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Cystatins are reversible, competitive inhibitors of cysteine proteinases. Their inhibitory profiles, as well as their affinities for target enzymes, vary with different cysteine proteinases. Human cystatin C and salivary cystatin SN are 120- and 121-amino-acid (a.a.) proteins, respectively, and both contain 2 disulfide bonds. In this study, we examined the structure/function relationship of cystatin SN with respect to the inhibition of papain, with particular emphasis on the role of cystatin SN's cysteine residues, and addressed the inhibitory profiles of these two human cystatins on several cysteine proteinases (papain, clostripain, and calpain II). The full-length recombinant cystatin C and cystatin SN, and cystatin SN variants (C-truncated C-tr; a.a. 1-102, delta 56-60 deletion, cysteine 74-->serine C74S, cys 84-->serine C84S, cysteine 98-->serine C98S, and cysteine 118-->serine C118S) were cloned, expressed, and produced in the pET30(b) and pGEX2T Escherichia coli expression systems. All recombinant proteins were tested for the inhibition of papain, and the full-length proteins were also tested for the inhibition of clostripain and calpain II. The secondary structures of the cystatins were also determined and compared. The results showed that the full-length cystatin C and cystatin SN, and the cystatin SN variants C98S and C118S inhibited the activity of papain. However, cystatin SN C-tr and delta 56-60 variants exhibited no inhibitory activity toward papain, while the cystatin SN variants C74S and C84S exhibited slight inhibition at higher concentrations. These results suggested that in the inhibition of papain by cystatin SN, the first disulfide loop is more important than the second. In addition, cystatin C, but not cystatin SN, inhibited calpain II, while neither cystatin inhibited clostripain, and these results, in conjunction with those from other studies, indicated that cystatin C is a broader-spectrum inhibitor of cysteine proteinases than cystatin SN.
Hiltkel et al. (Sun,) studied this question.