Elucidating the activation mechanism of botulinum neurotoxin a: role of α-clostripain and NTNH

Botulinum neurotoxin A (BoNT/A) is one of the most toxic substances known. The bacterium Clostridium botulinum produces it as a 150-kDa polypeptide with relatively low potency. Surprisingly, the endogenous protease that transforms this polypeptide into an activated, full-potency toxin, consisting of a 100-kDa heavy chain connected to a 50-kDa light chain (LC) by a disulfide bond, is still unknown. This study aimed to identify the BoNT/A-activating protease. We screened cation-exchange chromatography fractions of C. botulinum A culture supernatant for activity using a toxin-simulating substrate comprising the LC and the translocation domain (HN). Proteomic analysis of the active fraction identified α-clostripain as a candidate BoNT/A-activating protease. Recombinant α-clostripain cleaved the simulating substrate between the toxin LC and HN. However, incubation of recombinant α-clostripain with recombinant inactivated BoNT/A (rBoNT/Ai) resulted in non-specific digestion of the toxin. Since similar non-specific digestion was observed also by C. botulinum A culture supernatant, we hypothesized that the toxin should be protected by an accessory protein to prevent non-specific cleavage. Indeed, incubation of rBoNT/Ai with α-clostripain or culture supernatant in the presence of recombinant NTNH (non-toxic non-hemagglutinin) resulted in specific cleavage of the toxin into 100- and 50-kDa fragments. Subsequently, we evaluated the activation of rBoNT/A by α-clostripain in a mouse model. Cleavage of rBoNT/A by α-clostripain in the presence of NTNH resulted in a 77-fold increase in toxicity, corresponding to toxin activation. To the best of our knowledge, these results elucidate the mechanism of BoNT/A activation for the first time.