title: Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium botulinum Expression System.
abstract: Clostridium botulinum subtype A4 neurotoxin (BoNT/A4) is naturally expressed in the dual-toxin-producing C. botulinum strain 657Ba at 100× lower titers than BoNT/B. In this study, we describe purification of recombinant BoNT/A4 (rBoNT/A4) expressed in a nonsporulating and nontoxigenic C. botulinum expression host strain. The rBoNT/A4 copurified with nontoxic toxin complex components provided in trans by the expression host and was proteolytically cleaved to the active dichain form. Activity of the recombinant BoNT/A4 in mice and in human neuronal cells was about 1,000-fold lower than that of BoNT/A1, and the recombinant BoNT/A4 was effectively neutralized by botulism heptavalent antitoxin. A previous report using recombinant truncated BoNT/A4 light chain (LC) expressed in Escherichia coli has indicated reduced stability and activity of BoNT/A4 LC compared to BoNT/A1 LC, which was surmounted by introduction of a single-amino-acid substitution, I264R. In order to determine whether this mutation would also affect the holotoxin activity of BoNT/A4, a recombinant full-length BoNT/A4 carrying this mutation as well as a second mutation predicted to increase solubility (L260F) was produced in the clostridial expression system. Comparative analyses of the in vitro, cellular, and in vivo activities of rBoNT/A4 and rBoNT/A4-L260F I264R showed 1,000-fold-lower activity than BoNT/A1 in both the mutated and nonmutated BoNT/A4. This indicates that these mutations do not alter the activity of BoNT/A4 holotoxin. In summary, a recombinant BoNT from a dual-toxin-producing strain was expressed and purified in an endogenous clostridial expression system, allowing analysis of this toxin.
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Marite
authors.initials: M
authors.lastname: Bradshaw
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: William H
authors.initials: WH
authors.lastname: Tepp
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Regina C M
authors.initials: RC
authors.lastname: Whitemarsh
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Sabine
authors.initials: S
authors.lastname: Pellett
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA eajohnso@wisc.edu.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Johnson
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000992:Antitoxins / Q000378:metabolism; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity*; D002478:Cells, Cultured; D003014:Clostridium botulinum / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006027:Glycosides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity*; D006801:Humans; D051379:Mice; D009474:Neurons / Q000187:drug effects*; D009500:Neutralization Tests; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity; D014315:Triterpenes / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity*
chemical_list: D000992:Antitoxins; D006027:Glycosides; D011994:Recombinant Proteins; D014315:Triterpenes; C001883:stichoposide; D019274:Botulinum Toxins, Type A