PSEUDOMONAS EXOTOXIN EXHIBITS INCREASED SENSITIVITY TO FURIN WHEN SEQUENCES AT THE CLEAVAGE SITE ARE MUTATED TO RESEMBLE THE ARGININE-RICH LOOP OF DIPHTHERIA-TOXIN

To be toxic for mammalian cells, Pseudomonas exotoxin (PE) requires pr oteolytic cleavage between Arg-279 and Gly-280, Cleavage, which is med iated by the cellular protease furin, generates an active C-terminal f ragment which translocates to the cytosol and inhibits protein synthes is, In vitro, furin-mediated cleavage is optimal at pH 5.5 with a rela tively slow turnover rate. Within cells, only 5-10% of cell-associated PE is cleaved, To investigate the reasons for this inefficient cleava ge, the amino acid composition near the cleavage site was altered to r esemble more closely the arginine-rich sequence from the functionally similar region of diphtheria toxin (DT). Four PE-DT mutants were gener ated, whereby 1, 5, 6 or 8 amino acids at the PE-cleavage site were ch anged to amino acids found at the DT-cleavage site, Mutant proteins we re expressed in Escherichia coil, purified and then analysed for their susceptibility to cleavage by furin and trypsin, susceptibility to ce ll-mediated cleavage, and cytotoxic activity relative to wild-type PE. At pH 5.5, the rate of both furin-mediated cleavage and trypsin-media ted cleavage increased dramatically when amino acids in PE were altere d to resemble the DT sequence. This increase did not alter the pH opti mum for furin-mediated cleavage of PE toxins, which remained at pH 5.0 -5.5, When radioactive versions of selected PE-DT proteins were added to intact cells, an increase in the percentage of molecules that were cleaved relative to wild-type PE was also seen, However, changes that favoured increased proteolysis apparently interfered with other import ant toxin functions because none of the PE-DT proteins exhibited enhan ced toxicity for cells when compared with the activity of wildtype PE.