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Membrane insertion by anthrax protective antigen in cultured cells.
Maen Qa'dan1, Kenneth A Christensen, Lei Zhang
1Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, Massachusetts 02115, USA.
Molecular and Cellular Biology
|June 21, 2005
Summary
This study confirms the beta-barrel model for anthrax toxin (PA) pore formation in mammalian cells. Fluorescently labeling protective antigen (PA) revealed how its structure inserts into the endosomal membrane to enable toxin entry.
Area of Science:
- Biochemistry
- Cell Biology
- Toxicology
Background:
- Anthrax toxin enters cells through a protective antigen (PA) pore in the endosomal membrane.
- Pore formation involves a pH-induced rearrangement of the PA prepore into a beta-barrel structure.
Purpose of the Study:
- To investigate the in vivo mechanism of protective antigen (PA) pore formation.
- To validate the proposed beta-barrel model of membrane insertion for anthrax toxin.
Main Methods:
- Protective antigen (PA) was labeled with a fluorescent probe (NBD) at specific cysteine residues within the 2beta2-2beta3 loop.
- Fluorescence was monitored in CHO cells using microscopy and cell suspensions after PA binding.
Main Results:
- NBD fluorescence increased significantly at positions predicted to face the lipid bilayer, supporting membrane insertion.
- Fluorescence changes were minimal at positions predicted to face the pore lumen.
- Specific labeling sites and the time course of membrane insertion were identified.
Conclusions:
- The findings support the beta-barrel model for protective antigen (PA) pore formation.
- This study provides insights into the dynamics of anthrax toxin membrane insertion in vivo.