Sheep scrapie susceptibility-linked polymorphisms do not modulate the initial binding of cellular to disease-associated prion protein prior to conversion

Alan Rigter and Alex Bossers

Central Institute for Animal Disease Control, Department of Bacteriology and TSEs, PO Box 2004, 8203 AA Lelystad, The Netherlands

Correspondence
Alex Bossers
alex.bossers{at}wur.nl


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Conversion of the host-encoded protease-sensitive cellular prion protein (PrPC) into the scrapie-associated protease-resistant isoform (PrPSc) of prion protein (PrP) is the central event in transmissible spongiform encephalopathies or prion diseases. Differences in transmissibility and susceptibility are largely determined by polymorphisms in PrP, but the exact molecular mechanism behind PrP conversion and the modulation by disease-associated polymorphisms is still unclear. To assess whether the polymorphisms in either PrPC or PrPSc modulate the initial binding of PrPC to PrPSc, several naturally occurring allelic variants of sheep PrPC and PrPSc that are associated with differential scrapie susceptibility and transmissibility [the phylogenetic wild-type (ARQ), the codon 136Val variant (VRQ) and the codon 171Arg variant (ARR)] were used. Under cell-free PrP conversion conditions known to reproduce the observed in vivo differential scrapie susceptibility, it was found that the relative amounts of PrPC allelic variants bound by various allelic PrPSc variants are PrP-specific and have comparable binding efficiencies. Therefore, the differential rate-limiting step in conversion of sheep PrP variants is not determined by the initial PrPC–PrPSc-binding efficiency, but seems to be an intrinsic property of PrPC itself. Consequently, a second step after PrPC–PrPSc-binding should determine the observed differences in PrP conversion efficiencies. Further study of this second step may provide a future tool to determine the mechanism underlying refolding of PrPC into PrPSc and supports the use of conversion-resistant polymorphic PrPC variants as a potential therapeutic approach to interfere with PrP conversion in transmissible spongiform encephalopathy development.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Transmissible spongiform encephalopathy (TSE) diseases are fatal neurodegenerative disorders and include (among others) familial, sporadic and variant Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE) in cattle and scrapie in sheep. TSEs (or prion diseases) are characterized by the formation and accumulation of protease-resistant prion protein (PrPSc) mainly in tissues of the central nervous system. Formation of PrPSc is a post-translational process and involves refolding (conversion) of the host-encoded prion protein (PrPC) into partially protease-resistant forms (PrPSc) (DeArmond & Prusiner, 2003).

Scrapie in small ruminants (e.g. sheep) is one of the best documented models for natural TSE transmission. Polymorphisms in PrP have been shown to be of importance in both interspecies and intraspecies transmissibilities (Bossers et al., 2003). Susceptibility of sheep to scrapie seems mainly dictated by polymorphisms in the gene encoding the prion protein itself, and to date over 20 different naturally occurring polymorphisms (only one mutation per allele) of PrP have been described (Goldmann et al., 1990, 1991; Belt et al., 1996; Bossers et al., 1996; Junghans et al., 1998; Elsen et al., 1999; Thorgeirsdottir et al., 1999; Tranulis et al., 1999; O'Rourke et al., 2000). The effects of polymorphisms in ovine PrP on the relative susceptibility of sheep to scrapie have been gauged in epidemiological studies of natural scrapie outbreaks, in experimental transmissions to and from sheep, and in cell-free conversion assays (Goldmann et al., 1994; Bossers et al., 1996, 1997, 1999, 2000; Hunter et al., 1996). Polymorphisms at sheep PrP aa 136, 154 and 171 have been shown to be most relevant in association with differential TSE susceptibility. Several studies have shown that an alanine at position 136, arginine at position 154 and glutamine at position 171 (ARQ) to be the phylogenetic wild-type (wt) PrP, with intermediate susceptibility to scrapie. The polymorphism associated with increased susceptibility to scrapie is the substitution of alanine with valine at codon 136 (VRQ; 136V) and thus far the only polymorphism shown to be associated with decreased susceptibility or even resistance to natural scrapie is the substitution of glutamine with arginine at codon 171 (ARR; 171R). Cell-free conversion of PrPC provides an excellent in vitro model, in which relative amounts of produced proteinase K (PK)-resistant PrP reflect important biological aspects of TSEs at the molecular level (Caughey et al., 1995; Bossers et al., 1997, 2000, 2003; Raymond et al., 1997, 2000; Bossers, 1999). In sheep scrapie, this technique has shown that 136V and wt-PrPC are readily converted into PK-resistant PrP by various types of PrPSc isolated from sheep having different PrP genotypes. In contrast, 171R-PrP is hardly converted into PK-resistant PrP (Bossers et al., 1997, 2000, 2003; Bossers, 1999; Raymond et al., 2000).

Studies on the conversion of hamster and mouse PrPC isoforms resulted in indications that diminished acquisition of PK resistance is not due to lack of binding of PrPC to PrPSc (Horiuchi et al., 2000). However, no data on binding efficiencies of ovine PrPC to PrPSc are available to date. Furthermore, whereas differences in susceptibility of- and transmissibility in sheep can entirely be explained at the molecular level by the effects of single polymorphisms in PrPC or PrPSc on PrP conversion, the exact molecular mechanism determining these differences is still unknown (Bossers et al., 2000; Dubois et al., 2002; Tranulis, 2002; Sabuncu et al., 2003).

In the present study, sheep scrapie susceptibility-linked polymorphisms were used to determine whether differential binding efficiencies of sheep PrPC to PrPSc determine the observed differential conversion efficiencies of sheep PrP (Bossers et al., 1997, 2000; Raymond et al., 1997, 2000).


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
PrPC constructs and expression.
The three sheep PrPC variants used (136V, wt and 171R) were cloned, expressed and characterized as described previously (Bossers et al., 1997). Briefly, PrP open reading frames (ORF) were subcloned into the eukaryotic expression vector pECV7. The vectors containing PrP ORF were stably transfected into Chinese hamster ovary (CHO) cells. High and stable expressing single-cell-clones were selected by immunoperoxidase monolayer assay and Western blotting, using rabbit anti-peptide antiserum R521-7 (van Keulen et al., 1995).

Radiolabelling and purification of PrPC.
Radiolabelling and purification of the three PrPC variants were performed as described previously (Raymond et al., 1997; Bossers et al., 2000). Briefly, single cell clones expressing the different PrPC variants were starved for 30–60 min in label medium and subsequently labelled with 1 mCi (37 MBq) [35S]methionine/cysteine TRAN35S-label (ICN Biomedicals). Cells were lysed in lysis buffer containing Triton X-100 (0·5 %; ICN Biomedicals) in the presence of protease inhibitors (1 nM Pefabloc SC, 1 nM leupeptine, 1 nM pepstatin and 0·15 nM aproprotin). 35S-labelled PrPC was immunopurified by PrP-specific antiserum R521-7 captured by protein A–Sepharose (10 % w/v), which was eluted in 0·1 M acetic acid.

Radiolabelling and purification of classical swine fever virus (CSFV) glycoprotein E2.
For labelling, an expression vector containing the gene encoding glycoprotein E2 of CSFV transfected into SK6 cells (van Gennip et al., 2002) was used. Radiolabelling and purification of E2 were essentially performed as described above, albeit on a larger scale. 35S-labelled E2 was immunopurified using monoclonal antibody V3 (Wensvoort, 1989) and eluted in 0·1 M acetic acid.

PrPSc purification and analysis.
PrPSc was isolated from brain tissue of clinically ill scrapie sheep with either homozygous alleles for 136V-PrP or wt-PrP. PrP genotypes were determined by Sanger sequencing of the full PrP ORF as described previously (Bossers et al., 1996). PrPSc was purified by ultracentrifugational pelleting from Sarkosyl-homogenated brains as described previously (Caughey et al., 1995; Bossers et al., 1997). The final pellets were sonicated in PBS containing 1·0 % zwitter-reagent (SB 3-14). Yields of PrPSc were quantified by SDS-PAGE (12 % NuPAGE; Invitrogen) and Western blotting using antiserum R521-7.

Conversion-binding assay.
Conversion and binding efficiencies were determined by double volume cell-free conversion reactions essentially as described previously (Horiuchi et al., 2000; Priola & Lawson, 2001) and adapted to ovine cell-free conversion conditions as used before (Caughey et al., 1995; Bossers et al., 1997, 2000). Briefly, PrPSc was partially denatured in 2·5 M guanidinium-hydrochloride (GdnHCl) for at least 2·5 h at 37 °C. Aliquots of denatured PrPSc (2–4 µg per reaction) were mixed with 10 000–20 000 c.p.m. purified 35S-labelled PrPC (~20–40 ng 35S-labelled PrPC) and further diluted to a final concentration of 1·0 M GdnHCl in conversion buffer (50 mM sodium citrate, pH 6·0, 5 mM cetylpyridinium chloride, 1 % N-lauroylsarcosine and protease inhibitors). Reactions were incubated for 3 days at 37 °C (or shorter for the kinetic experiments). After incubation, the reaction volume was split in two equal aliquots in separate siliconized tubes. One aliquot was used for binding analysis and centrifuged for 30 min at 17 500 g at room temperature, the supernatant was transferred to a separate siliconized tube (unbound fraction) and the pellet (bound fraction) dissolved in 1 % SDS by sonication. From the second aliquot, 1/10 volume was transferred to a separate siliconized tube (reference fraction) and the remaining 9/10 volume was treated with 35 µg PK ml–1 for 1 h at 37 °C. PK was inactivated by the addition of Pefabloc-SC (Roche).

All the samples were methanol-precipitated and the pellet was dried and dissolved in Laemmli SDS-PAGE sample buffer containing 5 % (v/v) 2-mercaptoethanol and 4 M urea. Samples were run on SDS-PAGE (12 % NuPAGE; Invitrogen), the dried gels were visualized by phosphorimaging and analysed using a STORM-840 imager and the ImageQuant 5.1 software (Molecular Dynamics). Binding percentages were calculated by dividing the amount of labelled PrPC (molecular mass between 24 and 28·5 kDa) of each fraction [pellet (p) and supernatant (s)] by the total amount of labelled PrPC (p+s). Conversion percentages were calculated by dividing the amount of labelled PrP left after PK digestion (molecular mass between 19 and 21·5 kDa) by the amount of labelled PrPC in the reference fraction (molecular mass between 24 and 28·5 kDa).

Statistical analysis of binding efficiencies.
Statistical calculations were performed using the GenStat 6.1 program. To compensate for differences in possible variance as a result of a fixed scale, variance stabilizing angular transformation of the binding percentages was utilized (section 4·1·3; McCullagh, 1983). Absolute amounts of bound 35S-labelled PrPC varied, probably as a result of the aggregated state of the PrPSc isolate used; therefore binding patterns were compared by the analysis of variance method to determine whether significant differences occurred in these binding patterns. Comparisons of binding patterns were made separately for the three PrPC variants and for the two different PrPSc isolate groups. In order to determine significant differences in binding patterns, the least significant difference (LSD; the minimum amount needed to demonstrate a significant difference) was calculated and compared with the observed differences between the mean binding percentages for either the PrPC variants or the PrPSc isolate groups.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Binding efficiencies of PrPC to PrPSc
In total, six independent PrPSc isolates from six sheep homozygous for 136V-PrPC and six independent PrPSc isolates from six sheep homozygous for wt-PrPC were isolated and tested for binding affinities to three natural allelic variants of sheep PrPC; 136V-PrPC (VRQ), wt-PrPC (ARQ) and 171R-PrPC (ARR). At least two independent reaction duplicates were analysed from each PrPSc isolate. Binding efficiencies of the individual PrPSc isolates were determined using the conversion-binding assay, in which cell-free conversion conditions used were identical to previous studies, showing significant differential conversion of sheep PrPC variants (Bossers et al., 2000). Because aggregates were pelleted by spinning for 30 min at 17 500 g, we needed to take into account that not all of the PrPSc is actually pelleted at this ‘low’ speed. However, most of the PrPSc was pelleted (~86·4 % of the total input). Therefore, it can be assumed that the amount of 35S-labelled PrPC found in the pellet fraction is representative of most if not all of the actual bound 35S-labelled PrPC. The addition of PrPSc, isolated from sheep homozygous for 136V-PrP (Fig. 1a) or isolated from sheep homozygous for wt-PrP (Fig. 1b), resulted in recovering most of the labelled PrPC in the bound pellet (p) fraction (Fig. 1a and b, lanes 1, 3 and 5) and only a small amount of labelled PrPC remained in the unbound supernatant (s) fraction (Fig. 1a and b, lanes 2, 4 and 6) for each PrPC tested (Table 1; 136V-PrPSc and wt-PrPSc isolates). Comparison between repeated measurements of a PrPSc isolate or between different PrPSc isolates showed that the absolute amount of bound PrPC was linked to the isolated batch of PrPSc [probably due to differences in preparation of PrPSc aliquots (sonication) or ‘age’ of an isolate]. Therefore, the absolute binding percentages (per measurement) of bound 35S-labelled PrPC were not compared, but rather the mean binding percentages of repeated measurements (Table 1).



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Fig. 1. Phosphorimage of SDS-PAGE showing an example of binding assay samples obtained with PrPSc isolated from a sheep homozygous for 136V-PrP allele (a) or from a sheep homozygous for wt-PrP allele (b). Lanes containing 14C-marker (Amersham Biosciences) are marked ‘m’, sizes of the marker bands are 30 and 21·5 kDa. Non-glycosylated PrP is indicated by the 27 kDa marker. Lanes 1 and 2 represent results with 136V-PrPC, lanes 3 and 4 represent results with wt-PrPC and lanes 5 and 6 represent results with 171R-PrPC of which the odd lanes contain the pelleted (bound) fraction (p) and the even lanes contain the supernatant (unbound) fraction (s).

 

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Table 1. Mean percentages of bound 35S-labelled PrPC determined for PrPSc isolates from sheep

 
Binding percentages were compared by variance analysis, after variance stabilizing angular transformation of the binding percentages. Firstly, binding patterns were compared between the PrPC variants and no significant differences were found (Fig. 2a) since the LSD between PrPC variants was calculated to be 4·5 %, which was higher than the maximum difference of 2·4 % between the mean binding percentages of 136V-PrPC, wt-PrPC and 171R-PrPC (88·0±1·6, 85·6±2·5 and 86·6±2·2 %, respectively). Secondly, binding patterns were compared between the PrPSc groups and again no significant differences were shown (Fig. 2b) since the LSD between PrPSc variants was 5·8 %, which is again higher than the maximum difference of 1·1 % between the mean binding percentages for the wt/wt (homozygous wt-PrP) and 136V/136V (homozygous 136V-PrP) PrPSc isolate groups (86·2±1·7 and 87·3±1·8 %, respectively).



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Fig. 2. Boxplots of binding patterns after angular transformation of binding percentages. The binding percentages have been plotted against the PrPC variants (a) and PrPSc isolate groups (b). The boxplot shows the total spread of all determined binding percentages, with the box representing 95 % of all measurements and the line in the box representing the mean value of the measurements. Analysis of variance of the binding percentages shows that neither the PrPC variant nor the PrPSc isolate group have a significant effect on the binding patterns obtained.

 
To gain insight into the dynamics of the binding reaction, binding percentages were also determined at shorter incubations (1 h, 1 day, 2 days and the standard 3 days). At each time point, binding percentages were determined of the three PrPC variants to PrPSc (n=4; two wt/wt and two 136V/136V isolates). No significant differences were found between the three PrPC variants and the overall mean binding percentages at the four time points (Fig. 3).



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Fig. 3. Kinetics of the binding efficiencies of the three PrPC variants to PrPSc homozygous 136V and homozygous wt. Mean binding percentages for each PrPC variant are indicated including the error bars (SEM) for the repeated experiments after incubation for the indicated time.

 
Binding specificity of PrPSc to PrPC
In order to exclude potential non-specific binding or aggregation features of PrPC, we performed several controls. First of all, 35S-labelled PrPC was incubated without addition of PrPSc to determine whether ‘self aggregation’ and spontaneous pelleting of PrPC occurred. Exclusion of PrPSc, by replacing with demineralized water (SQ), did not result in significant amounts of 35S-labelled PrPC in the pellet fraction, leaving on average 93·6±1·3 % of the 35S-labelled PrPC variants in the supernatant fraction (Fig. 4a, lanes 1 and 2). Therefore, self-aggregation of PrPC is not responsible for recovering significant amounts of labelled PrPC in the pellet. Furthermore, it shows that the presence of aggregated protein (PrPSc) is a prerequisite for pelleting 35S-labelled PrPC under conditions maintaining cell-free conversion specificity.



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Fig. 4. Examples of control reactions for determining specificity of PrPC–PrPSc interaction within the binding assay. (a) Phosphorimage of SDS-PAGE (indicated in pseudo intensity staining) analysis of samples obtained when PrPSc was replaced with either water (SQ; lanes 1 and 2), KLH (lanes 3 and 4) or TG (lanes 5 and 6). (b) Sypro Orange total protein staining of SDS-PAGE (indicated in pseudo intensity staining) containing conversion/binding samples having KLH (lanes 1 and 2) and TG (lanes 3 and 4) instead of PrPSc under the specific conversion conditions used. (c) Phosphorimage of SDS-PAGE (indicated in pseudo intensity staining) containing samples obtained when 35S-labelled PrPC was replaced with 35S-labelled E2 protein of CSFV. All odd lanes contain the pelleted (p) (bound) fraction and all even lanes contain the supernatant (s) (unbound) fraction.

 
To determine whether 35S-labelled PrPC could be non-specifically ‘captured’ and pelleted by any aggregated protein, PrPSc was replaced in the binding assay with keyhole limpet haemocyanin (KLH), a very large mainly aggregated protein. A surplus of KLH was added (about 5 µg per reaction) to favour KLH aggregation. No significant amounts of 35S-labelled PrPC were detected in the pellet fraction. On average 94·3±2·3 % of the 35S-labelled PrPC remained in the unbound fraction (Fig. 4a, lanes 3 and 4), which is about the same as the amount of PrPC in the pellet fraction of assays without any aggregated protein (see above). To ensure that KLH remained aggregated under the specific conversion conditions used, soluble and pellet fractions were also analysed on SDS-PAGE by total protein staining (Sypro Orange; Molecular Probes). On average 79·8±1·0 % of the added KLH was recovered in the pellet fraction (Fig. 4b, lanes 1 and 2), which is comparable to the percentage of aggregation determined for KLH (75·9±2·1 %) in storing buffer (0·1 M sodium phosphate buffer, pH 7). Since no increase in PrPC pelleting was observed, and although only one other aggregated protein was tested for non-specific capture of PrPC, this indicates that 35S-labelled PrPC is probably not a ‘sticky’ protein binding to any aggregate.

To determine whether 35S-labelled PrPC could non-specifically bind to other large soluble proteins, resulting in significant amounts of precipitation, PrPSc was replaced by thyroglobulin (TG), a large unrelated soluble protein frequently used as a carrier in protein precipitation methodologies. A surplus of TG was added (about 5 µg per reaction) but no significant amounts of 35S-labelled PrPC were detected in the pellet fraction; on average 90·6±2·6 % remained in the supernatant fraction (Fig. 4a, lanes 5 and 6). To ensure that TG remained largely soluble under the specific conversion conditions used, soluble and pellet fractions were also analysed on SDS-PAGE by total protein staining (Sypro Orange). On average 94·4±2·5 % of the TG remained in the supernatant (Fig. 4b, lanes 3 and 4) under the specific conversion conditions used. Even though only one large soluble protein was tested, this indicates that 35S-labelled PrPC is not significantly precipitated by binding to any other large soluble heterologous protein like TG.

To determine whether any labelled soluble protein would bind to the added aggregated PrPSc, 35S-labelled PrPC was replaced with 35S-labelled E2 of CSFV, an unrelated but similarly processed protein (membrane bound partially N-glycosylated protein of about 51–54 kDa). No significant amounts of 35S-labelled E2 protein were found in the bound fraction, while 91·9±3·5 % of the 35S-labelled E2 protein remained in the unbound fraction (Fig. 4c, lanes 1 and 2). This indicates that binding of 35S-labelled PrPC by PrPSc is PrP-specific.

In summary, we have shown that PrPC binds efficiently to PrPSc with no significant differences in binding patterns between PrPC variants and PrPSc isolate groups, under conditions maintaining cell-free conversion specificity (Fig. 5). Furthermore, we have shown that PrPC does not spontaneously aggregate due to the specific conversion condition used, does not stick to unrelated aggregated protein like KLH and does not precipitate with other large soluble proteins like TG. Additionally, we have shown that PrPSc does not bind to unrelated labelled soluble protein (E2). Therefore, we can conclude that PrPSc-associated pelleting of 35S-labelled PrPC represents a PrPC–PrPSc-specific interaction and that addition of aggregated protein (PrPSc) is a prerequisite for pelleting 35S-labelled PrPC under conditions maintaining cell-free conversion specificity. Since no differences were detected in the binding patterns of the tested PrPC variants to the different PrPSc isolates, the rate-limiting step determining the observed differential conversion efficiencies of PrPC variants has to be during a subsequent step in the conversion after binding of PrPC to PrPSc (Fig. 6).



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Fig. 5. Observed relative normalized conversion efficiencies of the combined conversion-binding reactions. Reactions were normalized to the most efficient homologous reactions. (a) Conversion efficiencies of the three PrPC variants induced by 136V-PrPSc. (b) Reactions induced by wt-PrPSc. Mean normalized efficiencies and the corresponding error bars (SEM) are indicated.

 


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Fig. 6. Schematic representation of PrPC conversion. (a) Conversion of homologous, conversion prone PrPC (wt, 136V): efficient binding (i), highly efficient conversion (ii) and ‘re-seeding’ (iii) possible. (b) Conversion of heterologous, conversion prone PrPC (wt, 136V): efficient binding (i), efficient conversion (ii) and ‘re-seeding’ (iii) possible. (c) Conversion of heterologous, conversion-resistant PrPC (171R): efficient binding (i), highly inefficient or blocked conversion (ii) and ‘re-seeding’ (iii) possibly blocked.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The aim of this study was to gain insight into the mechanism underlying the modulation of sheep scrapie susceptibility by polymorphisms in PrPC or PrPSc. Bossers et al. (2000) showed that the in vitro conversion assay is a representative tool for assessing modulating effects of scrapie-associated polymorphisms. Other studies have shown that the conversion of PrPC by PrPSc is induced by the aggregated forms of PrPSc (Caughey et al., 1995, 1997). These aggregates can be pelleted by high-speed centrifugation. The amount of 35S-labelled PrPC that is bound by PrPSc and subsequently recovered from the pellet should give an indication of whether the disease-associated polymorphisms modulate binding of PrPC by PrPSc or whether these polymorphisms have their modulating effects in a subsequent step after the initial binding during the conversion.

By applying a conversion-binding assay (Horiuchi et al., 2000), binding efficiencies of sheep PrPC variants to sheep PrPSc variants have been measured. Since no significant differences in binding efficiencies were measured between any of the variants, the initial binding efficiencies cannot account for the observed differential conversion efficiencies of sheep scrapie susceptibility-linked variants of PrP. We show for instance that 171R-PrPC binds to PrPSc as efficiently as 136V-PrPC or wt-PrPC, whereas conversion efficiencies differ remarkably. Therefore, a second (or) further step in the conversion process, in which the disease-associated polymorphisms have their modulating effect, seems to be involved in the conversion of the PrP protein (Fig. 6). These findings are corroborated by a study in which interactions between heterologous forms of prion protein have been studied in vitro using mouse and hamster PrP isoforms (Horiuchi et al., 2000), in which is shown that PrPC of different species (hamster and mouse) bind equally efficiently to PrPSc of mouse while preserving conversion specificity, also indicating that a second step in the conversion after initial binding should determine the species specificity.

In this study, we also show that PrPC does not precipitate spontaneously, does not stick to unrelated aggregated protein (KLH), and does not precipitate with other large soluble proteins (TG). In addition, we showed that PrPSc does not bind unrelated labelled soluble protein CSFV E2. Therefore, we conclude that PrPSc-associated pelleting of 35S-labelled PrPC represents a PrPC–PrPSc-specific interaction and that addition of aggregated PrPSc is a prerequisite for pelleting 35S-labelled PrPC under conditions maintaining cell-free conversion specificity. These results are in conjunction with results in which no spontaneous PK-resistant PrP was formed under the cell-free conversion conditions when PrPSc was replaced by SQ water (Bossers et al., 1997, 2000). The inability of PrPSc to significantly bind 35S-labelled E2 additionally confirms that binding of 35S-labelled PrPC to PrPSc is PrP-specific and does not solely depend on post-translational modifications or non-specific ‘sticky’ properties of PrP.

Since 171R-PrPC seems to bind as efficiently to PrPSc as wt-PrPC and 136V-PrPC, the 171R-PrPC variant may be valuable in firstly, providing clues for designing new therapeutic strategies by determination of the mechanism underlying the refolding process of PrPC into PrPSc, for example, by using the 171R-PrPC variant to determine sites involved in binding and/or conversion or by comparing protein properties (i.e. stability, unfolding/refolding kinetics). Secondly, since conversion-resistant 171R-PrPC binds efficiently to PrPSc it may provide a future tool to block prion conversion through direct interference or blocking of PrPSc polymer growth as hypothesized before by Bossers et al. (1999). In addition, results from literature show that heterozygosity for PrP is a protective factor against TSE development as demonstrated by studies in vitro (Priola et al., 1994; Holscher et al., 1998; Horiuchi et al., 2000) or in vivo for sheep (Goldmann et al., 1994; Belt et al., 1995; Clouscard et al., 1995; Bossers et al., 1996; Hunter et al., 1996) and humans (Collinge et al., 1991; Palmer et al., 1991). This is in conjunction with our results showing that various differentially converting PrPC variants bind equally efficiently to PrPSc but have different conversion efficiencies. In heterozygotes, this ‘inhibition’ of conversion by heterologous PrP variants might explain why heterozygotes have longer incubation times than their homozygous counterparts. This is corroborated by the fact that resistance in heterozygous sheep is not caused by preferential allelic use (Caplazi et al., 2004).

The coupled in vitro cell-free conversion efficiencies (Fig. 5) reflect results as described before (Bossers et al., 2000), where susceptibility to scrapie was linked to the modulating effects of polymorphisms on the conversion of sheep PrP. In addition to these in vitro cell-free conversion assays, PrP polymorphisms have been shown to tightly control sheep prion replication in cultured cells (Sabuncu et al., 2003). Furthermore, it has been shown that polymorphisms in PrP determine both interspecies and intraspecies transmissibilities (Bossers, 1999; Bossers et al., 2003) and/or the stability of the PrPC molecule itself (Rezaei et al., 2002).

By correlating conversion- and binding-patterns, we showed that 171R-PrPC binds to PrPSc as efficiently as conversion prone variants like wt (ARQ) and 136V-PrPC. Since naturally occurring polymorphisms of sheep PrPC seem not to have a significant modulating effect on the initial binding of PrPC to PrPSc, these could somehow modulate a subsequent step in the conversion process (Fig. 6). Both 136V and 171R are polymorphisms that affect PrPC stability and are close to the region that supposedly is involved in refolding of PrPC to PrPSc (Rezaei et al., 2002; Eghiaian et al., 2004). This region is composed of the two small {beta}-sheets [sheep aa 129–134 (S1) and 163–167 (S2)], which are the positions from where the first {alpha}-helix [aa 146–158 (H1)] is converted into an anti-parallel organized {beta}-sheeted structure. It could also be that the 171R polymorphism results in increased protease sensitivity of 171R-PrPC itself due to destabilization of the PrPC molecule (Rezaei et al., 2002), thus resulting in slower amyloidogenesis because the 171R-PrPC molecule could internalize and be degraded by the PrP-expressing cell more rapidly than the other variants before the actual polymerization can take place. In contrast, the 136V polymorphism could stabilize the PrPC molecule, resulting in an elongation of the survival of 136V-PrPC and thereby supporting the subsequent conversion.

Since disease-associated polymorphisms of sheep PrP do not have an effect on binding properties of PrPC to PrPSc, dominant-negative inhibition of the 171R polymorphism on prion conversion (Bossers, 1999; Bossers et al., 1999, 2000; Perrier et al., 2002) is therefore not due to lack of interaction between PrP variants as suggested by Perrier et al. (2002), but more probably due to a more subtle mode of modulation by the PrP polymorphism on the conversion or on the interaction with chaperone proteins under natural conditions.

This study shows that the interaction between PrPC and PrPSc in the conversion-binding assay is PrP-specific. Whether PrPC binds to the so-called nucleation site of PrPSc only or whether it can bind to other sites of PrPSc aggregates is under investigation. The next logical step, currently under investigation, is to find out whether conversion-resistant (natural or artificial) PrP variants can effectively interfere with the process of PrP conversion and thereby therapeutically block or significantly delay TSE development.


   ACKNOWLEDGEMENTS
 
This work was supported by grant 903-51-177 from the Dutch Organization for Scientific Research (NWO) and a grant from the Dutch Ministry of Agriculture, Nature Management and Fisheries (LNV). We thank Dr J. P. M. Langeveld for providing antibody R521-7 used for radioimmune precipitation of 35S-labelled PrPC, H. G. P. van Gennip (BSc.) for kindly providing the tissue culture cell line expressing E2 of CSFV and Dr M. M. Hulst for providing mAb V3 used for radioimmune precipitation of 35S-labelled E2. Statistical analysis was carried out with the aid of Dr J. de Bree, ASG-Lelystad. We thank Dr J. P. M. Langeveld for critically reading the manuscript.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Belt, P. B., Muileman, I. H., Schreuder, B. E., Bos-de Ruijter, J., Gielkens, A. L. & Smits, M. A. (1995). Identification of five allelic variants of the sheep PrP gene and their association with natural scrapie. J Gen Virol 76, 509–517.[Abstract]

Belt, P. B. G. M., Bossers, A., Schreuder, B. E. C. & Smits, M. A. (1996). PrP allelic variants associated with natural scrapie. In Bovine Spongiform Encephalopathy; The BSE Dilemma, pp. 294–305. Edited by C. J. Gibbs, Jr. New York: Springer.

Bossers, A. (1999). Prion Disease: Susceptibility and Transmissibility; In Vivo and In Vitro Studies with Sheep Scrapie. University of Utrecht.

Bossers, A., Schreuder, B. E., Muileman, I. H., Belt, P. B. & Smits, M. A. (1996). PrP genotype contributes to determining survival times of sheep with natural scrapie. J Gen Virol 77, 2669–2673.[Abstract]

Bossers, A., Belt, P. B. G. M., Raymond, G. J., Caughey, B., de Vries, R. & Smits, M. A. (1997). Scrapie susceptibility-linked polymorphisms modulate the in vitro conversion of sheep prion protein to protease-resistant forms. Proc Natl Acad Sci U S A 94, 4931–4936.[Abstract/Free Full Text]

Bossers, A., Harders, F. L. & Smits, M. A. (1999). PrP genotype frequencies of the most dominant sheep breed in a country free from scrapie. Arch Virol 144, 829–834.[CrossRef][Medline]

Bossers, A., de Vries, R. & Smits, M. A. (2000). Susceptibility of sheep for scrapie as assessed by in vitro conversion of nine naturally occurring variants of PrP. J Virol 74, 1407–1414.[Abstract/Free Full Text]

Bossers, A., Rigter, A., de Vries, R. & Smits, M. A. (2003). In vitro conversion of normal prion protein into pathologic isoforms. In Clin Lab Med, pp. 227–247. Edited by B. Ghetti, MD & P. Piccardo, MD. W. B. Saunders Company, a division of Elsevier Science.

Caplazi, P. A., O'Rourke, K. I. & Baszler, T. V. (2004). Resistance to scrapie in PrP ARR/ARQ heterozygous sheep is not caused by preferential allelic use. J Clin Pathol 57, 647–650.[Abstract/Free Full Text]

Caughey, B., Kocisko, D. A., Raymond, G. J. & Lansbury, P. T., Jr (1995). Aggregates of scrapie-associated prion protein induce the cell-free conversion of protease-sensitive prion protein to the protease-resistant state. Chem Biol 2, 807–817.[CrossRef][Medline]

Caughey, B., Raymond, G. J., Kocisko, D. A. & Lansbury, P. T., Jr (1997). Scrapie infectivity correlates with converting activity, protease resistance, and aggregation of scrapie-associated prion protein in guanidine denaturation studies. J Virol 71, 4107–4110.[Abstract]

Clouscard, C., Beaudry, P., Elsen, J. M. & 7 other authors (1995). Different allelic effects of the codons 136 and 171 of the prion protein gene in sheep with natural scrapie. J Gen Virol 76, 2097–2101.[Abstract]

Collinge, J., Palmer, M. S. & Dryden, A. J. (1991). Genetic predisposition to iatrogenic Creutzfeldt–Jakob disease. Lancet 337, 1441–1442.[CrossRef][Medline]

DeArmond, S. J. & Prusiner, S. B. (2003). Perspectives on prion biology, prion disease pathogenesis, and pharmacologic approaches to treatment. Clin Lab Med 23, 1–41.[CrossRef][Medline]

Dubois, M. A., Sabatier, P., Durand, B., Calavas, D., Ducrot, C. & Chalvet-Monfray, K. (2002). Multiplicative genetic effects in scrapie disease susceptibility. C R Biol 325, 565–570.[Medline]

Eghiaian, F., Grosclaude, J., Lesceu, S., Debey, P., Doublet, B., Treguer, E., Rezaei, H. & Knossow, M. (2004). Insight into the PrPC->PrPSc conversion from the structures of antibody-bound ovine prion scrapie-susceptibility variants. Proc Natl Acad Sci U S A 101, 10254–10259.[Abstract/Free Full Text]

Elsen, J. M., Amigues, Y., Schelcher, F. & 7 other authors (1999). Genetic susceptibility and transmission factors in scrapie: detailed analysis of an epidemic in a closed flock of Romanov. Arch Virol 144, 431–445.[CrossRef][Medline]

Goldmann, W., Hunter, N., Foster, J. D., Salbaum, J. M., Beyreuther, K. & Hope, J. (1990). Two alleles of a neural protein gene linked to scrapie in sheep. Proc Natl Acad Sci U S A 87, 2476–2480.[Abstract/Free Full Text]

Goldmann, W., Hunter, N., Benson, G., Foster, J. D. & Hope, J. (1991). Different scrapie-associated fibril proteins (PrP) are encoded by lines of sheep selected for different alleles of the Sip gene. J Gen Virol 72, 2411–2417.[Abstract]

Goldmann, W., Hunter, N., Smith, G., Foster, J. & Hope, J. (1994). PrP genotypes and the Sip gene in Cheviot sheep form the basis for scrapie strain typing in sheep. Ann N Y Acad Sci 724, 296–299.[Medline]

Holscher, C., Delius, H. & Burkle, A. (1998). Overexpression of nonconvertible PrPC {Delta}114–121 in scrapie-infected mouse neuroblastoma cells leads to trans-dominant inhibition of wild-type PrPSc accumulation. J Virol 72, 1153–1159.[Abstract/Free Full Text]

Horiuchi, M., Priola, S. A., Chabry, J. & Caughey, B. (2000). Interactions between heterologous forms of prion protein: binding, inhibition of conversion, and species barriers. Proc Natl Acad Sci U S A 97, 5836–5841.[Abstract/Free Full Text]

Hunter, N., Foster, J. D., Goldmann, W., Stear, M. J., Hope, J. & Bostock, C. (1996). Natural scrapie in a closed flock of Cheviot sheep occurs only in specific PrP genotypes. Arch Virol 141, 809–824.[Medline]

Junghans, F., Teufel, B., Buschmann, A., Steng, G. & Groschup, M. H. (1998). Genotyping of German sheep with respect to scrapie susceptibility. Vet Rec 143, 340–341.[Free Full Text]

McCullagh, P. N. J. A. (1983). Genized linear models, 1st edn. Edited by P. McCullagh & J. A. Nelder. London, England: Chapman & Hall.

O'Rourke, K. I., Baszler, T. V., Besser, T. E. & 9 other authors (2000). Preclinical diagnosis of scrapie by immunohistochemistry of third eyelid lymphoid tissue. J Clin Microbiol 38, 3254–3259.[Abstract/Free Full Text]

Palmer, M. S., Dryden, A. J., Hughes, J. T. & Collinge, J. (1991). Homozygous prion protein genotype predisposes to sporadic Creutzfeldt–Jakob disease. Nature 352, 340–342.[CrossRef][Medline]

Perrier, V., Kaneko, K., Safar, J., Vergara, J., Tremblay, P., DeArmond, S. J., Cohen, F. E., Prusiner, S. B. & Wallace, A. C. (2002). Dominant-negative inhibition of prion replication in transgenic mice. Proc Natl Acad Sci U S A 99, 13079–13084.[Abstract/Free Full Text]

Priola, S. A. & Lawson, V. A. (2001). Glycosylation influences cross-species formation of protease-resistant prion protein. EMBO J 20, 6692–6699.[Abstract/Free Full Text]

Priola, S. A., Caughey, B., Race, R. E. & Chesebro, B. (1994). Heterologous PrP molecules interfere with accumulation of protease-resistant PrP in scrapie-infected murine neuroblastoma cells. J Virol 68, 4873–4878.[Abstract]

Raymond, G. J., Hope, J., Kocisko, D. A. & 12 other authors (1997). Molecular assessment of the potential transmissibilities of BSE and scrapie to humans. Nature 388, 285–288.[CrossRef][Medline]

Raymond, G. J., Bossers, A., Raymond, L. D. & 7 other authors (2000). Evidence of a molecular barrier limiting susceptibility of humans, cattle and sheep to chronic wasting disease. EMBO J 19, 4425–4430.[Abstract/Free Full Text]

Rezaei, H., Choiset, Y., Eghiaian, F., Treguer, E., Mentre, P., Debey, P., Grosclaude, J. & Haertle, T. (2002). Amyloidogenic unfolding intermediates differentiate sheep prion protein variants. J Mol Biol 322, 799–814.[CrossRef][Medline]

Sabuncu, E., Petit, S., Le Dur, A., Lan Lai, T., Vilotte, J. L., Laude, H. & Vilette, D. (2003). PrP polymorphisms tightly control sheep prion replication in cultured cells. J Virol 77, 2696–2700.[Abstract/Free Full Text]

Thorgeirsdottir, S., Sigurdarson, S., Thorisson, H. M., Georgsson, G. & Palsdottir, A. (1999). PrP gene polymorphism and natural scrapie in Icelandic sheep. J Gen Virol 80, 2527–2534.[Abstract/Free Full Text]

Tranulis, M. A. (2002). Influence of the prion protein gene, Prnp, on scrapie susceptibility in sheep. APMIS 110, 33–43.[CrossRef][Medline]

Tranulis, M. A., Osland, A., Bratberg, B. & Ulvund, M. J. (1999). Prion protein gene polymorphisms in sheep with natural scrapie and healthy controls in Norway. J Gen Virol 80, 1073–1077.[Abstract]

van Gennip, H. G., Bouma, A., van Rijn, P. A., Widjojoatmodjo, M. N. & Moormann, R. J. (2002). Experimental non-transmissible marker vaccines for classical swine fever (CSF) by trans-complementation of Erns or E2 of CSFV. Vaccine 20, 1544–1556.[CrossRef][Medline]

van Keulen, L. J., Schreuder, B. E., Meloen, R. H., Poelen-van den Berg, M., Mooij-Harkes, G., Vromans, M. E. & Langeveld, J. P. (1995). Immunohistochemical detection and localization of prion protein in brain tissue of sheep with natural scrapie. Vet Pathol 32, 299–308.[Abstract]

Wensvoort, G. (1989). Topographical and functional mapping of epitopes on hog cholera virus with monoclonal antibodies. J Gen Virol 70, 2865–2876.[Abstract]

Received 18 January 2005; accepted 15 June 2005.



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