Structure/function analysis of human cystatin SN and comparison of the cysteine proteinase inhibitory profiles of human cystatins C and SN

Cystatins are reversible, competitive inhibitors of cysteine proteinases. T heir inhibitory profiles, as well as their affinities for target enzymes, v ary with different cysteine proteinases. Human cystatin C and salivary cyst atin SN are 120- and 121-amino-acid (a.a.) proteins, respectively, and both contain 2 disulfide bonds. In this study, we examined the structure/functi on relationship of cystatin SN with respect to the inhibition of papain, wi th particular emphasis on the role of cystatin SN's cysteine residues, and addressed the inhibitory profiles of these two human cystatins on several c ysteine proteinases (papain, clostripain, and calpain II). The full-length recombinant cystatin C and cystatin SN, and cystatin SN variants (C-truncat ed [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) an d pGEX2T Escherichia coli expression systems. AU. recombinant proteins were tested for the inhibition of papain, and the full-length proteins were als o tested for the inhibition of clostripain and calpain II. The secondary st ructures of the cystatins were also determined and compared. The results sh owed that the full-length cystatin C and cystatin SN, and the cystatin SN v ariants C98S and C118S inhibited the activity of papain. However, cystatin SN C-tr and Delta 56-60 variants exhibited no inhibitory activity toward pa pain, while the cystatin SN variants C74S and C84S exhibited slight inhibit ion at higher concentrations. These results suggested that in the inhibitio n of papain by cystatin SN, the first disulfide loop is more important than the second. In addition, cystatin C, but not cystatin SN, inhibited calpai n II, while neither cystatin inhibited clostripain, and these results, in c onjunction with those from other studies, indicated that cystatin C is a br oader-spectrum inhibitor of cysteine proteinases than cystatin SN.