并在尾蚴的卵黄腺和体表检测到。综合这些结果,CsRab8对于寄生虫的生存和发育是不可或缺的,特别是对于调节排泄/分泌产物的分泌。

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