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 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 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 W(1257)Ycdots, three dots, centeredG(1270)cdots, three dots, centeredH(1282) plays an 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.
Tsukamoto
Kentaro
K
Department of Microbiology, Fujita Health University School of Medicine, Toyoake, Aichi 4701192, Japan.
Kozai
Yuiko
Y
Ihara
Hideshi
H
Kohda
Tomoko
T
Mukamoto
Masafumi
M
Tsuji
Takao
T
Kozaki
Shunji
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001905:Botulinum Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020740:Chimerin Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003014:Clostridium botulinum / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D050600:SNARE Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D013574:Synaptosomes / Q000378:metabolism
D020740:Chimerin Proteins; D050600:SNARE Proteins; C096325:botulinum toxin type D; D001905:Botulinum Toxins; C096324:botulinum toxin type C