Skip to main content
SpringerLink
Log in

Methods for Studying Prion Protein (PrP) Metabolism and the Formation of Protease-Resistant PrP in Cell Culture and Cell-Free Systems

  • Protocol
Prion Diseases

Part of the book series: Methods in Molecular Medicine ((MIMM,volume 3))

Abstract

The pathogenesis of scrapie and other transmissible spongiform encephalopathies (TSEs) appears to be based on the posttranslational conversion of the host’s protease-sensitive prion protein (PrP-sen or PrPc) to abnormal protease-resistant forms (PrP-res or PrPSc). In vitro studies using both scrapie-infected tissue culture cells and cell-free reactions have been used effectively to help define the cellular and molecular details of this process and how it might be inhibited. Here we discuss experimental approaches that we have used in these studies

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Caughey, B. (1991) Cellular metabolism of normal and scrapie-associated forms of PrP. Sem Virol 2, 189–196

    CAS  Google Scholar 

  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 gene cloned from scrapie-infected mouse brain. Proc Natl Acad Sci USA 85, 4657–4661

    Article  PubMed  CAS  Google Scholar 

  3. Caughey, B. (1993) Scrapte associated PrP accumulation and its prevention insights from cell culture. Br Med Bull 49, 860–872

    PubMed  CAS  Google Scholar 

  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 Przon 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. Memer, Z., Halimi, M., Polaktewicz, 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

    Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  7. Race, R. E., Fadness, L. H., and Chesebro, B. (1987) Charactertzation of scrapie infection in mouse neuroblastoma cells. J Gen Virol 68, 1391–1399

    Article  PubMed  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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-reststant prion proteins. J Virol. 62, 1558–1564

    PubMed  CAS  Google Scholar 

  10. Caughey, B. and Raymond, G. J. (1993) Sulfated polyanion inhibition of scrapteassociated PrP accumulation in cultured cells. J Virol 67, 643–650

    PubMed  CAS  Google Scholar 

  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 Ceil Biol 110, 743–752

    Article  CAS  Google Scholar 

  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) imphcations regarding the site of conversion of PrP to the protease-reststant state. J Virol 65, 6597–6603

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  15. Caughey, B. and Race, R. E. (1992) Potent inhibition of scrapie-associated PrP accumulation by Congo red. J Neurochem 59, 768–771

    Article  PubMed  CAS  Google Scholar 

  16. Stahl, N., Borchelt, D. R., Hsiao, K., and Prusiner, S. B. (1987) Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell 51, 229–240

    Article  PubMed  CAS  Google Scholar 

  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

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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 scrapieor Creutzfeldt-Jakob disease-infected brains. J Infect Dis 163, 488–494

    Article  PubMed  CAS  Google Scholar 

  20. Bolton, D. C., Meyer, R. K., and Prusmer, S. (1985) Scrapie PrP 27–30 is a sialoglycoprotein. J Virol 53, 596–606.

    PubMed  CAS  Google Scholar 

  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

    Article  PubMed  CAS  Google Scholar 

  22. Oesch, B., Westaway, D., Walchli, 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

    Article  PubMed  CAS  Google Scholar 

  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 scrapte agent. Proc Natl Acad Sci USA 83, 6372–6376

    Article  PubMed  CAS  Google Scholar 

  24. Endo, T., Groth, D., Prusiner, S. B., and Kobata, A. (1989) Diversity of oligosac-charide structures linked to asparagines of the scrapie prion protein. Biochemistry 28, 8380–8388.

    Article  PubMed  CAS  Google Scholar 

  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 scrapieinfected cells does not require asparagine-linked glycosylation. Proc Natl Acad Sci USA 87, 8262–8266

    Article  PubMed  CAS  Google Scholar 

  26. Thotakura, N. R. and Bahl, O. P. (1987) Enzymatic deglycosylation of glycoproteins. Methods Enzymol 138, 350–359

    Article  PubMed  CAS  Google Scholar 

  27. Somerville, R. A. and Ritchie, L. A. (1990) Differential glycosylation of the protein (PrP) forming scrapte-associated fibrils. J Gen Virol 71, 833–839

    Article  PubMed  CAS  Google Scholar 

  28. Elbein, A. D. (1987) Inhibitors of the biosynthesis and processing of N-linked oligo saccharide chains. Annu Rev Biochem 56, 497–534

    Article  PubMed  CAS  Google Scholar 

  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

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  31. Shyng, S.-L., Heuser, J. E., and Harris, D. A. (1994) A glycolipid-anchored prion protem is endocytosed via clathrin coated pits. J. Cell Biol 125, 1239–1250

    Article  PubMed  CAS  Google Scholar 

  32. Jeffrey, M., Goodsir, 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.

    Article  PubMed  CAS  Google Scholar 

  33. Jeffrey, M., Goodsir, 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

    Article  PubMed  CAS  Google Scholar 

  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

    PubMed  CAS  Google Scholar 

  35. Taraboulos, A., Raeber, A. J., Borchelt, D. R., Serban, D., and Prusiner, S. B. (1992) Synthesis and trafticking of prion proteins in cultured cells. Mol Biol Cell 3, 851–863

    PubMed  CAS  Google Scholar 

  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

    PubMed  CAS  Google Scholar 

  37. Caughey, B., Ernst, D., and Race, R. E. (1993) Congo red inhibition of scrapie agent replication. J Virol 67, 6270–6272

    PubMed  CAS  Google Scholar 

  38. Caughey, B., Brown, K., Raymond, G. J., Katzenstien, 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

    PubMed  CAS  Google Scholar 

  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

    Article  PubMed  CAS  Google Scholar 

  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

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  43. Ingrosso, L., Ladogana, A., and Pocchiari, M. (1995) Congo red prolongs the incubation period in scrapie-infected hamsters. J Virol 69, 506–508

    PubMed  CAS  Google Scholar 

  44. Scott, M. R. D., Butler, D. A., Bredesen, D. E., Walchli, M., Hsiao, K. K., and Prusiner, S. (1988) Prion protein gene expression in cultured cells. Prot Eng 2, 69–76.

    Article  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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

    PubMed  CAS  Google Scholar 

  47. Harris, D. A., Huber, M. T., vanDijken, 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

    Article  PubMed  CAS  Google Scholar 

  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

    PubMed  CAS  Google Scholar 

  49. Priola, S. A., Caughey, B., Race, R. E., and Chesebro, B. (1994) Heterologous PrP molecules interfere with accumulation of protease-resistant PrP in scrapieinfected murine neuroblastoma cells. J Virol 68, 4873–4878

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  51. Raeber, A. J., Borchelt, D. R., Scott, M., and Prusmer, S. B. (1992) Attempts to convert the cellular prion protein into the scrapre isoform in cell-free systems. J Virol 66, 6155–6163.

    PubMed  CAS  Google Scholar 

  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 protem. Nature 370, 471–474

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  54. 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.

    Article  PubMed  CAS  Google Scholar 

  55. 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.

    Article  PubMed  CAS  Google Scholar 

  56. Caughey, B. W., Dong, A., Bhat, K. S., Ernst, D., Hayes, S. F., and Caughey, W. S. (1991) Secondary structure analysis of the scrapre-associated protein PrP 27–30 in water by infrared spectroscopy. Biochemistry 30, 7672–7680

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institutes of Allergies and Infectious Diseases, Hamilton, MT

    Byron Caughey, David A. Kocisko, Suzette A. Priola, Gregory J. Raymond, Richard E. Race, Richard A. Bessen & Bruce Chesebro

  2. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA

    David A. Kocisko & Peter T. Lansbury Jr.

Authors
  1. Byron Caughey
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David A. Kocisko
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Suzette A. Priola
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gregory J. Raymond
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Richard E. Race
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Richard A. Bessen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Peter T. Lansbury Jr.
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Bruce Chesebro
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Cambridge University, Cambridge, UK

    Harry F. Baker  & Rosalind M. Ridley  & 

Rights and permissions

Reprints and permissions

Copyright information

© 1996 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Caughey, B. et al. (1996). Methods for Studying Prion Protein (PrP) Metabolism and the Formation of Protease-Resistant PrP in Cell Culture and Cell-Free Systems. In: Baker, H.F., Ridley, R.M. (eds) Prion Diseases. Methods in Molecular Medicine, vol 3. Springer, Totowa, NJ. https://doi.org/10.1385/0-89603-342-2:285

Download citation

  • DOI: https://doi.org/10.1385/0-89603-342-2:285

  • Publisher Name: Springer, Totowa, NJ

  • Print ISBN: 978-0-89603-342-9

  • Online ISBN: 978-1-59259-587-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation