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Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A-F Employing Taguchi Design-of-Experiments

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Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A-F Employing Taguchi Design-of-Experiments

Laura von Berg et al. Toxins (Basel).

Abstract

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.

Figures

Figure 1
Figure 1
Work-flow of Taguchi experiments. After formulating a specific aim (here, e.g., “Increase the cleavage of synaptosome-associated protein of 25 kDa (SNAP-25) by botulinum neurotoxin/A (BoNT/A)”), fixed and variable test parameters, the measuring method, and an adequate array layout are determined. If all parameters have been determined, experiments are carried out. Finally, the data are analyzed by statistical means using an analysis of mean (ANOM) (to determine the optimal factor levels) and an ANOVA (to determine the impact of each factor). Eventually, the outcome of Taguchi experiments should be controlled to validate the results.
Figure 2
Figure 2
Substrate cleavage of BoNT serotypes A–F in different buffer compositions according to the L9-Array 1 (left image of each panel) and L9-Array 2 (right image of each panel). Full-length SNAP-25 or vesicle-associated membrane protein-2 (VAMP-2), respectively, were incubated with BoNT for 30 min (BoNT/A, B, D, E, F) or 18 h (BoNT/C) at 37 °C in the assigned buffer (Table 1). Cleavage was analyzed densitometrically via SDS-PAGE and Coomassie staining. One representative gel image out of two experiments is shown. Bars indicate normalized averaged cleavage (n = 2; standard deviation (SD) is shown). NC = Uncleaved negative control in 50 mM HEPES.
Figure 3
Figure 3
ANOM of L9-Array 1 (left image of each panel) and L9-Array 2 (right image of each panel). signal-to-noise (S/N)-ratios (larger-the-better) in db (decibel) for each experiment were determined. Graphs display the mean S/N-ratio for each factor level and the overall mean out of two independent experiments. Factor levels (from lowest to highest level as indicated with triangle): pH: 6.5, 7, 7.5; ZnCl2: 10, 50, and 250 µM; DTT (dithiothreitol): 1, 5, and 25 mM; NaCl: 0 mM, 20, and 100 mM; BSA (bovine serum albumin): 0, 0.5, and 1 mg/mL, trimethylamine N-oxide (TMAO): 0, 0.75, and 1.5 M; Tween 20: 0%, 0.5%, 1%; C = Control (empty factor). A maximum in each factor indicates the optimal level.
Figure 4
Figure 4
Comparison of optimized buffer conditions for substrate cleavage by each serotype with two reference buffers and our consensus buffer. Either full-length SNAP-25 or VAMP-2 were incubated in a 1:2 dilution series of BoNT for 30 min (BoNT/A, B, D, E, F) or 18 h (BoNT/C) at 37 °C in the serotype-optimized buffer developed in this work (colored circles; see Table 2 for buffer compositions), the Jones buffer (light grey circles), the Evans buffer (dark grey circles), or the consensus buffer (open black circles). Km values (indicated as dashed lines for each curve) were calculated using Michaelis–Menten kinetics (Y = Vmax × X/(Km + X)) assuming a Km > 0 and Vmax equal to 100. Cleavage was analyzed via SDS-PAGE (n = 2; ±SD), as described in the method section.

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