Identification of protein receptor for Clostridium botulinum type B neurotoxin in rat brain synaptosomes.
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.
Nishiki
T
T
Department of Veterinary Science, College of Agriculture, University of Osaka Prefecture, Japan.
Kamata
Y
Y
Nemoto
Y
Y
Omori
A
A
Ito
T
T
Takahashi
M
M
Kozaki
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001905:Botulinum Toxins / Q000378:metabolism*; D001923:Brain Chemistry; D002135:Calcium-Binding Proteins; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005732:Gangliosides / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000032:analysis*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis*; D051381:Rats; D013574:Synaptosomes / Q000737:chemistry*; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D005732:Gangliosides; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D050857:Synaptotagmins; D001905:Botulinum Toxins