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. 1991 Oct;59(10):3381–3386. doi: 10.1128/iai.59.10.3381-3386.1991

Anthrax protective antigen interacts with a specific receptor on the surface of CHO-K1 cells.

V Escuyer 1, R J Collier 1
PMCID: PMC258895  PMID: 1909998

Abstract

The interaction of protective antigen (PA), a component of the anthrax toxin, with receptors on the Chinese hamster ovary cell line CHO-K1 was characterized. Protective antigen binding at 4 degrees C is highly specific, concentration dependent, saturable (Kd = 0.9 nM), and reversible. Scatchard analysis indicates the presence of a single class of PA binding sites at a concentration of 10,000 +/- 2,000 per cell. Pretreatment of cells with a number of different proteases strongly inhibits PA binding, suggesting that the receptor may be at least partially proteinaceous. Direct chemical cross-linking of radioiodinated PA to the cell surface results in the appearance of a major band exhibiting an apparent molecular mass of 170 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The appearance of this band is completely inhibited by a 200-fold molar excess of unlabeled PA, indicating a high specificity for this interaction. Our results suggest that a cell surface protein(s) of 85 to 90 kDa is, or constitutes a portion of, a specific receptor for the PA.

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  1. BEALL F. A., TAYLOR M. J., THORNE C. B. Rapid lethal effect in rats of a third component found upon fractionating the toxin of Bacillus anthracis. J Bacteriol. 1962 Jun;83:1274–1280. doi: 10.1128/jb.83.6.1274-1280.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blaustein R. O., Koehler T. M., Collier R. J., Finkelstein A. Anthrax toxin: channel-forming activity of protective antigen in planar phospholipid bilayers. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2209–2213. doi: 10.1073/pnas.86.7.2209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cieplak W., Gaudin H. M., Eidels L. Diphtheria toxin receptor. Identification of specific diphtheria toxin-binding proteins on the surface of Vero and BS-C-1 cells. J Biol Chem. 1987 Sep 25;262(27):13246–13253. [PubMed] [Google Scholar]
  4. Cuatrecasas P. Commentary. Insulin receptors, cell membranes and hormone action. Biochem Pharmacol. 1974 Sep 1;23(17):2353–2361. doi: 10.1016/0006-2952(74)90224-x. [DOI] [PubMed] [Google Scholar]
  5. Cuatrecasas P. Gangliosides and membrane receptors for cholera toxin. Biochemistry. 1973 Aug 28;12(18):3558–3566. doi: 10.1021/bi00742a032. [DOI] [PubMed] [Google Scholar]
  6. Eidels L., Proia R. L., Hart D. A. Membrane receptors for bacterial toxins. Microbiol Rev. 1983 Dec;47(4):596–620. doi: 10.1128/mr.47.4.596-620.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Escuyer V., Duflot E., Sezer O., Danchin A., Mock M. Structural homology between virulence-associated bacterial adenylate cyclases. Gene. 1988 Nov 30;71(2):293–298. doi: 10.1016/0378-1119(88)90045-5. [DOI] [PubMed] [Google Scholar]
  8. Fish D. C., Klein F., Lincoln R. E., Walker J. S., Dobbs J. P. Pathophysiological changes in the rat associated with anthrax toxin. J Infect Dis. 1968 Feb;118(1):114–124. doi: 10.1093/infdis/118.1.114. [DOI] [PubMed] [Google Scholar]
  9. FitzGerald D., Morris R. E., Saelinger C. B. Receptor-mediated internalization of Pseudomonas toxin by mouse fibroblasts. Cell. 1980 Oct;21(3):867–873. doi: 10.1016/0092-8674(80)90450-x. [DOI] [PubMed] [Google Scholar]
  10. Friedlander A. M. Macrophages are sensitive to anthrax lethal toxin through an acid-dependent process. J Biol Chem. 1986 Jun 5;261(16):7123–7126. [PubMed] [Google Scholar]
  11. Gordon V. M., Leppla S. H., Hewlett E. L. Inhibitors of receptor-mediated endocytosis block the entry of Bacillus anthracis adenylate cyclase toxin but not that of Bordetella pertussis adenylate cyclase toxin. Infect Immun. 1988 May;56(5):1066–1069. doi: 10.1128/iai.56.5.1066-1069.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hranitzky K. W., Durham D. L., Hart D. A., Eidels L. Role of glycosylation in expression of functional diphtheria toxin receptors. Infect Immun. 1985 Aug;49(2):336–343. doi: 10.1128/iai.49.2.336-343.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Keen J. H., Maxfield F. R., Hardegree M. C., Habig W. H. Receptor-mediated endocytosis of diphtheria toxin by cells in culture. Proc Natl Acad Sci U S A. 1982 May;79(9):2912–2916. doi: 10.1073/pnas.79.9.2912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Keusch G. T., Jacewicz M. Pathogenesis of Shigella diarrhea. VII. Evidence for a cell membrane toxin receptor involving beta1 leads to 4-linked N-acetyl-D-glucosamine oligomers. J Exp Med. 1977 Aug 1;146(2):535–546. doi: 10.1084/jem.146.2.535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Koehler T. M., Collier R. J. Anthrax toxin protective antigen: low-pH-induced hydrophobicity and channel formation in liposomes. Mol Microbiol. 1991 Jun;5(6):1501–1506. doi: 10.1111/j.1365-2958.1991.tb00796.x. [DOI] [PubMed] [Google Scholar]
  16. Leppla S. H. Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells. Proc Natl Acad Sci U S A. 1982 May;79(10):3162–3166. doi: 10.1073/pnas.79.10.3162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Leppla S. H. Bacillus anthracis calmodulin-dependent adenylate cyclase: chemical and enzymatic properties and interactions with eucaryotic cells. Adv Cyclic Nucleotide Protein Phosphorylation Res. 1984;17:189–198. [PubMed] [Google Scholar]
  18. Leppla S. H. Production and purification of anthrax toxin. Methods Enzymol. 1988;165:103–116. doi: 10.1016/s0076-6879(88)65019-1. [DOI] [PubMed] [Google Scholar]
  19. Manhart M. D., Morris R. E., Bonventre P. F., Leppla S., Saelinger C. B. Evidence for pseudomonas exotoxin A receptors on plasma membrane of toxin-sensitive lm fibroblasts. Infect Immun. 1984 Sep;45(3):596–603. doi: 10.1128/iai.45.3.596-603.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mekada E., Okada Y., Uchida T. Identification of diphtheria toxin receptor and a nonproteinous diphtheria toxin-binding molecule in Vero cell membrane. J Cell Biol. 1988 Aug;107(2):511–519. doi: 10.1083/jcb.107.2.511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Middlebrook J. L., Dorland R. B. Bacterial toxins: cellular mechanisms of action. Microbiol Rev. 1984 Sep;48(3):199–221. doi: 10.1128/mr.48.3.199-221.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mock M., Labruyère E., Glaser P., Danchin A., Ullmann A. Cloning and expression of the calmodulin-sensitive Bacillus anthracis adenylate cyclase in Escherichia coli. Gene. 1988 Apr 29;64(2):277–284. doi: 10.1016/0378-1119(88)90342-3. [DOI] [PubMed] [Google Scholar]
  23. Novick D., Orchansky P., Revel M., Rubinstein M. The human interferon-gamma receptor. Purification, characterization, and preparation of antibodies. J Biol Chem. 1987 Jun 25;262(18):8483–8487. [PubMed] [Google Scholar]
  24. Pilch P. F., Czech M. P. Interaction of cross-linking agents with the insulin effector system of isolated fat cells. Covalent linkage of 125I-insulin to a plasma membrane receptor protein of 140,000 daltons. J Biol Chem. 1979 May 10;254(9):3375–3381. [PubMed] [Google Scholar]
  25. Proia R. L., Eidels L., Hart D. A. Diphtheria toxin-binding glycoproteins on hamster cells: candidates for diphtheria toxin receptors. Infect Immun. 1979 Sep;25(3):786–791. doi: 10.1128/iai.25.3.786-791.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Robertson D. L., Leppla S. H. Molecular cloning and expression in Escherichia coli of the lethal factor gene of Bacillus anthracis. Gene. 1986;44(1):71–78. doi: 10.1016/0378-1119(86)90044-2. [DOI] [PubMed] [Google Scholar]
  27. Robertson D. L., Tippetts M. T., Leppla S. H. Nucleotide sequence of the Bacillus anthracis edema factor gene (cya): a calmodulin-dependent adenylate cyclase. Gene. 1988 Dec 20;73(2):363–371. doi: 10.1016/0378-1119(88)90501-x. [DOI] [PubMed] [Google Scholar]
  28. STANLEY J. L., SMITH H. Purification of factor I and recognition of a third factor of the anthrax toxin. J Gen Microbiol. 1961 Sep;26:49–63. doi: 10.1099/00221287-26-1-49. [DOI] [PubMed] [Google Scholar]
  29. Singh Y., Chaudhary V. K., Leppla S. H. A deleted variant of Bacillus anthracis protective antigen is non-toxic and blocks anthrax toxin action in vivo. J Biol Chem. 1989 Nov 15;264(32):19103–19107. [PubMed] [Google Scholar]
  30. Singh Y., Leppla S. H., Bhatnagar R., Friedlander A. M. Internalization and processing of Bacillus anthracis lethal toxin by toxin-sensitive and -resistant cells. J Biol Chem. 1989 Jul 5;264(19):11099–11102. [PubMed] [Google Scholar]
  31. Stenmark H., Olsnes S., Sandvig K. Requirement of specific receptors for efficient translocation of diphtheria toxin A fragment across the plasma membrane. J Biol Chem. 1988 Sep 15;263(26):13449–13455. [PubMed] [Google Scholar]
  32. Takatsuki A., Tamura G. Effect of tunicamycin on the synthesis of macromolecules in cultures of chick embryo fibroblasts infected with Newcastle disease virus. J Antibiot (Tokyo) 1971 Nov;24(11):785–794. doi: 10.7164/antibiotics.24.785. [DOI] [PubMed] [Google Scholar]
  33. Thompson M. R., Forristal J., Kauffmann P., Madden T., Kozak K., Morris R. E., Saelinger C. B. Isolation and characterization of Pseudomonas aeruginosa exotoxin A binding glycoprotein from mouse LM cells. J Biol Chem. 1991 Feb 5;266(4):2390–2396. [PubMed] [Google Scholar]
  34. Vodkin M. H., Leppla S. H. Cloning of the protective antigen gene of Bacillus anthracis. Cell. 1983 Sep;34(2):693–697. doi: 10.1016/0092-8674(83)90402-6. [DOI] [PubMed] [Google Scholar]
  35. Welkos S. L., Lowe J. R., Eden-McCutchan F., Vodkin M., Leppla S. H., Schmidt J. J. Sequence and analysis of the DNA encoding protective antigen of Bacillus anthracis. Gene. 1988 Sep 30;69(2):287–300. doi: 10.1016/0378-1119(88)90439-8. [DOI] [PubMed] [Google Scholar]
  36. Wood C. L., O'Dorisio M. S. Covalent cross-linking of vasoactive intestinal polypeptide to its receptors on intact human lymphoblasts. J Biol Chem. 1985 Jan 25;260(2):1243–1247. [PubMed] [Google Scholar]

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