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Propagation of RML prions in mice expressing PrP devoid of GPI anchor leads to formation of a novel, stable prion strain.

Mahal SP, Jablonski J, Suponitsky-Kroyter I, Oelschlegel AM, Herva ME, Oldstone M, Weissmann C - PLoS Pathog. (2012)

Bottom Line: PrP(C), a host protein which in prion-infected animals is converted to PrP(Sc), is linked to the cell membrane by a GPI anchor.We found that RML and ME7, but not 22L prions propagated in tgGPI⁻ brain developed novel cell tropisms, as determined by the Cell Panel Assay (CPA).Surprisingly, the levels of proteinase K-resistant PrP(Sc) (PrP(res)) in RML- or ME7-infected tgGPI⁻ brain were 25-50 times higher than in wild-type brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectology, Scripps Florida, Jupiter, Florida, United States of America.

ABSTRACT
PrP(C), a host protein which in prion-infected animals is converted to PrP(Sc), is linked to the cell membrane by a GPI anchor. Mice expressing PrP(C) without GPI anchor (tgGPI⁻ mice), are susceptible to prion infection but accumulate anchorless PrP(Sc) extra-, rather than intracellularly. We investigated whether tgGPI⁻ mice could faithfully propagate prion strains despite the deviant structure and location of anchorless PrP(Sc). We found that RML and ME7, but not 22L prions propagated in tgGPI⁻ brain developed novel cell tropisms, as determined by the Cell Panel Assay (CPA). Surprisingly, the levels of proteinase K-resistant PrP(Sc) (PrP(res)) in RML- or ME7-infected tgGPI⁻ brain were 25-50 times higher than in wild-type brain. When returned to wild-type brain, ME7 prions recovered their original properties, however RML prions had given rise to a novel prion strain, designated SFL, which remained unchanged even after three passages in wild-type mice. Because both RML PrP(Sc) and SFL PrP(Sc) are stably propagated in wild-type mice we propose that the two conformations are separated by a high activation energy barrier which is abrogated in tgGPI⁻ mice.

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PrPres in RML- and ME7-infected wild-type and tgGPI- mouse brains, quantified by Western blot and sandwich ELISA.(A) Western blot of RML− and ME7-infected brain homogenates before and after PK treatment. “µg”, total protein loaded. ‘r’, 2.5 ng recombinant murine PrP (recPrP). GPI−[RML] (lanes 5, 7) and GPI−/GPI−[ME7] (lane 17) brain homogenates gave rise to ladders reflecting multimers with molecular weights extending to >250 kDa, which were reduced to monomers by PK digestion, while GPI−[ME7] homogenate gave no detectable signals. (B) Western blot of PK-digested brain homogenates treated or not with PNGase. Lanes 3, 5, 7, 9 show, from top to bottom, diglycosylated, monoglycosylated and unglycosylated, truncated PrP; PNGase-treated samples (lanes 4, 6, 8, 10) show a single band corresponding to unglycosylated, truncated PrP. Samples from tgGPI− mice (lanes 1, 11) show two bands, corresponding to truncated, monoglycosylated (top) and unglycosylated PrP, and, after PNGase treatment, a single band corresponding to truncated, deglycosylated PrP (lanes 2, 12). Because anchorless PrP is retained inefficiently by PVDF membranes [69], 24 times more total protein was loaded for GPI− than for C57 samples, to give about the same signal strength. (C) Sandwich ELISA of PK-treated samples. Absorbance of quadruplicate samples is plotted against log[input protein]. Samples a to g are identified in panel D; h, uninfected C57 brain homogenate. The abundance of a sample relative to that of RML can be read off by comparing the amounts of protein required to give the same absorbance. For example, an absorbance of 0.1 is given by 0.01 µg GPI−[RML] and 0.5 µg C57[RML] brain homogenate (total protein prior to PK treatment), therefore the abundance of GPI−[RML] is about 50 times higher than that of C57[RML] PrPres. In Figure S2 absorbance of the same samples is plotted against input protein on a linear scale, to show that the response is almost linear up to a protein input of 1.5 µg/well. (D) The PrPres signals (“pix”) from the western blots of Figure 1A were quantified relative to C57[RML] and the log of the ratio was plotted (red bars). For the sandwich ELISA, the plot shows the log of the absorbance (A) relative to that of C57[RML] (blue bars).
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ppat-1002746-g001: PrPres in RML- and ME7-infected wild-type and tgGPI- mouse brains, quantified by Western blot and sandwich ELISA.(A) Western blot of RML− and ME7-infected brain homogenates before and after PK treatment. “µg”, total protein loaded. ‘r’, 2.5 ng recombinant murine PrP (recPrP). GPI−[RML] (lanes 5, 7) and GPI−/GPI−[ME7] (lane 17) brain homogenates gave rise to ladders reflecting multimers with molecular weights extending to >250 kDa, which were reduced to monomers by PK digestion, while GPI−[ME7] homogenate gave no detectable signals. (B) Western blot of PK-digested brain homogenates treated or not with PNGase. Lanes 3, 5, 7, 9 show, from top to bottom, diglycosylated, monoglycosylated and unglycosylated, truncated PrP; PNGase-treated samples (lanes 4, 6, 8, 10) show a single band corresponding to unglycosylated, truncated PrP. Samples from tgGPI− mice (lanes 1, 11) show two bands, corresponding to truncated, monoglycosylated (top) and unglycosylated PrP, and, after PNGase treatment, a single band corresponding to truncated, deglycosylated PrP (lanes 2, 12). Because anchorless PrP is retained inefficiently by PVDF membranes [69], 24 times more total protein was loaded for GPI− than for C57 samples, to give about the same signal strength. (C) Sandwich ELISA of PK-treated samples. Absorbance of quadruplicate samples is plotted against log[input protein]. Samples a to g are identified in panel D; h, uninfected C57 brain homogenate. The abundance of a sample relative to that of RML can be read off by comparing the amounts of protein required to give the same absorbance. For example, an absorbance of 0.1 is given by 0.01 µg GPI−[RML] and 0.5 µg C57[RML] brain homogenate (total protein prior to PK treatment), therefore the abundance of GPI−[RML] is about 50 times higher than that of C57[RML] PrPres. In Figure S2 absorbance of the same samples is plotted against input protein on a linear scale, to show that the response is almost linear up to a protein input of 1.5 µg/well. (D) The PrPres signals (“pix”) from the western blots of Figure 1A were quantified relative to C57[RML] and the log of the ratio was plotted (red bars). For the sandwich ELISA, the plot shows the log of the absorbance (A) relative to that of C57[RML] (blue bars).

Mentions: We first analyzed the prion-infected brain samples for their content of PrPres. Western blot analysis of C57[RML] and C57[ME7] brain homogenates showed three PrP-specific bands, attributed to di-, mono- and unglycosylated species, whose mobility increased after truncation by PK digestion (Figure 1A, lanes 1,2 and 9,10 respectively). The same amount or threefold higher of GPI−[ME7] sample gave no detectable PrP signal (lanes 11–14), while GPI−/GPI−[ME7] (lane 17) and GPI−[RML] (lane 5) samples gave rise to a ladder of bands with mobilities corresponding to about 28 to >250 kDa. Treatment with PK converted the ladders to 2 bands (lanes 6, 8, 18), the major one corresponding to unglycosylated and the minor one to monoglycosylated GPI−PrP, as shown by the fact that PNGase digestion abrogates the slower-moving band (Figure 1B, lanes 2 and 12). The aggregated forms of GPI−-PrP are likely derived from the abundant amyloid plaques described earlier [60]. Although GPI−[ME7] brain homogenate from mice culled at 300 dpi showed barely a trace, if any at all, of PrPres on western blots (Figure 1A, lanes 12, 14) it nonetheless caused clinical disease by about 170 and 450 dpi when inoculated into wild-type and GPI− mice, respectively (Table S1, #20, 21), raising the suspicion that quantitation by western blot was erroneous. Nishina and Supattapone have reported that PrPC deprived of a GPI anchor by PIPLC-treatment was retained by the PVDF membranes used for western blotting at less than about 5% the efficiency of its GPI-linked counterpart [69]. We therefore compared the levels of PrPres in PK-treated samples from GPI− and C57 mice by western blotting and by sandwich ELISA, in which PK-treated, denatured samples were bound to wells coated with anti-PrP antibody D18 and visualized with biotinylated antibody D13. Astonishingly, sandwich ELISA revealed levels of PrPres in GPI−[RML] and GPI−/GPI−[ME7] brain 50- and 25-fold higher, respectively, than those in C57[RML] brain (Figure 1C) rather than 30% and >50% lower, as indicated by western blotting (Figure 1A). Figure 1D shows that quantitation of PrPres by western blotting and sandwich ELISA are consistent for wild-type brain but highly discordant for GPI− brain. Moreover, sandwich ELISA showed that the GPI−[ME7] samples, which were negative by western blotting, in fact contained PrPres at about 12% the level in C57[ME7] brains. Brains from GPI− mice infected with 22L, 79A and 139A were not examined by western blotting or sandwich ELISA.


Propagation of RML prions in mice expressing PrP devoid of GPI anchor leads to formation of a novel, stable prion strain.

Mahal SP, Jablonski J, Suponitsky-Kroyter I, Oelschlegel AM, Herva ME, Oldstone M, Weissmann C - PLoS Pathog. (2012)

PrPres in RML- and ME7-infected wild-type and tgGPI- mouse brains, quantified by Western blot and sandwich ELISA.(A) Western blot of RML− and ME7-infected brain homogenates before and after PK treatment. “µg”, total protein loaded. ‘r’, 2.5 ng recombinant murine PrP (recPrP). GPI−[RML] (lanes 5, 7) and GPI−/GPI−[ME7] (lane 17) brain homogenates gave rise to ladders reflecting multimers with molecular weights extending to >250 kDa, which were reduced to monomers by PK digestion, while GPI−[ME7] homogenate gave no detectable signals. (B) Western blot of PK-digested brain homogenates treated or not with PNGase. Lanes 3, 5, 7, 9 show, from top to bottom, diglycosylated, monoglycosylated and unglycosylated, truncated PrP; PNGase-treated samples (lanes 4, 6, 8, 10) show a single band corresponding to unglycosylated, truncated PrP. Samples from tgGPI− mice (lanes 1, 11) show two bands, corresponding to truncated, monoglycosylated (top) and unglycosylated PrP, and, after PNGase treatment, a single band corresponding to truncated, deglycosylated PrP (lanes 2, 12). Because anchorless PrP is retained inefficiently by PVDF membranes [69], 24 times more total protein was loaded for GPI− than for C57 samples, to give about the same signal strength. (C) Sandwich ELISA of PK-treated samples. Absorbance of quadruplicate samples is plotted against log[input protein]. Samples a to g are identified in panel D; h, uninfected C57 brain homogenate. The abundance of a sample relative to that of RML can be read off by comparing the amounts of protein required to give the same absorbance. For example, an absorbance of 0.1 is given by 0.01 µg GPI−[RML] and 0.5 µg C57[RML] brain homogenate (total protein prior to PK treatment), therefore the abundance of GPI−[RML] is about 50 times higher than that of C57[RML] PrPres. In Figure S2 absorbance of the same samples is plotted against input protein on a linear scale, to show that the response is almost linear up to a protein input of 1.5 µg/well. (D) The PrPres signals (“pix”) from the western blots of Figure 1A were quantified relative to C57[RML] and the log of the ratio was plotted (red bars). For the sandwich ELISA, the plot shows the log of the absorbance (A) relative to that of C57[RML] (blue bars).
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getmorefigures.php?uid=PMC3369955&req=5

ppat-1002746-g001: PrPres in RML- and ME7-infected wild-type and tgGPI- mouse brains, quantified by Western blot and sandwich ELISA.(A) Western blot of RML− and ME7-infected brain homogenates before and after PK treatment. “µg”, total protein loaded. ‘r’, 2.5 ng recombinant murine PrP (recPrP). GPI−[RML] (lanes 5, 7) and GPI−/GPI−[ME7] (lane 17) brain homogenates gave rise to ladders reflecting multimers with molecular weights extending to >250 kDa, which were reduced to monomers by PK digestion, while GPI−[ME7] homogenate gave no detectable signals. (B) Western blot of PK-digested brain homogenates treated or not with PNGase. Lanes 3, 5, 7, 9 show, from top to bottom, diglycosylated, monoglycosylated and unglycosylated, truncated PrP; PNGase-treated samples (lanes 4, 6, 8, 10) show a single band corresponding to unglycosylated, truncated PrP. Samples from tgGPI− mice (lanes 1, 11) show two bands, corresponding to truncated, monoglycosylated (top) and unglycosylated PrP, and, after PNGase treatment, a single band corresponding to truncated, deglycosylated PrP (lanes 2, 12). Because anchorless PrP is retained inefficiently by PVDF membranes [69], 24 times more total protein was loaded for GPI− than for C57 samples, to give about the same signal strength. (C) Sandwich ELISA of PK-treated samples. Absorbance of quadruplicate samples is plotted against log[input protein]. Samples a to g are identified in panel D; h, uninfected C57 brain homogenate. The abundance of a sample relative to that of RML can be read off by comparing the amounts of protein required to give the same absorbance. For example, an absorbance of 0.1 is given by 0.01 µg GPI−[RML] and 0.5 µg C57[RML] brain homogenate (total protein prior to PK treatment), therefore the abundance of GPI−[RML] is about 50 times higher than that of C57[RML] PrPres. In Figure S2 absorbance of the same samples is plotted against input protein on a linear scale, to show that the response is almost linear up to a protein input of 1.5 µg/well. (D) The PrPres signals (“pix”) from the western blots of Figure 1A were quantified relative to C57[RML] and the log of the ratio was plotted (red bars). For the sandwich ELISA, the plot shows the log of the absorbance (A) relative to that of C57[RML] (blue bars).
Mentions: We first analyzed the prion-infected brain samples for their content of PrPres. Western blot analysis of C57[RML] and C57[ME7] brain homogenates showed three PrP-specific bands, attributed to di-, mono- and unglycosylated species, whose mobility increased after truncation by PK digestion (Figure 1A, lanes 1,2 and 9,10 respectively). The same amount or threefold higher of GPI−[ME7] sample gave no detectable PrP signal (lanes 11–14), while GPI−/GPI−[ME7] (lane 17) and GPI−[RML] (lane 5) samples gave rise to a ladder of bands with mobilities corresponding to about 28 to >250 kDa. Treatment with PK converted the ladders to 2 bands (lanes 6, 8, 18), the major one corresponding to unglycosylated and the minor one to monoglycosylated GPI−PrP, as shown by the fact that PNGase digestion abrogates the slower-moving band (Figure 1B, lanes 2 and 12). The aggregated forms of GPI−-PrP are likely derived from the abundant amyloid plaques described earlier [60]. Although GPI−[ME7] brain homogenate from mice culled at 300 dpi showed barely a trace, if any at all, of PrPres on western blots (Figure 1A, lanes 12, 14) it nonetheless caused clinical disease by about 170 and 450 dpi when inoculated into wild-type and GPI− mice, respectively (Table S1, #20, 21), raising the suspicion that quantitation by western blot was erroneous. Nishina and Supattapone have reported that PrPC deprived of a GPI anchor by PIPLC-treatment was retained by the PVDF membranes used for western blotting at less than about 5% the efficiency of its GPI-linked counterpart [69]. We therefore compared the levels of PrPres in PK-treated samples from GPI− and C57 mice by western blotting and by sandwich ELISA, in which PK-treated, denatured samples were bound to wells coated with anti-PrP antibody D18 and visualized with biotinylated antibody D13. Astonishingly, sandwich ELISA revealed levels of PrPres in GPI−[RML] and GPI−/GPI−[ME7] brain 50- and 25-fold higher, respectively, than those in C57[RML] brain (Figure 1C) rather than 30% and >50% lower, as indicated by western blotting (Figure 1A). Figure 1D shows that quantitation of PrPres by western blotting and sandwich ELISA are consistent for wild-type brain but highly discordant for GPI− brain. Moreover, sandwich ELISA showed that the GPI−[ME7] samples, which were negative by western blotting, in fact contained PrPres at about 12% the level in C57[ME7] brains. Brains from GPI− mice infected with 22L, 79A and 139A were not examined by western blotting or sandwich ELISA.

Bottom Line: PrP(C), a host protein which in prion-infected animals is converted to PrP(Sc), is linked to the cell membrane by a GPI anchor.We found that RML and ME7, but not 22L prions propagated in tgGPI⁻ brain developed novel cell tropisms, as determined by the Cell Panel Assay (CPA).Surprisingly, the levels of proteinase K-resistant PrP(Sc) (PrP(res)) in RML- or ME7-infected tgGPI⁻ brain were 25-50 times higher than in wild-type brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectology, Scripps Florida, Jupiter, Florida, United States of America.

ABSTRACT
PrP(C), a host protein which in prion-infected animals is converted to PrP(Sc), is linked to the cell membrane by a GPI anchor. Mice expressing PrP(C) without GPI anchor (tgGPI⁻ mice), are susceptible to prion infection but accumulate anchorless PrP(Sc) extra-, rather than intracellularly. We investigated whether tgGPI⁻ mice could faithfully propagate prion strains despite the deviant structure and location of anchorless PrP(Sc). We found that RML and ME7, but not 22L prions propagated in tgGPI⁻ brain developed novel cell tropisms, as determined by the Cell Panel Assay (CPA). Surprisingly, the levels of proteinase K-resistant PrP(Sc) (PrP(res)) in RML- or ME7-infected tgGPI⁻ brain were 25-50 times higher than in wild-type brain. When returned to wild-type brain, ME7 prions recovered their original properties, however RML prions had given rise to a novel prion strain, designated SFL, which remained unchanged even after three passages in wild-type mice. Because both RML PrP(Sc) and SFL PrP(Sc) are stably propagated in wild-type mice we propose that the two conformations are separated by a high activation energy barrier which is abrogated in tgGPI⁻ mice.

Show MeSH
Related in: MedlinePlus