A recombinant form of Pseudomonas exotoxin directed at the epidermal growth factor receptor that is cytotoxic without requiring proteolytic processing.
Pseudomonas exotoxin A is composed of three structural domains that mediate cell recognition (I), membrane translocation (II), and ADP-ribosylation (III). Within the cell, the toxin is cleaved within domain II to produce a 37-kDa carboxyl-terminal fragment, containing amino acids 280-613, which is translocated to the cytosol and causes cell death. In this study, we constructed a mutant protein (PE37), composed of amino acids 280-613 of Pseudomonas exotoxin A, which does not require proteolysis to translocate. PE37 was targeted specifically to cells with epidermal growth factor receptors by inserting transforming growth factor-alpha (TGF-alpha) after amino acid 607 near the carboxyl terminus of Pseudomonas exotoxin A. PE37/TGF-alpha was very cytotoxic to cells with epidermal growth factor receptors. It was severalfold more cytotoxic than a derivative of full-length Pseudomonas exotoxin A containing TGF-alpha in the same position, probably because the latter requires intracellular proteolytic processing to exhibit its cytotoxicity, and proteolytic processing is not 100% efficient. Deletion of 2, 4, or 7 amino acids from the amino terminus of PE37/TGF-alpha greatly diminished cytotoxic activity, indicating the need for a proper amino-terminal sequence. In addition, a mutant containing an internal deletion of amino acids 314-380 was minimally active, indicating that other regions of domain II are also required for the cytotoxic activity of Pseudomonas exotoxin A.
Theuer
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Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
FitzGerald
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Pastan
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D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D001483:Base Sequence; D001667:Binding, Competitive; D001943:Breast Neoplasms; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects*; D002872:Chromosome Deletion; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000187:drug effects / Q000378:metabolism*; D005098:Exotoxins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D005260:Female; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis / Q000187:drug effects; D011499:Protein Processing, Post-Translational; D011550:Pseudomonas aeruginosa / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000494:pharmacology; D016211:Transforming Growth Factor alpha / Q000235:genetics / Q000494:pharmacology; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D005098:Exotoxins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D016211:Transforming Growth Factor alpha; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A