A secreted cystatin from the tick Haemaphysalis longicornis and its distinct expression patterns in relation to innate immunity.
A secreted cystatin from the tick Haemaphysalis longicornis and its distinct expression patterns in relation to innate immunity.
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DOI：
10.1016/j.ibmb.2006.03.003
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发表时间：
2006-07
影响因子：
3.8
通讯作者：
Jinlin Zhou;M. Ueda;R. Umemiya;B. Battsetseg;D. Boldbaatar;X. Xuan;K. Fujisaki
中科院分区：
文献类型：
--
作者：
Jinlin Zhou;M. Ueda;R. Umemiya;B. Battsetseg;D. Boldbaatar;X. Xuan;K. Fujisaki
能够选择性和特异性抑制半胱氨酸蛋白酶的蛋白质已被鉴定为半胱氨酸蛋白酶抑制剂，并且从多种微生物和动物和植物的组织中分离。这些蛋白的生理功能被认为是调节蛋白质周转和防御病原体以及平衡宿主-寄生虫免疫关系。已在几种蜱中发现了编码半胱氨酸蛋白酶抑制剂的基因，但对半胱氨酸蛋白酶抑制剂在蜱中的功能尚不清楚。我们从蜱H中克隆了编码半胱氨酸蛋白酶抑制剂的基因。longicornis，命名为Hlcyst-2（H.长角线虫cystatin-2）。其cDNA全长为569 bp，编码一个133个氨基酸的蛋白质，并含有一个明显的信号肽。序列分析表明，该基因与已知的半胱氨酸蛋白酶抑制剂有很高的同源性。重组蛋白在大肠杆菌中以GST融合的可溶性形式表达，并通过亲和层析纯化。通过荧光底物分析鉴定重组蛋白对木瓜蛋白酶、组织蛋白酶L和组织蛋白酶B的抑制活性。半胱氨酸蛋白酶抑制剂主要表达于蜱的中肠和血细胞。血液喂养诱导中肠中的表达显著增加。Real-time PCR证实，注射LPS的成蜱表达Hlcyst-2是注射PBS的对照蜱的1.6倍，吉氏巴氏巴贝虫感染的幼蜱表达Hlcyst-2是正常幼蜱的1.8倍。重组蛋白对体外培养的牛Babybovis也表现出明显的生长抑制作用。这些结果表明，该半胱氨酸蛋白酶抑制剂Hlcyst-2参与蜱天然免疫。
Proteins capable of selective and specific inhibition of cysteine protease have been identified as cystatins and are isolated from a variety of microbes and tissues of animals and plants. The physiological function of these proteins has been proposed to be the regulation of protein turnover and defense against pathogens as well as the balance of the host–parasite immune relationship. Genes encoding cystatins have been found in several species of ticks, but the function of cystatin in ticks is not understood. We cloned a gene encoding cystatin from tick H. longicornis and designated it as Hlcyst-2 (H. longicornis cystatin-2). Its full-length cDNA is 569bp, and it encodes a putative 133 amino acid protein with an obvious signal peptide. Sequence analysis demonstrated that it has significant homology with the known cystatin. The recombinant protein was expressed in a GST-fused soluble form in Escherichia coli and purified by affinity chromatography. The inhibitory activity of the recombinant protein against papain, cathepsin L, and cathepsin B was identified by fluorogenic substrate analysis. Cystatin was mostly expressed in the tick midgut and hemocyte. Blood feeding induced significantly increased expression in the midgut. Real-time PCR confirmed that LPS-injected adult ticks expressed Hlcyst-2 1.6 more times than the PBS-injected control; Babesia gibsoni-infected larvae ticks expressed Hlcyst-2 1.8 more times than normal larvae ticks. The recombinant protein also showed a significant growth-inhibitory effect on Babesia bovis cultured in vitro. These results indicated this cystatin Hlcyst-2 is involved in tick innate immunity.