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Glycosaminoglycan sulphation affects the seeded misfolding of a mutant prion protein.

Lawson VA, Lumicisi B, Welton J, Machalek D, Gouramanis K, Klemm HM, Stewart JD, Masters CL, Hoke DE, Collins SJ, Hill AF - PLoS ONE (2010)

Bottom Line: Enzymatic depletion of endogenous nucleic acids or heparan sulphate (HS) from the PrP(C) substrate was found to specifically prevent PrP(res) formation seeded by mouse derived PrP(Sc).Cofactor requirements for PrP(res) formation are host species and prion strain specific and affected by disease associated mutations of the prion protein.It further highlights the challenge of designing effective therapeutics against a disease which effects a range of mammalian species, caused by range of aetiologies and prion strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Melbourne, Parkville, Victoria, Australia. vlawson@unimelb.edu.au

ABSTRACT

Background: The accumulation of protease resistant conformers of the prion protein (PrP(res)) is a key pathological feature of prion diseases. Polyanions, including RNA and glycosaminoglycans have been identified as factors that contribute to the propagation, transmission and pathogenesis of prion disease. Recent studies have suggested that the contribution of these cofactors to prion propagation may be species specific.

Methodology/principal finding: In this study a cell-free assay was used to investigate the molecular basis of polyanion stimulated PrP(res) formation using brain tissue or cell line derived murine PrP. Enzymatic depletion of endogenous nucleic acids or heparan sulphate (HS) from the PrP(C) substrate was found to specifically prevent PrP(res) formation seeded by mouse derived PrP(Sc). Modification of the negative charge afforded by the sulphation of glycosaminoglycans increased the ability of a familial PrP mutant to act as a substrate for PrP(res) formation, while having no effect on PrP(res) formed by wildtype PrP. This difference may be due to the observed differences in the binding of wild type and mutant PrP for glycosaminoglycans.

Conclusions/significance: Cofactor requirements for PrP(res) formation are host species and prion strain specific and affected by disease associated mutations of the prion protein. This may explain both species and strain dependent propagation characteristics and provide insights into the underlying mechanisms of familial prion disease. It further highlights the challenge of designing effective therapeutics against a disease which effects a range of mammalian species, caused by range of aetiologies and prion strains.

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Conversion activity of brain derived PrPC in the CAA seeded with infected brain homogenates.(A) UBH from balb/c (WT) mice were subjected to the CAA in the presence of IBH for differing periods of time (0–24 hours). B) The CAA was performed for 16 hours using IBH added to DPBS, or UBH from KO, WT or PrP over expressing Tga20 (TG) mice. DPBS represents total (DPBS− without PK treatment), and protease resistant (DPBS+ with PK treatment) PrP present in the IBH used to seed the CAA. Relative PrPC expression (without PK treatment) is shown in right of panel for KO, WT and TG mice. Conversion activity was determined as the fold increase in immunoreactive signal of WT relative to KO reactions after overnight (or as indicated) incubation at 37°C and treatment with PK (100µg/ml, 1hr at 37°C). Blots developed with 03R19. Molecular weights (kDa) are shown. Western blots are representative of replicated experiments, quantification is based on at least three experiments, mean and SEM are shown. *p<0.05, **p<0.01, ***p<0.001 using one-way analysis of variance (ANOVA) with Tukey's multiple comparison test (GraphPad, Prism).
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pone-0012351-g001: Conversion activity of brain derived PrPC in the CAA seeded with infected brain homogenates.(A) UBH from balb/c (WT) mice were subjected to the CAA in the presence of IBH for differing periods of time (0–24 hours). B) The CAA was performed for 16 hours using IBH added to DPBS, or UBH from KO, WT or PrP over expressing Tga20 (TG) mice. DPBS represents total (DPBS− without PK treatment), and protease resistant (DPBS+ with PK treatment) PrP present in the IBH used to seed the CAA. Relative PrPC expression (without PK treatment) is shown in right of panel for KO, WT and TG mice. Conversion activity was determined as the fold increase in immunoreactive signal of WT relative to KO reactions after overnight (or as indicated) incubation at 37°C and treatment with PK (100µg/ml, 1hr at 37°C). Blots developed with 03R19. Molecular weights (kDa) are shown. Western blots are representative of replicated experiments, quantification is based on at least three experiments, mean and SEM are shown. *p<0.05, **p<0.01, ***p<0.001 using one-way analysis of variance (ANOVA) with Tukey's multiple comparison test (GraphPad, Prism).

Mentions: The Conversion Activity Assay (CAA) generates PrPres from a PrPC substrate derived from an uninfected brain homogenate (UBH) seeded with a prion infected brain homogenate (IBH). Using the M1000 mouse adapted prion strain [41] as the IBH seed, PrPres formation occurs in a time (Figure 1A) and PrPC dependent manner with PrPres generated from the balb/c (WT) but not Prnp−/− (KO) mouse brain homogenates (Figure 1B). While the PrPC contained within the WT UBH was efficiently converted, there is evidence of further limiting, non-PrP factors in the process as only a small proportion of the available PrPC substrate (24±9%, n = 8) is converted in the reaction using UBH derived from PrPC over expressing Tga20 mice [42]. That an increase in PrPC does not significantly increase conversion efficiency suggests that factors other than PrPC in the UBH may limit the output of the assay (Figure 1B).


Glycosaminoglycan sulphation affects the seeded misfolding of a mutant prion protein.

Lawson VA, Lumicisi B, Welton J, Machalek D, Gouramanis K, Klemm HM, Stewart JD, Masters CL, Hoke DE, Collins SJ, Hill AF - PLoS ONE (2010)

Conversion activity of brain derived PrPC in the CAA seeded with infected brain homogenates.(A) UBH from balb/c (WT) mice were subjected to the CAA in the presence of IBH for differing periods of time (0–24 hours). B) The CAA was performed for 16 hours using IBH added to DPBS, or UBH from KO, WT or PrP over expressing Tga20 (TG) mice. DPBS represents total (DPBS− without PK treatment), and protease resistant (DPBS+ with PK treatment) PrP present in the IBH used to seed the CAA. Relative PrPC expression (without PK treatment) is shown in right of panel for KO, WT and TG mice. Conversion activity was determined as the fold increase in immunoreactive signal of WT relative to KO reactions after overnight (or as indicated) incubation at 37°C and treatment with PK (100µg/ml, 1hr at 37°C). Blots developed with 03R19. Molecular weights (kDa) are shown. Western blots are representative of replicated experiments, quantification is based on at least three experiments, mean and SEM are shown. *p<0.05, **p<0.01, ***p<0.001 using one-way analysis of variance (ANOVA) with Tukey's multiple comparison test (GraphPad, Prism).
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Related In: Results  -  Collection

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pone-0012351-g001: Conversion activity of brain derived PrPC in the CAA seeded with infected brain homogenates.(A) UBH from balb/c (WT) mice were subjected to the CAA in the presence of IBH for differing periods of time (0–24 hours). B) The CAA was performed for 16 hours using IBH added to DPBS, or UBH from KO, WT or PrP over expressing Tga20 (TG) mice. DPBS represents total (DPBS− without PK treatment), and protease resistant (DPBS+ with PK treatment) PrP present in the IBH used to seed the CAA. Relative PrPC expression (without PK treatment) is shown in right of panel for KO, WT and TG mice. Conversion activity was determined as the fold increase in immunoreactive signal of WT relative to KO reactions after overnight (or as indicated) incubation at 37°C and treatment with PK (100µg/ml, 1hr at 37°C). Blots developed with 03R19. Molecular weights (kDa) are shown. Western blots are representative of replicated experiments, quantification is based on at least three experiments, mean and SEM are shown. *p<0.05, **p<0.01, ***p<0.001 using one-way analysis of variance (ANOVA) with Tukey's multiple comparison test (GraphPad, Prism).
Mentions: The Conversion Activity Assay (CAA) generates PrPres from a PrPC substrate derived from an uninfected brain homogenate (UBH) seeded with a prion infected brain homogenate (IBH). Using the M1000 mouse adapted prion strain [41] as the IBH seed, PrPres formation occurs in a time (Figure 1A) and PrPC dependent manner with PrPres generated from the balb/c (WT) but not Prnp−/− (KO) mouse brain homogenates (Figure 1B). While the PrPC contained within the WT UBH was efficiently converted, there is evidence of further limiting, non-PrP factors in the process as only a small proportion of the available PrPC substrate (24±9%, n = 8) is converted in the reaction using UBH derived from PrPC over expressing Tga20 mice [42]. That an increase in PrPC does not significantly increase conversion efficiency suggests that factors other than PrPC in the UBH may limit the output of the assay (Figure 1B).

Bottom Line: Enzymatic depletion of endogenous nucleic acids or heparan sulphate (HS) from the PrP(C) substrate was found to specifically prevent PrP(res) formation seeded by mouse derived PrP(Sc).Cofactor requirements for PrP(res) formation are host species and prion strain specific and affected by disease associated mutations of the prion protein.It further highlights the challenge of designing effective therapeutics against a disease which effects a range of mammalian species, caused by range of aetiologies and prion strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Melbourne, Parkville, Victoria, Australia. vlawson@unimelb.edu.au

ABSTRACT

Background: The accumulation of protease resistant conformers of the prion protein (PrP(res)) is a key pathological feature of prion diseases. Polyanions, including RNA and glycosaminoglycans have been identified as factors that contribute to the propagation, transmission and pathogenesis of prion disease. Recent studies have suggested that the contribution of these cofactors to prion propagation may be species specific.

Methodology/principal finding: In this study a cell-free assay was used to investigate the molecular basis of polyanion stimulated PrP(res) formation using brain tissue or cell line derived murine PrP. Enzymatic depletion of endogenous nucleic acids or heparan sulphate (HS) from the PrP(C) substrate was found to specifically prevent PrP(res) formation seeded by mouse derived PrP(Sc). Modification of the negative charge afforded by the sulphation of glycosaminoglycans increased the ability of a familial PrP mutant to act as a substrate for PrP(res) formation, while having no effect on PrP(res) formed by wildtype PrP. This difference may be due to the observed differences in the binding of wild type and mutant PrP for glycosaminoglycans.

Conclusions/significance: Cofactor requirements for PrP(res) formation are host species and prion strain specific and affected by disease associated mutations of the prion protein. This may explain both species and strain dependent propagation characteristics and provide insights into the underlying mechanisms of familial prion disease. It further highlights the challenge of designing effective therapeutics against a disease which effects a range of mammalian species, caused by range of aetiologies and prion strains.

Show MeSH
Related in: MedlinePlus