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<p>Botulinum neurotoxin A (BoNT/A) is one of the most toxic substances known. The bacterium <i>Clostridium botulinum</i> 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 <i>C. botulinum</i> 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 <i>C. botulinum</i> 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年10月24日

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  • Shani Pitel,
  • Ada Barnea,
  • Eran Diamant,
  • Amram Torgeman,
  • Tal Noy-Porat,
  • Reut Falach,
  • Ran Zichel,
  • Zvi Hayouka,
  • Alon Ben David

摘要

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.