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    • Materials and methods br Results Assay of botulinum toxin ac

    2020-11-20

    Materials and methods
    Results Assay of botulinum toxin activity is critical for the risk-assessment, diagnosis, and treatment of botulism cases. The frequently encountered active forms of the toxin are; L chain (inside intoxicated neuronal cells), BoNT (in the blood stream of a botulism patient, and in the form of a complex (in food samples, gastric juices, and intestinal aspirate). It has already been reported that there is a difference between the endopeptidase activity of the BoNT/A toxin and BoNT/A complex, former showing optimal endopeptidase activity only under the conditions when the disulfide bond between the L and H chain is reduced, whereas BoNT/A complex showing nearly full endopeptidase activity without the reduction of the disulfide bond (Cai et al., 1999). In other words, the botulinum toxin complex is fully active without reduction. An intriguing question arises, why there is no need for the reduction of the disulfide bond in the complex? Do associated Anacardic acid synthesis help toxin get the right conformational state for nearly optimal endopeptidase activity? A toxin detection platform based on the endopeptidase activity needs characterization of the endopeptidase activity of the different samples, using a common substrate. Similarly, development of antidotes against the endopeptidase activity requires assay of the enzyme activity of all the forms of botulinum toxin.
    Discussion The results demonstrate that the reduction of the disulfide bond is one of the requirements for getting better endopeptidase activity as the nonreduced states show lesser activity. BoNT/A LC is equally active in reduced and non-reduced conditions in the first 20 min with SNAPtide as the substrate due to the absence of the disulfide bond. BoNT/A LC in the reduced state with SNAPtide (88%) as the substrate is observed to have similar activity as the BoNT/A complex with full-length His-SNAG (83%) substrate. (Table 1, Table 2). Since the SNAPtide consists of a cleavage site along with short segments of neighboring peptide residues, the LC cleaves the SNAPtide almost instantly, and we observed the rise in fluorescence reporter signal. But endopeptidase activity under natural conditions involves three different events; a) recognition and binding of the substrate, which involves domains other than the peptide segment containing the cleavage site of the substrate and the enzyme active site, b) exposure of active site as a result of substrate binding, and c) cleavage of the substrate. Peptide substrate lacks recognition and binding to distal sites, its relatively small size allows it to reach the enzyme active site for specific binding and cleavage. The fact that the peptide substrate (SNAPtide) exhibits even slightly higher enzymatic cleavage than the full-length SNAP-25 with light chain implies that in the absence of the heavy chain it has higher accessibility to the active site. This would imply that the additional binding of the substrate to exosites in the BoNT/A toxin pushes the active site out from the obfuscation of the heavy chain. Furthermore, the presence of NAPs apparently makes the active site less accessible to the peptide substrate, as shown by the significantly lower endopeptidase activity of BoNT/A complex with the peptide substrate compared to even BoNT/A toxin (Table 1, Table 2). Interestingly, the full-length substrate exhibits the highest activity with the complex and lowest with the light chain. These observations suggest that there may be synergistic effects of the substrate binding to exosites in the presence of either heavy chain and/or more so NAPs. Further studies are needed to understand this unique and novel observation. Notably, the full-length SNAP-25 is tagged with GFP. Although GFP tag is not in the vicinity, of the binding site of the substrate or α & β exosites, it is possible that GFP tag can impede binding and recognition of the substrate. These observations should be confirmed with the SNAP-25 substrate without any tag to better mimic the in vivo conditions.