Botulinum neurotoxin C mutants reveal different effects of syntaxin or SNAP-25 proteolysis on neuromuscular transmission

PLoS Pathog. 2017 Aug 11;13(8):e1006567. doi: 10.1371/journal.ppat.1006567. eCollection 2017 Aug.

Abstract

Botulinum neurotoxin serotype C (BoNT/C) is a neuroparalytic toxin associated with outbreaks of animal botulism, particularly in birds, and is the only BoNT known to cleave two different SNARE proteins, SNAP-25 and syntaxin. BoNT/C was shown to be a good substitute for BoNT/A1 in human dystonia therapy because of its long lasting effects and absence of neuromuscular damage. Two triple mutants of BoNT/C, namely BoNT/C S51T/R52N/N53P (BoNT/C α-51) and BoNT/C L200W/M221W/I226W (BoNT/C α-3W), were recently reported to selectively cleave syntaxin and have been used here to evaluate the individual contribution of SNAP-25 and syntaxin cleavage to the effect of BoNT/C in vivo. Although BoNT/C α-51 and BoNT/C α-3W toxins cleave syntaxin with similar efficiency, we unexpectedly found also cleavage of SNAP-25, although to a lesser extent than wild type BoNT/C. Interestingly, the BoNT/C mutants exhibit reduced lethality compared to wild type toxin, a result that correlated with their residual activity against SNAP-25. In spite of this, a local injection of BoNT/C α-51 persistently impairs neuromuscular junction activity. This is due to an initial phase in which SNAP-25 cleavage causes a complete blockade of neurotransmission, and to a second phase of incomplete impairment ascribable to syntaxin cleavage. Together, these results indicate that neuroparalysis of BoNT/C at the neuromuscular junction is due to SNAP-25 cleavage, while the proteolysis of syntaxin provides a substantial, but incomplete, neuromuscular impairment. In light of this evidence, we discuss a possible clinical use of BoNT/C α-51 as a botulinum neurotoxin endowed with a wide safety margin and a long lasting effect.

MeSH terms

  • Animals
  • Botulinum Toxins / genetics
  • Botulinum Toxins / toxicity*
  • Evoked Potentials / drug effects
  • Immunoblotting
  • Immunohistochemistry
  • Mice
  • Mutation
  • Neuromuscular Junction / drug effects
  • Patch-Clamp Techniques
  • Proteolysis
  • Qa-SNARE Proteins / metabolism*
  • Rats
  • Synaptic Transmission / drug effects*
  • Synaptosomal-Associated Protein 25 / metabolism*

Substances

  • Qa-SNARE Proteins
  • Synaptosomal-Associated Protein 25
  • Botulinum Toxins
  • botulinum toxin type C

Grant support

This work was supported by grants from the University of Padova to OR (grant DOR1692572/16) and in part by the Bundesministerium für Wirtschaft und Technologie (BMWi) Zentrales Innovationsprogramm Mittelstand (KF2437603MD3) to AR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.