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. 1992 Sep 2;118(6):1389–1399. doi: 10.1083/jcb.118.6.1389

The 27-kD diphtheria toxin receptor-associated protein (DRAP27) from vero cells is the monkey homologue of human CD9 antigen: expression of DRAP27 elevates the number of diphtheria toxin receptors on toxin- sensitive cells

PMCID: PMC2289621  PMID: 1522113

Abstract

Diphtheria toxin (DT) receptor associates with a 27-kD membrane protein (DRAP27) in monkey Vero cells. A cDNA encoding DRAP27 was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence revealed that DRAP27 is the monkey homologue of human CD9 antigen. DRAP27 is recognized by CD9 antibodies. A human-mouse hybrid cell line (3279-10) possessing human chromosome 5, sensitive to DT, but not expressing CD9 antigen, was used for transfection experiments with DRAP27. When the cloned cDNA encoding DRAP27 was transiently expressed in 3279-10 cells, the total DT binding capacity was three to four times higher than that of untransfected controls. Transfectants stably expressing DRAP27 have an increased number of DT binding sites on the cell surface. Furthermore, the transfectants are 3-25 times more sensitive to DT than untransfected cells, and the sensitivity of these cells to DT is correlated with the number of DRAP27 molecules on the surface. However, when the cloned cDNA was introduced into mouse cell lines that do not express DT receptors, neither an increased DT binding nor enhancement of DT sensitivity was observed. Hence, we conclude that DRAP27 itself does not bind DT, but serves to increase DT binding and consequently enhances DT sensitivity of cells that have DT receptors. 12 proteins related to DRAP27/CD9 antigen were found through homology search analysis. These proteins appear to belong to a new family of transmembrane proteins.

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Selected References

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  1. Angelisová P., Vlcek C., Stefanová I., Lipoldová M., Horejsí V. The human leucocyte surface antigen CD53 is a protein structurally similar to the CD37 and MRC OX-44 antigens. Immunogenetics. 1990;32(4):281–285. doi: 10.1007/BF00187099. [DOI] [PubMed] [Google Scholar]
  2. Aruffo A., Seed B. Molecular cloning of a CD28 cDNA by a high-efficiency COS cell expression system. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8573–8577. doi: 10.1073/pnas.84.23.8573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Athwal R. S., Searle B. M., Jansons V. K. Diphtheria toxin sensitivity in a monochromosomal hybrid containing human chromosome 5. J Hered. 1985 Sep-Oct;76(5):329–334. [PubMed] [Google Scholar]
  4. Atkinson B., Ernst C. S., Ghrist B. F., Herlyn M., Blaszczyk M., Ross A. H., Herlyn D., Steplewski Z., Koprowski H. Identification of melanoma-associated antigens using fixed tissue screening of antibodies. Cancer Res. 1984 Jun;44(6):2577–2581. [PubMed] [Google Scholar]
  5. Atkinson B., Ernst C. S., Ghrist B. F., Ross A. H., Clark W. H., Herlyn M., Herlyn D., Maul G., Steplewski Z., Koprowski H. Monoclonal antibody to a highly glycosylated protein reacts in fixed tissue with melanoma and other tumors. Hybridoma. 1985 Fall;4(3):243–255. doi: 10.1089/hyb.1985.4.243. [DOI] [PubMed] [Google Scholar]
  6. Begy C., Bridges C. D. Nucleotide and predicted protein sequence of rat retinal degeneration slow (rds). Nucleic Acids Res. 1990 May 25;18(10):3058–3058. doi: 10.1093/nar/18.10.3058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bellacosa A., Lazo P. A., Bear S. E., Tsichlis P. N. The rat leukocyte antigen MRC OX-44 is a member of a new family of cell surface proteins which appear to be involved in growth regulation. Mol Cell Biol. 1991 May;11(5):2864–2872. doi: 10.1128/mcb.11.5.2864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Benoit P., Gross M. S., Frachet P., Frézal J., Uzan G., Boucheix C., Nguyen V. C. Assignment of the human CD9 gene to chromosome 12 (region P13) by use of human specific DNA probes. Hum Genet. 1991 Jan;86(3):268–272. doi: 10.1007/BF00202407. [DOI] [PubMed] [Google Scholar]
  9. Blewitt M. G., Chung L. A., London E. Effect of pH on the conformation of diphtheria toxin and its implications for membrane penetration. Biochemistry. 1985 Sep 24;24(20):5458–5464. doi: 10.1021/bi00341a027. [DOI] [PubMed] [Google Scholar]
  10. Boquet P., Silverman M. S., Pappenheimer A. M., Jr, Vernon W. B. Binding of triton X-100 to diphtheria toxin, crossreacting material 45, and their fragments. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4449–4453. doi: 10.1073/pnas.73.12.4449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Boucheix C., Benoit P. CD9 antigen: will platelet physiology help to explain the function of a surface molecule during hemopoietic differentiation? Nouv Rev Fr Hematol. 1988;30(4):201–202. [PubMed] [Google Scholar]
  12. Boucheix C., Soria C., Mirshahi M., Soria J., Perrot J. Y., Fournier N., Billard M., Rosenfeld C. Characteristics of platelet aggregation induced by the monoclonal antibody ALB6 (acute lymphoblastic leukemia antigen p 24). Inhibition of aggregation by ALB6Fab. FEBS Lett. 1983 Sep 19;161(2):289–295. doi: 10.1016/0014-5793(83)81027-8. [DOI] [PubMed] [Google Scholar]
  13. Cabiaux V., Brasseur R., Wattiez R., Falmagne P., Ruysschaert J. M., Goormaghtigh E. Secondary structure of diphtheria toxin and its fragments interacting with acidic liposomes studied by polarized infrared spectroscopy. J Biol Chem. 1989 Mar 25;264(9):4928–4938. [PubMed] [Google Scholar]
  14. 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]
  15. Classon B. J., Williams A. F., Willis A. C., Seed B., Stamenkovic I. The primary structure of the human leukocyte antigen CD37, a species homologue of the rat MRC OX-44 antigen. J Exp Med. 1989 Apr 1;169(4):1497–1502. doi: 10.1084/jem.169.4.1497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Classon B. J., Williams A. F., Willis A. C., Seed B., Stamenkovic I. The primary structure of the human leukocyte antigen CD37, a species homologue of the rat MRC OX-44 antigen. J Exp Med. 1990 Sep 1;172(3):1007–1007. doi: 10.1084/jem.172.3.1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Collier R. J. Diphtheria toxin: mode of action and structure. Bacteriol Rev. 1975 Mar;39(1):54–85. doi: 10.1128/br.39.1.54-85.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Connell G. J., Molday R. S. Molecular cloning, primary structure, and orientation of the vertebrate photoreceptor cell protein peripherin in the rod outer segment disk membrane. Biochemistry. 1990 May 15;29(19):4691–4698. doi: 10.1021/bi00471a025. [DOI] [PubMed] [Google Scholar]
  19. Creagan R. P., Chen S., Ruddle F. H. Genetic analysis of the cell surface: association of human chromosome 5 with sensitivity to diphtheria toxin in mouse-human somatic cell hybrids. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2237–2241. doi: 10.1073/pnas.72.6.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Davern K. M., Wright M. D., Herrmann V. R., Mitchell G. F. Further characterisation of the Schistosoma japonicum protein Sj23, a target antigen of an immunodiagnostic monoclonal antibody. Mol Biochem Parasitol. 1991 Sep;48(1):67–75. doi: 10.1016/0166-6851(91)90165-3. [DOI] [PubMed] [Google Scholar]
  21. Gorman D. J., Castaldi P. A., Zola H., Berndt M. C. Preliminary functional characterization of a 24,000 dalton platelet surface protein involved in platelet activation. Nouv Rev Fr Hematol. 1985;27(4):255–259. [PubMed] [Google Scholar]
  22. Hato T., Sumida M., Yasukawa M., Watanabe A., Okuda H., Kobayashi Y. Induction of platelet Ca2+ influx and mobilization by a monoclonal antibody to CD9 antigen. Blood. 1990 Mar 1;75(5):1087–1091. [PubMed] [Google Scholar]
  23. Hayes H., Kaneda Y., Uchida T., Okada Y. Regional assignment of the gene for diphtheria toxin sensitivity using subchromosomal fragments in microcell hybrids. Chromosoma. 1987;96(1):26–32. doi: 10.1007/BF00285879. [DOI] [PubMed] [Google Scholar]
  24. Hein J. Unified approach to alignment and phylogenies. Methods Enzymol. 1990;183:626–645. doi: 10.1016/0076-6879(90)83041-7. [DOI] [PubMed] [Google Scholar]
  25. Higashihara M., Maeda H., Shibata Y., Kume S., Ohashi T. A monoclonal anti-human platelet antibody: a new platelet aggregating substance. Blood. 1985 Feb;65(2):382–391. [PubMed] [Google Scholar]
  26. Hotta H., Ross A. H., Huebner K., Isobe M., Wendeborn S., Chao M. V., Ricciardi R. P., Tsujimoto Y., Croce C. M., Koprowski H. Molecular cloning and characterization of an antigen associated with early stages of melanoma tumor progression. Cancer Res. 1988 Jun 1;48(11):2955–2962. [PubMed] [Google Scholar]
  27. Iwamoto R., Senoh H., Okada Y., Uchida T., Mekada E. An antibody that inhibits the binding of diphtheria toxin to cells revealed the association of a 27-kDa membrane protein with the diphtheria toxin receptor. J Biol Chem. 1991 Oct 25;266(30):20463–20469. [PubMed] [Google Scholar]
  28. Jennings L. K., Fox C. F., Kouns W. C., McKay C. P., Ballou L. R., Schultz H. E. The activation of human platelets mediated by anti-human platelet p24/CD9 monoclonal antibodies. J Biol Chem. 1990 Mar 5;265(7):3815–3822. [PubMed] [Google Scholar]
  29. Kersey J. H., LeBien T. W., Abramson C. S., Newman R., Sutherland R., Greaves M. P-24: a human leukemia-associated and lymphohemopoietic progenitor cell surface structure identified with monoclonal antibody. J Exp Med. 1981 Mar 1;153(3):726–731. doi: 10.1084/jem.153.3.726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kohno K., Uchida T., Mekada E., Okada Y. Characterization of diphtheria-toxin-resistant mutants lacking receptor function or containing nonribosylatable elongation factor 2. Somat Cell Mol Genet. 1985 Sep;11(5):421–431. doi: 10.1007/BF01534836. [DOI] [PubMed] [Google Scholar]
  31. Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  33. Lanza F., Wolf D., Fox C. F., Kieffer N., Seyer J. M., Fried V. A., Coughlin S. R., Phillips D. R., Jennings L. K. cDNA cloning and expression of platelet p24/CD9. Evidence for a new family of multiple membrane-spanning proteins. J Biol Chem. 1991 Jun 5;266(16):10638–10645. [PubMed] [Google Scholar]
  34. Mekada E., Kohno K., Ishiura M., Uchida T., Okada Y. Methylamine facilitates demonstration of specific uptake of diphtheria toxin by CHO cell and toxin-resistant CHO cell mutants. Biochem Biophys Res Commun. 1982 Dec 15;109(3):792–799. doi: 10.1016/0006-291x(82)92009-5. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Mekada E., Senoh H., Iwamoto R., Okada Y., Uchida T. Purification of diphtheria toxin receptor from Vero cells. J Biol Chem. 1991 Oct 25;266(30):20457–20462. [PubMed] [Google Scholar]
  37. Mekada E., Uchida T. Binding properties of diphtheria toxin to cells are altered by mutation in the fragment A domain. J Biol Chem. 1985 Oct 5;260(22):12148–12153. [PubMed] [Google Scholar]
  38. Metzelaar M. J., Wijngaard P. L., Peters P. J., Sixma J. J., Nieuwenhuis H. K., Clevers H. C. CD63 antigen. A novel lysosomal membrane glycoprotein, cloned by a screening procedure for intracellular antigens in eukaryotic cells. J Biol Chem. 1991 Feb 15;266(5):3239–3245. [PubMed] [Google Scholar]
  39. Middlebrook J. L., Dorland R. B., Leppla S. H. Association of diphtheria toxin with Vero cells. Demonstration of a receptor. J Biol Chem. 1978 Oct 25;253(20):7325–7330. [PubMed] [Google Scholar]
  40. Middlebrook J. L., Dorland R. B. Response of cultured mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae: differential cytotoxicity. Can J Microbiol. 1977 Feb;23(2):183–189. doi: 10.1139/m77-026. [DOI] [PubMed] [Google Scholar]
  41. Morris R. E., Gerstein A. S., Bonventre P. F., Saelinger C. B. Receptor-mediated entry of diphtheria toxin into monkey kidney (Vero) cells: electron microscopic evaluation. Infect Immun. 1985 Dec;50(3):721–727. doi: 10.1128/iai.50.3.721-727.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Moskaug J. O., Sletten K., Sandvig K., Olsnes S. Translocation of diphtheria toxin A-fragment to the cytosol. Role of the site of interfragment cleavage. J Biol Chem. 1989 Sep 15;264(26):15709–15713. [PubMed] [Google Scholar]
  43. Moskaug J. O., Stenmark H., Olsnes S. Insertion of diphtheria toxin B-fragment into the plasma membrane at low pH. Characterization and topology of inserted regions. J Biol Chem. 1991 Feb 5;266(4):2652–2659. [PubMed] [Google Scholar]
  44. Moya M., Dautry-Varsat A., Goud B., Louvard D., Boquet P. Inhibition of coated pit formation in Hep2 cells blocks the cytotoxicity of diphtheria toxin but not that of ricin toxin. J Cell Biol. 1985 Aug;101(2):548–559. doi: 10.1083/jcb.101.2.548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Naglich J. G., Eidels L. Isolation of diphtheria toxin-sensitive mouse cells from a toxin-resistant population transfected with monkey DNA. Proc Natl Acad Sci U S A. 1990 Sep;87(18):7250–7254. doi: 10.1073/pnas.87.18.7250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Oren R., Takahashi S., Doss C., Levy R., Levy S. TAPA-1, the target of an antiproliferative antibody, defines a new family of transmembrane proteins. Mol Cell Biol. 1990 Aug;10(8):4007–4015. doi: 10.1128/mcb.10.8.4007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Pappenheimer A. M., Jr Diphtheria toxin. Annu Rev Biochem. 1977;46:69–94. doi: 10.1146/annurev.bi.46.070177.000441. [DOI] [PubMed] [Google Scholar]
  48. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Rendu F., Boucheix C., Lebret M., Bourdeau N., Benoit P., Maclouf J., Soria C., Levy-Toledano S. Mechanisms of the mAb ALB6(CD9) induced human platelet activation: comparison with thrombin. Biochem Biophys Res Commun. 1987 Aug 14;146(3):1397–1404. doi: 10.1016/0006-291x(87)90805-9. [DOI] [PubMed] [Google Scholar]
  50. Sandvig K., Olsnes S. Diphtheria toxin entry into cells is facilitated by low pH. J Cell Biol. 1980 Dec;87(3 Pt 1):828–832. doi: 10.1083/jcb.87.3.828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sandvig K., Olsnes S. Interactions between diphtheria toxin entry and anion transport in Vero cells. IV. Evidence that entry of diphtheria toxin is dependent on efficient anion transport. J Biol Chem. 1986 Feb 5;261(4):1570–1575. [PubMed] [Google Scholar]
  52. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Schwartz-Albiez R., Dörken B., Hofmann W., Moldenhauer G. The B cell-associated CD37 antigen (gp40-52). Structure and subcellular expression of an extensively glycosylated glycoprotein. J Immunol. 1988 Feb 1;140(3):905–914. [PubMed] [Google Scholar]
  54. Seed B. An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2. 1987 Oct 29-Nov 4Nature. 329(6142):840–842. doi: 10.1038/329840a0. [DOI] [PubMed] [Google Scholar]
  55. Slupsky J. R., Seehafer J. G., Tang S. C., Masellis-Smith A., Shaw A. R. Evidence that monoclonal antibodies against CD9 antigen induce specific association between CD9 and the platelet glycoprotein IIb-IIIa complex. J Biol Chem. 1989 Jul 25;264(21):12289–12293. [PubMed] [Google Scholar]
  56. Takahashi S., Doss C., Levy S., Levy R. TAPA-1, the target of an antiproliferative antibody, is associated on the cell surface with the Leu-13 antigen. J Immunol. 1990 Oct 1;145(7):2207–2213. [PubMed] [Google Scholar]
  57. Tedder T. F., Klejman G., Schlossman S. F., Saito H. Structure of the gene encoding the human B lymphocyte differentiation antigen CD20 (B1). J Immunol. 1989 Apr 1;142(7):2560–2568. [PubMed] [Google Scholar]
  58. Travis G. H., Brennan M. B., Danielson P. E., Kozak C. A., Sutcliffe J. G. Identification of a photoreceptor-specific mRNA encoded by the gene responsible for retinal degeneration slow (rds). Nature. 1989 Mar 2;338(6210):70–73. doi: 10.1038/338070a0. [DOI] [PubMed] [Google Scholar]
  59. Travis G. H., Christerson L., Danielson P. E., Klisak I., Sparkes R. S., Hahn L. B., Dryja T. P., Sutcliffe J. G. The human retinal degeneration slow (RDS) gene: chromosome assignment and structure of the mRNA. Genomics. 1991 Jul;10(3):733–739. doi: 10.1016/0888-7543(91)90457-p. [DOI] [PubMed] [Google Scholar]
  60. Travis G. H., Sutcliffe J. G., Bok D. The retinal degeneration slow (rds) gene product is a photoreceptor disc membrane-associated glycoprotein. Neuron. 1991 Jan;6(1):61–70. doi: 10.1016/0896-6273(91)90122-g. [DOI] [PubMed] [Google Scholar]
  61. Uchida T., Yamaizumi M., Okada Y. Reassembled HVJ (Sendai virus) envelopes containing non-toxic mutant proteins of diphtheria toxin show toxicity to mouse L cell. Nature. 1977 Apr 28;266(5605):839–840. doi: 10.1038/266839a0. [DOI] [PubMed] [Google Scholar]
  62. Worthington R. E., Carroll R. C., Boucheix C. Platelet activation by CD9 monoclonal antibodies is mediated by the Fc gamma II receptor. Br J Haematol. 1990 Feb;74(2):216–222. doi: 10.1111/j.1365-2141.1990.tb02568.x. [DOI] [PubMed] [Google Scholar]
  63. Wright M. D., Henkle K. J., Mitchell G. F. An immunogenic Mr 23,000 integral membrane protein of Schistosoma mansoni worms that closely resembles a human tumor-associated antigen. J Immunol. 1990 Apr 15;144(8):3195–3200. [PubMed] [Google Scholar]

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