Dataset Information

Mechanism of scrapie prion precipitation with phosphotungstate anions.

ABSTRACT: The phosphotungstate anion (PTA) is widely used to facilitate the precipitation of disease-causing prion protein (PrP(Sc)) from infected tissue for applications in structural studies and diagnostic approaches. However, the mechanism of this precipitation is not understood. In order to elucidate the nature of the PTA interaction with PrP(Sc) under physiological conditions, solutions of PTA were characterized by NMR spectroscopy at varying pH. At neutral pH, the parent [PW12O40](3-) ion decomposes to give a lacunary [PW11O39](7-) (PW11) complex and a single orthotungstate anion [WO4](2-) (WO4). To measure the efficacy of each component of PTA, increasing concentrations of PW11, WO4, and mixtures thereof were used to precipitate PrP(Sc) from brain homogenates of scrapie prion-infected mice. The amount of PrP(Sc) isolated, quantified by ELISA and immunoblotting, revealed that both PW11 and WO4 contribute to PrP(Sc) precipitation. Incubation with sarkosyl, PTA, or individual components of PTA resulted in separation of higher-density PrP aggregates from the neuronal lipid monosialotetrahexosylganglioside (GM1), as observed by sucrose gradient centrifugation. These experiments revealed that yield and purity of PrP(Sc) were greater with polyoxometalates (POMs), which substantially supported the separation of lipids from PrP(Sc) in the samples. Interaction of POMs and sarkosyl with brain homogenates promoted the formation of fibrillar PrP(Sc) aggregates prior to centrifugation, likely through the separation of lipids like GM1 from PrP(Sc). We propose that this separation of lipids from PrP is a major factor governing the facile precipitation of PrP(Sc) by PTA from tissue and might be optimized further for the detection of prions.

SUBMITTER: Levine DJ

PROVIDER: S-EPMC4437617 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Mechanism of scrapie prion precipitation with phosphotungstate anions.

Levine Dana J DJ Stöhr Jan J Falese Lillian E LE Ollesch Julian J Wille Holger H Prusiner Stanley B SB Long Jeffrey R JR

ACS chemical biology 20150219 5

The phosphotungstate anion (PTA) is widely used to facilitate the precipitation of disease-causing prion protein (PrP(Sc)) from infected tissue for applications in structural studies and diagnostic approaches. However, the mechanism of this precipitation is not understood. In order to elucidate the nature of the PTA interaction with PrP(Sc) under physiological conditions, solutions of PTA were characterized by NMR spectroscopy at varying pH. At neutral pH, the parent [PW12O40](3-) ion decomposes ...[more]

PMID: 25695325

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Project description:It is widely known that prion strains can mutate in response to modification of the replication environment and we have recently reported that prion mutations can occur in vitro during amplification of vole-adapted prions by Protein Misfolding Cyclic Amplification on bank vole substrate (bvPMCA). Here we exploited the high efficiency of prion replication by bvPMCA to study the in vitro propagation of natural scrapie isolates. Although in vitro vole-adapted PrPSc conformers were usually similar to the sheep counterpart, we repeatedly isolated a PrPSc mutant exclusively when starting from extremely diluted seeds of a single sheep isolate. The mutant and faithful PrPSc conformers showed to be efficiently autocatalytic in vitro and were characterized by different PrP protease resistant cores, spanning aa ?155-231 and ?80-231 respectively, and by different conformational stabilities. The two conformers could thus be seen as different bona fide PrPSc types, putatively accounting for prion populations with different biological properties. Indeed, once inoculated in bank vole the faithful conformer was competent for in vivo replication while the mutant was unable to infect voles, de facto behaving like a defective prion mutant. Overall, our findings confirm that prions can adapt and evolve in the new replication environments and that the starting population size can affect their evolutionary landscape, at least in vitro. Furthermore, we report the first example of "authentic" defective prion mutant, composed of brain-derived PrPC and originating from a natural scrapie isolate. Our results clearly indicate that the defective mutant lacks of some structural characteristics, that presumably involve the central region ?90-155, critical for infectivity but not for in vitro replication. Finally, we propose a molecular mechanism able to account for the discordant in vitro and in vivo behavior, suggesting possible new paths for investigating the molecular bases of prion infectivity.

Project description:BackgroundExisting mathematical models for scrapie dynamics in sheep populations assume that the PrP gene is only associated with scrapie susceptibility and with no other fitness related traits. This assumption contrasts recent findings of PrP gene associations with post-natal lamb survival in scrapie free Scottish Blackface populations. Lambs with scrapie resistant genotypes were found to have significantly lower survival rates than those with susceptible genotypes. The present study aimed to investigate how these conflicting PrP gene associations may affect the dynamic patterns of PrP haplotype frequencies and disease prevalence.Methodology/principal findingsA deterministic mathematical model was developed to explore how the associations between PrP genotype and both scrapie susceptibility and postnatal lamb mortality affect the prevalence of scrapie and the associated change in PrP gene frequencies in a closed flock of sheep. The model incorporates empirical evidence on epidemiological and biological characteristics of scrapie and on mortality rates induced by causes other than scrapie. The model results indicate that unfavorable associations of the scrapie resistant PrP haplotypes with post-natal lamb mortality, if sufficiently strong, can increase scrapie prevalence during an epidemic, and result in scrapie persisting in the population. The range of model parameters, for which such effects were observed, is realistic but relatively narrow.Conclusions/significanceThe results of the present model suggest that for most parameter combinations an unfavourable association between PrP genotype and post-natal lamb mortality does not greatly alter the dynamics of scrapie and, hence, would not have an adverse impact on a breeding programme. There were, however, a range of scenarios, narrow, but realistic, in which such an unfavourable association resulted in an increased prevalence and in the persistence of infection. Consequently, associations between PrP genotypes and fitness traits should be taken into account when designing future models and breeding programmes.

Dataset Information

Mechanism of scrapie prion precipitation with phosphotungstate anions.

Publications

Mechanism of scrapie prion precipitation with phosphotungstate anions.

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