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

Laura von Berg,Martin Bernhard Dorner,Brigitte Gertrud Dorner,Jasmin Weisemann,Andreas Rummel,Daniel Stern

doi:10.3390/toxins11100588

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.

Highlights

The anaerobic spore forming bacteria of the genus Clostridia produce botulinum neurotoxins (BoNTs), the most poisonous toxins known and cause of the life-threatening disease botulism [1,2].BoNTs are synthesized as 150 kDa holotoxins and become activated by cleavage into a 50 kDa light chain (LC) and a 100 kDa heavy chain (HC) which remain connected via a single disulfide bond [3]
This is in agreement with the results of Shone et al, who observed that increasing NaCl concentrations had an inhibitory effect on BoNT/B substrate cleavage for unknown physiological reasons [58]
We presented, for the first time, a rational analysis of the factors influencing substrate cleavage, covering all clinically relevant BoNT serotypes

Summary

Introduction

BoNTs are synthesized as 150 kDa holotoxins and become activated by cleavage into a 50 kDa light chain (LC) and a 100 kDa heavy chain (HC) which remain connected via a single disulfide bond [3]. The LC represents the enzymatic subunit enabling the toxin-mediated cleavage of soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins by a Zn2+ -dependent endopeptidase activity. The mechanism of BoNT action involves three main steps: First, the binding of the HC to specific receptors on synaptic membranes and subsequent internalization into recycling synaptic vesicles. The third step is the LC-catalyzed hydrolysis of the proteins of the SNARE complex, a hydrolysis which prevents the fusion of neurotransmitter-loaded vesicles with synaptic membranes, thereby blocking neurotransmitter release into the synaptic cleft [4,5]

Objectives

Methods

Results

Discussion

Conclusion