Abstract
The prion diseases occur following the conversion of the cellular prion protein (PrP(C)) into disease-related isoforms (PrP(Sc)). In this study, the role of the glycosylphosphatidylinositol (GPI) anchor attached to PrP(C) in prion formation was examined using a cell painting technique. PrP(Sc) formation in two prion-infected neuronal cell lines (ScGT1 and ScN2a cells) and in scrapie-infected primary cortical neurons was increased following the introduction of PrP(C). In contrast, PrP(C) containing a GPI anchor from which the sialic acid had been removed (desialylated PrP(C)) was not converted to PrP(Sc). Furthermore, the presence of desialylated PrP(C) inhibited the production of PrP(Sc) within prion-infected cortical neurons and ScGT1 and ScN2a cells. The membrane rafts surrounding desialylated PrP(C) contained greater amounts of sialylated gangliosides and cholesterol than membrane rafts surrounding PrP(C). Desialylated PrP(C) was less sensitive to cholesterol depletion than PrP(C) and was not released from cells by treatment with glimepiride. The presence of desialylated PrP(C) in neurons caused the dissociation of cytoplasmic phospholipase A2 from PrP-containing membrane rafts and reduced the activation of cytoplasmic phospholipase A2. These findings show that the sialic acid moiety of the GPI attached to PrP(C) modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrP(Sc) formation. These results suggest that pharmacological modification of GPI glycosylation might constitute a novel therapeutic approach to prion diseases.
Highlights
The prion diseases are fatal neurodegenerative disorders that include scrapie in sheep and goats, bovine spongiform encephalopathy in cattle, and Creutzfeldt-Jakob disease in man
Deglycosylated PrPC Is Converted to PrPC into disease-associated isoforms (PrPSc)—Initial studies were performed to determine whether the N-linked glycans that are attached to PrPC affect PrPSc formation in ScGT1 cells
The addition of PrPC or endoglycosidase F-digested PrPC increased the PrPSc content of ScGT1 cells in a dose-dependent manner (Fig. 1C); there were no significant differences in the amounts of PrPSc in cells incubated with PrPC or with deglycosylated PrPC
Summary
Cell Lines—Prion-infected ScN2a and ScGT1 cells were grown in Ham’s F-12 medium containing 2 mM glutamine and 2% fetal calf serum. Cell Extracts—Treated cells were washed three times with ice-cold PBS and homogenized in an extraction buffer containing 10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, and 0.2% SDS at 106 cells/ml, and nuclei and large fragments were removed by centrifugation (1000 ϫ g for 5 min). Isolation of Detergent-resistant Membranes (DRMs)/Rafts— The cells were homogenized in an ice-cold buffer containing 1% Triton X-100, 10 mM Tris-HCl, pH 7.2, 100 mM NaCl, 10 mM EDTA, and mixed protease inhibitors (106 cells/ml), and nuclei and large fragments were removed by centrifugation (1000 ϫ g for 5 min). Any bound GPI was detected by the addition of mAb 5AB3-11 [15], followed by a biotinylated anti-mouse IgM (Sigma), extravidin-alkaline phosphatase and 1 mg/ml 4-nitrophenyl phosphate. Correlations between data sets were analyzed using Pearson’s bivariate coefficient (IBM SPSS statistics 20)
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