Characterization of membrane translocation by anthrax protective antigen

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• The protective antigen (PA) heptamer of anthrax toxin facilitates membrane translocation of toxic moieties.
• Understanding the mechanism of this translocation is crucial for developing countermeasures.

Purpose of the Study:

• To develop and utilize a novel assay to quantify the translocation efficiency of anthrax toxin components across the plasma membrane.
• To investigate the requirements for protein translocation mediated by protective antigen.

Main Methods:

• Development of a cell-based assay using radiolabeled ligands and proteolytically activated PA (PA63).
• Induction of translocation by pH change, followed by Pronase E treatment and SDS-PAGE analysis.
• Assessment of translocation efficiency for various anthrax toxin fragments and fusion proteins.

Main Results:

• Translocation efficiency varied significantly, with the N-terminal PA binding domain of lethal factor (LFN) showing the highest efficiency (35%-50%).
• Intact lethal factor (LF), edema factor (EF), and certain fusion proteins exhibited lower translocation rates (15%-20%).
• Evidence suggests proteins must unfold for translocation, as disulfide bond formation or ligand binding blocked the process.

Conclusions:

• The developed assay effectively measures anthrax toxin translocation.
• Protein unfolding is a critical requirement for translocation mediated by protective antigen.
• The acid-induced translocation mechanism shares similarities with diphtheria toxin, despite distinct pore formation pathways.