Prion diseases are characterised by the conversion of a cellular prion protein (PrP C ) by its misfolded, hence pathogenic, isoform (PrP Sc ). The efficiency of this transition depends on the molecular similarities between both interaction partners and on the intrinsic convertibility of PrP C . Transgenic mice expressing chimeric murine/ovine PrP C (Tgmushp mice) are susceptible to BSE and/or scrapie prions of bovine or ovine origin while transgenic mice expressing similar murine/bovine PrP C chimera (Tgmubo mice) are essentially resistant. We have studied this phenomenon by cell-free conversion on procaryotically expressed chimeric PrP C . Mouse passaged scrapie or BSE PrP Sc was used as a seed and the conversion reaction was carried out under semi-native conditions. The results obtained in this assay were similar to those of our in vivo experiments. Since mubo-and mushp-PrP C differ only at four amino acid positions (S96G, N142S, Y154H and Q185E), single or double point mutations of mushp-PrP C were examined in the cell-free conversion assay. While the scrapie Me7 prion induced conversion was largely reduced by the N142S and Q185E but not by the S96G and Y154H mutation, the BSE induced conversion was retained in all mutants. Newly formed PrP res exhibited strain specific characteristics, such as the localisation of the proteinase K cleavage site, even in the chimeric PrP C mutants. We therefore postulate that the efficiency of the conversion of chimeric PrP C depends on the amino acid sequence as well as on prion strain specific effects.

Introduction

Transmissible spongiform encephalopathies (TSEs) or prion diseases are fatal neurological disorders which include among others, scrapie in small ruminants, BSE in cattle and Creutzfeldt-Jacob disease in humans. Prion diseases are supposedly caused by the conversion of the cellular prion protein (PrP C ) into an abnormal conformational isoform (PrP Sc ) [START_REF] Prusiner | Novel proteinaceous infectious particles cause scrapie[END_REF][START_REF] Prusiner | Molecular biology and pathogenesis of prion diseases[END_REF]. However, the molecular mechanism of this conversion process is still not fully understood. The formation of PrP Sc is associated with an increase in the ß-sheet content of the prion protein. This results in a partial resistance to proteases such as proteinase K (PK) and the tendency to form amyloid fibrils. Due to this insolubility, a high-resolution structure of PrP Sc can not be analysed to date, whereas the structure of the α-helical and hence more soluble PrP C has been revealed by nuclear magnetic resonance analysis [START_REF] Hornemann | Recombinant full-length murine prion protein, mPrP(23-231): purification and spectroscopic characterization[END_REF].

Various studies of familial human TSEs have indicated a coherence between the amino acid sequence of the host PrP C and both the incubation time and the disposition to develop a spongiform encephalopathy [START_REF] Mastrianni | Inherited prion disease caused by the V210I mutation: transmission to transgenic mice[END_REF]. There are about 20 point mutations or other polymorphisms known to be associated with the occurrence of human TSEs [START_REF] Prusiner | Genetics of prions[END_REF]. It has also been demonstrated, that the substitutions of few or even single amino acids have the ability to alter either the incubation time or actually the probability to develop clinical disease [START_REF] Korth | Abbreviated incubation times for human prions in mice expressing a chimeric mouse-human prion protein transgene[END_REF]. In sheep nine different alleles/polymorphisms out of the 256 amino acids of the prion protein have been described. Out of these, the polymorphisms at amino acids 136, 154 and 171 have been linked with the susceptibility to classical scrapie and BSE infections and are currently used in the European Union for breeding programs to generate resistant sheep [START_REF] Hunter | Natural scrapie and PrP genotype: case control studies in British sheep[END_REF][START_REF] Goldmann | Frequencies of PrP gene haplotypes in British sheep flocks and the implications for breeding programmes[END_REF]. Another polymorphism at amino acid 141 is linked to the susceptibility to atypical scrapie [START_REF] Moum | Polymorphisms at codons 141 and 154 in the ovine prion protein gene are associated with scrapie Nor98 cases[END_REF]. Other than in sheep only one amino acid polymorphism (introduction of an additional sixt octapeptide sequence) has been revealed in cattle which, however, is not modulating the BSE prion susceptibility [START_REF] Prusiner | Immunologic and molecular biologic studies of prion proteins in bovine spongiform encephalopathy[END_REF].

The molecular mechanism by which these mutations in PrP control the incubation times is not as yet understood. The species barrier phenomenon, i.e. the reduced efficiency of prion transmission to a new host species, is primarily believed to result from the inefficient

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interaction between PrP Sc and PrP C from the two different species. Moreover, independent from this effect a variety of different prion strains has been found. Some strains propagate only inefficiently in some animal species, while others replicate very well. Prion strains can also be distinguished by distinct incubation times in different host species, by distinct neuropathological lesion profiles in defined mouse lines and, as revealed by western blot analysis, by differences in the PK resistance and in the glycosylation patterns of the abnormal PrP Sc [START_REF] Groschup | Die TSE-Erregerstämme[END_REF][START_REF] Bruce | TSE strain variation[END_REF].

In order to overcome the species barrier for the transmission of human or ruminant derived prions to mice, a number of transgenic mouse models have been developed in which the human, bovine or ovine PrP C is overexpressed [START_REF] Buschmann | Detection of cattle-derived BSE prions using transgenic mice overexpressing bovine PrP(C)[END_REF][START_REF] Crozet | Efficient Transmission of Two Different Sheep Scrapie Isolates in Transgenic Mice Expressing the Ovine PrP Gene[END_REF][START_REF] Telling | Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein[END_REF][START_REF] Telling | Transmission of Creutzfeldt-Jakob Disease from humans to transgenic mice expressing chimeric human-mouse prion protein[END_REF]. For example, Tgbov XV mice exhibit significantly shorter incubation times following inoculation with cattle derived BSE prions (220-240 days) than conventional RIII mice (well over 330 days) [START_REF] Buschmann | Detection of cattle-derived BSE prions using transgenic mice overexpressing bovine PrP(C)[END_REF]. Moreover, these incubation times are not further shortened during the secondary passage. Furthermore, by parallel end point titrations it was shown that these mice are approximately 10.000 times more susceptible to a BSE infection than RIII mice and are about ten times more susceptible than cattle [START_REF] Buschmann | Highly bovine spongiform encephalopathy-sensitive transgenic mice confirm the essential restriction of infectivity to the nervous system in clinically diseased cattle[END_REF]. However, in humanized and bovinized transgenic mice this PrP C replacement strategy only worked well when the endogenous mouse PrP C expression was ablated [START_REF] Telling | Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein[END_REF]. On the other hand, human prions could also be efficiently transmitted into transgenic mice expressing a chimeric PrP C in which the core part of murine PrP C (amino acids 94-188 of hamster PrP) was substituted by human sequences when they coexpressed wild-type murine PrP C [START_REF] Telling | Transmission of Creutzfeldt-Jakob Disease from humans to transgenic mice expressing chimeric human-mouse prion protein[END_REF]. This phenomenon gave rise to the protein X hypothesis which postulates that murine cofactors obligatorily needed for the human PrP C conversion would bind with high affinity to murine PrP C , if coexpressed and thereby block the conversion of the transgeneencoded PrP C . As human/murine chimeric PrP C expressing transgenic mice were equally susceptible to a CJD infection, it was concluded that the authentic binding sites for protein X would also be provided by the chimeric protein.

Chimeric mushp-PrP C overexpressing transgenic mice were almost equally susceptible to ovine and bovine TSE isolates as wild-type ovine PrP C overexpressing transgenic mice (results described in this paper). However, a different effect was observed in bovine/murine chimeric PrP C overexpressing transgenic mice which were almost completely resistant to a BSE infection, even in the absence of an endogenous murine PrP C expression [START_REF] Scott | Propagation of prion strains through specific conformers of the prion protein[END_REF][START_REF] Buschmann | Highly bovine spongiform encephalopathy-sensitive transgenic mice confirm the essential restriction of infectivity to the nervous system in clinically diseased cattle[END_REF]. Interestingly, mubo-PrP C and mushp-PrP C differ in just four amino acids at residues 96, 142, 154 and 185 (Fig. 1). Therefore we used these models to examine the influence of the primary structure of PrP C on the protein interaction and the convertibility during the initial steps of the prion cascade. For this analysis we used a cell-free conversion assay in which the conversion reaction can be mimicked by incubating highly purified PrP C molecules together with PrP Sc seeds in an appropriate conversion buffer [START_REF] Caughey | Methods for studying prion protein (PrP) metabolism and the formation of protease-resistant PrP in cell culture and cell-free systems. An update[END_REF]. Under these conditions it was shown that PrP Sc itself is able to induce the conversion of PrP C into a partially proteinase K resistant form which is designated PrP res . This newly formed PrP res can be selectively detected and quantified by an antibody that reacts to an epitope tag which is absent in the original PrP Sc seed [START_REF] Vorberg | A Novel Epitope for the Specific Detection of Exogenous Prion Proteins in Transgenic Mice and Transfected Murine Cell Lines[END_REF][START_REF] Eiden | Synergistic and strain specific effects of BSE and scrapie prions in the cell-free conversion of recombinant prion protein[END_REF]. As the results obtained using the cell-free assay were generally in line with in vivo data, this method offers the possibility to simulate -in a first approach-PrP C conversion reactions involved in prion diseases without performing more time, work and life consuming transgenic mouse challenge experiments.

Materials and methods

Generation of chimeric transgenic mice

The generation and characterization of murine/bovine PrP transgenic mice (Tgmubo XIII) has been described earlier [START_REF] Buschmann | Detection of cattle-derived BSE prions using transgenic mice overexpressing bovine PrP(C)[END_REF].

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Murine/ovine PrP transgenic mice (Tgmushp XIX) were prepared accordingly. Briefly, the open reading frame sequence coding for the core region of the ovine PrP from aa 94 to 188 of murine PrP (98 to 192 of ovine PrP) with the amino acids alanine (A), arginine (R), and glutamine (Q) at positions 136, 154 and 171 (PrP ARQ allele) was PCR amplified. This PCR step introduced a Kpn I digestion site and at the same time a point mutation from serine (ovine sequence) to threonine (murine sequence) at codon 98 of the ovine PrP sequence (codon 94 of the murine PrP) by PCR-mutagenesis using the primers ovPrPHG2.seq 5' GGG GTC AAG GTG GTA CCC ACA GTC 3' and ovPrPHG3.rev 5' GGT GGT GGT GAC CGT GTG TTG C 3' leaving the Bst EII digestion site at codon 189 (codon 193 of ovine PrP) intact. The fragment was then cloned into the vector muboPrPHG coding for the murine/bovine chimeric PrP [START_REF] Buschmann | Detection of cattle-derived BSE prions using transgenic mice overexpressing bovine PrP(C)[END_REF] that had been digested with Kpn I and Bst EII. The resulting transgene construct mushpPrPHG was microinjected into oocytes of PrP 0/0 mice. Five transgenic mouse lines were identified that expressed the murine/ovine chimeric PrP in their brains at a level that was at least as high as in the ovine brain, out of which the mouse line Tgmushp XIX displayed with 8-16 fold the highest overexpression. Therefore this mouse line was selected for comparative mouse bioassays.

Transgenic mouse challenge studies

Challenge studies were performed to compare the susceptibility of Tgmubo and Tgmushp mice with that of the respective mouse lines expressing full-length ovine and bovine PrP. One line of each transgene construct with the highest brain expression level was selected for comparative transmission studies. These were the lines Tgbov XV (16 fold overexpression), Tgmubo XIII (16 fold overexpression), Tgshp XI (4-8 fold overexpression) and Tgmushp XIX (8-16 fold overexpression). All challenged mice were on a PrP-ablated background. Ten or 15 mice (depending on the availability of inoculum and on the expected survival times of the mice) were inoculated intracerebrally (i.c.) with 20 µl plus 100 µl intraperitoneally (i.p.) of a 10% brain homogenate of the following BSE and scrapie isolates: BSE BBP12/92 which had already been used for a parallel BSE titration experiment in Tgbov XV mice, a German field scrapie isolate designated 'Potsdam' derived from an animal carrying the PrP ARQ/ARQ allele, and the mouse passaged scrapie strains Me7 (passaged in C57Bl mice), 22A (passaged in C57Bl mice), and Chandler (passaged in CD1 mice).

Mice were supervised at least twice weekly for the onset of clinical symptoms. Animals showing signs of disease were sacrificed and the mouse brains were tested for the accumulation of PrP Sc by western blot or immunohistochemical methods.

Detection of PrP Sc accumulation in the brains of challenged transgenic mice Preparation of scrapie-associated fibrils (SAF) and immunoblot

Two hundred microliters of a 10% mouse brain homogenate were incubated with 50 µg/ml proteinase K (PK; Roche, Mannheim, Germany) for 60 min at 37°C to completely digest all PrP C . The reaction was stopped by the addition of 10 mM PMSF (phenylmethylsulfonylfluoride) and incubated at 95°C for 5 min. After adding sarcosine to a final concentration of 10% and Tris-HCl (pH 7.4) to 10 mM, the samples were incubated at room temperature for 15 min. The samples were then carefully loaded over 250 µl of a 10 mM Tris-HCl solution (pH 7.4) containing 10% sucrose and centrifuged at 540 000 g for 45 min. The pellets were resuspended in 40 µl gel loading buffer containing 2% SDS and incubated at 95°C for 5 min.

Immunohistochemical examination

Mouse brain sections were pre-treated (incubation of the slides in formic acid for 15 min, incubation with 4 µg/ml Proteinase K at 37°C for 15 min and hydrated autoclaving for 15 min) in order to improve the accessibility of the antibody epitopes. Slides were then incubated with mab L42 [START_REF] Harmeyer | Synthetic peptide vaccines yield monoclonal antibodies to cellular and pathological prion proteins of ruminants[END_REF] at an appropriate concentration. A biotinylated goat-anti-mouse conjugate (Vector, Burnigame, CA, USA) visualized the binding of the primary antibody. For signal

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detection, an avidin-biotin-complex system (Vectastain ABC Elite Kit) in combination with the VIP substrate (Vector, Burlingame) was applied.

Mutation of Prnp

Amino acids at residues 96, 142, 154 and 185 of mushp-PrP C were substituted by those of mubo-PrP C . The mutations were carried out by a two step PCR using first internal primers for the particular substitution and external primers for the amplification of the mutant template in a second step. The mutant mushp-PrP C was appropriately named depending on the particular substitution.

Using the primers which are listed below and Prnp mushp as a template, point mutations were introduced to alter residues 96 (mushp-PrP C S96G), 142 (mushp-PrP C N142S), 154 (mushp-PrP C Y154H) and 185 (mushp-PrP C E185Q). Mutation at residue 96 was carried out by the use of 5'-GGT ACC CAC GGT CAG TGG AAC-3' as N-terminal internal primer and 5'-GTT CCA CTG ACC GTG GGT ACC-3' as Cterminal internal primer. Mutation at residue 142 was carried out using 5'-CAT TTT GGC AGT GAC TAT GAG-3' as N-terminal internal and 5'-CTC ATA GTC ACT GCC AAA ATG-3' as C-terminal internal primer. Mutation at residue 154 was carried out using 5'-GAA AAC ATG CAC CGT TAC CCC-3' as N-terminal internal primer and 5'-GGG GTA ACG GTG CAT GTT TTC-3' as C-terminal internal primer. Finally mutation at residue 185 was carried out using 5'-ACA GTC AAG GAA CAC ACA GTC-3' as N-terminal internal primer and 5'-GAC TGT GTG TTC CTT GAC TGT-3' as C-terminal internal primer. 5'-CG GGA TCC AAA AAG CGG CCA-3' and 5'-GG AAG CTT GGA TCT TCT CCC GTC G-3' were used as external primers. Successive mutations were accomplished by the use of PrnP mushp S96G as a template and the primers for substitution of residue 142 (mushp-PrP C S96G/N142S) and mushp-PrnP C S96G/N142S as a template and the primers for mutation at residue 154 respectively mushp-PrP C S96G/N142S/Y154H).

Expression and purification of recombinant PrP C

The gene coding for either mubo-PrP C or mushp-PrP C was amplified from the ORF 23-231 by PCR using the primers 5'-CG GGA TCC AAA AAG CGG CCA-3' and 5'-GG AAG CTT GGA TCT TCT CCC GTC G-3'. Using the Bam HI and Hind III cleavage sites, the amplified gene was cloned into the E. coli vector pQE 40 (Qiagen) which harbours a N-terminal histidine (his)-tag. Expression was carried out in Mi 5-cells (Qiagen) which were cultivated at 37°C in 500 ml LB medium to an optical density of 0.6 at 600 nm. After the addition of Isopropyl-ß-d-thiogalactopyranoside (to a final concentration of 1 mM) the cultures were grown for another 4.5 h and the bacteria harvested by centrifugation at 3600g and 4°C for 10 min. Purification was carried out according to the manufactures instruction under denaturing conditions (8 M Urea). Refolding was accomplished by dialysis in 20 mM HEPES (Roth) containing 0.2% 5 M EDTA and 0,1% N-lauroylsarcosine sodium salt (Sigma Aldrich).

Preparation of PrP Sc

PrP Sc was purified from terminally scrapie diseased mouse brains (C57Bl/6) (mouse scrapie Me7 as well as mouse passaged BSE). The preparation was performed according to the method described by Caughey et al. [START_REF] Caughey | Methods for studying prion protein (PrP) metabolism and the formation of protease-resistant PrP in cell culture and cell-free systems. An update[END_REF] with some modifications. Briefly, 1g mouse brain samples were homogenised on ice in 15 ml TEND buffer (10 mM Tris, pH 8,3, 1mM EDTA, 130 mM NaCl) supplemented with 10 % N-lauroylsarcosine sodium salt as well as protease inhibitors (1 mM DTT, 0,1 mM Pefabloc, 0,5µg/ml Leupeptin, 1 µg/ml Aprotinin, 0,7 µg/ml Pepstatin) and centrifuged for 30 min at 23 000 rpm (4°C) in a TLA 100.4 rotor (Beckman).

The supernatant was collected and centrifuged for 2.5 h at 58 000 rpm (4°C). The resulting pellet was resuspended in 15 ml TEND buffer containing 10% NaCl and 1% Sulfobetain SB-14 and centrifuged at 68 000 rpm for 1.5 h at 20°C. The supernatant was decanted and the pellet was resuspended in 8.8 ml TMS buffer which was supplemented with 10% NaCl and 0.5% SB-14. After homogenisation the suspension was carefully loaded onto a sucrose cushion (1.0 M sucrose, 0.1 M NaCl, 0.5% SB-14) and centrifuged at 68 000 rpm for 1.5 h at 20°C. The remaining pellet was resuspended in 200 µl PBS containing 0.5% SB-14, homogenised, sonified and finally stored at -20°C.

PrP cell-free conversion assay

The conversion reaction was carried out using equimolar amounts of PrP C and PrP Sc as determined by immunoblotting using a recombinant PrP C standard. Generally, the amounts of protein used were between 500-800 µg. After an incubation for three days at 37°C in a defined semi-native buffer (50 mM citrate pH 6.0, 200 mM KCl, 5 mM MgCl 2 , 1,25% Nlauroylsarcosine) the samples were treated with proteinase K (PK) diluted in TN buffer (0.15 M NaCl, 0.05 M TrisHCl pH 7.4) to a concentration of 30 µg/ml. After one hour at 37°C the reaction was stopped with 10 mM phenyl methansulfonyl fluoride (PMSF). A carrier protein (20 µg thyreoglobuline) was added to the samples prior to an incubation with 200 µl methanol at -20°C for at least one hour. Precipitated proteins were pelleted by centrifugation at 13 000 rpm for 15 min (20°C) in a table top centrifuge. Each experiment included control samples which did not contain any PrP Sc (Fig. 2, lanes 1, 2), which were not digested with PK (lane 1) or which were not incubated for three days at 37°C but instead immediately stored at -20°C (lane 3).

SDS-PAGE and immunoblotting

Following centrifugation the pellets were resuspended in a loading buffer (1% SDS, 25mM Tris/HCl pH7.4, 0.5% mercaptoethanol, 0.001% bromphenolblue) and incubated at 95°C for 5 min. 15 µl of each sample was separated in a 16% acrylamide gel. Proteins were then transferred onto PVDF membranes by semi-dry electroblotting. The membranes were incubated with blocking buffer (PBS, 0.1% Tween, 5% skimmed milk powder) at room temperature for 30 min, followed by an incubation for 1 hour with the monoclonal antibody mab L42 [START_REF] Harmeyer | Synthetic peptide vaccines yield monoclonal antibodies to cellular and pathological prion proteins of ruminants[END_REF] that specifically binds to ruminant PrP but not to murine PrP in order to detect the newly formed PrP res . Afterwards, the membranes were washed three times for ten minutes in PBS, 0.1% Tween. The proteins were visualised by using an alkaline phosphatase conjugated goat anti-mouse secondary antibody and the c hemiluminescence detection substrate CDP-Star (Tropix).

After treating the membranes with power strip buffer (0.2 M glycin, 1% SDS, pH 2) two times for ten minutes PrP Sc was detected by using a polyclonal antibody pab Ra10 [START_REF] Groschup | The major species specific epitope in prion proteins of ruminants[END_REF][START_REF] Groschup | Antigenic features of prion proteins of sheep and of other mammalian species[END_REF] that does not detect ruminant PrP, but specifically binds to murine PrP, and visualised by goat anti-rabbit AP and CDP-Star.

Quantification and determination of molecular mass

Chemiluminescence signals were detected using the BioRad VersaDoc TM photo imaging system and analysed by the Quantity One quantification software (BioRad). The conversion rate was defined as the average rate of five independent reactions. Efficiencies were calculated as the percentages of PrP C converted into newly formed PrP res . Determination of molecular masses was carried out using a custom-designed protein ladder, which ranged between 16 and 23 kDa in steps of 1 kDa (FLI marker). Each of the proteins harboured an amino-terminal his-tag and could therefore be detected by an anti-histidine antibody (RGS-His., Qiagen).

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Results

Generation and challenge of transgenic mice

Chimeric murine/ovine (Tgmushp XI) and murine/bovine PrP C (Tgmubo XIII) overexpressing transgenic mice were generated in which residues 94-188 represented ruminant PrP C . Transgenic lines were selected in which the transgene expression was about 8-16fold higher than the PrP C expression in nontransgenic mice. All transgenic mice used in these challenge experiments were on a wild-type PrP C ablated genetic background. These challenge experiments showed that chimeric Tgmushp XIX mice are almost as susceptible to cattle derived BSE, to sheep derived scrapie as well as to mouse passaged scrapie 22A as their non-chimeric counterpart, Tgshp XI. Meanwhile, their susceptibility to the mouse passaged scrapie strains Me7 and Chandler was significantly higher than that of Tgshp XI mice. On the other hand, Tgmubo XIII mice displayed long incubation times of over 550 days after being challenged with BSE and field scrapie as well as the mouse passaged scrapie strain 22A. These mice proved to be essentially resistant to an infection with the mouse passaged scrapie strains ME7, where no mouse developed disease at all, and Chandler, where two out of seven mice developed disease after 749 days. Comparing the two transgenic mouse lines expressing the different chimeric PrP constructs, Tgmushp XIX mice show distinctly shorter incubation times than Tgmubo XIII mice after challenge with all five inocula. The most interesting difference, however, was seen after challenge with the scrapie strains ME7 and Chandler, that induced very short incubation times of 182 and 152 days in Tgmushp XIX mice and that hardly induced disease at all in Tgmubo XIII mice although these two mouse lines only differ in four amino acids of the expressed prion protein (Tab. 1).

Mouse Me7-PrP Sc induced conversion of chimeric PrP C to PrP res

The intrinsic convertibility of chimeric mubo-PrP C and mushp-PrP C was probed in a cell-free conversion assay under semi-native (i.e. non-denaturing) conditions, where purified mouse Me7-PrP Sc was used as a seed and structurally renatured and affinity purified procaryotic PrP C was used as a template. PrP C from sheep carrying the PrP ARQ (easily convertible into the pathogenic isoform) or the PrP ARR (not convertible under the applied conditions) allele in homozygosity were used as positive and negative controls for the conversion assay (Fig. 2,3 A -D). After an incubation of mushp-PrP C as a template and mouse Me7-PrP Sc as a seed for three days at 37°C, a PrP res fragment of about 17 kDa was detected in western blot analysis using mab L42. In contrast, no PrP res was detectable after the same treatment when mubo-PrP C was used as a template (Fig. 3 E -H). These cell-free conversion results were in accordance with the in vivo findings described above.

Conversion of mushp-PrP C mutants

Mushp-and mubo-PrP C differ in only four amino acids, at positions 96, 142, 154 and 185 (Fig. 1). In order to find out which of these four polymorphisms were responsible for the resistance of mubo-PrP C , we mutated each of these amino acids in mushp-PrP C , either alone or in different combinations. Residue 96 is positioned in the flexible N-terminal region of PrP C , which is not well structured. Serine was substituted by glycine in mushp-PrP C S96G.

Residue 142 is located in the vicinity of the first a-helix in the loop close to the third helix. Asparagine was replaced by serine in mushp-PrP C N142S. Mushp-PrP C Y154H was created by the substitution of tyrosine by histidine. This residue is localised at the end of the first a-helix and the amino acid side chain points to the outside of the PrP C molecule. Glutamine was replaced by glutamic acid in mushp-PrP C Q185E at residue 185 which is located in the second half of a-helix two. In the three-dimensional structure of PrP, glutamine as well as glutamic acid point towards the first structured parts of PrP C . The convertibility of mutant mushp-PrP C into its PrP res counterpart was not affected when serine was changed to glycine at residue 96 (mushp-PrP C S96G) (Fig. 4 A) or when tyrosine was changed to histidine at residue 154 (mushp-PrP C Y154H) (Fig. 4 E). However, when residue 142 was changed from asparagine to serine (mushp-PrP C N142S) (Fig. 4 B) or when residue 185 was changed from glutamine to glutamic acid (mushp-PrP C Q185E) (Fig. 4 F), the formation of a PrP res fragment could not be detected.

In the next step, we inserted combinations of several mutations in one protein. After the substitution from serine to glycine at residue 96 and asparagine to serine at residue 142 (mushp-PrP C 96-142) there was no conversion detectable (Fig. 6 B). Moreover, when tyrosine was additionally changed to histidine at residue 154 (mushp-PrP C 96-142-154), PrP C was also inconvertible (Fig. 6 B) These results indicate that point mutations at amino acid residues 142 and 185, separately or in combinations, have a negative influence on the convertibility of chimeric mushp-PrP C to PrP res when Me7-PrP Sc is used as a seed (Fig. 4 B,D; F,H).

Mouse derived BSE-PrP Sc induced conversion of chimeric PrP C to PrP res

Results obtained with mouse passaged BSE-PrP Sc seeds and mushp-PrP C or mubo-PrP C were identical to those obtained with ME7-PrP Sc , i.e. mushp-PrP C was convertible, while mubo-PrP C was not (Fig. 3 E,F).

Mouse derived BSE-PrP Sc conversion of chimeric PrP C mutants

Unlike mouse scrapie Me7-PrP Sc mouse passaged BSE induced a conversion of all mutant chimeric PrP C constructs (Fig. 4). The strain specific difference of approximately 1 kDa in molecular mass after PK digestion (Fig. 5) was maintained after the in vitro conversion. The newly formed PrP res was approximately 1 kDa lower in molecular mass when BSE-PrP Sc was used as a seed than the PK digested PrP res that was newly formed after incubation with Me7-PrP Sc as a seed (Fig. 3; Fig. 4).

The results of the convertibility assays of all PrP constructs with Me7-PrP Sc and BSE-PrP Sc are summarized in Table 2.

Quantitative amounts of PrP res

The relative ratio of converted prion protein was up to seven percent of the amount of PrP C that was initially set into the reaction (Fig. 6A). Mushp-PrP C showed almost equal conversion rates after incubation with Me7-PrP Sc or BSE-PrP Sc . Mushp-PrP C S96G as well as mushp-PrP C S154H were more efficiently converted by BSE-PrP Sc than by Me7-PrP Sc as the ratio of BSE-PrP Sc induced PrP res was about 1,5 times higher than that of Me7-PrP Sc induced PrP res . This was even more obvious for the mutants mushp-PrP C N142S, mushp PrP C Q185E, mushp-PrP C 96-142 and mushp-PrP C 96-142-154, since Me7-PrP Sc did not provoke any conversion of theses mutants, while the conversion rate was approximately 2% of the total PrP when the reaction was carried out using BSE-PrP Sc as a seed (Fig. 6 B).

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Discussion

We have generated transgenic mice overexpressing either wild-type bovine (Tgbov XV) or ovine (Tgshp XI) PrP C or murine/bovine (Tgmubo XIII) or murine/ovine (Tgmushp XIX) PrP C chimera. These animals were challenged with either murine or ovine scrapie or bovine BSE prions. Although mubo-PrP C and mushp-PrP C differ in only four amino acids (Fig. 1), Tgmushp XIX mice were highly susceptible, whereas Tgmubo XIII mice were almost resistant to an infection with mouse scrapie ME7 and Chandler. They also displayed a low susceptibility (incubation times of at least 560 days) to bovine BSE, sheep scrapie, and the scrapie strain 22A (Tab. 1). We therefore postulate the occurrence of strain-specific interactions of PrP Sc with certain amino acid positions of PrP, since the scrapie strains ME7 and Chandler induced very short incubation times of 182 and 152 days in Tgmushp XIX mice while Tgmubo XIII mice hardly developed the disease at all. The resistance of Tgmubo XIII mice is highly unlikely to be caused by a mismatch at the postulated protein X binding sites at residues 183 and 185 of PrP C since the highly susceptible Tgbov XV mice encode a protein in which these amino acids are identical to that of Tgmubo XIII mice. Since all mouse lines used in this study showed an expression rate of 8-16 fold, this difference must be caused by the differing four amino acids at positions 96, 142, 154, and 185 between Tgbov XV and Tgmubo XIII PrP C .

Similar effects were also seen, when a cell-free PrP conversion system was employed.

Although this system does not fully reflect the in vitro situation, since a procaryotic PrP C is used which is not posttranslationally modified in the same way as eucaryotically expressed PrP, the applied system is considered appropriate to study the general effects of point mutations on the convertibility of the protein. However, a distinct quantification of the conversion efficiency is difficult within this system. Further studies using more authentical systems such as PMCA are anticipated for the future. Me7-PrP Sc as well as BSE-PrP Sc were able to induce a conversion of mushp-PrP C to PrP res in the cell-free assay. In contrast, it was not possible to detect any PrP res when the conversion experiment was carried out with mubo-PrP C as a template. Thus the difference of the four amino acids at positions 96, 142, 154 and 185 appears to be also responsible for the inconvertibility of mubo-PrP C in vitro. The influence of the amino acid sequence on the convertibility and therefore on the development of disease has already be shown in numerous studies [START_REF] Priola | A Single Hamster PrP Amino Acid Blocks Conversion to Protease-Resistant PrP in Scrapie-Infected Mouse Neuroblastoma Cells[END_REF][START_REF] Manson | A single amino acid alteration in murine PrP dramatically alters TSE incubation time[END_REF]. Scrapie susceptibility of sheep is known to be linked to certain PrP genotypes [START_REF] Goldmann | Two alleles of a neuronal protein gene linken to scrapie in sheep[END_REF]. Especially polymorphisms at codons 136, 154 and 171 are known to be associated with susceptibility and disease phenotype [START_REF] Hunter | Natural scrapie and PrP genotype: case control studies in British sheep[END_REF]. These findings have been successfully reproduced in in vitro systems, either in cell culture or in cell-free conversion assays [START_REF] Bossers | Scrapie susceptibility-linked polymorphisms modulate the in vitro conversion of sheep prion protein to protease-resistant forms[END_REF][START_REF] Eiden | Synergistic and strain specific effects of BSE and scrapie prions in the cell-free conversion of recombinant prion protein[END_REF]. Since prion proteins of different species only show minor structural modifications [START_REF] Lysek | Prion protein NMR structures of cats, dogs, pigs and sheep[END_REF], it can be assumed that mubo-PrP C and mushp-PrP C also do not display major differences in their three dimensional structure. Even though single or few amino acid substitutions do not necessarily change the structural properties or the overall stability of PrP C [START_REF] Hosszu | The Residue 129 Polymorphism in Human Prion Protein Does Not Confer Susceptibility to Creutzfeldt-Jakob Disease by Altering the Structure or Global Stability of PrP C[END_REF], they may have an impact on the protein interactions prior to and/or during the conversion process as well as on the binding affinity to yet unknown cofactors. Moreover, the amino acid sequence determines the intrinsic stability of PrP C and therefore its propensity to conversion and the formation of amyloid fibrils. Such effects have been proposed regarding the allele specific polymorphisms particularly within ovine PrP C in sheep [START_REF] Eghiaian | Insight into the PrP C ? PrP Sc conversion from the structures of antibody-bound ovine prion scrapie-susceptibility variants[END_REF].

As the in vitro results were very well in accordance with the in vivo findings we tried to elucidate the molecular mechanisms of the conversion reaction by introducing relevant mutations (singular or in combination) at positions 96, 142, 154, or 185 of mushp-PrP C . These mutants were co-incubated in a cell-free conversion assay using Me7-PrP Sc as a seed. These studies revealed that the mutations at residues 96 and 154 had no effect (Fig. 4 A, E), while altering positions 142 and 185 rendered mushp-PrP C inconvertible by Me7-PrP Sc seeds (Fig. 4 B, F).

In mushp-PrP C S96G serine (uncharged polar side chain) is replaced by glycin (non-polar with a very short side chain). This residue is located in the flexible, structurally not well defined part of PrP C . This is probably the reason why this substitution did not have a negative effect on the convertibility of PrP C in our experiments. However, it has already been reported that the N-terminus of PrP C does play a role in the prion conversion, as the deletion of amino acids up to residue 124 of hamster PrP resulted in a reduced in vivo susceptibility and a lower in vitro efficiency of PrP res formation [START_REF] Lawson | Flexible Nterminal Region of Prion Protein Influences Conformation of Protease-resistant Prion Protein Isoforms Associated with Cross-species Scrapie Infection in Vivo and in Vitro[END_REF].

The Y154H substitution which also had no effect on the cell-free conversion efficiency of mushp-PrP C in our study using Me7-PrP Sc is located just after the first a-helix. However, it can be assumed that the dipolar character of the a-helix is stronger supported by the presence of tyrosine than it is by histidine, because tyrosine has a charged polar side chain (phenolic hydroxy-group), whereas histidine carries an uncharged polar side chain.

In contrast, the two mutations N142S and Q185E rendered mushp-PrP C inconvertible using Me7-PrP Sc (Fig. 4 B,F). Asparagine and glutamine at residues 142 (mushp-PrP C N142S) and 185 (mushp-PrP C Q185E) are two amino acids which are both known to stabilise secondary structure elements via H-bonds. Residue 142 is located close to the first a-helix residue, whereas 185 lies within the second a-helix. In mushp-PrP C N142S asparagine is substituted by serine. Both amino acids exhibit an uncharged polar side chain but serine is noticeably smaller than asparagine. Furthermore, serine misses the terminal carboxyamid group which enables asparagine to form H-bonds, which again enhance the intrinsic stability of the molecule. Thus, the exchange might result in a lower stability of mushp-PrP C N142S and therefore lead to negative results in cell free conversion experiments, as unstable proteins are more easily degraded by endogenous proteases [START_REF] Eghiaian | Insight into the PrP C ? PrP Sc conversion from the structures of antibody-bound ovine prion scrapie-susceptibility variants[END_REF]. On the other hand in mushp-PrP C Q185E glutamine carrying an uncharged polar side chain was substituted by glutamic acid with a charged polar side chain. Like in mushp-PrP C N142S the replacement of a stabilising amino acid at residue 185 hampered the conversion of this mutated protein into its PrPres counterpart. This is due to the terminally located carboxyamide group present in glutamine, since H-bonds can be built both from the oxygen and from the amino group. In addition to these single point mutations we gradually mutated mushp-PrP C to mubo-PrP C by consecutively introducing the amino acids substitutions S96G, N142S, Y154H and Q185E. However, as soon as the S96G substitution was combined with the N142S mutation, mushp-PrP C lost its convertibility and did not regain it by the additional replacement of Y154H.

It has been reported that the A136V and Q171R mutations in the PrP ARQ , PrP ARR and PrP VRQ alleles which modulate the scrapie resistance of sheep, seem to alter the structural stability of ovine PrP C by preventing the formation of stabilizing H-bonds at the surface of the protein [START_REF] Rezaei | Amyloidogenic Unfolding Intermediates Differentiate Sheep Prion Protein Variants[END_REF]. In accordance with this model we could show that PrP ARQ but not PrP ARR is convertible in the in the cell free conversion assay, independent of whether BSE or Me7 prions are used [START_REF] Eiden | Synergistic and strain specific effects of BSE and scrapie prions in the cell-free conversion of recombinant prion protein[END_REF].

All mushp-PrP C mutants were eventually incubated with mouse passaged BSE-PrP Sc . Surprisingly, the detection of a PrP res fragment was possible in every mutant but not in mubo-PrP C (Fig. 3,4). Although Me7-PrP Sc and BSE-PrP Sc harbour the same amino acid sequence (both derived from passages in C57Bl/6 mice), they differ in strain specific characteristics: the incubation time for mouse BSE in C57Bl/6 mice is about 177 days whereas the incubation time for mouse scrapie Me7 is 139 days in the same mouse line [START_REF] Junghans | Scrapie: Untersuchungen zum genetischen Status deutscher Schafe und Charakterisierung deutscher und anderer europaeischer Feldisolate[END_REF]. Moreover, BSE and scrapie display distinct differences in their glycosylation pattern [START_REF] Groschup | Die TSE-Erregerstämme[END_REF][START_REF] Gretzschel | Strain Typing of German Transmissible Spongiform Encephalopathies Field Cases in Small Ruminants by Biochemical Methods[END_REF] as well as the molecular mass after PK digestion (Fig. 5): The PK cleavage site of BSE-PrP Sc is located further N-terminally than in Me7-PrP Sc . Therefore, the residual Me7-PrP Sc after PK treatment has a slightly higher molecular mass than the PK resistant part of BSE-PrP Sc [START_REF] Hayashi | The N-terminal cleavage site of PrPSc from BSE differs from that of PrPSc from scrapie[END_REF].

Both distinctive features might refer to an agent specific three dimensional structure of PrP Sc , which obviously enables the pathogen to transmit its specific characteristics to the newly formed PrP res during the conversion process: PrP res showed a by one kDa reduced molecular mass after PK-digestion when the conversion reaction was carried out with BSE-PrP Sc as a seed as compared to the banding pattern that was obtained after incubation with Me7-PrP Sc as a seed. The mechanism by which such strain specific features are imparted on PrP res still remains unclear. Most probably this question will not be able to be answered as long as no high resolution structural analysis of PrP Sc is available.

The observation that BSE-PrP Sc but not Me7-PrP Sc was capable to convert mushp-PrP C N142S and mushp-PrP C Q185E shows that the conversion efficency of a PrP mutant can also strongly depend on strain specific effects. Although the precise underlying mechanism for this specific preference still remains to be elucidated, it already points at the importance of single amino acid positions for the interaction between the PrP C template and the PrP Sc seed prior to and/or during the conversion process.

A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T

Figures and Tables

Fig. 1: Amino acid sequence alignment of mouse, hamster, human bovine and sheep prion protein (residues 90-232) as well as of bovine/mouse and ovine/mouse chimera. Residues demarked 'X' specify the proposed protein X binding sites, while residues 183 and 185 are supposed to determine the primary PrP interaction sites which are responsible for intra-and inter-species binding and barrier effects in bovine PrP. Chimeric PrP C s are composed of murine sequences (blue) into which sheep sequences (mushp, yellow) or bovine sequences (mubo, green) were introduced between amino acids 94 and 188. Mubo and mushp differ in four amino acids (red), whose effects on the convertibility were examined.

Fig. 2:

A) Immunoblot of a cell-free conversion reaction using ARQ. The newly formed PrP res fragment is detected by mab L42, if recombinant PrP C (lane 1) changed its conformation into a partially PK resistant form, which is characterised by a molecular mass of about 17 kDa after PK treatment (lanes 4-6). As mab L42 does not detect mouse PrP Sc , the blotting membranes were washed with glycine buffer pH 2,0 to wash off mab L42 prior to an incubation with the polyclonal antibody Ra10 (Fig. 2 B) which detects the murine Me7-PrP Sc seed (lanes 3-6).

Fig. 3:

Immunoblots of recombinant sheep prion protein allelic variants PrP ARQ (A) and PrP ARR (B) after incubation with Me7-PrP Sc (C,D: lanes 2,3) and BSE-PrP Sc (C,D: lanes 4,5). The difference of about 1 kDa in the molecular mass of the PrP Sc seed is transmitted to the particular PrP res . Immunoblots of recombinant chimeric mubo-PrP C and mushp-PrP C (E,F) after incubation with Me7-PrP Sc (G,H: lanes 2,3) and BSE-PrP Sc (G,H: lanes 4,5). PrP res can be detected in the case of mushp-PrP C (F: lanes 2-5) whereas there is no comparable fragment detected in the case of mubo-PrP C (E).

Fig. 4:

Immunoblots of recombinant chimeric mushp-PrP C S96G (A), mushp-PrP C N142S (B), mushp-PrP C Y154H (E) and mushp-PrP C Q185E (F) after incubation with Me7-PrP Sc (lanes 2,3) or BSE-PrP Sc (lanes 4,5). PK resistant PrP is visualized by using mab L42. Immunoblot lanes on panels C, D, G and H depict the corresponding PrP Sc used as seed, which was visualized by a specific polyclonal antibody Ra10. Even though the BSE-PrP Sc signal on the immunoblot is not as strong as the one of Me7-PrP Sc (C,D), BSE-PrP Sc induces a conversion of mushp-PrP C N142S and mushp-PrP C Q185E into a partially PK resistant fragment (B,F: lanes 4,5), whiles Me7-PrP Sc was not able to induce a conversion in mushp-PrP C N142S or mushp-PrP C Q185E into PrP res (B,F: lanes 2,3). Mushp-PrP C S96G and mushp-PrP C Y154H were converted by both seeds, Me7-PrP Sc as well as BSE-PrP Sc (A,E: lanes 2-5). The slight difference in the molecular mass between Me7-PrP Sc and BSE-PrP Sc is also transferred to the PrP res of the mutant chimeric mushp-PrP C .

Fig.5:

Immunoblot of Me7-PrP Sc and BSE-PrP Sc after treatment with proteinase K. Due to a difference in the PK cleavage site, which is located further C-terminally in BSE-PrP Sc than it is in Me7-PrP Sc a slight shift in the molecular mass of about 1 kDa can be detected (small arrows) between Me7-PrP Sc and BSE-PrP Sc .

Fig. 6:

Quantitative amounts of newly formed PrP res in percent as compared to the total amount of PrP C which was used in the conversion assay. Concerning the mutant chimeric mushp-PrP C S96G/mushp-PrP C Y154H the effectivity of conversion is higher if BSE-PrP Sc is used as seed than if Me7-PrP Sc is used. In contrast, the positive control PrP ARQ shows higher amounts of newly formed PrP res if the incubation is carried out with Me7-PrP Sc instead of BSE-PrP Sc (A). Even though Me7-PrP Sc is not able to induce a conversion of the mutant chimeric mushp-PrP C s, the quantitative amount of newly formed PrP res which can be detected after an incubation with BSE-PrP Sc ranged at least up to 2% compared to the volume of undigested PrP C (B).

Tab. 1:

Average incubation times in days of transgenic mice that had accumulated PrP Sc in their brains (as detected by immunoblot and/or immunohistochemistry) after i.c. and i.p. inoculation of BSE and scrapie. All animals were euthanized after the onset of clinical symptoms or after 800 days at the end of the experiment.

Tab.2:

Overview of the chimeric PrP C s which were examined for their convertibility using either Me7-PrP Sc or BSE-PrP Sc as seeds. Mubo-PrP C was the only construct which was not converted by any of the PrP Sc seeds. Mushp WGQGGGTHSQWNKPSKPKTNMKHVAGAAAAGAVVGGLGGYMLGSAMSRPLIHFGNDYEDRYYRENMYRYPNQVYYRPVDQYSNQNNFVHDCVNITVKQH TVTTTTKGENF Mubo --------G---------------------------------------------S-----------H------------------------------E------------

Mouse

--------N-----------L----------------------------M------W--------------------------------------I--------------

  Fig.1

  Fig. 2

A C C E P T E D

M A N U S C R I P T


Matteo Bertelli
email: matteo.bertelli@birdfoundation.org

Stefano Cecchin

Cristina Lapucci

Paola De Gemmis

Daniela Danieli

Emanuele S G D'amore

Luciano Buttolo

Filippo Giunta

Pietro Mortini

Massimo Pandolfo

Corrigendum to "Quantification of chloride channel 2 (CLCN2) gene isoforms in normal versus lesion-and epilepsy-associated brain tissue" [

Please cite this article as: Matteo Bertelli, Stefano Cecchin, Cristina Lapucci, Paola de Gemmis, Daniela Danieli, Emanuele S.G. d'Amore, Luciano Buttolo, Filippo Giunta, Pietro Mortini, Massimo Pandolfo, Corrigendum to "Quantification of chloride channel 2 (CLCN2) gene isoforms in normal versus lesion-and epilepsy-associated brain tissue" [Biochem. Biophys. Acta 1772 (2007) 15-20], BBA -Molecular Basis of Disease (2007Disease ( ), doi: 10.1016Disease ( /j.bbadis.2006.10.020 .10.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.