Differential Effect toward Inhibition of Papain and Cathepsin C by Recombinant Human Salivary Cystatin SN and Its Variants Produced by a Baculovirus System

Human salivary cystatin SN (CsnSN) is a member of the cystatin superfamily of cysteine proteinase inhibitors. In this study we used a baculovirus expression system to produce a full-length unaltered CsnSN and its variants. The variants were constructed with the changes in the three predicted proteinase-binding regions: the N-terminus (variant N12–13, G12A–G13A), β-hairpin loop I (variant L56–58, Q56G–T57G–V58G) and β-hairpin loop II (variant L106–107, P106G–W107G). The secreted CsnSNs were purified using sequential spiral cartridge ultrafiltration and DE-52 radial flow chromatography. The purified proteins were examined for papain- and cathepsin C-inhibition. The wild-type CsnSN, and variants N12–13 and L106–107 bound tightly to papain (Ki < 10 pM), whereas mutation in the loop I reduced binding affinity 5700-fold (Ki = 57 nM). On the other hand, the wild-type CsnSN bound to cathepsin C less tightly (Ki = 100 nM). The mutation in the N-terminus or loop I reduced binding affinity by 16 (Ki = 1.6 μM)- and 19-fold (Ki = 1.9 μM), respectively, while mutation in loop II resulted in an ineffective cathepsin C inhibitor (Ki = 14 μM). Collectively, these results suggest that the N-terminal G12–G13 residues of CsnSN are not essential for papain inhibition but play a role in cathepsin C inhibition; residues Q56–T57–V58 in the loop I are essential for both papain and cathepsin C inhibitions, and residues P106–W107 in the loop II are not important for papain inhibition but essential for cathepsin C inhibition. These results demonstrated that CsnSN variants have different effects toward different cysteine proteinases.