CLOSTRIDIUM-SEPTICUM ALPHA-TOXIN IS PROTEOLYTICALLY ACTIVATED BY FURIN

Clostridium septicum alpha-toxin is secreted as an inactive 46,450-Da protoxin. The protoxin is activated by proteolytic cleavage near the C terminus, which eventually causes the release of a 45-amino-acid frag ment, Proteoytic activation and loss of the propeptide allow alpha-tox in to oligomerize and form pores on the plasma membrane, which results in colloidal-osmotic lysis, Activation may be accomplished in vitro b y cleavage with trypsin at Arg(367) (J, Ballard, Y, Sokolov, W, L, Yua n, B, L, Kagan, and R Ii, Tweten, Mol, Microbiol. 10:627-634, 1993), w hich is located within the sequence KKRRGKR(367)S. A conspicuous featu re of this site is a recognition site (RGKR) for the eukaryotic protea se furin, Pro-alpha-toxin (AT(pro)) that was digested with trypsin or recombinant soluble furin yielded the 41,327-Da active form (AT(act)). A mutated alpha-toxin in which the furin consensus site was altered t o KKRSGSRS at the cleavage site (AT(SGSR)) was cleaved and activated b y trypsin but not by furin, In cytotoxicity assays, wild-type Chinese hamster ovary (CHO) and furin-deficient CHO (FD11) cells were killed b y AT(pro) but not by AT(SGSR), Both cell types were killed by AT(SGSR) that was preactivated with trypsin, Propidium iodide uptake assays re vealed that FD11 cells were approximately 22% less sensitive to AT(pro ) than were CHO cells, AT(pro)-induced cell lysis of FD11 cells, asses sed by propidium iodide uptake,,vas partially prevented by leupeptin ( 5 mM) and completely prevented by antipain (2.5 mM). The inhibition by antipain suggested the presence of cysteine or serine proteases that could also activate AT(pro), These findings demonstrate that furin is involved in the activation of C, septicum alpha-toxin on the cell surf ace but that alternate eukaryotic proteases can also activate the toxi n, Regardless of the activating protease, the furin consensus site app ears to be essential for the activation of alpha-toxin on the cell sur face.