Characterization of the functional activity of botulinum neurotoxin subtype B6

Clostridium botulinum produces the highly potent neurotoxin, botulinum neurotoxin (BoNT), which is classified into seven serotypes, A-G, and the subtype classification is confirmed by the diversity of the amino acid sequence among the serotypes. The BoNT from the Osaka05 strain is associated with type B infant botulism and has been divided into BoNT/B subtype B6 (BoNT/B6) by phylogenetic analysis and the antigenicity of its C-terminal heavy chain (HC) domain. However, the molecular basis for its properties, including the potency, is poorly understood. In this study, we purified the BoNT/B6 holotoxin and compared the biological activity and receptor binding activity of BoNT/B6 to those of the previously-characterized BoNT/B1 and BoNT/B2 subtypes. The derivative BoNT/B6 was found to be already nicked and in an activated form, thus indicating that endogenous protease production in this strain may be higher than in the other two strains. BoNT/B1 exhibited the highest lethal activity in mice, followed by BoNT/B6, which is consistent with the sensitivity levels of PC12 cells. No significant differences were seen in the enzymatic activities of the BoNT/Bs against their substrate. HC/B1 and HC/B6 exhibited similar binding affinities to synaptotagmin II (SytII), which is a specific protein receptor for BoNT/B. Binding to the SytII/ganglioside complex is functionally related to the toxic action, but the receptor recognition sites are conserved. These results suggest that the distinct characteristics and the difference in biological sensitivity of BoNT/B6 may be attributable to the function of its N-terminal heavy chain domain.