1887

Abstract

Human prion diseases, such as Creutzfeldt–Jakob disease (CJD), a lethal, neurodegenerative condition, occur in sporadic, genetic and transmitted forms. CJD is associated with the conversion of normal cellular prion protein (PrP) into a protease-resistant isoform (PrP). The mechanism of the conversion has not been studied in human cell cultures, due to the lack of a model system. In this study, such a system has been developed by culturing cell lines. Human glioblastoma cell line T98G had no coding-region mutations of the prion protein gene, which was of the 129 M/V genotype, and expressed endogenous PrP constitutively. T98G cells produced a form of proteinase K (PK)-resistant prion protein fragment following long-term culture and high passage number; its deglycosylated form was approximately 18 kDa. The PK-treated PrP was detected by immunoblotting with the mAb 6H4, which recognizes residues 144–152, and a polyclonal anti-C-terminal antibody, but not by the mAb 3F4, which recognizes residues 109–112, or the anti-N-terminal mAb HUC2-13. These results suggest that PrP was converted into a proteinase-resistant form of PrP in T98G cells.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.80043-0
2004-11-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/jgv/85/11/vir853449.html?itemId=/content/journal/jgv/10.1099/vir.0.80043-0&mimeType=html&fmt=ahah

References

  1. Archer F., Bachelin C., Andreoletti O. 8 other authors 2004; Cultured peripheral neuroglial cells are highly permissive to sheep prion infection. J Virol 78:482–490 [CrossRef]
    [Google Scholar]
  2. Béranger F., Mangé A., Goud B., Lehmann S. 2002; Stimulation of PrPC retrograde transport toward the endoplasmic reticulum increases accumulation of PrPSc in prion-infected cells. J Biol Chem 277:38972–38977 [CrossRef]
    [Google Scholar]
  3. Birkett C. R., Hennion R. M., Bembridge D. A., Clarke M. C., Chree A., Bruce M. E., Bostock C. J. 2001; Scrapie strains maintain biological phenotypes on propagation in a cell line in culture. EMBO J 20:3351–3358 [CrossRef]
    [Google Scholar]
  4. Brown D. R., Hafiz F., Glasssmith L. L., Wong B.-S., Jones I. M., Clive C., Haswell S. J. 2000; Consequences of manganese replacement of copper for prion protein function and proteinase resistance. EMBO J 19:1180–1186 [CrossRef]
    [Google Scholar]
  5. Brun A., Castilla J., Ramírez M. A. 8 other authors 2004; Proteinase K enhanced immunoreactivity of the prion protein-specific monoclonal antibody 2A11. Neurosci Res 48:75–83 [CrossRef]
    [Google Scholar]
  6. Buschmann A., Kuczius T., Bodemer W., Groschup M. H. 1998; Cellular prion proteins of mammalian species display an intrinsic partial proteinase K resistance. Biochem Biophys Res Commun 253:693–702 [CrossRef]
    [Google Scholar]
  7. Butler D. A., Scott M. R. D., Bockman J. M., Borchelt D. R., Taraboulos A., Hsiao K. K., Kingsbury D. T., Prusiner S. B. 1988; Scrapie-infected murine neuroblastoma cells produce protease-resistant prion proteins. J Virol 62:1558–1564
    [Google Scholar]
  8. Capellari S., Parchi P., Russo C. M., Sanford J., Sy M. S., Gambetti P., Petersen R. B. 2000; Effect of the E200K mutation on prion protein metabolism. Comparative study of a cell model and human brain. Am J Pathol 157:613–622 [CrossRef]
    [Google Scholar]
  9. Caughey B., Raymond G. J. 1993; Sulfated polyanion inhibition of scrapie-associated PrP accumulation in cultured cells. J Virol 67:643–650
    [Google Scholar]
  10. Caughey B., Raymond G. J., Ernst D., Race R. E. 1991; N-terminal truncation of the scrapie-associated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistant state. J Virol 65:6597–6603
    [Google Scholar]
  11. Caughey B., Horiuchi M., Demaimay R., Raymond G. J. 1999; Assays of protease-resistant prion protein and its formation. Methods Enzymol 309:122–133
    [Google Scholar]
  12. Caughey B., Raymond G. J., Callahan M. A., Wong C., Baron G. S., Xiong L.-W. 2001; Interactions and conversions of prion protein isoforms. Adv Protein Chem 57:139–169
    [Google Scholar]
  13. Chen S. G., Teplow D. B., Parchi P., Teller J. K., Gambetti P., Autilio-Gambetti L. 1995; Truncated forms of the human prion protein in normal brain and in prion diseases. J Biol Chem 270:19173–19180 [CrossRef]
    [Google Scholar]
  14. Clarke M. C., Haig D. A. 1970; Evidence for the multiplication of scrapie agent in cell culture. Nature 225:100–101 [CrossRef]
    [Google Scholar]
  15. Doh-Ura K., Iwaki T., Caughey B. 2000; Lysosomotropic agents and cysteine protease inhibitors inhibit scrapie-associated prion protein accumulation. J Virol 74:4894–4897 [CrossRef]
    [Google Scholar]
  16. Follet J., Lemaire-Vieille C., Blanquet-Grossard F. 8 other authors 2002; PrP expression and replication by Schwann cells: implications in prion spreading. J Virol 76:2434–2439 [CrossRef]
    [Google Scholar]
  17. Harris D. A. 2001; Biosynthesis and cellular processing of the prion protein. Adv Protein Chem 57:203–228
    [Google Scholar]
  18. Hill A. F., Antoniou M., Collinge J. 1999; Protease-resistant prion protein produced in vitro lacks detectable infectivity. J Gen Virol 80:11–14
    [Google Scholar]
  19. Kascsak R. J., Rubenstein R., Merz P. A., Tonna-DeMasi M., Fersko R., Carp R. I., Wisniewski H. M., Diringer H. 1987; Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins. J Virol 61:3688–3693
    [Google Scholar]
  20. Kikuchi Y., Irie M., Yoshimatsu K. 8 other authors 1991; A monoclonal antibody to scopolamine and its use for competitive enzyme-linked immunosorbent assay. Phytochemistry 30:3273–3276 [CrossRef]
    [Google Scholar]
  21. Kikuchi Y., Kakeya T., Yamazaki T. 7 other authors 2002; G1-dependent prion protein expression in human glioblastoma cell line T98G. Biol Pharm Bull 25:728–733 [CrossRef]
    [Google Scholar]
  22. Korth C., Stierli B., Streit P. 14 other authors 1997; Prion (PrPSc)-specific epitope defined by a monoclonal antibody. Nature 390:74–77 [CrossRef]
    [Google Scholar]
  23. Kovács G. G., Trabattoni G., Hainfellner J. A., Ironside J. W., Knight R. S. G., Budka H. 2002; Mutations of the prion protein gene phenotypic spectrum. J Neurol 249:1567–1582 [CrossRef]
    [Google Scholar]
  24. Ladogana A., Liu Q., Xi Y. G., Pocchiari M. 1995; Proteinase-resistant protein in human neuroblastoma cells infected with brain material from Creutzfeldt-Jakob patient. Lancet 345:594–595
    [Google Scholar]
  25. Lehmann S., Harris D. A. 1996; Mutant and infectious prion proteins display common biochemical properties in cultured cells. J Biol Chem 271:1633–1637 [CrossRef]
    [Google Scholar]
  26. Lehmann S., Harris D. A. 1997; Blockade of glycosylation promotes acquisition of scrapie-like properties by the prion protein in cultured cells. J Biol Chem 272:21479–21487 [CrossRef]
    [Google Scholar]
  27. Ma J., Lindquist S. 1999; De novo generation of a PrPSc-like conformation in living cells. Nat Cell Biol 1:358–361 [CrossRef]
    [Google Scholar]
  28. Ma J., Lindquist S. 2001; Wild-type PrP and a mutant associated with prion disease are subject to retrograde transport and proteasome degradation. Proc Natl Acad Sci U S A 98:14955–14960 [CrossRef]
    [Google Scholar]
  29. Ma J., Lindquist S. 2002; Conversion of PrP to a self-perpetuating PrPSc-like conformation in the cytosol. Science 298:1785–1788 [CrossRef]
    [Google Scholar]
  30. Mastrianni J. A., Nixon R., Layzer R., Telling G. C., Han D., DeArmond S. J., Prusiner S. B. 1999; Prion protein conformation in a patient with sporadic fatal insomnia. N Engl J Med 340:1630–1638 [CrossRef]
    [Google Scholar]
  31. Matsuda H., Mitsuda H., Nakamura N., Furusawa S., Mohri S., Kitamoto T. 1999; A chicken monoclonal antibody with specificity for the N-terminal of human prion protein. FEMS Immunol Med Microbiol 23:189–194 [CrossRef]
    [Google Scholar]
  32. Matsunaga Y., Peretz D., Williamson A., Burton D., Mehlhorn I., Groth D., Cohen F. E., Prusiner S. B., Baldwin M. A. 2001; Cryptic epitopes in N-terminally truncated prion protein are exposed in the full-length molecule: dependence of conformation on pH. Proteins 44:110–118 [CrossRef]
    [Google Scholar]
  33. Moser M., Colello R. J., Pott U., Oesch B. 1995; Developmental expression of the prion protein gene in glial cells. Neuron 14:509–517 [CrossRef]
    [Google Scholar]
  34. Naslavsky N., Stein R., Yanai A., Friedlander G., Taraboulos A. 1997; Characterization of detergent-insoluble complexes containing the cellular prion protein and its scrapie isoform. J Biol Chem 272:6324–6331 [CrossRef]
    [Google Scholar]
  35. Nishida N., Harris D. A., Vilette D., Laude H., Frobert Y., Grassi J., Casanova D., Milhavet O., Lehmann S. 2000; Successful transmission of three mouse-adapted scrapie strains to murine neuroblastoma cell lines overexpressing wild-type mouse prion protein. J Virol 74:320–325 [CrossRef]
    [Google Scholar]
  36. Paramithiotis E., Pinard M., Lawton T. 19 other authors 2003; A prion protein epitope selective for the pathologically misfolded conformation. Nat Med 9:893–899 [CrossRef]
    [Google Scholar]
  37. Parchi P., Capellari S., Chen S. G. 8 other authors 1997; Typing prion isoforms. Nature 386:232–234 [CrossRef]
    [Google Scholar]
  38. Parchi P., Giese A., Capellari S. 15 other authors 1999a; Classification of sporadic Creutzfeldt-Jakob disease based on molecular and phenotypic analysis of 300 subjects. Ann Neurol 46:224–233 [CrossRef]
    [Google Scholar]
  39. Parchi P., Capellari S., Chin S. 7 other authors 1999b; A subtype of sporadic prion disease mimicking fatal familial insomnia. Neurology 52:1757–1763 [CrossRef]
    [Google Scholar]
  40. Parchi P., Zou W., Wang W. 10 other authors 2000; Genetic influence on the structural variations of the abnormal prion protein. Proc Natl Acad Sci U S A 97:10168–10172 [CrossRef]
    [Google Scholar]
  41. Petersen R. B., Tabaton M., Chen S. G. 10 other authors 1995; Familial progressive subcortical gliosis: presence of prions and linkage to chromosome 17. Neurology 45:1062–1067 [CrossRef]
    [Google Scholar]
  42. Prusiner S. B. 2001; Prions. In Fields Virology . , 4th edn. pp  3063–3087 Edited by Knipe D. M., Howley P. M. Philadelphia, PA: Lippincott Williams & Wilkins;
  43. Race R. E., Fadness L. H., Chesebro B. 1987; Characterization of scrapie infection in mouse neuroblastoma cells. J Gen Virol 68:1391–1399 [CrossRef]
    [Google Scholar]
  44. Rubenstein R., Carp R. I., Callahan S. M. 1984; In vitro replication of scrapie agent in a neuronal model: infection of PC12 cells. J Gen Virol 65:2191–2198 [CrossRef]
    [Google Scholar]
  45. Satoh J., Kurohara K., Yukitake M., Kuroda Y. 1998; Constitutive and cytokine-inducible expression of prion protein gene in human neural cell lines. J Neuropathol Exp Neurol 57:131–139 [CrossRef]
    [Google Scholar]
  46. Schätzl H. M., Laszlo L., Holtzman D. M., Tatzelt J., DeArmond S. J., Weiner R. I., Mobley W. C., Prusiner S. B. 1997; A hypothalamic neuronal cell line persistently infected with scrapie prions exhibits apoptosis. J Virol 71:8821–8831
    [Google Scholar]
  47. Stahl N., Borchelt D. R., Hsiao K., Prusiner S. B. 1987; Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell 51:229–240 [CrossRef]
    [Google Scholar]
  48. Stein G. H. 1979; T98G: an anchorage-independent human tumor cell line that exhibits stationary phase G1 arrest in vitro. J Cell Physiol 99:43–54 [CrossRef]
    [Google Scholar]
  49. Takekida K., Kikuchi Y., Yamazaki T. 7 other authors 2002; Quantitative analysis of prion protein by immunoblotting. J Health Sci 48:288–291 [CrossRef]
    [Google Scholar]
  50. Taraboulos A., Serban D., Prusiner S. B. 1990; Scrapie prion proteins accumulate in the cytoplasm of persistently infected cultured cells. J Cell Biol 110:2117–2132 [CrossRef]
    [Google Scholar]
  51. van Keulen L. J., Schreuder B. E., Meloen R. H., Poelen-van den Berg M., Mooij-Harkes G., Vromans M. E., Langeveld J. P. 1995; Immunohistochemical detection and localization of prion protein in brain tissue of sheep with natural scrapie. Vet Pathol 32:299–308 [CrossRef]
    [Google Scholar]
  52. Vey M., Pilkuhn S., Wille H., Nixon R., DeArmond S. J., Smart E. J., Anderson R. G. W., Taraboulos A., Prusiner S. B. 1996; Subcellular colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains. Proc Natl Acad Sci U S A 93:14945–14949 [CrossRef]
    [Google Scholar]
  53. Yedidia Y., Horonchik L., Tzaban S., Yanai A., Taraboulos A. 2001; Proteasomes and ubiquitin are involved in the turnover of the wild-type prion protein. EMBO J 20:5383–5391 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.80043-0
Loading
/content/journal/jgv/10.1099/vir.0.80043-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error