title: Botulinum neurotoxin types B and E: purification, limited proteolysis by endoproteinase Glu-C and pepsin, and comparison of their identified cleaved sites relative to the three-dimensional structure of type A neurotoxin.
abstract: Botulinum neurotoxin (NT) serotypes B and E are approximately 150 kDa proteins. Isolated from the liquid culture of Clostridium botulinum the NT type E is a single chain protein while the NT type B, from the proteolytic strain of the bacteria, is a mixture of dichain (nicked within a disulfide loop located about one-third the way from the N-terminus to the C-terminus) protein and its precursor single-chain protein. Endoproteinase Glu-C (EC 3.4.21.19) and pepsin (EC 3.4.23.1) were used for controlled digestion of NT types B and E; the amino acid residues flanking many of the cleavable peptide bonds were identified and the corresponding proteolytic fragments partially characterized. Chemical identification of 82 and 108 residues of types B and E NT, respectively, revealed that the residue 738 and 1098 in type E NT, identified as Leu and Asn, respectively, differ from those deduced from nucleotide sequences. Several fragments overlapped spanning various segments of the NT's functional domains; they appear to have potential for structure-function studies of the NT. The cleavage sites were compared with the previously determined proteolyzed sites on NT types A and E. The cleavage sites of the NT types A, B and E, all exposed on the protein surface, were scrutinized in the context of the three-dimensional structure of crystallized NT type A [Lacy, D.B., Stevens, R.C., 1999. J. Mol. Biol. 291, 1091-1104]. Detailed procedures for isolation of pure NT types B and E in large quantities (average yield 92 and 62 mg, respectively) suitable for crystallization are reported.
authors.affiliation: Department of Food Microbiology and Toxicology, University of Wisconsin, Madison, 53706, USA.
authors.forename: S
authors.initials: S
authors.lastname: Prabakaran
authors.forename: W
authors.initials: W
authors.lastname: Tepp
authors.forename: B R
authors.initials: BR
authors.lastname: DasGupta
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001905:Botulinum Toxins / Q000302:isolation & purification* / Q000378:metabolism*; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000378:metabolism*; D002847:Chromatography, Agarose; D002848:Chromatography, DEAE-Cellulose; D003014:Clostridium botulinum / Q000472:pathogenicity*; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D009465:Neuromuscular Agents / Q000378:metabolism; D010434:Pepsin A / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000378:metabolism*
chemical_list: D009465:Neuromuscular Agents; D010446:Peptide Fragments; C096323:rimabotulinumtoxinB; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D010434:Pepsin A; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096326:botulinum toxin type E