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

• Published in: The Journal of biological chemistry 1994-04, Vol.269 (14), p.10498-10503
• Main Authors: NISHIKI, T.-I, KAMATA, Y, OMORI, A, ITO, T, TAKAHASHI, M, KOZAKI, S
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Biological and medical sciences; Botulinum Toxins - metabolism; Brain Chemistry; Calcium-Binding Proteins; Cell receptors; Cell structures and functions; Chromatography, Ion Exchange; Clostridium botulinum; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Gangliosides - metabolism; Male; Membrane Glycoproteins - analysis; Mice; Mice, Inbred BALB C; Miscellaneous; Molecular and cellular biology; Molecular Sequence Data; Nerve Tissue Proteins - analysis; Rats; Synaptosomes - chemistry; Synaptotagmins
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(17)34087-5

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 GT1b or GD1a. 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 GT1b or GD1a may be a natural receptor for C. botulinum type B neurotoxin at the nerve terminals.