Brady C P, Dowd A J, Brindley P J, Ryan T, Day S R, Dalton J P
School of Biological Sciences, Dublin City University, Ireland.
Infect Immun. 1999 Jan;67(1):368-74. doi: 10.1128/IAI.67.1.368-374.1999.
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 of Schistosoma 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 transforming Saccharomyces 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; kcat/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.
血吸虫表达的半胱氨酸蛋白酶似乎在宿主血红蛋白的消化过程中发挥关键作用，血红蛋白是这些寄生虫利用的氨基酸营养的主要来源。我们之前已经表明，曼氏血吸虫和日本血吸虫成虫的可溶性提取物及排泄/分泌（ES）产物中的主要半胱氨酸蛋白酶活性，在底物特异性方面类似于组织蛋白酶L。然而，由于血吸虫组织蛋白酶L至少存在两种不同形式，即SmCL1和SmCL2，对血吸虫提取物和ES产物中的组织蛋白酶L活性进行生化分析变得复杂。我们现在报告了活性重组SmCL1的纯化及酶学特性，该重组蛋白是通过用编码SmCL1前体酶的表达质粒转化酿酒酵母而获得的。重组SmCL1由转化后的酵母分泌到培养基中，通过凝胶过滤和离子交换色谱法从培养基中进行纯化。纯化后的酶对合成肽底物（如Boc-Val-Leu-Lys-NHMec和Z-Phe-Arg-NHMec；kcat/Km分别为17.25和6.24 mM-1 s-1）以及明胶和血红蛋白表现出底物特异性，这是组织蛋白酶L的特征。使用针对重组SmCL1产生的抗血清进行免疫印迹分析表明，33 kDa的天然SmCL1存在于曼氏血吸虫的ES产物和可溶性提取物中。利用这种抗血清和薄组织切片，我们将天然SmCL1定位到成虫血吸虫的胃皮层和体表。重组SmCL1能够在pH 4.0至4.5时降解人血红蛋白，但在更高pH时则不能，这表明在成虫血吸虫盲肠中发现的低pH导致的血红蛋白变性，可能是其被组织蛋白酶L和其他肠道相关蛋白酶催化所必需的。总之，这些结果支持SmCL1在血吸虫肠道内宿主血红蛋白降解中的作用。
