title: Differential contribution of the residues in C-terminal half of the heavy chain of botulinum neurotoxin type B to its binding to the ganglioside GT1b and the synaptotagmin 2/GT1b complex.
abstract: Clostridium botulinum type B neurotoxin was effectively bound to synaptotagmin 2 (Stg2) associated with ganglioside GT1b, however, the molecular interaction between the neurotoxin and the Stg2/GT1b complex has not been identified. Previously, we found that infant botulism-related strain 111 generated a low activity of the neurotoxin (111/NT), which differed in some amino acid residues, especially in the carboxyl terminal half of the heavy chain (H(C)), from the original neurotoxin of strain Okra (Okra/NT) associated with a food-borne botulism. In this study, we evaluated the binding capabilities of site-directed mutants of Okra/H(C) to the Stg2/GT1b complex and to GT1b alone, and investigated the relationship between the toxic action and receptor binding. Replacement of K1187 and E1190 with glutamic acid and lysine, respectively, which substituted for the 111/NT residues, caused a reduction of binding affinity to the Stg2/GT1b complex, suggesting that both these residues contribute to the different binding affinity between Okra/NT and 111/NT. Substitution of four residues, H1240, S1259, W1261 and Y1262, which form a ganglioside pocket, drastically decreased the binding of H(C) to the Stg2/GT1b complex and to GT1b. Mutation in the residues, K1186, E1189, K1191 and K1260 reduced the binding of H(C) to GT1b alone, but not to the Stg2/GT1b complex. Analyses of effects of mutant toxins on toxicity of BoNT/B to cerebellar granule cells suggest the association of cell toxicity with binding to Stg2/GT1b complex but not that to GT1b alone.
authors.affiliation: Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Kohda
authors.forename: Hideshi
authors.initials: H
authors.lastname: Ihara
authors.forename: Yukiji
authors.initials: Y
authors.lastname: Seto
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Tsutsuki
authors.forename: Masafumi
authors.initials: M
authors.lastname: Mukamoto
authors.forename: Shunji
authors.initials: S
authors.lastname: Kozaki
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001905:Botulinum Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D019274:Botulinum Toxins, Type A; D002478:Cells, Cultured; D002531:Cerebellum; D003014:Clostridium botulinum / Q000737:chemistry / Q000201:enzymology; D005732:Gangliosides / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism / Q000633:toxicity; D016415:Sequence Alignment; D013045:Species Specificity; D050861:Synaptotagmin II / Q000378:metabolism*
chemical_list: D005732:Gangliosides; D011994:Recombinant Proteins; D050861:Synaptotagmin II; C096323:rimabotulinumtoxinB; C016481:trisialoganglioside GT1; D008666:Metalloendopeptidases; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A