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Effect of glycans and the glycophosphatidylinositol anchor on strain dependent conformations of scrapie prion protein: improved purifications and infrared spectra.

ABSTRACT: Mammalian prion diseases involve conversion of normal prion protein, PrP(C), to a pathological aggregated state (PrP(res)). The three-dimensional structure of PrP(res) is not known, but infrared (IR) spectroscopy has indicated high, strain-dependent ?-sheet content. PrP(res) molecules usually contain a glycophosphatidylinositol (GPI) anchor and large Asn-linked glycans, which can also vary with strain. Using IR spectroscopy, we tested the conformational effects of these post-translational modifications by comparing wild-type PrP(res) with GPI- and glycan-deficient PrP(res) produced in GPI-anchorless PrP transgenic mice. These analyses required the development of substantially improved purification protocols. Spectra of both types of PrP(res) revealed conformational differences between the 22L, ME7, and Chandler (RML) murine scrapie strains, most notably in bands attributed to ?-sheets. These PrP(res) spectra were also distinct from those of the hamster 263K scrapie strain. Spectra of wild-type and anchorless 22L PrP(res) were nearly indistinguishable. With ME7 PrP(res), modest differences between the wild-type and anchorless spectra were detected, notably an ?2 cm(-1) shift in an apparent ?-sheet band. Collectively, the data provide evidence that the glycans and anchor do not grossly affect the strain-specific secondary structures of PrP(res), at least relative to the differences observed between strains, but can subtly affect turns and certain ?-sheet components. Recently reported H-D exchange analyses of anchorless PrP(res) preparations strongly suggested the presence of strain-dependent, solvent-inaccessible ?-core structures throughout most of the C-terminal half of PrP(res) molecules, with no remaining ?-helix. Our IR data provide evidence that similar core structures also comprise wild-type PrP(res).

SUBMITTER: Baron GS

PROVIDER: S-EPMC3101284 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Effect of glycans and the glycophosphatidylinositol anchor on strain dependent conformations of scrapie prion protein: improved purifications and infrared spectra.

Baron Gerald S GS Hughson Andrew G AG Raymond Gregory J GJ Offerdahl Danielle K DK Barton Kelly A KA Raymond Lynne D LD Dorward David W DW Caughey Byron B

Biochemistry 20110503 21

Mammalian prion diseases involve conversion of normal prion protein, PrP(C), to a pathological aggregated state (PrP(res)). The three-dimensional structure of PrP(res) is not known, but infrared (IR) spectroscopy has indicated high, strain-dependent β-sheet content. PrP(res) molecules usually contain a glycophosphatidylinositol (GPI) anchor and large Asn-linked glycans, which can also vary with strain. Using IR spectroscopy, we tested the conformational effects of these post-translational modifi ...[more]

PMID: 21539311

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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.

Dataset Information

Effect of glycans and the glycophosphatidylinositol anchor on strain dependent conformations of scrapie prion protein: improved purifications and infrared spectra.

Publications

Effect of glycans and the glycophosphatidylinositol anchor on strain dependent conformations of scrapie prion protein: improved purifications and infrared spectra.

Similar Datasets

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