The cellular prion protein with a monoacylated glycosylphosphatidylinositol anchor modifies cell membranes,inhibits cell signaling and reduces prion formation

The prion diseases occur following the conversion of the cellular prion protein (PrP^C) into a disease-related isoform (PrP^Sc). In this study a cell painting technique was used to examine the role of the glycosylphosphatidylinositol (GPI) anchor attached to PrP^C in prion formation. The introduction of PrP^C to infected neuronal cells increased the cholesterol content of cell membranes, increased activation of cytoplasmic phospholipase A₂ (cPLA₂) and increased PrP^Sc formation. In contrast, PrP^C with a monoacylated GPI anchor did not alter the amount of cholesterol in cell membranes, was not found within lipid rafts and did not activate cPLA₂. Although monoacylated PrP^C remains within cells for longer than native PrP^C it was not converted to PrP^Sc. Moreover, the presence of monoacylated PrP^C displaced cPLA₂ from PrP^Sc-containing lipid rafts, reducing the activation of cPLA₂ and PrP^Sc formation. We conclude that acylation of the GPI anchor attached to PrP^C modifies the local membrane microenvironments that control some cell signaling pathways, the trafficking of PrP^C and PrP^Sc formation. In addition, such observations raise the possibility that the pharmacological modification of GPI anchors might constitute a novel therapeutic approach to prion diseases.Key words: cholesterol, glycosylphosphatidylinositol, lipid rafts, membranes, phospholipase A₂, prion, trafficA key event in the prion diseases is the conversion of a normal host protein (PrP^C) into a disease-associated isoform (PrP^Sc).¹ Although the presence of PrP^C is essential for prion formation,^2,3 not all cells that express PrP^C are permissive for PrP^Sc replication. The reasons why some cells that express PrP^C do not replicate PrP^Sc are not fully understood. Reports that the targeting of PrP^C to specific membranes is required for efficient PrP^Sc formation⁴ indicate that the factors that affect the cellular targeting and intracellular trafficking of PrP^C are critical in determining PrP^Sc replication.Our study examined the effects of the glycosylphosphatidylinositol (GPI) anchor that links the majority of PrP^C molecules to cell membranes⁵ on PrP^Sc formation. Originally GPI anchors were seen as a simple method of attaching proteins to cell membranes. However, there is increasing interest in the role of GPI anchors in complex biological functions including the regulation of membrane composition, cell signaling and protein trafficking.⁶ To examine the role of the GPI anchor PrP^C preparations were digested with phosphatidylinositol-phospholipase C (PI-PLC) (deacylated PrP^C) or phospholipase A₂ (PLA₂) (monoacylated PrP^C) (Fig. 1) and isolated by reverse phase chromatography. These digestions, coupled with a cell painting technique, allowed us to examine modifications of the GPI anchor that could not be achieved by genetic manipulation methods. Controversy surrounds the role of the GPI anchor in PrP^Sc formation; the seminal observation that transgenic mice producing anchorless PrP^C produced large amounts of extracellular PrP^Sc,⁷ suggests that the GPI has little effect upon PrP^Sc replication. In contrast, a recent study showed that cells that produce anchorless PrP^C were not permissive to PrP^Sc formation⁸ and in our study deacylated PrP^C did not affect PrP^Sc production. Although at first glance these results appear contradictory, they may be explained by reference to the site of conversion of PrP^C to PrP^Sc. Clearly anchorless PrP^C can be converted to PrP^Sc in a process that occurs within the extracellular milieu. However, as anchorless PrP^C is rapidly secreted from cells⁷ it has little contact with cell-associated PrP^Sc. Similarly we found that deacylated PrP^C was fully soluble and did not readily associate with cells.Open in a separate windowFigure 1Phospholipase digestion of PrP^C affects the acylation of the GPI anchor. Cartoon showing the putative GPI anchor attached to PrP^C, monoacylated PrP^C and deacylated PrP^C. Glycan residues shown include inositol (Inos), mannose (Man), sialic acid (SA), galactose (Gal), N-acetyl galactosamine (GalNAc) and glucosamine (GlcN) as well as phosphate (P).Native PrP^C is rapidly transferred to cells⁹ and we showed that the addition of PrP^C caused a dose-dependent increase in the PrP^Sc content of all prion-infected cell lines tested. We used this cell painting technique to introduce monoacylated PrP^C to recipient cells. Our paper describes three major observations; firstly that monoacylated PrP^C behaves differently to native PrP^C with regards to cellular distribution, intracellular trafficking and cell signaling; secondly, that monoacylated PrP^C was not converted to PrP^Sc and thirdly, that monoacylated PrP^C inhibited the conversion of endogenous PrP^C to PrP^Sc.The presence of GPI anchors targets proteins including PrP^C and PrP^Sc to specialized membrane micro-domains that are commonly called lipid rafts.^10,11 Lipid rafts are patches of membranes that are highly enriched in cholesterol and sphingolipids and which are operationally defined by their insolubility in cold non-ionic detergents and floatation as low density membranes on sucrose density gradients. The importance of lipid rafts in prion diseases is based upon studies showing that treatment with cholesterol synthesis inhibitors reduced cellular cholesterol and the formation of PrP^Sc.¹² Since the cholesterol content of cell membranes is critical for the formation of lipid rafts¹³ it is assumed that the integrity of these lipid rafts is necessary for efficient PrP^Sc formation. The presence of GPI-anchored proteins is thought to help lipid rafts form as the saturated fatty acids that are contained within GPI anchors facilitate the solubilisation of cholesterol in the membrane¹⁴ and the glycan component protect cholesterol from water.¹⁵ Thus the nature and number of the acyl chains contained within GPI anchors are factors that affect lipid raft formation (Fig. 2).Open in a separate windowFigure 2Acylation of PrP^C affects the underlying cell membrane. Cartoon showing the proposed membranes surrounding native PrP^C and monoacylated PrP^C, including cholesterol (), lyso-phospholipids (), saturated phospholipids () and unsaturated phospholipids (). Monoacylated PrP^C is not directed to lipid rafts and the membrane surrounding contains less cholesterol and more unsaturated phospholipids.We observed that the addition of native PrP^C significantly increased the amount of cholesterol in cell membranes. This result was unexpected as the amount of cholesterol in cell membranes is tightly controlled by an esterification and hydrolysis cycle which releases cholesterol from stored cholesterol esters.¹⁶ The PrP^C-induced increase in cholesterol was accompanied by a reduction in cholesterol esters suggesting that it was derived from the hydrolysis of cholesterol esters. Pharmacological inhibition of cholesterol ester hydrolysis not only blocked the PrP^C-induced increase in cholesterol and the reduction in cholesterol esters, but also reduced the PrP^C-induced increase in PrP^Sc formation. Collectively, these results indicate that cells respond to the introduction of PrP^C by the hydrolysis of cholesterol esters; which provides cholesterol to stabilize PrP^C within the lipid rafts that are necessary to facilitate PrP^Sc formation.The differences in the cellular distribution of PrP^C and monoacylated PrP^C were examined using neurons from Prnp knockout mice. While PrP^C was targeted to lipid rafts, monoacylated PrP^C was found predominantly within the normal (non-raft) cell membranes. Unlike native PrP^C, monoacylated PrP^C did not affect the cholesterol content of cell membranes; an observation that highlights the critical role of the presence of two saturated fatty acids contained within the GPI anchor to sequester cholesterol and precipitate the formation of lipid rafts.¹⁴ Critically, monoacylated PrP^C was unable to solubilize cholesterol or precipitate lipid raft formation and was consequently found outside lipid rafts (Fig. 2).Since many raft-associated proteins including PrP^C traffic within cells via specific pathways,¹⁷ we argued that monoacylated PrP^C might undergo alternative trafficking pathways to those utilized by native PrP^C. Our observations, that greater amounts of monoacylated PrP^C than native PrP^C were expressed at the cell surface, and that while most of the native PrP^C was removed from these cells within 24 h, monoacylated PrP^C remained in neurons for longer, are indicative of altered intracellular trafficking. It is possible that the cellular location and/or pathway(s) used by monoacylated PrP^C may be physically segregated from PrP^Sc. This hypothesis would explain our observation that the addition of monoacylated PrP^C to prion-infected cell lines did not increase PrP^Sc formation indicating that it was not converted to PrP^Sc.Since monoacylated phospholipids exist only transiently within cell membranes, they are rapidly reacylated by esterases,¹⁸ we wondered whether the monoacylated PrP^C could also be reacylated to form the native, diacylated PrP^C. We found no evidence that the monoacylated PrP^C added to Prnp knockout neurons was reacylated suggesting that the enzymes involved in reacylation of membrane phospholipids do not recognize phosphatidylinositol when it is incorporated as part of the GPI anchor. In addition we were unable to detect monoacylated PrP^C occurring naturally within Prnp wild-type neurons.While these theories explain why monoacylated PrP^C was not readily converted to PrP^Sc; a more refined hypothesis is required to explain why monoacylated PrP^C reduced PrP^Sc production. One possibility is that monoacylated PrP^C is converted to monoacylated PrP^Sc which in turn acts as an inefficient template for PrP^C to PrP^Sc conversion.¹⁹ It is also possible that monoacylated PrP^C competes with endogenous PrP^C for specific partner proteins involved in endocytosis. The depletion of these partner proteins could consequently alter the trafficking of endogenous PrP^C and hence PrP^Sc formation. In our paper we explored the idea that the binding of monoacylated PrP^C to PrP^Sc modifies the lipid rafts that are involved in PrP^Sc formation. Both the composition and function of lipid rafts is dynamic and controlled by an induced fit model.²⁰ Since monoacylated PrP^C does not sequester cholesterol, the membrane surrounding a complex between PrP^Sc and monoacylated PrP^C might be expected to contain less cholesterol than membranes formed following the interaction between PrP^Sc and PrP^C (Fig. 3). Thus, the binding of monoacylated PrP^C to PrP^Sc may reduce the cholesterol content of local membranes to a level below that required for the conversion of PrP^C to PrP^Sc. This hypothesis is consistent with observations that formation of PrP^Sc was affected by the lipid composition of membranes²¹ and that lipids were essential co-factors in prion formation.²²Open in a separate windowFigure 3Monoacylated PrP^C affects the capture of cPLA₂ in PrP^Sc-containing lipid rafts. (A) Cartoon showing the proposed membranes surrounding PrP^Sc and PrP^C including the capture of cPLA₂ in lipid rafts that are dense in cholesterol () and saturated phospholipids (). (B) Cartoon showing the proposed interactions between PrP^Sc and monoacylated PrP^C which reduces the solubility of membranes to cholesterol, increases the concentration of unsaturated phospholipids () and prevents the capture of cPLA₂ into PrP^Sc-containing lipid rafts.Our studies raise the question “why are lipid rafts important in PrP^Sc formation?” Lipid rafts are enriched with signaling molecules and can act as domains in which the GPI anchors attached to PrP^C can interact with cell signaling pathways.²³ Although PrP^C has been reported to interact with many cell signaling pathways we concentrated upon its effects on the activation of cPLA₂, based upon studies showing that the activation of cPLA₂ correlates strongly with the amounts of cholesterol and PrP^Sc,²⁴ and that the inhibition of cPLA₂ reduces PrP^Sc formation.²⁵ These observations underpin the hypothesis that it is the clustering of GPI anchors attached to PrP proteins that leads to the activation of cPLA₂. This hypothesis was tested by incubating Prnp knockout neurons with PrP^C or monoacylated PrP^C and then adding the anti-PrP mAb 4F2. We found that the cross-linkage of PrP^C by mAb 4F2 caused the activation of cPLA₂, whereas the cross-linkage of monoacylated PrP^C by mAb 4F2 had no significant affect. The activation of cPLA₂ is associated with multiple phosphorylation events and the translocation of cPLA₂ to specific membranes.²⁶ In scrapie-infected GT1 (ScGT1) cells most of the cPLA₂ was found within lipid rafts consistent with reports that the activation of cPLA₂ is dependent upon cholesterol-sensitive lipid rafts.²⁷ More specifically, immunoprecipitation studies showed that cPLA₂ was targeted to PrP^Sc-containing lipid rafts.²⁴ Collectively, these observations suggest that PrP^C binds to PrP^Sc in cholesterol-dense lipid rafts, where it activates the cPLA₂ that facilitates the conversion of PrP^C to PrP^Sc (Fig. 3).We found that the presence of monoacylated PrP^C reduced the activation of cPLA₂ within prion-infected cells. As cPLA₂ can be activated by multiple different stimuli we sought to determine whether the inhibitory effect of monoacylated PrP^C was stimulus specific. We reported that monoacylated PrP^C did not affect the activation of cPLA₂ by a phospholipase A₂-activating peptide indicating that monoacylated PrP^C did not have a direct inhibitory effect upon cPLA₂. Rather we found that the addition of monoacylated PrP^C to prion-infected cells caused the dissociation of some cPLA₂ from PrP^Sc-containing lipid rafts. The targeting of cPLA₂ to membranes containing their endogenous substrates can regulate cell signaling, including for the formation of second messengers such as platelet-activating factor that facilitate PrP^Sc formation.²⁵ We propose that the binding of monoacylated PrP^C to PrP^Sc changed the composition of the underlying membrane so that it no longer captured and activated cPLA₂ (Fig. 3).In conclusion our study showed that the addition of monoacylated PrP^C modified cell membranes thus reducing the activation of cPLA₂ and PrP^Sc formation in prion-infected cells. We propose that the 2 acyl chains attached to the GPI anchor is a critical factor that facilitates the conversion to PrP^C to PrP^Sc within cell membranes and that the presence of monoacylated PrP^C disrupts lipid raft micro-domains that are essential for efficient PrP^Sc formation. Moreover, these results raise the possibility that targeting the GPI anchor attached to PrP^C may reveal novel therapeutics/treatments for prion diseases.