title: Mechanism of action of Pseudomonas exotoxin. Identification of a rate-limiting step.
abstract: Pseudomonas exotoxin (PE) enters cells by receptor-mediated endocytosis and is cleaved by a cellular protease between Arg279 and Gly280 to produce an NH2-terminal fragment of 28 kDa which contains the toxin's binding domain and a COOH-terminal fragment of 37 kDa which has translocating and ADP-ribosylating activity. After proteolysis, the COOH-terminal fragment reaches the endoplasmic reticulum by retrograde transport where it translocates to the cytosol and inhibits protein synthesis by ADP-ribosylating elongation factor 2. To understand how the 37-kDa fragment functions, we focused on the role of specific amino acids located near its NH2 terminus. We found that there was a 4-250-fold loss in toxic activity when tryptophan 281, leucine 284, or tyrosine 289 were changed to other residues. Mutations at these three positions did not interfere with the receptor binding, cell-mediated proteolytic cleavage, or ADP-ribosylating activity. To determine the role of these amino acids, a competition assay was devised in which the addition of excess PE delta 553, a mutant form of PE that lacks ADP-ribosylation activity, competed efficiently for the toxicity of PE. Excess PE with mutations near the NH2 terminus of the 37-kDa fragment competed poorly. This competition occurred after proteolysis since PEGly276, a mutant form of PE that is not cleaved, did not complete. We conclude that specific amino acids at the NH2 terminus of the 37-kDa fragment interact in a saturable manner with an unknown intracellular component.
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: A G
authors.initials: AG
authors.lastname: Zdanovsky
authors.forename: M
authors.initials: M
authors.lastname: Chiron
authors.forename: I
authors.initials: I
authors.lastname: Pastan
authors.forename: D J
authors.initials: DJ
authors.lastname: FitzGerald
mesh_terms: D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001427:Bacterial Toxins; D001483:Base Sequence; D001667:Binding, Competitive; D002478:Cells, Cultured; D005098:Exotoxins / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose; D000596:Amino Acids; D001427:Bacterial Toxins; D005098:Exotoxins; D009838:Oligodeoxyribonucleotides; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A