Cell-mediated cleavage of Pseudomonas exotoxin between Arg279 and Gly280 generates the enzymatically active fragment which translocates to the cytosol.
Pseudomonas exotoxin (PE) is a three-domain toxin which is cleaved by a cellular protease within cells and then reduced to generate two prominent fragments (Ogata, M., Chaudhary, V. K., Pastan, I., and FitzGerald, D. J. (1990) J. Biol. Chem. 265, 20678-20685). The N-terminal fragment is 28 kDa in size and contains the binding domain. The 37-kDa C-terminal fragment, which translocates to the cytosol, contains the translocation domain and the ADP-ribosylation domain. Cleavage followed by reduction is essential for toxicity since mutant forms of the toxin that cannot be cleaved by cells are nontoxic. Previous results with these mutants suggest that cleavage occurred in an arginine-rich (arginine residues are at positions 274, 276, and 279) disulfide loop near the beginning of the translocation domain, but the exact site of cleavage was not determined. Since very few molecules of the 37-kDa fragment are generated within cells it was not possible to determine the site of cleavage by performing a conventional N-terminal sequence analysis of the 37-kDa fragment. Two experimental approaches were used to overcome this limitation. First, existing amino acids near the cleavage sites were replaced with methionine residues; this was followed by the addition of [35S]methionine-labeled versions of these toxins to cells. The pattern of radioactive toxin fragments recovered from the cells indicated that the toxin was cleaved either just before or just after Arg279. Second, [3H]leucine-labeled toxin was produced and added to the cells. Sequential Edman degradations were performed on the small amount of radioactive 37-kDa fragment that could be recovered from toxin-treated cells. A peak of radioactivity in the fifth fraction indicated that leucine was the 5th amino acid on the C-terminal side of the cleavage site. This result confirmed that cleavage was between Arg279 and Gly280.
Ogata
M
M
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Fryling
C M
CM
Pastan
I
I
FitzGerald
D J
DJ
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D002470:Cell Survival / Q000187:drug effects; D003600:Cytosol / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D005098:Exotoxins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D005998:Glycine; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D015183:Restriction Mapping; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001120:Arginine; D001425:Bacterial Outer Membrane Proteins; D001427:Bacterial Toxins; D010450:Endopeptidases; D005098:Exotoxins; D005998:Glycine; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A