title: Botulinum neurotoxin types A, B, and E: fragmentations by autoproteolysis and other mechanisms including by O-phenanthroline-dithiothreitol, and association of the dinucleotides NAD(+)/NADH with the heavy chain of the three neurotoxins.
abstract: The first evidence of autoproteolytic activity of the approximately 50-kDa light chain of the clostridial neurotoxins (NT) is traceable to the observations that the light chains of botulinum NT serotypes A and E, separated from their approximately 100-kDa heavy chain conjugate, were found cleaved at the amino side of Tyr250 and Arg244, respectively [DasGupta and Foley (1989). Biochimie 71: 1183-1200]. Specific cleavages of the recombinant light chain of NT type A, including at Tyr249-Tyr250, firmly established that the cleavages reported earlier were due to autoproteolysis [Ahmed et al. (2001). J. Protein Chem. 20: 221-231; Ahmed et al. (2003). Biochemistry 42:12539-12549] and not by contaminating proteases or non-enzymatic. We now report many cleavages in the NT types A, B and E and also in their separated light and heavy chains, and identification of several of the peptide bonds cleaved. None of the identified cleaved bonds (-P1-P1' -) in one serotype (except Asp-Pro) was found common in other serotypes or cleaved within itself at a second site. After separation from the heavy chain self-cleavages of the light chains of type A, B and E at Tyr249-Tyr250, Gln258-Ser259 and Ile243-Arg244, respectively indicate an intriguing feature (in the aligned sequences these bonds of type A and B are 2 and type A and E are 4 peptide bonds apart) that may have some role in the NT's structure-function relationship yet to be understood. We point out that autoproteolysis of a single peptide bond (Phe418-Thr419 or Phe422-Glu423) in NT type A reported by Ahmed et al. (2001) can potentially generate proteolytically active light chain freed of the heavy chain; this is an efficient pathway, that by-passes nicking by a trypsin-like protease(s) inside the intrachain disulfide bridge and its reductive cleavage. We offer probable explanations for the observed cleavages such as acid- and metal-mediated (non-catalytic and non-stoichiometric) reactions in addition to autoproteolysis but cannot predict which mechanism(s) of cleavage occur or prevail following NT's entry in the body as poison or therapeutic agent. The metal chelator O-phenanthroline (above critical miceller concentration) in the presence of dithiothreitol cleaved type E NT at limited sites generating discrete 114-, 87-, 49-, 42-, and 31-kDa fragments but degraded NTs type A and B extensively. The limited cleavage of type E NT was dependent on the presence of metal ion(s) bound to the protein and its native (urea sensitive) conformation. The self-cleavage of the NTs at specific sites prompted us to search for specific binding sites on the NTs analogous to SNARE-motifs-the 9-residuelong motifs present on the NT's natural substrates (SNAP-25, syntaxin, VAMP/synaptobrevin); such putative binding motifs (sites) noted on all clostridial NTs are reported here. Their relationship to the observed autoproteolysis remains to be determined experimentally. The dinucleotide NAD(+)/NADH associated with the NTs type A, B and E (2-3 NADH per protein molecule) via their H-chains, and a portion of the H-chain (toward the C-terminus) appears to exhibit limited amino acid sequence homology with lactate dehydrogenase-a representative NAD(+)/NADH binding protein.
authors.affiliation: Department of Food Microbiology and Toxicology, University of Wisconsin - Madison, 1925 Willow Drive, Madison, WI 53706, USA. brdasgup@wisc.edu
authors.forename: Bibhuti R
authors.initials: BR
authors.lastname: Dasgupta
authors.forename: Babu S
authors.initials: BS
authors.lastname: Antharavally
authors.forename: William
authors.initials: W
authors.lastname: Tepp
authors.forename: Mary L
authors.initials: ML
authors.lastname: Evenson
mesh_terms: D000595:Amino Acid Sequence; D001905:Botulinum Toxins / Q000378:metabolism*; D019274:Botulinum Toxins, Type A / Q000378:metabolism; D004229:Dithiothreitol; D007770:L-Lactate Dehydrogenase / Q000737:chemistry; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000378:metabolism; D010618:Phenanthrolines; D017386:Sequence Homology, Amino Acid
chemical_list: D010446:Peptide Fragments; D010618:Phenanthrolines; D009243:NAD; C096323:rimabotulinumtoxinB; D007770:L-Lactate Dehydrogenase; D010447:Peptide Hydrolases; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096326:botulinum toxin type E; D004229:Dithiothreitol; C025205:1,10-phenanthroline