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A role for PACE4 in the proteolytic activation of anthrax toxin protective antigen
V M Gordon1, A Rehemtulla, S H Leppla
1Laboratory of Microbial Ecology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892, USA. gordon@yoda.nidr.nih.gov
Abstract:
Several bacterial protein toxins require activation by eukaryotic proteases. Previous studies have shown that anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) are cleaved by furin C-terminal to the sequences RKKR, RQPR, and RVRR, respectively. Because furin-deficient cells retain some sensitivity to PA and DT, it is evident that other cellular proteases can activate these toxins. Whereas furin has been shown to require arginine residues at positions -1 and -4 for substrate recognition, another protease with an activity which could substitute for furin in toxin activation, the furin-related protease PACE4, requires basic residues in the -1, -2, and -4 positions of the substrate sequence. To examine the relative roles of furin and PACE4 in toxin activation, we used furin-deficient CHO cells (FD11 cells) transfected with either the furin (FD11/furin cells) or PACE4 (FD11/PACE4 cells) gene. Mutant PA proteins containing the cleavage sequence RAAR or KR were cytotoxic toward cells expressing only PACE4. In vitro cleavage data demonstrated that PACE4 can recognize RAAR and, to a much lesser extent, KR and RR. When extracts from PACE4-transfected cells were used as a source of proteases, PACE4 had minimal activity, indicating that it had been partially inactivated or did not remain associated with the cell membranes. Cleavage of iodinated PA containing the sequence RKKR or RAAR was detected on the surface of all cell types tested, but cleavage of a dibasic sequence was detected only intracellularly and only in cells that expressed furin or PACE4. The data provide evidence that PACE4 is present at the exterior of cells, that it plays a role in the proteolytic activation of anthrax toxin PA, and that PACE4 can activate substrates at the sequence RAAR or KR.
Insights
Protease PACE4 activates anthrax toxin protective antigen (PA) on cell surfaces, recognizing specific cleavage sequences. This protease plays a key role in bacterial toxin activation beyond furin.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Bacterial protein toxins often require host cell proteases for activation.
- Furin is a known protease that cleaves toxins like anthrax protective antigen (PA), but other proteases can also mediate this activation.
- Furin and PACE4 proteases have distinct substrate recognition requirements.
Purpose of the Study:
- To investigate the distinct roles of furin and PACE4 in the proteolytic activation of bacterial toxins, specifically anthrax toxin PA.
- To determine if PACE4 can activate PA and identify the specific cleavage sequences recognized by PACE4.
Main Methods:
- Utilized furin-deficient Chinese Hamster Ovary (CHO) cells (FD11) transfected with either furin or PACE4 genes.
- Created mutant PA proteins with altered cleavage sequences (RAAR, KR) to assess cytotoxicity.
- Performed in vitro cleavage assays and analyzed PA cleavage on cell surfaces and intracellularly.
Main Results:
- Mutant PA proteins were cytotoxic to cells expressing PACE4, indicating PACE4's role in activation.
- PACE4 recognized the RAAR sequence and, to a lesser extent, KR and RR sequences in vitro.
- PA cleavage occurred on the cell surface for RKKR and RAAR sequences, but dibasic sequence cleavage was intracellular and dependent on furin or PACE4 expression.
Conclusions:
- PACE4 is located on the cell exterior and contributes to the proteolytic activation of anthrax toxin PA.
- PACE4 can activate PA at RAAR or KR sequences, expanding the known repertoire of toxin activation pathways.
- This study highlights the functional redundancy and distinct substrate specificities of proteases like furin and PACE4 in toxin activation.