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 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.
Highlights
B neurotoxin was purified 340-fold fromratsynapto- such as GDlaland GTlb,interact with C. botulinum neurotoxin somes by successive chromatography onDM-Sepha- (8-10).the direct interaction of the neurotoxin with
In the previous study(12),we demonstrated that both protease and sialidase treatmeonftsynaptosomes markedly decreasedthe binding of type B neurotoxin, againstthepurified 58-kDa receptorproteinand a a n d the reduced toxin binding activityof sialidase-treated, but monoclonal antibody against the heavy chain (1k0D3 a) not protease-treated synaptosomes, was restoredby incorporaof the neurotoxin reacted with the cross-linked produticotn of exogenous gangliosides G, and GTlb,suggesting that of 161 kDa in immunoblotting experiments
We deter- type B neurotoxin may bind to a protein receptor-ganglioside mined partial amino acid sequenceosf the 58-kDa pro- complex
Summary
B neurotoxin was purified 340-fold fromratsynapto- such as GDlaland GTlb,interact with C. botulinum neurotoxin somes by successive chromatography onDM-Sepha- (8-10).the direct interaction of the neurotoxin with. We deter- type B neurotoxin may bind to a protein receptor-ganglioside mined partial amino acid sequenceosf the 58-kDa pro- complex. The fractions containing the toxin binding activity were referred to as the heparin eluate.
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