title: Characterization of dominant-negative forms of anthrax protective antigen.
abstract: Certain mutations within the protective antigen (PA) moiety of anthrax toxin endow the protein with a dominant-negative (DN) phenotype, converting it into a potent antitoxin. Proteolytically activated PA oligomerizes to form ring-shaped heptameric complexes that insert into the membrane of an acidic intracellular compartment and promote translocation of bound edema factor and/or lethal factor to the cytosol. DN forms of PA co-oligomerize with the wild-type protein and block the translocation process. We prepared and characterized 4 DN forms: a single, a double, a triple, and a quadruple mutant. The mutants were made by site-directed mutation of the cloned form of PA in Escherichia coli and tested by various assays conducted on CHO cells or in solution. All 4 mutant PAs were competent for heptamerization and ligand binding but were defective in the pH-dependent functions: pore formation, ability to convert to the SDS-resistant heptamer, and ability to translocate bound ligand. The single mutant (F427K) showed less attenuation than the others in the pH-dependent functions and lower DN activity in a CHO cell assay. The quadruple (K397D + D425K + F427A + 2beta2-2beta3) deletion showed the most potent DN activity at low concentrations but also gave indications of low stability in a urea-mediated unfolding assay. The double mutant (K397D + D425K) and the triple (K397D + D425K + F427A) showed strong DN activity and slight reduction in stability relative to the wild-type protein. The properties of the double and the triple mutants make these forms worthy of testing in vivo as a new type of antitoxic agent for treatment of anthrax.
authors.affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.
authors.forename: Ming
authors.initials: M
authors.lastname: Yan
authors.forename: R John
authors.initials: RJ
authors.lastname: Collier
mesh_terms: D000818:Animals; D000881:Anthrax / Q000517:prevention & control*; D000942:Antigens, Bacterial / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics*; D016466:CHO Cells; D006224:Cricetinae; D005453:Fluorescence; D005799:Genes, Dominant / Q000502:physiology*; D008854:Microscopy, Electron; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020011:Protective Agents
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D020011:Protective Agents; C030325:anthrax toxin