Delaria K, Fiorentino L, Wallace L, Tamburini P, Brownell E, Muller D
Miles, Inc., Pharmaceutical Division, West Haven, Connecticut 06516-4175.
J Biol Chem. 1994 Oct 7;269(40):25172-7.
The protein sequence of cytotoxic T-lymphocyte antigen-2 beta (CTLA-2 beta) is 36% identical to the proregion of mouse cathepsin L (Denizot, F., Brunet, J.F., Roustan, P., Harper, K., Suzan, M., Luciani, M. F., Mattei, M. G., and Goldstein, P. (1989) Eur. J. Immunol. 19, 631-635). Here we report the expression, purification, and characterization of recombinant murine CTLA-2 beta. The protein was purified by consecutive gel-filtration, anion-exchange, and reverse-phase (C4) chromatography. Purified CTLA-2 beta exists in solution primarily as a dimer but also as a disulfide-linked tetramer as judged by size exclusion chromatography. Circular dichroism studies suggest that the dimeric form of the protein contains 8% alpha-helix, 67% beta-sheet, and 21% random coil and also indicates that there is a conformational change upon formation of the tetramer. The protein is a competitive inhibitor of certain cysteine proteases including papain (Ki = 25 nM), cathepsins L (Ki = 24 nM) and H (IC50 = 67 nM) but not cathepsin B. CTLA-2 beta forms a noncovalent complex with cathepsin L and has a stoichiometry of binding to papain of 1 mol of CTLA-2 beta/mol of papain. There is no homology between CTLA-2 beta and any of the known cysteine protease inhibitors, including the kininogens and cystatins. Therefore, CTLA-2 beta represents a novel class of cysteine protease inhibitor that is specific for the cathepsin L family of proteases.
细胞毒性T淋巴细胞抗原2β（CTLA-2β）的蛋白质序列与小鼠组织蛋白酶L的前区域有36%的同源性（德尼佐特，F.，布鲁内，J.F.，鲁斯坦，P.，哈珀，K.，苏珊，M.，卢西亚尼，M.F.，马泰，M.G.，和戈尔茨坦，P.（1989年）《欧洲免疫学杂志》19，631 - 635）。在此我们报告重组小鼠CTLA-2β的表达、纯化及特性鉴定。该蛋白通过连续的凝胶过滤、阴离子交换和反相（C4）色谱法进行纯化。通过尺寸排阻色谱法判断，纯化后的CTLA-2β在溶液中主要以二聚体形式存在，但也有二硫键连接的四聚体形式。圆二色性研究表明，该蛋白的二聚体形式含有8%的α-螺旋、67%的β-折叠和21%的无规卷曲，同时也表明四聚体形成时存在构象变化。该蛋白是某些半胱氨酸蛋白酶的竞争性抑制剂，包括木瓜蛋白酶（Ki = 25 nM）、组织蛋白酶L（Ki = 24 nM）和H（IC50 = 67 nM），但对组织蛋白酶B无抑制作用。CTLA-2β与组织蛋白酶L形成非共价复合物，与木瓜蛋白酶的结合化学计量比为1摩尔CTLA-2β/摩尔木瓜蛋白酶。CTLA-2β与任何已知的半胱氨酸蛋白酶抑制剂，包括激肽原和胱抑素，均无同源性。因此，CTLA-2β代表了一类新型的半胱氨酸蛋白酶抑制剂，对组织蛋白酶L家族的蛋白酶具有特异性。
