Protease-resistant prion protein amplification reconstituted with partially purified substrates and synthetic polyanions.

Little is currently known about the biochemical mechanism by which induced prion protein (PrP) conformational change occurs during mammalian prion propagation. In this study, we describe the reconstitution of PrPres amplification in vitro using partially purified and synthetic components. Overnight incubation of purified PrP27-30 and PrPC molecules at a molar ratio of 1:250 yielded approximately 2-fold baseline PrPres amplification. Addition of various polyanionic molecules increased the level of PrPres amplification to approximately 10-fold overall. Polyanionic compounds that stimulated purified PrPres amplification to varying degrees included synthetic, homopolymeric nucleic acids such as poly(A) and poly(dT), as well as non-nucleic acid polyanions, such as heparan sulfate proteoglycan. Size fractionation experiments showed that synthetic poly(A) polymers must be >0.2 kb in length to stimulate purified PrPres amplification. Thus, one possible set of minimal components for efficient conversion of PrP molecules in vitro may be surprisingly simple, consisting of PrP27-30, PrPC, and a stimulatory polyanionic compound.


From the Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755
Little is currently known about the biochemical mechanism by which induced prion protein (PrP) conformational change occurs during mammalian prion propagation. In this study, we describe the reconstitution of PrPres amplification in vitro using partially purified and synthetic components. Overnight incubation of purified PrP27-30 and PrP C molecules at a molar ratio of 1:250 yielded ϳ2-fold baseline PrPres amplification. Addition of various polyanionic molecules increased the level of PrPres amplification to ϳ10-fold overall. Polyanionic compounds that stimulated purified PrPres amplification to varying degrees included synthetic, homopolymeric nucleic acids such as poly(A) and poly(dT), as well as non-nucleic acid polyanions, such as heparan sulfate proteoglycan. Size fractionation experiments showed that synthetic poly(A) polymers must be >0.2 kb in length to stimulate purified PrPres amplification. Thus, one possible set of minimal components for efficient conversion of PrP molecules in vitro may be surprisingly simple, consisting of PrP27-30, PrP C , and a stimulatory polyanionic compound.
Transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease, bovine spongiform encephalopathy (BSE), 1 chronic wasting disease, and scrapie are fatal infectious diseases of the central nervous system with an unusual etiology. Many biochemical and biophysical experiments have shown that the infectious agents of transmissible spongiform encephalopathies, termed prions, lack informational nucleic acids (1). Furthermore, the replication of infectious prions in vivo and in cultured cells is generally accompanied by the transformation of the normal cellular isoform of a neuronal membrane protein (PrP C ) into a protease-resistant state (PrP Sc or PrPres) (2,3), and PrPres co-purifies with prion infectivity (4). These observations can be explained by the protein-only hypothesis, which contends that infectious prions are exclusively composed of misfolded PrP molecules such as PrPres (5). In strong support of this hypothesis, Legname et al. (6) recently demonstrated the generation of infectious prions in vitro by refolding purified recombinant PrP.
Little is known about the molecular mechanism that mediates the self-propagating conversion of PrP C to PrPres. Several investigators have used a biochemical approach to investigate this process in vitro. Caughey and colleagues (7)(8)(9)(10) developed the first successful cell-free PrP conversion system by using a radiolabeled PrP substrate and showed that the efficiency of PrP C to PrPres conversion in vitro was dependent upon PrP sequence and prion strain in a manner that precisely modeled the specificity of transmissible spongiform encephalopathy transmission in vivo. Later, Saborio and Soto (11) developed the protein-misfolding cyclic amplification technique, in which crude brain homogenates are intermittently sonicated to generate much more efficient conversion of PrP C to PrPres than the radiolabel technique, resulting in high level amplification of PrPres levels. Even without sonication, a mixture of crude normal and diluted scrapie brain homogenates generates Ͼ6fold amplification of PrPres after overnight incubation (11,12). In contrast, a 50-fold stoichiometric excess of PrPres template is required to drive the conversion of radiolabeled PrP C molecules in a cell-free system containing only purified prion proteins (9). This discrepancy in the efficiencies of crude versus purified systems suggests that factors other than PrP molecules are required for efficient PrP conversion in vitro, and that crude brain homogenates contain such factors. Furthermore, several genetic and biochemical experiments have provided evidence for the existence of PrP conversion cofactors (13,14). Deleault et al. (15) demonstrated that treatment of crude brain homogenates with RNase abolished PrPres amplification in vitro and that efficient PrPres amplification could be reconstituted by addition of exogenous mammalian RNA. These results showed that RNA molecules within crude brain homogenates promote efficient PrPres amplification in non-purified systems such as protein-misfolding cyclic amplification. However, studies in crude homogenates cannot determine whether RNA molecules alone are sufficient to drive efficient PrPres amplification, because it is possible that other factors within the homogenates are also required for the amplification process. Furthermore, it is not known whether RNA molecules stimulate PrPres amplification directly by binding to prion proteins, or indirectly, for instance by sequestering a reaction inhibitor or activating a catalyst (16). To investigate the molecular requirements for PrPres amplification in vitro, we developed a series of protocols to isolate PrP C and PrP Sc molecules under * This work was supported by the Burroughs Wellcome Fund and by National Institutes of Health Grants AI058979 and NS046478. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
conditions that preserve their ability to reconstitute efficient PrPres amplification in vitro. Using these preparations, we have generated and characterized an efficient in vitro PrPres amplification system using only purified and synthetic components. Our results show that one possible set of minimal components for efficient amplification of PrPres in vitro may be surprisingly simple, consisting of PrP C , PrP Sc , and a polyanionic compound.
All synthetic polynucleotides were purchased from Sigma. The size distributions of these various commercial preparations were determined by a combination of agarose gel electrophoresis and size exclusion high performance liquid chromatography techniques (data not shown). The preparations assayed were: poly(A) catalog number P9403 (0.2-6 kb by agarose gel electrophoresis), poly(C) catalog number P4903 (ϳ6 kb by size exclusion high performance liquid chromatography), poly(G) catalog number P4404 (ϳ0.2 kb by agarose gel electrophoresis), poly(U) catalog number P9528, (0.3-1 kb by size exclusion high performance liquid chromatography), poly(dA) catalog number P0087 (ϳ1.5-4 kb by agarose gel electrophoresis), poly(dT) catalog number P6905 (ϳ1.5-4 kb by agarose gel electrophoresis), and poly(dC) catalog number P5444 (0.39 kb, according to manufacturer). Stock solutions of synthetic polynucleotides were prepared in 1ϫ TE pH 8.0, and concentrations were confirmed by A 260 nm .
Preparation of IgG Cross-linked Protein A-Agarose Beads-All procedures were performed at room temperature. Four hundred microliters of ImmunoPure Immobilized Protein A Plus 50% slurry (Pierce) was mixed with 16 g IgG per microliter of packed resin for 2 h. Following incubation, agarose beads were recovered by centrifugation at 1000 ϫ g for 1 min and washed twice with 1 ml of 200 mM triethanolamine, pH 8.0 (Acros Organics, Geel, Belgium). Antibodies were cross-linked by incubation in 1 ml of 10 mM dimethyl pimelimidate hydrochloride (Pierce), 200 mM triethanolamine, pH 8.0, for 30 min. The reaction was quenched by the addition of 50 l of 1 M Tris, pH 8.0, and beads were recovered by centrifugation at 1000 ϫ g for 1 min. Cross-linked beads were then washed three times, once in phosphate-buffered saline without calcium or magnesium (PBS), 1% Triton X-100, and twice in PBS. Beads were resuspended in 200 l of PBS and stored at 4°C.
Immunopurification of PrP C from Hamster Brain-All procedures were performed at 4°C. Four brains, including cerebellum and brainstem, from 8-to 12-week-old specific-pathogen-free Golden Syrian hamsters of either sex were homogenized in 10 volumes (w/v) of ice-cold PBS plus Complete® protease inhibitors (Roche Applied Science) using a Biohomogenizer Mixer (Biospec Products, Bartlesville, OK) at 7,000 rpm for 30 -60 s. The homogenate was centrifuged at 3,200 ϫ g for 20 min, and the pellet was resuspended in 40 ml of PBS, 1% sodium deoxycholate, 1% Triton X-100, and Complete® protease inhibitors using a Wheaton glass Dounce homogenizer (10 strokes with pestle B). The sample was incubated on ice for 30 min and then centrifuged at 100,000 ϫ g for 30 min. The solubilized supernatant was removed and placed into a 50-ml conical tube with 400 l of either D13 or 3F4 cross-linked protein A-agarose beads (50% slurry) and incubated endover-end for 2 h. Beads were centrifuged at 1,000 ϫ g for 2 min, and the supernatant was discarded. The beads were then washed once with 50 ml of Immunopure Gentle Ag/Ab Binding Buffer (Pierce), transferred to a microcentrifuge tube, and washed in 1 ml of the same buffer. The beads were eluted twice with 500 l of Immunopure Gentle Ag/Ab Elution Buffer (Pierce). The two eluate volumes were combined, diluted with 47 ml of IMAC-CuSO 4 wash buffer (20 mM MOPS, pH 7.0, 0.15 M NaCl, 10 mM imidazole, 1% Triton) plus EDTA-free Complete® protease inhibitors (Roche Applied Science), incubated with 2 ml of pre-equilibrated IMAC-CuSO 4 resin (Amersham Biosciences) on an end-over-end rotator for 30 min, and centrifuged for 2 min at 1,000 ϫ g. The supernatant was removed and discarded, and the resin was washed twice in 50 ml of IMAC-CuSO 4 wash buffer. PrP C was then eluted in 6 ml of IMAC-CuSO 4 elution buffer (20 mM MOPS pH 7.5, 0.15 M NaCl, 0.15 M imidazole, 1% Triton X-100) containing EDTA-free Complete® protease inhibitors. Typically, the yield of PrP C was ϳ10-fold higher when crosslinked 3F4 beads were used than when cross-linked D13 beads were used.
Purification of PrP C from Hamster Brain by Conventional Affinity Chromatography-An alternative protocol for purifying PrP C from hamster brain was based on a modification of the method described by Pan et al. (17,18). All procedures were performed at 4°C. Ten hamster brains were homogenized in 5 volumes (w/v) of ice-cold PBS with Com-plete® protease inhibitors using a Potter homogenizer. The homogenate was centrifuged at 100 ϫ g for 30 s, and the post-nuclear supernatant was removed and centrifuged at 3,200 ϫ g for 20 min. The resulting pellet was resuspended in 45 ml of 20 mM MOPS, pH 7.0, 0.15 M NaCl, 1% sodium deoxycholate, 1% Triton X-100, 10 mM imidazole, containing EDTA-free Complete® protease inhibitors, homogenized with a Dounce homogenizer, and incubated on ice for 30 min to allow for membrane solubilization. The solubilized homogenate was centrifuged at 100,000 ϫ g for 30 min, and the supernatant was applied to a preequilibrated 2-ml IMAC-CuSO 4 column (Amersham Biosciences). The column was washed with 20 ml of IMAC-CuSO 4 wash buffer and eluted with10 ml of IMAC-CuSO 4 elution buffer containing EDTA-free Com-plete® protease inhibitors. The eluate was applied to a pre-equilibrated 2-ml wheat germ agglutinin column (Vector Laboratories, Burlingame, CA), washed with 20 ml of 20 mM MOPS, pH 7.5, 0.15 M NaCl, 1% Triton X-100, and eluted with 10 ml of 20 mM MOPS, pH 7.5, 0.15 M NaCl, 50 mM N-acetylglucosamine, 1% Triton X-100 containing EDTA-free Com-plete® protease inhibitors.
Preparation of PrP 27-30 -Post-nuclear brain supernatants were prepared from Sc237 scrapie-infected brains as previously described (12). One milliliter of Sc237 post-nuclear brain supernatant (0.4% w/v) in PBS-1% Triton X-100 was incubated with 10 g/ml Proteinase K (PK, specific activity, 30 units/mg, Roche Applied Science) for 30 min at 37°C. Protease digestion was terminated by addition of 5 mM phenylmethylsulfonyl fluoride (from a 0.3 M stock solution in methanol). The digested sample was centrifuged for 1 h at 100,000 ϫ g at 4°C, and resuspended in 100 l of ice-cold PBS-1% Triton X-100 by 10 ϫ 5 s pulses of direct sonication with a Bandelin Sonopuls ultrasonicator delivering ϳ55% power to the probe tip (Amtrex Technologies, Saint-Laurent, Canada). After addition of 100 l of ice cold PBS-1% Triton X-100, the sample was sonicated for an additional 10 pulses as above. An additional 800 l of ice-cold PBS-1% Triton X-100 was added, and the sample was centrifuged at 100,000 ϫ g for 30 min at 4°C. The pellet was subjected to another identical round of resuspension and sonication to generate the final 1 ml of sample containing ϳ10 ng/ml PrP27-30 in PBS plus 1% Triton X-100.
In Vitro Amplification of PrPres-For experiments testing purified or synthetic compounds, each 100-l amplification reaction contained 2.5 g/ml PrP C and 10 ng/ml PrP27-30 in 0.75ϫ PBS, 0.25ϫ TE, 0.75% Triton X-100, 2 mM EDTA. In experiments testing the effects of Prnp 0/0 mouse brain homogenate, each sample contained 5 g/ml PrP C and 10 ng/ml PrP27-30 in PBS, 0.75% Triton X-100, 2 mM EDTA plus 2.5% (w/v) Prnp 0/0 mouse brain post-nuclear supernatant or buffer. Samples were mixed at 37°C and shaken overnight at 800 rpm (Eppendorf Thermomixer, Fisher Scientific). To detect PrPres, each sample was incubated with 60 g/ml PK for 40 min at 37°C, boiled in SDS sample buffer, and subjected to Western blotting using 3F4 monoclonal antibody as described previously (12).
Quantitation of PrP C , PrP27-30, and Western Blot Signals-PrP C and PrP27-30 were quantified by comparing dilutions of these preparations against known amounts of recombinant PrP C on Western blots (Prionics, Schlieren, Switzerland). Densitometric measurement of membrane marker film signals was performed through the analysis of multiple film exposures to ensure that comparisons were made within the linear range of the film. Signals within the linear range were quantified using the histogram functions in Adobe Photoshop and calibrated against the background signal. Serial dilutions of normal hamster brain were used to calibrate densitometric measurements.
RNA Purification-All total RNA preparations were isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Poly(A) ϩ RNA was isolated from hamster liver total RNA using the Poly(A) Purist Kit (Ambion), and the sample that did not bind to the oligo(dT) column was designated Poly(A) Ϫ RNA. All RNA preparations were resuspended in 1ϫ TE, pH 8.0, and the concentration and quality of each preparation were determined by agarose gel electrophoresis and by measuring A 260 nm /A 280 nm .
Size Fractionation of Poly(A)-Synthetic poly(A) (Sigma catalog number P9403) was resuspended in 1ϫ TE, pH 8.0, and the concentration was confirmed by A 260 nm . A sample containing 200 g of this preparation was electrophoresed on a 1% agarose gel. Unstained slices corresponding to different mobility ranges were excised and extracted using a gel-extraction kit (Qiagen, Valencia, CA). Poly(A) 45-, 25-, and 10-mer oligonucleotides were purchased from IDT (Coralville, IA) and resuspended in 1ϫ TE, pH 8.0. Concentrations of each poly(A) fraction were determined by A 260 nm .

RESULTS
Because post-translational modifications of PrP C might affect its ability to convert efficiently to PrPres, we chose to purify mature, mammalian PrP C directly from normal brain tissue using detergent solubilization (19). To ensure that our results did not depend upon any peculiarities of one purification method, we developed two different protocols to generate PrP C molecules capable of undergoing efficient conversion to PrPres (see "Experimental Procedures"). In the first protocol, adapted from the method of Pan et al. (17,18), PrP C molecules were purified from solubilized brain membranes by sequential adsorption to copper and lectin affinity columns. This procedure produced a preparation that contained conversion-competent PrP C molecules, but also contained many contaminating proteins, resulting in Ͻ10% purity (data not shown). In the second protocol, PrP C was immunopurified using immobilized anti-PrP antibodies. This procedure generates samples containing PrP C with ϳ50% purity; we identified one major contaminant as the immunoglobulin heavy chain (Fig. 1A). The three glycoforms of PrP C are identifiable as bands with molecular masses in the range from 30 to 33 kDa. As expected, these bands are more abundant in a sample purified from Tga20 transgenic PrP-overexpressing mice and absent from a sample prepared from Prnp 0/0 mice (Fig. 1A).
A preparation of immunopurified hamster PrP C was incubated overnight with purified Syrian hamster Sc237 PrP27-30 template at a molar ratio of 250:1, and PrPres amplification was measured. Co-incubation of these two purified prion proteins alone yielded ϳ2-fold amplification of PrPres (Fig. 1B). Addition of Prnp 0/0 mouse brain homogenate lacking PrP C to the mixture of purified proteins increased the PrPres amplification level to ϳ10-fold (Fig. 1B), similar to the level of PrPres amplification in crude brain homogenates (12). In control reactions, Prnp 0/0 brain homogenate did not affect the protease resistance of either PrP C or PrP27-30 molecules in isolation (Fig. 1B). These results confirm that crude brain homogenates contain one or more cofactor(s) that promote the efficiency of PrPres amplification.
We previously found that RNA molecules are required for efficient PrPres amplification in crude brain homogenates (15). Therefore, we analyzed whether isolated RNA molecules might stimulate PrPres amplification from purified prion proteins. We found that addition of total hamster liver RNA to a mixture of PrP27-30 and immunopurified PrP C molecules yielded ϳ10fold PrPres amplification (Fig. 1C). This result indicates that RNA molecules can act directly upon prion proteins without intermediary molecules and that purified PrP C , PrP Sc , and RNA molecules are sufficient reconstitute PrPres amplification to the same level as crude brain homogenates. The efficiency of purified PrPres amplification stimulated by RNA is further increased by protein-misfolding cyclic amplification (11), resulting in Ͼ20-fold total PrPres amplification after 24 cycles, which again is similar to the level of PrPres amplification obtained with protein-misfolding cyclic amplification using reconstituted brain homogenate (Supplemental Fig. S1).
In crude brain homogenates, RNA concentrations between 100 and 500 g/ml stimulate PrPres amplification in a speciesspecific manner (15). For example, addition of total RNA prepared from hamster or mouse tissues increases PrPres ampli-fication in crude brain homogenates, but addition of the total RNA prepared from a variety of non-mammalian species, such as Caenorhabditis elegans and Escherichia coli, to crude homogenates does not affect amplification levels. To study the species specificity and potency of RNA stimulation in our purified system, we tested the ability of varying concentrations of total RNA prepared from a variety of species to stimulate purified PrPres amplification. Unexpectedly, we found that total RNA prepared from every species tested, including C. elegans and E. coli, potently stimulated PrPres amplification in our purified system (Fig. 2). For each preparation, the threshold RNA concentration for stimulation of purified PrPres amplification was ϳ1 g/ml, and stimulation was optimal at an RNA concentration of ϳ10 g/ml (Fig. 2). In contrast, the threshold concen-FIG. 1. Reconstitution of PrPres amplification using purified PrP molecules. A, silver stain of purified PrP C preparation. Brain homogenates prepared from normal Syrian hamsters, Tga20 mice overexpressing mouse PrP C (45), and Prnp 0/0 mice lacking PrP (Ablated) (46) were immunopurified using cross-linked D13 beads as described under "Experimental Procedures" (similar results were obtained with cross-linked 3F4 beads, data not shown). Samples were concentrated by the method of Wessel and Flugge (47) for SDS-PAGE. A control sample of hamster brain homogenate was mock purified using unconjugated Protein A-agarose beads (Protein A). A sample of crude solubilized Syrian brain homogenate (Crude) is shown for comparison. B, Western blot of PrPres amplification assays using purified PrP molecules and Prnp 0/0 (Ablated) brain post-nuclear supernatant. Samples containing the indicated components were incubated for 16 h at 37°C and subjected to proteinase K digestion except where indicated (-PK), as described under "Experimental Procedures." Protease-digested samples were prepared in duplicate. C, Western blot of PrPres amplification assays using purified PrP molecules and total hamster liver RNA. Samples containing the indicated components were incubated for 16 h at 37°C and subjected to proteinase K digestion, except where indicated (-PK), as described under "Experimental Procedures." Total RNA was used at a concentration of 10 g/ml. Protease-digested samples were prepared in duplicate. tration of total hamster liver RNA required to stimulate PrPres amplification in crude homogenates is ϳ100 g/ml, and the concentration required for optimal stimulation is ϳ500 g/ml (data not shown). Thus, RNA stimulation of PrPres amplification is both more potent and less specific in the purified system than in homogenate mixtures.
These results suggest that no specific RNA species is uniquely responsible for stimulating purified PrPres amplification. To confirm this hypothesis, we compared the potencies of poly(A) ϩ and poly(A) Ϫ RNA for stimulation of purified PrPres amplification. The results indicate that, despite ϳ100fold enrichment of mRNA molecules in the poly(A) ϩ fraction compared with the poly(A) Ϫ fraction, the two preparations stimulated purified PrPres amplification with equal potency (Supplemental Fig. S2). In addition, we found no difference in stimulation potency between brain and liver total RNA, indicating that stimulatory RNA molecules are not specifically enriched in brain tissue (data not shown).
Based on these results, we speculated that perhaps a broad range of polyanions might stimulate purified PrPres amplification, and therefore we tested a variety of pure compounds for their ability to stimulate PrPres amplification. We first assayed several commercially available preparations of synthetic homopolymeric nucleotides with overlapping size distributions. Among the compounds tested, poly(A) and poly(dT) stimulated PrPres amplification at a concentration of 1 g/ml; poly(dA) stimulated PrPres amplification at a concentration of 100 g/ ml; and poly(C) failed to stimulate PrPres amplification at all concentrations tested (Fig. 3). Double-stranded plasmid DNA also stimulated amplification at a concentration of ϳ10 g/ml (Supplemental Fig. S3). Taken together, these results indicate that RNA, single-stranded DNA, and double-stranded DNA molecules can stimulate purified PrPres amplification and that homopolymeric nucleotide preparations with overlapping size distributions differ in their ability to stimulate purified PrPres amplification, according to the rank order poly(A) ϭ poly(dT) Ͼ poly(dA) Ͼ poly(C).
To study in isolation the effect of polynucleotide size upon stimulatory activity, we tested discrete size fractions of poly(A) for their ability to stimulate PrPres amplification. The results indicate that poly(A) oligonucleotides Յ 45 bases in length were unable to stimulate PrPres amplification; a fraction containing poly(A) polymers between 0.2 and 0.4 kb in length partially stimulated PrPres amplification; and poly(A) polymers Ͼ 4 kb in length strongly stimulated PrPres amplification (Fig. 4). Furthermore, monomeric nucleotides did not stimulate PrPres amplification at concentrations between 1 ng/ml and 100 g/ml (Fig. 6). Taken together, these results indicate that the threshold size required for stimulation of purified PrPres amplification by poly(A) is ϳ300 bases. Consistent with this estimate of threshold size, a uniform preparation of poly(G)-containing polymers ϳ0.2 kb in length did not stimulate PrPres amplification, whereas a preparation of poly(U)-containing polymers 0.39 kb in length potently stimulated PrPres amplification (Supplemental Fig. S3).
Several independent lines of investigation have implicated proteoglycans and glycosaminoglycans, particularly heparin sulfate proteoglycan (HSPG), in the pathogenesis of prion diseases (20 -22), and Wong et al. (23) showed that heparan sulfate and pentosan sulfate stimulate cell-free conversion of radiolabeled PrP. Therefore, we tested the compounds heparan sulfate (molecular mass, ϳ12-14 kDa), pentosan sulfate, and HSPG (molecular mass, Ͼ400 kDa) for their ability to stimulate purified PrPres amplification. Pentosan sulfate and HSPG stimulated PrPres amplification only moderately at a concentration of 100 g/ml, whereas heparan sulfate had no effect (Fig. 5). Other investigators have shown that copper affects the affinity of heparin binding to PrP (24), and therefore we also measured heparan sulfate stimulation of purified PrPres amplification in the presence of copper. We found no apparent stimulation of PrPres amplification by heparan sulfate in the presence of 1-100 M CuCl 2 (data not shown). The levels of stimulation induced by pentosan sulfate and HSPG were both Ͻ30% the level of control stimulation by total hamster liver RNA (Fig. 5, compare lanes 3, 4, and 7). An artificial polyanionic compound, polyglutamate (molecular mass ϳ50 -100 kDa), also stimulated purified PrPres amplification over a broad range of concentrations from 0.1 to 100 g/ml (Fig. 5). However, the level of stimulation induced by polyglutamate was again less than the level of control stimulation induced by total hamster RNA, and some of the apparent increase in PrPres signal caused by polyglutamate may be attributable to a direct effect of this compound on the inherent protease resistance of PrP27-30 (Fig. 5, bottom panel, lane 6).
Some studies suggest a role for charged lipids in PrP struc-FIG. 2. Western blot of purified PrPres amplification assays testing stimulation by total RNA prepared from various animal species. Samples containing purified hamster PrP C , Sc237 PrP27-30, and varying concentrations of total RNA were incubated for 16 h at 37°C and subjected to proteinase K digestion, except where indicated (-PK), as described under "Experimental Procedures." Hamster and mouse RNA were prepared from livers, and worm and bacterial RNA were prepared from whole animals.

FIG. 3. Western blot of purified PrPres amplification assays testing stimulation by synthetic homopolymeric nucleic acids.
Samples containing purified hamster PrP C , Sc237 PrP27-30, and varying concentrations of synthetic nucleic acids were incubated for 16 h at 37°C and subjected to proteinase K digestion, except where indicated (-PK), as described under "Experimental Procedures." tural conversion (25). Therefore, we tested whether an extract of brain gangliosides could stimulate purified PrPres amplification in vitro. The results show that brain gangliosides did not affect the efficiency of PrPres amplification in this system (Fig.  6). Detailed analytical studies have shown that purified prion rods contain a glycogen-like scaffold composed primarily of 1,4-linked glucose units (26). Therefore, we also tested whether glycogen could stimulate purified PrPres amplification and found that this compound also did not stimulate the formation of PrPres (Fig. 6). DISCUSSION In this report, we describe the first in vitro PrP conversion system using partially purified substrates capable of amplifying PrPres levels. The essential components for efficient con-version in this system include PrP C , PrP Sc , and a polyanionic scaffold. The molar ratio of PrP Sc -to-PrP C used in this purified PrPres amplification system is ϳ1:250, and ϳ10-fold amplification of PrPres is observed after a 16-h incubation period. In contrast, the radiolabel cell-free conversion assay requires a 50:1 molar ratio of PrP Sc -to-PrP C to trigger conversion (9). Reconstitution experiments show that the difference in PrP conversion efficiency between the two systems can be attributed mainly to the stimulatory effect of the polyanions (Fig.  1C). Another difference between the two systems is that the PrP C substrate used in the cell-free conversion assay lacks a glycophosphatidylinositol anchor (9); additional work will be required to determine whether presence of the glycophosphatidylinositol anchor affects the efficiency of PrP conversion in vitro. Earlier experiments performed with the cell-free conversion assay as well as some of the experiments reported here show that the interaction of purified PrP C and PrP Sc molecules alone can produce a moderate level of PrP conversion (9). This baseline conversion observed with purified PrP molecules is consistent with the concept that infectious PrP Sc molecules can directly bind to PrP C molecules to promote conformational change and that this bimolecular reaction is the fundamental biochemical event in the propagation of infectious prions.
Our reconstitution experiments demonstrate that a number of different polyanions are able to stimulate purified PrPres amplification above the baseline level. Some of the polyanion preparations that successfully stimulate purified PrPres amplification in vitro include total RNA prepared from both mammalian and invertebrate species, synthetic RNA and DNA molecules, and, less potently, HSPG. Thus, the stimulation of PrPres amplification using purified substrates is notably less selective than the equivalent process in crude brain homogenates, which appears to require mammalian RNA specifically. Furthermore, total hamster liver RNA is ϳ100 times more potent at stimulating PrPres amplification in the purified system than the crude system. The precise nature of the interaction between polyanions and components of the crude homogenate that causes lower potency and higher specificity in stimulating PrPres amplification remains to be determined. Consistent with the low specificity of PrPres amplification by polyanions in our purified system, other investigators have reported that purified, recombinant PrP molecules bind to a several classes of polyanionic molecules (24,(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37). It is important to note that our results only identify one possible set of molecular components that is able to generate efficient PrPres amplification. It is possible that polyanions are not the only FIG. 5. Western blot of purified PrPres amplification assays testing stimulation by non-nucleic acid polyanions. Samples containing purified hamster PrP C , Sc237 PrP27-30, and varying concentrations of polyanions were incubated for 16 h at 37°C and subjected to proteinase K digestion, except where indicated (-PK), as described under "Experimental Procedures." FIG. 6. Western blot of purified PrPres amplification assays testing stimulation by free nucleotides, brain gangliosides, and glycogen. Samples containing purified hamster PrP C , Sc237 PrP27-30, and varying concentrations of each compound were incubated for 16 h at 37°C and subjected to proteinase K digestion, except where indicated (-PK), as described under "Experimental Procedures." factors present in the brain homogenate that contribute to the efficiency of the reaction. Furthermore, because our PrP C preparations are not 100% pure, it is possible that some of the additional polypeptides that co-purify with PrP C may also be required for PrPres amplification. Nonetheless, our current results significantly extend our previous work in homogenates (15). In particular, the observation that polyanion stimulation of PrPres amplification is less selective in a purified system raises the possibility that polyanions other than RNA, or perhaps even multiple polyanions, can act as endogenous stimulators of PrPres formation.
Our observations are consistent with the explanation that endogenous polyanions may accelerate the rate of prion disease progression by acting as scaffolds or surfaces that facilitate interaction between PrP C and PrP Sc molecules. This explanation was originally proposed by Wong et al. (23) to explain the stimulation of PrPres formation by glycosaminoglycans. Candidate stimulatory endogenous polyanions include proteoglycans and host-encoded cellular nucleic acids, which could be released from dying cells into the extracellular space. It is also possible that the polyanionic compounds able to stimulate PrPres amplification in vitro mimic negatively charged surfaces of specific accessory proteins and that such proteins facilitate prion propagation. Our poly(A) size fractionation experiments indicate that a minimum molecular size of ϳ300 bases is required for full stimulation activity. There are several potential explanations for this observation as follows: 1) Optimal stimulation of PrP conversion may require the polyanion to adopt a particular surface or three-dimensional structure. This could explain why synthetic poly(C) failed to stimulate PrPres amplification (Fig. 3). It is known that, unlike other homopolymeric polynucleotides, poly(C) does not easily acquire secondary structure (38). 2) A scaffold length of at least 300 bases may be required to accommodate the minimum PrP C /PrP Sc "conversion unit," the subunit composition of which remains unknown. 3) Large polyanions may disaggregate prion rods and thereby increase the number of infectious particles available to drive conversion. Further studies will be required to distinguish between these possibilities.
Our biochemical approach is inherently limited in its ability to model the process of prion propagation in vivo. However, the hypothesis that endogenous polyanions play a pathogenic role in prion disease is also supported by the observation that both small polyanionic compounds and polycationic dendrimers block prion propagation. Small polyanionic compounds may block prion propagation by competitively inhibiting endogenous stimulatory polyanions (20,39,40), and dendrimers may bind to and sequester endogenous polyanions, preventing their interaction with PrP molecules. Metabolic inhibitor and enzyme degradation studies in scrapie-infected neuroblastoma cells provide additional evidence that heparan sulfate molecules play a rate-limiting role in prion propagation (22). In our studies, HSPG molecules were significantly less potent than endogenous and synthetic nucleic acids in stimulating purified PrPres amplification. However, the HSPG preparation used in our studies was specifically prepared from the basement membrane of Engelbreth-Holm-Swarm mouse sarcoma cells, and it is possible that other proteoglycans would be more potent in stimulating PrPres amplification.
Our reconstitution of PrPres amplification using purified and synthetic components may also contribute to the development of sensitive prion detection assays such as protein-misfolding cyclic amplification (11,41). Preparations of purified PrP C and synthetic polyanions can be prepared more uniformly and quantitatively than crude homogenates, leading to more consistent amplification assays. Furthermore, defined compo-nents could be chemically manipulated to simplify assay formats. For instance, it may be possible to attach either PrP C or a synthetic polyanion to a solid surface, where it could act as both an amplification substrate as well as a capture reagent for PrP Sc .
The observation that certain polyanions directly stimulate induced PrP misfolding may also have relevance to other disease processes. It is interesting to speculate that polyanions such as nucleic acids and HSPG may play roles in the pathogenesis of other neurodegenerative diseases associated with protein misfolding. Notably, HSPG and specific neuronal RNA molecules accumulate in extracellular plaques associated with Alzheimer's disease (42)(43)(44). The interaction between endogenous polyanions and misfolded neuronal polypeptides, such as PrP or A␤ 1-42 , may eventually prove to be a common therapeutic target for a broad range of neurodegenerative diseases.