High Conservation of Tetanus and Botulinum Neurotoxins Cleavage Sites on Human SNARE Proteins Suggests That These Pathogens Exerted Little or No Evolutionary Pressure on Humans

Abstract

The Genome Aggregation Database presently contains >120,000 human genomes. We searched in this database for the presence of mutations at the sites of tetanus (TeNT) and botulinum neurotoxins (BoNTs) cleavages of the three SNARE proteins: VAMP, SNAP-25 and Syntaxin. These mutations could account for some of the BoNT/A resistant patients. At the same time, this approach was aimed at testing the possibility that TeNT and BoNT may have acted as selective agents in the development of resistance to tetanus or botulism. We found that mutations of the SNARE proteins are very rare and concentrated outside the SNARE motif required for the formation of the SNARE complex involved in neuroexocytosis. No changes were found at the BoNT cleavage sites of VAMP and syntaxins and only one very rare mutation was found in the essential C-terminus region of SNAP-25, where Arg198 was replaced with a Cys residue. This is the P1' cleavage site for BoNT/A and the P1 cleavage site for BoNT/C. We found that the Arg198Cys mutation renders SNAP-25 resistant to BoNT/A. Nonetheless, its low frequency (1.8 × 10^-5) indicates that mutations of SNAP-25 at the BoNT/A cleavage site are unlikely to account for the existence of BoNT/A resistant patients. More in general, the present findings indicate that tetanus and botulinum neurotoxins have not acted as selective agents during human evolution as it appears to have been the case for tetanus in rats and chicken.

Keywords: ExAC; SNAP-25; VAMP-1/2; botulinum neurotoxin; gnomAD; syntaxin-1A/1B; tetanus neurotoxin.

Figure 1

Mutation frequency maps of human Syntaxin-1A/1B. The figure displays the sequences of STX-1A (top) and STX-1B (bottom) with respective mutations found in the gnomAD database (red dots) together with those exclusively reported in the ExAC database (black squares). Only changes with respect to the canonical sequence of the corresponding protein are shown. Below each diagram a schematization of the protein primary structure and of its domains is given. Arrows indicate the cleavage sites by BoNT/C occurring at K253-A254 of STX-1A and at K252-A253 of STX-1B. No mutations within these peptide bonds were found.

Figure 2

Mutation frequency maps of human VAMP-1/2. The figure displays the sequences of VAMP-1 (left) and VAMP-2 (right) with respective mutations found in the gnomAD database (blue dots) together with those reported exclusively in the ExAC database (black squares). Only changes with respect to the canonical sequence of the corresponding protein are shown. Frequency values higher than 8 × 10⁻⁵ are reported in the graph. Below each diagram a schematization of the protein primary structure and of its domains is given. Arrows indicate the proteolytic sites by the indicated neurotoxins: in VAMP-1 BoNT/F5 and BoNT/FA (L56-E57), BoNT/F (Q60-K61), BoNT/D and /DC (K61-L62), BoNT/B and TeNT (Q78-F79) and BoNT/G (A83-A84); R68 at the cleavage site of BoNT/X in VAMP-1 is substituted by Q; in VAMP-2 BoNT/F5 and BoNT/FA (L54-E55), BoNT/F1 (Q58-K59), BoNT/D and /DC (K59-L60), TeNT and BoNT/B (Q76-F77) and BoNT/G (A81-A82).

Figure 3

Mutation frequency maps of human SNAP-25 and SNAP-23 proteins. The figure displays the sequences of SNAP-25 (top) and SNAP-23 (bottom) with respective mutations found in the gnomAD database (green dots) together with those reported exclusively in the ExAC database (black squares). Only changes with respect to the canonical sequence of the corresponding protein are shown. Mutations with frequency higher than 8 × 10⁻⁵ are reported in the graph. Below each diagram a schematization of the protein primary sequence, of its domains and of the four palmitoylated cysteines (in yellow) is given. Arrows indicate the cleavage site of indicated neurotoxins. BoNT/E cleaves at R180-I181, BoNT/A cleaves at Q197-R198 peptide and BoNT/C cleaves at R198-A199. The red box indicates the R198C mutation.

Figure 4

Western blot analysis of BoNT/A and /E cleavage of wild type (WT) and mutant (R198C) SNAP-25 in vitro. (A) Scheme of the fusion protein used to test SNAP-25 and SNAP-25 R198C cleavability. Recombinant SNAP-25 substrate (300 ng/sample) was incubated with reduced full length BoNT/A (B) or BoNT/E (C) (10 ng/sample). At indicated time points, the reaction was blocked by adding loading sample buffer and heat denaturation. Samples were separated by SDS-PAGE and proteins stained with Coomassie Blue. Ratio of uncleaved to total SNAP-25 was evaluated by densitometric analysis. Ratios are shown in the bar chart as mean with respective standard deviation of 3 independent experiments. In some experiments, after electrophoresis, samples were immunoblotted for His (recognizing both intact and truncated SNAP-25), BoNT/A-cleaved SNAP-25 or whole SNAP-25 (recognizing only native SNAP-25) detection. Antibodies were visualized using fluorescent secondary antibodies for detection by the Odyssey Imaging System. The assays were performed at least 3 times and representative blots are shown in the bottom panels.