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 proteolytic cleavage between Arg-279 and Gly-280. Cleavage, which is mediated by the cellular protease furin, generates an active C-terminal fragment which translocates to the cytosol and inhibits protein synthesis. In vitro, furin-mediated cleavage is optimal at pH 5.5 with a relatively slow turnover rate. Within cells, only 5-10% of cell-associated PE is cleaved. To investigate the reasons for this inefficient cleavage, the amino acid composition near the cleavage site was altered to resemble more closely the arginine-rich sequence from the functionally similar region of diphtheria toxin (DT). Four PE-DT mutants were generated, whereby 1, 5, 6 or 8 amino acids at the PE-cleavage site were changed to amino acids found at the DT-cleavage site. Mutant proteins were expressed in Escherichia coli, purified and then analysed for their susceptibility to cleavage by furin and trypsin, susceptibility to cell-mediated cleavage, and cytotoxic activity relative to wild-type PE. At pH 5.5, the rate of both furin-mediated cleavage and trypsin-mediated cleavage increased dramatically when amino acids in PE were altered to resemble the DT sequence. This increase did not alter the pH optimum 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 important toxin functions because none of the PE-DT proteins exhibited enhanced toxicity for cells when compared with the activity of wild-type PE.
Chiron
M F
MF
Biotherapy Section, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Ogata
M
M
FitzGerald
D J
DJ
D000818:Animals; D001120:Arginine; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D003603:Cytotoxins / Q000235:genetics / Q000378:metabolism*; D004167:Diphtheria Toxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005098:Exotoxins / Q000235:genetics / Q000378:metabolism*; D045683:Furin; D006801:Humans; D007700:Kinetics; D051379:Mice; D016296:Mutagenesis; D011499:Protein Processing, Post-Translational; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D001427:Bacterial Toxins; D003603:Cytotoxins; D004167:Diphtheria Toxin; D005098:Exotoxins; D011993:Recombinant Fusion Proteins; D001120:Arginine; D013381:Subtilisins; D014357:Trypsin; D045683:Furin