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

Botulinum neurotoxin Type A is synthesized by Clostridium botulinum as a approximately 150 kD single chain polypeptide. The posttranslationa l 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 m ature 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., 19 90, 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 f orm. After confirming the predicted number of Cys residues by amino ac id analysis, the positions of the 5 Cys residues carrying sulfhydryl g roups and the 4 involved in disulfide bridges were determined by compa ring the elution patterns in reversed-phase HPLC of the cyanogen bromi de mixtures of the exclusively alkylated and the mercaptolyzed-alkylat ed neurotoxin. The chromatographically isolated components were identi fied by N-terminal amino acid sequence analysis. The HPLC patterns sho wed characteristic differences. The Cys residues predicted in position s 133, 164, 790, %6, and 1059 were found in the sulfhydryl form; Cys 4 29 and 453 were found disulfide-bridged connecting the light and heavy chains, and Cys 1234 and 1279 were found in an intrachain disulfide-b ridge near the C-terminus in the heavy chain. Ten amino acid residues, Thr 438-Lys 447, predicted to be present in the single chain neurotox in were not found in the dichain neurotoxin. Nicking of single-chain n eurotoxin by the protease(s) endogenous to the bacteria therefore appe ars to excise these 10 amino acid residues from the nicking region whi ch leaves Lys 437 as the C-terminus of the light chain and Ala 448 as the N-terminus of the heavy chain. The N-terminal Pro 1 and C-terminal Leu 1295, predicted from the nucleotide sequence, remain conserved af ter nicking. Residues Pro 1-Lys 437 and Ala 448-Leu 1295 constitute th e light and heavy chains, respectively. The C-termini were determined by isolation of short C-terminal peptide fragments and subsequent sequ ence analysis by Edman degradation. About 20% of the amino acid sequen ce predicted from DNA analysis was confirmed in these studies by prote in-chemical methods.