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

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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.