Identification of protein receptor for Clostridium botulinum type B neurotoxin in rat brain synaptosomes

Tei Ichi Nishiki, Yoichi Kamata, Yasuo Nemoto, Akira Omori, Tomoko Ito, Masami Takahashi, Shunji Kozaki

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274 Citations (Scopus)

Abstract

The protein receptor for Clostridium botulinum type B neurotoxin was purified 340-fold from rat synaptosomes by successive chromatography on DEAE- Sepharose, phenyl-Toyopearl, and heparin-Toyopearl columns. 125I-Labeled neurotoxin bound to lipid vesicles containing the protein receptor and ganglioside G(T1b) or G(D1a). The reconstituted receptor showed the same affinities as the native receptor on synaptosomes. Chemical cross-linking of 125I-toxin to the receptor in the presence of gangliosides resulted in formation of a cross-linked product of 161 kDa under reducing conditions. Cross-linking was specific, as it was inhibited by the presence of excess unlabeled toxin. A monoclonal antibody against the purified 58-kDa receptor protein and a monoclonal antibody against the heavy chain (103 kDa) of the neurotoxin reacted with the cross-linked product of 161 kDa in immunoblotting experiments. We determined partial amino acid sequences of the 58-kDa protein, which were identical to synaptotagmin, a synaptic vesicle membrane protein. In addition, the monoclonal antibody against the 58-kDa receptor protein recognized recombinant rat synaptotagmin. These results suggest that synaptotagmin in association with ganglioside G(T1b) or G(D1a) may be a natural receptor for C. botulinum type B neurotoxin at the nerve terminals.

Original languageEnglish
Pages (from-to)10498-10503
Number of pages6
JournalJournal of Biological Chemistry
Volume269
Issue number14
Publication statusPublished - Apr 8 1994

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Nishiki, T. I., Kamata, Y., Nemoto, Y., Omori, A., Ito, T., Takahashi, M., & Kozaki, S. (1994). Identification of protein receptor for Clostridium botulinum type B neurotoxin in rat brain synaptosomes. Journal of Biological Chemistry, 269(14), 10498-10503.