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.
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - Apr 8 1994|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology