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首页> 外文期刊>Infection and immunity >Recombinant Expression and Localization ofSchistosoma mansoni Cathepsin L1 Support Its Role in the Degradation of Host Hemoglobin
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Recombinant Expression and Localization ofSchistosoma mansoni Cathepsin L1 Support Its Role in the Degradation of Host Hemoglobin

机译：曼氏血吸虫组织蛋白酶L1的重组表达和定位支持其在宿主血红蛋白降解中的作用

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Cysteine proteinases expressed by schistosomes appear to play key roles in the digestion of host hemoglobin, the principal source of amino acid nutrients utilized by these parasites. We have shown previously that the predominant cysteine proteinase activity in soluble extracts and excretory/secretory (ES) products of adults ofSchistosoma mansoni and S. japonicum is cathepsin L-like in its substrate specificity. However, biochemical analysis of the cathepsin L activity in extracts and ES products of schistosomes has been complicated by the presence of at least two distinct forms of schistosome cathepsin L, termed SmCL1 and SmCL2. We now report the purification and enzyme characteristics of active, recombinant SmCL1 which was obtained by transformingSaccharomyces cerevisiae with an expression plasmid encoding the preproenzyme of SmCL1. Recombinant SmCL1 was secreted by the transformed yeast into the culture media from which it was purified by gel filtration and ion-exchange chromatography. The purified enzyme exhibited substrate specificity against synthetic peptidyl substrates (e.g., Boc-Val-Leu-Lys-NHMec and Z-Phe-Arg-NHMec;k cat/Km = 17.25 and 6.24 mM?1 s?1, respectively) and against gelatin and hemoglobin, characteristic of cathepsin L. Immunoblot analysis using antiserum raised against recombinant SmCL1 demonstrated that native SmCL1 of 33 kDa was present in ES products and soluble extracts of S. mansoni. Using this antiserum and thin tissue sections, we localized the native SmCL1 to the gastrodermis and to the tegument of adult schistosomes. Recombinant SmCL1 was capable of degrading human hemoglobin at pH 4.0 to 4.5 but not higher, suggesting that denaturation of hemoglobin by low pH, as found in the cecum of the adult schistosome, may be necessary for its catalysis by cathepsin L and other gut-associated proteinases. Together, these results support a role for SmCL1 in the degradation of host hemoglobin within the gut of the schistosome.
机译：血吸虫所表达的半胱氨酸蛋白酶似乎在消化宿主血红蛋白中起关键作用，宿主血红蛋白是这些寄生虫利用的氨基酸营养的主要来源。我们以前已经证明，曼氏血吸虫和 S的成年人的可溶性提取物和分泌/分泌（ES）产物中半胱氨酸蛋白酶的主要活性。 japonicum 在组织底物特异性上类似于组织蛋白酶L。然而，由于存在至少两种不同形式的血吸虫组织蛋白酶L，即SmCL1和SmCL2，对血吸虫的提取物和ES产物中的组织蛋白酶L活性进行了生化分析。现在，我们报道了通过用编码SmCL1前酶的表达质粒转化啤酒酵母（Saccharomyces cerevisiae）而获得的活性重组SmCL1的纯化和酶学特征。重组的SmCL1由转化的酵母分泌到培养基中，并通过凝胶过滤和离子交换色谱从中纯化。纯化的酶对合成的肽基底物（例如Boc-Val-Leu-Lys-NHMec和Z-Phe-Arg-NHMec; k cat / K m = 17.25和6.24 mM ？1 s ？1 ），并针对明胶和血红蛋白，这是组织蛋白酶的特征L.使用针对重组SmCL1产生的抗血清进行的免疫印迹分析表明，ES产品和 S的可溶性提取物中存在33 kDa的天然SmCL1。 mansoni 。使用这种抗血清和薄组织切片，我们将天然SmCL1定位于胃真皮和成人血吸虫的外皮。重组SmCL1能够在pH 4.0到4.5的条件下降解人血红蛋白，但不能更高，这表明在成人血吸虫盲肠中发现的低pH引起的血红蛋白变性可能是由组织蛋白酶L和其他肠相关的催化作用所必需的。蛋白酶。在一起，这些结果支持SmCL1在血吸虫的肠道内宿主血红蛋白降解中的作用。

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