PROTEOLYTIC ACTIVATION OF BACTERIAL TOXINS BY EUKARYOTIC CELLS IS PERFORMED BY FURIN AND BY ADDITIONAL CELLULAR PROTEASES

Before intoxication can occur, anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) must be activat ed by proteolytic cleavage at specific amino acid sequences. Previousl y, it was shown that PA and DT can be activated by furin. In Chinese h amster ovary (CHO) cells, wild-type (RKKR) and cleavage site mutants o f PA, each administered with a modified form of anthrax toxin lethal f actor (the N terminus of lethal factor fused to PE domain III), had th e following potencies: RKKR (wild type) (concentration causing 50% cel l death [EC(50)]= 12 ng/ml) greater than or equal to RAAR (EC(50) = 18 ng/ml) > FTKR (EC(50) = 24 ng/ml) > STRR (EC(50) = 49 ng/ml). In vitr o cleavage of PA and cleavage site mutants of PA by furin demonstrated that native PA (RKKR) and PA with the cleavage sequence RAAR are subs trates for furin. To characterize eukaryotic proteases that play a rol e in activating bacterial toxins, furin deficient CHO cells were selec ted after chemical mutagenesis. Furin-deficient cells were resistant t o PE, whose cleavage site, RQPR, constitutes a furin recognition site and to all PA cleavage site mutants, but were sensitive to DT (EC(50) = 2.9 ng/ml) and PA (EC(50) = 23 ng/ml), whose respective cleavage sit es, RKKR and RVRR, contain additional basic residues. Furin-deficient cells that were transfected with the furin gene regained sensitivity t o PE and PA cleavage site mutants. These studies provide evidence that furin can activate the three toxins and that one or more additional p roteases contribute to the activation of DT and PA.