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Functional characterization of protease-treated Bacillus anthracis protective antigen
J M Novak1, M P Stein, S F Little
1Bacteriology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21702-5011.
The Journal of Biological Chemistry
|August 25, 1992
Summary
Protease treatment of Bacillus anthracis protective antigen (PA) generated fragments that bind cells and internalize lethal factor (LF). Chymotrypsin-treated PA, though active in binding and internalization, is biologically inactive, indicating a later defect in anthrax toxin processing.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Bacillus anthracis toxins, edema toxin (ET) and lethal toxin (LT), mediate anthrax pathogenesis.
- Protective antigen (PA) is crucial for cellular entry of both ET and LT.
- Understanding PA's functional domains is key to deciphering anthrax toxin mechanisms.
Purpose of the Study:
- To characterize functional domains of Bacillus anthracis protective antigen (PA).
- To investigate the biological activity of PA fragments generated by protease treatment.
- To determine the stage at which chymotrypsin-treated PA function is impaired.
Main Methods:
- Proteolytic digestion of PA using trypsin and chymotrypsin.
- Assessment of biological activity by cell killing assays.
- Analysis of PA-receptor binding and PA-mediated lethal factor (LF) binding and internalization using radiolabeled assays.
- Cellular localization studies of internalized LF.
Main Results:
- Trypsin digestion yielded an active 63-kDa PA fragment.
- Chymotrypsin digestion produced inactive 37- and 47-kDa PA fragments.
- All PA preparations bound cells and internalized 125I-LF, indicating the defect occurs post-internalization.
Conclusions:
- Chymotrypsin-treated PA is defective after cell binding and LF internalization.
- The defect likely involves later steps in toxin routing or intracellular processing.
- Protease-generated PA fragments are valuable tools for studying anthrax toxin mechanisms and structure-function relationships.