A protease resistant PrP isoform is present in urine of animals and humans affected with prion diseases

A protease resistant PrP isoform is present in urine of animals and humans affected with prion diseases

June 21, 2001 Journal of Biological Chemistry by Shaked GM, 
Shaked Y, Kariv Z, Halimi M, Avraham I, Gabizon R.


Since the appearance of BSE in 1985 (1,2) the need for an in-vivo diagnostic test for prion diseases has become acute. In the absence of such a method, an extensive slaughtering of cattle was required once an affected animal was identified within a herd. The need for such an in-vivo test was reinforced since the first cases of variant Creutzfeldt Jakob disease (vCJD) were reported in 1996 (3-5). vCJD is a fatal neurodegenerative disease believed to be caused by the consumption of BSE contaminated meat, and the incubation time between infection to clinical symptoms may be as long as decades (6). As opposed to cattle, the incubating individuals, (which at this point can be any of us), will be present for many years, donating blood and in some cases other organs to the non-affected population. The only identified component of the prion, the agent causing prion diseases (also referred to as transmissible spongiform encephalopaties or TSEs), is PrP Sc , a protease resistant abnormal isoform of PrP C . PrP C is a GPI anchored glycoprotein of unknown function (7,8). Although some other markers for prion diseases have been suggested (9),(10,11), PrP Sc remains not only an obligatory prion component, but also the only reliable and universally accepted marker for this family of diseases(12-14). We now show that a protease resistant isoform of PrP, hereby denominated UPrP Sc , can be detected, following a specific enrichment procedure, in the urine of scrapie infected hamsters, BSE infected cattle and humans suffering from CJD. These results pave the way for the development of a simple in-vivo test for prion diseases. [...]


Why is UPrP Sc excreted into urine? Since most urine proteins originate from blood, we speculate that some PrP Sc , either from brain or from a peripheral organ, is released during the disease incubation into the blood serum in a non-aggregated form, although at a low and undetectable concentrations. Due to its protease resistance, this PrP Sc is not digested by blood proteases. However, and since the MW of PrP is below the cut off size for filtering through kidney cells (about 40kDA)(32), PrP may subsequently be secreted into the urine and thereby be concentrated, as other proteins, at about 120 times over its concentration in blood(32). The concentration by the kidney, makes possible to detect PrP Sc in urine much easily than in blood. Since dialysis of the urine seems to be a necessary step in our detection procedure, we propose that UPrP Sc is present in a semi denatured form, probably due to the relative high concentrations of urine denaturing agents, and is subsequently renatured during the dialysis step. This denaturation/ renaturation effect, may also happen in the field due to absorption of the urea by the soil. UPrP Sc may differ in its conformation from brain PrP Sc , thereby explaining the fact that inoculation of comparable amounts of both protease resistant PrP isoforms produced such different results. It is to be remembered however that not all protease resistant PrP molecules carry prion infectivity. Indeed, in-vitro conversion experiments of PrP C to PrP Sc , in which protease resistance was achieved by a denaturation/renaturation procedure, resulted in a protease resistant but not infectious PrP isoform(33,34). Contrarily, UPrP Sc may resemble the new protease resistant soluble isoform we have identified lately, which is associated with very low levels of infectivity, if any(35,36). The latest possibility is consistent with the fact that UPrP Sc is found in urine since an aggregated molecule could not filter through the kidney. It can also be speculated that UPrP Sc is a component of a new prion strain, less virulent than the original 263K strain, which may produce not a fatal but a sub clinical or a carrier state prion disease. Recent publications indicate the presence of low levels of PrP Sc in the brains of animals which did not succumb to prion disease (37,38), suggesting a subclinical state of prion disease may exist. In some cases, the brains of these animals transmitted a fatal prion disease to other rodents, suggesting apparently healthy carriers of prions disease can transmit disease. To summarize, we have identified a prion specific protease resistant PrP isoform in the urine of prion infected animals and humans (UPrP Sc ), which may be used for the in-vivo early diagnosis of ill as well as seemingly healthy but prion infected individuals. Our findings, in addition to their practical aspects, may also open new avenues to investigate the metabolism and clearance mechanism of PrP Sc during prion infection and disease.

1: Shaked GM, Shaked Y, Kariv Z, Halimi M, Avraham I, Gabizon R. A protease resistant PrP isoform is present in urine of animals and humans affected with prion diseases. J Biol Chem. 2001 Jun 21 [epub ahead of print] PMID: 11423531 [PubMed - as supplied by publisher]

2: Rosenmann H, Talmor G, Halimi M, Yanai A, Gabizon R, Meiner Z. Prion protein with an E200K mutation displays properties similar to those of the cellular isoform PrP(C). J Neurochem. 2001 Mar;76(6):1654-62. PMID: 11259483 [PubMed - indexed for MEDLINE]

3: Shaked GM, Meiner Z, Avraham I, Taraboulos A, Gabizon R. Reconstitution of prion infectivity from solubilized protease-resistant PrP and nonprotein components of prion rods. J Biol Chem. 2001 Apr 27;276(17):14324-8. PMID: 11152454 [PubMed - indexed for MEDLINE]

4: Keshet GI, Bar-Peled O, Yaffe D, Nudel U, Gabizon R. The cellular prion protein colocalizes with the dystroglycan complex in the brain. J Neurochem. 2000 Nov;75(5):1889-97. PMID: 11032878 [PubMed - indexed for MEDLINE]

5: Meiner Z, Gabizon R. [A new variant of Creutzfeldt-Jakob disease and its relation to bovine spongiform encephalopathy (BSE) in Great Britain]. Harefuah. 1998 Mar 15;134(6):465-8. Review. Hebrew. No abstract available. PMID: 10909579 [PubMed - indexed for MEDLINE]

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7: Yanai A, Meiner Z, Gahali I, Gabizon R, Taraboulos A. Subcellular trafficking abnormalities of a prion protein with a disrupted disulfide loop. FEBS Lett. 1999 Oct 22;460(1):11-6. PMID: 10571052 [PubMed - indexed for MEDLINE]

8: Shaked Y, Rosenmann H, Talmor G, Gabizon R. A C-terminal-truncated PrP isoform is present in mature sperm. J Biol Chem. 1999 Nov 5;274(45):32153-8. PMID: 10542251 [PubMed - indexed for MEDLINE]

9: Rosenmann H, Kahana E, Korczyn AD, Kahana I, Chapman J, Gabizon R. Preliminary evidence for anticipation in genetic E200K Creutzfeldt-Jakob disease. Neurology. 1999 Oct 12;53(6):1328-9. PMID: 10522892 [PubMed - indexed for MEDLINE]

10: Shaked GM, Fridlander G, Meiner Z, Taraboulos A, Gabizon R. Protease-resistant and detergent-insoluble prion protein is not necessarily associated with prion infectivity. J Biol Chem. 1999 Jun 18;274(25):17981-6. PMID: 10364247 [PubMed - indexed for MEDLINE]

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14: Rosenmann H, Halimi M, Kahana I, Biran I, Gabizon R. Differential allelic expression of PrP mRNA in carriers of the E200K mutation. Neurology. 1997 Sep;49(3):851-6. PMID: 9305353 [PubMed - indexed for MEDLINE]

15: Rosenmann H, Meiner Z, Kahana E, Halimi M, Lenetsky E, Abramsky O, Gabizon R. Detection of 14-3-3 protein in the CSF of genetic Creutzfeldt-Jakob disease. Neurology. 1997 Aug;49(2):593-5. PMID: 9270603 [PubMed - indexed for MEDLINE]

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17: Gabizon R, Taraboulos A. Of mice and (mad) cows--transgenic mice help to understand prions. Trends Genet. 1997 Jul;13(7):264-9. Review. PMID: 9242048 [PubMed - indexed for MEDLINE]

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19: Ovadia H, Rosenmann H, Shezen E, Halimi M, Ofran I, Gabizon R. Effect of scrapie infection on the activity of neuronal nitric-oxide synthase in brain and neuroblastoma cells. J Biol Chem. 1996 Jul 12;271(28):16856-61. PMID: 8663207 [PubMed - indexed for MEDLINE]

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21: Kaneko K, Peretz D, Pan KM, Blochberger TC, Wille H, Gabizon R, Griffith OH, Cohen FE, Baldwin MA, Prusiner SB. Prion protein (PrP) synthetic peptides induce cellular PrP to acquire properties of the scrapie isoform. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):11160-4. PMID: 7479957 [PubMed - indexed for MEDLINE]

22: Telling GC, Scott M, Mastrianni J, Gabizon R, Torchia M, Cohen FE, DeArmond SJ, Prusiner SB. Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein. Cell. 1995 Oct 6;83(1):79-90. PMID: 7553876 [PubMed - indexed for MEDLINE]

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24: Gabizon R, Rosenman H, Meiner Z, Kahana I, Kahana E, Shugart Y, Ott J, Prusiner SB. Mutation in codon 200 and polymorphism in codon 129 of the prion protein gene in Libyan Jews with Creutzfeldt-Jakob disease. Philos Trans R Soc Lond B Biol Sci. 1994 Mar 29;343(1306):385-90. PMID: 7913755 [PubMed - indexed for MEDLINE]

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26: Goldhammer Y, Gabizon R, Meiner Z, Sadeh M. An Israeli family with Gerstmann-Straussler-Scheinker disease manifesting the codon 102 mutation in the prion protein gene. Neurology. 1993 Dec;43(12):2718-9. PMID: 7902971 [PubMed - indexed for MEDLINE]

27: Gabizon R, Meiner Z, Halimi M, Ben-Sasson SA. Heparin-like molecules bind differentially to prion-proteins and change their intracellular metabolic fate. J Cell Physiol. 1993 Nov;157(2):319-25. PMID: 7901226 [PubMed - indexed for MEDLINE]

28: Gabizon R, Rosenmann H, Meiner Z, Kahana I, Kahana E, Shugart Y, Ott J, Prusiner SB. Mutation and polymorphism of the prion protein gene in Libyan Jews with Creutzfeldt-Jakob disease (CJD). Am J Hum Genet. 1993 Oct;53(4):828-35. PMID: 8105682 [PubMed - indexed for MEDLINE]

29: Prusiner SB, Groth D, Serban A, Stahl N, Gabizon R. Attempts to restore scrapie prion infectivity after exposure to protein denaturants. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2793-7. PMID: 8464892 [PubMed - indexed for MEDLINE]

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33: Gabizon R, Prusiner SB. Prion liposomes. Biochem J. 1990 Feb 15;266(1):1-14. Review. No abstract available. PMID: 1968741 [PubMed - indexed for MEDLINE]

34: Gabizon R, McKinley MP, Groth D, Westaway D, DeArmond SJ, Carlson GA, Prusiner SB. Immunoaffinity purification and neutralization of scrapie prions. Prog Clin Biol Res. 1989;317:583-600. Review. PMID: 2574871 [PubMed - indexed for MEDLINE]

35: Gabizon R, McKinley MP, Groth D, Prusiner SB. Immunoaffinity purification and neutralization of scrapie prion infectivity. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6617-21. PMID: 3137571 [PubMed - indexed for MEDLINE]

36: Bellinger-Kawahara CG, Kempner E, Groth D, Gabizon R, Prusiner SB. Scrapie prion liposomes and rods exhibit target sizes of 55,000 Da. Virology. 1988 Jun;164(2):537-41. PMID: 3130718 [PubMed - indexed for MEDLINE]

37: Gabizon R, McKinley MP, Groth DF, Kenaga L, Prusiner SB. Properties of scrapie prion protein liposomes. J Biol Chem. 1988 Apr 5;263(10):4950-5. PMID: 3350818 [PubMed - indexed for MEDLINE]

38: Gabizon R, McKinley MP, Prusiner SB. Properties of scrapie prion proteins in liposomes and amyloid rods. Ciba Found Symp. 1988;135:182-96. Review. PMID: 2900719 [PubMed - indexed for MEDLINE]

39: Gabizon R, McKinley MP, Prusiner SB. Purified prion proteins and scrapie infectivity copartition into liposomes. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4017-21. PMID: 3108886 [PubMed - indexed for MEDLINE]

40: Prusiner SB, Gabizon R, McKinley MP. On the biology of prions. Acta Neuropathol (Berl). 1987;72(4):299-314. Review. PMID: 3554880 [PubMed - indexed for MEDLINE]

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