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• W2079435449 abstract "Glycosylphosphatidylinositol-anchored prion protein and Thy-1, found in adjacent microdomains or “rafts” on the neuronal surface, traffic very differently and show distinctive differences in their resistance to detergent solubilization. Monovalent immunogold labeling showed that the two proteins were largely clustered in separate domains on the neuronal surface: 86% of prion protein was clustered in domains containing no Thy-1, although 40% of Thy-1 had a few molecules of prion protein associated with it. Only 1% of all clusters contained appreciable levels of both proteins (≤3 immunogold label for both). In keeping with this distribution, immunoaffinity isolation of detergent-resistant membranes (DRMs) using the non-ionic detergent Brij 96 yielded prion protein DRMs with little Thy-1, whereas Thy-1 DRMs contained ∼20% of prion protein. The lipid content of prion protein and Thy-1 DRMs was measured by quantitative nano-electrospray ionization tandem mass spectrometry. In four independent preparations, the lipid content was highly reproducible, with Thy-1 and prion protein DRMs differing markedly from each other and from the total DRM pool from which they were immunoprecipitated. Prion protein DRMs contained significantly more unsaturated, longer chain lipids than Thy-1 DRMs and had 5-fold higher levels of hexosylceramide. The different lipid compositions are in keeping with the different trafficking dynamics and solubility of the two proteins and show that, under the conditions used, DRMs can isolate individual membrane microenvironments. These results further identify unsaturation and glycosylation of lipids as major sources of diversity of raft structure. Glycosylphosphatidylinositol-anchored prion protein and Thy-1, found in adjacent microdomains or “rafts” on the neuronal surface, traffic very differently and show distinctive differences in their resistance to detergent solubilization. Monovalent immunogold labeling showed that the two proteins were largely clustered in separate domains on the neuronal surface: 86% of prion protein was clustered in domains containing no Thy-1, although 40% of Thy-1 had a few molecules of prion protein associated with it. Only 1% of all clusters contained appreciable levels of both proteins (≤3 immunogold label for both). In keeping with this distribution, immunoaffinity isolation of detergent-resistant membranes (DRMs) using the non-ionic detergent Brij 96 yielded prion protein DRMs with little Thy-1, whereas Thy-1 DRMs contained ∼20% of prion protein. The lipid content of prion protein and Thy-1 DRMs was measured by quantitative nano-electrospray ionization tandem mass spectrometry. In four independent preparations, the lipid content was highly reproducible, with Thy-1 and prion protein DRMs differing markedly from each other and from the total DRM pool from which they were immunoprecipitated. Prion protein DRMs contained significantly more unsaturated, longer chain lipids than Thy-1 DRMs and had 5-fold higher levels of hexosylceramide. The different lipid compositions are in keeping with the different trafficking dynamics and solubility of the two proteins and show that, under the conditions used, DRMs can isolate individual membrane microenvironments. These results further identify unsaturation and glycosylation of lipids as major sources of diversity of raft structure. The separation of membrane lipids into different phases creates diverse microenvironments within a biological membrane (1Rietveld A. Simons K. Biochim. Biophys. Acta. 1998; 1376: 467-479Crossref PubMed Scopus (452) Google Scholar, 2Brown D.A. London E. J. Membr. Biol. 1998; 164: 103-114Crossref PubMed Scopus (831) Google Scholar). In particular, cholesterol is believed to condense with saturated phosphatidylcholine (PC) 1The abbreviations used are: PC, phosphatidylcholine; DRM, detergent-resistant membrane; ESI-MS/MS, electrospray ionization tandem mass spectrometry; GPI, glycosylphosphatidylinositol; HexCer, hexosylceramide; PrP, prion protein; SM, sphingomyelin. 1The abbreviations used are: PC, phosphatidylcholine; DRM, detergent-resistant membrane; ESI-MS/MS, electrospray ionization tandem mass spectrometry; GPI, glycosylphosphatidylinositol; HexCer, hexosylceramide; PrP, prion protein; SM, sphingomyelin. and sphingomyelin (SM) to form minute patches (40–100 nm wide) of lipids in a liquid-ordered phase (3Recktenwald D.J. McConnell H.M. Biochemistry. 1981; 20: 4505-4510Crossref PubMed Scopus (197) Google Scholar, 4Ipsen J.H. Karlstrom G. Mouritsen O.G. Wennerstrom H. Zuckermann M.J. Biochim. Biophys. Acta. 1987; 905: 162-172Crossref PubMed Scopus (896) Google Scholar, 5Vist M.R. Davis J.H. Biochemistry. 1990; 29: 451-464Crossref PubMed Scopus (1010) Google Scholar, 6Ahmed S.N. Brown D.A. London E. Biochemistry. 1997; 36: 10944-10953Crossref PubMed Scopus (609) Google Scholar, 7Schroeder R.J. Ahmed S.N. Zhu Y. London E. Brown D.A. J. Biol. Chem. 1998; 273: 1150-1157Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar), creating specialized lipid microenvironments called “rafts” within the disordered fluid phase formed by unsaturated lipids (8Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8018) Google Scholar). These ordered microdomains control the access and egress of subsets of membrane proteins, regulating signaling systems at the cell surface (9Simons K. Toomre D. Nat. Rev.: Mol. Cell Biol. 2000; 1: 31-39Crossref PubMed Scopus (5110) Google Scholar). liquid-ordered domains resist solubilization in non-ionic detergents (6Ahmed S.N. Brown D.A. London E. Biochemistry. 1997; 36: 10944-10953Crossref PubMed Scopus (609) Google Scholar, 7Schroeder R.J. Ahmed S.N. Zhu Y. London E. Brown D.A. J. Biol. Chem. 1998; 273: 1150-1157Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 10London E. Brown D.A. Biochim. Biophys. Acta. 2000; 1508: 182-195Crossref PubMed Scopus (572) Google Scholar, 11Wang T.Y. Leventis R. Silvius J.R. Biochemistry. 2001; 40: 13031-13040Crossref PubMed Scopus (84) Google Scholar, 12Xu X. Bittman R. Duportail G. Heissler D. Vilcheze C. London E. J. Biol. Chem. 2001; 276: 33540-33546Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 13Li X.M. Momsen M.M. Smaby J.M. Brockman H.L. Brown R.E. Biochemistry. 2001; 40: 5954-5963Crossref PubMed Scopus (134) Google Scholar), enabling them to be isolated as detergent-resistant membranes (DRMs) that float at low density upon gradient centrifugation (14Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2598) Google Scholar). Lipid-anchored proteins partition into both leaflets of these domains, the glycosylphosphatidylinositol (GPI)-anchored proteins into the outer (surface) layer and the diacylated cytoplasmic proteins into the inner layer (9Simons K. Toomre D. Nat. Rev.: Mol. Cell Biol. 2000; 1: 31-39Crossref PubMed Scopus (5110) Google Scholar, 15Moffett S. Brown D.A. Linder M.E. J. Biol. Chem. 2000; 275: 2191-2198Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 16Morris R.J. Cox H.M. Mombelli E. Quinn P.J. Quinn P.J. Membrane Dynamics and Domains. Kluwer Academic/Plenum Publishers, London2004Google Scholar).The membrane environment of GPI-anchored prion protein (PrP) is of particular interest since it is a candidate for chaperoning the conversion of PrP to the altered pathogenic conformation associated with prion disease (17Sanghera N. Pinheiro T.J. J. Mol. Biol. 2002; 315: 1241-1256Crossref PubMed Scopus (166) Google Scholar, 18Morillas M. Swiezicki W. Gambetti P. Surewicz W.K. J. Biol. Chem. 1999; 274: 36859-36865Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 19Kazlauskaite J. Sanghera N. Sylvester I. Venien-Bryan C. Pinheiro T.J. Biochemistry. 2003; 42: 3295-3304Crossref PubMed Scopus (152) Google Scholar). Immunolabeling shows PrP to be present on the neuronal surface in different, albeit often closely adjacent, domains to those occupied by Thy-1, the major GPI-anchored protein of mature neurons (20Madore N. Smith K.L. Graham C.H. Jen A. Brady K. Hall S. Morris R. EMBO J. 1999; 18: 6917-6926Crossref PubMed Scopus (330) Google Scholar). These differences in surface localization are reflected in the different functions and trafficking of these proteins. Thy-1 inhibits the activity of Src family kinases attached to the inner leaflet of rafts (21Hueber A.O. Bernard A.M. Battari C.L. Marguet D. Massol P. Foa C. Brun N. Garcia S. Stewart C. Pierres M. He H.T. Curr. Biol. 1997; 7: 705-708Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), undergoes relatively slow internalization (22Sunyach C. Jen A. Deng J. Fitzgerald K. Frobert Y. McCaffrey M. Morris R.J. EMBO J. 2003; 22: 3591-3601Crossref PubMed Scopus (245) Google Scholar), and has a half-life of >100 h (23Lemansky P. Fatemi S.H. Gorican B. Meyale S. Rossero R. Tartakoff A.M. J. Cell Biol. 1990; 110: 1525-1531Crossref PubMed Scopus (47) Google Scholar). PrP has a half-life of a few hours (24Parizek P. Roeckl C. Weber J. Flechsig E. Aguzzi A. Raeber A.J. J. Biol. Chem. 2001; 276: 44627-44632Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 25Shyng S.-L. Huber M.T. Harris D.A. J. Biol. Chem. 1993; 268: 15922-15928Abstract Full Text PDF PubMed Google Scholar); it is rapidly and constitutively endocytosed on neurons, leaving rafts (as defined by their insolubility in standard non-ionic detergents) to enter more soluble membrane domains on the cell surface, and thus coated pits and endosomes (22Sunyach C. Jen A. Deng J. Fitzgerald K. Frobert Y. McCaffrey M. Morris R.J. EMBO J. 2003; 22: 3591-3601Crossref PubMed Scopus (245) Google Scholar).The rafts occupied by PrP are distinctly more soluble than those of Thy-1 (20Madore N. Smith K.L. Graham C.H. Jen A. Brady K. Hall S. Morris R. EMBO J. 1999; 18: 6917-6926Crossref PubMed Scopus (330) Google Scholar), a result that could indicate differences in lipid composition in the membrane surrounding the two GPI-anchored proteins. The primary goal of this study is to characterize the lipid composition in immunoaffinity-isolated PrP and Thy-1 DRMs, to determine whether the lipid environment of these functionally different GPI-anchored proteins differs. We have examined in detail cholesterol, as well as three lipids that are found predominantly on the outer leaflet of the plasma membrane (26Holthuis J.C. Pomorski T. Raggers R.J. Sprong H. Van Meer G. Physiol. Rev. 2001; 81: 1689-1723Crossref PubMed Scopus (252) Google Scholar): PC as the major glycerolipid, SM as the major sphingolipid, and hexosylceramide (HexCer) as a glycosphingolipid.The approach followed here, of analyzing lipid composition of immunoisolated DRMs, is valid only if the detergent fractionates the membrane into discrete lipid microenvironments that maintain their separate identity during solubilization and purification (16Morris R.J. Cox H.M. Mombelli E. Quinn P.J. Quinn P.J. Membrane Dynamics and Domains. Kluwer Academic/Plenum Publishers, London2004Google Scholar, 27Munro S. Cell. 2003; 115: 377-388Abstract Full Text Full Text PDF PubMed Scopus (1318) Google Scholar). We have shown that the detergent commonly used for such studies, Triton X-100, promotes mixing of domains from totally different membranes, a problem not found with the non-ionic detergent, Brij 96 (20Madore N. Smith K.L. Graham C.H. Jen A. Brady K. Hall S. Morris R. EMBO J. 1999; 18: 6917-6926Crossref PubMed Scopus (330) Google Scholar). Here we have used Brij 96 to solubilize brain membranes, a preparation in which both PrP and Thy-1 are expressed almost exclusively on neuronal membrane (28Morris R. BioEssays. 1992; 14: 715-722Crossref PubMed Scopus (42) Google Scholar, 29Ford M.L. Burton L.J. Li H. Graham C.H. Frobert Y. Grassi J. Hall S.M. Morris R.J. Neuroscience. 2002; 111: 533-551Crossref PubMed Scopus (80) Google Scholar, 30Ford M.L. Burton L.J. Morris R.J. Hall S.M. Neuroscience. 2002; 113: 177-192Crossref PubMed Scopus (167) Google Scholar).EXPERIMENTAL PROCEDURESPreparation of DRMs (20Madore N. Smith K.L. Graham C.H. Jen A. Brady K. Hall S. Morris R. EMBO J. 1999; 18: 6917-6926Crossref PubMed Scopus (330) Google Scholar)—For a source of rat brain, four 3-monthold virgin female Sprague-Dawley littermates were used. All steps in the procedure were carried out at 4 °C independently for each brain with only the buffer/detergent solutions in common.Freshly removed brain was homogenized in 0.32 m sucrose/buffer S (10 mm Tris-Cl, pH 8.0/0,.02% NaN3) with protease inhibitors (1 mm phenylmethylsulfonyl fluoride (Sigma), added from 100× stock solution in dry ethanol immediately before homogenization, with Complete Mini protease inhibitor mixture (Roche Applied Science)). The postnuclear membrane pellet (18,000 × g, 40 min) was resuspended at 5 mg of protein/ml. This was diluted 1:1 in 1% Brij 96 (Fluka, Lot Number 402329/1) in buffer S, rotated gently for 30 min before a 1-ml aliquot was diluted 1:1 in 80% sucrose/buffer S and overlain with 8 ml of a continuous 30–5% sucrose gradient in buffer S/0.5% Brij 96 and centrifuged in a Beckman SW41 rotor (200,000 × g, 18 h). Sequential 1-ml fractions were removed, the position of the DRM fraction, identified by its opacity, was confirmed by immunoblotting for PrP and Thy-1, and an aliquot of the fraction (usually number 3) with the highest content of both proteins was subject to immunoaffinity purification using IgG antibody directly coupled to M-280 Tosyl-activated Dynabeads (Dynal Biotech). For PrP, the antibodies were immunoaffinity-purified MoPL1S and MoPa1S (29Ford M.L. Burton L.J. Li H. Graham C.H. Frobert Y. Grassi J. Hall S.M. Morris R.J. Neuroscience. 2002; 111: 533-551Crossref PubMed Scopus (80) Google Scholar)), and for Thy-1, antibodies were OX-7 monoclonal (31Mason D.W. Williams A.F. Biochem. J. 1980; 187: 1-20Crossref PubMed Scopus (446) Google Scholar).The DRM fraction from the gradient was divided into two aliquots from which either PrP or Thy-1 was immunoprecipitated. To isolate PrP DRMs, an aliquot was precleared by two 30-min incubations with anti-Thy-1 beads (200 and then 400 μg of IgG/ml DRM) followed by a 6-h incubation with 800 μg of anti-PrP IgG/ml DRM. To isolate Thy-1 DRMs, the other aliquot was similarly precleared with anti-PrP beads (400 and then 800 μg of IgG/ml DRM) prior to a 6-h incubation with 400 μg of anti-Thy-1 IgG/ml DRM. An aliquot (5 μl) was removed for immunoblot analysis, and the was remainder snap-frozen under nitrogen and stored at –20 °C prior to lipid analysis. Immunoblots were quantitated using alkaline phosphatase-coupled secondary antibody, the enzymatic reaction was developed within its linear range, and blots were scanned on a Heidelberg 1200 flatbed scanner and analyzed with NIH Image.Lipid Analysis—Lipid extractions in the presence of internal standards were performed according to the method of Bligh and Dyer (32Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42153) Google Scholar) as described previously (33Brügger B. Sandhoff R. Wegehingel S. Gorgas K. Malsam J. Helms J.B. Lehmann W.D. Nickel W. Wieland F.T. J. Cell Biol. 2000; 151: 507-518Crossref PubMed Scopus (184) Google Scholar). After solvent evaporation, samples were resuspended in methanol and further processed for mass spectrometry as described (33Brügger B. Sandhoff R. Wegehingel S. Gorgas K. Malsam J. Helms J.B. Lehmann W.D. Nickel W. Wieland F.T. J. Cell Biol. 2000; 151: 507-518Crossref PubMed Scopus (184) Google Scholar, 34Brügger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2339-2344Crossref PubMed Scopus (725) Google Scholar). Nano-ESI-MS/MS analysis was performed on a Micromass QII triple-stage quadrupole tandem mass spectrometer equipped with a nano-ESI source (Z spray) from Micromass. Argon was used as collision gas at a nominal pressure of 2.5 × 10–3 millibars. The cone voltage was set to 30 V. Resolution of Q1 and Q3 was set to achieve isotope resolution. Detection of PC and SM was performed by parent ion scanning for fragment ion m/z 184 at a collision energy of 32 eV. HexCer scanning was performed by parent ion scanning for fragment ion 264 at a collision energy of 44 eV, and ceramide scanning was performed by parent ion scanning for fragment ion 264 at a collision energy of 30 eV. SM detection in negative ion mode was done by applying a cone voltage of 100 V and a collision energy of 33 eV, selecting for a fragmentation of m/z 168. Cholesterol quantitation was performed as described (35Sandhoff R. Brügger B. Jeckel D. Lehmann W.D. Wieland F.T. J. Lipid Res. 1999; 40: 126-132Abstract Full Text Full Text PDF PubMed Google Scholar) with d6-cholesterol (Cambridge Isotope Laboratories Inc., Andover, MA) in negative ion mode, selecting for fragment ions of m/z 80 at a cone voltage of 50 V and a collision energy of 130 eV. Synthetic lipid standards were obtained from Avanti Polar Lipids (Alabaster, AL), standard synthesis of HexCer standards (18:1; 14:0/18:1; 19:0/18:1; 26:0), and ceramide standards (18:1; 14:0/18:1; 17:0/18:1; 25:0) were performed as described for sphingomyelin (33Brügger B. Sandhoff R. Wegehingel S. Gorgas K. Malsam J. Helms J.B. Lehmann W.D. Nickel W. Wieland F.T. J. Cell Biol. 2000; 151: 507-518Crossref PubMed Scopus (184) Google Scholar). Sphingosylphosphorylcholine, sphingosine, and psychosine were obtained from Matreya Inc. (Pleasant Gap, PA) and Avanti Polar Lipids, and fatty acids were from Merck (Darmstadt, Germany). Quantitative analyses were performed as described (33Brügger B. Sandhoff R. Wegehingel S. Gorgas K. Malsam J. Helms J.B. Lehmann W.D. Nickel W. Wieland F.T. J. Cell Biol. 2000; 151: 507-518Crossref PubMed Scopus (184) Google Scholar, 34Brügger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2339-2344Crossref PubMed Scopus (725) Google Scholar). Phosphate determination was performed according to Rouser et al. (36Rouser G. Fleischer S. Yamamoto A. Lipids. 1970; 5: 494-496Crossref PubMed Scopus (2858) Google Scholar). The significance of data was tested by analysis of variance with repeated measures; data that differed at p < 0.05 were then analyzed by paired, two-tailed t tests.Electron Microscopy—Adult mouse sensory neurons (37Lindsay R.M. Harmar A.J. Nature. 1989; 337: 362-364Crossref PubMed Scopus (839) Google Scholar), maintained in culture for 3–5 days, were immunolabeled for 30 min at 10 °C with 5 nm (PrP) or 10 nm (Thy-1) of gold, to which the appropriate Fab was directly coupled in a limiting amount to achieve monovalent binding to surface antigen; the gold was titered to a level at which it bound at >80% saturation of surface antigen (22Sunyach C. Jen A. Deng J. Fitzgerald K. Frobert Y. McCaffrey M. Morris R.J. EMBO J. 2003; 22: 3591-3601Crossref PubMed Scopus (245) Google Scholar). Cells were then fixed in 1% glutaraldehyde/1% paraformaldehyde in 0.1 m sodium phosphate buffer, pH 7.4, and processed for viewing 80–100-nm-thick sections in a transmission electron microscope (Hitachi 7600 at 75 kV). Specimens were digitally photographed at ×100,000 (after viewing at ×400,000 if necessary to resolve the size/number of closely clustered grains). Label (>2,000 grains counted for both PrP and Thy-1) that was within 20 nm of another label on the same membrane was scored as within a single cluster. Control experiments using neurons taken from mice genetically null for Thy-1 (38Nosten-Bertrand M. Errington M.L. Murphy K.P.S.J. Tokugawa Y. Barboni E. Kozlova E. Michalovich D. Morris R.G.M. Silver J. Stewart C.L. Bliss T.V.P. Morris R.J. Nature. 1996; 379: 826-829Crossref PubMed Scopus (202) Google Scholar) or PrP (39Manson J.C. Clarke A. Hooper M. Aitchison L. McConnell I. Hope J. Mol. Neurobiol. 1994; 8: 121-127Crossref PubMed Scopus (492) Google Scholar) showed label only for the expressed protein. With the 10 nm of gold used for Thy-1 labeling, smaller grains that would be scored as 5 nm (PrP) of label were occasionally seen, at a frequency of <1% of that seen for PrP label on wild type neurons.RESULTSSeparation of PrP and Thy-1 on the Membrane and in DRMs—To determine the extent to which immunolabel for PrP and Thy-1 identify separate microdomains on the neuronal surface, primary cultured sensory neurons were labeled with Fab directly coupled to 5 nm (anti-PrP) or 10 nm (anti-Thy-1) of gold and viewed in the transmission electron microscope. Label for each GPI-anchored protein was present in clusters largely devoid of the other (Fig. 1, A–C). However, one or two immunogold labels for PrP were frequently found at the border of Thy-1 clusters (Fig. 1, A, B, and E), and Thy-1 label was occasionally found beside a cluster of PrP (Fig. 1D).To assess the extent of overlap between PrP and Thy-1, a cluster was defined operationally as any group whose individual gold grains were within 20 nm of another. 2This is quite a stringent criterion, 20 nm being approximately the distance spanned by a single diglycosylated molecule of PrP (surface area ∼16 × 11 nm for the C-terminal domain (40Rudd P.M. Endo T. Colominas C. Groth D. Wheeler S.F. Harvey D.J. Wormald M.R. Serban H. Prusiner S.B. Kobata A. Dwek R.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13044-13049Crossref PubMed Scopus (242) Google Scholar) to which the unstructured 68 N-terminal residues must be added) or by two molecules of Thy-1 (surface area ∼8 × 12nm (41Perkins S.J. Williams A.F. Rademacher T.W. Dwek R.A. Trends Biochem. Sci. 1988; 13: 302-303Abstract Full Text PDF PubMed Scopus (20) Google Scholar)). Nonetheless, it placed >85% of both PrP and Thy-1 within an individual cluster of at least three immunogold labels; 34% of PrP and 45% of Thy-1 occurred in clusters containing >10 immunogold labels (calculated from data shown in Fig. 2). Most PrP (86.1%) was found within clusters of 1–43 immunogold grains that contained no Thy-1; the remainder occurred primarily as 1–3 grains of PrP label associated with Thy-1 clusters of varying size (Fig. 2). On the other hand, a few grains of PrP label were often associated with Thy-1 clusters, so that 40% of the latter contained some PrP. Clusters containing label for both proteins appear in Fig. 2, in the upper right quadrant. Very few clusters (8/746, or 1.1%) contained >3 immunogold particles for both proteins (Fig. 2).Fig. 2Extent of co-localization of PrP and Thy-1 label on sensory neurons. Each point represents a cluster of label for PrP (x axis) and Thy-1 (y axis). PrP-only clusters appear in the bottom right quadrant (the arrow indicates 3 points denoting clusters of 22 PrP immunogold labels), and Thy-1-only clusters appear in the top left quadrant (the arrow here indicates 2 points denoting clusters of 20 Thy-1 immunogold labels), and mixed clusters containing label for both GPI-anchored proteins are in the upper right quadrant (the arrow here indicates 2 points denoting 5 immunogold labels each for Thy-1 and PrP within the same cluster). For display purposes, a log/log plot has been used, with populous values (e.g. for clusters of 1 label) offset so as not to coincide.View Large Image Figure ViewerDownload Hi-res image Download (PPT)If the organization of PrP and Thy-1 on the neuronal membrane in vivo reflects that found with cultured neurons, it may be possible to immunoaffinity-purify a relatively pure population of PrP DRMs, whereas a substantial proportion of Thy-1 DRMs would have some PrP associated with them. In practice, immunoprecipitation would be unlikely to yield such clear separation since, for example, a few molecules of PrP within an otherwise Thy-1-containing DRM could cause the whole to be immunoprecipitated as a PrP DRM. Nonetheless, we previously observed purification broadly in keeping with the extent of separation of PrP and Thy-1 seen ultrastructurally; immunoprecipitation of DRMs with anti-PrP Immunobeads isolated 90% of PrP with 20% Thy-1, whereas anti-Thy-1 Immunobeads isolated >95% of the Thy-1 along with nearly 70% of PrP (20Madore N. Smith K.L. Graham C.H. Jen A. Brady K. Hall S. Morris R. EMBO J. 1999; 18: 6917-6926Crossref PubMed Scopus (330) Google Scholar).In this study, to maximize the yield of DRMs in which either PrP or Thy-1 was dominant, the total DRM pool (Fig. 3A) was divided in two, and each was precleared twice with the reciprocal antibody before immunoprecipitating PrP or Thy-1 (Fig. 3B). The preclears were done rapidly with limiting amounts of antibody to favor removal, from the pool from which PrP would be immunoprecipitated, of DRMs expressing the highest levels of Thy-1, and vice versa. This strategy was successful. Preclearing with anti-Thy-1 enabled PrP DRMs that contained 40% of the PrP, with <2% of the Thy-1, to be isolated, and over 50% of Thy-1 DRMs were obtained that contained 20% of the PrP (Fig. 3B). Since Thy-1 is >10-fold more abundant than PrP (assessed by the degree of purification required from brain; (42Hope J. Morton L. Farquhar C. Multhaup G. Beyreuther K. Kimberlin R.H. EMBO J. 1986; 5: 2591-2597Crossref PubMed Scopus (257) Google Scholar, 43Stahl B. Muller B. von B.Y. Cox E.C. Bonhoeffer F. Neuron. 1990; 5: 735-743Abstract Full Text PDF PubMed Scopus (206) Google Scholar, 44Barclay A.N. Letarte-Muirhead M. Williams A.F. Biochem. J. 1975; 151: 699-706Crossref PubMed Scopus (85) Google Scholar)), the lipids associated with 20% of PrP present in the Thy-1 DRMs would be a minor contaminant when compared with those associated with the more abundant Thy-1.Fig. 3The distribution of PrP and Thy-1 at stages during DRM purification.A, immunoblot showing the profile of PrP and Thy-1 across a density gradient after solubilization in 0.5% Brij 96. The low density fractions (2–5) contain the DRMs, and the 40% sucrose fractions (9–12) contain fully soluble membrane. The total DRM pool was the low density fraction (usually fraction 3) that contained the highest levels of PrP and Thy-1. As shown in B, PrP and Thy-1 present in the total DRM pool, and removed by the Thy-1 or PrP, preclears prior to the main immunoprecipitation (IP) for lipid analysis (indicated by PrP IP and Thy-1 IP). Residual protein not immunoprecipitated is shown in the Unbound fraction.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Significant Differences in Lipid Composition between PrP and Thy-1 DRMs—For each of the four independent preparations of PrP and Thy-1 DRMs, and the total pool of DRMs from which they were immunoprecipitated, the levels of cholesterol, ceramide, 3Only the major ceramide species, N-stearoylceramide, was measured, as a control for the HexCer measurements. PC, SM, and HexCer, as well as the amount of fatty acid of different mass (i.e. chain length and saturation) attached to the latter three lipids, were measured. For each sample, duplicate lipid analyses were done, and the mean was used as the value for that sample.The relative proportions of the lipids in Thy-1, PrP, and total DRMs is shown in Table I. The values are expressed as a percentage of the total lipid measured ([cholesterol] + [PC] + [SM] + [HexCer] + [ceramide]), as it is the relative proportions of these components that is of interest. The results overall were remarkably consistent between the four preparations, giving standard deviations that were small when compared with the differences in mean values for the different DRMs. The proportion of most lipids differed significantly between the two immunoaffinity-purified DRMs and between each of these and the total DRM pool from which they were purified (Table I). The cholesterol level of PrP DRMs was slightly (1.12-fold), albeit significantly, higher than that of Thy-1 DRMs, although both were significantly lower than the cholesterol level of the total DRM. The same trend was evident with HexCer, although the level of this glycosphingolipid in Thy-1 DRMs was markedly (6.6-fold) lower than in the total DRMs, whereas PrP DRMs were only slightly (0.78-fold) lower than the total pool. The higher levels of HexCer and cholesterol in PrP DRMs were unexpected, given that they are more soluble than Thy-1 DRMs (20Madore N. Smith K.L. Graham C.H. Jen A. Brady K. Hall S. Morris R. EMBO J. 1999; 18: 6917-6926Crossref PubMed Scopus (330) Google Scholar, 22Sunyach C. Jen A. Deng J. Fitzgerald K. Frobert Y. McCaffrey M. Morris R.J. EMBO J. 2003; 22: 3591-3601Crossref PubMed Scopus (245) Google Scholar). Thy-1 DRMs had higher (1.4-fold) levels of PC than either PrP or total DRMs, and both Thy-1 and PrP had 2-fold higher levels of SM than the total DRMs. In addition, ceramide level was highest in Thy-1 DRMs (2- and 1.4-fold enriched over total and PrP DRMs, respectively).Table IRelative proportions of lipids in total, and immunopurified, DRMsView Large Image Figure ViewerDownload Hi-res image Download (PPT)4Levels refer to the major ceramide species, N-stearoylceramide, only. Open table in a new tab The immunoaffinity-purified, and total, DRMs also differed significantly in the individual fatty acids attached to PC and SM, but not to HexCer. Values for the major lipid species are given in Table II. 4The full range of fatty acids detected, showing their proportion in the individual samples as well as mean values, is listed in Table I of the supplementary material. Thy-1 DRMs were significantly enri" @default.
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