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Cytotoxic activity of a recombinant chimaeric protein between Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae diphtheria toxin
Chantal Guidi-Rontani,
Chantal Guidi-Rontani
Unité des Toxines Microbiennes, CNRS URA557, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France.
Search for more papers by this authorChantal Guidi-Rontani,
Chantal Guidi-Rontani
Unité des Toxines Microbiennes, CNRS URA557, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France.
Search for more papers by this authorTel. (1) 45 68 80 00, ext. 7230; Fax (1) 45 68 84 56.
Summary
A segment of the exotoxin A gene of Pseudomonas aeruginosa, coding for the N-terminal end of domain I and domain II of the toxin (ETA), was genetically fused to the diphtheria toxin gene of Corynebacterium diphtheriae, coding for the N-terminal end of A fragment of diphtheria toxin (DT). The resulting hybrid protein (termed CED1) was produced in large amounts and exported to the periplasm in Escherichia coli. This chimaeric protein reacted with both anti-ETA and anti-DT antisera. Furthermore, the chimaeric protein displayed ADP-ribosylation activity and exhibited cytotoxicity to mouse 3T6 fibroblasts. These results demonstrated that the chimaeric protein is cytotoxic, and that the toxic potential of DTA can be selectively internalized and translocated via domains I and II of exotoxin A, which are thus sufficient to direct and translocate an enzymatically active heterologous polypeptide segment into the cytosol of sensitive cells.
References
- Allured, V.S., Collier, R.J., Carroll, S.F., and McKay, D.B. (1986) Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution. Proc Natl Acad Sci USA 83: 1320–1324.
- Birnboim, H.C, and Doly, J. (1979) A rapid extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7: 1513–1525.
- Bradford, M. (1976) A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.
- Carroll, S.F., and Collier, R.J. (1987) Active site of Pseudomonas aeruginosa exotoxin A. J Biol Chem 262: 8707–8711.
- Chaudhary, V.K., Xu, Y.H., FitzGerald, D., Adhya, S., and Pastan, I. (1988) Role of domain II of Pseudomonas exotoxin in the secretion of proteins into the periplasm and medium by E. coli. Proc Natl Acad Sci USA 85: 2939–2943.
- Chung, D.W., and Collier, R.J. (1977) Enzymatically active peptide from the adenosine diphosphate-ribosylating toxin of Pseudomonas aeruginosa. Infect Immun 16: 832–841.
- Collier, R.J., and Kandel, J. (1971) Structure and activity of diphtheria toxin. J Biol Chem 246: 1496–1503.
- Domenighini, M., Wontecucco, C., Ripka, W.C., and Rappuoli, R. (1991) Computer modelling of the NAD binding site of ADP-ribosylating toxins: active-site structure and mechanism of NAD binding. Mol Microbiol 5: 23–31.
- Douglas, C., Guidi-Rontani, C., and Collier, R.J. (1987) Exotoxin A of Pseudomonas aeruginosa: active, cloned toxin is secreted into the periplasmic space of Escherichia coli. J Bacteriol 169: 4962–4966.
- Drazin, R., Kandel, J., and Collier, R.J. (1971) Structure and activity of diphtheria toxin. J Biol Chem 246: 1504–1510.
- Fairbanks, G., Steck, T.L., and Wallach, D.F.H. (1971) Electrophoretic analysis of major polypeptides of the human erythrocyte membrane. Biochemistry 10: 2606–2617.
- Gill, D.M., and Dinius, L.L. (1971) Observations on the structure of diphtheria toxin. J Biol Chem 246: 1485–1491.
- Gill, D.M., and Pappenheimer, A.M. (1971) Structure-activity relationships in diphtheria toxin. J Biol Chem 246: 1492–1495.
- Gill, D.M., Pappenheimer, A.M., Brown, R., and Kurnick, J.J. (1969) Studies on the mode of action of diphtheria toxin. VII. Toxin-stimulated hydrolysis of nicotinamide adenine dinucleotide in mammalian cell extracts. J Exp Med 129: 1–21.
- Gray, G.L, Smith, D.H., Baldridge, J.S., Harkins, R.N., Vasil, M.L, Chen, E.Y., and Heyneker, H.L. (1984) Cloning, nucleotide sequence and expression in Escherichia coli of the exotoxin A structural gene of Pseudomonas aeruginosa. Proc Natl Acad Sci USA 81: 2645–2649.
- Greenfield, G.L., Bjorn, M.J., Horn, G., Fong, D., Buck, G.A., Collier, R.J., and Kaplan, D.A. (1983) Nucleotide sequence of the structural gene for diphtheria toxin carried by corynebacteriophage beta. Proc Natl Acad Sci USA 80: 6853–6857.
- Guidi-Rontani, C. (1991) Functional analysts of exotoxin A-related protein of Pseudomonas aeruginosa lacking residues 225–412. FEMS Microbiol Letts 80: 103–110.
- Guidi-Rontani, C., and Collier, R.J. (1987) Exotoxin A of Pseudomonas aeruginosa: evidence that domain I functions in receptor binding. Mol Micro Biol 1: 67–72.
- Honjo, T., Nishizuka, Y., Hayaishi, O., and Kato, I. (1968) Diphtheria toxin-dependent adenosine diphosphate ribosylation of aminoacyl transferase II and inhibition of protein synthesis. J Biol Chem 243: 3553–3555.
- Hwang, J., Fitzgerald, D.J., Adhya, S., and Pastan, I. (1987) Functional domains of Pseudomonas exotoxin identified by deletion analysis of the gene expressed in E. coli. Cell 48: 129–136.
- Iglewski, B.H., and Kabat, D. (1975) NAD-dependent inhibition of protein synthesis by Pseudomonas aeruginosa toxin A. Proc Natl Acad Sci USA 72: 2284–2288.
- Iglewski, B.H., Liu, P.V., and Kabat, D. (1977) Mechanism of action of Pseudomonas aeruginosa exotoxin A: adenosine diphosphate-ribosylation of mammalian elongation factor 2 in vitro and in vivo. Infect Immun 15: 138–144.
- Kohno, K., Uchida, T., Ohkubo, H., Nakanishi, S., Nakanishi, T., Fukui, T., Ohtsuka, E., Ikehara, M., and Okada, Y. (1986)Amino acid sequence of mammalian elongation factor 2 deduced from the cDNA sequence: homology with GTP-binding proteins. Proc Natl Acad Sci USA 83: 4978–4982.
- Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
- Mandel, M., and Higa, A. (1970) Calcium-dependent bacteriophage DNA infection. J Mol Biol 53: 159–162.
- Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor , New York : Cold Spring Harbor Laboratory Press.
- Murphy, J.R., Bishai, W., Borowski, M., Miyanohara, A., Boyd, J., and Nagle, S. (1986) Genetic construction, expression, and melanoma-selective cytotoxicity of a diphtheria toxin-related alpha-melanocyte stimulating hormone fusion protein. Proc Natl Acad Sci USA 83: 8258–8262.
- Ogata, M., Chaudhary, V.K., FitzGerald, D.J., and Pastan, I. (1989) Cytotoxic activity of a recombinant fusion protein between interleukin 4 and Pseudomonas exotoxin. Proc Natl Acad Sci USA 86: 4215–4219.
- Prior, T.I., FitzGerald, D.J., and Pastan, I. (1991) Barnase toxin: a new chimeric toxin composed of Pseudomonas exotoxin A and Barnase. Cell 64: 1017–1023.
- Russell, D.R., and Bennett, G.N. (1982) Construction and analysis of in vivo activity of E. coli promoter hybrids and promoter mutants that alter the — 35 to —10 spacing. Gene 20: 231–243.
- Siegall, CB., Chaudhary, V.K., FitzGerald, D.J., and Pastan, I. (1989) Functional analysis of Domains II, Ib, and III of Pseudomonas exotoxin. J Biol Chem 624; 14256–14261.
- Tweten, R.K., and Collier, R.J. (1983) Molecular cloning and expression of gene fragments from Corynebacterium phage beta encoding enzymatically active peptides of diphtheria toxin. J Bacteriol 156: 680–685.
- Uchida, T., Pappenheimer, Jr, A.M., and Harper, A.A. (1972) Reconstitution of diphtheria toxin from two nontoxic crossreacting mutant proteins. Science 175: 901–903.
- van Ness, B.G., Howard, J.B., and Bodley, J.W. (1980) ADP-ribosylation of elongation factor by diphtheria toxin. J Biol Chem 255: 10710–10720.
- Wilson, B.A., Reich, K.A., Weinstein, B.R., and Collier, R.J. (1990) Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine. Biochemistry 29: 8643–8651.
- Zucker, D.R., and Murphy, J.R. (1984) Monoclonal antibody analysis of diphtheria toxin-l: Localization of epitopes and neutralization of cytotoxicity. Mol Immunol 21: 785–793.
- Zucker, D.R., Murphy, J.R., and Pappenheimer, A.M. (1984) Monoclonal antibody analysis of diphtheria toxin II: inhibition of ADP-ribosyl-transferase activity. Mol Immunol 21: 795–800.
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