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

Research output: Contribution to journalArticle

270 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
Externally publishedYes

Fingerprint

Clostridium botulinum type B
Clostridium
Synaptosomes
Neurotoxins
Synaptotagmins
Rats
Brain
Gangliosides
Monoclonal Antibodies
Proteins
Recombinant proteins
Synaptic Membranes
Synaptic Vesicles
Chromatography
Recombinant Proteins
Immunoblotting
Heparin
Amino Acid Sequence
Membrane Proteins
Association reactions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Nishiki, T., 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.

Identification of protein receptor for Clostridium botulinum type B neurotoxin in rat brain synaptosomes. / Nishiki, Tei-ichi; Kamata, Yoichi; Nemoto, Yasuo; Omori, Akira; Ito, Tomoko; Takahashi, Masami; Kozaki, Shunji.

In: Journal of Biological Chemistry, Vol. 269, No. 14, 08.04.1994, p. 10498-10503.

Research output: Contribution to journalArticle

Nishiki, T, 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, vol. 269, no. 14, pp. 10498-10503.
Nishiki T, Kamata Y, Nemoto Y, Omori A, Ito T, Takahashi M et al. Identification of protein receptor for Clostridium botulinum type B neurotoxin in rat brain synaptosomes. Journal of Biological Chemistry. 1994 Apr 8;269(14):10498-10503.
Nishiki, Tei-ichi ; Kamata, Yoichi ; Nemoto, Yasuo ; Omori, Akira ; Ito, Tomoko ; Takahashi, Masami ; Kozaki, Shunji. / Identification of protein receptor for Clostridium botulinum type B neurotoxin in rat brain synaptosomes. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 14. pp. 10498-10503.
@article{5db3747c170a446a873b57341db10400,
title = "Identification of protein receptor for Clostridium botulinum type B neurotoxin in rat brain synaptosomes",
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.",
author = "Tei-ichi Nishiki and Yoichi Kamata and Yasuo Nemoto and Akira Omori and Tomoko Ito and Masami Takahashi and Shunji Kozaki",
year = "1994",
month = "4",
day = "8",
language = "English",
volume = "269",
pages = "10498--10503",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "14",

}

TY - JOUR

T1 - Identification of protein receptor for Clostridium botulinum type B neurotoxin in rat brain synaptosomes

AU - Nishiki, Tei-ichi

AU - Kamata, Yoichi

AU - Nemoto, Yasuo

AU - Omori, Akira

AU - Ito, Tomoko

AU - Takahashi, Masami

AU - Kozaki, Shunji

PY - 1994/4/8

Y1 - 1994/4/8

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0028341442&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028341442&partnerID=8YFLogxK

M3 - Article

C2 - 8144634

AN - SCOPUS:0028341442

VL - 269

SP - 10498

EP - 10503

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 14

ER -