Formation of protease-resistant prion protein in cell-free systems

In transmissible spongiform encephalopathies (TSE) or prion diseases, the endogenous protease-sensitive prion protein (PrP-sen) of the host is converted to an abnormal pathogenic form that has a characteristic partial protease resistance (PrP-res). Studies with cell-free reactions indicate that the PrP-res itself can directly induce this conversion of PrP-sen. This PrP-res induced conversion reaction is highly specific in ways that might account at the molecular level for TSE species barriers, polymorphism barriers, and strains. Not only has this reaction been observed using mostly purified PrP-sen and PrP-res reactants, but also in TSE-infected brain slices. The conversion mechanism appears to involve both the binding of PrP-sen to polymeric PrP-res and a conformational change that results in incorporation into the PrP-res polymer.     

Glycosylation influences cross-species formation of protease-resistant prion protein.

A key event in the transmissible spongiform encephalopathies (TSEs) is the formation of aggregated and protease-resistant prion protein, PrP-res, from a normally soluble, protease-sensitive and glycosylated precursor, PrP-sen. While amino acid sequence similarity between PrP-sen and PrP-res influences both PrP-res formation and cross-species transmission of infectivity, the influence of co- or post-translational modifications to PrP-sen is unknown. Here we report that, if PrP-sen and PrP-res are derived from different species, PrP-sen glycosylation can significantly affect PrP-res formation. Glycosylation affected PrP-res formation by influencing the amount of PrP-sen bound to PrP-res, while the amino acid sequence of PrP-sen influenced the amount of PrP-res generated in the post-binding conversion step. Our results show that in addition to amino acid sequence, co- or post-translational modifications to PrP-sen influence PrP-res formation in vitro. In vivo, these modifications might contribute to the resistance to infection associated with transmission of TSE infectivity across species barriers.     

Methods for studying prion protein (PrP) metabolism and the formation of protease-resistant PrP in cell culture and cell-free systems

Transmissible spongiform encephalopathies (TSE) or prion diseases result in aberrant metabolism of prion protein (PrP) and the accumulation of a protease-resistant, insoluble, and possibly infectious form of PrP, PrP-res. Studies of PrP biosynthesis, intracellular trafficking, and degradation has been studied in a variety of tissue culture cells. Pulse-chase metabolic labeling studies in scrapie-infected cells indicated that PrP-res is made posttranslationally from an apparently normal protease sensitive precursor, PrP-sen, after the latter reaches the cell surface. Cell-free reactions have provided evidence that PrP-res itself can induce the conversion of PrP-sen to PrP-res in a highly species- and strain-specific manner. These studies have shed light on the mechanism of PrP-res formation and suggest molecular bases for TSE species barrier effects and agent strain propagation.     

The role of the prion protein membrane anchor in prion infection

Normal cellular and abnormal disease-associated forms of prion protein (PrP) contain a C-terminal glycophosphatidyl-inositol (GPI) membrane anchor. The importance of the GPI membrane anchor in prion diseases is unclear but there are data to suggest that it both is and is not required for abnormal prion protein formation and prion infection. Utilizing an in vitro model of prion infection we have recently demonstrated that, while the GPI anchor is not essential for the formation of abnormal prion protein in a cell, it is necessary for the establishment of persistent prion infection. In combination with previously published data, our results suggest that GPI anchored PrP is important in the amplification and spread of prion infectivity from cell to cell.Key words: prion, GPI anchor, PrP, prion spread, scrapieIn transmissible spongiform encephalopathies (TSE or prion diseases) such as sheep scrapie, bovine spongiform encephalopathy and human Creutzfeldt-Jakob disease, normally soluble and protease-sensitive prion protein (PrP-sen or PrP^C) is converted to an abnormal, insoluble and protease-resistant form termed PrP-res or PrP^Sc. PrP-res/PrP^Sc is believed to be the main component of the prion, the infectious agent of the TSE/prion diseases. Its precursor, PrP-sen, is anchored to the cell surface at the C-terminus by a co-translationally added glycophosphatidyl-inositol (GPI) membrane anchor which can be cleaved by the enzyme phosphatidyl-inositol specific phospholipase (PIPLC). The GPI anchor is also present in PrP-res, but is inaccessible to PIPLC digestion suggesting that conformational changes in PrP associated with PrP-res formation have blocked the PIPLC cleavage site.¹ Although the GPI anchor is present in both PrP-sen and PrP-res, its precise role in TSE diseases remains unclear primarily because there are data to suggest that it both is and is not necessary for PrP-res formation and prion infection.In tissue culture cells infected with mouse scrapie, PrP-res formation occurs at the cell surface and/or along the endocytic pathway^2–4 and may be dependent upon the membrane environment of PrP-sen. For example, localization via the GPI anchor to caveolae-like domains favors PrP-res formation⁵ while substitution of the GPI anchor addition site with carboxy termini favoring transmembrane anchored PrP-sen inhibits formation of PrP-res.^5,6 Other studies have shown that localization of both PrP-sen and PrP-res to lipid rafts, cholesterol and sphingolipid rich membrane microdomains where GPI anchored proteins can be located, is important in PrP-res formation.^6–9However, there are also data which suggest that such localization is not necessarily essential for PrP-res formation. Anchorless PrP-sen isolated from cells by immunoprecipitation or wild-type PrP-sen purified by immunoaffinity column followed by cation exchange chromatography are efficiently converted into PrP-res in cell-free systems.^10,11 Furthermore, recombinant PrP-sen derived from E. coli, which has no membrane anchor or glycosylation, can be induced to form protease-resistant PrP in vitro when reacted with prion-infected brain homogenates.^12–14 Finally, in at least one instance, protease-resistant recombinant PrP-res generated in the absence of infected brain homogenate was reported to cause disease when inoculated into transgenic mice.¹⁵The data concerning the role of the PrP-sen GPI anchor in susceptibility to TSE infection are similarly contradictory. Transgenic mice expressing anchorless mouse PrP-sen are susceptible to infection with mouse scrapie and accumulate both PrP-res and prion infectivity.¹⁶ Thus, the GPI anchor is clearly not needed for PrP-res formation or productive TSE infection in vivo. However, we recently published data demonstrating that, in vitro, anchored PrP-sen is in fact required to persistently infect cells.¹⁷ Given that anchorless PrP-sen is not present on the cell surface but is released into the cell medium, we speculated that the differences between the in vitro and in vivo data were related to the location of PrP-res formation. In the mice expressing anchorless PrP-sen, environments conducive to PrP-res formation are present in certain areas of the complex extracellular milieu of the brain where anchorless, secreted PrP-sen can accumulate and come into contact with PrP-res from the infectious inoculum. Since similar environments are missing in vitro, any PrP-res formation in cells expressing anchorless PrP-sen must be cell-associated. While this explanation addresses how extracellular PrP-res could be generated in an unusual transgenic mouse model of TSE infection, it does not really help to define how the GPI anchor is involved in normal prion infection of a cell.As with other infectious organisms such as viruses, TSE infection can be roughly divided into three steps: uptake, replication and spread. Over the last several years, data derived from new techniques as well as new cell lines susceptible to prion infection have increased our knowledge of some of the basic events that occur during each of these steps. In order to try to tease out the role of the GPI anchor in normal TSE pathogenesis, it is therefore useful to consider the process of TSE infection of a cell and how the GPI anchor might be involved in each stage.In a conventional viral infection, binding and uptake of the virus is essential to establish infection. Studying PrP-res uptake has been complicated by the lack of an antibody that can specifically distinguish PrP-res from PrP-sen in live cells and by the difficulty of detecting the input PrP-res from the PrP-res made de novo by the cell. Recently, however, several groups have been able to study PrP-res uptake using input PrP-res that was either fluorescently labeled^18–20 or tagged with the epitope to the monoclonal antibody 3F4,²¹ or cell lines that express little or no PrP-sen.^19,21–23 The data show that PrP-res uptake is independent of scrapie strain or cell type but is influenced by the PrP-res microenvironment as well as PrP-res aggregate size.²¹ Importantly, these studies demonstrated that PrP-sen expression was not required.^19,21–23 Given these data, it is clear that GPI anchored PrP-sen is not involved in the initial uptake of PrP-res into the cell.The next stage of prion infection involves replication of infectivity which is typically assayed by following cellular PrP-res formation. Once again, however, the issue of how to distinguish PrP-res in the inoculum from newly formed PrP-res in the cells has made it difficult to study the early stages of prion replication. To overcome this difficulty, we developed a murine tissue culture system that utilizes cells expressing mouse PrP-sen tagged with the epitope to the 3F4 antibody (Mo3F4 PrP-sen).²⁴ Wild-type mouse PrP does not have this epitope. As a result, following exposure to an infected mouse brain homogenate, de novo PrP-res formation can be followed by assaying for 3F4 positive PrP-res. Our studies showed that there were two stages of PrP-res formation: (1) an initial acute burst within the first 96 hours post-infection that was cell-type and scrapie strain independent and, (2) persistent PrP-res formation (i.e., formation of PrP-res over multiple cell passages) that was dependent on cell-type and scrapie strain and associated with long-term infection.²⁴ Acute PrP-res formation did not necessarily lead to persistent PrP-res formation suggesting that other cell-specific factors or processes are needed for PrP-res formation to persist.²⁴When cells expressing Mo3F4 PrP-sen without the GPI anchor (Mo3F4 GPI-PrP-sen) were exposed to mouse scrapie infected brain homogenates, GPI negative, 3F4 positive PrP-res (Mo3F4 GPI-PrP-res) was detected within 96 hours indicating that acute PrP-res formation had occurred.¹⁷ Thus, despite the fact that Mo3F4 GPI-PrP-sen is not expressed on the cell surface¹⁶ (Fig. 1A), it was still available for conversion to PrP-res. These results are consistent with data from cell-free systems and demonstrate that, at least acutely, membrane anchored PrP is not necessary for PrP-res formation in a cell.Open in a separate windowFigure 1Persistent infection of cells in vitro requires the expression of GPI-anchored cell surface PrP-sen. PrP knockout cells (CF10)²¹ were transduced with 3F4 epitope tagged mouse PrP-sen (Mo3F4), 3F4 epitope tagged mouse PrP-sen without the GPI anchor (Mo3F4 GPI-), or Mo3F4 GPI-PrP-sen plus wild-type, GPI anchored mouse PrP-sen (MoPrP). The cells were then exposed to the mouse scrapie strain 22L and passaged. (A) The presence of 3F4 epitope tagged, cell surface mouse PrP-sen was assayed by FACS analysis of fixed, non-permeabilized cells. CF10 cells expressing the following mouse PrP-sen molecules were assayed: Mo3F4 (solid line); Mo3F4 GPI⁻ (dashed line); Mo3F4 GPI⁻ + MoPrP (dotted and dashed line); Mo3F4 GPI⁻ + MoPrP infected with 22L scrapie (dotted line). Only cells expressing Mo3F4 PrP-sen were positive for cell surface, 3F4 epitope tagged PrP. (B) Persistent infection was analyzed by inoculating the cells intracranially into transgenic mice overexpressing MoPrP (Tga20 mice). Only cells expressing anchored mouse PrP-sen were susceptible to scrapie infection. Cells expressing anchorless mouse PrP-sen did not contain detectable infectivity in either the cells or the cellular supernatant (data not shown). Data in (B) are adapted from McNally 2009.¹⁷In terms of persistent PrP-res formation, however, our data suggest that the GPI anchor is important. Despite an initial burst of PrP-res formation within the first 96 hours post-infection, Mo3F4 GPI-PrP-res was not observed following passage of the cells nor did the cells become infected. This effect was not due either to resistance of the cells to scrapie infection or to an inability of the scrapie strain used to infect cells. When the same cells expressed anchored Mo3F4 PrP-sen and were exposed to the same mouse scrapie strain, both acute and persistent PrP-res formation were detected and the cells were persistently infected with scrapie (Fig. 1B).¹⁷ Taken together, these data demonstrate that cells expressing anchorless PrP-sen do not support persistent PrP-res formation. Furthermore, the data strongly suggest that GPI-anchored PrP-sen is required during the transition from acute to persistent scrapie infection. In support of this hypothesis, the resistance of cells expressing Mo3F4 GPI-PrP-sen to persistent prion infection could be overcome if wild-type GPI anchored PrP-sen was co-expressed in the same cell. When both forms of PrP-sen were expressed, anchored and anchorless forms of PrP-res were made and the cells became persistently infected (Fig. 1B).¹⁷ Thus, the data suggest that GPI anchored PrP is necessary to establish prion infection within a cell.How could GPI membrane anchored PrP be involved in the establishment and maintenance of persistent prion infection? Several studies have suggested that the GPI anchor is needed to localize PrP-sen to specific membrane environments where PrP-res formation is favored.^5–8 However, if this localization was essential for PrP-res formation, GPI-PrP-sen would presumably never form PrP-res. Lacking the GPI anchor, it would not be in the correct membrane environment to support conversion. As a result, neither acute nor persistent prion infection could occur. This is obviously not the case. Transgenic mice expressing only anchorless PrP-sen generate PrP-res and can be infected with scrapie even though (1) flotation gradients showed that anchorless PrP-sen was not in the same membrane environment as anchored PrP-sen and, (2) flow cytometry analysis demonstrated that anchorless PrP-sen was not present on the cell surface.¹⁶ Thus, the GPI anchor is not needed to target PrP-sen to a conversion friendly membrane environment.Consistent with the idea that the GPI anchor is not essential for PrP-res formation, in our studies anchorless PrP-sen could form PrP-res in cells acutely infected with scrapie despite the fact that it is processed differently than anchored PrP-sen, is not present on the cell surface (Fig. 1A), and is secreted.¹⁷ Persistent formation of anchorless PrP-res only occurred when both anchored and anchorless forms of PrP were expressed in the same cell.¹⁷ For this to happen both types of PrP must share a cellular compartment where PrP-res formation occurs, presumably either on the cell surface or in a specific location along the endocytic pathway^2,3 such as the endosomal recycling compartment.⁴ Analysis of infected and uninfected cells co-expressing Mo3F4 GPI-PrP-sen and wild-type PrP-sen demonstrated that Mo3F4 GPI-PrP-sen was not present on the cell surface (Fig. 1A). Thus, it is unlikely that GPI-PrP-res formation is occurring on the cell surface. We speculate that the anchored form of PrP-res encounters anchorless PrP-sen along either a secretory or endocytic pathway, allowing for the formation of anchorless PrP-res. Regardless of the precise location, the in vitro and in vivo data strongly suggest that the role of the anchor in persistent prion infection is not simply to localize PrP-sen to an environment compatible with PrP-res formation.However, the data are consistent with the idea that GPI anchored PrP is absolutely essential for the establishment of persistent infection in vitro. This is likely related to the spread of infectivity within a culture that is necessary for maintaining a persistent infection over time. Evidence suggests that PrP-res can be transferred between cells in a variety of ways including mother-daughter cell division,²⁵ cell-to-cell contact^26,27 and exosomes.²⁸ Tunneling nanotubes have also been hypothesized to be involved in intercellular prion spread¹⁹ and recent data suggest that spread can occur via these structures.²⁰ Any of these processes could involve the cell-to-cell transfer of PrP-res in membrane containing particles as has been observed in cell-free⁷ and cell-based systems.²⁹ If cell-to-cell contact were required, for example via simple physical proximity or perhaps tunneling nanotubes,^19,20 then the conversion of cell surface PrP-sen on the naïve cell by cell surface PrP-res on the infected cell would transfer infection to the naïve cell. In this instance, GPI membrane anchored, cell surface PrP-sen would be essential as it would allow for PrP-res formation on the cell surface. If spread is via cell division, then GPI-anchored, cell surface PrP-sen would be important for its role as a precursor to PrP-res formation.² In this instance, cell surface PrP-sen would be an essential intermediate in the continuous formation of PrP-res necessary for the accumulation and amplification of PrP-res within the cell. It would also help to cycle PrP between the cell surface and intracellular compartments where PrP-res can be formed.⁴ In either case, GPI-anchored PrP-sen would facilitate the accumulation of intracellular PrP-res to high enough levels to maintain both persistent infection in the mother cell and enable the transfer of organelles containing sufficient PrP-res to initiate infection in the daughter cell. Thus, we would suggest that efficient spread of infectivity requires not just the passive transfer of PrP-res from cell-to-cell but the concurrent initiation of conversion and amplification of PrP-res via cell surface, GPI anchored PrP-sen.In vivo, several lines of evidence suggest that the spread of scrapie infectivity also requires de novo PrP-res formation in the recipient cell and not simply transfer of PrP-res from one cell to another. For example, when neurografts from PrP expressing mice were placed in the brains of PrP knockout mice and the mice were challenged intracranially with scrapie, the graft showed scrapie pathology, but the surrounding tissue did not.³⁰ Furthermore, PrP-res from the graft migrated to the host tissue demonstrating that simple transfer of PrP-res was not sufficient and that PrP-sen expression was required for the spread of scrapie pathology.³⁰ In fact, these mice did not develop scrapie pathology following peripheral infection even when peripheral lymphoid tissues were reconstituted with PrP-sen expressing cells.³¹ Even though PrP-sen expressing cells were present in both the brain and spleen, in order for infectivity to spread from the lymphoreticular system to the central nervous system PrP-sen expression was also required in an intermediate tissue such as peripheral nerve.^31,32 Given that PrP-res uptake and trafficking do not require PrP-sen, the most obvious explanation for the requirement of PrP-sen in contiguous tissues is that de novo PrP-res formation in naïve cells is necessary for (1) infectivity to move from cell to cell within a tissue and, (2) infectivity to move from tissue to tissue.Another study demonstrated that peripheral expression of heterologous mouse PrP significantly increased the incubation time and actually prevented clinical disease in the majority of transgenic mice expressing hamster PrP in neurons of the brain.³³ Once again, if simple transfer and uptake of PrP-res were sufficient for spread, the presence of heterologous PrP molecules should not interfere because cellular uptake of PrP-res is independent of PrP-sen expression.^19,21–23 Clinical disease in these mice was likely prevented by the heterologous PrP molecule interfering with conversion of PrP-sen to PrP-res suggesting that prevention of de novo PrP-res formation inhibits spread of PrP-res and infectivity. These in vivo data, when combined with our recent in vitro data,¹⁷ provide evidence to support the importance of cell surface, and by extension GPI-anchored, PrP in the spread of prion infection.Our data demonstrate that the GPI anchor plays a role in the establishment of persistent scrapie infection in vitro. In our tissue culture system,²¹ as well as others where spread of infectivity by cell to cell contact appears to be limited,^25,34 the role of GPI anchored PrP-sen would be to amplify PrP-res to enable the efficient transfer of infectivity from mother to daughter cell. In cell systems where spread of prion infectivity may require cell to cell contact,^26,27 we propose that the role of GPI anchored PrP-sen is to facilitate the spread of prion infection via a chain of conversion from cell-to-cell, a “domino” type spread of infection that has been previously hypothesized.^35,36In vivo, such a mechanism might explain why neuroinvasion does not necessarily require axonal transport^32,37,38 and can occur independently of the axonal neurofilament machinery.³⁹ It would likely vary with cell type²⁷ and be most important in areas where infectivity is transferred from the periphery to the nervous system as well as in areas where cell division may be limited. It is also possible, if the location of PrP-res formation differs for different scrapie strains,⁴⁰ that the relative importance of a domino-like spread of infectivity in vivo would vary with the scrapie strain.Of course, spread of infectivity via a “wave” of GPI anchored, PrP mediated conversion would not preclude the spread of infectivity by other intracellular means such as axonal transport (reviewed in ref. ⁴¹). Furthermore, spread of infectivity may still also occur extracellularly such as in the unique case of mice which express anchorless PrP-sen,¹⁶ where our in vitro data would suggest that the cells themselves are not infected. In such a case, spread would require neither GPI anchored PrP-sen nor amplification of PrP-res in cells but would likely occur via other means such as blood⁴¹ or interstitial fluid flow.⁴²     

Conformational change, aggregation and fibril formation induced by detergent treatments of cellular prion protein

The conversion of protease-sensitive prion protein (PrP-sen) to a high beta-sheet, protease-resistant and often fibrillar form (PrP-res) is a central event in transmissible spongiform encephalopathies (TSE) or prion diseases. This conversion can be induced by PrP-res itself in cell-free conversion reactions. The detergent sodium N-lauroyl sarkosinate (sarkosyl) is a detergent that is widely used in PrP-res purifications and is known to stimulate the PrP-res-induced conversion reaction. Here we report effects of sarkosyl and other detergents on recombinant hamster PrP-sen purified from mammalian cells under oxidizing conditions that maintain the single native disulfide bond. Low concentrations of sarkosyl (0.001-0.1%) induced aggregation of PrP-sen molecules, increased light scattering, altered fluorescence excitation and emission spectra, and enhanced the proportion of beta-sheet secondary structure according to circular dichroism and infrared spectroscopies. An enhancement of beta-sheet content was also seen with 0.001% sodium dodecyl sulfate (SDS) but not several other types of detergents. Electron microscopy revealed that sarkosyl induced the formation of both amorphous and fibrillar aggregates. The fibrils appeared to be constructed from spherical bead-like protofibrils. Neither TSE infectivity nor the characteristic partial proteinase K resistance of PrP-res was detected in the sarkosyl-induced PrP aggregates. We conclude that certain anionic detergents can disrupt the conformation of PrP-sen and induce high beta-sheet aggregates that are distinct from scrapie-associated PrP-res in terms of protease-resistance, infrared spectrum and infectivity. These results reinforce the idea that not all high-beta aggregates of PrP are equivalent to the pathologic form, PrP-res.     

Octapeptide repeat insertions increase the rate of protease-resistant prion protein formation

A central feature of transmissible spongiform encephalopathies (TSE or prion diseases) involves the conversion of a normal, protease-sensitive glycoprotein termed prion protein (PrP-sen) into a pro-tease-resistant form, termed PrP-res. The N terminus of PrP-sen has five copies of a repeating eight amino acid sequence (octapeptide repeat). The presence of one to nine extra copies of this motif is associated with a heritable form of Creutzfeld-Jakob disease (CJD) in humans. An increasing number of octapeptide repeats correlates with earlier CJD onset, suggesting that the rate at which PrP-sen misfolds into PrP-res may be influenced by these mutations. In order to determine if octapeptide repeat insertions influence the rate at which PrP-res is formed, we used a hamster PrP amyloid-forming peptide (residues 23-144) into which two to 10 extra octapeptide repeats were inserted. The spontaneous formation of protease-resistant PrP amyloid from these peptides was more rapid in response to an increased number of octapeptide repeats. Furthermore, experiments using full-length glycosylated hamster PrP-sen demonstrated that PrP-res formation also occurred more rapidly from PrP-sen molecules expressing 10 extra copies of the octapeptide repeat. The rate increase for PrP-res formation did not appear to be due to any influence of the octapeptide repeat region on PrP structure, but rather to more rapid binding between PrP molecules. Our data from both models support the hypothesis that extra octapeptide repeats in PrP increase the rate at which protease resistant PrP is formed which in turn may affect the rate of disease onset in familial forms of CJD.     

Multiple amino acid residues within the rabbit prion protein inhibit formation of its abnormal isoform

Transmissible spongiform encephalopathies (TSEs) are neurological diseases that are associated with the conversion of the normal host-encoded prion protein (PrP-sen) to an abnormal protease-resistant form, PrP-res. Transmission of the TSE agent from one species to another is usually inefficient and accompanied by a prolonged incubation time. Species barriers to infection by the TSE agent are of particular importance given the apparent transmission of bovine spongiform encephalopathy to humans. Among the few animal species that appear to be resistant to infection by the TSE agent are rabbits. They survive challenge with the human kuru and Creutzfeldt-Jakob agents as well as with scrapie agent isolated from sheep or mice. Species barriers to the TSE agent are strongly influenced by the PrP amino acid sequence of both the donor and recipient animals. Here we show that rabbit PrP-sen does not form PrP-res in murine tissue culture cells persistently infected with the mouse-adapted scrapie agent. Unlike other TSE species barriers that have been studied, critical amino acid residues that inhibit PrP-res formation are located throughout the rabbit PrP sequence. Our results suggest that the resistance of rabbits to infection by the TSE agent is due to multiple rabbit PrP-specific amino acid residues that result in a PrP structure that is unable to refold to the abnormal isoform associated with disease.     

Factors affecting interactions between prion protein isoforms

Interactions between normal, protease-sensitive prion protein (PrP-sen or PrP(C)) and its protease-resistant isoform (PrP-res or PrP(Sc)) are critical in transmissible spongiform encephalopathy (TSE) diseases. To investigate the propagation of PrP-res between cells we tested whether PrP-res in scrapie brain microsomes can induce the conversion of PrP-sen to PrP-res if the PrP-sen is bound to uninfected raft membranes. Surprisingly, no conversion was observed unless the microsomal and raft membranes were fused or PrP-sen was released from raft membranes. These results suggest that the propagation of infection between cells requires transfer of PrP-res into the membranes of the recipient cell. To assess potential cofactors in PrP conversion, we used cell-free PrP conversion assays to show that heparan sulphate can stimulate PrP-res formation, supporting the idea that endogenous sulphated glycosaminoglycans can act as important cofactors or modulators of PrP-res formation in vivo. In an effort to develop therapeutics, the antimalarial drug quinacrine was identified as an inhibitor of PrP-res formation in scrapie-infected cell cultures. Confirmation of the latter result by others has led to the initiation of human clinical trials as a treatment for Creutzfeldt-Jakob disease. PrP-res formation can also be inhibited using a variety of other types of small molecule, specific synthetic PrP peptides, and an antiserum directed at the C-terminus of PrP-sen. The latter results help to localize the sites of interaction between PrP-sen and PrP-res. Disruption of those interactions with antibodies or peptidomimetic drugs may be an attractive therapeutic strategy. The likelihood that PrP-res inhibitors can rid TSE-infected tissues of PrP-res would presumably be enhanced if PrP-res formation were reversible. However, our attempts to measure dissociation of PrP-sen from PrP-res have failed under non-denaturing conditions. Finally, we have attempted to induce the spontaneous conversion of PrP-sen into PrP-res using low concentrations of detergents. A conformational conversion from alpha-helical monomers into high-beta-sheet aggregates and fibrils was induced by low concentrations of the detergent sarkosyl; however, the aggregates had neither infectivity nor the characteristic protease-resistance ofPrP-res.     

N-terminal truncation of prion protein affects both formation and conformation of abnormal protease-resistant prion protein generated in vitro

Transmissible spongiform encephalopathy diseases are characterized by conversion of the normal protease-sensitive host prion protein, PrP-sen, to an abnormal protease-resistant form, PrP-res. In the current study, deletions were introduced into the flexible tail of PrP-sen (23) to determine if this region was required for formation of PrP-res in a cell-free assay. PrP-res formation was significantly reduced by deletion of residues 34-94 relative to full-length hamster PrP. Deletion of another nineteen amino acids to residue 113 further reduced the amount of PrP-res formed. Furthermore, the presence of additional proteinase K cleavage sites indicated that deletion to residue 113 generated a protease-resistant product with an altered conformation. Conversion of PrP deletion mutants was also affected by post-translational modifications to PrP-sen. Conversion of unglycosylated PrP-sen appeared to alter both the amount and the conformation of protease-resistant PrP-res produced from N-terminally truncated PrP-sen. The N-terminal region also affected the ability of hamster PrP to block mouse PrP-res formation in scrapie-infected mouse neuroblastoma cells. Thus, regions within the flexible N-terminal tail of PrP influenced interactions required for both generating and disrupting PrP-res formation.     

Molecular basis of scrapie strain glycoform variation

Transmissible spongiform encephalopathies (TSE) are characterized by the conversion of a protease-sensitive host glycoprotein, prion protein or PrP-sen, to a protease-resistant form (PrP-res). PrP-res molecules that accumulate in the brain and lymphoreticular system of the host consist of three differentially glycosylated forms. Analysis of the relative amounts of the PrP-res glycoforms has been used to discriminate TSE strains and has become increasingly important in the differential diagnosis of human TSEs. However, the molecular basis of PrP-res glycoform variation between different TSE agents is unknown. Here we report that PrP-res itself can dictate strain-specific PrP-res glycoforms. The final PrP-res glycoform pattern, however, can be influenced by the cell and significantly altered by subtle changes in the glycosylation state of PrP-sen. Thus, strain-specific PrP-res glycosylation profiles are likely the consequence of a complex interaction between PrP-res, PrP-sen, and the cell and may indicate the cellular compartment in which the strain-specific formation of PrP-res occurs.     

Deletion of beta-strand and alpha-helix secondary structure in normal prion protein inhibits formation of its protease-resistant isoform

A fundamental event in the pathogenesis of transmissible spongiform encephalopathies (TSE) is the conversion of a normal, proteinase K-sensitive, host-encoded protein, PrP-sen, into its protease-resistant isoform, PrP-res. During the formation of PrP-res, PrP-sen undergoes conformational changes that involve an increase of beta-sheet secondary structure. While previous studies in which PrP-sen deletion mutants were expressed in transgenic mice or scrapie-infected cell cultures have identified regions in PrP-sen that are important in the formation of PrP-res, the exact role of PrP-sen secondary structures in the conformational transition of PrP-sen to PrP-res has not yet been defined. We constructed PrP-sen mutants with deletions of the first beta-strand, the second beta-strand, or the first alpha-helix and tested whether these mutants could be converted to PrP-res in both scrapie-infected neuroblastoma cells (Sc(+)-MNB cells) and a cell-free conversion assay. Removal of the second beta-strand or the first alpha-helix significantly altered both processing and the cellular localization of PrP-sen, while deletion of the first beta-strand had no effect on these events. However, all of the mutants significantly inhibited the formation of PrP-res in Sc(+)-MNB cells and had a greatly reduced ability to form protease-resistant PrP in a cell-free assay system. Thus, our results demonstrate that deletion of the beta-strands and the first alpha-helix of PrP-sen can fundamentally affect PrP-res formation and/or PrP-sen processing.     

Efficient conversion of normal prion protein (PrP) by abnormal hamster PrP is determined by homology at amino acid residue 155

In the transmissible spongiform encephalopathies, disease is closely associated with the conversion of the normal proteinase K-sensitive host prion protein (PrP-sen) to the abnormal proteinase K-resistant form (PrP-res). Amino acid sequence homology between PrP-res and PrP-sen is important in the formation of new PrP-res and thus in the efficient transmission of infectivity across species barriers. It was previously shown that the generation of mouse PrP-res was strongly influenced by homology between PrP-sen and PrP-res at amino acid residue 138, a residue located in a region of loop structure common to PrP molecules from many different species. In order to determine if homology at residue 138 also affected the formation of PrP-res in a different animal species, we assayed the ability of hamster PrP-res to convert a panel of recombinant PrP-sen molecules to protease-resistant PrP in a cell-free conversion system. Homology at amino acid residue 138 was not critical for the formation of protease-resistant hamster PrP. Rather, homology between PrP-sen and hamster PrP-res at amino acid residue 155 determined the efficiency of formation of a protease-resistant product induced by hamster PrP-res. Structurally, residue 155 resides in a turn at the end of the first alpha helix in hamster PrP-sen; this feature is not present in mouse PrP-sen. Thus, our data suggest that PrP-res molecules isolated from scrapie-infected brains of different animal species have different PrP-sen structural requirements for the efficient formation of protease-resistant PrP.     

Effect of glycosylphosphatidylinositol anchor-dependent and -independent prion protein association with model raft membranes on conversion to the protease-resistant isoform

Prion protein (PrP) is usually bound to membranes by a glycosylphosphatidylinositol (GPI) anchor that associates with detergent-resistant membranes, or rafts. To examine the effect of membrane association on the interaction between the normal protease-sensitive PrP isoform (PrP-sen) and the protease-resistant isoform (PrP-res), a model system was employed using PrP-sen reconstituted into sphingolipid-cholesterol-rich raft-like liposomes (SCRLs). Both full-length (GPI(+)) and GPI anchor-deficient (GPI(-)) PrP-sen produced in fibroblasts stably associated with SCRLs. The latter, alternative mode of membrane association was not detectably altered by glycosylation and was markedly reduced by deletion of residues 34-94. The SCRL-associated PrP molecules were not removed by treatments with either high salt or carbonate buffer. However, only GPI(+) PrP-sen resisted extraction with cold Triton X-100. PrP-sen association with SCRLs was pH-independent. PrP-sen was also one of a small subset of phosphatidylinositol-specific phospholipase C (PI-PLC)-released proteins from fibroblast cells found to bind SCRLs. A cell-free conversion assay was used to measure the interaction of SCRL-bound PrP-sen with exogenous PrP-res as contained in microsomes. SCRL-bound GPI(+) PrP-sen was not converted to PrP-res until PI-PLC was added to the reaction or the combined membrane fractions were treated with the membrane-fusing agent polyethylene glycol (PEG). In contrast, SCRL-bound GPI(-) PrP-sen was converted to PrP-res without PI-PLC or PEG treatment. Thus, of the two forms of raft membrane association by PrP-sen, only the GPI anchor-directed form resists conversion induced by exogenous PrP-res.     

Evidence of a molecular barrier limiting susceptibility of humans, cattle and sheep to chronic wasting disease

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of deer and elk, and little is known about its transmissibility to other species. An important factor controlling interspecies TSE susceptibility is prion protein (PrP) homology between the source and recipient species/genotypes. Furthermore, the efficiency with which the protease-resistant PrP (PrP-res) of one species induces the in vitro conversion of the normal PrP (PrP-sen) of another species to the protease-resistant state correlates with the cross-species transmissibility of TSE agents. Here we show that the CWD-associated PrP-res (PrP(CWD)) of cervids readily induces the conversion of recombinant cervid PrP-sen molecules to the protease-resistant state in accordance with the known transmissibility of CWD between cervids. In contrast, PrP(CWD)-induced conversions of human and bovine PrP-sen were much less efficient, and conversion of ovine PrP-sen was intermediate. These results demonstrate a barrier at the molecular level that should limit the susceptibility of these non-cervid species to CWD.     

Filipin prevents pathological prion protein accumulation by reducing endocytosis and inducing cellular PrP release

Conversion of the normal membrane-bound prion protein (PrP-sen) to its pathological isoform (PrP-res) is a key event in the pathogenesis of transmissible spongiform encephalopathies. Although the subcellular sites of conversion are poorly characterized, several lines of evidence have suggested the involvement of membrane lipid rafts in the conversion process. Here we report that copper stimulates the endocytosis of PrP-sen via a caveolin-dependent pathway in both microglia and neuroblastoma cells. We show that the polyene antibiotic filipin both limits endocytosis of PrP-sen and dramatically reduces the amount of membrane-bound PrP-sen. This reduction results from a rapid and massive release of full matured PrP-sen into the culture medium. Finally, we demonstrate that filipin is a potent inhibitor of PrP-res formation into chronically infected neuroblastoma cells. Our results reinforce the role of rafts in PrP trafficking and raise the possibility that the release of PrP-sen from the plasma membrane decreases the amount of available substrate PrP-sen at the conversion sites.     

Specific binding of normal prion protein to the scrapie form via a localized domain initiates its conversion to the protease-resistant state.

In the transmissible spongiform encephalopathies, normal prion protein (PrP-sen) is converted to a protease-resistant isoform, PrP-res, by an apparent self-propagating activity of the latter. Here we describe new, more physiological cell-free systems for analyzing the initial binding and subsequent conversion reactions between PrP-sen and PrP-res. These systems allowed the use of antibodies to map the sites of interaction between PrP-sen and PrP-res. Binding of antibodies (alpha219-232) to hamster PrP-sen residues 219-232 inhibited the binding of PrP-sen to PrP-res and the subsequent generation of PK-resistant PrP. However, antibodies to several other parts of PrP-sen did not inhibit. The alpha219-232 epitope itself was not required for PrP-res binding; thus, inhibition by alpha219-232 was likely due to steric blocking of a binding site that is close to, but does not include the epitope in the folded PrP-sen structure. The selectivity of the binding reaction was tested by incubating PrP-res with cell lysates or culture supernatants. Only PrP-sen was observed to bind PrP-res. This highly selective binding to PrP-res and the localized nature of the binding site on PrP-sen support the idea that PrP-sen serves as a critical ligand and/or receptor for PrP-res in the course of PrP-res propagation and pathogenesis in vivo.     

Conversion of raft associated prion protein to the protease-resistant state requires insertion of PrP-res (PrP(Sc)) into contiguous membranes

Prion protein (PrP) is usually attached to membranes by a glycosylphosphatidylinositol-anchor that associates with detergent-resistant membranes (DRMs), or rafts. To model the molecular processes that might occur during the initial infection of cells with exogenous transmissible spongiform encephalopathy (TSE) agents, we examined the effect of membrane association on the conversion of the normal protease-sensitive PrP isoform (PrP-sen) to the protease-resistant isoform (PrP-res). A cell-free conversion reaction approximating physiological conditions was used, which contained purified DRMs as a source of PrP-sen and brain microsomes from scrapie-infected mice as a source of PrP-res. Interestingly, DRM-associated PrP-sen was not converted to PrP-res until the PrP-sen was either released from DRMs by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), or the combined membrane fractions were treated with the membrane-fusing agent polyethylene glycol (PEG). PEG-assisted conversion was optimal at pH 6--7, and acid pre-treating the DRMs was not sufficient to permit conversion without PI-PLC or PEG, arguing against late endosomes/lysosomes as primary compartments for PrP conversion. These observations raise the possibility that generation of new PrP-res during TSE infection requires (i) removal of PrP-sen from target cells; (ii) an exchange of membranes between cells; or (iii) insertion of incoming PrP-res into the raft domains of recipient cells.     

Inhibition of interactions and interconversions of prion protein isoforms by peptide fragments from the C-terminal folded domain

The formation of protease-resistant prion protein (PrP-res or PrP(Sc)) involves selective interactions between PrP-res and its normal protease-sensitive counterpart, PrP-sen or PrP(C). Previous studies have shown that synthetic peptide fragments of the PrP sequence corresponding to residues 119-136 of hamster PrP (Ha119-136) can selectively block PrP-res formation in cell-free systems and scrapie-infected tissue culture cells. Here we show that two other peptides corresponding to residues 166-179 (Ha166-179) and 200-223 (Ha200-223) also potently inhibit the PrP-res induced cell-free conversion of PrP-sen to the protease-resistant state. In contrast, Ha121-141, Ha180-199, and Ha218-232 were much less effective as inhibitors. Mechanistic analyses indicated that Ha166-179, Ha200-223, and peptides containing residues 119-136 inhibit primarily by binding to PrP-sen and blocking its binding to PrP-res. Circular dichroism analyses indicated that Ha117-141 and Ha200-223, but not non-inhibitory peptides, readily formed high beta-sheet structures when placed under the conditions of the conversion reaction. We conclude that these inhibitory peptides may mimic contact surfaces between PrP-res and PrP-sen and thereby serve as models of potential therapeutic agents for transmissible spongiform encephalopathies.     

Sulfated glycans and elevated temperature stimulate PrP(Sc)-dependent cell-free formation of protease-resistant prion protein

A conformational conversion of the normal, protease- sensitive prion protein (PrP-sen or PrP(C)) to a protease-resistant form (PrP-res or PrP(Sc)) is commonly thought to be required in transmissible spongiform encephalopathies (TSEs). Endogenous sulfated glycosaminoglycans are associated with PrP-res deposits in vivo, suggesting that they may facilitate PrP-res formation. On the other hand, certain exogenous sulfated glycans can profoundly inhibit PrP-res accumulation and serve as prophylactic anti-TSE compounds in vivo. To investigate the seemingly paradoxical effects of sulfated glycans on PrP-res formation, we have assayed their direct effects on PrP conversion under physiologically compatible cell-free conditions. Heparan sulfate and pentosan polysulfate stimulated PrP-res formation. Conversion was stimulated further by increased temperature. Both elevated temperature and pentosan polysulfate promoted interspecies PrP conversion. Circular dichroism spectropolarimetry measurements showed that pentosan polysulfate induced a conformational change in PrP-sen that may potentiate its PrP-res-induced conversion. These results show that certain sulfated glycosaminoglycans can directly affect the PrP conversion reaction. Therefore, depending upon the circumstances, sulfated glycans may be either cofactors or inhibitors of this apparently pathogenic process.     

Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein

The scrapie prion protein isoform, PrPSc, is a prion-associated marker that seeds the conformational conversion and polymerization of normal protease-sensitive prion protein (PrP-sen). This seeding activity allows ultrasensitive detection of PrPSc using cyclical sonicated amplification (PMCA) reactions and brain homogenate as a source of PrP-sen. Here we describe a much faster seeded polymerization method (rPrP-PMCA) which detects >or=50 ag of hamster PrPSc (approximately 0.003 lethal dose) within 2-3 d. This technique uses recombinant hamster PrP-sen, which, unlike brain-derived PrP-sen, can be easily concentrated, mutated and synthetically tagged. We generated protease-resistant recombinant PrP fibrils that differed from spontaneously initiated fibrils in their proteolytic susceptibility and by their infrared spectra. This assay could discriminate between scrapie-infected and uninfected hamsters using 2-microl aliquots of cerebral spinal fluid. This method should facilitate the development of rapid, ultrasensitive prion assays and diagnostic tests, in addition to aiding fundamental studies of structure and mechanism of PrPSc formation.