Dichain structure of botulinum neurotoxin: Identification of cleavage sites in types C, D, and F neurotoxin molecules

Botulinum neurotoxin (NT) is synthesized by Clostridium botulinum as about a 150-kDa single-chain polypeptide. Posttranslational modification by bacte rial or exogenous proteases yielded dichain structure which formed a disulf ide 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 regio n 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 th e N-terminal sequence of He. Cleavage was found at one or two sites at Arg4 44/Ser445 and Lys449/Thr450 for type C, and Lys442/Asn443 and Arg445/Asp446 for type D, respectively. In culture fluid of mildly proteolytic strains o f type C and D, therefore, NT exists as a mixture of at least three forms o f 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 resid ue of He, 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 th e amino acid sequence. In addition, the observed correlation may also be ad apted to the cleavage sites of the other botulinum toxin types, A, B, E, an d G.