A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers

Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by \u3e105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure

A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers

Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by \u3e105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure

A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers.

Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure

Cofactor and glycosylation preferences for in vitro prion conversion are predominantly determined by strain conformation.

Prion diseases are caused by the misfolding of a host-encoded glycoprotein, PrPC, into a pathogenic conformer, PrPSc. Infectious prions can exist as different strains, composed of unique conformations of PrPSc that generate strain-specific biological traits, including distinctive patterns of PrPSc accumulation throughout the brain. Prion strains from different animal species display different cofactor and PrPC glycoform preferences to propagate efficiently in vitro, but it is unknown whether these molecular preferences are specified by the amino acid sequence of PrPC substrate or by the conformation of PrPSc seed. To distinguish between these two possibilities, we used bank vole PrPC to propagate both hamster or mouse prions (which have distinct cofactor and glycosylation preferences) with a single, common substrate. We performed reconstituted sPMCA reactions using either (1) phospholipid or RNA cofactor molecules, or (2) di- or un-glycosylated bank vole PrPC substrate. We found that prion strains from either species are capable of propagating efficiently using bank vole PrPC substrates when reactions contained the same PrPC glycoform or cofactor molecule preferred by the PrPSc seed in its host species. Thus, we conclude that it is the conformation of the input PrPSc seed, not the amino acid sequence of the PrPC substrate, that primarily determines species-specific cofactor and glycosylation preferences. These results support the hypothesis that strain-specific patterns of prion neurotropism are generated by selection of differentially distributed cofactors molecules and/or PrPC glycoforms during prion replication

Raman spectroscopy of cofactor and protein-only PrP^Sc, focusing on spectral regions assigned to tyrosine side chains.

<p>The primary sequence of the region examined by DXMS is shown, with residues 144–163 boxed and tyrosine residues highlighted in green. Raman shifts corresponding to the ν(C = C) ring mode (~1620 cm^-1) and the tyrosine Fermi-doublet (~850 and 830 cm^-1) are shown.</p

Cofactor and protein-only PrP^Sc are stably propagating recombinant PrP conformers that differ in their ability to template the conversion of native PrP^C.

<p>(<i>A</i>) Western blot showing three-round sPMCA reactions using recombinant PrP as the substrate and seeded with full-length or PK-digested cofactor and protein-only PrP^Sc, as indicated. (<i>B</i>) Western blot showing three-round sPMCA reactions using normal mouse brain homogenate as the substrate and seeded with full-length or PK-digested cofactor and protein-only PrP^Sc, as indicated.</p

Regional solvent accessibility of cofactor and protein-only PrP^Sc conformers.

<p>Purified PrP^Sc conformers were incubated in D₂O-containing exchange buffer for 30 min, 2 h, or 24 h and the reaction products were quenched and subjected to LC/MS to measure peptide-specific deuterium incorporation. Data from overlapping peptides was used to quantify localized deuterium exchange and construct ribbon diagrams from a representative experiment. (<i>A</i>) Regional solvent accessibility comparison between cofactor and protein-only PrP^Sc conformers derived from the same OSU prion strain. The discontinuous epitope of mAb 15B3, which selectively binds infectious PrP^Sc, is indicated with black bars [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005017#ppat.1005017.ref018" target="_blank">18</a>]. (<i>B</i>) Regional solvent accessibility of two additional cofactor PrP^Sc conformers, derived from the 301C and ME7 prion strains. (<i>C</i>) Map of the 188 high quality deuterated peptides, including different peptide charge states, identified in all four PrP^Sc experimental samples and used to construct ribbon diagrams in parts (<i>A</i>) and (<i>B</i>). Alternating shades of blue in part (<i>C</i>) are used to highlight neighboring peptides. Despite complete coverage of amino acids 89–230 by overlapping peptides, gaps exist in the ribbon diagrams shown in parts (<i>A</i>) and (<i>B</i>) for two reasons: 1. Deuterium incorporation cannot be quantified for the two N-terminal residues of a peptide as a result of rapid back exchange [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005017#ppat.1005017.ref019" target="_blank">19</a>], and 2. Proline residues lack amide hydrogen atoms and therefore do not contribute to deuterium incorporation measurements.</p

Cofactor and protein-only PrP^Sc differ in their ability to template the conversion of PrP substrates containing a GPI anchor.

<p>Western blot showing three round sPMCA reactions using partially purified and deglycosylated PrP^C as the substrate and seeded with protein-only PrP^Sc, cofactor PrP^Sc, or prion-infected brain homogenate, as indicated. A replicate of this experiment is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005017#ppat.1005017.s009" target="_blank">S9 Fig</a></p

Immunoprecipitation with conformation-specific mAb 15B3 distinguishes between cofactor and protein-only PrP^Sc.

<p>Converted sPMCA products were purified by ultracentrifugation with nOG washes to remove unconverted α-PrP and excess lipid, and immunoprecipation was performed using 15B3-coated or uncoated rat anti-mouse IgM-conjugated magnetic beads, as indicated. The location of the discontinuous, 15B3 conformational epitope is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005017#ppat.1005017.g002" target="_blank">Fig 2A</a>. Arrows indicate an M_r of ~23 kDa, the expected mobility of full-length recombinant PrP. By densitometry, the efficiency of 15B3 immunoprecipitation in this experiment is 79%, 71% and 74% for 301C-seeded, ME7-seeded, and OSU-seeded cofactor PrP^Sc, respectively, and 15% for OSU-seeded protein-only PrP^Sc.</p