Isolation of synaptotagmin as a receptor for types A and E botulinum neurotoxin and analysis of their comparative binding using a new microtiter plate assay.
Clostridium botulinum neurotoxin acts on nerve endings to block acetylcholine release. Binding of the neurotoxin to a membrane receptor through its heavy chain is the first essential step in its mode of toxin action. Type E botulinum neurotoxin (BoNT/E) or type A botulinum neurotoxin (BoNT/A) receptor was purified from rat brain synaptosomes employing a neurotoxin affinity column chromatography. The protein fraction eluted from the affinity column with 0.5 M NaCl contained a 57 kDa protein as a major eluant. Immunoblotting the eluant with anti-synaptotagmin antibodies revealed that the 57 kDa protein was synaptotagmin I. Rat synaptotagmin I has been suggested as the receptor for BoNT/B (Nishiki et al., J. Biol. Chem. 269, 10498-10503, 1994) in rat brain. In this study, binding of BoNT/A and BoNT/E to synaptotagmin I was studied by a microtiter plate-based method. Binding of synaptotagmin I to BoNT/A coated on the plate was competitively reduced upon preincubation of the proteins with BoNT/E, suggesting a competitive binding of BoNT/A and BoNT/E to the receptor. Taken together, these results suggest that the same receptor protein binds to all three BoNT serotypes tested.
Li
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Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth 02747, USA.
Singh
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BR
D000818:Animals; D001667:Binding, Competitive; D001905:Botulinum Toxins / Q000378:metabolism*; D019274:Botulinum Toxins, Type A / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D002135:Calcium-Binding Proteins; D002846:Chromatography, Affinity; D003014:Clostridium botulinum / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification*; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification*; D051381:Rats; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D013574:Synaptosomes / Q000378:metabolism; D050863:Synaptotagmin I; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D011956:Receptors, Cell Surface; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D050857:Synaptotagmins; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096326:botulinum toxin type E