Functional analysis of domains II, Ib, and III of Pseudomonas exotoxin.
Pseudomonas exotoxin is composed of three structural domains that are responsible for cell recognition, membrane translocation, and ADP-ribosylation. The substitution of the cell recognition domain (domain Ia) with a growth factor such as transforming growth factor alpha (TGF alpha), creates a cell-specific cytotoxic agent, TGF alpha-PE40, which kills cells bearing epidermal growth factor (EGF) receptors. We have used TGF alpha-PE40 to define the role of sequences in domains II, Ib, and III. Various mutations were made in these domains and mutant forms of TGF alpha-PE40 expressed in Escherichia coli. Mutant proteins were then tested for their ADP-ribosylation, EGF receptor-binding, and cell-killing activities. Additionally, the amino boundary of domain III, which contains the ADP-ribosylation activity, was determined by deletion analysis. Data indicate that (i) the functional amino terminus of domain III is near amino acid 400; (ii) deletion of various regions in domain II or conversion of cysteines 265 and 268 to serines results in a loss of cytotoxicity which ranged from 10-fold to more than 150-fold, indicating that domain II is essential for full expression of cytotoxicity; (iii) deletion of the amino terminus of domain Ib results in a molecule with somewhat increased cytotoxic activity, indicating that domain Ib is not essential for the cytotoxic effect of TGF alpha-PE40; and (iv) TGF alpha-PE40, produced by denaturing and refolding of insoluble material from inclusion bodies, binds better to EGF receptors and is about 10-fold more cytotoxic to cells bearing EGF receptors than is the secreted form of soluble TGF alpha-PE40.
Siegall
C B
CB
Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
Chaudhary
V K
VK
FitzGerald
D J
DJ
Pastan
I
I
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D002294:Carcinoma, Squamous Cell / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D005098:Exotoxins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D005798:Genes, Bacterial; D006801:Humans; D007457:Iodine Radioisotopes; D009154:Mutation; D011500:Protein Synthesis Inhibitors / Q000633:toxicity; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000633:toxicity; D014407:Tumor Cells, Cultured / Q000378:metabolism
D005098:Exotoxins; D007457:Iodine Radioisotopes; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D000246:Adenosine Diphosphate Ribose; D004815:Epidermal Growth Factor; D066246:ErbB Receptors