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The N-terminal sequence of prion protein consists an epitope specific to the abnormal isoform of prion protein (PrP(Sc)).

Masujin K, Kaku-Ushiki Y, Miwa R, Okada H, Shimizu Y, Kasai K, Matsuura Y, Yokoyama T - PLoS ONE (2013)

Bottom Line: Epitope analysis revealed that mAbs 8D5 and 6A12 recognized the PrP subregions between amino acids 31-39 and 41-47, respectively.This indicates that a PrP(Sc)-specific epitope exists in the N-terminal region of PrP(Sc), and mAbs 6A12 and 8D5 are powerful tools with which to detect native and intact PrP(Sc).We found that the ratio of proteinase K (PK)-sensitive PrP(Sc) to PK-resistant PrP(Sc) was constant throughout the disease time course.

View Article: PubMed Central - PubMed

Affiliation: Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki, Japan.

ABSTRACT
The conformation of abnormal prion protein (PrP(Sc)) differs from that of cellular prion protein (PrP(C)), but the precise characteristics of PrP(Sc) remain to be elucidated. To clarify the properties of native PrP(Sc), we attempted to generate novel PrP(Sc)-specific monoclonal antibodies (mAbs) by immunizing PrP-deficient mice with intact PrP(Sc) purified from bovine spongiform encephalopathy (BSE)-affected mice. The generated mAbs 6A12 and 8D5 selectivity precipitated PrP(Sc) from the brains of prion-affected mice, sheep, and cattle, but did not precipitate PrP(C) from the brains of healthy animals. In histopathological analysis, mAbs 6A12 and 8D5 strongly reacted with prion-affected mouse brains but not with unaffected mouse brains without antigen retrieval. Epitope analysis revealed that mAbs 8D5 and 6A12 recognized the PrP subregions between amino acids 31-39 and 41-47, respectively. This indicates that a PrP(Sc)-specific epitope exists in the N-terminal region of PrP(Sc), and mAbs 6A12 and 8D5 are powerful tools with which to detect native and intact PrP(Sc). We found that the ratio of proteinase K (PK)-sensitive PrP(Sc) to PK-resistant PrP(Sc) was constant throughout the disease time course.

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Epitope analysis.(A) The epitopes of 6A12 and 8D5 in the PrP amino acid sequence. The epitope positions of mAbs 6A12 and 8D5 are represented by boxes under the amino acid sequence of mouse PrP (residues 1–254) (Supplemental Fig. 2). The dashed line indicates the octapeptide repeat region. Sequence numbers are shown on the right. (B) Peptide competition assay. P31–39 and P41–47 are synthetic peptides. MAbs were pre-incubated with (P31–39 or P41–47) and without (−) synthetic peptide prior to use in immunoprecipitation. The brain homogenate [0.05% (w/v)] from BSE-affected mice was incubated with the antibody-peptide complex and immunoprecipitated with protein G-coupled magnetic beads. The immunoprecipitated PrP was western blotted and detected with HPR-conjugated mAb T2. Total PrP in the brain homogenate was detected by routine western blotting (WB). Molecular weight markers are shown on the left (kDa). P31–39 blocked the reactivity of mAb 8D5 to PrPSc, but not the reactivity of mAb 6A12 to PrPSc. Conversely, P41–47 blocked the reactivity of mAb 6A12 to PrPSc, while P31–39 did not.
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pone-0058013-g004: Epitope analysis.(A) The epitopes of 6A12 and 8D5 in the PrP amino acid sequence. The epitope positions of mAbs 6A12 and 8D5 are represented by boxes under the amino acid sequence of mouse PrP (residues 1–254) (Supplemental Fig. 2). The dashed line indicates the octapeptide repeat region. Sequence numbers are shown on the right. (B) Peptide competition assay. P31–39 and P41–47 are synthetic peptides. MAbs were pre-incubated with (P31–39 or P41–47) and without (−) synthetic peptide prior to use in immunoprecipitation. The brain homogenate [0.05% (w/v)] from BSE-affected mice was incubated with the antibody-peptide complex and immunoprecipitated with protein G-coupled magnetic beads. The immunoprecipitated PrP was western blotted and detected with HPR-conjugated mAb T2. Total PrP in the brain homogenate was detected by routine western blotting (WB). Molecular weight markers are shown on the left (kDa). P31–39 blocked the reactivity of mAb 8D5 to PrPSc, but not the reactivity of mAb 6A12 to PrPSc. Conversely, P41–47 blocked the reactivity of mAb 6A12 to PrPSc, while P31–39 did not.

Mentions: MAbs 6A12 and 8D5 recognized PrPSc both in solution and in situ (acetone-fixed tissues). To elucidate the molecular basis for these observations, we determined the epitopes of these mAbs by a synthetic peptide assay (Fig. S1). MAb 8D5 recognized WNTGGSRYP, at amino acid positions 31–39. MAb 6A12 recognized QGSPGGN, at positions 41–47, and RYPNQVYY, at positions 155–162 (Figs. 4A and S1). To confirm the specific reactivity of the mAbs to PrPSc, we next performed peptide competition assays. We synthesized peptides corresponding to PrP amino acids 31–39 (P31–39) and 41–47 (P41–47). In the peptide competition assays, the homologous synthetic peptide completely blocked the reactivity of mAbs 8D5 and 6A12 to PrPSc, while the heterogeneous peptide did not (Fig. 4B). Further, mAbs 6A12 and 8D5 did not react with PK-digested brain homogenates, which contain PrPcore (Fig. S2). MAb 6A12 also demonstrated faint immunoreactivity against PrP amino acids 155–162 (Figs. 4A and S1) in peptide spots. However, mAb 6A12 did not react with PrPcore (Fig. S2), and the synthetic peptide (P41–47) eliminated the reactivity of mAb 6A12 to PrPSc (Fig. 4B). This suggests that PrP 155–162 is not essential to the immunoreactivity of mAb 6A12. We next immunoprecipitated denatured PrPSc to examine the properties of the epitopes for mAbs 6A12 and 8D5 on PrPSc. The brain homogenates of BSE-affected mice were pretreated with SDS (denatured PrPSc) prior to immunoprecipitation. The immunoreactivity of mAb 6H4 against PrP increased after SDS denaturation of the brain samples. MAb SAF32 reacted with PrP in both SDS-denatured and native brain homogenates. In contrast, the immunoreactivity of mAbs 6A12 and 8D5 against PrPSc was markedly decreased by denaturation (Fig. S3).


The N-terminal sequence of prion protein consists an epitope specific to the abnormal isoform of prion protein (PrP(Sc)).

Masujin K, Kaku-Ushiki Y, Miwa R, Okada H, Shimizu Y, Kasai K, Matsuura Y, Yokoyama T - PLoS ONE (2013)

Epitope analysis.(A) The epitopes of 6A12 and 8D5 in the PrP amino acid sequence. The epitope positions of mAbs 6A12 and 8D5 are represented by boxes under the amino acid sequence of mouse PrP (residues 1–254) (Supplemental Fig. 2). The dashed line indicates the octapeptide repeat region. Sequence numbers are shown on the right. (B) Peptide competition assay. P31–39 and P41–47 are synthetic peptides. MAbs were pre-incubated with (P31–39 or P41–47) and without (−) synthetic peptide prior to use in immunoprecipitation. The brain homogenate [0.05% (w/v)] from BSE-affected mice was incubated with the antibody-peptide complex and immunoprecipitated with protein G-coupled magnetic beads. The immunoprecipitated PrP was western blotted and detected with HPR-conjugated mAb T2. Total PrP in the brain homogenate was detected by routine western blotting (WB). Molecular weight markers are shown on the left (kDa). P31–39 blocked the reactivity of mAb 8D5 to PrPSc, but not the reactivity of mAb 6A12 to PrPSc. Conversely, P41–47 blocked the reactivity of mAb 6A12 to PrPSc, while P31–39 did not.
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pone-0058013-g004: Epitope analysis.(A) The epitopes of 6A12 and 8D5 in the PrP amino acid sequence. The epitope positions of mAbs 6A12 and 8D5 are represented by boxes under the amino acid sequence of mouse PrP (residues 1–254) (Supplemental Fig. 2). The dashed line indicates the octapeptide repeat region. Sequence numbers are shown on the right. (B) Peptide competition assay. P31–39 and P41–47 are synthetic peptides. MAbs were pre-incubated with (P31–39 or P41–47) and without (−) synthetic peptide prior to use in immunoprecipitation. The brain homogenate [0.05% (w/v)] from BSE-affected mice was incubated with the antibody-peptide complex and immunoprecipitated with protein G-coupled magnetic beads. The immunoprecipitated PrP was western blotted and detected with HPR-conjugated mAb T2. Total PrP in the brain homogenate was detected by routine western blotting (WB). Molecular weight markers are shown on the left (kDa). P31–39 blocked the reactivity of mAb 8D5 to PrPSc, but not the reactivity of mAb 6A12 to PrPSc. Conversely, P41–47 blocked the reactivity of mAb 6A12 to PrPSc, while P31–39 did not.
Mentions: MAbs 6A12 and 8D5 recognized PrPSc both in solution and in situ (acetone-fixed tissues). To elucidate the molecular basis for these observations, we determined the epitopes of these mAbs by a synthetic peptide assay (Fig. S1). MAb 8D5 recognized WNTGGSRYP, at amino acid positions 31–39. MAb 6A12 recognized QGSPGGN, at positions 41–47, and RYPNQVYY, at positions 155–162 (Figs. 4A and S1). To confirm the specific reactivity of the mAbs to PrPSc, we next performed peptide competition assays. We synthesized peptides corresponding to PrP amino acids 31–39 (P31–39) and 41–47 (P41–47). In the peptide competition assays, the homologous synthetic peptide completely blocked the reactivity of mAbs 8D5 and 6A12 to PrPSc, while the heterogeneous peptide did not (Fig. 4B). Further, mAbs 6A12 and 8D5 did not react with PK-digested brain homogenates, which contain PrPcore (Fig. S2). MAb 6A12 also demonstrated faint immunoreactivity against PrP amino acids 155–162 (Figs. 4A and S1) in peptide spots. However, mAb 6A12 did not react with PrPcore (Fig. S2), and the synthetic peptide (P41–47) eliminated the reactivity of mAb 6A12 to PrPSc (Fig. 4B). This suggests that PrP 155–162 is not essential to the immunoreactivity of mAb 6A12. We next immunoprecipitated denatured PrPSc to examine the properties of the epitopes for mAbs 6A12 and 8D5 on PrPSc. The brain homogenates of BSE-affected mice were pretreated with SDS (denatured PrPSc) prior to immunoprecipitation. The immunoreactivity of mAb 6H4 against PrP increased after SDS denaturation of the brain samples. MAb SAF32 reacted with PrP in both SDS-denatured and native brain homogenates. In contrast, the immunoreactivity of mAbs 6A12 and 8D5 against PrPSc was markedly decreased by denaturation (Fig. S3).

Bottom Line: Epitope analysis revealed that mAbs 8D5 and 6A12 recognized the PrP subregions between amino acids 31-39 and 41-47, respectively.This indicates that a PrP(Sc)-specific epitope exists in the N-terminal region of PrP(Sc), and mAbs 6A12 and 8D5 are powerful tools with which to detect native and intact PrP(Sc).We found that the ratio of proteinase K (PK)-sensitive PrP(Sc) to PK-resistant PrP(Sc) was constant throughout the disease time course.

View Article: PubMed Central - PubMed

Affiliation: Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki, Japan.

ABSTRACT
The conformation of abnormal prion protein (PrP(Sc)) differs from that of cellular prion protein (PrP(C)), but the precise characteristics of PrP(Sc) remain to be elucidated. To clarify the properties of native PrP(Sc), we attempted to generate novel PrP(Sc)-specific monoclonal antibodies (mAbs) by immunizing PrP-deficient mice with intact PrP(Sc) purified from bovine spongiform encephalopathy (BSE)-affected mice. The generated mAbs 6A12 and 8D5 selectivity precipitated PrP(Sc) from the brains of prion-affected mice, sheep, and cattle, but did not precipitate PrP(C) from the brains of healthy animals. In histopathological analysis, mAbs 6A12 and 8D5 strongly reacted with prion-affected mouse brains but not with unaffected mouse brains without antigen retrieval. Epitope analysis revealed that mAbs 8D5 and 6A12 recognized the PrP subregions between amino acids 31-39 and 41-47, respectively. This indicates that a PrP(Sc)-specific epitope exists in the N-terminal region of PrP(Sc), and mAbs 6A12 and 8D5 are powerful tools with which to detect native and intact PrP(Sc). We found that the ratio of proteinase K (PK)-sensitive PrP(Sc) to PK-resistant PrP(Sc) was constant throughout the disease time course.

Show MeSH
Related in: MedlinePlus