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Molecular Biotechnology

, Volume 13, Issue 1, pp 45–55 | Cite as

Methods for studying prion protein (PrP) metabolism and the formation of protease-resistant PrP in cell culture and cell-free systems

An update
  • Byron Caughey
  • Gregory J. Raymond
  • Suzette A. Priola
  • David A. Kocisko
  • Richard E. Race
  • Richard A. Bessen
  • Peter T. LansburyJr.
  • Bruce Chesebro
Protocol

Abstract

Transmissible spongiform encephalopathies (TSE) or prion diseases result in aberrant metabolism of prion protein (PrP) and the accumulation of a protease-resistant, insoluble, and possibly infectious form of PrP, PrP-res. Studies of PrP biosynthesis, intracellular trafficking, and degradation has been studied in a variety of tissue culture cells. Pulse-chase metabolic labeling studies in scrapie-infected cells indicated that PrP-res is made posttranslationally from an apparently normal protease sensitive precursor, PrP-sen, after the latter reaches the cell surface. Cell-free reactions have provided evidence that PrP-res itself can induce the conversion of PrP-sen to PrP-res in a highly species- and strain-specific manner. These studies have shed light on the mechanism of PrP-res formation and suggest molecular bases for TSE species barrier effects and agent strain propagation.

Index Entries

Prion protein PrP scrapie transmissible spongiform encephalopathy 

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References

  1. 1.
    Caughey, B. (1991) Cellular metabolism of normal and scrapie-associated forms of PrP. Sem. Virol. 2, 189–196.Google Scholar
  2. 2.
    Caughey, B., Race, R. E., Vogel, M., Buchmeier, M. J., and Chesebro, B. (1988) In vitro expression in eukaryotic cells of the prion protein gone cloned from scrapie-infected mouse brain. Proc. Natl. Acad. Sci. USA 85, 4657–4661.PubMedCrossRefGoogle Scholar
  3. 3.
    Caughey, B. (1993) Scrapie associated PrP accumulation and its prevention: insights from cell culture. Br. Med. Bull. 49, 860–872.PubMedGoogle Scholar
  4. 4.
    Taraboulos, A., Borchelt, D. R., McKinley, M. P., Raeber, A., Serban, D., DeArmond, S. J., et al. (1992) Dissecting the pathway of scrapie prion synthesis in cultured cells, in Prion Diseases of Humans and Animals (Prusiner, S. B., Collinge, J., Powell, J., and Anderton, B., eds.), Ellis Horwood, Chichester, pp. 435–444.Google Scholar
  5. 5.
    Meiner, Z., Halimi, M., Polakiewicz, R. D., Prusiner, S. B., and Gabizon, R. (1992) Presence of prion protein in peripheral tissues of Libyan Jews with Creutzfeldt-Jakob disease. Neurology 42, 1355–1360.PubMedGoogle Scholar
  6. 6.
    Cashman, N. R., Loertscher, R., Nalbantoglu, J., Shaw, I., Kascsak, R. J., Bolton, D. C., et al. (1990) Cellular isoform of the scrapie agent protein participates in lymphocyte activation. Cell 61, 185–192.PubMedCrossRefGoogle Scholar
  7. 7.
    Race, R. E., Fadness, L. H., and Chesebro, B. (1987) Characterization of scrapie infection in mouse neuroblastoma cells. J. Gen. Virol. 68, 1391–1399.PubMedGoogle Scholar
  8. 8.
    Race, R. E., Caughey, B., Graham, K., Ernst, D., and Chesebro, B. (1988) Analyses of frequency of infection, specific infectivity, and prion protein biosynthesis in scrapie-infected neuroblastoma cell clones. J. Virol. 62, 2845–2849.PubMedGoogle Scholar
  9. 9.
    Butler, D. A., Scott, M. R. D., Bockman, J. M., Borchelt, D. R., Taraboulos, A., Hsiao, K. K., et al. (1988) Scrapie-infected murine neuroblastoma cells produce protease-resistant prion proteins. J. Virol. 62, 1558–1564.PubMedGoogle Scholar
  10. 10.
    Caughey, B., and Raymond, G. J. (1993) Sulfated polyanion inhibition of scrapie-associated PrP accumulation in cultured cells. J. Virol. 67, 643–650.PubMedGoogle Scholar
  11. 11.
    Borchelt, D. R., Scott, M., Taraboulos, A., Stahl, N., and Prusiner, S. B. (1990) Scrapie and cellular prion proteins differ in the kinetics of synthesis and topology in cultured cells. J. Cell Biol. 110, 743–752.PubMedCrossRefGoogle Scholar
  12. 12.
    Caughey, B., Raymond, G. J., Ernst, D., and 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.PubMedGoogle Scholar
  13. 13.
    Caughey, B., Race, R. E., Ernst, D., Buchmeier, M. J., and Chesebro, B. (1989) Prion protein (PrP) biosynthesis in scrapie-infected and uninfected neuroblastoma cells. J. Virol. 63, 175–181.PubMedGoogle Scholar
  14. 14.
    Caughey, B. and Raymond, G. J. (1991) The scrapie-associated form of PrP is made from a cell surface precursor that is both protease- and phospholipase-sensitive. J. Biol. Chem. 266, 18,217–18,223.Google Scholar
  15. 15.
    Caughey, B. and Race, R. E. (1992) Potent inhibition of scrapie-associated PrP accumulation by Congo red. J. Neurochem. 59, 768–771.PubMedCrossRefGoogle Scholar
  16. 16.
    Stahl, N., Borchelt, D. R., Hsiao, K., and Prusiner, S. B. (1987) Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell 51, 229–240.PubMedCrossRefGoogle Scholar
  17. 17.
    Caughey, B., Neary, K., Buller, R., Ernst, D., Perry, L., Chesebro, B., et al. (1990) Normal and scrapie-associated forms of prion protein differ in their sensitivities to phospholipase and proteases in intact neuroblastoma cells. J. Virol. 64, 1093–1101.PubMedGoogle Scholar
  18. 18.
    Stahl, N., Borchelt, D. R., and Prusiner, S. B. (1990) Differential release of cellular and scrapie prion proteins from cellular membranes by phosphatidylinositol-specific phospholipase C. Biochemistry 29, 5405–5412.PubMedCrossRefGoogle Scholar
  19. 19.
    Safar, J., Ceroni, M., Gajdusek, D. C., and Gibbs, C. J., Jr. (1991) Differences in the membrane interaction of scrapie amyloid precursor proteins in normal and scrapie- or Creutzfeldt-Jakob disease-infected brains. J. Infect. Dis. 163, 488–494.PubMedGoogle Scholar
  20. 20.
    Bolton, D. C., Meyer, R. K., and Prusiner, S. B. (1985) Scrapie PrP 27–30 is a sialoglycoprotein. J. Virol. 53, 596–606.PubMedGoogle Scholar
  21. 21.
    Manuelidis, L., Valley, S., and Manuelidis, E. E. (1985) Specific proteins associated with Creutzfeldt-Jakob disease and scrapie share antigenic and carbohydrate determinants. Proc. Natl. Acad. Sci. USA 82, 4263–4267.PubMedCrossRefGoogle Scholar
  22. 22.
    Oesch, B., Westaway, D., Wälchli, M., McKinley, M. P., Kent, S. B. H., Aebersold, R., et al. (1985) A cellular gene encodes scrapie PrP 27–30 protein. Cell 40, 735–746.PubMedCrossRefGoogle Scholar
  23. 23.
    Locht, C., Chesebro, B., Race, R., and Keith, J. M. (1986) Molecular cloning and complete sequence of prion protein cDNA from mouse brain infected with the scrapie agent. Proc. Natl. Acad. Sci. USA 83, 6372–6376.PubMedCrossRefGoogle Scholar
  24. 24.
    Endo, T., Groth, D., Prusiner, S. B., and Kobata, A. (1989) Diversity of oligosaccharide structures linked to asparagines of the scrapie prion protein. Biochemistry 28, 8380–8388.PubMedCrossRefGoogle Scholar
  25. 25.
    Taraboulos, A., Rogers, M., Borchelt, D. R., McKinley, M. P., Scott, M., Serban, D., et al. (1990) Acquisition of protease resistance by prion proteins in scrapie-infected cells does not require asparagine-linked glycosylation. Proc. Natl. Acad. Sci. USA 87, 8262–8266.PubMedCrossRefGoogle Scholar
  26. 26.
    Thotakura, N. R. and Bahl, O. P. (1987) Enzymatic deglycosylation of glycoproteins. Methods Enzymol. 138, 350–359.PubMedGoogle Scholar
  27. 27.
    Somerville, R. A. and Ritchie, L. A. (1990) Differential glycosylation of the protein (PrP) forming scrapie-associated fibrils. J. Gen. Virol. 71, 833–839.PubMedGoogle Scholar
  28. 28.
    Elbein, A. D. (1987) Inhibitors of the biosynthesis and processing of N-linked oligo saccharide chains. Annu. Rev. Biochem. 56, 497–534.PubMedCrossRefGoogle Scholar
  29. 29.
    Shyng, S.-L., Huber, M. T., and Harris, D. A. (1993) A prion protein cycles between the cell surface and an endocytic compartment in cultured neuroblastoma cells. J. Biol. Chem. 268, 15,922–15,928.Google Scholar
  30. 30.
    Jeffrey, M., Goodsir, C. M., Bruce, M. E., McBride, P. A., Scott, J. R., and Halliday, W. G. (1992) Infection specific prion protein (PrP) accumulates on neuronal plasmalemma in scrapie infected mice. Neurosci. Lett. 147, 106–109.PubMedCrossRefGoogle Scholar
  31. 31.
    Shyng, S.-L., Heuser, J. E., and Harris, D. A. (1994) A glycolipid-anchored prion protein is endocytosed via clathrin coated pits. J. Cell Biol. 125, 1239–1250.PubMedCrossRefGoogle Scholar
  32. 32.
    Jeffrey, M., Goodsir, C. M., Bruce, M. E., McBride, P. A., Fowler, N., and Scott, J. R. (1994) Murine scrapie-infected neurons in vivo release excess prion protein into the extracellular space. Neurosci. Lett. 174, 39–42.PubMedCrossRefGoogle Scholar
  33. 33.
    Jeffrey, M., Goodsir, C. M., Bruce, M., McBride, P. A., Scott, J. R., and Halliday, W. G. (1994) Correlative light and electron microscopy studies of PrP localisation in 87V scrapie. Brain Res. 656, 329–343.PubMedCrossRefGoogle Scholar
  34. 34.
    Borchelt, D. R., Taraboulos, A., and Prusiner, S. B. (1992) Evidence for synthesis of scrapie prion protein in the endocytic pathway. J. Biol. Chem. 267, 16,188–16,199.Google Scholar
  35. 35.
    Taraboulos, A., Raeber, A. J., Borchelt, D. R., Serban, D., and Prusiner, S. B. (1992) Synthesis and trafficking of prion proteins in cultured cells. Mol. Biol. Cell 3, 851–863.PubMedGoogle Scholar
  36. 36.
    McKinley, M. P., Taraboulos, A., Kenaga, L., Serban, D., Stieber, A., DeArmond, S. J., et al. (1991) Ultrastructural localization of scrapie prion proteins in cytoplasmic vesicles of infected cultured cells. Lab. Invest. 65, 622–630.PubMedGoogle Scholar
  37. 37.
    Caughey, B., Ernst, D., and Race, R. E. (1993) Congo red inhibition of scrapie agent replication. J. Virol. 67, 6270–6272.PubMedGoogle Scholar
  38. 38.
    Caughey, B., Brown, K., Raymond, G. J., Katzenstein, G. E., and Thresher, W. (1994) Binding of the protease-sensitive form of PrP (prion protein) to sulfated glycosaminoglycan and Congo red. J. Virol. 68, 2135–2141.PubMedGoogle Scholar
  39. 39.
    Ehlers, B. and Diringer, H. (1984) Dextran sulphate 500 delays and prevents mouse scrapie by impairment of agent replication in spleen. J. Gen. Virol. 65, 1325–1330.PubMedGoogle Scholar
  40. 40.
    Farquhar, C. F. and Dickinson, A. G. (1986) Prolongation of scrapie incubation period by an injection of dextran sulphate 500 within the month before or after infection. J. Gen. Virol. 67, 463–473.PubMedGoogle Scholar
  41. 41.
    Kimberlin, R. H. and Walker, C. A. (1986) Suppression of scrapie infection in mice by heteropolyanion 23, dextran sulfate, and some other polyanions. Antimicrob. Agents Chemother. 30, 409–413.PubMedGoogle Scholar
  42. 42.
    Ladogana, A., Casaccia, P., Ingrosso, L., Cibati, M., Salvatore, M., Xi, Y. G., et al. (1992) Sulphate polyanions prolong the incubation period of scrapie-infected hamsters. J. Gen. Virol. 73, 661–665.PubMedCrossRefGoogle Scholar
  43. 43.
    Ingrosso, L., Ladogana, A., and Pocchiari, M. (1995) Congo red prolongs the incubation period in scrapie-infected hamsters. J. Virol. 69, 506–508.PubMedGoogle Scholar
  44. 44.
    Scott, M. R. D., Butler, D. A., Bredesen, D. E., Walchli, M., Hsiao, K. K., and Prusiner, S. B. (1988) Prion protein gene expression in cultured cells. Prot. Eng. 2, 69–76.CrossRefGoogle Scholar
  45. 45.
    Robertson, M. N., Spangrude, G. J., Hasenkrug, K., Perry, L., Nishio, J., Wehrly, K., et al. (1992) Role and specificity of T-cell subsets in spontaneous recovery from friend virus-induced leukemia in mice. J. Virol. 66, 3271–3277.PubMedGoogle Scholar
  46. 46.
    Chesebro, B., Wehrly, K., Caughey, B., Nishio, J., Ernst, D., and Race, R. (1993) Foreign PrP expression and scrapie infection in tissue culture cell lines. Dev. Biol. Stand. 80, 131–140.PubMedGoogle Scholar
  47. 47.
    Harris, D. A., Huber, M. T., van Dijken, P., Shyng, S.-L., Chait, B. T., and Wang, R. (1993) Processing of a cellular prion protein: identification of N- and C-terminal cleavage sites. Biochemistry 32, 1009–1016.PubMedCrossRefGoogle Scholar
  48. 48.
    Lynch, C. M. and Miller, A. D. (1991) Production of high-titer helper virus-free retroviral vectors by cocultivation of packaging cells with different host ranges. J. Virol. 65, 3887–3890.PubMedGoogle Scholar
  49. 49.
    Priola, S. A., Caughey, B., Race, R. E., and Chesebro, B. (1994) Heterologous PrP molecules interfere with accumulation of protease-resistant PrP in scrapie-infected murine neuroblastoma cells. J. Virol. 68, 4873–4878.PubMedGoogle Scholar
  50. 50.
    Priola, S. A., Caughey, B., Wehrly, K., and Chesebro, B. (1995) A 60-kDa prion protein (PrP) with properties of both the normal and scrapie-associated forms of PrP. J. Biol. Chem. 270, 3299–3305.PubMedCrossRefGoogle Scholar
  51. 51.
    Raeber, A. J., Borchelt, D. R., Scott, M., and Prusiner, S. B. (1992) Attempts to convert the cellular prion protein into the scrapie isoform in cell-free systems. J. Virol. 66, 6155–6163.PubMedGoogle Scholar
  52. 52.
    Kocisko, D. A., Come, J. H., Priola, S. A., Chesebro, B., Raymond, G. J., Lansbury, P. T., et al. (1994) Cell-free formation of protease-resistant prion protein. Nature 370, 471–474.PubMedCrossRefGoogle Scholar
  53. 53.
    Kocisko, D. A., Priola, S. A., Raymond, G. J., Chesebro, B., Lansbury, P. T., Jr., and Caughey, B. (1995) Species specificity in the cell-free conversion of prion protein to protease-resistant forms: a model for the scrapie species barrier. Proc. Natl. Acad. Sci. USA 92, 3923–3927.PubMedCrossRefGoogle Scholar
  54. 54.
    Raymond, G. J., Hope, J., Kocisko, D. A., Priola, S. A., Raymond, L. D., Bossers, A., Ironside, J., Will, R. G., Chen, S. G., Petersen, R. B., Gambetti, P., Rubenstein, R., Smits, M. A., Lansbury, P. T., Jr., Caughey, B. (1997) Molecular assessment of the transmissibilities of BSE and scrapie to humans. Nature 388, 285–288.PubMedCrossRefGoogle Scholar
  55. 55.
    Bossers, A., Belt, P. B. G. M., Raymond, G. J., Caughey, B., de Vries, R., Smits, M. A. (1997) Scrapie susceptibility-linked polymorphisms modulate the in vitro conversion of sheep prion protein to protease-resistant forms. Proc. Natl. Acad. Sci. USA 94, 4931–4936.PubMedCrossRefGoogle Scholar
  56. 56.
    Bessen, R. A., Kocisko, D. A., Raymond, G. J., Nandan, S., Lansbury, P. T., Jr., and Caughey, B. (1995) Nongenetic propagation of strain-specific phenotypes of scrapie prion protein. Nature 375, 698–700.PubMedCrossRefGoogle Scholar
  57. 57.
    Bessen, R. A., Raymond, G. J., Caughey, B. (1997) In situ formation of protease-resistant prion protein in transmissible spongiform encephalopathy-infected brain slices. J. Biol. Chem. 272, 15,227–15,231.CrossRefGoogle Scholar
  58. 58.
    DebBurman, S. K., Raymond, G. J., Caughey, B., Lindquist, S. (1997) Chaperone-supervised conversion of prion protein to its protease-resistant form. Proc. Natl. Acad. Sci. USA 94, 13,938–13,943.CrossRefGoogle Scholar
  59. 59.
    Chabry, J., Caughey, B., Chesebro, B. (1998) Specific inhibition of in vitro formation of protease-resistant prion protein by synthetic peptides. J. Biol. Chem. 273, 13,203–13,207.CrossRefGoogle Scholar
  60. 60.
    Demaimay, R., Harper, J., Gordon, H., Weaver, D., Chesebro, B., Caughey, B. (1998) Structural aspects of Congo read as an inhibitor of protease-resistant PrP formation. J. Neurochem. (In Press).Google Scholar
  61. 61.
    Herrmann, L. M. and Caughey, B. (1998) The importance of the disulfide bond in prion protein conversion. Neuroreport 9, (in press).Google Scholar
  62. 62.
    Caughey, B., Raymond, G. J., Kocisko, D. A., Lansbury, P. T., Jr. (1997) Scrapie infectivity correlates with converting activity, protease resistance, and aggregation of scrapie-associated prion protein in guanidine denaturation studies. J. Virol. 71, 4107–4110.PubMedGoogle Scholar
  63. 63.
    Bolton, D. C., Bendheim, P. E., Marmostein, A. D., and Potempska, A. (1987) Isolation and structural studies of the intact scrapie agent protein. Arch. Biochem. Biophys. 258, 579–590.PubMedCrossRefGoogle Scholar
  64. 64.
    Caughey, B. W., Dong, A., Bhat, K. S., Ernst, D., Hayes, S. F., and Caughey, W. S. (1991) Secondary structure analysis of the scrapie-associated protein PrP 27–30 in water by infrared spectroscopy. Biochemistry 30, 7672–7680.PubMedCrossRefGoogle Scholar
  65. 65.
    Caughey, B., Kocisko, D.A., Priola, S.A. Raymond, G.J., Race, R.E., Bessen, R.A., Lansbury, P.T., Jr., Chesebro, B. Methods for studying prion protein (PrP) metabolism and the formation of protease-resistant PrP in cell culture and cell-free systems, in Methods in Molecular Medicine: Prion Diseases (Baker, H. and Ridley, R.M., eds.) Humana Press, Totowa, NJ, pp. 285–299.Google Scholar

Copyright information

© Humana Press Inc 1999

Authors and Affiliations

  • Byron Caughey
    • 1
  • Gregory J. Raymond
  • Suzette A. Priola
  • David A. Kocisko
  • Richard E. Race
  • Richard A. Bessen
  • Peter T. LansburyJr.
  • Bruce Chesebro
  1. 1.NIH Rocky Mountain LaboratoriesHamilton

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