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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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Dynamics of PrPSc in BSE-affected mice.BSE-affected mice were killed at 0, 40, 80, 120, and 150 dpi (terminal stage). (A) PrP detection by routine western blotting in disease stages. Brain homogenate [0.05% (w/v)] from BSE-affected mice was treated with (+) or without (−) 50 µg/mL PK for 1 h followed by western blotting with mAb T2. (B) PrPSc detection by immunoprecipitation. PrPSc was precipitated by mAb 6A12 from the same samples as in (A). The precipitated PrPSc was treated with (+) or without (−) PK, and PrP signals were detected by HRP-conjugated mAb T2. PK (−) samples indicate the total amount of PrPSc, and PK (+) samples represent the amount of PrPcore. Molecular weights are shown on the left (kDa). (C) Quantification of PrPSc signals in (B). The band intensity relative to total PrPSc (%) at the terminal stage is shown. Black bar: PrPcore. White bar: total PrPSc. All values were calculated as the mean ± standard deviation of at least 3 independent experiments.
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pone-0058013-g006: Dynamics of PrPSc in BSE-affected mice.BSE-affected mice were killed at 0, 40, 80, 120, and 150 dpi (terminal stage). (A) PrP detection by routine western blotting in disease stages. Brain homogenate [0.05% (w/v)] from BSE-affected mice was treated with (+) or without (−) 50 µg/mL PK for 1 h followed by western blotting with mAb T2. (B) PrPSc detection by immunoprecipitation. PrPSc was precipitated by mAb 6A12 from the same samples as in (A). The precipitated PrPSc was treated with (+) or without (−) PK, and PrP signals were detected by HRP-conjugated mAb T2. PK (−) samples indicate the total amount of PrPSc, and PK (+) samples represent the amount of PrPcore. Molecular weights are shown on the left (kDa). (C) Quantification of PrPSc signals in (B). The band intensity relative to total PrPSc (%) at the terminal stage is shown. Black bar: PrPcore. White bar: total PrPSc. All values were calculated as the mean ± standard deviation of at least 3 independent experiments.

Mentions: We successfully isolated native and intact PrPSc by using the newly generated mAbs. Next, we examined the dynamics of native PrPSc in BSE-affected mice over the disease time course using these mAbs. PrPSc signals were detected at 80 dpi by routine western blotting (Fig. 6A). In immunoprecipitation assays with mAb 6A12, the PrPSc signal was detected at 80, 120, and 150 dpi (Fig. 6B). The dynamics of PrPcore detected after PK digestion of 6A12-precipitated PrPSc were similar to those of PrPcore detected by western blotting (Figs. 6A, B). To estimate the amount of PrPcore relative to the amount of total PrPSc, the signal intensity of 6A12-precipitated PrPSc with PK digestion (PrPcore) was compared to that of 6A12-precipitated PrPSc without PK digestion (total PrPSc). The ratios of PrPcore to total PrPSc were 3.9∶12.2, 15.4∶57.7, and 27.0∶100 at 80, 120, and 150 dpi, respectively (Fig. 6C). Interestingly, the percentage of PrPcore was constant (27–30% of total PrPSc) throughout the course of the disease.


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)

Dynamics of PrPSc in BSE-affected mice.BSE-affected mice were killed at 0, 40, 80, 120, and 150 dpi (terminal stage). (A) PrP detection by routine western blotting in disease stages. Brain homogenate [0.05% (w/v)] from BSE-affected mice was treated with (+) or without (−) 50 µg/mL PK for 1 h followed by western blotting with mAb T2. (B) PrPSc detection by immunoprecipitation. PrPSc was precipitated by mAb 6A12 from the same samples as in (A). The precipitated PrPSc was treated with (+) or without (−) PK, and PrP signals were detected by HRP-conjugated mAb T2. PK (−) samples indicate the total amount of PrPSc, and PK (+) samples represent the amount of PrPcore. Molecular weights are shown on the left (kDa). (C) Quantification of PrPSc signals in (B). The band intensity relative to total PrPSc (%) at the terminal stage is shown. Black bar: PrPcore. White bar: total PrPSc. All values were calculated as the mean ± standard deviation of at least 3 independent experiments.
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Related In: Results  -  Collection

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pone-0058013-g006: Dynamics of PrPSc in BSE-affected mice.BSE-affected mice were killed at 0, 40, 80, 120, and 150 dpi (terminal stage). (A) PrP detection by routine western blotting in disease stages. Brain homogenate [0.05% (w/v)] from BSE-affected mice was treated with (+) or without (−) 50 µg/mL PK for 1 h followed by western blotting with mAb T2. (B) PrPSc detection by immunoprecipitation. PrPSc was precipitated by mAb 6A12 from the same samples as in (A). The precipitated PrPSc was treated with (+) or without (−) PK, and PrP signals were detected by HRP-conjugated mAb T2. PK (−) samples indicate the total amount of PrPSc, and PK (+) samples represent the amount of PrPcore. Molecular weights are shown on the left (kDa). (C) Quantification of PrPSc signals in (B). The band intensity relative to total PrPSc (%) at the terminal stage is shown. Black bar: PrPcore. White bar: total PrPSc. All values were calculated as the mean ± standard deviation of at least 3 independent experiments.
Mentions: We successfully isolated native and intact PrPSc by using the newly generated mAbs. Next, we examined the dynamics of native PrPSc in BSE-affected mice over the disease time course using these mAbs. PrPSc signals were detected at 80 dpi by routine western blotting (Fig. 6A). In immunoprecipitation assays with mAb 6A12, the PrPSc signal was detected at 80, 120, and 150 dpi (Fig. 6B). The dynamics of PrPcore detected after PK digestion of 6A12-precipitated PrPSc were similar to those of PrPcore detected by western blotting (Figs. 6A, B). To estimate the amount of PrPcore relative to the amount of total PrPSc, the signal intensity of 6A12-precipitated PrPSc with PK digestion (PrPcore) was compared to that of 6A12-precipitated PrPSc without PK digestion (total PrPSc). The ratios of PrPcore to total PrPSc were 3.9∶12.2, 15.4∶57.7, and 27.0∶100 at 80, 120, and 150 dpi, respectively (Fig. 6C). Interestingly, the percentage of PrPcore was constant (27–30% of total PrPSc) throughout the course of the disease.

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