J Korean Med Sci.  2014 Dec;29(12):1604-1609. 10.3346/jkms.2014.29.12.1604.

Evaluation of Infective Property of Recombinant Prion Protein Amyloids in Cultured Cells Overexpressing Cellular Prion Protein

Affiliations
  • 1Department of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Korea. cryou2@hanyang.ac.kr

Abstract

Misfolded isoform of prion protein (PrP), termed scrapie PrP (PrP(Sc)), tends to aggregate into various fibril forms. Previously, we reported various conditions that affect aggregation of recombinant PrP into amyloids. Because amyloidogenesis of PrP is closely associated with transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, we investigated infectivity of recombinant PrP amyloids generated in vitro. Using cultured cell lines which overexpress cellular PrP of different species, we measured the level of de novo synthesized PrP(Sc) in cells inoculated with recombinant mouse PrP amyloids. While PrP-overexpressing cells were susceptible to mouse-adapted scrapie prions used as the positive control, demonstrating the species barrier effect, infection with amyloids made of truncated recombinant PrP (PrP[89-230]) failed to form and propagate PrP(Sc) even in the cells that express mouse cellular PrP. This suggests that infectivity of PrP amyloids generated in vitro is different from that of natural prions. Recombinant PrP (89-230) amyloids tested in the current study retain no or a minute level, if any, of prion infectivity.

Keyword

Prions; Recombinant Prion Protein; Aggregates; Amyloid; Infectivity

MeSH Terms

Animals
Cell Line
Kidney/*metabolism/*pathology
Mice
PrPSc Proteins/*metabolism
Prion Diseases/*metabolism/*pathology
Prions/*metabolism
Rabbits
Recombinant Proteins/*metabolism
Up-Regulation
PrPSc Proteins
Prions
Recombinant Proteins

Figure

  • Fig. 1 Expression of Hu, Mo and Bo PrPC in stably transfected RK13 cells. Western blotting of RK13-HuPrP, RK13-MoPrP, RK13-BoPrP, and RK13-pIRES (control) cell lyates was performed using D13 (A), R1 (B) and 6H4 (C) anti-PrP antibodies.

  • Fig. 2 rPrP amyloids generated in vitro. Recombinant MoPrP (89-230) was used to form amyloids in PAFA. In situ fluorescence detection of ThT-bound amyloids over 48 hr time course is presented. AFU, arbitrary fluorescence unit.

  • Fig. 3 Infection of PrPC-overexpressing cells with rPrP amyloids. Western blotting of PK-resistant PrPSc from cell lysate of RK13-HuPrP, RK13-MoPrP, RK13-BoPrP, and RK13-pIRES at the fourth (A) and eighth (B) passages post-infection was performed using 6H4 anti-PrP antibody. PK-digested N2a and ScN2a cell lysates are analyzed as controls. Am, infected with rPrP amyloids.

  • Fig. 4 Infection of PrPC-overexpressing cells with with RML prions. (A) Western blotting of PrPC and PK-resistant PrPSc from cell lysate of RK13-HuPrP, RK13-MoPrP, and RK13-BoPrP cells at the fourth passage post-infection. (B) Cell blotting of RK13-MoPrP inoculated with normal (N) and RML-sick (RML) brain homogenates. Western and cell blotting analyses were performed using 6H4 anti-PrP antibody. RML, infected with RML-sick brain homogenate; N, infected with uninfected normal brain homogenate; PK, undigested with PK; + PK, digested with PK.


Reference

1. Prusiner SB. Prion diseases and the BSE crisis. Science. 1997; 278:245–251.
2. Gambetti P, Kong Q, Zou W, Parchi P, Chen SG. Sporadic and familial CJD: classification and characterisation. Br Med Bull. 2003; 66:213–239.
3. Belay ED. Transmissible spongiform encephalopathies in humans. Annu Rev Microbiol. 1999; 53:283–314.
4. Beekes M, McBride PA. The spread of prions through the body in naturally acquired transmissible spongiform encephalopathies. FEBS J. 2007; 274:588–605.
5. Prusiner SB. Novel proteinaceous infectious particles cause scrapie. Science. 1982; 216:136–144.
6. Eghiaian F, Grosclaude J, Lesceu S, Debey P, Doublet B, Tréguer E, Rezaei H, Knossow M. Insight into the PrPC-->PrPSc conversion from the structures of antibody-bound ovine prion scrapie-susceptibility variants. Proc Natl Acad Sci U S A. 2004; 101:10254–10259.
7. Basu S, Mohan ML, Luo X, Kundu B, Kong Q, Singh N. Modulation of proteinase K-resistant prion protein in cells and infectious brain homogenate by redox iron: implications for prion replication and disease pathogenesis. Mol Biol Cell. 2007; 18:3302–3312.
8. Baldwin MA, Cohen FE, Prusiner SB. Prion protein isoforms, a convergence of biological and structural investigations. J Biol Chem. 1995; 270:19197–19200.
9. Prusiner SB, McKinley MP, Bowman KA, Bolton DC, Bendheim PE, Groth DF, Glenner GG. Scrapie prions aggregate to form amyloid-like birefringent rods. Cell. 1983; 35:349–358.
10. Aguzzi A, Calella AM. Prions: protein aggregation and infectious diseases. Physiol Rev. 2009; 89:1105–1152.
11. Zhang Z, Zhang Y, Wang F, Wang X, Xu Y, Yang H, Yu G, Yuan C, Ma J. De novo generation of infectious prions with bacterially expressed recombinant prion protein. FASEB J. 2013; 27:4768–4775.
12. Legname G, Baskakov IV, Nguyen HO, Riesner D, Cohen FE, DeArmond SJ, Prusiner SB. Synthetic mammalian prions. Science. 2004; 305:673–676.
13. Caughey B, Baron GS, Chesebro B, Jeffrey M. Getting a grip on prions: oligomers, amyloids, and pathological membrane interactions. Annu Rev Biochem. 2009; 78:177–204.
14. Groveman BR, Dolan MA, Taubner LM, Kraus A, Wickner RB, Caughey B. Parallel in-register intermolecular beta-sheet architectures for prion-seeded prion protein (PrP) amyloids. J Biol Chem. 2014; 289:24129–24142.
15. Makarava N, Kovacs GG, Savtchenko R, Alexeeva I, Budka H, Rohwer RG, Baskakov IV. Genesis of mammalian prions: from non-infectious amyloid fibrils to a transmissible prion disease. PLoS Pathog. 2011; 7:e1002419.
16. Jeffrey M, McGovern G, Chambers EV, King D, González L, Manson JC, Ghetti B, Piccardo P, Barron RM. Mechanism of PrP-amyloid formation in mice without transmissible spongiform encephalopathy. Brain Pathol. 2012; 22:58–66.
17. Kim SG, Lee HM, Ryou C. Parameters that affect macromolecular self-assembly of prion protein. Protein J. 2014; 33:243–252.
18. Smirnovas V, Kim JI, Lu X, Atarashi R, Caughey B, Surewicz WK. Distinct structures of scrapie prion protein (PrPSc)-seeded versus spontaneous recombinant prion protein fibrils revealed by hydrogen/deuterium exchange. J Biol Chem. 2009; 284:24233–24241.
19. Diaz-Espinoza R, Soto C. High-resolution structure of infectious prion protein: the final frontier. Nat Struct Mol Biol. 2012; 19:370–377.
20. Mays CE, Yeom J, Kang HE, Bian J, Khaychuk V, Kim Y, Bartz JC, Telling GC, Ryou C. In vitro amplification of misfolded prion protein using lysate of cultured cells. PLoS One. 2011; 6:e18047.
21. Chandler RL. Encephalopathy in mice produced by inoculation with scrapie brain material. Lancet. 1961; 1:1378–1379.
22. Mays CE, Kang HE, Kim Y, Shim SH, Bang JE, Woo HJ, Cho YH, Kim JB, Ryou C. CRBL cells: establishment, characterization and susceptibility to prion infection. Brain Res. 2008; 1208:170–180.
23. Peretz D, Williamson RA, Kaneko K, Vergara J, Leclerc E, Schmitt-Ulms G, Mehlhorn IR, Legname G, Wormald MR, Rudd PM, et al. Antibodies inhibit prion propagation and clear cell cultures of prion infectivity. Nature. 2001; 412:739–743.
24. Safar JG, Scott M, Monaghan J, Deering C, Didorenko S, Vergara J, Ball H, Legname G, Leclerc E, Solforosi L, et al. Measuring prions causing bovine spongiform encephalopathy or chronic wasting disease by immunoassays and transgenic mice. Nat Biotechnol. 2002; 20:1147–1150.
25. Korth C, Stierli B, Streit P, Moser M, Schaller O, Fischer R, Schulz-Schaeffer W, Kretzschmar H, Raeber A, Braun U, et al. Prion (PrPSc)-specific epitope defined by a monoclonal antibody. Nature. 1997; 390:74–77.
26. Bosque PJ, Prusiner SB. Cultured cell sublines highly susceptible to prion infection. J Virol. 2000; 74:4377–4386.
27. Carlson GA, Ebeling C, Yang SL, Telling G, Torchia M, Groth D, Westaway D, DeArmond SJ, Prusiner SB. Prion isolate specified allotypic interactions between the cellular and scrapie prion proteins in congenic and transgenic mice. Proc Natl Acad Sci U S A. 1994; 91:5690–5694.
28. Legname G, Nguyen HO, Baskakov IV, Cohen FE, Dearmond SJ, Prusiner SB. Strain-specified characteristics of mouse synthetic prions. Proc Natl Acad Sci U S A. 2005; 102:2168–2173.
29. Piccardo P, Manson JC, King D, Ghetti B, Barron RM. Accumulation of prion protein in the brain that is not associated with transmissible disease. Proc Natl Acad Sci U S A. 2007; 104:4712–4717.
30. Chesebro B, Trifilo M, Race R, Meade-White K, Teng C, LaCasse R, Raymond L, Favara C, Baron G, Priola S, et al. Anchorless prion protein results in infectious amyloid disease without clinical scrapie. Science. 2005; 308:1435–1439.
31. Colby DW, Giles K, Legname G, Wille H, Baskakov IV, DeArmond SJ, Prusiner SB. Design and construction of diverse mammalian prion strains. Proc Natl Acad Sci U S A. 2009; 106:20417–20422.
32. Makarava N, Kovacs GG, Bocharova O, Savtchenko R, Alexeeva I, Budka H, Rohwer RG, Baskakov IV. Recombinant prion protein induces a new transmissible prion disease in wild-type animals. Acta Neuropathol. 2010; 119:177–187.
33. Barria MA, Mukherjee A, Gonzalez-Romero D, Morales R, Soto C. De novo generation of infectious prions in vitro produces a new disease phenotype. PLoS Pathog. 2009; 5:e1000421.
34. Deleault NR, Harris BT, Rees JR, Supattapone S. Formation of native prions from minimal components in vitro. Proc Natl Acad Sci U S A. 2007; 104:9741–9746.
35. Dear DV, Young DS, Kazlauskaite J, Meersman F, Oxley D, Webster J, Pinheiro TJ, Gill AC, Bronstein I, Lowe CR. Effects of post-translational modifications on prion protein aggregation and the propagation of scrapie-like characteristics in vitro. Biochim Biophys Acta. 2007; 1774:792–802.
36. Wang F, Wang X, Yuan CG, Ma J. Generating a prion with bacterially expressed recombinant prion protein. Science. 2010; 327:1132–1135.
37. Turnbaugh JA, Unterberger U, Saá P, Massignan T, Fluharty BR, Bowman FP, Miller MB, Supattapone S, Biasini E, Harris DA. The N-terminal, polybasic region of PrP(C) dictates the efficiency of prion propagation by binding to PrP(Sc). J Neurosci. 2012; 32:8817–8830.
38. Ostapchenko VG, Makarava N, Savtchenko R, Baskakov IV. The polybasic N-terminal region of the prion protein controls the physical properties of both the cellular and fibrillar forms of PrP. J Mol Biol. 2008; 383:1210–1224.
Full Text Links
  • JKMS
Actions
Cited
CITED
export Copy
Close
Share
  • Twitter
  • Facebook
Similar articles
Copyright © 2024 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: koreamed@kamje.or.kr