Proteolytic activation of receptor-bound anthrax protective antigen on macrophages promotes its internalization

Beauregard, Kathryn; Collier, Ann R.; Swanson, Joel A.

doi:10.1046/j.1462-5822.2000.00052.x

Cited by 98 publications

(78 citation statements)

References 28 publications

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“…Alternatively, PA is cleaved by a serum protease(s) [11] before binding to the cell receptor. The resulting cell-bound PA63 heptamers [12] competitively bind lethal factor (LF) or edema factor (EF), forming lethal toxin (LeTx) and edema toxin [13], respectively, also promote internalization of LF and EF into the cytosol [14]. Previous studies demonstrated that a quantitative anti-PA IgG ELISA and toxin neutralizing antibody (TNA) assay served as immunological correlates to immunity in New Zealand white rabbits previously inoculated with AVA [15].…”

Section: Introductionmentioning

confidence: 99%

Development of an in vitro-based potency assay for anthrax vaccine

Little

Webster

Ivins

et al. 2004

Vaccine

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Section: Introductionmentioning

confidence: 99%

Development of an in vitro-based potency assay for anthrax vaccine

Little

Webster

Ivins

et al. 2004

Vaccine

View full text Add to dashboard Cite

“…11 and 12). Whereas native PA persists on the cell surface, the heptamer is endocytosed (13), presumably because oligomerization aggregates anthrax toxin receptor. The endocytosed toxic complexes are trafficked to endosomal compartments where the low pH causes the PA 63 heptamer to insert into the membrane and form a water-filled channel (14,15).…”

mentioning

confidence: 99%

The lethal and edema factors of anthrax toxin bind only to oligomeric forms of the protective antigen

Mogridge

Cunningham

Lacy

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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171

160

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The three proteins that comprise anthrax toxin, edema factor (EF), lethal factor (LF), and protective antigen (PA), assemble at the mammalian cell surface into toxic complexes. After binding to its receptor, PA is proteolytically activated, yielding a carboxyl-terminal 63-kDa fragment (PA63) that coordinates assembly of the complexes, promotes their endocytosis, and translocates EF and LF to the cytosol. PA63 spontaneously oligomerizes to form symmetric ring-shaped heptamers that are capable of binding three molecules of EF and͞or LF as competing ligands. To determine whether binding of these ligands depends on oligomerization of PA63, we prepared two oligomerization-deficient forms of this protein, each mutated on a different PA63-PA63 contact face. In solution or when bound to receptors on Chinese hamster ovary K1 cells, neither mutant alone bound ligand, but a mixture of them did. After the two mutants were proteolytically activated and mixed with ligand in solution, a ternary complex was isolated containing one molecule of each protein. Thus EF and LF bind stably only to PA63 dimers or higher order oligomers. These findings are relevant to the kinetics and pathways of assembly of anthrax toxin complexes.

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“…EF and LF bind competitively to identical sites on oligomeric PA 63 (12,13), yielding a series of toxic complexes containing one to three molecules of EF and͞or LF bound per PA 63 heptamer (14). Oligomerization of PA 63 triggers endocytosis (15,16) and trafficking to an endosomal compartment (17,18). After acidification of the endosomal compartment, the PA 63 heptamer undergoes a conformational change that allows it to insert into the membrane, form a cation-selective pore, and translocate EF͞LF to the cytosol (19)(20)(21).…”

mentioning

confidence: 99%

Mapping dominant-negative mutations of anthrax protective antigen by scanning mutagenesis

Mourez

Yan

Lacy

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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109

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The protective antigen (PA) moiety of anthrax toxin transports edema factor and lethal factor to the cytosol of mammalian cells by a mechanism that depends on its ability to oligomerize and form pores in the endosomal membrane. Previously, some mutated forms of PA, designated dominant negative (DN), were found to coassemble with wild-type PA and generate defective heptameric pore-precursors (prepores). Prepores containing DN-PA are impaired in pore formation and in translocating edema factor and lethal factor across the endosomal membrane. To create a more comprehensive map of sites within PA where a single amino acid replacement can give a DN phenotype, we used automated systems to generate a Cys-replacement mutation for each of the 568 residues of PA63, the active 63-kDa proteolytic fragment of PA. Thirty-three mutations that reduced PA's ability to mediate toxicity at least 100-fold were identified in all four domains of PA63. A majority (22) were in domain 2, the pore-forming domain. Seven of the domain-2 mutations, located in or adjacent to the 2␤6 strand, the 2␤7 strand, and the 2␤10-2␤11 loop, gave the DN phenotype. This study demonstrates the feasibility of high-throughput scanning mutagenesis of a moderate sized protein. The results show that DN mutations cluster in a single domain and implicate 2␤6 and 2␤7 strands and the 2␤10-2␤11 loop in the conformational rearrangement of the prepore to the pore. They also add to the repertoire of mutations available for structure-function studies and for designing new antitoxic agents for treatment of anthrax.

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Proteolytic activation of receptor-bound anthrax protective antigen on macrophages promotes its internalization

Cited by 98 publications

References 28 publications

Development of an in vitro-based potency assay for anthrax vaccine

Development of an in vitro-based potency assay for anthrax vaccine

The lethal and edema factors of anthrax toxin bind only to oligomeric forms of the protective antigen

Mapping dominant-negative mutations of anthrax protective antigen by scanning mutagenesis

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