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
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ABSTRACT |
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INTRODUCTION |
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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
).
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METHODS |
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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 3060 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 ASepharose (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 (24 µg per reaction) were mixed with 10 00020 000 c.p.m. purified 35S-labelled PrPC (
2040 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 ml1 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.
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RESULTS |
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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 5154 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 PrPCPrPSc-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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DISCUSSION |
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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 PrPCPrPSc-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
-sheets [sheep aa 129134 (S1) and 163167 (S2)], which are the positions from where the first
-helix [aa 146158 (H1)] is converted into an anti-parallel organized
-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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 18 January 2005;
accepted 15 June 2005.
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