SmCL3, a gastrodermal cysteine protease of the human blood fluke Schistosoma mansoni.
SmCL3, a gastrodermal cysteine protease of the human blood fluke Schistosoma mansoni.
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DOI：
10.1371/journal.pntd.0000449
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发表时间：
2009-06-02
影响因子：
3.8
通讯作者：
Caffrey CR
中科院分区：
文献类型：
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作者：
Dvorák J;Mashiyama ST;Sajid M;Braschi S;Delcroix M;Schneider EL;McKerrow WH;Bahgat M;Hansell E;Babbitt PC;Craik CS;McKerrow JH;Caffrey CR
血吸虫属的血吸虫是感染全世界2亿人的扁平蠕虫寄生虫。从宿主血流中消化营养物质对寄生虫的发育和繁殖至关重要。蛋白水解酶（蛋白酶）的网络促进宿主血红蛋白和血清蛋白的水解。我们利用基于S. mansoni EST序列数据。日本血吸虫中存在一种直系同源物。SmCL 3作为一种活性酶在毕赤酵母中异源表达。重组SmCL 3对肽基底物具有广泛的pH活性范围，并被Clan CA蛋白酶抑制剂抑制。与降解宿主蛋白的功能一致，SmCL 3水解血清白蛋白和血红蛋白，定位于成体胃真皮，主要在感染哺乳动物宿主的生命阶段表达。SmCL 3在寄生虫中的主要形式是作为酶原存在，这对于蛋白酶来说是不寻常的。这种酶原包括一个异常长的前结构域，具有α螺旋二级结构基序。SmCL 3的显着特异性的氨基酸与大的芳香族侧链（色氨酸和酪氨酸）在P2底物的位置，确定与位置扫描合成组合库，是一致的分子模型，显示了一个大而深的S2口袋。一个序列相似性网络（SSN）的视图集群SmCL 3和其他组织蛋白酶L按照以前的大规模系统发育分析，确定六个超级王国。SmCL 3是一种肠道相关的组织蛋白酶L，可能有助于参与降解宿主血液蛋白作为营养物质的蛋白酶网络。此外，这种酶表现出一些不寻常的序列和生物物理特征，可能会导致额外的功能。组织蛋白酶L之间的网络相互关系的可视化表明，这些酶是合适的“标记序列”，包括在未来的系统发育分析。由血吸虫属血吸虫引起的寄生虫感染是一个主要的全球性健康问题。超过2亿人被感染。鉴定和表征寄生虫生化途径的组成酶应该揭示开发新疗法的机会（即，疫苗、药物）。血吸虫以宿主血液为食，许多蛋白水解酶（蛋白酶）有助于这一过程。我们已经确定并表征了一种新的蛋白酶，SmCL 3（血吸虫组织蛋白酶L3），这是发现在肠道组织的寄生虫。我们已经采用了各种生化和分子生物学方法和序列相似性分析，以表征SmCL 3，并获得洞察其可能的功能，在寄生虫，以及它的组织蛋白酶L蛋白酶之间的进化地位一般。SmCL 3水解主要的宿主血液蛋白（血清白蛋白和血红蛋白），并在感染哺乳动物宿主的寄生虫生命阶段中表达。通过分子模拟证实了位置扫描-合成组合文库检测的酶底物特异性。根据以前的系统发育分析，序列分析将SmCL 3置于其他组织蛋白酶L的簇中。
Blood flukes of the genus Schistosoma are platyhelminth parasites that infect 200 million people worldwide. Digestion of nutrients from the host bloodstream is essential for parasite development and reproduction. A network of proteolytic enzymes (proteases) facilitates hydrolysis of host hemoglobin and serum proteins. We identified a new cathepsin L termed SmCL3 using PCR strategies based on S. mansoni EST sequence data. An ortholog is present in Schistosoma japonicum. SmCL3 was heterologously expressed as an active enzyme in the yeast, Pichia pastoris. Recombinant SmCL3 has a broad pH activity range against peptidyl substrates and is inhibited by Clan CA protease inhibitors. Consistent with a function in degrading host proteins, SmCL3 hydrolyzes serum albumin and hemoglobin, is localized to the adult gastrodermis, and is expressed mainly in those life stages infecting the mammalian host. The predominant form of SmCL3 in the parasite exists as a zymogen, which is unusual for proteases. This zymogen includes an unusually long prodomain with alpha helical secondary structure motifs. The striking specificity of SmCL3 for amino acids with large aromatic side chains (Trp and Tyr) at the P2 substrate position, as determined with positional scanning-synthetic combinatorial library, is consistent with a molecular model that shows a large and deep S2 pocket. A sequence similarity network (SSN) view clusters SmCL3 and other cathepsins L in accordance with previous large-scale phylogenetic analyses that identify six super kingdoms. SmCL3 is a gut-associated cathepsin L that may contribute to the network of proteases involved in degrading host blood proteins as nutrients. Furthermore, this enzyme exhibits some unusual sequence and biophysical features that may result in additional functions. The visualization of network inter-relationships among cathepsins L suggests that these enzymes are suitable ‘marker sequences’ for inclusion in future phylogenetic analyses. Parasitic infection caused by blood flukes of the genus Schistosoma is a major global health problem. More than 200 million people are infected. Identifying and characterizing the constituent enzymes of the parasite's biochemical pathways should reveal opportunities for developing new therapies (i.e., vaccines, drugs). Schistosomes feed on host blood, and a number of proteolytic enzymes (proteases) contribute to this process. We have identified and characterized a new protease, SmCL3 (for Schistosoma mansoni cathepsin L3), that is found within the gut tissue of the parasite. We have employed various biochemical and molecular biological methods and sequence similarity analyses to characterize SmCL3 and obtain insights into its possible functions in the parasite, as well as its evolutionary position among cathepsin L proteases in general. SmCL3 hydrolyzes major host blood proteins (serum albumin and hemoglobin) and is expressed in parasite life stages infecting the mammalian host. Enzyme substrate specificity detected by positional scanning-synthetic combinatorial library was confirmed by molecular modeling. A sequence analysis placed SmCL3 to the cluster of other cathepsins L in accordance with previous phylogenetic analyses.