The detection of catalytically active botulinum neurotoxins (BoNTs) can be achieved by monitoring the enzymatic cleavage of soluble NSF (N-ethylmaleimide-sensitive-factor) attachment protein receptor (SNARE) proteins by the toxins' light chains (LC) in cleavage-based assays. Thus, for sensitive BoNT detection, optimal cleavage conditions for the clinically relevant A-F serotypes are required. Until now, a systematic evaluation of cleavage conditions for the different BoNT serotypes is still lacking. To address this issue, we optimized cleavage conditions for BoNT/A-F using the Taguchi design-of-experiments (DoE) method. To this aim, we analyzed the influence of buffer composition (pH, Zn2+, DTT (dithiothreitol), NaCl) as well as frequently used additives (BSA (bovine serum albumin), Tween 20, trimethylamine N-oxide (TMAO)) on BoNT substrate cleavage. We identified major critical factors (DTT, Zn2+, TMAO) and were able to increase the catalytic efficiency of BoNT/B, C, E, and F when compared to previously described buffers. Moreover, we designed a single consensus buffer for the optimal cleavage of all tested serotypes. Our optimized buffers are instrumental to increase the sensitivity of cleavage-based assays for BoNT detection. Furthermore, the application of the Taguchi DoE approach shows how the method helps to rationally improve enzymatic assays.
Keywords: SNARE; botulinum neurotoxins; detection; substrate cleavage; taguchi design-of-experiments.
Conflict of interest statement
The authors declare no conflict of interest.
The in vitro detection of botulinum neurotoxin-cleaved endogenous VAMP is epitope-dependent.Toxicol In Vitro. 2018 Apr;48:255-261. doi: 10.1016/j.tiv.2018.01.016. Epub 2018 Jan 31. Toxicol In Vitro. 2018. PMID: 29373835
Substrate recognition mechanism of VAMP/synaptobrevin-cleaving clostridial neurotoxins.J Biol Chem. 2008 Jul 25;283(30):21145-52. doi: 10.1074/jbc.M800610200. Epub 2008 May 29. J Biol Chem. 2008. PMID: 18511418 Free PMC article.
Structure of botulinum neurotoxin type D light chain at 1.65 A resolution: repercussions for VAMP-2 substrate specificity.Biochemistry. 2006 Mar 14;45(10):3255-62. doi: 10.1021/bi052518r. Biochemistry. 2006. PMID: 16519520
Light Chain Diversity among the Botulinum Neurotoxins.Toxins (Basel). 2018 Jul 2;10(7):268. doi: 10.3390/toxins10070268. Toxins (Basel). 2018. PMID: 30004421 Free PMC article. Review.
[Clostridium botulinum and botulinum neurotoxin].Brain Nerve. 2011 Jul;63(7):755-61. Brain Nerve. 2011. PMID: 21747146 Review. Japanese.