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. 2017 Aug 3;8:14130. doi: 10.1038/ncomms14130

Figure 4. Full-length BoNT/X is active on cultured neurons and in vivo in mice.

Figure 4

(a) A schematic drawing of the sortase ligation method. (b) Sortase ligation reaction mixtures were analysed by SDS–PAGE and Coomassie Blue staining. The asterisk marks the proteins aggregates due to inter-molecular disulfide bonds. Full-length BoNT/X (X-FL) appeared only in the sortase ligation mixture. (c) Neurons exposed to the sortase ligation mixture (15 μl) or control mixtures for 12 h in culture medium. Cell lysates were analysed by immunoblot. The mixture containing both X-LC-HN and X-HC (but not sortase) cleaved slightly more VAMP2 than X-LC-HN alone. Ligating X-LC-HN and X-HC by sortase further enhanced cleavage of VAMP2, demonstrating that ligated X-FL is functional on neurons. (d) BoNT/A-G, BoNT/DC and BoNT/X were subjected to the dot blot assay, using four horse antisera (trivalent anti-BoNT/A, B and E, anti-BoNT/C, anti-BoNT/DC and anti-BoNT/F), as well as two goat antisera (anti-BoNT/G and anti-BoNT/D). BoNT/X is composed of X-LC-HN and X-HC at 1:1 molar ratio. These antisera recognized their corresponding target toxins, yet none recognized BoNT/X. The antisera against BoNT/DC and BoNT/C cross-react, as these two toxins share a high degree of similarity within their HC domains. (e) Cultured rat cortical neurons were exposed to ligated X-FL in culture medium for 12 h, with or without two combinations of anti-sera. Ab1: trivalent anti-BoNT/A/B/E, anti-BoNT/C and anti-BoNT/F. Ab2: anti-BoNT/G and anti-BoNT/D. The trivalent anti-BoNT/A/B/E was used at 1:50 dilution. All other anti-sera were used at 1:100 dilution. None of the antisera affected the cleavage of VAMP2 and VAMP4 by X-FL. The specificity and potency of these antisera were validated for their ability to neutralize target serotypes in the same assay as described in Supplementary Fig. 7. (f) X-FL linked by sortase reaction (0.5 μg) was injected into the gastrocnemius muscles of the right hind limb of mice (n=4). The injected limb developed typical flaccid paralysis, and the toes failed to spread within 12 h. The left limb was not injected with toxins, serving as a control. (g) Full-length inactive form of BoNT/X (BoNT/XRY) was purified as a His6-tagged recombinant protein in E. coli. Further purified BoNT/XRY is shown in Supplementary Fig. 8b. One of two (e) or three (c,d) independent experiments is shown.