COVALENT STRUCTURE OF BOTULINUM NEUROTOXIN TYPE-E - LOCATION OF SULFHYDRYL-GROUPS, AND DISULFIDE BRIDGES AND IDENTIFICATION OF C-TERMINI OFLIGHT AND HEAVY-CHAINS

Botulinum neurotoxin (NT) serotype E is synthesized by Clostridium bot ulinum as an similar to 150-kDa single-chain polypeptide of 1252 amino acid residues of which 8 are Cys residues [Puolet et al. (1992); Bioc hem. Biophys. Res. Commun. 183, 107-113]. The posttranslational proces sing of the gene product removes only the initiating methionine. A ver y narrow segment of this 1251-residue-long mature protein-at one-third the distance from the N-terminus (between residues Lys 418 and Arg 42 1)-is highly sensitive to proteases, such as trypsin. The single-chain NT easily undergoes an exogenous posttranslational modification by tr ypsin; residues 419-421 (Gly-Ile-Arg) are excised. The proteolytically processed NT is a dichain protein in which Pro l-Lys 418 constitute t he similar to 50-kDa light chain, Lys 422-Lys 1251 constitute the simi lar to 100-kDa heavy chain; Cys 411-Cys 425 and Cys 1196-Cys 1237 form the interchain and intrachain disulfide bonds, respectively; the othe r four Cys residues at positions 25, 346, 941, and 1035 remain as free sulfhydryl groups. The similar to 150-kDa dichain NT, and separated l ight and heavy chains, were fragmented with CNBr and endoproteases (pe psin and clostripain); some of these fragments were carboxymethylated with iodoacetamide (with or without C-14 label) before and after fragm entation. The fragments were separated and analyzed for amino acid com positions and sequences by Edman degradation to determine the complete covalent structure of the dichain type E NT. A total of 208 amino aci d residues, i.e., 16.5% of the entire protein's sequence deduced from nucleotide sequence, was identified. Direct chemical identification of these amino acids was in complete agreement with that deduced from nu cleotide sequence.