Glycosylphosphatidylinositol anchors regulate glycosphingolipid levels
2012; Elsevier BV; Volume: 53; Issue: 8 Linguagem: Inglês
10.1194/jlr.m025692
ISSN1539-7262
AutoresUrsula Loizides‐Mangold, Fabrice David, Victor J. Nesatyy, Taroh Kinoshita, Howard Riezman,
Tópico(s)Trypanosoma species research and implications
ResumoGlycosylphosphatidylinositol (GPI) anchor biosynthesis takes place in the endoplasmic reticulum (ER). After protein attachment, the GPI anchor is transported to the Golgi where it undergoes fatty acid remodeling. The ER exit of GPI-anchored proteins is controlled by glycan remodeling and p24 complexes act as cargo receptors for GPI anchor sorting into COPII vesicles. In this study, we have characterized the lipid profile of mammalian cell lines that have a defect in GPI anchor biosynthesis. Depending on which step of GPI anchor biosynthesis the cells were defective, we observed sphingolipid changes predominantly for very long chain monoglycosylated ceramides (HexCer). We found that the structure of the GPI anchor plays an important role in the control of HexCer levels. GPI anchor-deficient cells that generate short truncated GPI anchor intermediates showed a decrease in very long chain HexCer levels. Cells that synthesize GPI anchors but have a defect in GPI anchor remodeling in the ER have a general increase in HexCer levels. GPI-transamidase-deficient cells that produce no GPI-anchored proteins but generate complete free GPI anchors had unchanged levels of HexCer. In contrast, sphingomyelin levels were mostly unaffected. We therefore propose a model in which the transport of very long chain ceramide from the ER to Golgi is regulated by the transport of GPI anchor molecules. Glycosylphosphatidylinositol (GPI) anchor biosynthesis takes place in the endoplasmic reticulum (ER). After protein attachment, the GPI anchor is transported to the Golgi where it undergoes fatty acid remodeling. The ER exit of GPI-anchored proteins is controlled by glycan remodeling and p24 complexes act as cargo receptors for GPI anchor sorting into COPII vesicles. In this study, we have characterized the lipid profile of mammalian cell lines that have a defect in GPI anchor biosynthesis. Depending on which step of GPI anchor biosynthesis the cells were defective, we observed sphingolipid changes predominantly for very long chain monoglycosylated ceramides (HexCer). We found that the structure of the GPI anchor plays an important role in the control of HexCer levels. GPI anchor-deficient cells that generate short truncated GPI anchor intermediates showed a decrease in very long chain HexCer levels. Cells that synthesize GPI anchors but have a defect in GPI anchor remodeling in the ER have a general increase in HexCer levels. GPI-transamidase-deficient cells that produce no GPI-anchored proteins but generate complete free GPI anchors had unchanged levels of HexCer. In contrast, sphingomyelin levels were mostly unaffected. We therefore propose a model in which the transport of very long chain ceramide from the ER to Golgi is regulated by the transport of GPI anchor molecules. cholesterol ester ceramide Chinese hamster ovary endoplasmic reticulum ethanolamine-phosphate glucosylceramide glycosylphosphatidylinositol hexylceramide phosphatidylcholine phosphatidyle-thanolamine phosphatidylinositol phosphatidylserine Lipid anchoring of proteins to the outer leaflet of the plasma membrane is essential for cellular function and development (1Maeda Y. Ashida H. Kinoshita T. CHO glycosylation mutants: GPI anchor.Methods Enzymol. 2006; 416: 182-205Crossref PubMed Scopus (34) Google Scholar). One prominent lipid anchor is a complex glycolipid called glycosylphosphatidylinositol (GPI). The GPI anchor has the core structure phosphatidylinositol (PI)-glucosamine (GlcN)-(Mannose)3-phosphoethanolamine (EtN-P), which is conserved among all species. After biosynthesis, the GPI anchor is attached posttranslationally to the newly generated C terminus of certain eukaryotic proteins destined for anchoring thereby tethering the protein to the membrane surface by the glycolipid moiety. GPI-anchored proteins can be released from the cell surface by phosphatidylinositol specific phospholipases and this cleavage event can induce major conformational changes on the GPI-anchored protein itself (2Bütikofer P. Malherbe T. Boschung M. Roditi I. GPI-anchored proteins: now you see 'em, now you don't.FASEB J. 2001; 15: 545-548Crossref PubMed Scopus (78) Google Scholar). At least three organelles, the endoplasmic reticulum (ER), Golgi, and peroxisomes, are involved in the biosynthesis and remodeling of the GPI anchor. The biosynthesis is initiated on the outer side of the ER membrane. After the first two reactions, the GPI anchor precursor is flipped and biosynthesis continues on the luminal side of the ER where the diacyl chains of phosphatidylinositol are then replaced by alkyl-acyl chains. This step is impaired in mutants of the peroxisomal alkyl phospholipid biosynthesis pathway (3Kanzawa N. Maeda Y. Ogiso H. Murakami Y. Taguchi R. Kinoshita T. Peroxisome dependency of alkyl-containing GPI-anchor biosynthesis in the endoplasmic reticulum.Proc. Natl. Acad. Sci. USA. 2009; 106: 17711-17716Crossref PubMed Scopus (39) Google Scholar). After protein attachment, the GPI anchor undergoes complex remodeling that begins in the ER with the removal of the inositol-linked acyl chain (4Tanaka S. Maeda Y. Tashima Y. Kinoshita T. Inositol deacylation of glycosylphosphatidylinositol-anchored proteins is mediated by mammalian PGAP1 and yeast Bst1p.J. Biol. Chem. 2004; 279: 14256-14263Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) and the remodeling of the GPI glycan part (5). Glycan remodeling is crucial for sorting GPI-anchored proteins into ER exit sites and their subsequent ER to Golgi transport (6Fujita M. Watanabe R. Jaensch N. Romanova-Michaelides M. Satoh T. Kato M. Riezman H. Yamaguchi Y. Maeda Y. Kinoshita T. Sorting of GPI-anchored proteins into ER exit sites by p24 proteins is dependent on remodeled GPI.J. Cell Biol. 2011; 194: 61-75Crossref PubMed Scopus (95) Google Scholar). In mammalian cells, remodeling of the GPI anchor is then continued in the Golgi where the unsaturated fatty acid of the GPI anchor is replaced by a saturated fatty acid chain (7Maeda Y. Tashima Y. Houjou T. Fujita M. Yoko-o T. Jigami Y. Taguchi R. Kinoshita T. Fatty acid remodeling of GPI-anchored proteins is required for their raft association.Mol. Biol. Cell. 2007; 18: 1497-1506Crossref PubMed Scopus (156) Google Scholar). From the Golgi compartment, GPI-anchored proteins are transported to the plasma membrane where they are thought to associate preferentially with glycosphingolipids and cholesterol to be enriched in lipid-ordered microdomains. Lipid remodeling is likely to be important for this association because unremodeled GPI-anchored proteins, which carry unsaturated fatty acids, are no longer enriched in detergent resistant membrane fractions (7Maeda Y. Tashima Y. Houjou T. Fujita M. Yoko-o T. Jigami Y. Taguchi R. Kinoshita T. Fatty acid remodeling of GPI-anchored proteins is required for their raft association.Mol. Biol. Cell. 2007; 18: 1497-1506Crossref PubMed Scopus (156) Google Scholar). Treatments that deplete either membrane cholesterol or sphingolipids also disrupt the association of GPI-anchored proteins with detergent-resistant membranes (DRMs) further supporting the notion that specialized domains are critical for the correct localization of this subset of proteins (8Schroeder R.J. Ahmed S.N. Zhu Y. London E. Brown D.A. Cholesterol and sphingolipid enhance the Triton X-100 insolubility of glycosylphosphatidylinositol-anchored proteins by promoting the formation of detergent-insoluble ordered membrane domains.J. Biol. Chem. 1998; 273: 1150-1157Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 9Hanada K. Nishijima M. Akamatsu Y. Pagano R.E. Both sphingolipids and cholesterol participate in the detergent insolubility of alkaline phosphatase, a glycosylphosphatidylinositol-anchored protein, in mammalian membranes.J. Biol. Chem. 1995; 270: 6254-6260Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Importantly, trafficking of GPI-anchored proteins is affected by alterations in sphingolipids and sterols. In yeast, the ER-to-Golgi transport of GPI-anchored proteins is rapidly reduced by inhibition of de novo sphingolipid biosynthesis without affecting the transport of soluble or transmembrane proteins (10Horvath A. Sutterlin C. Manning-Krieg U. Movva N.R. Riezman H. Ceramide synthesis enhances transport of GPI-anchored proteins to the Golgi apparatus in yeast.EMBO J. 1994; 13: 3687-3695Crossref PubMed Scopus (183) Google Scholar, 11Watanabe R. Funato K. Venkataraman K. Futerman A.H. Riezman H. Sphingolipids are required for the stable membrane association of glycosylphosphatidylinositol-anchored proteins in yeast.J. Biol. Chem. 2002; 277: 49538-49544Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). However, GPI-anchored proteins might play an important role in the transport of membrane proteins such as Tat2 and Fur4p, which are no longer associated with DRMs and are retained in the ER in cells that are deficient at an early stage of GPI anchor biosynthesis (12Okamoto M. Yoko-o T. Umemura M. Nakayama K. Jigami Y. Glycosylphosphatidylinositol-anchored proteins are required for the transport of detergent-resistant microdomain-associated membrane proteins Tat2p and Fur4p.J. Biol. Chem. 2006; 281: 4013-4023Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In mammalian cells, it was shown that inhibition of sphingolipid biosynthesis affects apical targeting of GPI-anchored proteins in Madin-Darby canine kidney (MDCK) cells (13Mays R.W. Siemers K.A. Fritz B.A. Lowe A.W. van Meer G. Nelson W.J. Hierarchy of mechanisms involved in generating Na/K-ATPase polarity in MDCK epithelial cells.J. Cell Biol. 1995; 130: 1105-1115Crossref PubMed Scopus (193) Google Scholar) and sorting of the axonal GPI-anchored protein Thy-1 in primary hippocampal neurons (14Ledesma M.D. Simons K. Dotti C.G. Neuronal polarity: essential role of protein-lipid complexes in axonal sorting.Proc. Natl. Acad. Sci. USA. 1998; 95: 3966-3971Crossref PubMed Scopus (199) Google Scholar). However unlike yeast, ER-to-Golgi transport of GPI-anchored proteins in mammalian cells does not depend on de novo sphingolipid biosynthesis (15Rivier A.S. Castillon G.A. Michon L. Fukasawa M. Romanova-Michaelides M. Jaensch N. Hanada K. Watanabe R. Exit of GPI-anchored proteins from the ER differs in yeast and mammalian cells.Traffic. 2010; 11: 1017-1033Crossref PubMed Scopus (27) Google Scholar). An important characteristic of sphingolipid trafficking is the coexistence of at least two different ceramide transport pathways, a major ATP and cytosol-dependent pathway and a minor ATP or cytosol-independent pathway (16Yamaji T. Kumagai K. Tomishige N. Hanada K. Two sphingolipid transfer proteins, CERT and FAPP2: their roles in sphingolipid metabolism.IUBMB Life. 2008; 60: 511-518Crossref PubMed Scopus (69) Google Scholar). Evidence for two different ceramide transport pathways was first obtained with the isolation of the Chinese hamster ovary (CHO) mutant cell line LY-A that shows a defect in SM but not in HexCer biosynthesis (17Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. Mammalian cell mutants resistant to a sphingomyelin-directed cytolysin. Genetic and biochemical evidence for complex formation of the LCB1 protein with the LCB2 protein for serine palmitoyltransferase.J. Biol. Chem. 1998; 273: 33787-33794Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar) and the subsequent identification of a ceramide transport protein called CERT (18Hanada K. Kumagai K. Yasuda S. Miura Y. Kawano M. Fukasawa M. Nishijima M. Molecular machinery for non-vesicular trafficking of ceramide.Nature. 2003; 426: 803-809Crossref PubMed Scopus (823) Google Scholar). Two pathways for ceramide transport also exist in yeast (19Funato K. Riezman H. Vesicular and nonvesicular transport of ceramide from ER to the Golgi apparatus in yeast.J. Cell Biol. 2001; 155: 949-959Crossref PubMed Scopus (152) Google Scholar). As changes in lipid composition affect GPI-anchored proteins, we asked whether a lack of GPI-anchored proteins together with the abnormal accumulation of GPI anchor intermediates would affect the lipid profile of mammalian cells. To address this question, we made use of a series of mutant CHO cell lines (1Maeda Y. Ashida H. Kinoshita T. CHO glycosylation mutants: GPI anchor.Methods Enzymol. 2006; 416: 182-205Crossref PubMed Scopus (34) Google Scholar) that have defects along the GPI anchor biosynthesis pathway. We determined the lipid profile of the GPI anchor deficient cells using a lipidomics approach. Lipidomics has emerged in the era of genomics and proteomics as a rapidly expanding research field due to recent advances in mass spectrometry and bioinformatics. Here, we applied tandem mass spectrometry coupled with multiple reaction monitoring to detect and quantify over 850 phospho- and sphingo-lipids from GPI anchor deficient cells. Using the same crude lipid extracts, we analyzed complex glycosphingolipids by a nontargeted mass spectrometry approach and also investigated the sterol composition of each sample by GC-MS. DLPC 12:0/12:0 (850335), PE 17:0/14:1 (PE31:1, LM-1104), PI 17:0/14:1 (PI31:1, LM-1504), PS 17:0/14:1 (PS31:1, LM-1304), C17:0 ceramide (860517), C12:0 SM (860583) and Glucosyl C8:0 Cer (860540) were used as internal lipid standards and were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). Ergosterol was used as sterol standard and was purchased from Fluka (Buchs, Switzerland). Methyl tert-butyl ether (MTBE) was from Fluka (Buchs). Methyl amine (33% in absolute ethanol) was from Sigma Aldrich (Steinheim, Germany). HPLC-grade chloroform was purchased from Acros (Geel, Belgium), LC-MS grade methanol and LC-MS grade ammonium acetate were from Fluka. LC-MS grade water was purchased from Biosolve (Valkenswaard, The Netherlands). All CHO cell lines used in this study were from the laboratory of Taroh Kinoshita (1Maeda Y. Ashida H. Kinoshita T. CHO glycosylation mutants: GPI anchor.Methods Enzymol. 2006; 416: 182-205Crossref PubMed Scopus (34) Google Scholar). Cells of the F21 background stably express the GPI anchor marker proteins CD59 and DAF and twelve proteins involved in GPI anchor biosynthesis (20Ashida H. Hong Y. Murakami Y. Shishioh N. Sugimoto N. Kim Y.U. Maeda Y. Kinoshita T. Mammalian PIG-X and yeast Pbn1p are the essential components of glycosylphosphatidylinositol-mannosyltransferase I.Mol. Biol. Cell. 2005; 16: 1439-1448Crossref PubMed Google Scholar) whereas cells of the C311 background express four proteins of the GPI anchor biosynthesis pathway in addition to the markers CD59 and DAF (21Hong Y. Kang J.Y. Kim Y.U. Shin D.J. Choy H.E. Maeda Y. Kinoshita T. New mutant Chinese hamster ovary cell representing an unknown gene for attachment of glycosylphosphatidylinositol to proteins.Biochem. Biophys. Res. Commun. 2005; 335: 1060-1069Crossref PubMed Scopus (5) Google Scholar–23Hong Y. Ohishi K. Kang J.Y. Tanaka S. Inoue N. Nishimura J. Maeda Y. Kinoshita T. Human PIG-U and yeast Cdc91p are the fifth subunit of GPI transamidase that attaches GPI-anchors to proteins.Mol. Biol. Cell. 2003; 14: 1780-1789Crossref PubMed Scopus (95) Google Scholar). Presence of those plasmids was verified by antibiotic resistance of cells to G418, hygromycin B and blasticidin S (F21 series) or resistance of cells to G418, hygromycin B and puromycin (C311 series). For lipid extraction, cells were maintained in Ham's F-12 medium (Invitrogen) supplemented with 10% fetal calf serum (FCS) and 1% PS (penicillin (50 U/ml) and streptomycin (50 U/ml), Invitrogen). HeLa cells were maintained in DMEM (Invitrogen) with 10% FCS supplemented with 1% PS. All cells were grown at 37°C and 5% CO2. HeLa cells were transiently transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. Human ON-TARGETplus SMARTpool siRNA for PIG-L (L-011953-01-0005), DPM3 (L-017492-02-0005), PIG-X (L-013784-02-0005), PIG-F (L-011753-01-0005), PIG-O (L-008728-01-0005), PIG-U (L-017428-00-0005), PGAP1 (L-008110-01-0005), PGAP5 (L-008547-01-0005), p23 (L-003718-00-0005), p24 (L-008074-01-0005), CERT (L-012101-00-0005) and control scrambled siRNA (ON-TARGETplus nontargeting Pool D-001810-10-05) were purchased from Thermo Scientific. GeneSolution siRNA against PGAP2 (1027416) was purchased from Qiagen. Stable CHO transfectants expressing human PIG-F or PIG-U respectively together with the Venus-FLAG-CD59 construct were established by transfection with Lipofectamine 2000 (Invitrogen) followed by selection with Zeocin (300 μg/ml, Invitrogen) for 2 weeks. Single clones were isolated, expanded and analyzed for CD59-Venus surface expression as a marker for restored GPI anchor biosynthesis. Using the EcoRI / NotI restriction sites human PIG-F and PIG-U were subcloned from pMEori-PIG-F and pME-PIG-U (20Ashida H. Hong Y. Murakami Y. Shishioh N. Sugimoto N. Kim Y.U. Maeda Y. Kinoshita T. Mammalian PIG-X and yeast Pbn1p are the essential components of glycosylphosphatidylinositol-mannosyltransferase I.Mol. Biol. Cell. 2005; 16: 1439-1448Crossref PubMed Google Scholar) into the pcDNATM3.1/Zeo(+) mammalian expression vector (Invitrogen). Constructs were verified by sequencing. Vector pME-puro-Venus-FLAG-CD59 (15Rivier A.S. Castillon G.A. Michon L. Fukasawa M. Romanova-Michaelides M. Jaensch N. Hanada K. Watanabe R. Exit of GPI-anchored proteins from the ER differs in yeast and mammalian cells.Traffic. 2010; 11: 1017-1033Crossref PubMed Scopus (27) Google Scholar) was obtained from the laboratory of Reika Watanabe (University of Geneva, Switzerland). Total RNA was isolated from HeLa cells 72 h after transfection using the RNeasy MINI kit (Qiagen) according to the manufacturer's instructions. RNA was converted into cDNA using random hexamers and Superscript II reverse transcriptase (Invitrogen). qRT-PCR was carried out on a BIO-RAD iCycler machine (BioRad, Hercules, CA) with the ABsoluteTMQPCR SYBR Green reagent (Thermo Scientific). Results were normalized against TBP expression. All primers except for PIG-U, PGAP1, and PGAP5 were designed using the NCBI Primer BLAST web tool. The following primers were used: PIGL.For 5′-GGGTGCTCTGTGCTCACGCT-3′, PIGL.Rev 5′-TG GCTTTCTTGGCCTGTGCCA-3′ DPM3.For 5′-GGCCACTGCCC GCCTACTTG-3′, DPM3.Rev 5′-GTCGGCTCGGGCCTCCTGTA-3′ PIGX.For 5′-GCTCTGACGCCGGCATAAGGG-3′, PIGX.Rev 5′-GA C GGCAGGTGTGCAAGTCCTC-3′ PIGF.For 5′-GCCGCCCGT C G TACCTGATG-3′, PIGF.Rev 5′-TGGCTAGCTAACTCTCCCT CC CG-3′ PIGO.For 5′-CACCACCATGCAGCGCCTCA-3′, PIGO.Rev 5′-CGCCTTCCTGCACTGGTGAGC-3′ PGAP2.For 5′-GCTG GAGTGTACACCATCTTTGCC-3′, PGAP2.Rev 5′-CCGAAGTCCC AC CAGGCCGT-3′ CERT.For 5′-AGGCTGTCATCACACCTCAC GA-3′, CERT.Rev 5′-AGCCATGTCGACGCAAGCTGG-3′ p23.For 5′-TGCGCAGCCACCTCAAGATCAC-3′, p23.Rev 5′-CGCCCTGTT C CCTTGCTCTCA-3′ p24.For 5′-TCGACGTGGAGATTACAGG A CCA-3′, p24.Rev 5′-TGGAGTCATGGTGGACATCCGGT-3′ TBP.For 5′-CCGAATATAATCCCAAGCGGT-3′, TBP.Rev 5′ AAATCAGTGCCGTGGTTCGT-3′. For PIG-U, PGAP1, and PGAP5 predesigned primersets from Qiagen (QuantiTect Primer Assay) were purchased. Relative CHOP and BiP mRNA levels were measured against TBP expression by qRT-PCR using primer: CHOP.For 5′-AGAACCA GGAAACGGAAACAGA-3′ CHOP.Rev 5′-TCTCCTTCATGCGCT GCTTT-3′ BiP.For 5′-TGTTCAACCAATTATCAGCAAACTC-3′ BiP.Rev 5′-TTCTGCTGTATCCTCTTCACCAGT-3′ (24Oslowski C.M. Urano F. Measuring ER stress and the unfolded protein response using mammalian tissue culture system.Methods Enzymol. 2011; 490: 71-92Crossref PubMed Scopus (573) Google Scholar). The efficiency of each primer set was determined to be between 90 and 100%. Lipid extracts were prepared using the MTBE protocol (25Matyash V. Liebisch G. Kurzchalia T.V. Shevchenko A. Schwudke D. Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics.J. Lipid Res. 2008; 49: 1137-1146Abstract Full Text Full Text PDF PubMed Scopus (1363) Google Scholar). Briefly, 2.5 × 106 cells were resuspended in 100 μl water. The cell suspension was transferred into a 2 ml Eppendorf tube. Three hundred and sixty microliters methanol and a mix of internal standards was added (400 pmol DLPC, 1000 pmol PE31:1, 1000 pmol PI31:1, 3300 pmol PS31:1, 2500 pmol C12SM, 500 pmol C17Cer and 100 pmol C8GC). Samples were vortexed and 1.2 ml of MTBE was added. Samples were placed for 10 min on a multitube vortexer at 4°C (Lab-tek International, Christchurch, New Zealand) followed by an incubation for 1 h at room temperature (RT) on a shaker. Phase separation was induced by addition of 200 μl MS-grade water. After 10 min of incubation at RT samples were centrifuged at 1,000 g for 10 min. The upper (organic) phase was transferred into a 13 mm glass tube (Corning) with a Teflon-lined cap and the lower phase was reextracted with 400 μl of a MTBE/MeOH/H2O mixture (10:3:1.5). Samples were vortexed, incubated for 10 min at RT, and centrifuged for 10 min at 1000 g. The upper phase was collected and the combined organic phases were dried in a CentriVap Vacuum Concentrator (Labconco, MO). In total, 1,500 μl of organic phase was recovered from each samples and split into three parts. One part was treated by alkaline hydrolysis to enrich for sphingolipids and the other two aliquots were used for glycerophospholipid and sterol analysis, respectively. Glycerophospholipids were deacylated according to the method by Clarke (26Clarke N.G. Dawson R.M. Alkaline O leads to N-transacylation. A new method for the quantitative deacylation of phospholipids.Biochem. J. 1981; 195: 301-306Crossref PubMed Scopus (186) Google Scholar). Briefly, 1 ml freshly prepared monomethylamine reagent [methylamine/H2O/n-butanol/methanol at 5/3/1/4 (v/v)] was added to the dried lipid extract and then incubated at 53°C for 1 h in a water bath. Lipids were cooled to RT and then dried in a CentriVap Vacuum Concentrator. For desalting, the dried lipid extracts were resuspended in 300 μl water-saturated n-butanol. The extracts were sonicated and then extracted with 150 µl H2O. The organic phase was collected, and the aqueous phase was rec xextracted twice with 300 µl water-saturated n-butanol. The organic phases were pooled and dried in a CentriVap Vacuum Concentrator. The dried glycerophospholipid extract was resuspended in 250 μl chloroform/methanol (1:1) and 100 μl were placed into a 13 mm disposable pyrex tube. The solvent was completely evaporated to avoid inhibition of the reaction by organic solvents. 0, 2, 5, 10, 20 μl of a 3 mM KH2PO4 standard solution were placed into separate pyrex tubes. To each tube, 20 μl of water and 140 μl of 70% perchloric acid were added. Tubes were heated at 180°C for 1 h in a hood. Tubes were removed from the heat block and kept at RT for 5 min. Then 800 μl of freshly prepared water/1.25% NH4Molybdate (50 mg/4 ml water)/1.67% ascorbic acid (100 mg/6 ml water) in the ratio of 5:2:1 were added. Tubes were heated at 100°C for 5 min with a marble on each tube to prevent evaporation during heating. Tubes were removed from the block and cooled at RT for 5 min. One hundred microliters of each sample was then transferred into a 96-well microplate and the absorbance at 820 nm was measured. Tandem mass spectrometry for the identification and quantification of phospho- and sphingo-lipid molecular species was performed using multiple reaction monitoring with a TSQ Vantage Triple Stage Quadrupole Mass Spectrometer (Thermo Scientific) equipped with a robotic nanoflow ion source, Nanomate HD (Advion Biosciences, Ithaca, NY). Each individual ion dissociation pathway was optimized with regard to collision energy. Lipid concentrations were calculated relative to the relevant internal standards as described in (27Epstein S. Kirkpatrick C.L. Castillon G.A. Muniz M. Riezman I. David F.P. Wollheim C.B. Riezman H. Activation of the unfolded protein response pathway causes ceramide accumulation in yeast and INS-1E insulinoma cells.J. Lipid Res. 2011; 53: 412-420Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) and then normalized to the total phosphate content of each total lipid extract to adjust for difference in cell size, membrane content, and extraction efficiency. The cellular ceramide glycosylation assay was performed as described previously (28Gupta V. Patwardhan G.A. Zhang Q.J. Cabot M.C. Jazwinski S.M. Liu Y.Y. Direct quantitative determination of ceramide glycosylation in vivo: a new approach to evaluate cellular enzyme activity of glucosylceramide synthase.J. Lipid Res. 2010; 51: 866-874Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Briefly, HeLa cells were treated with siRNA against PIG-F, PIG-O, or PIG-U for 70 h. Scrambled (SCR) siRNA was used as control. Cells were then switched to 950 μl of 1% BSA DMEM medium containing 10 μM NBD C6-ceramide for 2 h. Cells were then rinsed with ice-cold PBS, scraped, and pelleted. Sphingolipids were extracted using the MTBE/methylamine protocol. Samples were resolved on partisil HPTLC plates with fluorescent indicator (Whatman). To distinguish GalCer and GlcCer the HPTLC plates were impregnated with borate as described previously (28Gupta V. Patwardhan G.A. Zhang Q.J. Cabot M.C. Jazwinski S.M. Liu Y.Y. Direct quantitative determination of ceramide glycosylation in vivo: a new approach to evaluate cellular enzyme activity of glucosylceramide synthase.J. Lipid Res. 2010; 51: 866-874Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). After dipping the plates into a 1% aqueous sodium tetraborate solution, the HPTLC plates were activated at 120°C for 30 min. Sphingolipids were resolved with the solvent system of chloroform/MeOH/water (100:30:4). Fluorescent lipids were visualized under UV exposure. NBD C6-Cer and NBD C6-Cer complexed to BSA were purchased from Invitrogen, NBD C6-GlcCer and NBD C6-GalCer were purchased from Matreya. Bands were quantified with the Image J software and values were calculated as percentage of input (NBD C6-Cer). Individual GM3 species were detected by high resolution mass spectrometry on the LTQ Orbitrap XL linear ion trap (Thermo Scientific). Sphingolipid-enriched extracts were infused at a low flow rate using the TriVersa NanoMate robotic ESI source (Advion Biosciences) equipped with a standard ESI chip (Advion Biosciences). Samples were analyzed in negative ion mode. Individual GM3 species were identified by their parental mass combined with fragmentation. Product ions of m/z 290 were obtained from HCD fragmentation of the GM3 precursor ions. These ions correspond to Neu5Ac fragments obtained after cleavage of the glycosidic bond. Extracts were analyzed by GC-MS as described (29Guan X.L. Riezman I. Wenk M.R. Riezman H. Yeast lipid analysis and quantification by mass spectrometry.Methods Enzymol. 2010; 470: 369-391Crossref PubMed Scopus (62) Google Scholar). Briefly, samples were injected into a VARIAN CP-3800 gas chromatograph equipped with a Factor Four Capillary Column VF-5ms 15 m × 0.32 mm i.d. DF = 0.10 and analyzed by a Varian 320 MS triple quadrupole with electron energy set to – 70 eV at 250°C. Samples were applied with the column oven at 45°C, held for 4 min, then raised to 195°C (20°C/min). Sterols were eluted with a linear gradient from 195 to 230°C (4°C / min), followed by raising to 320°C (10°C / min). Finally, the column temperature was raised to 350°C (6°C / min) to elute sterol esters. Cholesterol and cholesterol esters were identified by their retention times (compared with standards) and fragmentation patterns, which were compared with the NIST library. All results are representative of at least three independent experiments. Statistical analyses were performed using an unpaired Student's t-test. Differences were considered significant for P < 0.05 (*), P < 0.01 (**), and P < 0.005 (***). The goal of this study was to establish the lipid profile of cells that have a defect in GPI anchor biosynthesis. CHO GPI anchor mutants have been very useful in the past to understand the GPI biosynthesis pathway and have allowed cloning of the majority of genes involved in this process (1Maeda Y. Ashida H. Kinoshita T. CHO glycosylation mutants: GPI anchor.Methods Enzymol. 2006; 416: 182-205Crossref PubMed Scopus (34) Google Scholar). We focused on CHO mutants that had either the F21 or the C311 genetic background. The F21 series includes cell lines that are defective in Dol-P-Mannose synthase (DPM3), GPI mannosyltransferase I (PIG-X), ethanolamine phosphate transferase II and III (PIG-F), and GPI lipid remodeling (PGAP2). From the C311 series, we analyzed cells defective in ethanolamine phosphate transferase III (PIG-O) and GPI transamidase (PIG-U). As can be seen in Fig. 1 we observed changes in HexCer levels in CHO mutant cells that have a defect in GPI anchor biosynthesis. Because CHO cells do not possess endogenous GalCer (30van der Bijl P. Strous G.J. Lopes-Cardozo M. Thomas-Oates J. van Meer G. Synthesis of non-hydroxy-galactosylceramides and galactosyldiglycerides by hydroxy-ceramide galactosyltransferase.Biochem. J. 1996; 317: 589-597Crossref PubMed Scopus (51) Google Scholar) the changes in HexCer are due to a difference in their glucosylceramide (GlcCer) levels. In detail, we observed a downregulation of total GlcCer levels in a subset of GPI anchor mutants (DPM3, PIG-X, and PIG-F) that generate short truncated GPI anchor intermediates. To our surprise, we did not see an effect in GPI anchor mutant PIG-U cells (Fig. 1B). Because PIG-U mutant cells (PA16.1) have no expression of the GPI marker protein CD59 and only 1% remaining DAF expression (supplementary Table I), this indicates that the observed GlcCer changes are not due to a general absence of GPI-anchored proteins (23Hong Y. Ohishi K. Kang J.Y. Tanaka S. Inoue N. Nishimura J. Maeda Y. Kinoshita T. Human PIG-U and yeast Cdc91p are the fifth subunit of GPI transamidase that attaches GPI-anchors to proteins.Mol. Biol. Cell. 2003; 14: 1780-1789Crossref PubMed Scopus (95) Google Scholar). Lipid
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