Identification of the receptor-binding sites in the carboxyl-terminal half of the heavy chain of botulinum neurotoxin types C and D

Botulinum neurotoxin (BoNT) binds to presynaptic neuronal cells and blocks neurotransmitter release. The carboxyl-terminal half of the heavy chain (H-C) of the neurotoxin recognizes its specific receptor oil 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 H-C. Chimeric mutants and site-directed mutants of BoNT/C-H-C. and BoNT/D-H-C were generated and their binding activities evaluated. The chimeric H-C that consisted of the amino-terminal half of BoNT/D-H-C and the carboxyl-terminal half of BoNT/C-H-C possessed activity similar to the authentic BoNT/C-H-C. suggesting that the carboxyl-terminal region of H-C is involved in the receptor recognition of BoNT/C. Moreover, analysis using site-directed mutants indicated that the peptide motif (WY)-Y-1257...G(1270)...H-1282 plays,in important role in the interaction between BoNT/C and gangliosides. In contrast, we revealed that two lysine residues of BoNT/D-H-C are involved in the formation of the critical binding site for receptor binding. (C) 2008 Elsevier Ltd. All rights reserved.