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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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Dominant inhibition of the cell-free conversion by alleles associated with low susceptibility. (A) PrPres fragments generated from 136A (lane 1), a 1:1 mxiture of 136A with BSA (lanes 2-3), a 1:1 mixture of 136A with 171R (lanes 4-5), a 1:1 mixture of 136A with 154H (lanes 6-7). PrPres fragments derived from IHNRRQ (lane 8), a 1:1 mixture of IHNRRQ with 146S (lanes 9-10), a 1:1 mixture of IHNRRQ with 146D (lanes 11-12) and a 1:1 mixture of IHNRRQ with 222K (lanes 13-14). Detection was carried out using mab P4. Arrow indicates PrPres fragments. (B) Mean relative conversion efficiencies (± SEM) for each set of at least four conversion reactions. Relative conversion rates were calculated in relation to the ovine 136A reference allele and the caprine IHNRRQ reference allele. 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 2: Dominant inhibition of the cell-free conversion by alleles associated with low susceptibility. (A) PrPres fragments generated from 136A (lane 1), a 1:1 mxiture of 136A with BSA (lanes 2-3), a 1:1 mixture of 136A with 171R (lanes 4-5), a 1:1 mixture of 136A with 154H (lanes 6-7). PrPres fragments derived from IHNRRQ (lane 8), a 1:1 mixture of IHNRRQ with 146S (lanes 9-10), a 1:1 mixture of IHNRRQ with 146D (lanes 11-12) and a 1:1 mixture of IHNRRQ with 222K (lanes 13-14). Detection was carried out using mab P4. Arrow indicates PrPres fragments. (B) Mean relative conversion efficiencies (± SEM) for each set of at least four conversion reactions. Relative conversion rates were calculated in relation to the ovine 136A reference allele and the caprine IHNRRQ reference allele. The differences were analyzed by unpaired student t-test. *: p < 0.05; **: p < 0.01; ***: p < 0.001. ns: not significant.

Mentions: To examine, whether the alleles 154H or 171R, which are linked to low susceptibility, had an inhibitory effect on the cell-free conversion of the ovine 136A allele, 1:1 mixtures of 136A with variants 154H or 171R (corresponding to heterozygous 136A/154H and 136A/171R animals) were carried out. Both combinations 136A/171R (Figure 2A, lanes 4-5) and 136A/154H (Figure 2A, lanes 6-7) showed a clear reduction to 17.5% (154H) and 12.3% (171R) in the conversion rates (Figure 2B) compared to 136A alone (Figure 2A, lane 1, Figure 2B). As a control a 1:1 mixture of 136A with bovine serum albumine as additive was used (retaining the same total amount of protein of the sample) in order to analyse the conversion rate of 50% of the original 136A PrP. The determined reduction was 59%, which correlates to the half of the amount of 136A (Figure 2A, lanes 2-3, Figure 2B). The significant reduction by 154H and 171R allele, which exceeded 50% by far, underlines their direct interference with the 136A allele and definitely shows an inhibitory effect of 171R and 154H alleles over the 136A alleles at the protein level.


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)

Dominant inhibition of the cell-free conversion by alleles associated with low susceptibility. (A) PrPres fragments generated from 136A (lane 1), a 1:1 mxiture of 136A with BSA (lanes 2-3), a 1:1 mixture of 136A with 171R (lanes 4-5), a 1:1 mixture of 136A with 154H (lanes 6-7). PrPres fragments derived from IHNRRQ (lane 8), a 1:1 mixture of IHNRRQ with 146S (lanes 9-10), a 1:1 mixture of IHNRRQ with 146D (lanes 11-12) and a 1:1 mixture of IHNRRQ with 222K (lanes 13-14). Detection was carried out using mab P4. Arrow indicates PrPres fragments. (B) Mean relative conversion efficiencies (± SEM) for each set of at least four conversion reactions. Relative conversion rates were calculated in relation to the ovine 136A reference allele and the caprine IHNRRQ reference allele. 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 2: Dominant inhibition of the cell-free conversion by alleles associated with low susceptibility. (A) PrPres fragments generated from 136A (lane 1), a 1:1 mxiture of 136A with BSA (lanes 2-3), a 1:1 mixture of 136A with 171R (lanes 4-5), a 1:1 mixture of 136A with 154H (lanes 6-7). PrPres fragments derived from IHNRRQ (lane 8), a 1:1 mixture of IHNRRQ with 146S (lanes 9-10), a 1:1 mixture of IHNRRQ with 146D (lanes 11-12) and a 1:1 mixture of IHNRRQ with 222K (lanes 13-14). Detection was carried out using mab P4. Arrow indicates PrPres fragments. (B) Mean relative conversion efficiencies (± SEM) for each set of at least four conversion reactions. Relative conversion rates were calculated in relation to the ovine 136A reference allele and the caprine IHNRRQ reference allele. The differences were analyzed by unpaired student t-test. *: p < 0.05; **: p < 0.01; ***: p < 0.001. ns: not significant.
Mentions: To examine, whether the alleles 154H or 171R, which are linked to low susceptibility, had an inhibitory effect on the cell-free conversion of the ovine 136A allele, 1:1 mixtures of 136A with variants 154H or 171R (corresponding to heterozygous 136A/154H and 136A/171R animals) were carried out. Both combinations 136A/171R (Figure 2A, lanes 4-5) and 136A/154H (Figure 2A, lanes 6-7) showed a clear reduction to 17.5% (154H) and 12.3% (171R) in the conversion rates (Figure 2B) compared to 136A alone (Figure 2A, lane 1, Figure 2B). As a control a 1:1 mixture of 136A with bovine serum albumine as additive was used (retaining the same total amount of protein of the sample) in order to analyse the conversion rate of 50% of the original 136A PrP. The determined reduction was 59%, which correlates to the half of the amount of 136A (Figure 2A, lanes 2-3, Figure 2B). The significant reduction by 154H and 171R allele, which exceeded 50% by far, underlines their direct interference with the 136A allele and definitely shows an inhibitory effect of 171R and 154H alleles over the 136A alleles at the protein level.

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