Botulinum neurotoxin (BoNT) binds to presynaptic neuronal cells and blocks neurotransmitter release. The carboxyl-terminal half of the heavy chain (HC) of the neurotoxin recognizes its specific receptor on the plasma membrane. We have previously demonstrated that BoNT/C binds to gangliosides GD1b and GT1b under physiological conditions, while BoNT/D interacts with phosphatidylethanolamine (PE). Here we report that the recognition sites for gangliosides and PE are present in the carboxyl-terminal domain of HC. Chimeric mutants and site-directed mutants of BoNT/C-HC and BoNT/D-HC were generated and their binding activities evaluated. The chimeric HC that consisted of the amino-terminal half of BoNT/D-HC and the carboxyl-terminal half of BoNT/C-HC possessed activity similar to the authentic BoNT/C-HC, suggesting that the carboxyl-terminal region of HC is involved in the receptor recognition of BoNT/C. Moreover, analysis using site-directed mutants indicated that the peptide motif W1257Y⋯G1270⋯H1282 plays an important role in the interaction between BoNT/C and gangliosides. In contrast, we revealed that two lysine residues of BoNT/D-HC are involved in the formation of the critical binding site for receptor binding.
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