Nga Bui Thi To, Luziga Claudius, Yamamoto Misa, Kusakabe Ken Takeshi, Yamamoto Yoshimi
a Joint Faculty of Veterinary Science , Yamaguchi University , Yamaguchi , Japan.
Biosci Biotechnol Biochem. 2015;79(4):587-97. doi: 10.1080/09168451.2014.991686. Epub 2014 Dec 17.
Cytotoxic T-lymphocyte antigen-2α (CTLA-2α) is a potent inhibitor of cathepsin L-like cysteine proteases. Recombinant CTLA-2α is known to be a potent, competitive inhibitor of cathepsin L-like cysteine proteases. In this study, cathepsin L, cathepsin C, and tubulointerstitial nephritis antigen-related protein 1 (TINAGL1) were identified as novel interactive proteins of CTLA-2α by the yeast two-hybrid screening system. The direct interactions and co-localization of these proteins with CTLA-2α were confirmed using co-immunoprecipitation and immunofluorescence staining, respectively. The disulfide-bonded CTLA-2α/cathepsin L complex was isolated from mouse tissue. CTLA-2α was found to be specific and consistently expressed on the maternal side of the mouse placenta. Double immunofluorescence analysis showed that CTLA-2α was co-localized with cathepsin L, cathepsin C, and TINAGL1 in placenta. A simple cell-based fluorescence assay revealed that CTLA-2α exhibited inhibitory activity toward cathepsin C in live cells, which indicated that CTLA-2α is a novel endogenous inhibitor of cathepsin C.
细胞毒性T淋巴细胞抗原2α（CTLA-2α）是组织蛋白酶L样半胱氨酸蛋白酶的强效抑制剂。已知重组CTLA-2α是组织蛋白酶L样半胱氨酸蛋白酶的强效竞争性抑制剂。在本研究中，通过酵母双杂交筛选系统，组织蛋白酶L、组织蛋白酶C和肾小管间质性肾炎抗原相关蛋白1（TINAGL1）被鉴定为CTLA-2α的新型相互作用蛋白。分别使用免疫共沉淀和免疫荧光染色证实了这些蛋白与CTLA-2α的直接相互作用和共定位。从小鼠组织中分离出二硫键连接的CTLA-2α/组织蛋白酶L复合物。发现CTLA-2α在小鼠胎盘的母体侧特异性且持续表达。双重免疫荧光分析表明，CTLA-2α与胎盘组织中的组织蛋白酶L、组织蛋白酶C和TINAGL1共定位。一种基于细胞的简单荧光测定法显示，CTLA-2α在活细胞中对组织蛋白酶C具有抑制活性，这表明CTLA-2α是组织蛋白酶C的新型内源性抑制剂。
