title: Identification of new dominant-negative mutants of anthrax protective antigen using directed evolution.
abstract: The anthrax toxin is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema toxin (EF). The PA moiety carries EF and LF into the cytosol of mammalian cells via a mechanism that depends on the oligomerization of PA and transmembrane pore formation by the PA oligomer. Certain mutants of PA, termed dominant-negative (DN) mutants, can co-oligomerize with wild-type PA and disrupt the translocation ability of the pore. Here, we constructed a PA mutant library by introducing random mutations into domain II of PA and screened three new DN mutants of PA: V377E, T380S, and I432C. All the mutants inhibited the anthrax toxin action against sensitive cells. V377E had the strongest inhibitory effect and was further confirmed to be able to protect mice against a challenge with anthrax lethal toxin. Furthermore, we functionally characterized these mutants. The result showed that these mutations did not impair proteolytic activation or oligomer formation of PA, but impeded the prepore-pore conversion of the oligomer. These DN mutants of PA identified in our study may provide valuable information for elucidating the structure-function relationship of PA and for designing therapeutics for anthrax treatment.
authors.affiliation: College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, People's Republic of China.
authors.forename: Gaobing
authors.initials: G
authors.lastname: Wu
authors.forename: Chunfang
authors.initials: C
authors.lastname: Feng
authors.forename: Sha
authors.initials: S
authors.lastname: Cao
authors.forename: Aizhen
authors.initials: A
authors.lastname: Guo
authors.forename: Ziduo
authors.initials: Z
authors.lastname: Liu
mesh_terms: D000818:Animals; D000881:Anthrax / Q000235:genetics / Q000276:immunology / Q000382:microbiology; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D001408:Bacillus anthracis / Q000235:genetics / Q000276:immunology / Q000472:pathogenicity; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D016466:CHO Cells; D006224:Cricetinae; D019020:Directed Molecular Evolution; D006801:Humans; D051379:Mice; D050505:Mutant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D009154:Mutation; D017434:Protein Structure, Tertiary
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D050505:Mutant Proteins; C030325:anthrax toxin