Zhou Ze-jun, Qiu Reng, Zhang Jian
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China.
Fish Physiol Biochem. 2015 Apr;41(2):473-83. doi: 10.1007/s10695-014-9998-4. Epub 2014 Oct 19.
Cathepsin B is an enzymatic protein belonging to the peptidase C1 family. It is involved in diverse physiological and pathological functions that include immune response. In this study, we identified and characterized a cathepsin B homolog (SmCatB) from turbot (Scophthalmus maximus). SmCatB is composed of 330 amino acid residues and possesses typical domain architecture of cathepsin B, which contains a propeptide region and a cysteine protease domain, and the latter processes four conserved residues (Q101, C107, H277, and N297) in the active site. SmCatB shares 80.6-87.6% overall sequence identities with the cathepsin B of a number of teleost. SmCatB expression was detected in a wide range of tissues and upregulated by bacterial infection in a time-dependent manner. Recombinant SmCatB (rSmCatB-WT) purified from Escherichia coli exhibited apparent protease activity, which was optimal at 50 °C and pH 5.5. Compared to rSmCatB-WT, the mutant proteins rSmCatB-C107S, rSmCatB-H277A, and rSmCatB-N297A, which bear C107S, H277A, and N297A mutations, respectively, were significantly reduced in protease activity, with the highest reduction observed with rSmCatB-N297A. These results indicate that SmCatB is a bioactive protease that depends on the conserved structural features and that SmCatB is involved in pathogen-induced immune response.
组织蛋白酶B是一种属于肽酶C1家族的酶蛋白。它参与多种生理和病理功能，包括免疫反应。在本研究中，我们从大菱鲆（Scophthalmus maximus）中鉴定并表征了一种组织蛋白酶B同源物（SmCatB）。SmCatB由330个氨基酸残基组成，具有组织蛋白酶B典型的结构域结构，其中包含一个前肽区域和一个半胱氨酸蛋白酶结构域，后者在活性位点有四个保守残基（Q101、C107、H277和N297）。SmCatB与多种硬骨鱼的组织蛋白酶B的整体序列同一性为80.6 - 87.6%。SmCatB在多种组织中均有表达，并在细菌感染后呈时间依赖性上调。从大肠杆菌中纯化得到的重组SmCatB（rSmCatB-WT）表现出明显的蛋白酶活性，其最适温度为50°C，最适pH为5.5。与rSmCatB-WT相比，分别携带C107S、H277A和N297A突变的突变蛋白rSmCatB-C107S、rSmCatB-H277A和rSmCatB-N297A的蛋白酶活性显著降低，其中rSmCatB-N297A的活性降低最为明显。这些结果表明，SmCatB是一种依赖于保守结构特征的生物活性蛋白酶，并且SmCatB参与了病原体诱导的免疫反应。
