Covalent structure of botulinum neurotoxin type E: location of sulfhydryl groups, and disulfide bridges and identification of C-termini of light and heavy chains.
Botulinum neurotoxin (NT) serotype E is synthesized by Clostridium botulinum as an approximately 150-kDa single-chain polypeptide of 1252 amino acid residues of which 8 are Cys residues [Puolet et al. (1992); Biochem. Biophys. Res. Commun. 183, 107-113]. The posttranslational processing of the gene product removes only the initiating methionine. A very narrow segment of this 1251-residue-long mature protein--at one-third the distance from the N-terminus (between residues Lys 418 and Arg 421)--is highly sensitive to proteases, such as trypsin. The single-chain NT easily undergoes an exogenous posttranslational modification by trypsin; residues 419-421 (Gly-Ile-Arg) are excised. The proteolytically processed NT is a dichain protein in which Pro 1-Lys 418 constitute the approximately 50-kDa light chain, Lys 422-Lys 1251 constitute the approximately 100-kDa heavy chain; Cys 411-Cys 425 and Cys 1196-Cys 1237 form the interchain and intrachain disulfide bonds, respectively; the other four Cys residues at positions 25, 346, 941, and 1035 remain as free sulfhydryl groups. The approximately 150-kDa dichain NT, and separated light and heavy chains, were fragmented with CNBr and endoproteases (pepsin and clostripain); some of these fragments were carboxymethylated with iodoacetamide (with or without 14C label) before and after fragmentation. The fragments were separated and analyzed for amino acid compositions and sequences by Edman degradation to determine the complete covalent structure of the dichain type E NT. A total of 208 amino acid 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 nucleotide sequence.
Antharavally
B S
BS
Department of Food Microbiology and Toxicology, University of Wisconsin, Madison 53706, USA.
DasGupta
B R
BR
D000595:Amino Acid Sequence; D001905:Botulinum Toxins / Q000737:chemistry* / Q000302:isolation & purification; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017421:Sequence Analysis; D013438:Sulfhydryl Compounds / Q000032:analysis
D004220:Disulfides; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D001905:Botulinum Toxins; C096326:botulinum toxin type E