Functional characterization of a cystatin from the tick Rhipicephalus haemaphysaloides.
Functional characterization of a cystatin from the tick Rhipicephalus haemaphysaloides.
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DOI：
10.1186/s13071-015-0725-5
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发表时间：
2015-03-03
影响因子：
3.2
通讯作者：
Zhou J
中科院分区：
文献类型：
--
作者：
Wang Y;Zhou Y;Gong H;Cao J;Zhang H;Li X;Zhou J
蜱和蜱传疾病影响世界范围内的动物和人类健康，并在畜牧业中造成重大的经济损失。功能分子研究对于在分子水平上了解蜱的生物学特性具有重要意义。酶和酶抑制分子在蜱生理学中起着非常重要的作用，而半胱氨酸蛋白酶抑制剂是木瓜蛋白酶样半胱氨酸蛋白酶的紧密结合抑制剂。为此，从蜱Rhipicephalus haemaphysaloides中分离出一种新的半胱氨酸蛋白酶抑制剂，命名为RHcyst-1。利用RACE技术克隆了RHcyst-1的全长基因。在大肠杆菌中以谷胱甘肽S-转移酶（GST）融合的可溶性形式表达重组蛋白RHcyst-1，并通过荧光底物分析鉴定其对组织蛋白酶L、B、C、H和S以及木瓜蛋白酶的抑制活性。通过定量逆转录- PCR（qRT-PCR）进行RHcyst-1在不同蜱阶段的表达分析。进行RHcyst-1的RNAi实验以确定其对蜱生理学的功能。RHcyst-1的全长cDNA为471 bp，包括一个完整的开放阅读框架，编码98个氨基酸的预期蛋白，不含信号肽，预测分子量约为11 kDa，等电点为5.66。序列分析表明，它与已知的1型半胱氨酸蛋白酶抑制剂具有显著的同源性。蛋白酶抑制实验结果表明，rRHcyst-1对6种半胱氨酸蛋白酶的酶活性均有抑制作用。对RHcyst-1基因表达谱的研究表明，它在胚胎（卵）阶段更丰富地转录。RHcyst-1基因的破坏表明蜱孵化率显着下降。我们的研究结果表明，RHcyst-1可能参与蜱的早期胚胎发育。
Ticks and tick-borne diseases affect animal and human health worldwide and cause significant economic losses in the animal industry. Functional molecular research is important to understand the biological characteristics of ticks at the molecular level. Enzymes and enzyme inhibitory molecules play very important roles in tick physiology, and the cystatins are tight-binding inhibitors of papain-like cysteine proteases. To this end, a novel cystatin, designated RHcyst-1, was isolated from the tick Rhipicephalus haemaphysaloides. The full-length gene of RHcyst-1 was cloning by RACE. The recombinant protein of RHcyst-1 was expressed in a glutathione S-transferase (GST)-fused soluble form in Escherichia coli, and its inhibitory activity against cathepsin L, B, C, H, and S, as well as papain, was identified by fluorogenic substrate analysis. Expression analysis of RHcyst-1 at different tick stages was performed by quantitative reverse transcription - PCR (qRT-PCR). An RNAi experiment for RHcyst-1 was performed to determine its function for tick physiology. The full-length cDNA of RHcyst-1 is 471 bp, including an intact open reading frame encoding an expected protein of 98 amino acids, without a signal peptide, having a predicted molecular weight of ~11 kDa and an isoelectric point of 5.66. A sequence analysis showed that it has significant homology with the known type 1 cystatins. The results of proteinase inhibition assays showed that rRHcyst-1 can effectively inhibit the six cysteine proteases’ enzyme activities. An investigation of the RHcyst-1 genes’ expression profile showed that it was more richly transcribed in the embryo (egg) stage. A disruption of the RHcyst-1 gene showed a significant decrease in the rate of tick hatching. Our results suggested that RHcyst-1 may be involved in the early embryonic development of ticks.
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影响因子：
4.8
作者：
Kotsyfakis, Michalis;Karim, Shahid;Ribeiro, Jose M. C.
通讯作者：
Ribeiro, Jose M. C.
影响因子：
1.2
作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
2.2
作者：
Gao, Xiao;Shi, Lei;Zhou, Jinlin
通讯作者：
Zhou, Jinlin
DOI：
10.1016/j.bbrc.2006.06.018
发表时间：
2006-08-18
影响因子：
3.1
作者：
Lima, Cassia A.;Sasaki, Sergio D.;Tanaka, Aparecida S.
通讯作者：
Tanaka, Aparecida S.