BOTULINUM TYPE-A NEUROTOXIN DIGESTED WITH PEPSIN YIELDS 132, 97, 72, 45, 42, AND 18 KD FRAGMENTS

Botulinum neurotoxin (NT) serotype A is a dichain protein made of a li ght and a heavy chain linked by at least one interchain disulfide; bas ed on SDS-polyacrylamide gel electrophoresis their molecular masses ap pear as 147, 52, and 93 kD, respectively. Digestion of the NT with pep sin under controlled pH (4.3 and 6.0), time (I and 24 hr), and tempera ture (25 and 30-degrees-C) produced 132, 97, 42, and 18 kD fragments. The three larger fragments were isolated by ion-exchange chromatograph y. The 132 and 97 kD fragments are composed of 52 kD light chain and 7 2 and 45 kD fragments of the heavy chain, respectively. The sequences of amino terminal residues of these fragments were determined to ident ify the pepsin cleavage sites in the NT, which based on nucleotide seq uence has 1295 amino acid residues (Binz et al., J. Biol. Chem. 265, 9 153, 1990). The 42 kD fragment, beginning with residue 866, is the C-t erminal half of the heavy chain. The 18 kD fragment, of which the firs t 72 residues were identified beginning with residue 1147, represents the C-terminal segment of the heavy chain. The 132 kD fragment (residu e 1 to approximately 1146) is thus a truncated version of the NT witho ut its 18 kD C-terminal segment. The 97 kD fragment (residue 1 to appr oximately 865) is also a truncated NT with its 42 kD C-terminal segmen t excised. These peptic fragments contain one or two of the three func tional domains of the NT (binds receptors, forms channels, and intrace llularly inhibits exocytosis of the neurotransmitter) that can be used for structure-function studies of the NT. This report also demonstrat es for the first time that of the six Cys residues 453, 790, 966, 1059 , 1234, and 1279 located in the heavy chain the later four do not form interchain disulfide links with the light chain; however, Cys 1234 an d 1279 contained within the 18 kD fragment form intrachain disulfide. The electrophoretic behaviors of type A NT and its fragments in native gels and their comparison with botulinum NT serotypes B and E as well as tetanus NT suggest that each NT forms dimers or other aggregates a nd the aggregation does not occur when the 42 kD C-terminal half of th e heavy chain is excised. Thus, the C-terminal half of the heavy chain appears important in the self-association to form dimers.