title: Dichain structure of botulinum neurotoxin: identification of cleavage sites in types C, D, and F neurotoxin molecules.
abstract: Botulinum neurotoxin (NT) is synthesized by Clostridium botulinum as about a 150-kDa single-chain polypeptide. Posttranslational modification by bacterial or exogenous proteases yielded dichain structure which formed a disulfide loop connecting a 50-kDa light chain (Lc) and 100-kDa heavy chain (Hc). We determined amino acid sequences around cleavage sites in the loop region of botulinum NTs produced by type C strain Stockholm, type D strain CB16, and type F strain Oslo by analysis of the C-terminal sequence of Lc and the N-terminal sequence of Hc. Cleavage was found at one or two sites at Arg444/Ser445 and Lys449/Thr450 for type C, and Lys442/Asn443 and Arg445/Asp446 for type D, respectively. In culture fluid of mildly proteolytic strains of type C and D, therefore, NT exists as a mixture of at least three forms of nicked dichain molecules. The NT of type F proteolytic strain Oslo showed the Arg435 as a C-terminal residue of Lc and Ala440 as an N-terminal residue of Hc, indicating that the bacterial protease cuts twice (Arg435/Lys436 and Lys439/Ala440), with excision of four amino acid residues. The location of cleavage and number of amino acid residue excisions in the loop region could be explained by the degree of exposure of amino acid residues on the surface of the molecule, which was predicted as surface probability from the amino acid sequence. In addition, the observed correlation may also be adapted to the cleavage sites of the other botulinum toxin types, A, B, E, and G.
authors.affiliation: Department of Food Science, Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Sagane
authors.forename: T
authors.initials: T
authors.lastname: Watanabe
authors.forename: H
authors.initials: H
authors.lastname: Kouguchi
authors.forename: H
authors.initials: H
authors.lastname: Sunagawa
authors.forename: K
authors.initials: K
authors.lastname: Inoue
authors.forename: Y
authors.initials: Y
authors.lastname: Fujinaga
authors.forename: K
authors.initials: K
authors.lastname: Oguma
authors.forename: T
authors.initials: T
authors.lastname: Ohyama
mesh_terms: D000595:Amino Acid Sequence; D001905:Botulinum Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides; D010450:Endopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D020539:Sequence Analysis, Protein
chemical_list: D004220:Disulfides; C096325:botulinum toxin type D; D010450:Endopeptidases; D001905:Botulinum Toxins; C096324:botulinum toxin type C; C096321:botulinum toxin type F