Serum protease cleavage of Bacillus anthracis protective antigen

Ezzell, John W.; Abshire, Teresa G.

doi:10.1099/00221287-138-3-543

Cited by 88 publications

(100 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…PA is the component of the anthrax exotoxins that binds to cellular receptors [9] and undergoes proteolysis by the cell surface protease furin [10]. 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].…”

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

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

“…The third, Protective Antigen (PA), is a receptor-binding and pore-forming protein that transports LF and EF to the cytosol (4). Initially, PA is activated by cellular proteases of the furin family, or by serum proteases (5)(6)(7)(8), which cleave the 83-kDa protein into N-terminal 20-kDa and C-terminal 63-kDa fragments. The larger fragment self-associates to form ring-shaped heptamers (prepores), which bind up to three molecules of LF and/or EF, and a smaller population of octamers, which bind up to four molecules of these moieties (9,10).…”

mentioning

confidence: 99%

Effects of Introducing a Single Charged Residue into the Phenylalanine Clamp of Multimeric Anthrax Protective Antigen

Janowiak¹,

Fischer²,

Collier

2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

Multimeric pores formed in the endosomal membrane by the Protective Antigen moiety of anthrax toxin translocate the enzymatic moieties of the toxin to the cytosolic compartment of mammalian cells. There is evidence that the side chains of the Phe 427 residues come into close proximity with one another in the lumen of the pore and form a structure, termed the Phe clamp, that catalyzes the translocation process. In this report we describe the effects of replacing Phe 427 in a single subunit of the predominantly heptameric pore with a basic or an acidic amino acid. Incorporating any charged residue at this position inhibited cytotoxicity >1,000-fold in our standard assay and caused strong inhibition of translocation in a planar phospholipid bilayer system. His and Glu were the most strongly inhibitory residues, ablating both cytotoxicity and translocation. Basic residues at position 427 prevented the Phe clamp from interacting with a translocation substrate to form a seal against the passage of ions and accelerated dissociation of the substrate from the pore. Acidic residues, in contrast, allowed the seal to form and the substrate to remain firmly bound, but blocked its passage, perhaps via electrostatic interactions with the positively charged N-terminal segment. Our findings are discussed in relation to the role of the Phe clamp in a Brownian ratchet model of translocation.

show abstract

“…2,3 PA can also be cleaved and activated by serum proteases. 4 Once activated, PA 63 self-assembles into a ring-shaped heptamer, termed the prepore, which can bind up to three molecules of EF and/or LF. 5 The resulting hetero-oligomeric complexes are then endocytosed and delivered to the endosome.…”

Section: Introductionmentioning

confidence: 99%

An approach to characterizing single‐subunit mutations in multimeric prepores and pores of anthrax protective antigen

2009

View full text Add to dashboard Cite

Heptameric pores formed by the protective antigen (PA) moiety of anthrax toxin translocate the intracellular effector moieties of the toxin across the endosomal membrane to the cytosol of mammalian cells. We devised a protocol to characterize the effects of individual mutations in a single subunit of heptameric PA prepores (pore precursors) or pores. We prepared monomeric PA containing a test mutation plus an innocuous Cys-replacement mutation at a second residue (Lys563, located on the external surface of the prepore). The introduced Cys was biotinylated, and the protein was allowed to cooligomerize with a 20-fold excess of wild-type PA. Finally, biotinylated prepores were freed from wild-type prepores by avidin affinity chromatography. For the proof of principle, we examined single-subunit mutations of Asp425 and Phe427, two residues where Ala replacements have been shown to cause strong inhibitory effects. The single-subunit D425A mutation inhibited pore formation by >10 4 and abrogated activity of PA almost completely in our standard cytotoxicity assay. The single-subunit F427A mutation caused $100-fold inhibition in the cytotoxicity assay, and this effect was shown to result from a combination of strong inhibition of translocation and smaller effects on pore formation and ligand affinity. Our results show definitively that replacing a single residue in one subunit of the heptameric PA prepore can inhibit the transport activity of the oligomer almost completely-and by different mechanisms, depending on the specific residue mutated.

show abstract

Serum protease cleavage of Bacillus anthracis protective antigen

Cited by 88 publications

References 33 publications

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

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

Effects of Introducing a Single Charged Residue into the Phenylalanine Clamp of Multimeric Anthrax Protective Antigen

An approach to characterizing single‐subunit mutations in multimeric prepores and pores of anthrax protective antigen

Contact Info

Product

Resources

About