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Print Email Share/bookmark CiteULike Facebook Twitter Delicious Digg Google+ LinkedIn Reddit StumbleUpon Recombinant Human Prion Protein Inhibits Prion Propagation in vitro Jue Yuan,1, 3, 15 Yi-An Zhan,1, 7, 15 Romany Abskharon,5, 6, 14, 15 Xiangzhu Xiao,1, 3 Manuel Camacho Martinez,1, 3 Xiaochen Zhou,1, 7 Geoff Kneale,8 Jacqueline Mikol,9 Sylvain Lehmann,10 Witold K. Surewicz,13 Joaquín Castilla,12 Jan Steyaert,5, 6 Shulin Zhang,1 Qingzhong Kong,1, 2, 3 Robert B. Petersen,1, 2, 11 Alexandre Wohlkonig5, 6 & Wen-Quan Zou1, 2, 3, 4, 7 Affiliations Contributions Corresponding authors Journal name: Scientific Reports Volume: 3, Article number: 2911 DOI: doi:10.1038/srep02911 Received 31 May 2013 Accepted 24 September 2013 Published 09 October 2013 Article tools PDF
Recombinant Human Prion Protein Inhibits Prion Propagation in vitro
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Prion diseases are associated with the conformational conversion of the cellular prion protein (PrPC) into the pathological scrapie isoform (PrPSc) in the brain. Both the in vivo and in vitro conversion of PrPC into PrPSc is significantly inhibited by differences in amino acid sequence between the two molecules. Using protein misfolding cyclic amplification (PMCA), we now report that the recombinant full-length human PrP (rHuPrP23-231) (that is unglycosylated and lacks the glycophosphatidylinositol anchor) is a strong inhibitor of human prion propagation. Furthermore, rHuPrP23-231 also inhibits mouse prion propagation in a scrapie-infected mouse cell line. Notably, it binds to PrPSc, but not PrPC, suggesting that the inhibitory effect of recombinant PrP results from blocking the interaction of brain PrPC with PrPSc. Our findings suggest a new avenue for treating prion diseases, in which a patient's own unglycosylated and anchorless PrP is used to inhibit PrPSc propagation without inducing immune response side effects.
Subject terms: Neurodegeneration Medical research
Previous Figures index Discussion Introduction• Results• Discussion• Methods• References• Acknowledgements• Author information• Supplementary information The in vitro and in vivo conversion efficiency of PrPC into PrPSc can be significantly affected by the presence of additional PrP molecules that differ from the endogenous PrPC by as little as one residue6, 28, 11, 12. The present study now demonstrates that a PrP molecule that shares the identical amino acid sequence with the PrPC substrate and PrPSc template also causes interference. It is worth noting that although the amino acid sequence is identical, recombinant PrP does not contain N-linked glycans or a GPI anchor. Our new results suggest that in addition to the amino acid sequence, glycosylation and the GPI anchor are important in mediating the conversion of PrPC into PrPSc. The unglycosylated and anchorless recombinant PrP appears to act as an inhibitor of the conversion process by preferentially binding to PrPSc.
PrPC is a glycoprotein with two non-obligatory, N-linked glycosylation sites at residues 181 and 197 and a GPI anchor24, 29, 30. Binding of heterologous PrPC to PrPSc can be influenced by PrPC glycosylation in a species-specific manner31, 32. Moreover, using PMCA and a scrapie cell assay, Nishina et al reported that the stoichiometry of host PrPC glycoforms modulates the efficiency of PrPSc formation in vitro17. Specifically, their study demonstrated that while unglycosylated PrPC is required to propagate mouse RML prions, in a similar reaction, amplification of hamster Sc237 prions is inhibited by substoichiometric levels of homologous unglycosylated PrPC. This study provides direct in vitro evidence that changes in the PrP glycoform ratios can affect the efficiency of PrPSc formation in a species-specific manner. Recently, we observed glycoform-selective prion formation in unique sporadic and inherited forms of Creutzfeldt-Jakob disease (CJD) including variably protease-sensitive prionopathy (VPSPr) and familial CJD linked to a valine to isoleucine mutation at residue 180 (fCJDV180I)16. Although all four glycoforms are present, including di-, monoglycosylated at residue 181 (mono-181), monoglycosylated at residue 197 (mono-197), and unglycosylated PrP forms in the brain of VPSPr and fCJDV180I, only the mono-197 and unglycosylated PrP species were converted into PrPSc. The mono-181 and diglycosylated PrP species were not converted into PrPSc in the cerebral cortical brain areas examined. Moreover, the level of the classic PK-resistant PrPSc probed with the 3F4 antibody was significantly decreased compared to typical sporadic CJD. Instead, a unique five-step ladder-like electrophoretic profile of PK-resistant PrPSc was detected in both diseases by the 1E4 antibody16. In contrast to the threonine to alanine mutation at residue 183 of PrP (PrPT183A), the PrPV180I mutation exhibits a typical PrP glycosylation profile, although there is no detectable mono-181 and diglycosylated PrPSc33, 16. However, using the N-linked glycosylation prediction algorithm NetNGlyc 1.0 at http://www.cbs.dtu.dk/services/NetNGlyc/34 , we predicted a slight decrease in the glycosylation potential at N181 in PrPV180I compared to PrPWt (0.597 vs 0.664) while no potential change was predicted at all for N181 in PrPT183A16. The prediction data suggests that although the T183A mutation completely eliminates the N181 glycosylation site, the V180I mutation may merely alter the glycan composition at N181, which modifies the ratio of the four PrP glycoforms in the PrP mixture. Further investigation into the mechanism, by which altered glycosylation affects both conversion efficiency of PrPC into PrPSc and PrPSc conformation, is warranted. Unglycosylated and anchorless recombinant human PrP may have greater affinity for PrPSc seeds compared to the brain PrPC, but it is a poor substrate for conversion into PrPres by the standard PMCA protocol or in ScN2a cells. Indeed, both recombinant hamster and mouse PrP are not converted into PK-resistant PrP by serial PMCA in the presence of hamster prion Sc237 or mouse prion RML, respectively17. It is worth noting, however, that the recombinant PrP could be converted into PrPres using a modified PMCA protocol in which the conversion buffer contained 0.1% SDS and the normal brain-derived PrPC was replaced by recombinant hamster PrP as a substrate35, 36 and the product of this reaction was proved to be infectious in animal bioassays37. Furthermore, in the absence of brain homogenates, recombinant PrP was converted by PMCA to highly infectious prions in the presence of additional cofactors such as phosphatidylglycerol and RNA38 or phosphatidylethanolamine39. Prion infectivity was also produced in Syrian hamsters by inoculating full-length recombinant hamster PrP that was converted into a cross-β-sheet amyloid conformation and subjected to an annealing procedure40.
We cannot rule out the possibility that the inhibition of human PrPSc amplification by recombinant human PrP results from the lack of the GPI anchor, although the GPI anchor of PrPC is believed to have little or no effect on the formation of PK-resistant PrP31, 32. Anchorless PrP generated in either cultured mammalian cells or E. coli are converted to PK-resistant PrP by a cell-free conversion approach41, 42, 43, 44. Moreover, it has been reported that anchorless prion protein induced an infectious amyloid disease in transgenic animals, although the animal themselves were asymptomatic45. However, amplification of hamster PrPSc using a standard PMCA protocol is inhibited when the substrate of normal hamster brain PrPC was pretreated with phosphatidylinositol-specific phospholipase C (PIPLC) to remove the GPI anchor19. Furthermore, recombinant hamster PrP was previously shown to inhibit PMCA of hamster PrPSc using the normal hamster brain homogenate as a substrate18. Kim et al proposed that both of these effects are due to the lack of the GPI anchor in PIPLC-treated PrPC or recombinant PrP19. On the other hand, the unglycosylated hamster PrPC purified from brains and containing an intact GPI anchor likewise inhibits amplification of Sc237 prions17. Therefore, the role of GPI anchor in the inhibition of human and mouse PrPSc propagation by recombinant human PrP observed in our study remains to be determined.
We demonstrated that recombinant human and other PrP that exhibited 50% or greater inhibition of PrPSc formation in a PMCA reaction bind to human PrPSc but not to PrPC. Although full-length, N- or C-terminally truncated recombinant PrP all bind to PrPSc efficiently, the full-length rHuPrP23-231 exhibits the highest inhibition efficiency compared to the two truncated forms, suggesting that the inhibition involves both N- and C-terminal domains. Moreover, antibodies including SAF32, 3F4, and 6H4 directed against PrP regions covering residues 59 to 152 showed less than 10% inhibition. The 8H4 antibody against human PrP175-185 exhibited virtually no inhibition. These results are in good agreement with a previous report by Horiuchi and Caughey1. In addition, it has been shown that the 3F4 and 6H4 antibodies preferentially bind to native PrPC, although they also detect denatured PrPSc on Western blots46, 47. Therefore, the interaction of inhibitors with PrPSc may be required for the inhibition of PrPC conversion. We observed that recombinant mouse PrP is also able to bind to human PrPSc (Figure S2), although it caused significantly less inhibition compared to recombinant human PrP. Moreover, the anti-DNA antibody that specifically captures PrPSc but not PrPC showed less than 10% inhibition while g5p caused more than 50% inhibition. This suggests that the different inhibitors have distinct binding sites on PrPSc: one class of sites is specifically associated with recruiting PrPC while the other is not. Recombinant human PrP is likely to compete with brain PrPC for the same site on the PrPSc molecule. Moreover, its affinity for PrPSc seems to be greater than that of brain-derived PrPC. Interestingly, a two-site model has been proposed by Horiuchi and co-workers to explain the molecular mechanism for sequence-difference interference28. According to this model, PrPSc has two types of PrPC binding sites: one is able to induce conversion to PrPSc while the other is not.
A recombinant mouse PrP with a substitution of lysine for glutamine at mouse codon 218 (rPrP-Q218K), corresponding to human PrPE219K, an Asian-specific polymorphism believed to be resistant to CJD infection, considerably prolonged incubation time of prion infection in an iatrogenic mouse model48. Recombinant mouse PrP was delivered into the mouse brain for 7 days by intracerebroventricular administration using an indwelling catheter connected to an implanted osmotic pump. The same group also found that rPrP-Q218K reduced PrPSc formation in ScN2a cells. However, using wild-type mouse PrP did not cause inhibition, which is different than our findings. This discrepancy may be due to different experimental conditions between the studies. We showed that murine PrPSc amplification was inhibited in both PMCA and ScN2a by unglycosylated and anchorless recombinant human PrP. Most importantly, since the amino acid sequence of recombinant human PrP is identical to that of human brain PrPC, it is expected that this protein would not elicit an immune response after intracerebroventricular administration while it inhibits PrPSc propagation. Therefore, our findings suggest a new therapeutic strategy for treating human prion diseases.
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Recombinant Human Prion Protein Inhibits Prion Propagation
Case Western Reserve Researchers Discover New Avenue for Preventing and Treating Fatal Brain Disease
News Release: October 9, 2013
Case Western Reserve University researchers today published findings that point to a promising discovery for the treatment and prevention of prion diseases, rare neurodegenerative disorders that are always fatal. The researchers discovered that recombinant human prion protein stops the propagation of prions, the infectious pathogens that cause the diseases.
“This is the very first time recombinant protein has been shown to inhibit diseased human prions,” said Wen-Quan Zou, MD, PhD, senior author of the study and associate professor of pathology and neurology at Case Western Reserve School of Medicine.
Recombinant human prion protein is generated in E. coli bacteria and it has the same protein sequence as normal human brain protein. But different in that, the recombinant protein lacks attached sugars and lipids. In the study, published online in Scientific Reports, researchers used a method called protein misfolding cyclic amplification which, in a test-tube, mimics the prions’ replication within the human brain. The propagation of human prions was completely inhibited when the recombinant protein was added into the test-tube. The researchers found that the inhibition is dose-dependent and highly specific in responding to the human-form of the recombinant protein, as compared to recombinant mouse and bovine prion proteins. They demonstrated that the recombinant protein works not only in the cell-free model but also in cultured cells, which are the first steps of translational research. Further, since the recombinant protein has an identical sequence to the brain protein, the application of the recombinant protein is less likely to cause side effects.
Prion diseases are a group of fatal transmissible brain diseases affecting both humans and animals. Prions are formed through a structural change of a normal prion protein that resides in all humans. Once formed, they continue to recruit other normal prion protein and finally cause spongiform-like damage in the brain. Currently, the diseases have no cure.
Previous outbreaks of mad cow disease and subsequent occurrences of the human form, variant Creutzfeldt–Jakob disease, have garnered a great deal of public attention. The fear of future outbreaks makes the search for successful interventions all the more urgent.
Zou, who also serves as the associate director of the National Prion Disease Pathology Surveillance Center at Case Western Reserve, and collaborators hope to extend their finding using transgenic mice expressing the human prion protein and patient-specific induced pluripotent stem cells (iPSCs)-derived neurons because they are made from human cells, offering an additional level of authenticity. The new animal models were generated in collaboration with Case Western Reserve School of Medicine faculty members, Robert Petersen, PhD, and Qingzhong Kong, PhD, who are the co-authors in this study. Further, patient-specific iPSCs-derived neurons have also just been generated in collaboration with fellow faculty, Paul Tesar, PhD, and Xin Qi, PhD.
About Case Western Reserve University School of Medicine Founded in 1843, Case Western Reserve University School of Medicine is the largest medical research institution in Ohio and is among the nation's top medical schools for research funding from the National Institutes of Health. The School of Medicine is recognized throughout the international medical community for outstanding achievements in teaching. The School's innovative and pioneering Western Reserve2 curriculum interweaves four themes--research and scholarship, clinical mastery, leadership, and civic professionalism--to prepare students for the practice of evidence-based medicine in the rapidly changing health care environment of the 21st century. Eleven Nobel Laureates have been affiliated with the school.
Annually, the School of Medicine trains more than 800 M.D. and M.D./Ph.D. students and ranks in the top 25 among U.S. research-oriented medical schools as designated by U.S. News & World Report's "Guide to Graduate Education."
The School of Medicine's primary affiliate is University Hospitals Case Medical Center and is additionally affiliated with MetroHealth Medical Center, the Louis Stokes Cleveland Department of Veterans Affairs Medical Center, and the Cleveland Clinic, with which it established the Cleveland Clinic Lerner College of Medicine of Case Western Reserve University in 2002.
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