Comparison of the catalytic properties of the botulinum neurotoxin subtypes A1 and A5

Biochim Biophys Acta. 2013 Dec;1834(12):2722-8. doi: 10.1016/j.bbapap.2013.09.007. Epub 2013 Oct 2.


Clostridium botulinum neurotoxins (BoNTs) cause the life-threatening disease botulism through the inhibition of neurotransmitter release by cleaving essential SNARE proteins. There are seven serologically distinctive types of BoNTs and many subtypes within a serotype have been identified. BoNT/A5 is a recently discovered subtype of type A botulinum neurotoxin which possesses a very high degree of sequence similarity and identity to the well-studied A1 subtype. In the present study, we examined the endopeptidase activity of these two BoNT/A subtypes and our results revealed significant differences in substrate binding and cleavage efficiency between subtype A5 and A1. Distinctive hydrolysis efficiency was observed between the two toxins during cleavage of the native substrate SNAP-25 versus a shortened peptide mimic. N-terminal truncation studies demonstrated that a key region of the SNAP-25, including the amino acid residues at 151 through 154 located in the remote binding region of the substrate, contributed to the differential catalytic properties between A1 and A5. Elevated binding affinity of the peptide substrate resulted from including these important residues and enhanced BoNT/A5's hydrolysis efficiency. In addition, mutations of these amino acid residues affect the proteolytic performance of the two toxins in different ways. This study provides a better understanding of the biological activity of these toxins, their performance characteristics in the Endopep-MS assay to detect BoNT in clinical samples and foods, and is useful for the development of peptide substrates.

Keywords: A; BoNT/A; Botulinum neurotoxin; D; E; G; K; LC; MS; Mass spectrometry; N-ethylmaleimide-sensitive factor; NSF; Q; R; S; SNAP-25; SNARE; V; VAMP2; alanine; arginine; aspartic acid; botulinum neurotoxin serotype A; glutamic acid; glutamine; glycine; hS; homoserine; light chain; lysine; mass spectrometry; serine; soluble NSF attachment protein 25; soluble NSF attachment receptor; valine; vesicle-associated membrane protein 2.

Publication types

  • Comparative Study

MeSH terms

  • Botulinum Toxins, Type A / chemistry*
  • Botulinum Toxins, Type A / genetics
  • Catalysis
  • Clostridium botulinum / enzymology*
  • Hydrolysis
  • Mutation
  • Protein Binding
  • Synaptosomal-Associated Protein 25 / chemistry*
  • Synaptosomal-Associated Protein 25 / genetics


  • Synaptosomal-Associated Protein 25
  • Botulinum Toxins, Type A