Fig 2. Recombinant SmKI-1 and its Kunitz domain inhibit serine proteases and protect S. mansoni against neutrophil elastase.

Recombinant SmKI-1, its Kunitz or C-terminal domains (100 nM) were tested as inhibitor of serino proteases: (a) Bovine Trypsin activity (100nM), (b) Human Neutrophil Elastase activity (300nM) and (c) Neutrophil-secreted elastase activity. In all experiments, bovine serum albumin (BSA, 300nM) was used as a negative control. Enzyme inhibition was detected over two-hour incubation with rSmKI-1 or its Kunitz domain. Bars indicate each enzyme activity mean ± standard deviation. (d) Protective effect of rSmKI-1 (0.15 mg/mL) in cultured schistosomula treated with purified elastase (0.05 mg/mL). Bars represent live parasites ± standard deviation. Data are representative of at least three independent experiments. For (a) and (b), an asterisk indicate statistically significant differences of rSmKI-1 or Kunitz domain compared to control group p< 0.05. For (c) and (d), ** asterisks indicate statistically significant differences of rSmKI-1, compared to control group or elastase group p< 0.005. (e) Binding mode of SmKI-1 Kunitz domain (purple) to neutrophil elastase (gray) predicted by docking with CLUSPRO 2.0. Residues from elastase catalytic triad (His⁷⁰, Asp¹¹⁷ and Ser²⁰²) are highlighted in orange sticks. (f) Detailed analysis of the docking predicted interface reveals residues involved in hydrogen bonds, a salt bridge and a π-stacking interaction (all interactions shown as green dashes). SmKI-1 residues are represented and labeled in purple, NE residues in gray.