Characterization of a Ten-Cysteine Peptide from Scorpion That Inhibits Classical Complement Activation via C4/C4b

Scorpion toxin peptides are useful bioactive substance resources, but no anticomplement peptides have been reported from scorpions. Here, we attempt to explore scorpion-derived anticomplement peptides by scanning a small and specific scorpion toxin peptide library. Given that complement activation constitutes a protease-driven proteolytic cascade reaction, we focused on two kinds of serine protease inhibitor-like peptides from scorpion Kunitz-type and Ascaris-type toxin subfamilies. Assays of complement-mediated erythrocyte lysis showed that one scorpion peptide SjAPI-2, with an Ascaris-type structural fold inhibited classical complement activation in a concentration-dependent manner. Complement-deficient experiments revealed that the anti-complement effect of SjAPI-2 was markedly enhanced in sera reconstituted with deficient complement proteins C3 or C4, and suggested that C3 and C4 are associated with the anticomplement effect of SjAPI-2. Combined ELISA and Co-IP assays further revealed that SjAPI-2 can directly bind to C4 and C4b. These results indicated that SjAPI-2 can inhibit classical complement activation via binding to C4 and C4b, which blocks the upstream proteolytic cleavage of C4 as well as the downstream cleavage and activation of C3. Alanine-scanning mutagenesis showed that SjAPI-2 suppressed complement activation via its functional loop from K30 to V38. Myocardial ischemia–reperfusion injury experiment showed that SjAPI-2 limited the extent of myocardial infarction and exerted a cardioprotective effect in vivo. To the best of our knowledge, SjAPI-2 is the first anticomplement toxin peptide from a scorpion, suggesting that scorpion venom peptides might be a new resource for anticomplement peptide discovery. The unique ten-cysteine peptide scaffold of SjAPI-2 also provided a novel lead drug template for C4/C4b associated complement diseases.