Novel cysteine proteinase inhibitor from Bombyx mori-its function and origin-

家蚕半胱氨酸蛋白酶抑制剂（BCPI）是一种新型半胱氨酸蛋白酶抑制剂。该蛋白序列与某些半胱氨酸蛋白酶的原区同源。本文报道其抑制几种半胱氨酸蛋白酶的机制。BCPI对家蚕半胱氨酸蛋白酶（BCP）活性有较强的抑制作用，Ki值为5.9 pM，对人组织蛋白酶L活性有较强的抑制作用，Ki值为36 pM。抑制符合慢结合动力学。对组织蛋白酶H的抑制作用较弱（Ki = 82 nM），而对木瓜蛋白酶（Ki > 1mM）和组织蛋白酶B （Ki > 4mM）的抑制作用可以忽略不计。与BCP孵育后，BCPI首先在c端被截断，然后逐渐降解，时间延长。截断主要发生在两个c端氨基酸残基上。缺乏两个c端氨基酸残基的重组BCPI仍具有显著的抑制活性。我们的研究结果表明，BCPI是一种稳定的、高选择性的组织蛋白酶l样半胱氨酸蛋白酶抑制剂。小鼠活化的t淋巴细胞表达细胞毒性t淋巴细胞抗原（CTLA-2），该抗原与小鼠组织蛋白酶l的前区同源。重组细胞毒性t淋巴细胞抗原（CTLA-2a）也表现出对组织蛋白酶l样半胱氨酸蛋白酶的选择性抑制。根据这些结果，我们提出BCPI和CTLA-2是半胱氨酸蛋白酶抑制剂的新成员，并且是组织蛋白酶l样半胱氨酸蛋白酶的高选择性抑制剂。基因组分析显示，果蝇和大鼠中表达了类似的前肽样蛋白，这表明在多种生物体中存在一类新的半胱氨酸蛋白酶抑制剂。基因结构的研究和系统发育分析表明，半胱氨酸蛋白酶抑制剂的基因来源于其亲本酶的祖先基因。

Bombyx cysteine proteinase inhibitor (BCPI) is a novel cysteine proteinase inhibitor. The protein sequence is homologous to the proregions of certain cysteine proteinases. Here we report its mechanism of inhibition of several cysteine proteinases. BCPI strongly inhibited Bombyx cysteine proteinase (BCP) activity with a Ki=5.9 pM, and human cathepsin L with a Ki=36 pM. The inhibition obeyed slow-binding kinetics. The inhibition of cathepsin H was much weaker (Ki = 82 nM), while inhibition of papain (Ki > 1mM) and cathepsin B (Ki > 4mM) was negligible. Following incubation with BCP, BCPI was first truncated at the C-terminal end, and then gradually degraded for prolonged time. The truncation occured mainly by two C-terminal amino acid residues. Recombinant BCPI lacking the two C-terminal amino acid residues still retained substantial inhibitory activity. Our results indicate that BCPI is a stable and highly selective inhibitor of cathepsin L-like cysteine proteinases. Mouse activated T-lymphocytes express cytotoxic T-lymphocyte antigen (CTLA-2), which is homologous to the proregion of mouse cathepsin L. Recombinant cytotoxic T-lymphocyte antigen (CTLA-2a) also exhibited selective inhibition of cathepsin L-like cysteine proteinases. From these results, we propose that the BCPI and CTLA-2 are a new member of cysteine proteinase inhibitors, and are highly selective inhibitors of cathepsin L-like cysteine proteinases. Genome analyses have shown the expression of similar propeptide-like proteins in Drosophila and rat, suggesting the presence of a novel class of cysteine proteinase inhibitors in a variety of organisms. Studies of the gene structures and phylogenetic analysis have shown that genes of the propeptide-like cysteine proteinase inhibitors have emerged from ancestor genes of their parental enzymes.