COVALENT STRUCTURE OF BOTULINUM NEUROTOXIN TYPE-B - LOCATION OF SULFHYDRYL-GROUPS AND DISULFIDE BRIDGE AND IDENTIFICATION OF C-TERMINI OF LIGHT AND HEAVY-CHAINS

Botulinum neurotoxin (NT) serotype B, produced by Clostridium botulinu m (proteolytic strain), is a similar to 150-kDa single-chain polypepti de of 1291 amino acids, of which 10 are Cys residues [Whelan et al. (1 992), Appl. Environ. Microbiol. 58, 2345-2354] The posttranslational m odifications of the gene product were found to consist of excision of only the initiating Met residue, limited proteolysis (nicking) of the 1290-residue-long protein between Lys 440 and Ala 441, and formation o f at least one disulfide bridge. The dichain (nicked) protein, in a mi xture with the precursor single-chain (unnicked) molecules, was found to have a similar to 50-kDa light chain (Pro 1 through Lys 440) and a similar to 100-kDa heavy chain (Ala 441 through Glu 1290). The limited in vivo nicking of the single-chain NT to the dichain form, by protea se endogenous to the bacteria, and the nonfacile in vitro cleavage by trypsin of the Lys 440-Ala 441 bond appear to be due to the adjacent A la 441-Pro 442 imide bond's probable cis configuration in a mixed popu lation of molecules with cis and trans configurations. The two chains were found connected by an interchain disulfide formed by Cys 436 and Cys 445. Six other Cys residues, at positions 70, 195, 308, 777, 954, and 1277, were found in sulfhydryl form. In addition, a Cys at positio n 1220 or 1257 appeared to be in sulfhydryl form, hence our experiment al results could not unambiguously identify presence of an intrachain disulfide bridge near the C-terminus of the NT. A total of 384 amino a cid residues, including the 6 Cys residues at positions 70, 195, 308, 436, 445, and 1277, were identified by direct protein-chemical analysi s; thus 29.7% of the protein's entire amino acid sequence predicted fr om the nucleotide sequence was confirmed. The 6 amino acids, residues 945-950, did not match with the sequence predicted in 1992, but did ma tch with a later report of 1995. The above determinations were made by a combination of chemical (CNBr and acidic cleavage at Asp-Pro) and e nzymatic (trypsin, clostripain, and pepsin) cleavages of the NT, and N T carboxymethylated with iodoacetamide (with or without C-14 label), s eparation and isolation of the fragments by SDS-PAGE (followed by elec troblotting onto PVDF membrane), and/or reversed-phase HPLC, and analy ses of the fragments for the N-terminal amino acid sequences by Edman degradation and amino acid compositions.