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Short Communication |
1 Institute for Animal Health, Compton Laboratories, Newbury, Berkshire RG20 7NN, UK
2 Neuropathogenesis Unit, Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh EH9 3JF, UK
Correspondence
Louise Kirby
louise.kirby{at}bbsrc.ac.uk
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ABSTRACT |
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; Hope et al., 1986; Meyer et al., 1986). Although the mechanism of conversion is unknown, interaction between PrPc and PrPSc is critically implicated by in vitro studies (Caughey & Chesebro, 1997) and features in the current models of replication (Prusiner, 1991; Jarrett & Lansbury, 1993).
Polymorphisms in PrP are associated with susceptibility to and pathology of TSEs. The major determinants controlling the susceptibility of sheep to scrapie are polymorphisms at PrP codons 136, 154 and 171 (Hunter et al., 1997). The PrP ARR allele (amino acids, in single-letter code, at positions 136, 154 and 171, respectively) is associated with resistance to classical scrapie. The PrP ARQ and VRQ alleles are associated with susceptibility to disease. Novel ovine PrP polymorphisms are identified regularly in sheep-genotyping programmes. Most of these novel polymorphisms occur with low frequency and their association with disease susceptibility is not known (Baylis & Goldmann, 2004). It is important to assess the disease association of such variants in the hope of identifying additional scrapie-resistant alleles, as not all breeds or populations have significant frequencies of the known resistant ARR allele and it has been reported that sheep homozygous for the ARR allele may be susceptible to atypical scrapie (Buschmann et al., 2004) and intracerebral (i.c.) inoculation with BSE (Houston et al., 2003). The identification of other resistant alleles may also help to protect against novel strains of scrapie or adaptation to a particular genotype.
Due to the long incubation time and high cost of animal experiments, the in vitro cell-free conversion assay has provided a quick, well-defined system in which to assess the disease association of such alleles (Kocisko et al., 1994). However, for natural infection, dose, route, strain of agent, influence of second allele and breed of sheep are all likely to play a role in TSE susceptibility, and additional evidence from experimental challenge would be required to support any association of particular alleles with resistance to TSEs. In the cell-free conversion assay, PrPSc, isolated from the brains of scrapie-infected animals, induces the conversion of radiolabelled recombinant PrP to a PK-resistant isoform, PrPres (Kocisko et al., 1994). The assay has been shown to replicate in vivo species specificity, strain properties and polymorphism barriers (Bessen et al., 1995; Kocisko et al., 1995; Bossers et al., 1997, 2000; Raymond et al., 1997; Horiuchi et al., 2000; Iniguez et al., 2000; Zhang et al., 2002) and has been used to study many aspects of molecular conversion. As yet, however, no in vitro, cell-free-generated PrPres has been shown to be infectious (Hill et al., 1999). More recently, we reported the use, as substrate, of mouse and hamster PrP purified biochemically from recombinant bacteria and demonstrated that the assay replicated several characteristics of in vivo disease (Kirby et al., 2003).
Evidence is presented here that bacterial recombinant ovine PrP can be converted to PrPres in the cell-free conversion assay and that the sheep polymorphism barriers of scrapie transmission are replicated. Full-length ovine PrP of the ARR, ARQ and VRQ genotypes, with the N-terminal signal sequence replaced with methionine and the C-terminal signal sequence encoding glycosylphosphatidylinisotol-anchor addition removed, corresponding to aa 25–233, was PCR-amplified from genomic DNA by using the 5' and 3' primers 5'-GGATCCATCATGAAGAAGCGACCAAAACCTGGC-3' and 5'-CCGAATTCTCATGCCCCCCTTTGGTAATAA-3', respectively. Plasmid pTrcHis B (Invitrogen) was digested with restriction enzymes NcoI and EcoRI to remove the six-histidine tag. PCR fragments were digested with restriction enzymes EcoRI and RcaI and ligated into the modified pTrcHis B plasmid. Therefore, the vector encodes full-length, untagged ovine PrP. Calcium chloride-competent Escherichia coli strain 1B392 (Wright et al., 1986) was transformed with the recombinant vectors. Ovine PrP variants were expressed, radiolabelled, purified, refolded and characterized by mass spectrometry and circular dichroism (CD) as described previously (Kirby et al., 2003). A representative autoradiograph is shown in Fig. 1, lanes 1–3. PrPSc was purified from the brainstems of VRQ-homozygous sheep clinically infected with scrapie (SSBP/1 source), from ARQ-homozygous sheep clinically infected with BSE and from BSE-infected cows, based on a method described by Hope et al. (1986). Cell-free conversion assays were carried out by using the three radiolabelled ovine PrP variants ([35S]rARRPrP, [35S]rARQPrP and [35S]rVRQPrP) as substrates and the three different PrPSc types as seeds, as described previously (Kirby et al., 2003). Briefly, 1 µg PrPSc was incubated with 200 ng [35S]rPrP for 24 h at 37 °C in a non-guanidine-containing conversion buffer. Following incubation, 5 % of the reaction was treated with 60 µg PK ml–1 for 1 h at 37 °C. PK digestion was stopped by adding Pefabloc (Roche) to 1 mM. All samples were methanol-precipitated and analysed by SDS-PAGE and autoradiography. Autoradiographs were quantifed by using Phoretix gel-analysis software. A typical autoradiograph is shown in Fig. 1(a). The experiment was repeated three times, efficiencies of conversion were determined by densitometric analysis and the mean conversion efficiencies were calculated (±SEM) by densitometric analysis of labelled PrP before and after PK treatment (Fig. 1b).
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shows that bacterial recombinant ovine PrP is converted into a PK-resistant form in the cell-free conversion assay. The conversion efficiencies obtained by using bacterial recombinant ovine PrP were low (Fig. 1b) in comparison to the efficiencies obtained by using bacterial recombinant rodent PrP (Kirby et al., 2003). Conversion efficiency may be increased by the addition of guanidine to the conversion buffer (Bossers et al., 1997). Indeed, it has recently been reported that the addition of guanidine to the assay buffer is essential to obtain any conversion of sheep PrP (Piening et al., 2006). However, this reduces the physiological relevance of the assay as a model of conversion. In addition, the conversion efficiencies between sets of experiments (a set is defined as the five types of [35S]rOvPrP and one type of PrPSc) varied enormously (Fig. 1b). This has been reported by others using a similar cell-free conversion assay (Bossers et al., 1997). The reason for this is unknown, but breed of sheep or the area of brain selected for purification of PrPSc may contribute. However, the relative conversion efficiencies within a set were similar each time that the experiment was repeated. Of the three frequently occurring ovine PrP variants, ARR, ARQ and VRQ, [35S]rARRPrP, the variant associated with resistance to classical scrapie, consistently produced the lowest amount of PrPres when used as substrate in the cell-free conversion reaction (Fig. 1a, lanes 6, 11 and 16). [35S]rARQPrP and [35S]rVRQPrP, variants associated with susceptibility to scrapie and short incubation times, converted with higher efficiencies (Fig. 1a, lanes 7, 8, 12, 13, 17 and 18). A switch in the convertibility of [35S]rARQPrP and [35S]rVRQPrP is evident with homologous and heterologous PrPSc. The homologous conversion reactions produced the greatest amount of PrPres (Fig. 1a, lanes 8 and 17). These results indicate that the known in vivo polymorphism barriers of scrapie transmission can be mimicked in the cell-free conversion assay using bacterial recombinant ovine PrP.
Recently, Goldmann et al. (2005) identified two novel ovine PrP alleles. Sheep carrying a PrP variant with a proline to leucine polymorphism at aa 168 (L168) were shown to have increased survival time after experimental infection with BSE in two independent experiments (Goldmann et al., 2006). ARL168Q occurs at a low frequency and, although it may be linked with resistance to experimental BSE, no data exist on its resistance to scrapie infection. The other variant, ARQE175, with a change from glutamine to glutamic acid at aa 175 (E175), is also rare and not yet associated with scrapie or BSE susceptibility. Therefore, the cell-free conversion assay was used to predict whether these ovine variants are associated with resistance to scrapie infection. Amino acid positions 168 and 175 are of interest, as they are located close to the putative factor X-binding site (Telling et al., 1995; Kaneko et al., 1997) and the resistance-associated R171 position.
In a further experiment, [35S]rARL168QPrP and [35S]rARQE175PrP were produced (Fig. 1a, lanes 4 and 5) as described above for the other ovine variants and used as substrates in the cell-free conversion assay, incubating with the three different PrPSc types. [35S]rARL168QPrP converted with low efficiency using all three types of PrPSc (Fig. 1a, lanes 9, 14 and 19), indicating that the ARL168Q effect on conversion is significant for scrapie as well as for BSE. [35S]rARQE175PrP converted with an efficiency similar to that of [35S]rARQPrP with the three different types of PrPSc (Fig. 1a, lanes 10, 15 and 20), suggesting that this amino acid change does not affect conversion. Table 1 provides a rank order of conversion efficiences of the [35S]rOvPrP variants with the three different PrPSc types.
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), suggesting that mouse PrP can be used to model the effects of PrP polymorphisms in other species.
The murine equivalent of ovine PrP aa 168 is aa 164. Full-length mouse PrP (aa 23–230) of the Prn-pa genotype with a proline to leucine mutation at aa 164 was constructed by site-directed mutatgenesis (QuikChange II kit; Stratagene) using the full-length mouse PrP clone, the production of which has been described previously (Kirby et al., 2003), as a template and the following primers: 5'-CCAAGTGTACTACAGGCTAGTGGATCAGTACAGC-3' and 5'-GCTGTACTGATCCACTAGCCTGTAGTACACTTGG-3'. Rosetta E. coli (Novagen), which overexpresses the rare leucine tRNA, were transformed with pTrcMoL164PrP. MoL164PrP and MoPrP were expressed, radiolabelled, purified and characterized as described for the ovine variants. PrPSc was purified from the brains of terminally ill 87V-infected VM mice, based on a method described by Hope et al. (1986). Cell-free conversion assays, using the two mouse PrP variants as substrates and 87V PrPSc as seed, were carried out in the absence of guanidine and analysed as described previously (Kirby et al., 2003). The experiment was repeated at least three times and a typical autoradiograph is shown in Fig. 2.
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shows that [35S]rMoPrP is converted efficiently to PrPres in the presence of 87V PrPSc (Fig. 2, lane 2). However, [35S]rMoL164 PrP is not converted to PrPres in the presence of 87V PrPSc (Fig. 2, lane 3), suggesting that this amino acid/position may be a significant site in the conversion process in other species.
It is not understood how substitution of different amino acids at certain positions within PrP has such a profound effect on susceptibility. It has been suggested that mutations can modulate the stability of PrPc, PrPSc or both, or can affect the binding of PrP to effector molecules. In the case of a proline to leucine mutation, the amino acids share similar hydrophobicity, but are structurally diverse. Proline, the only imino acid, has backbone torsion angles that are controlled tightly as a result of its cyclic structure. Because of this, it results in turns in protein backbones and often occurs at the end of 
-sheets. A change from proline to leucine, which has a greater range of flexibility in its backbone angles, may reduce the propensity of PrP to form -sheets and explain the significantly protective effect of this mutation. Alternatively, leucine is an amino acid capable of involvement in many types of secondary structure, including 
-helices, and a change to a leucine may aid the stabilization of PrPc. Interestingly, the same mutation, proline to leucine, is associated with apparently spontaneous disease in humans when it occurs at position 102 in human PrP, the reverse of what we find for ovine position 168 and its equivalent mouse position. This is likely to be a reflection of the very different tertiary structures in the different parts of the molecule and the different involvement of these areas in conversion of PrPc to PrPSc. In addition, extensive gene-targeted transgenic-mouse experiments show that L101 mice have altered susceptibility to a range of TSE isolates compared with wild-type mice (Barron et al., 2001), further complicating interpretation. The neutral phenotype of the ovine PrP E175 polymorphism in our assay suggests that not every change in this region of PrP will affect susceptibility and indicates that the underlying mechanism may be highly positional and residue-specific.
To determine the molecular mechanisms responsible for the protective effect of the L168 mutation, substitution of a range of different amino acids at position 168 would be required and their effect on conversion assessed. Such experiments are currently under way in our laboratory using the murine cell-free conversion assay as a model. In addition, the ovine and murine version of the cell-free conversion assay using bacterial recombinant PrP can be used to assess the link between novel ovine polymorphisms, as they are identified, with classical scrapie, atypical scrapie, such as Nor98, and BSE.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 30 March 2006;
accepted 9 August 2006.
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