Figure 4. SMSs inhibit mammalian proteases and form serpin/protease complexes.

(A) Inhibitory profile of mammalian and mite proteases by wild type SMSs (overview). Proteases were pre-incubated with different concentrations of SMSB3 or SMSB4 at 37°C for 10 min in the appropriate reaction buffer. The reaction was started by addition of the corresponding fluorescent aminomethyl-coumarin substrate and enzyme inhibition was measured as change in fluorescence in comparison with controls. Shown are means ± SD (n = 3). (B) Stoichiometry of inhibition for SMSB3. The inhibitory activity was assessed by measuring residual enzyme activities of trypsin, chymotrypsin and leucocyte elastase. Enzymes were incubated with a 2-120:1 molar excess of SMSB3 at 37°C for 10 min in the appropriate reaction buffer. Residual enzyme activity was measured in triplicate (SD ≤5%) by adding the appropriate aminomethyl-coumarin peptide substrate and determining the reaction velocity as change in fluorescence. The fractional activity was the ratio of the velocity of inhibited enzyme (v_i) over the velocity of uninhibited control (v_o). The initial inhibitor/enzyme ratios ([I₀]/[E₀]) represent the molar excess of serpin over enzyme. (C) Complex formation between SMSB3 and serine proteases. Serpin/protease complexes were analyzed by SDS-PAGE under non-reducing conditions after pre-incubation of SMSB3 (black arrow) with each protease at 37°C for 15 min at serpin/protease ratios of 4∶1. Covalent complex formation was found with chymotrypsin (grey arrow), while SMSB3 was degraded by trypsin, elastase and Der p 3 (white arrows). C, chymotrypsin; D, house dust mite Der p 3; E, neutrophil elastase; S, SMSB3; S3, scabies mite Sar s 3; T, trypsin. (D) Effects of SMSB3 and two mutants on chymotrypsin activity.