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 expr ession system to produce a full-length unaltered CsnSN and its variants. Th e variants were constructed with the changes in the three predicted protein ase-binding regions: the N-terminus (variant N12-13, G12A-G13A), beta-hairp in loop I (variant L56-58, Q56G-T57G-V58G) and beta-hairpin loop II (varian t L106-107, P106G-W107G). The secreted CsnSNs were purified using sequentia l spiral cartridge ultrafiltration and DE-52 radial how chromatography, The purified proteins were examined for papain- and cathepsin C-inhibition, Th e wild-type CsnSN, and variants N12-13 and L106-107 bound tightly to papain (K-i < 10 pM), whereas mutation in the loop I reduced binding affinity 570 0-fold (K-i = 57 nM). On the other hand, the wild-type CsnSN bound to cathe psin C less tightly (K-i = 100 nM). The mutation in the N-terminus or loop I reduced binding affinity by 16 (K-i = 1.6 mu M)- and 19-fold (K-i = 1.9 m u M), respectively, while mutation in loop II resulted in an ineffective ca thepsin C inhibitor (K-i = 14 mu M). Collectively, these results suggest th at the N-terminal G12-G13 residues of CsnSN are not essential for papain in hibition 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 b ut essential for cathepsin C inhibition. These results demonstrated that Cs nSN variants have different effects toward different cysteine proteinases. (C) 2000 Academic Press.