PrPC is sorted to the basolateral membrane of epithelial cells independently of its association with rafts

PrP(C) is a glycosylphosphatidylinositol-anchored protein expressed in neurons as well as in the cells of several peripheral tissues. Although the normal function of PrP(C) remains unknown, a conformational isoform called PrP(Sc) (scrapie) has been proposed to be the infectious agent of transmissible spongiform encephalopathies in animals and humans. Where and how the PrP(C) to PrP(Sc) conversion occurs in the cells is not yet known. Therefore, dissecting the intracellular trafficking of the wild-type prion protein, as well as of the scrapie isoform, can be of major relevance to the pathogenesis of the diseases. In this report we have analyzed the exocytic pathway of transfected mouse PrP(C) in thyroid and kidney polarized epithelial cells. In contrast to the majority of glycosylphosphatidylinositol-anchored proteins, we found that PrP(C) is localized mainly on the basolateral domain of the plasma membrane of both cell lines. This is reminiscent of the predominant somatodendritic localization found in neurons. However, similarly to apical glycosylphosphatidylinositol-proteins, PrP(C) associates with detergent-resistant microdomains, which have been suggested to have a role in apical sorting of glycosylphosphatidylinositol-proteins, as well as in the conversion process of PrP(C) to PrP(Sc). In order to discriminate whether detergent-resistant microdomains have a direct role in PrP(Sc) conversion, or whether they are involved in the transport of the protein to the site of its conversion, we have examined the effect of disruption of detergent-resistant microdomain association on PrP(C) intracellular traffic. Consistent with the unusual basolateral localization of this glycosylphosphatidylinositol-linked protein, our data exclude a classical role for detergent-resistant microdomains in the post-trans-Golgi network sorting and transport of PrP(C) to the plasma membrane.     

PrP(C) association with lipid rafts in the early secretory pathway stabilizes its cellular conformation

The pathological conversion of cellular prion protein (PrP(C)) into the scrapie prion protein (PrP(Sc)) isoform appears to have a central role in the pathogenesis of transmissible spongiform encephalopathies. However, the identity of the intracellular compartment where this conversion occurs is unknown. Several lines of evidence indicate that detergent-resistant membrane domains (DRMs or rafts) could be involved in this process. We have characterized the association of PrP(C) to rafts during its biosynthesis. We found that PrP(C) associates with rafts already as an immature precursor in the endoplasmic reticulum. Interestingly, compared with the mature protein, the immature diglycosylated form has a different susceptibility to cholesterol depletion vs. sphingolipid depletion, suggesting that the two forms associate with different lipid domains. We also found that cholesterol depletion, which affects raft-association of the immature protein, slows down protein maturation and leads to protein misfolding. On the contrary, sphingolipid depletion does not have any effect on the kinetics of protein maturation or on the conformation of the protein. These data indicate that the early association of PrP(C) with cholesterol-enriched rafts facilitates its correct folding and reinforce the hypothesis that cholesterol and sphingolipids have different roles in PrP metabolism.     

Monoacylated cellular prion protein modifies cell membranes, inhibits cell signaling, and reduces prion formation

Prion diseases occur following the conversion of the cellular prion protein (PrP(C)) into a disease related, protease-resistant isoform (PrP(Sc)). In these studies, a cell painting technique was used to introduce PrP(C) to prion-infected neuronal cell lines (ScGT1, ScN2a, or SMB cells). The addition of PrP(C) resulted in increased PrP(Sc) formation that was preceded by an increase in the cholesterol content of cell membranes and increased activation of cytoplasmic phospholipase A(2) (cPLA(2)). In contrast, although PrP(C) lacking one of the two acyl chains from its glycosylphosphatidylinositol (GPI) anchor (PrP(C)-G-lyso-PI) bound readily to cells, it did not alter the amount of cholesterol in cell membranes, was not found within detergent-resistant membranes (lipid rafts), and did not activate cPLA(2). It remained within cells for longer than PrP(C) with a conventional GPI anchor and was not converted to PrP(Sc). Moreover, the addition of high amounts of PrP(C)-G-lyso-PI displaced cPLA(2) from PrP(Sc)-containing lipid rafts, reduced the activation of cPLA(2), and reduced PrP(Sc) formation in all three cell lines. In addition, ScGT1 cells treated with PrP(C)-G-lyso-PI did not transmit infection following intracerebral injection to mice. We propose that that the chemical composition of the GPI anchor attached to PrP(C) modified the local membrane microenvironments that control cell signaling, the fate of PrP(C), and hence PrP(Sc) formation. In addition, our observations raise the possibility that pharmacological modification of GPI anchors might constitute a novel therapeutic approach to prion diseases.     

Neurodegeneration induced by clustering of sialylated glycosylphosphatidylinositols of prion proteins

The transmissible spongiform encephalopathies, more commonly known as the prion diseases, are associated with the production and aggregation of disease-related isoforms of the prion protein (PrP(Sc)). The mechanisms by which PrP(Sc) accumulation causes neurodegeneration in these diseases are poorly understood. In cultured neurons, the addition of PrP(Sc) alters cell membranes, increasing cholesterol, activating cytoplasmic phospholipase A(2) (cPLA(2)), and triggering synapse damage. These effects of PrP(Sc) are dependent upon its glycosylphosphatidylinositol (GPI) anchor, suggesting that it is the increased density of GPIs that occurs following the aggregation of PrP(Sc) molecules that triggers neurodegeneration. This hypothesis was supported by observations that cross-linkage of the normal cellular prion protein (PrP(C)) also increased membrane cholesterol, activated cPLA(2), and triggered synapse damage. These effects were not seen after cross-linkage of Thy-1, another GPI-anchored protein, and were dependent on the GPI anchor attached to PrP(C) containing two acyl chains and sialic acid. We propose that the aggregation of PrP(Sc), or the cross-linkage of PrP(C), causes the clustering of sialic acid-containing GPI anchors at high densities, resulting in altered membrane composition, the pathological activation of cPLA(2), and synapse damage.     

Misfolding of the prion protein at the plasma membrane induces endocytosis, intracellular retention and degradation

Suramin induces misfolding of the cellular prion protein (PrP(C)) and interferes with the propagation of infectious scrapie prions. A mechanistic analysis of this effect revealed that suramin-induced misfolding occurs at the plasma membrane and is dependent on the proximal region of the C-terminal domain (aa 90-158) of PrP(C). The conformational transition induces rapid internalization, mediated by the unstructured N-terminal domain, and subsequent intracellular degradation of PrP(C). As a consequence, PrP Delta N adopts a misfolded conformation at the plasma membrane; however, internalization is significantly delayed. We also found that misfolding and intracellular retention of PrP(C) can be induced by copper and that, moreover, copper interferes with the propagation of the pathogenic prion protein (PrP(Sc)) in scrapie-infected N2a cells. Our study revealed a quality control pathway for aberrant PrP conformers present at the plasma membrane and identified distinct PrP domains involved.     

Synthesis and structural characterization of a mimetic membrane-anchored prion protein

During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrP(Sc)) of the host encoded prion protein (PrP(C)) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrP(C) and PrP(Sc) have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrP(C) to PrP(Sc), but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C-terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP-GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane-anchored PrP. We show that the structure of PrP-GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor-free forms of PrP represent the structure of cellular, membrane-anchored PrP. The availability of a lipid-anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP.     

Scrapie-induced defects in learning and memory of transgenic mice expressing anchorless prion protein are associated with alterations in the gamma aminobutyric acid-ergic pathway

After infection with RML murine scrapie agent, transgenic (tg) mice expressing prion protein (PrP) without its glycophosphatidylinositol (GPI) membrane anchor (GPI(-/-) PrP tg mice) continue to make abundant amounts of the abnormally folded disease-associated PrPres but have a normal life span. In contrast, all age-, sex-, and genetically matched mice with a GPI-anchored PrP become moribund and die due to a chronic progressive neurodegenerative disease by 160 days after RML scrapie agent infection. We report here that infected GPI(-/-) PrP tg mice, although free from progressive neurodegenerative disease of the cerebellum and extrapyramidal and pyramidal systems, nevertheless suffer defects in learning and memory, long-term potentiation, and neuronal excitability. Such dysfunction increases over time and is associated with an increase in gamma aminobutyric acid (GABA) inhibition but not loss of excitatory glutamate/N-methyl-d-aspartic acid. Enhanced deposition of abnormally folded infectious PrP (PrPsc or PrPres) in the central nervous system (CNS) localizes with GABAA receptors. This occurs with minimal evidence of CNS spongiosis or apoptosis of neurons. The use of monoclonal antibodies reveals an association of PrPres with GABAA receptors. Thus, the clinical defects of learning and memory loss in vivo in GPI(-/-) PrP tg mice infected with scrapie agent may likely involve the GABAergic pathway.     

Interaction of the cellular prion protein with raft-like lipid membranes

Conversion of the cellular isoform of the prion protein (PrP(C)) into the disease-associated isoform (PrP(Sc)) plays a key role in the development of prion diseases. Within its cellular pathway, PrP(C) undergoes several posttranslational modifications, i.e., the attachment of two N-linked glycans and a glycosyl phosphatidyl inositol (GPI) anchor, by which it is linked to the plasma membrane on the exterior cell surface. To study the interaction of PrP(C) with model membranes, we purified posttranslationally modified PrP(C) from transgenic Chinese hamster ovary (CHO) cells. The mono-, di- and oligomeric states of PrP(C) free in solution were analyzed by analytical ultracentrifugation. The interaction of PrP(C) with model membranes was studied using both lipid vesicles in solution and lipid bilayers bound to a chip surface. The equilibrium and mechanism of PrP(C) association with the model membranes were analyzed by surface plasmon resonance. Depending on the degree of saturation of binding sites, the concentration of PrP(C) released from the membrane into aqueous solution was estimated at between 10(-9) and 10(-7) M. This corresponds to a free energy of the insertion reaction of -48 kJ/mol. Consequences for the conversion of PrP(C) to PrP(Sc) are discussed.     

Effect of glycosylphosphatidylinositol anchor-dependent and -independent prion protein association with model raft membranes on conversion to the protease-resistant isoform

Prion protein (PrP) is usually bound to membranes by a glycosylphosphatidylinositol (GPI) anchor that associates with detergent-resistant membranes, or rafts. To examine the effect of membrane association on the interaction between the normal protease-sensitive PrP isoform (PrP-sen) and the protease-resistant isoform (PrP-res), a model system was employed using PrP-sen reconstituted into sphingolipid-cholesterol-rich raft-like liposomes (SCRLs). Both full-length (GPI(+)) and GPI anchor-deficient (GPI(-)) PrP-sen produced in fibroblasts stably associated with SCRLs. The latter, alternative mode of membrane association was not detectably altered by glycosylation and was markedly reduced by deletion of residues 34-94. The SCRL-associated PrP molecules were not removed by treatments with either high salt or carbonate buffer. However, only GPI(+) PrP-sen resisted extraction with cold Triton X-100. PrP-sen association with SCRLs was pH-independent. PrP-sen was also one of a small subset of phosphatidylinositol-specific phospholipase C (PI-PLC)-released proteins from fibroblast cells found to bind SCRLs. A cell-free conversion assay was used to measure the interaction of SCRL-bound PrP-sen with exogenous PrP-res as contained in microsomes. SCRL-bound GPI(+) PrP-sen was not converted to PrP-res until PI-PLC was added to the reaction or the combined membrane fractions were treated with the membrane-fusing agent polyethylene glycol (PEG). In contrast, SCRL-bound GPI(-) PrP-sen was converted to PrP-res without PI-PLC or PEG treatment. Thus, of the two forms of raft membrane association by PrP-sen, only the GPI anchor-directed form resists conversion induced by exogenous PrP-res.     

The N-terminal region of the prion protein ectodomain contains a lipid raft targeting determinant

The association of the prion protein (PrP) with sphingolipid- and cholesterol-rich lipid rafts is instrumental in the pathogenesis of the neurodegenerative prion diseases. Although the glycosylphosphatidylinositol (GPI) anchor is an exoplasmic determinant of raft association, PrP remained raft-associated in human neuronal cells even when the GPI anchor was deleted or substituted for a transmembrane anchor indicating that the ectodomain contains a raft localization signal. The raft association of transmembrane-anchored PrP occurred independently of Cu(II) binding as it failed to be abolished by either deletion of the octapeptide repeat region (residues 51-90) or treatment of cells with a Cu(II) chelator. Raft association of transmembrane-anchored PrP was only abolished by the deletion of the N-terminal region (residues 23-90) of the ectodomain. This region was sufficient to confer raft localization when fused to the N terminus of a non-raft transmembrane-anchored protein and suppressed the clathrin-coated pit localization signal in the cytoplasmic domain of the amyloid precursor protein. These data indicate that the N-terminal region of PrP acts as a cellular raft targeting determinant and that residues 23-90 of PrP represent the first proteinaceous raft targeting signal within the ectodomain of a GPI-anchored protein.     

Removal of the glycosylphosphatidylinositol anchor from PrP(Sc) by cathepsin D does not reduce prion infectivity

According to the protein-only hypothesis of prion propagation, prions are composed principally of PrP(Sc), an abnormal conformational isoform of the prion protein, which, like its normal cellular precursor (PrP(C)), has a GPI (glycosylphosphatidylinositol) anchor at the C-terminus. To date, elucidating the role of this anchor on the infectivity of prion preparations has not been possible because of the resistance of PrP(Sc) to the activity of PI-PLC (phosphoinositide-specific phospholipase C), an enzyme which removes the GPI moiety from PrP(C). Removal of the GPI anchor from PrP(Sc) requires denaturation before treatment with PI-PLC, a process that also abolishes infectivity. To circumvent this problem, we have removed the GPI anchor from PrP(Sc) in RML (Rocky Mountain Laboratory)-prion-infected murine brain homogenate using the aspartic endoprotease cathepsin D. This enzyme eliminates a short sequence at the C-terminal end of PrP to which the GPI anchor is attached. We found that this modification has no effect (i) on an in vitro amplification model of PrP(Sc), (ii) on the prion titre as determined by a highly sensitive N2a-cell based bioassay, or (iii) in a mouse bioassay. These results show that the GPI anchor has little or no role in either the propagation of PrP(Sc) or on prion infectivity.     

Structural changes of the prion protein in lipid membranes leading to aggregation and fibrillization

Prion diseases are associated with a major refolding event of the normal cellular prion protein, PrP(C), where the predominantly alpha-helical and random coil structure of PrP(C) is converted into a beta-sheet-rich aggregated form, PrP(Sc). Under normal physiological conditions PrP(C) is attached to the outer leaflet of the plasma membrane via a GPI anchor, and it is plausible that an interaction between PrP and lipid membranes could be involved in the conversion of PrP(C) into PrP(Sc). Recombinant PrP can be refolded into an alpha-helical structure, designated alpha-PrP isoform, or into beta-sheet-rich states, designated beta-PrP isoform. The current study investigates the binding of beta-PrP to model lipid membranes and compares the structural changes in alpha- and beta-PrP induced upon membrane binding. beta-PrP binds to negatively charged POPG membranes and to raft membranes composed of DPPC, cholesterol, and sphingomyelin. Binding of beta-PrP to raft membranes results in substantial unfolding of beta-PrP. This membrane-associated largely unfolded state of PrP is slowly converted into fibrils. In contrast, beta-PrP and alpha-PrP gain structure with POPG membranes, which instead leads to amorphous aggregates. Furthermore, binding of beta-PrP to POPG has a disruptive effect on the integrity of the lipid bilayer, leading to total release of vesicle contents, whereas raft vesicles are not destabilized upon binding of beta-PrP.     

Dual mechanisms for shedding of the cellular prion protein

The cellular prion protein (PrP(C)) is essential for the pathogenesis and transmission of prion diseases. Whereas the majority of PrP(C) is bound to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor, a secreted form of the protein has been identified. Here we show that PrP(C) can be shed into the medium of human neuroblastoma SH-SY5Y cells by both protease- and phospholipase-mediated mechanisms. The constitutive shedding of PrP(C) was inhibited by a range of hydroxamate-based zinc metalloprotease inhibitors in a manner identical to the alpha-secretase-mediated shedding of the amyloid precursor protein, indicating a proteolytic shedding mechanism. Like amyloid precursor protein, this zinc metalloprotease-mediated shedding of PrP(C) could be stimulated by phorbol myristate acetate and by copper ions. The lipid raft-disrupting agents filipin and methyl-beta-cyclodextrin promoted the shedding of PrP(C) via a distinct mechanism that was not inhibited by hydroxamate-based inhibitors. Filipin-mediated shedding of PrP(C) is likely to occur via phospholipase cleavage of the GPI anchor, since a transmembrane polypeptide-anchored PrP construct was not shed in response to filipin treatment. Collectively, our data indicate that shedding of PrP(C) can occur via both secretase-like proteolytic cleavage of the protein and phospholipase cleavage of the GPI anchor moiety.     

Glycosylphosphatidylinositol anchor-dependent stimulation pathway required for generation of baculovirus-derived recombinant scrapie prion protein

The pathogenic isoform (PrP(Sc)) of the host-encoded cellular prion protein (PrP(C)) is considered to be an infectious agent of transmissible spongiform encephalopathy (TSE). The detailed mechanism by which the PrP(Sc) seed catalyzes the structural conversion of endogenous PrP(C) into nascent PrP(Sc) in vivo still remains unclear. Recent studies reveal that bacterially derived recombinant PrP (recPrP) can be used as a substrate for the in vitro generation of protease-resistant recPrP (recPrP(res)) by protein-misfolding cyclic amplification (PMCA). These findings imply that PrP modifications with a glycosylphosphatidylinositol (GPI) anchor and asparagine (N)-linked glycosylation are not necessary for the amplification and generation of recPrP(Sc) by PMCA. However, the biological properties of PrP(Sc) obtained by in vivo transmission of recPrP(res) are unique or different from those of PrP(Sc) used as the seed, indicating that the mechanisms mediated by these posttranslational modifications possibly participate in reproductive propagation of PrP(Sc). In the present study, using baculovirus-derived recombinant PrP (Bac-PrP), we demonstrated that Bac-PrP is useful as a PrP(C) substrate for amplification of the mouse scrapie prion strain Chandler, and PrP(Sc) that accumulated in mice inoculated with Bac-PrP(res) had biochemical and pathological properties very similar to those of the PrP(Sc) seed. Since Bac-PrP modified with a GPI anchor and brain homogenate of Prnp knockout mice were both required to generate Bac-PrP(res), the interaction of GPI-anchored PrP with factors in brain homogenates is essential for reproductive propagation of PrP(Sc). Therefore, the Bac-PMCA technique appears to be extremely beneficial for the comprehensive understanding of the GPI anchor-mediated stimulation pathway.     

Thioredoxin converts the Syrian hamster (29-231) recombinant prion protein to an insoluble form

The prion protein (PrP) is an essential, and probably the only, component of the infectious agent responsible for the transmissible spongiform encephalopathies. In its cellular (PrP(C)) form, it is a soluble, alpha-helix-rich protein of yet unknown function attached to the outer membrane of neurons through a glycosylphosphatidyl inositol anchor. In its pathogenic, "scrapie" form (PrP(Sc)), it appears as an aggregate showing no detectable covalent modifications but displaying a profoundly altered conformation enriched in beta-sheet structure. Reduction of the single disulfide bridge in the prion protein with millimolar concentrations of dithiothreitol results in transformation of the alpha-helix-rich to the beta-sheet-rich conformation, with concomitant decrease in solubility. We report here that thioredoxin coupled with thioredoxin reductase and NADPH efficiently reduces recombinant Syrian hamster (29-231) prion protein under physiologically relevant conditions. The reduced prion protein immediately becomes insoluble and precipitates, although it does not gain significant resistance to proteinase K. The thioredoxin/thioredoxin reductase system is approximately 7000 times more efficient than dithiothreitol.     

The role of the prion protein membrane anchor in prion infection

In transmissible spongiform encephalopathies (TSE or prion diseases) such as sheep scrapie, bovine spongiform encephalopathy and human Creutzfeldt-Jakob disease, normally soluble and protease-sensitive prion protein (PrP-sen or PrPC) is converted to an abnormal, insoluble and protease-resistant form termed PrP-res or PrPSc. PrP-res/PrPSc is believed to be the main component of the prion, the infectious agent of the TSE/prion diseases. Its precursor, PrP-sen, is anchored to the cell surface at the C-terminus by a co-translationally added glycophosphatidyl-inositol (GPI) membrane anchor which can be cleaved by the enzyme phosphatidyl-inositol specific phospholipase (PIPLC). The GPI anchor is also present in PrP-res, but is inaccessible to PIPLC digestion suggesting that conformational changes in PrP associated with PrP-res formation have blocked the PIPLC cleavage site. Although the GPI anchor is present in both PrP-sen and PrP-res, its precise role in TSE diseases remains unclear primarily because there are data to suggest that it both is and is not necessary for PrP-res formation and prion infection.     

Effect of amphotericin B on wild-type and mutated prion proteins in cultured cells: putative mechanism of action in transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies form a group of fatal neurodegenerative disorders that have the unique property of being infectious, sporadic, or genetic in origin. Although some doubts remain on the nature of the responsible agent of these diseases, it is clear that a protein called PrP(Sc) [the scrapie isoform of prion protein (PrP)] plays a central role. PrP(Sc) represents a conformational variant of PrP(C) (the cellular isoform of PrP), the normal host protein. Polyene antibiotics, such as amphotericin B, have been shown to delay the accumulation of PrP(Sc) and to increase the incubation time of the disease after experimental transmission in laboratory animals. Unlike agents such as Congo red, the inhibitory effect of amphotericin B on PrP(Sc) generation has not been observed in infected cultures. Using transfected cells expressing wild-type or mutated mouse PrPs, we show here that amphotericin B is able to interfere with the generation of abnormal PrP isoforms in culture. Its action seems related to a modification of PrP trafficking through the association of this glycosylphosphatidylinositol-anchored protein with detergent-resistant microdomains. These results represent a first step toward the comprehension of the mechanism of action of amphotericin B in transmissible spongiform encephalopathies.     

Molecular dynamics simulation of human prion protein including both N-linked oligosaccharides and the GPI anchor

Although glycosylation appears to protect prion protein (PrP(C)) from the conformational transition to the disease-associated scrapie form (PrP(Sc)), available NMR structures are for non-glycosylated PrP(C), only. To investigate the influence of both the two N-linked glycans, Asn181 and Asn197, and of the GPI anchor attached to Ser230, on the structural, dynamical and electrostatic behavior of PrP, we have undertaken molecular dynamics simulations on the C-terminal region of human prion protein HU:PrP(90-230), with and without the three glycans. The simulations used the AMBER94 force field in a periodic box model with explicit water molecules, considering all long-range electrostatic interactions. The results suggest the structured part of the protein, HU:PrP(127-227) is stabilized overall from addition of the glycans, specifically by extensions of Helix-B and Helix-C and reduced flexibility of the linking turn containing Asn197, although some regions such as residues in the turn (165-170) between Strand-B and Helix-B have increased flexibility. The stabilization appears indirect, by reducing the mobility of the surrounding water molecules, and not from specific interactions such as H bonds or ion pairs. The results are consistent with glycosylation at Asn197 having a stabilizing role, while that at Asn181, in a region with already stable secondary structure, having a more functional role, in agreement with literature suggestions. Due to three negatively charged SiaLe(x) groups per N-glycan, the surface electrostatic properties change to a negative electrostatic field covering most of the C-terminal part, including the surface of Helix-B and Helix-C, while the positively charged N-terminal part PrP(90-126) of undefined structure creates a positive potential. The unusual hydrophilic Helix-A (144-152) is not covered by either of these dominant electrostatic fields, and modeling shows it could readily dimerize in anti parallel fashion. In combination with separate simulations of the GPI anchor in a membrane model, the results show the GPI anchor is highly flexible and would maintain the protein at a distance between 9 and 13 A from the membrane surface, with little influence on its structure or orientational freedom.     

The Fate of PrP GPI‐Anchor Signal Peptide is Modulated by P238S Pathogenic Mutation

Glycosylphosphatidylinositol (GPI)‐anchored proteins are localized to the plasma membrane via a C‐terminally linked GPI anchor. The GPI anchor is added concomitantly to the cleavage of the carboxy‐terminal GPI‐anchor signal sequence, thereby causing the release of a C‐terminal hydrophobic peptide, whose fate has not yet been investigated. Here we followed the fate of the GPI‐attachment signal of the prion protein (PrP), a protein implicated in various types of transmissible neurodegenerative spongiform encephalopathies (TSE). The PrP GPI‐anchor signal sequence shows a remarkable and unusual degree of conservation across the species and contains two point mutations (M232R/T and P238S) that are responsible for genetic forms of prion disorders. We show that the PrP GPI‐anchor signal peptide (SP), but not the one from an unrelated GPI‐anchored protein (folate receptor), undergoes degradation via the proteasome. Moreover, the P238S point mutation partially protects the PrP GPI‐anchor SP from degradation. Our data provide the first attempt to address the fate of a GPI‐anchor SP and identify a role for the P238S mutation, suggesting the possibility that the PrP GPI‐anchor SP could play a role in neurodegenerative prion diseases.      

Differential release of cellular and scrapie prion proteins from cellular membranes by phosphatidylinositol-specific phospholipase C

The abnormal isoform of the scrapie prion protein PrPSc is both a host-derived protein and a component of the infectious agent causing scrapie. PrPSc and the normal cellular isoform PrPC have different physical properties that apparently arise from a posttranslational event. Both PrP isoforms are covalently modified at the carboxy terminus by a glycoinositol phospholipid. Using preparations of dissociated cells derived from normal and scrapie-infected hamster brain tissue, we find that the majority of PrPC is released from membranes by phosphatidylinositol-specific phospholipase C (PIPLC), while PrPSc is resistant to release. In contrast, purified denatured PrP 27-30 (which is formed from PrPSc during purification by proteolysis of the amino terminus) is completely cleaved by PIPLC. Incubation of the cell preparations with proteinase K cleaves PrPSc to form PrP 27-30, demonstrating that PrPSc is accessible to added enzymes. We have also developed a protocol involving biotinylation that gives a quantitative estimate of the fraction of a protein exposed to the cell exterior. Using this strategy, we find that a large portion of PrPSc in the cell preparations reacts with a membrane-impermeant biotinylation reagent. Whether alternative membrane anchoring of PrPSc, inaccessibility of the glycoinositol phospholipid anchor to PIPLC, or binding to another cellular component is responsible for the differential release of prion proteins from cells remains to be determined.