Pseudomonas exotoxin exhibits increased sensitivity to furin when sequences at the cleavage site are mutated to resemble the arginine-rich loop of diphtheria toxin

Molecular Microbiology
M F ChironD J FitzGerald

Abstract

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 alte...Continue Reading

Citations

Apr 22, 2009·Infection and Immunity·Juliette Morlon-GuyotBruno Beaumelle
Mar 1, 2014·Blood·Alan S WayneIra Pastan
Nov 25, 2004·Progress in Biophysics and Molecular Biology·Michael W Parker, Susanne C Feil
Nov 12, 2016·Toxins·Marta BorowiecKrzysztof Ginalski
Aug 3, 2005·Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology·Robert J KreitmanIra Pastan
Feb 12, 1998·The Journal of Biological Chemistry·M F ChironD FitzGerald
Mar 18, 2011·Bioconjugate Chemistry·Roberta Traini, Robert J Kreitman

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