Cross-Class Inhibition of Elastolytic Cysteine Proteinases by the Human Serpin SCCA1. • 1824

In disorders such as asthma, cystic fibrosis and bronchitis, the bronchial mucosa is injured by neutrophil-derived degradative enzymes such as cathepsin G (catG) and elastase. Damaged epithelial cells exacerbate the injury by releasing lysosomal cysteine proteinases (LCPs) capable of degrading extracellular matrix. The LCPs catK and S are of particular concern, since they are potent elastolytic agents and are active at both acidic and neutral pH. We hypothesize that the bronchial mucosa has evolved an antiproteinase defense system to protect against excessive LCP activity. We identified two high-molecular-weight serine proteinase inhibitors (serpins), SCCA1 and SCCA2. These molecules are 92% identical at the amino acid level and are highly expressed in cells of the bronchial epithelium and glands. Kinetic analysis shows that SCCA2 is a potent inhibitor of the chymotrypsin-like serine proteinases, catG and mast cell chymase. In contrast, SCCA1 inhibited members of the papain-like LCP family. To confirm that SCCA1 was a specific inhibitor and not just a preferred substrate for LCPs, we performed a kinetic analysis. Typical of serpins, SCCA1 formed tight complexes with catS, L, and K at a stoichiometry of 1:1 and with second order rate constants(k_ass)=5, 3, and 1 × 105 M¹ sec^-1, respectively. These rate constants are comparable to those measured for serpin-serine proteinase interactions. To determine whether SCCA1 could inhibit degradation of complex substrates, we measured the inhibition of³ H-elastin degradation by catK. SCCA1 inhibited elastolytic activity by 80% and on a molar basis was more potent than the archetypal cysteine proteinase inhibitor, cystatin C (cysC). Finally, we compared the first order rate constants(k_diss) and k_ass for the interactions of catS with either cysC vs. SCCA1 by progress curves. The catS-cysC complex association and dissociation = 4 × 10⁵M¹ sec^-1 and 2 × 10^-4 sec, respectively. The complex half-life (t_1/2) was 55 minutes. For the catS-SCCA1 interactions, the k_ass=1 × 10⁵M¹ sec^-1, but the k_diss was not measured since no dissociation of the complex was detected. Thus, a minimal estimate of the catS-SCCA1 t_1/2 was 1155 minutes. These results show that relative to cysC, SCCA1 was slightly slower to form complexes with catS, but once formed were extremely long lived. We conclude that SCCA1 is a bona fide cross-class inhibitor of elastolytic LCPs and that its location in the bronchial mucosa may serve to limit mucosal injury mediated the leakage of endogenous proteinases.