Covalent structure of botulinum neurotoxin type A: location of sulfhydryl groups, and disulfide bridges and identification of C-termini of light and heavy chains.
Botulinum neurotoxin Type A is synthesized by Clostridium botulinum as a approximately 150 kD single chain polypeptide. The posttranslational processing of the 1296 amino acid residue long gene product involves removal of the initiating methionine, formation of disulfide bridges, and limited proteolysis (nicking) by the bacterial protease(s). The mature dichain neurotoxin is made of a approximately 50-kD light chain and a approximately 100-kD heavy chain connected by a disulfide bridge. DNA derived amino acid sequence predicted a total of 9 Cys residues (Binz et al., 1990, J. Biol. Chem. 265, 9153-9158; Thompson et al., 1990, Eur. J. Biochem. 189, 73-81). Treatment of the dichain neurotoxin, dissolved in 6 M guanidine. HCl, with 4-vinylpyridine converted 5 Cys residues into S-pyridylethyl cysteine residues; but alkylation after mercaptolysis converted all 9 Cys residues in the S-pyridylethylated form. After confirming the predicted number of Cys residues by amino acid analysis, the positions of the 5 Cys residues carrying sulfhydryl groups and the 4 involved in disulfide bridges were determined by comparing the elution patterns in reversed-phase HPLC of the cyanogen bromide mixtures of the exclusively alkylated and the mercaptolyzed-alkylated neurotoxin. The chromatographically isolated components were identified by N-terminal amino acid sequence analysis. The HPLC patterns showed characteristic differences. The Cys residues predicted in positions 133, 164, 790, 966, and 1059 were found in the sulfhydryl form; Cys 429 and 453 were found disulfide-bridge connecting the light and heavy chains, and Cys 1234 and 1279 were found in an intrachain disulfide-bridge near the C-terminus in the heavy chain.(ABSTRACT TRUNCATED AT 250 WORDS)
Krieglstein
K G
KG
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
DasGupta
B R
BR
Henschen
A H
AH
D000595:Amino Acid Sequence; D001905:Botulinum Toxins / Q000096:biosynthesis / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003014:Clostridium botulinum / Q000378:metabolism; D003488:Cyanogen Bromide; D004220:Disulfides / Q000032:analysis; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D013438:Sulfhydryl Compounds / Q000032:analysis; D014357:Trypsin
D004220:Disulfides; D046911:Macromolecular Substances; D009498:Neurotoxins; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D014357:Trypsin; D001905:Botulinum Toxins; D003488:Cyanogen Bromide