Simple modifications of the serpin reactive site loop convert SCCA2 into acysteine proteinase inhibitor: A critical role for the P3 ' proline in facilitating RSL cleavage

The human squamous cell carcinoma antigens (SCCA) 1 and 2 are members of th e serpin family that are 92% identical in their amino acid sequence. Despit e this similarity, they inhibit distinct classes of proteinases. SCCA1 neut ralizes the papain-like cysteine proteinases, cathepsins (cat) S, L, and K; and SCCA2 inhibits the chymotrypsin-like serine proteinases, catG and huma n mast cell chymase. SCCA2 also can inhibit catS, as well as other papain-l ike cysteine proteinases, albeit at a rate 50-fold less than that of SCCA1. Analysis of the mechanism of inhibition by SCCA1 revealed that the reactiv e site loop (RSL) is important for cysteine proteinase inhibition. The inhi bition of catS by a mutant SCCA2 containing the RSL of SCCA1 is comparable to that of wild-type SCCA1. This finding suggested that there were no motif s outside and only eight residues within the RSL that were directing catS-s pecific inhibition. The purpose of this study was to determine which of the se residues might account for the marked difference in the ability of SCCA 1 and SCCA2 to inhibit papain-like cysteine proteinases. SCCA2 molecules co ntaining different RSL mutations showed that no single amino acid substitut ion could convert SCCA2 into a more potent cysteine proteinase inhibitor. R ather, different combinations of mutations led to incremental increases in catS inhibitory activity with residues in four positions (P1, P3', P4', and P11') accounting for 80% of the difference in activity between SCCA1 and S CCA2. Interestingly, the RSL cleavage site differed between wild-type SCCA2 and this mutant. Moreover, these data established the importance of a Pro residue in the P3' position for efficient inhibition of catS by both wild-t ype SCCA1 and mutated SCCA2. Molecular modeling studies suggested that this residue might facilitate positioning of the RSL within the active site of the cysteine proteinase.