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Effects of polymorphisms in ovine and caprine prion protein alleles on cell-free conversion.

Eiden M, Soto EO, Mettenleiter TC, Groschup MH - Vet. Res. (2011)

Bottom Line: In sheep polymorphisms of the prion gene (PRNP) at the codons 136, 154 and 171 strongly influence the susceptibility to scrapie and bovine spongiform encephalopathy (BSE) infections.With this approach it was possible to assign reduced conversion efficiencies to specific polymorphisms, which are associated to low frequency in scrapie-affected goats or found only in healthy animals.Moreover, we could demonstrate a dominant-negative inhibition of prion polymorphisms associated with high susceptibility by alleles linked to low susceptibility in vitro.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Novel and Emerging Infectious Diseases at the Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, D-17493 Greifswald-Insel Riems, Germany. martin.groschup@fli.bund.de.

ABSTRACT
In sheep polymorphisms of the prion gene (PRNP) at the codons 136, 154 and 171 strongly influence the susceptibility to scrapie and bovine spongiform encephalopathy (BSE) infections. In goats a number of other gene polymorphisms were found which are suspected to trigger similar effects. However, no strong correlation between polymorphisms and TSE susceptibility in goats has yet been obtained from epidemiological studies and only a low number of experimental challenge data are available at present. We have therefore studied the potential impact of these polymorphisms in vitro by cell-free conversion assays using mouse scrapie strain Me7. Mouse scrapie brain derived PrPSc served as seeds and eleven recombinant single mutation variants of sheep and goat PrPC as conversion targets. With this approach it was possible to assign reduced conversion efficiencies to specific polymorphisms, which are associated to low frequency in scrapie-affected goats or found only in healthy animals. Moreover, we could demonstrate a dominant-negative inhibition of prion polymorphisms associated with high susceptibility by alleles linked to low susceptibility in vitro.

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Cell-free conversion of ovine and caprine PrPC variants by scrapie prions. (A) Detection of cell-free converted PrPres. Non-proteinase K (PK) treated wildtype 136A and wildtype IHNRRQ samples are shown in lane 1 and lane 5 respectively. PrPres fragments of 136A (lane 2) and its variants 154H (lane 3) and 171R (lane 4) are depicted. PrPres fragments of caprine IHNRRQ (lane 6) and its variants 142M (lane 7), 143R (lane 8), 146S (lane 9), 146D (lane 10), 151H (lane 11), 211R (lane 12) and 222K (lane 13). The ovine and caprine PrP was detected using monoclonal antibody (mab) P4. Molecular mass marker is indicated on the left. Arrow indicates PrPres fragments. (B) Mean relative conversion efficiencies (± standard error of the mean, SEM) for each set of conversion reaction. Relative conversion rates of ovine PrP variants were calculated in relation to the ovine 136A reference allele and the goat derived variants in relation to the caprine IHNRRQ reference allele. Bars depict the SEM of at least 4 reactions. The differences were analyzed by unpaired student t-test. *: p < 0.05; **: p < 0.01; ***: p < 0.001. ns: not significant.
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Figure 1: Cell-free conversion of ovine and caprine PrPC variants by scrapie prions. (A) Detection of cell-free converted PrPres. Non-proteinase K (PK) treated wildtype 136A and wildtype IHNRRQ samples are shown in lane 1 and lane 5 respectively. PrPres fragments of 136A (lane 2) and its variants 154H (lane 3) and 171R (lane 4) are depicted. PrPres fragments of caprine IHNRRQ (lane 6) and its variants 142M (lane 7), 143R (lane 8), 146S (lane 9), 146D (lane 10), 151H (lane 11), 211R (lane 12) and 222K (lane 13). The ovine and caprine PrP was detected using monoclonal antibody (mab) P4. Molecular mass marker is indicated on the left. Arrow indicates PrPres fragments. (B) Mean relative conversion efficiencies (± standard error of the mean, SEM) for each set of conversion reaction. Relative conversion rates of ovine PrP variants were calculated in relation to the ovine 136A reference allele and the goat derived variants in relation to the caprine IHNRRQ reference allele. Bars depict the SEM of at least 4 reactions. The differences were analyzed by unpaired student t-test. *: p < 0.05; **: p < 0.01; ***: p < 0.001. ns: not significant.

Mentions: Figure 1 shows the conversion of recombinant prokaryotic ovine prion protein (136A allele) into its PK resistant form, PrPres (Figure 1A, lane 2). The shift of approximately 6-7 kDa (in comparison to the nontreated control) (Figure 1A, lane 1) results from the partial resistance of PrPres, i.e. removal of the nonstructured N-terminus. The relative ratio of converted PrPC was approx. 5% of the PrPC that was initially used in the reaction. Conversions were carried out with mouse passaged scrapie strain Me7 as described before [19]. The physiological relevance of the assay was demonstrated with ovine PrP polymorphisms 154H and 171R, which strongly affect the scrapie susceptibility in sheep. In vitro, the conversion of the 154H (Figure 1A, lane 3) and 171R alleles were almost completely absent (Figure 1A, lane 4) compared to 136A conveying susceptibility in vivo (Figure 1A, lane 2). The relative conversion rates were 1.9% and 2.14% respectively as shown in Figure 1B.


Effects of polymorphisms in ovine and caprine prion protein alleles on cell-free conversion.

Eiden M, Soto EO, Mettenleiter TC, Groschup MH - Vet. Res. (2011)

Cell-free conversion of ovine and caprine PrPC variants by scrapie prions. (A) Detection of cell-free converted PrPres. Non-proteinase K (PK) treated wildtype 136A and wildtype IHNRRQ samples are shown in lane 1 and lane 5 respectively. PrPres fragments of 136A (lane 2) and its variants 154H (lane 3) and 171R (lane 4) are depicted. PrPres fragments of caprine IHNRRQ (lane 6) and its variants 142M (lane 7), 143R (lane 8), 146S (lane 9), 146D (lane 10), 151H (lane 11), 211R (lane 12) and 222K (lane 13). The ovine and caprine PrP was detected using monoclonal antibody (mab) P4. Molecular mass marker is indicated on the left. Arrow indicates PrPres fragments. (B) Mean relative conversion efficiencies (± standard error of the mean, SEM) for each set of conversion reaction. Relative conversion rates of ovine PrP variants were calculated in relation to the ovine 136A reference allele and the goat derived variants in relation to the caprine IHNRRQ reference allele. Bars depict the SEM of at least 4 reactions. The differences were analyzed by unpaired student t-test. *: p < 0.05; **: p < 0.01; ***: p < 0.001. ns: not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3050705&req=5

Figure 1: Cell-free conversion of ovine and caprine PrPC variants by scrapie prions. (A) Detection of cell-free converted PrPres. Non-proteinase K (PK) treated wildtype 136A and wildtype IHNRRQ samples are shown in lane 1 and lane 5 respectively. PrPres fragments of 136A (lane 2) and its variants 154H (lane 3) and 171R (lane 4) are depicted. PrPres fragments of caprine IHNRRQ (lane 6) and its variants 142M (lane 7), 143R (lane 8), 146S (lane 9), 146D (lane 10), 151H (lane 11), 211R (lane 12) and 222K (lane 13). The ovine and caprine PrP was detected using monoclonal antibody (mab) P4. Molecular mass marker is indicated on the left. Arrow indicates PrPres fragments. (B) Mean relative conversion efficiencies (± standard error of the mean, SEM) for each set of conversion reaction. Relative conversion rates of ovine PrP variants were calculated in relation to the ovine 136A reference allele and the goat derived variants in relation to the caprine IHNRRQ reference allele. Bars depict the SEM of at least 4 reactions. The differences were analyzed by unpaired student t-test. *: p < 0.05; **: p < 0.01; ***: p < 0.001. ns: not significant.
Mentions: Figure 1 shows the conversion of recombinant prokaryotic ovine prion protein (136A allele) into its PK resistant form, PrPres (Figure 1A, lane 2). The shift of approximately 6-7 kDa (in comparison to the nontreated control) (Figure 1A, lane 1) results from the partial resistance of PrPres, i.e. removal of the nonstructured N-terminus. The relative ratio of converted PrPC was approx. 5% of the PrPC that was initially used in the reaction. Conversions were carried out with mouse passaged scrapie strain Me7 as described before [19]. The physiological relevance of the assay was demonstrated with ovine PrP polymorphisms 154H and 171R, which strongly affect the scrapie susceptibility in sheep. In vitro, the conversion of the 154H (Figure 1A, lane 3) and 171R alleles were almost completely absent (Figure 1A, lane 4) compared to 136A conveying susceptibility in vivo (Figure 1A, lane 2). The relative conversion rates were 1.9% and 2.14% respectively as shown in Figure 1B.

Bottom Line: In sheep polymorphisms of the prion gene (PRNP) at the codons 136, 154 and 171 strongly influence the susceptibility to scrapie and bovine spongiform encephalopathy (BSE) infections.With this approach it was possible to assign reduced conversion efficiencies to specific polymorphisms, which are associated to low frequency in scrapie-affected goats or found only in healthy animals.Moreover, we could demonstrate a dominant-negative inhibition of prion polymorphisms associated with high susceptibility by alleles linked to low susceptibility in vitro.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Novel and Emerging Infectious Diseases at the Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, D-17493 Greifswald-Insel Riems, Germany. martin.groschup@fli.bund.de.

ABSTRACT
In sheep polymorphisms of the prion gene (PRNP) at the codons 136, 154 and 171 strongly influence the susceptibility to scrapie and bovine spongiform encephalopathy (BSE) infections. In goats a number of other gene polymorphisms were found which are suspected to trigger similar effects. However, no strong correlation between polymorphisms and TSE susceptibility in goats has yet been obtained from epidemiological studies and only a low number of experimental challenge data are available at present. We have therefore studied the potential impact of these polymorphisms in vitro by cell-free conversion assays using mouse scrapie strain Me7. Mouse scrapie brain derived PrPSc served as seeds and eleven recombinant single mutation variants of sheep and goat PrPC as conversion targets. With this approach it was possible to assign reduced conversion efficiencies to specific polymorphisms, which are associated to low frequency in scrapie-affected goats or found only in healthy animals. Moreover, we could demonstrate a dominant-negative inhibition of prion polymorphisms associated with high susceptibility by alleles linked to low susceptibility in vitro.

Show MeSH
Related in: MedlinePlus