Covalent structure of botulinum neurotoxin type A: Location of sulfhydryl groups, and disulfide bridges and identification of C-termini of light and heavy chains

Abstract

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)

Mapping of the antibody-binding regions on botulinum neurotoxin H-chain domain 855–1296 with antitoxin antibodies from three host species

“…Recently, however, the complete primary structures of BoNT types A (Binz et al, 1990a;Thompson et al, 1990), B (Jung et al, 1992;Whelan et al, 1992a), C1 (Hauser et al, 1990), D (Binz et al, 1990b;Sunagawa et al, 1992), E (Poulet et al, 1992;Whelan et al, 1992b), F (East et al, 1992) and G (Campbell et al, 1993) have been determined. In addition the disulfide pairing in BoNT/A has been established (Krieglstein et al, 1990(Krieglstein et al, , 1994. BoNT/A has been crystallized and a preliminary X-ray analysis of its structure has been reported (Stevens et al, 1991).…”

Epitope Mapping of Neutralizing Botulinum Neurotoxin A Antibodies by Phage Display

“…Recently, the crystal structure of BoNT/A (14) revealed that holotoxin is composed of three distinct functional domains: catalytic (residues 1 to 437), translocation (residues 448 to 872), and receptor binding (H C ; residues 873 to 1295) (12). A molecular model overlay of selected epitopes corresponds to the three-dimensional H C .…”

Therapy and prophylaxis of inhaled biological toxins

“…unsaturation; comparable pyrrole ester attached to a 20α-OH (Tokuyama et al, 1969) 539 D Receptor may be site 2 of the five neurotoxin receptor sites on the voltage-gated Na + channels of excitable membranes Botulinum toxins*** Proteins, each of two peptide chains -heavy (HC) and light (LC) -connected by a disulphide bridge (Krieglstein et al, 1994); LC has Zn 2+ endopeptidase activity (Fujii et al, 1992). Toxins have channel-forming capability HC: 100 kD LC: 50 kD Primary: HC → nerve ending membrane protein (synaptotagmin) (Nishiki et al, 1994;Li and Singh, 1998) and complex phospholipid and gangliosides (GT1b) (Kozaki et al, 1998) Secondary: LC → various synaptic vesicle proteins (e.g.…”