Secretion and processing of a novel multi-domain cystatin-like protein by intracellular stages of Trichinella spiralis
Secretion and processing of a novel multi-domain cystatin-like protein by intracellular stages of Trichinella spiralis
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DOI：
10.1016/j.molbiopara.2006.09.008
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发表时间：
2007-01-01
影响因子：
1.5
通讯作者：
Connolly, Bernadette
中科院分区：
文献类型：
--
作者：
Robinson, Mark W.;Massie, Diane H.;Connolly, Bernadette
线虫寄生虫的排泄-分泌（ES）蛋白受到人们的主要关注，因为它们在宿主-寄生虫界面发挥作用，并且可能对成功寄生发挥至关重要的作用。此外，细胞内线虫（如旋毛虫）的 ES 蛋白也可能发挥调节宿主细胞基因表达的作用。在最近的蛋白质组学分析中，我们从旋毛虫 L1 肌肉幼虫中鉴定出一种新型分泌型半胱氨酸蛋白酶抑制剂样蛋白。在这里，我们发现蛋白质 MCD-1（多半胱氨酸蛋白酶抑制剂样结构域蛋白 1）包含三个重复的半胱氨酸蛋白酶抑制剂样结构域，并且对 mcd-1 基因结构的分析表明，重复结构域源自祖先半胱氨酸蛋白酶抑制剂基因的重复。半胱氨酸蛋白酶抑制剂是一组不同的半胱氨酸蛋白酶抑制剂，由寄生线虫分泌的半胱氨酸蛋白酶抑制剂是重要的免疫调节因子。半胱氨酸蛋白酶抑制剂超家族还包括不具有半胱氨酸蛋白酶抑制活性的半胱氨酸蛋白酶抑制剂样蛋白。在大肠杆菌中表达为 GST 融合蛋白的重组 MCD-1 蛋白在体外未能抑制木瓜蛋白酶，这表明螺旋毛螺旋体蛋白是非抑制性半胱氨酸蛋白酶抑制剂相关蛋白的新成员。 T.spiralis 分泌的 MCD-1 以高分子量和低分子量亚型存在，我们表明 HeLa 细胞分泌的重组 MCD-1 蛋白经历 pH 依赖性加工，可能导致单个半胱氨酸蛋白酶抑制剂样结构域的释放。此外，我们发现 mcd-1 基因表达很大程度上局限于细胞内阶段，在成虫中表达水平最高。该蛋白质的主要作用很可能是在螺旋毛虫感染的肠道阶段。 (c) 2006 Elsevier B.V. 保留所有权利。
The excretory-secretory (ES) proteins of nematode parasites are of major interest as they function at the host-parasite interface and are likely to have roles crucial for successful parasitism. Furthermore, the ES proteins of intracellular nematodes such as Trichinella spiralis may also function to regulate gene expression in the host cell. In a recent proteomic analysis we identified a novel secreted cystatin-like protein from T. spiralis L1 muscle larva. Here we show that the protein, MCD-1 (multi-cystatin-like domain protein 1), contains three repeating cystatin-like domains and analysis of the mcd-1 gene structure suggests that the repeated domains arose from duplication of an ancestral cystatin gene. Cystatins are a diverse group of cysteine protease inhibitors and those secreted by parasitic nematodes are important immuno-modulatory factors. The cystatin superfamily also includes cystatin-like proteins that have no cysteine protease inhibitory activity. A recombinant MCD-1 protein expressed as a GST-fusion protein in Escherichia coli failed to inhibit papain in vitro suggesting that the T. spiralis protein is a new member of the non-inhibitory cystatin-related proteins. MCD-1 secreted from T. spiralis exists as high- and low-molecular weight isoforms and we show that a recombinant MCD-1 protein secreted by HeLa cells undergoes pH-dependent processing that may result in the release of individual cystatin-like domains. Furthermore, we found that mcd-1 gene expression is largely restricted to intracellular stages with the highest levels of expression in the adult worms. It is likely that the major role of the protein is during the intestinal stage of T. spiralis infections. (c) 2006 Elsevier B.V. All rights reserved.