Functional characterization of a new non-Kunitz serine protease inhibitor from the scorpion Lychas mucronatus

Abstract

Serine protease inhibitors have been widely discovered from different animal venoms, but most of them belong to Kunitz-type toxin subfamily. Here, by screening scorpion venom gland cDNA libraries, we identified four new non-Kunitz serine protease inhibitors with a conserved Ascaris-type structural fold: Ascaris-type toxins Lychas mucronatus Ascaris-type protease inhibitor (LmAPI), Pandinus cavimanus Ascaris-type protease inhibitor (PcAPI), Pandinus cavimanus Ascaris-type protease inhibitor 2 (PcAPI-2), and Hottentotta judaicus Ascaris-type protease inhibitor (HjAPI). The detailed characterization of one Ascaris-type toxin LmAPI was further carried out, which contains 60 residues and possesses a classical Ascaris-type cysteine framework reticulated by five disulfide bridges. Enzyme and inhibitor reaction kinetics experiments showed that recombinant LmAPI inhibits the activity of chymotrypsin potently with a Ki value of 15.5 nM, but has little effect on trypsin and elastase. Bioinformatics analyses suggested that LmAPI contains unique functional residues "TQD" and might be a useful template to produce specific protease inhibitors. Our results indicated that animal venoms are a natural source of new type of protease inhibitors, which will accelerate the development of diagnostic and therapeutic agents for human diseases that target diverse proteases.

Keywords: Ascaris-type toxin; Molecular diversity; cDNA libraries.