| Summary: | The extraordinarily potent clostridial neurotoxins (CNTs) comprise tetanus neurotoxin (TeNT) and the seven established botulinum neurotoxin serotypes (BoNT/A-G). They are composed of four structurally independent domains: the roles of the catalytically active light chain, the translocation domain H<sub>N</sub>, and the C-terminal receptor binding domain H<sub>CC</sub> are largely resolved, but that of the H<sub>CN</sub> domain sandwiched between H<sub>N</sub> and H<sub>CC</sub> has remained unclear. Here, mutants of BoNT/A, BoNT/B, and TeNT were generated by deleting their H<sub>CN</sub> domains or swapping H<sub>CN</sub> domains between each other. Both deletion and replacement of TeNT H<sub>CN</sub> domain by H<sub>CN</sub>A and H<sub>CN</sub>B reduced the biological activity similarly, by ~95%, whereas BoNT/A and B deletion mutants displayed >500-fold reduced activity in the mouse phrenic nerve hemidiaphragm assay. Swapping H<sub>CN</sub> domains between BoNT/A and B hardly impaired their biological activity, but substitution with H<sub>CN</sub>T did. Binding assays revealed that in the absence of H<sub>CN</sub>, not all receptor binding sites are equally well accessible. In conclusion, the presence of H<sub>CN</sub> is vital for CNTs to exert their neurotoxicity. Although structurally similar, the H<sub>CN</sub> domain of TeNT cannot equally substitute those of BoNT and vice versa, leaving the possibility that H<sub>CN</sub>T plays a different role in the intoxication mechanism of TeNT.
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