Abstract
Botulinum neurotoxin (BoNT) is used for the treatment of a number of ailments. The activity of the toxin that is isolated from bacterial cultures is frequently tested in the mouse lethality assay. Apart from the ethical concerns inherent to this assay, species-specific differences in the affinity for different BoNT serotypes give rise to activity results that differ from the activity in humans. Thus, BoNT/B is more active in mice than in humans. The current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma–based reporter cell line (SIMA-hPOMC1-26-Gluc) was inhibited by clostridial and recombinant BoNT/A to the same extent, whereas both clostridial and recombinant BoNT/B inhibited the release to a lesser extent and only at much higher concentrations, reflecting the low activity of BoNT/B in humans. By contrast, the genetically modified BoNT/B-MY, which has increased affinity for human synaptotagmin, and the BoNT/B protein receptor inhibited luciferase release effectively and with an EC50 comparable to recombinant BoNT/A. This was due to an enhanced uptake into the reporter cells of BoNT/B-MY in comparison to the recombinant wild-type toxin. Thus, the SIMA-hPOMC1-26-Gluc cell assay is a versatile tool to determine the activity of different BoNT serotypes providing human-relevant dose-response data.
Highlights
Apart from the ethical concerns inherent to this assay, species-specific differences in the affinity for different Botulinum neurotoxin (BoNT) serotypes give rise to activity results that differ from the activity in humans
The SIMA-hPOMC1-26-Gluc cell assay is a versatile tool to determine the activity of different BoNT serotypes providing human-relevant dose-response data
The human-specific difference in activity between the two toxins that is not detectable in murine systems was a result of an enhanced uptake of BoNT/B-MY into the reporter cell line
Summary
Apart from the ethical concerns inherent to this assay, species-specific differences in the affinity for different BoNT serotypes give rise to activity results that differ from the activity in humans. The assay was shown to detect the human-specific increase in activity of a mutant BoNT/B-MY over the corresponding wild-type toxin. The human-specific difference in activity between the two toxins that is not detectable in murine systems was a result of an enhanced uptake of BoNT/B-MY into the reporter cell line. The heavy chain mediates the uptake of the exotoxin into the target cells as well as the translocation of the light chain of toxin into the cell’s cytosol, where the highly specific protease activity of the small subunits cleaves and inactivates so-called SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins that are essential for the vesicle fusion and neurotransmitter release. G cleave synaptobrevin on the vesicle [1] With their heavy chain, the different serotypes bind to different protein receptors to enter the neurons. While BoNT/A, D, E, and F bind to published maps and institutional affiliations
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