Title: Characterization of the Properties and Trafficking of an Anchorless Form of the Prion Protein
Abstract: Conversion of PrPC into PrPSc is the central event in the pathogenesis of transmissible prion diseases. Although the molecular basis of this event and the intracellular compartment where it occurs are not yet understood, the association of PrP with cellular membranes and in particular its presence in detergent-resistant microdomains appears to be of critical importance. In addition it appears that scrapie conversion requires membrane-bound glycosylphosphatidylinositol (GPI)-linked PrP. The GPI anchor may affect either the conformation, the intracellular localization, or the association of the prion protein with specific membrane domains. However, how this occurs is not known. To understand the relevance of the GPI anchor for the cellular behavior of PrP, we have studied the biosynthesis and localization of a PrP version which lacks the GPI anchor attachment signal (PrPΔGPI). We found that PrPΔGPI is tethered to cell membranes and associates to membrane detergent-resistant microdomains but does not assume a transmembrane topology. Differently to PrPC, this protein does not localize at the cell surface but is mainly released in the culture media in a fully glycosylated soluble form. The cellular behavior of anchorless PrP explains why PrPΔGPI Tg mice can be infected but do not show the classical signs of the disorder, thus indicating that the plasma membrane localization of PrPC and/or of the converted scrapie form might be necessary for the development of a symptomatic disease. Conversion of PrPC into PrPSc is the central event in the pathogenesis of transmissible prion diseases. Although the molecular basis of this event and the intracellular compartment where it occurs are not yet understood, the association of PrP with cellular membranes and in particular its presence in detergent-resistant microdomains appears to be of critical importance. In addition it appears that scrapie conversion requires membrane-bound glycosylphosphatidylinositol (GPI)-linked PrP. The GPI anchor may affect either the conformation, the intracellular localization, or the association of the prion protein with specific membrane domains. However, how this occurs is not known. To understand the relevance of the GPI anchor for the cellular behavior of PrP, we have studied the biosynthesis and localization of a PrP version which lacks the GPI anchor attachment signal (PrPΔGPI). We found that PrPΔGPI is tethered to cell membranes and associates to membrane detergent-resistant microdomains but does not assume a transmembrane topology. Differently to PrPC, this protein does not localize at the cell surface but is mainly released in the culture media in a fully glycosylated soluble form. The cellular behavior of anchorless PrP explains why PrPΔGPI Tg mice can be infected but do not show the classical signs of the disorder, thus indicating that the plasma membrane localization of PrPC and/or of the converted scrapie form might be necessary for the development of a symptomatic disease. According to the "protein only" hypothesis (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5194) Google Scholar), the infectious agent of transmissible spongiform encephalopathies is an abnormally folded β-sheet-enriched conformer of the cellular prion protein (PrPC), 2The abbreviations used are: PrPC, cellular prion protein; PrPSc, scrapie prion protein; βCD, methyl-β-cyclodextrin; CNX, calnexin; DRM, detergent-resistant membrane domain; EndoH, endoglycosidase H; ER, endoplasmic reticulum; FRT, Fischer rat thyroid; GPI, glycosylphosphatidylinositol; PM, plasma membrane; PK, proteinase K; TX-100, Triton X-100; PGNaseF, peptide N-glycosidase F; PBS, phosphate-buffered saline; TRITC, tetramethyl-rhodamine isothiocyanate. called PrP scrapie (PrPSc) or prion (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5194) Google Scholar). PrPSc is able to replicate and propagate itself by transferring its altered conformation to the endogenous PrPC, a cell surface-enriched protein that becomes partially resistant to proteases and accumulates in plaques in the brain (2Horwich A.L. Weissman J.S. Cell. 1997; 89: 499-510Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). The molecular basis of PrPC-PrPSc conversion, the intracellular compartment where the conversion occurs, and how the process leads to neurological dysfunction are still very open and debated questions (3Campana V. Sarnataro D. Zurzolo C. Trends Cell Biol. 2005; 15: 102-111Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Several studies indicate that PrPC-PrPSc conversion is a post-translational event that occurs after the protein reaches the cell surface (4Caughey B. Raymond G.J. J. Biol. Chem. 1991; 266: 18217-18223Abstract Full Text PDF PubMed Google Scholar, 5Borchelt D.R. Taraboulos A. Prusiner S.B. J. Biol. Chem. 1992; 267: 16188-16199Abstract Full Text PDF PubMed Google Scholar, 6Taraboulos A. Raeber A.J. Borchelt D.R. Serban D. Prusiner S.B. Mol. Biol. Cell. 1992; 3: 851-863Crossref PubMed Scopus (240) Google Scholar). Indeed, it is possible to impair PrPSc formation in infected cells either by preventing PrPC transport to the plasma membrane (7Lee K.S. Magalhaes A.C. Zanata S.M. Brentani R.R. Martins V.R. Prado M.A. J. Neurochem. 2001; 79: 79-87Crossref PubMed Scopus (110) Google Scholar), by exposing PrPC to specific antibodies, or by releasing it from the cell surface by different methods (4Caughey B. Raymond G.J. J. Biol. Chem. 1991; 266: 18217-18223Abstract Full Text PDF PubMed Google Scholar, 5Borchelt D.R. Taraboulos A. Prusiner S.B. J. Biol. Chem. 1992; 267: 16188-16199Abstract Full Text PDF PubMed Google Scholar) (for review, see Ref. 3Campana V. Sarnataro D. Zurzolo C. Trends Cell Biol. 2005; 15: 102-111Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). However, these data do not distinguish whether scrapie conversion occurs on the plasma membrane or later during its internalization. In infected cells PrPSc accumulates in late endosomes, and inhibition of endocytosis reduces scrapie production (5Borchelt D.R. Taraboulos A. Prusiner S.B. J. Biol. Chem. 1992; 267: 16188-16199Abstract Full Text PDF PubMed Google Scholar, 8Marella M. Lehmann S. Grassi J. Chabry J. J. Biol. Chem. 2002; 277: 25457-25464Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), thus indicating that the endolysosomal pathway could be also involved in scrapie formation. Although the exact nature of the compartment of prion conversion is still debated, the membrane domains (also called rafts) with which PrP associates seems to be important in the conversion process (3Campana V. Sarnataro D. Zurzolo C. Trends Cell Biol. 2005; 15: 102-111Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 9Naslavsky N. Stein R. Yanai A. Friedlander G. Taraboulos A. J. Biol. Chem. 1997; 272: 6324-6331Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 10Naslavsky N. Shmeeda H. Friedlander G. Yanai A. Futerman A.H. Barenholz Y. Taraboulos A. J. Biol. Chem. 1999; 274: 20763-20771Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 11Taraboulos A. Scott M. Semenov A. Avrahami D. Laszlo L. Prusiner S.B. Avraham D. J. Cell Biol. 1995; 129: 121-132Crossref PubMed Scopus (519) Google Scholar). Rafts are membrane domains enriched in cholesterol and sphingolipids that have been proposed to have a central role in many cellular processes (12Zurzolo C. van Meer G. Mayor S. EMBO Rep. 2003; 4: 1117-1121Crossref PubMed Scopus (47) Google Scholar), including membrane sorting and trafficking, cell polarization, and signal transduction (13Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Crossref PubMed Scopus (5207) Google Scholar, 14Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8188) Google Scholar, 15Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2560) Google Scholar). Like other GPI-anchored proteins, PrPC and PrPSc associate with rafts because of the affinity of their GPI anchor for saturated lipid species (16Sarnataro D. Paladino S. Campana V. Grassi J. Nitsch L. Zurzolo C. Traffic. 2002; 3: 810-821Crossref PubMed Scopus (91) Google Scholar, 17Sarnataro D. Campana V. Paladino S. Stornaiuolo M. Nitsch L. Zurzolo C. Mol. Biol. Cell. 2004; 15: 4031-4042Crossref PubMed Scopus (115) Google Scholar, 18Vey M. Pilkuhn S. Wille H. Nixon R. DeArmond S.J. Smart E.J. Anderson R.G. Taraboulos A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14945-14949Crossref PubMed Scopus (490) Google Scholar, 19Madore N. Smith K.L. Graham C.H. Jen A. Brady K. Hall S. Morris R. EMBO J. 1999; 18: 6917-6926Crossref PubMed Scopus (333) Google Scholar). In prion-infected N2a cells, perturbation of PrP raft association by modifying the cellular levels of cholesterol affects PrPSc formation (9Naslavsky N. Stein R. Yanai A. Friedlander G. Taraboulos A. J. Biol. Chem. 1997; 272: 6324-6331Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 10Naslavsky N. Shmeeda H. Friedlander G. Yanai A. Futerman A.H. Barenholz Y. Taraboulos A. J. Biol. Chem. 1999; 274: 20763-20771Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 11Taraboulos A. Scott M. Semenov A. Avrahami D. Laszlo L. Prusiner S.B. Avraham D. J. Cell Biol. 1995; 129: 121-132Crossref PubMed Scopus (519) Google Scholar). Moreover, removal of PrPC from rafts by the substitution of its GPI anchor with a transmembrane domain prevents the formation of PrPSc (11Taraboulos A. Scott M. Semenov A. Avrahami D. Laszlo L. Prusiner S.B. Avraham D. J. Cell Biol. 1995; 129: 121-132Crossref PubMed Scopus (519) Google Scholar, 20Kaneko K. Vey M. Scott M. Pilkuhn S. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2333-2338Crossref PubMed Scopus (236) Google Scholar), thus suggesting a role for these microdomains in scrapie replication. From these observations it also seems evident that scrapie conversion requires membrane-bound GPI-linked PrP. However, the role of the GPI-anchor in prion conversion is still debated. In cell-free experiments, PrP lacking the GPI moiety can be converted to the PrPSc form (21Kocisko D.A. Come J.H. Priola S.A. Chesebro B. Raymond G.J. Lansbury P.T. Caughey B. Nature. 1994; 370: 471-474Crossref PubMed Scopus (800) Google Scholar, 22Lawson V.A. Priola S.A. Wehrly K. Chesebro B. J. Biol. Chem. 2001; 276: 35265-35271Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), whereas in scrapie-infected cells the absence of the GPI moiety reduces conversion (4Caughey B. Raymond G.J. J. Biol. Chem. 1991; 266: 18217-18223Abstract Full Text PDF PubMed Google Scholar, 23Rogers M. Yehiely F. Scott M. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3182-3186Crossref PubMed Scopus (159) Google Scholar). Furthermore, it has been recently shown that a PrP mutant lacking the GPI anchor (anchorless PrP or PrPΔGPI) supports scrapie replication in transgenic mice, although the infected mice do not show any of the clinical signs of the disease until death (24Chesebro B. Trifilo M. Race R. Meade-White K. Teng C. LaCasse R. Raymond L. Favara C. Baron G. Priola S. Caughey B. Masliah E. Oldstone M. Science. 2005; 308: 1435-1439Crossref PubMed Scopus (541) Google Scholar). A possible explanation for these differences between PrPC and PrPΔGPI could be that PrPΔGPI is per se able to sustain conversion into PrPSc but that other factors present in specific compartments of the cell are also required for conversion and for the pathogenesis of the disease. These findings have prompted us to analyze the biosynthesis, intracellular trafficking, and biological properties of an anchorless version of PrP (PrPΔGPI) in transfected cells to better understand the role of the GPI anchor in the behavior of the prion protein. Differently to what was published before, we found that PrPΔGPI is fully glycosylated, but in contrast to the GPI-anchored version, it does not localize on the plasma membrane and is mainly secreted. Interestingly, despite the lack of the GPI anchor, PrPΔGPI associates to intracellular membranes but does not acquire a transmembrane topology. Furthermore, we found that a significant amount of the protein associates to detergent-resistant domains (DRMs), supporting previous evidence that PrPC could associate to lipid rafts in a GPI anchor-independent manner (25Walmsley A.R. Zeng F. Hooper N.M. J. Biol. Chem. 2003; 278: 37241-37248Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). We propose that the differences in metabolism and intracellular trafficking compared with PrPC are relevant for the development of prion diseases and might lead to a better understanding of their pathogenic mechanisms. Reagents and Antibodies—Cell culture reagents were purchased from Invitrogen. The α-PrP antibodies PRI308, SAF32, and SAF61 were a kind gift of Dr. J. Grassi (Commissariat a l'Energie Atomique, Saclay, France). The antibodies against α-calnexin (CNX) and early endosomal antigen 1 were from StressGen Biotechnologies Corp. (Victoria, BC, Canada). The antibody against Giantin was from Berkeley Antibody Co., Inc. (Richmond, CA). Lysotracker Red DND-99 was from Molecular Probes (Eugene, OR). Endoglycosidase H (EndoH) was from Roche Diagnostic, and peptide N-glycosidase F (PGNaseF) and neuraminidase were from Roche Applied Science. GM6001 (Ilomastat) matrix metalloprotease inhibitor was from Chemicon International. Protein-A-Sepharose was from GE Healthcare. Sulfo-H-hydroxy-biotin (S-NHS-biotin) was from Pierce. Methyl-β-cyclodextrin (βCD), mevinolin, fumonisin B1 and all other reagents were obtained from Sigma. PrP Constructs and Transfection—Fischer rat thyroid (FRT) cells were transfected with a cDNA encoding 3F4-tagged PrPΔGPI (a kind gift of Dr. Sylvain Lehmann, UPR CNRS1142, Montpellier, France) with the calcium phosphate procedure as previously described (26Zurzolo C. Lisanti M.P. Caras I.W. Nitsch L. Rodriguez-Boulan E. J. Cell Biol. 1993; 121: 1031-1039Crossref PubMed Scopus (140) Google Scholar), and single stable clones were selected by G418 resistance and used for the following experiments. Cell Culture and Drug Treatments—FRT cells stably expressing PrPΔGPI were grown in F-12 Coon's modified medium containing 5% fetal bovine serum. Tunicamycin (10 μg/ml) was added to the cell culture medium for 16 h. Mevinolin/βCD and fumonisin B1 treatments were carried out as described elsewhere (16Sarnataro D. Paladino S. Campana V. Grassi J. Nitsch L. Zurzolo C. Traffic. 2002; 3: 810-821Crossref PubMed Scopus (91) Google Scholar, 17Sarnataro D. Campana V. Paladino S. Stornaiuolo M. Nitsch L. Zurzolo C. Mol. Biol. Cell. 2004; 15: 4031-4042Crossref PubMed Scopus (115) Google Scholar). Cellular cholesterol levels before and after depletion were determined by a colorimetric assay (Infinity Cholesterol reagent; Sigma) according to the suggested Sigma protocol number 401, as previously described (17Sarnataro D. Campana V. Paladino S. Stornaiuolo M. Nitsch L. Zurzolo C. Mol. Biol. Cell. 2004; 15: 4031-4042Crossref PubMed Scopus (115) Google Scholar). The samples were then read in a spectrophotometer at 550 nm. Phorbol 12-myristate 13-acetate (1 μm), GM6001 (25 μm), and βCD (5 mm) were added to the culture medium for 7 h before collecting cell-free media. Immunoprecipitation—Cells grown in 60- or 100-mm dishes were washed 3 times with and lysed in Triton/DOC buffer (0.5% Triton X-100, 0.5% DOC, 150 mm NaCl, 50 mm Tris-HCl, pH 7.5) with protease inhibitor mixture (leupeptin, antipain, pepstatin, and 1 mm phenylmethylsulfonyl fluoride) for 20 min. Lysates were then precleared with protein-A-Sepharose beads (5 mg/sample) for 30 min and incubated overnight at 4 °C with α-PrP antibody coupled with protein-A-Sepharose beads (10 mg/sample). For media analysis cell media were collected at 7 h or after overnight incubation and immunoprecipitated. The pellets were washed twice with cold lysis buffer and three times with PBS. The samples were then boiled with SDS sample buffer, loaded on polyacrylamide gels, and revealed by Western blotting against PrP and ECL. For direct coupling of antibody to protein A-Sepharose beads, 10 mg/sample of protein A-Sepharose beads was incubated with the antibody for 1 h at room temperature with gentle rocking. The beads were then washed and incubated in 20 mm methyl pimelimidate in 0.2 m sodium borate, pH 9.0, for 30 min at room temperature. The reaction was stopped with 0.2 m ethanolamine. Peptide N-glycosidase F, Endoglycosidase H, and Neuraminidase Treatment—PGNaseF, EndoH, and neuraminidase digestions were performed on immunoprecipitated samples. For PGNaseF treatment the immunoprecipitated samples were resuspended and boiled for 5 min in 10 mm EDTA, 1% Triton X-100, 0.1% SDS, 1% β-mercaptoethanol and incubated with PGNaseF (5 units/sample) for 16 h at 37 °C. For EndoH and neuraminidase (5 milliunits/sample) digestion, the immunoprecipitated samples were first boiled for 3 min in 50 μl of 0.1 m sodium citrate, pH 5.5, containing 0.1% SDS and then treated with the specific enzyme for 16 h at 37 °C. The samples were then analyzed by SDS-PAGE and Western blotting. Pulse-Chase Analysis—FRT cells expressing PrPΔGPI and grown on 100-mm dishes were pulsed for 20 min with 100 μCi/ml [35S]methionine and chased for various times at 37 °C, as indicated in Fig. 1C. At the end of the chase times cells were washed with cold PBS and lysed for 20 min on ice in Triton/DOC buffer. PrPΔGPI immunoprecipitation was performed overnight using the α-PrP SAF32 antibody coupled to protein-A-Sepharose beads. The pellets were washed twice with cold lysis buffer and three times with PBS. The samples were then boiled with SDS sample buffer, loaded on 12% polyacrylamide gels, and revealed by phosphorimaging scanning. Assays for Scrapie-like Properties—Proteinase K (PK) digestion and Triton/DOC insolubility assays were performed as previously described (16Sarnataro D. Paladino S. Campana V. Grassi J. Nitsch L. Zurzolo C. Traffic. 2002; 3: 810-821Crossref PubMed Scopus (91) Google Scholar, 27Lehmann S. Harris D.A. J. Biol. Chem. 1997; 272: 21479-21487Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Fluorescence Microscopy—FRT cells stably expressing PrPΔGPI were grown for 4-5 days both on coverslips and on Transwell filters (not shown), washed with PBS, fixed in 2% paraformaldehyde, permeabilized with 0.075% saponin, and processed for indirect immunofluorescence using specific antibodies. PrPΔGPI was visualized with a fluorescein isothiocyanate-conjugated secondary antibody, whereas CNX, giantin, and early endosomal antigen 1 were revealed by TRITC-conjugated secondary antibodies using a Zeiss laser scanning confocal microscope (LSCM 510). For lysosome staining, cells were incubated for 1 h with Lysotracker (1:10,000) in complete medium before fixing. Biotinylation Assays—Confluent monolayers on Transwells were biotinylated and processed for immunoprecipitation as previously described (26Zurzolo C. Lisanti M.P. Caras I.W. Nitsch L. Rodriguez-Boulan E. J. Cell Biol. 1993; 121: 1031-1039Crossref PubMed Scopus (140) Google Scholar). To recover the immunoprecipitated PrP, the samples were boiled for 10 min and then loaded on 12% gels and revealed by Western blotting with horseradish peroxidase-conjugated streptavidin. Triton X-114 Phase Separation—Cells were lysed in Tris-buffered saline 1% Triton X-114 (10 mm Tris-HCl, pH 7.4, 150 mm NaCl. and 1 mm EDTA) for 1 h at 4°C. Post-nuclear supernatants were incubated for 3 min at 37 °C and centrifuged for 1 min at room temperature for phase separation. An aqueous and a detergent phase were collected and trichloroacetic acid-precipitated. PrPs were revealed by Western blotting. Topology Assays; Digitonin Permeabilization—FRT cells grown for 4-5 days on coverslips were washed twice with Buffer 1 (20 mm Hepes-KOH, pH 7.2, 110 mm potassium acetate, 2 mm magnesium acetate) and then incubated on ice for 5 min with digitonin (20 μg/ml) in Buffer 1. After washing, coverslips were fixed in 2% paraformaldehyde and, where indicated, permeabilized with 0.075% saponin, processed for indirect immunofluorescence, and analyzed by a Zeiss laser scanning confocal microscope (LSCM 510) as described above. PK Protection Assay—Membrane topology of PrPΔGPI was determined as previously described (28Campana V. Sarnataro D. Fasano C. Casanova P. Paladino S. Zurzolo C. J. Cell Sci. 2006; 119: 433-442Crossref PubMed Scopus (47) Google Scholar, 29Drisaldi B. Stewart R.S. Adles C. Stewart L.R. Quaglio E. Biasini E. Fioriti L. Chiesa R. Harris D.A. J. Biol. Chem. 2003; 278: 21732-21743Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Cells were lysed in 0.25 m sucrose, 10 mm HEPES, pH 7.4, by 10 passages through 26-gauge needles. The post-nuclear supernatant was divided into three samples; one untreated, the second digested with 250 μg/ml PK for 30 min at 22 °C in 50 mm Tris-HCl, pH 7.5, and the third digested with PK at the same concentration in the presence of 0.5% Triton X-100 (TX-100). Samples were immunoprecipitated both with SAF32 or SAF61 antibodies, divided in two aliquots digested (+) or not (-) with PGNaseF, and analyzed by Western blotting. Sodium Carbonate Extraction—Cells were homogenized in 0.25 m sucrose with either 0.1 m Tris-HCl, pH 7.5, or 0.2 m sodium carbonate, pH 11, for 30 min on ice by 10 passages through 26-gauge needles and centrifuged for 30 min at 4 °C at 61,000 rpm (in a MLA 130 rotor from TLA 100, Beckman Instruments, Fullerton, CA). Soluble and insoluble phases were collected and trichloroacetic acid-precipitated, and PrPs were revealed by Western blotting. DRM Analysis by Sucrose Density Gradients—Control and mevinolin/βCD- and fumonisin B1-treated cells grown to confluence in 150-mm dishes were harvested in cold PBS and resuspended in 1 ml of lysis buffer (1% TX-100, 10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA), left in ice for 20 min, and passaged 10 times through 22-gauge needles. Lysates were mixed with an equal volume of 85% sucrose (w/v) in 10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA, placed at the bottom of a discontinuous sucrose gradient (30-5%) in the same buffer, and ultracentrifuged at 200,000 × g for 17 h at 4 °C in an ultracentrifuge (SW41 rotor from Beckman Instruments). Twelve fractions were harvested from the top of the gradient. A white light-scattering band, identified in fraction 5 at the interface between 5 and 30% sucrose, contained DRM domains. Samples were trichloroacetic acid-precipitated, and proteins were analyzed by Western blotting. Lipid Analysis—Aliquots of fraction 5 (800 μl) of sucrose density gradients diluted with 200 μl of lysis buffer were precleared twice with Dynabeads for 2 h and incubated overnight at 4 °C with an α-PrP antibody. Immunoprecipitates were recovered using protein A-coupled magnetic beads (30Prinetti A. Chigorno V. Tettamanti G. Sonnino S. J. Biol. Chem. 2000; 275: 11658-11665Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). ⅕ of the samples were analyzed by SDS-PAGE. Lipids were extracted from the immunoprecipitates and analyzed as described below. Cholesterol was quantified after separation on high performance TLC by visualization with 15% concentrated sulfuric acid in 1-butanol (30Prinetti A. Chigorno V. Tettamanti G. Sonnino S. J. Biol. Chem. 2000; 275: 11658-11665Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Phospholipids and sphingomyelin were separated by a two-run mono-dimensional high performance TLC using the solvent system chloroform/methanol 9:1 (v/v) followed by the solvent system chloroform/methanol/acetic acid/water 30:20:2:1 (v/v/v/v) and quantified after separation on a high performance TLC followed by specific detection with a molybdate reagent. Lipids were quantified by densitometry and compared with known amounts of standard lipids (Molecular Analyst program, Bio-Rad). Preparation of Detergent-free Lipid Rafts—Detergent-free extraction and gradients were performed as previously published (31Macdonald J.L. Pike L.J. J. Lipid Res. 2005; 46: 1061-1067Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). Four 150-mm plates of cells were washed and scraped into 20 mm Tris-HCl, pH 7.8, 250 mm sucrose to which had been added 1 mm CaCl2 and 1 mm MgCl2. Cells were pelleted by centrifugation for 2 min at 250 × g and resuspended in 1 ml of 20 mm Tris-HCl, pH 7.8, 250 mm sucrose containing 1 mm CaCl2, 1 mm MgCl2, and protease inhibitors at final concentrations of 0.2 mm aminoethylbenzenesulfonyl fluoride, 1 μg/ml aprotinin, 10 μm bestatin, 3 μm E-64, 10 μg/ml leupeptin, 2 μm pepstatin, and 50 μg/ml calpain inhibitor I. The cells were then lysed by passage through a 22-gauge needle 20 times. Lysates were centrifuged at 1000 × g for 10 min. The resulting post-nuclear supernatant was collected and transferred to a separate tube. The pellet was again lysed by the addition of 1 ml of base buffer plus divalent cations and protease inhibitors followed by sheering 20 times through a needle and syringe. After centrifugation at 1000 × g for 10 min, the second post-nuclear supernatant was combined with the first. An equal volume (2 ml) of 20 mm Tris-HCl, pH 7.8, 250 mm sucrose containing 50% OptiPrep was added to the combined post-nuclear supernatants and placed in the bottom of a 12-ml centrifuge tube. An 8-ml gradient of 0-20% OptiPrep in base buffer was poured on top of the lysate, which was now 25% in OptiPrep. Gradients were centrifuged for 90 min at 52,000 × g using an SW-41 rotor in a Beckman ultracentrifuge. Gradients were fractionated into 0.67-ml fractions, and the distribution of various proteins was assessed by Western blotting. Differential Centrifugation and Secreted Vesicle Isolation—Cell culture media from 20 × 106 FRT cells was submitted to differential centrifugation in the absence or in the presence of 1% TX-100. Media were centrifuged at 1,000 × g for 5 min and at 10,000 × g for 30 min and ultracentrifuged at 100,000 × g for 1 h. Pellet and soluble fractions were recovered at each ultracentrifugation step and trichloroacetic acid-precipitated. PrPs were analyzed by Western blotting. Expression and Characterization of PrPΔGPI in Transfected FRT Cells—Polarized epithelial FRT (Fischer rat thyroid) cells, previously used to characterize the exocytic pathway of PrPC (16Sarnataro D. Paladino S. Campana V. Grassi J. Nitsch L. Zurzolo C. Traffic. 2002; 3: 810-821Crossref PubMed Scopus (91) Google Scholar, 17Sarnataro D. Campana V. Paladino S. Stornaiuolo M. Nitsch L. Zurzolo C. Mol. Biol. Cell. 2004; 15: 4031-4042Crossref PubMed Scopus (115) Google Scholar) and of an inherited pathological PrP mutant (28Campana V. Sarnataro D. Fasano C. Casanova P. Paladino S. Zurzolo C. J. Cell Sci. 2006; 119: 433-442Crossref PubMed Scopus (47) Google Scholar), were stably transfected with a PrP version lacking the GPI anchor attachment signal (PrPΔGPI) (23Rogers M. Yehiely F. Scott M. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3182-3186Crossref PubMed Scopus (159) Google Scholar). Although PrPΔGPI was previously shown to be predominantly unglycosylated in both cells and animals (23Rogers M. Yehiely F. Scott M. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3182-3186Crossref PubMed Scopus (159) Google Scholar, 24Chesebro B. Trifilo M. Race R. Meade-White K. Teng C. LaCasse R. Raymond L. Favara C. Baron G. Priola S. Caughey B. Masliah E. Oldstone M. Science. 2005; 308: 1435-1439Crossref PubMed Scopus (541) Google Scholar), in our hands it migrated as three major bands corresponding to unglycosylated (U), monoglycosylated (M), and highly diglycosylated isoforms (H), similarly to PrPC (Fig. 1A). This discrepancy could be explained by different conformations of the different isoforms of PrPΔGPI or by particular sugar modifications that could mask the epitopes recognized by some antibodies. Indeed, although all isoforms of PrPC were equally well immunoprecipitated by the three different antibodies used (α-N-terminal (SAF32, Fig. 1, lane 1), α-C-terminal (SAF61, lane 3), and α-3F4 tag (PRI308, lane 2)), in the case of PrPΔGPI only SAF32 and SAF61 antibodies recognized all glycoforms, whereas PRI308 specifically recognized only the unglycosylated form (Fig. 1A). Because the α-3F4 antibody recognizing the same epitope of PRI308 was previously used to characterize PrPΔGPI (23Rogers M. Yehiely F. Scott M. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3182-3186Crossref PubMed Scopus (159) Google Scholar), this could explain why it was previously thought to be unglycosylated (23Rogers M. Yehiely F. Scott M. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3182-3186Crossref PubMed Scopus (159) Google Scholar, 24Chesebro B. Trifilo M. Race R. Meade-White K. Teng C. LaCasse R. Raymond L. Favara C. Baron G. Priola S. Caughey B. Masliah E. Oldstone M. Science. 2005; 308: 1435-1439Crossref PubMed Scopus (541) Google Scholar). In support of our findings, inhibition of N-glycosylation either with PGNaseF digestion or tunicamycin treatment reduced PrPΔGPI to a single band corresponding to the unglycosylated PrP isoform (Fig. 1B) similar to the wild-type protein as previously shown (28Campana V. Sarnataro D. Fasano C. Casanova P. Paladino S. Zurzolo C. J. Cell Sci. 2006; 119: 433-442Crossref PubMed Scopus (47) Google Scholar). To characterize the oligosaccharide chains of PrPΔGPI, we performed a deglycosylation assay using either EndoH or neuraminidase (27Lehmann S. Harris D.A. J. Biol. Chem. 1997; 272: 21479-21487Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar) (Fig. 1B). Differently from PrPC, which in the same cells was resistant to EndoH and sensitive to neuraminidase dige