Demonstration of an oligosaccharide-diphosphodolichol diphosphatase activity whose subcellular localization is different than those of dolichyl-phosphate-dependent enzymes of the dolichol cycle
2016; Elsevier BV; Volume: 57; Issue: 6 Linguagem: Inglês
10.1194/jlr.m067330
ISSN1539-7262
AutoresAhmad Massarweh, Michaël Bosco, Soria Iatmanen-Harbi, Clarice Tessier, Nicolas Auberger, Patricia Busca, Isabelle Chantret, Christine Gravier‐Pelletier, Stuart Moore,
Tópico(s)Lysosomal Storage Disorders Research
ResumoOligosaccharyl phosphates (OSPs) are hydrolyzed from oligosaccharide-diphosphodolichol (DLO) during protein N-glycosylation by an uncharacterized process. An OSP-generating activity has been reported in vitro, and here we asked if its biochemical characteristics are compatible with a role in endoplasmic reticulum (ER)-situated DLO regulation. We demonstrate a Co2+-dependent DLO diphosphatase (DLODP) activity that splits DLO into dolichyl phosphate and OSP. DLODP has a pH optimum of 5.5 and is inhibited by vanadate but not by NaF. Polyprenyl diphosphates inhibit [3H]OSP release from [3H]DLO, the length of their alkyl chains correlating positively with inhibition potency. The diphosphodiester GlcNAc2-PP-solanesol is hydrolyzed to yield GlcNAc2-P and inhibits [3H]OSP release from [3H]DLO more effectively than the diphosphomonoester solanesyl diphosphate. During subcellular fractionation of liver homogenates, DLODP codistributes with microsomal markers, and density gradient centrifugation revealed that the distribution of DLODP is closer to that of Golgi apparatus-situated UDP-galactose glycoprotein galactosyltransferase than those of dolichyl-P-dependent glycosyltransferases required for DLO biosynthesis in the ER. Therefore, a DLODP activity showing selectivity toward lipophilic diphosphodiesters such as DLO, and possessing properties distinct from other lipid phosphatases, is identified. Separate subcellular locations for DLODP action and DLO biosynthesis may be required to prevent uncontrolled DLO destruction. Oligosaccharyl phosphates (OSPs) are hydrolyzed from oligosaccharide-diphosphodolichol (DLO) during protein N-glycosylation by an uncharacterized process. An OSP-generating activity has been reported in vitro, and here we asked if its biochemical characteristics are compatible with a role in endoplasmic reticulum (ER)-situated DLO regulation. We demonstrate a Co2+-dependent DLO diphosphatase (DLODP) activity that splits DLO into dolichyl phosphate and OSP. DLODP has a pH optimum of 5.5 and is inhibited by vanadate but not by NaF. Polyprenyl diphosphates inhibit [3H]OSP release from [3H]DLO, the length of their alkyl chains correlating positively with inhibition potency. The diphosphodiester GlcNAc2-PP-solanesol is hydrolyzed to yield GlcNAc2-P and inhibits [3H]OSP release from [3H]DLO more effectively than the diphosphomonoester solanesyl diphosphate. During subcellular fractionation of liver homogenates, DLODP codistributes with microsomal markers, and density gradient centrifugation revealed that the distribution of DLODP is closer to that of Golgi apparatus-situated UDP-galactose glycoprotein galactosyltransferase than those of dolichyl-P-dependent glycosyltransferases required for DLO biosynthesis in the ER. Therefore, a DLODP activity showing selectivity toward lipophilic diphosphodiesters such as DLO, and possessing properties distinct from other lipid phosphatases, is identified. Separate subcellular locations for DLODP action and DLO biosynthesis may be required to prevent uncontrolled DLO destruction. Protein N-glycosylation is essential for embryonic development (1Haltiwanger R.S. Lowe J.B. Role of glycosylation in development.Annu. Rev. Biochem. 2004; 73: 491-537Crossref PubMed Scopus (645) Google Scholar), and children born with deficits in this metabolic pathway often have severe multisystemic diseases called congenital disorders of glycosylation (CDG) (2Jaeken J. Congenital disorders of glycosylation (CDG): it's (nearly) all in it!.J. Inherit. Metab. Dis. 2011; 34: 853-858Crossref PubMed Scopus (106) Google Scholar). N-glycosylation begins by transfer of the oligosaccharide, Glc3Man9GlcNAc2, from oligosaccharide-diphosphodolichol (DLO; Glc3Man9GlcNAc2-PP-dolichol) onto nascent polypeptides in the endoplasmic reticulum (ER) by oligosaccharyltransferase (OST) (3Aebi M. Hennet T. Congenital disorders of glycosylation: genetic model systems lead the way.Trends Cell Biol. 2001; 11: 136-141Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Dolichyl diphosphate (DolPP), the by-product of OST-mediated protein glycosylation, is recycled to yield dolichyl phosphate (DolP) (4Frank C.G. Sanyal S. Rush J.S. Waechter C.J. Menon A.K. Does Rft1 flip an N-glycan lipid precursor?.Nature. 2008; 454: E3-E5Crossref PubMed Scopus (57) Google Scholar), which is required for the synthesis of GlcNAc-PP-dolichol, dolichyl-P-Man, and dolichyl-P-Glc (5Rosenwald A.G. Stoll J. Krag S.S. Regulation of glycosylation. Three enzymes compete for a common pool of dolichyl phosphate in vivo.J. Biol. Chem. 1990; 265: 14544-14553Abstract Full Text PDF PubMed Google Scholar). The former molecule is elongated to yield Glc3Man9GlcNAc2-PP-dolichol by UDP-GlcNAc-, GDP-Man-, dolichyl-P-Man- and dolichyl-P-Glc-requiring glycosyltransferases. These ER-situated reactions constitute the dolichol cycle and are controlled at many levels including feedback inhibition (6Kean E.L. Rush J.S. Waechter C.J. Activation of GlcNAc-P-P-dolichol synthesis by mannosylphosphoryldolichol is stereospecific and requires a saturated alpha-isoprene unit.Biochemistry. 1994; 33: 10508-10512Crossref PubMed Scopus (24) Google Scholar) and DLO deglucosylation and hydrolysis (7Spiro M.J. Spiro R.G. Potential regulation of N-glycosylation precursor through oligosaccharide-lipid hydrolase action and glucosyltransferase-glucosidase shuttle.J. Biol. Chem. 1991; 266: 5311-5317Abstract Full Text PDF PubMed Google Scholar). Neutral free oligosaccharides (nfOSs) can be hydrolyzed from mature DLO by OST (7Spiro M.J. Spiro R.G. Potential regulation of N-glycosylation precursor through oligosaccharide-lipid hydrolase action and glucosyltransferase-glucosidase shuttle.J. Biol. Chem. 1991; 266: 5311-5317Abstract Full Text PDF PubMed Google Scholar, 8Harada Y. Buser R. Ngwa E.M. Hirayama H. Aebi M. Suzuki T. Eukaryotic oligosaccharyltransferase generates free oligosaccharides during N-glycosylation.J. Biol. Chem. 2013; 288: 32673-32684Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), and oligosaccharyl phosphates (OSPs) derived from DLO have been shown to occur at low levels in mammalian tissues and cultured cells. OSPs with truncated oligosaccharide structures occur at elevated levels when the corresponding truncated DLOs accumulate in cells with defects in the dolichol cycle, including those from CDG patients, and glucose-starved cells (9Cacan R. Villers C. Belard M. Kaiden A. Krag S.S. Verbert A. Different fates of the oligosaccharide moieties of lipid intermediates.Glycobiology. 1992; 2: 127-136Crossref PubMed Scopus (52) Google Scholar, 10Cacan R. Duvet S. Kmiecik D. Labiau O. Mir A.M. Verbert A. 'Glyco-deglyco' processes during the synthesis of N-glycoproteins.Biochimie. 1998; 80: 59-68Crossref PubMed Scopus (25) Google Scholar, 11Peric D. Durrant-Arico C. Delenda C. Dupre T. De Lonlay P. de Baulny H.O. Pelatan C. Bader-Meunier B. Danos O. Chantret I. et al.The compartmentalisation of phosphorylated free oligosaccharides in cells from a CDG Ig patient reveals a novel ER-to-cytosol translocation process.PLoS One. 2010; 5: e11675Crossref PubMed Scopus (21) Google Scholar, 12Vleugels W. Duvet S. Peanne R. Mir A.M. Cacan R. Michalski J.C. Matthijs G. Foulquier F. Identification of phosphorylated oligosaccharides in cells of patients with a congenital disorders of glycosylation (CDG-I).Biochimie. 2011; 93: 823-833Crossref PubMed Scopus (21) Google Scholar, 13Harada Y. Nakajima K. Masahara-Negishi Y. Freeze H.H. Angata T. Taniguchi N. Suzuki T. Metabolically programmed quality control system for dolichol-linked oligosaccharides.Proc. Natl. Acad. Sci. USA. 2013; 110: 19366-19371Crossref PubMed Scopus (30) Google Scholar). Attempts to understand the significance of OSP generation have been hampered because enzymes responsible for this process have not been characterized at the biochemical or molecular levels. Membrane preparations of yeast and mammalian origins have been reported to contain Ca2+- and Mn2+-activated enzymes, respectively, capable of liberating OSP from DLO. Although these activities have been called pyrophosphatases [International Union of Biochemistry and Molecular Biology (IUBMB) nomenclature: oligosaccharide-diphosphodolichol diphosphatase (DLODP), EC 3.6.1.44] (14Belard M. Cacan R. Verbert A. Characterization of an oligosaccharide-pyrophosphodolichol pyrophosphatase activity in yeast.Biochem. J. 1988; 255: 235-242PubMed Google Scholar), a formal demonstration that this type of reaction actually occurs has yet to be presented. In fact, DLO could be split by either a diphosphatase to yield OSP and DolP or a phospholipase-like enzyme to yield dolichol and an oligosaccharyl diphosphate (OSPP) intermediate that could subsequently be rapidly dephosphorylated to yield OSP (15Dwivedi R. Nothaft H. Reiz B. Whittal R.M. Szymanski C.M. Generation of free oligosaccharides from bacterial protein N-linked glycosylation systems.Biopolymers. 2013; 99: 772-783Crossref PubMed Scopus (19) Google Scholar). Finally, whether the biochemical profile of this in vitro activity is compatible with a role in DLO regulation has not been addressed. In the present report, we describe a membrane-associated, Co2+-activated, DLO-hydrolyzing activity whose pH optimum is 5.5. This activity has a biochemical profile that distinguishes it from other DolP and DolPP phosphatases and hydrolyzes both truncated and mature DLO to yield OSP and DolP, demonstrating for the first time DLODP activity. The selective interaction of DLODP with lipophilic diphosphodiesters indicates that DLOs are potential physiological substrates for this enzyme. Nevertheless, subcellular fractionation studies suggest that DLO biosynthetic reactions and DLODP action are compartmentalized differently. d-[2-3H(N)]mannose (24.7 Ci/mmol), d-[6-3H(N)]glucosamine (25.9 Ci/mmol), guanosine 5′-diphosphate[1-3H]mannose, [5-3H]mevanolactone (20 Ci/mmol), UDP-[6-3H]N-acetylglucosamine (37.0 Ci/mmol), and En3hance spray were from PerkinElmer Life Sciences (Zaventem, Belgium). Dolichyl C95[1-3H]monophosphate (20 Ci/mmol), RS-[5-3H]mevalonolactone (50–60 Ci/mmol), and UDP-[1-3H]galactose (15–30 Ci/mmol) were from BIOTREND Chemikalien GmbH(Köln, Germany). UDP-[U-14C]glucose (348 mCi/mmol) was from Amersham International plc. TLC plates were from Merck (Darmstadt, Germany). Dowex resins, fucose, 2-acetamido-2-deoxy-1,3,4,6-tetra-O-acetyl-α-d-glucopyranose, endo-β-N-acetylglucosaminidase H (endo H) from Streptomyces plicatus, protease inhibitor cocktail, protease, Kodak X-Omat film, and alkaline phosphatase (ALP) were purchased from Sigma-Aldrich SARL (St. Quentin Fallavier, France). Castanospermine (Cst), kifunensin (Kif), and swainsonine (Sw) were from Toronto Research Chemicals Inc. (Toronto, Ontario, Canada). The antibodies used are anti-calnexin (CNX) (BD Biosciences, product number 610524) and anti-protein disulphide isomerase (PDI) (Cell Signaling Technology, product number 2446). NuPage LDS sample buffer was from Life Technologies (Cergy Pontoise, France). The Luminol ECL detection system was from Millipore. Solanesol- and citronellol-based DLO analog syntheses were performed according to literature procedures (16Bernardes G.J. Kikkeri R. Maglinao M. Laurino P. Collot M. Hong S.Y. Lepenies B. Seeberger P.H. Design, synthesis and biological evaluation of carbohydrate-functionalized cyclodextrins and liposomes for hepatocyte-specific targeting.Org. Biomol. Chem. 2010; 8: 4987-4996Crossref PubMed Scopus (73) Google Scholar, 17Busca P. Martin O.R. Synthesis of UDP-GalNAc analogues as probes for the study of polypeptide-alpha-GalNAc-transferases. Part 2.Tetrahedron Lett. 2004; 45: 4433-4436Crossref Scopus (12) Google Scholar, 18Fang X. Gibbs B.S. Coward J.K. Synthesis and evaluation of synthetic analogues of dolichyl-P-P-chitobiose as oligosaccharyltransferase substrates.Bioorg. Med. Chem. Lett. 1995; 5: 2701-2706Crossref Scopus (25) Google Scholar, 19Mathiselvam M. Srivastava A. Varghese B. Perez S. Loganathan D. Synthesis and X-ray crystallographic investigation of N-(beta-D-glycosyl)butanamides derived from GlcNAc and chitobiose as analogs of the conserved chitobiosylasparagine linkage of N-glycoproteins.Carbohydr. Res. 2013; 380: 37-44Crossref PubMed Scopus (4) Google Scholar, 20Scholte A.A. Vederas J.C. Incorporation of deuterium-labelled analogs of isopentenyl diphosphate for the elucidation of the stereochemistry of rubber biosynthesis.Org. Biomol. Chem. 2006; 4: 730-742Crossref PubMed Scopus (9) Google Scholar, 21Shpirt A.M. Kononov L.O. Maltsev S.D. Shibaev V.N. Chemical synthesis of polyprenyl sialyl phosphate, a probable biosynthetic intermediate of bacterial polysialic acid.Carbohydr. Res. 2011; 346: 2849-2854PubMed Google Scholar, 22Tai V.W. Imperiali B. Substrate specificity of the glycosyl donor for oligosaccharyl transferase.J. Org. Chem. 2001; 66: 6217-6228Crossref PubMed Scopus (43) Google Scholar, 23Zamyatina A. Gronow S. Puchberger M. Graziani A. Hofinger A. Kosma P. Efficient chemical synthesis of both anomers of ADP l-glycero- and d-glycero-d-manno-heptopyranose.Carbohydr. Res. 2003; 338: 2571-2589Crossref PubMed Scopus (53) Google Scholar) and will be described in detail in a separate study. HepG2 cells and the Thy−1, DPM1-deficient, mouse lymphoma cell line (24Chapman A. Trowbridge I.S. Hyman R. Kornfeld S. Structure of the lipid-linked oligosaccharides that accumulate in class E Thy-1-negative mutant lymphomas.Cell. 1979; 17: 509-515Abstract Full Text PDF PubMed Scopus (70) Google Scholar) were from ATCC (Rockville, MD) and were cultivated at 37°C under an atmosphere containing 5% CO2 in RPMI 1640 GlutamaxTM medium containing 10% FCS and 1% penicillin/streptomycin. For generation of Glc3-0[3H]Man9-8GlcNAc2-PP-dolichol, or Glc3-0Man9-8[3H]GlcNAc2-PP-dolichol, HepG2 cells were rinsed with RPMI 1640 medium containing 0.5 mM glucose, 2 mM fucose, and 2% dialyzed FCS and then incubated for 30 min in the same medium containing either 100 µCi/ml [2-3H]mannose or 100 µCi/ml [6-3H]glucosamine, respectively. For Glc3-0[3H]Man5GlcNAc2-PP-dolichol: 8 × 107 Thy−1 cells were harvested and then rinsed with glucose-free RPMI 1640 medium containing 0.5 mM glucose, 2.0 mM fucose, and 2% dialyzed FCS and then incubated in 1 ml of the same medium containing 100 µCi [2-3H]mannose for 30 min. Glc3-0Man9-8GlcNAc2-PP-[3H]dolichol was generated by cultivating HepG2 cells in normal growth medium containing 10 µM mevinolin and 100 µCi/ml [5-3H]mevanolactone for 47 h. These methods are based on previously described procedures (25Spiro M.J. Spiro R.G. Bhoyroo V.D. Lipid-saccharide intermediates in glycoprotein biosynthesis. I. Formation of an oligosaccharide-lipid by thyroid slices and evaluation of its role in protein glycosylation.J. Biol. Chem. 1976; 251: 6400-6408Abstract Full Text PDF PubMed Google Scholar, 26Anumula K.R. Spiro R.G. Release of glucose-containing polymannose oligosaccharides during glycoprotein biosynthesis. Studies with thyroid microsomal enzymes and slices.J. Biol. Chem. 1983; 258: 15274-15282Abstract Full Text PDF PubMed Google Scholar, 27Moore S.E. Spiro R.G. Intracellular compartmentalization and degradation of free polymannose oligosaccharides released during glycoprotein biosynthesis.J. Biol. Chem. 1994; 269: 12715-12721Abstract Full Text PDF PubMed Google Scholar). Washed cells were suspended in 4 ml of methanol/100 mM Tris HCl (pH 7.4) containing 4 mM MgCl2, 2:1. Four milliliters of CHCl3 was added, and the mixture shaken. After centrifugation, DLO was recovered from the lower CHCl3 phase from the CHCl3/methanol/water 10:10:3 extracts of the interphase proteins. These DLO preparations were kept at −20°C until required. Where indicated, oligosaccharides were released from DLO after mild acid hydrolysis with 0.02 N HCl for 30 min at 100°C. Seventy percent to 100% confluent HepG2 cell monolayers were rinsed and scraped into PBS. Pelleted cells were taken up in 10 mM Tris/HCl pH 7.4 containing protease inhibitors. After 20 min, cells were homogenized in the same buffer using a Dounce homogenizer, and postnuclear supernatants were then centrifuged at 100,000 gAv for 35 min. The pellets were taken up in 100 mM Tris/HCl pH 7.4 containing protease inhibitors, aliquoted, and frozen at −80°C. Livers from fasted C57BL6J mice were homogenized and fractionated according to a protocol devised for rat liver (28Fleischer S. Kervina M. Subcellular fractionation of rat liver.Methods Enzymol. 1974; 31: 6-41Crossref PubMed Scopus (362) Google Scholar). The mice were euthanized by cervical dislocation in accordance with the Debré-Bichat Ethics Committee on Animal Experimentation Protocol Number 2012-15/773-0091 and in accordance with French law on the protection of animals. Livers were removed, weighed, and transferred to ice-cold homogenization buffer (HB: 10 mM HEPES/NaOH pH 7.4 containing 250 mM sucrose, 1 mM EDTA, and protease inhibitors). The livers were cut into small pieces, rinsed with HB, and homogenized with 10 strokes in a Potter-Elvehjem homogenizer (800 rpm) with 4 vol of HB. In some experiments, a loose-fitting Dounce apparatus was used to homogenize the tissue. Homogenates were centrifuged at 750 gAv for 5 min, 2,500 gAv for 10 min, 12,500 gAv for 10 min, and 100,000 gAv for 45 min to give P1, P2, P3, and P4, respectively, and a final supernatant (S). Pellets and supernatant were aliquoted and frozen at −80°C. Mouse liver P4 was prepared as described above with exception that EDTA was omitted from the HB. Optiprep solutions were diluted into 10 mM HEPES/NaOH pH 7.40, containing 250 mM sucrose. Mouse liver P4 was centrifuged at 350,000 gAv for 90 min on the Optiprep gradient described in (29Plonne D. Cartwright I. Linss W. Dargel R. Graham J.M. Higgins J.A. Separation of the intracellular secretory compartment of rat liver and isolated rat hepatocytes in a single step using self-generating gradients of iodixanol.Anal. Biochem. 1999; 276: 88-96Crossref PubMed Scopus (24) Google Scholar) using a Beckman VTi 65.2 rotor. Protein and enzymes were detected using the following methods: protein BCA reagent (30Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Measurement of protein using bicinchoninic acid.Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18586) Google Scholar), lactate dehydrogenase (31Meister A. Reduction of alpha gamma-diketo and alpha-keto acids catalyzed by muscle preparations and by crystalline lactic dehydrogenase.J. Biol. Chem. 1950; 184: 117-129Abstract Full Text PDF PubMed Google Scholar), citrate synthase (32Srere P.A. Matsuoka Y. Inhibition of rat citrate synthase by acetoacetyl CoA and NADH.Biochem. Med. 1972; 6: 262-266Crossref PubMed Scopus (16) Google Scholar), phosphodiesterase I (33Razzell W.E. Tissue and intracellular distribution of two phosphodiesterases.J. Biol. Chem. 1961; 236: 3028-3030Abstract Full Text PDF PubMed Google Scholar), acid phosphatase (34Andersch M.A. Szczypinski A.J. Use of p-nitrophenylphosphate as the substrate in determination of serum acid phosphatase.Am. J. Clin. Pathol. 1947; 17: 571-574Crossref PubMed Scopus (258) Google Scholar), UDP-galactose glycoprotein galactosyltransferase (UGT) (35Wagner R.R. Cynkin M.A. Glycoprotein metabolism: a UDP-galactose-glycoprotein galactosyltransferase of rat serum.Biochem. Biophys. Res. Commun. 1971; 45: 57-62Crossref PubMed Scopus (26) Google Scholar), dolichyl-P-mannose synthase (DPMS) (36Richards J.B. Hemming F.W. The transfer of mannose from guanosine diphosphate mannose to dolichol phosphate and protein by pig liver endoplasmic reticulum.Biochem. J. 1972; 130: 77-93Crossref PubMed Scopus (88) Google Scholar), dolichyl-P-glucose synthase (DPGS) (37Chantret I. Dancourt J. Dupre T. Delenda C. Bucher S. Vuillaumier-Barrot S. Ogier de Baulny H. Peletan C. Danos O. Seta N. et al.A deficiency in dolichyl-P-glucose:Glc1Man9GlcNAc2-PP-dolichyl alpha3-glucosyltransferase defines a new subtype of congenital disorders of glycosylation.J. Biol. Chem. 2003; 278: 9962-9971Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), NADPH cytochrome c reductase (NCCR) (38Williams Jr., C.H. Kamin H. Microsomal triphosphopyridine nucleotide-cytochrome c reductase of liver.J. Biol. Chem. 1962; 237: 587-595Abstract Full Text PDF PubMed Google Scholar). UDP-GlcNAc-DolP-GlcNAc 1-P phosphotransferase (DPAGT) (39Lehrman M.A. Zhu X.Y. Khounlo S. Amplification and molecular cloning of the hamster tunicamycin-sensitive N-acetylglucosamine-1-phosphate transferase gene. The hamster and yeast enzymes share a common peptide sequence.J. Biol. Chem. 1988; 263: 19796-19803Abstract Full Text PDF PubMed Google Scholar). Samples prepared from density gradient fractions were diluted to the same protein concentration, heated with NuPAGE LDS sample buffer under reducing conditions according to the manufacturer's instructions, and subjected to SDS-PAGE; the primary antibodies were detected using horseradish-peroxidase-coupled secondary antibodies. Metabolically radiolabeled DLO (3 × 10−3 to 50 × 10−3 cpm) was dried down into 1.5 ml centrifuge tubes before being resuspended in 5 µl 1% NP-40. Further components were added to give final 50 µl reaction mixtures containing 100 mM MES, pH 5.5, 1 mM CoCl2, 0.1% NP-40, and various quantities of membrane protein that are indicated in the figure legends. Incubations were carried out at 37°C for various times, as indicated in the figure legends, and stopped by the addition of 150 µl ice-cold 10 mM MgCl2, 400 µl ice-cold methanol and 600 µl CHCl3 (25Spiro M.J. Spiro R.G. Bhoyroo V.D. Lipid-saccharide intermediates in glycoprotein biosynthesis. I. Formation of an oligosaccharide-lipid by thyroid slices and evaluation of its role in protein glycosylation.J. Biol. Chem. 1976; 251: 6400-6408Abstract Full Text PDF PubMed Google Scholar). After vigorous shaking, the phases were separated as described above, and after removal of organic solvent, the radioactivity associated with the CHCl3 phase was assayed by scintillation counting (cpm DLO). The upper phase was dried down under vacuum and loaded onto coupled 500 µl Dowex 50WX2 (H+ form) and Dowex 1 × 2 (acetate form) ion-exchange columns in water. The eluate and water washes containing neutral radioactive components were dried under vacuum and assayed by scintillation counting. The Dowex 1 × 2 (acetate form) columns were eluted with 5 ml 3.0 M formic acid, and after drying under vacuum, negatively charged components were assayed by scintillation counting (cpm formic acid). DLODP activity is defined as cpm formic acid/(cpm DLO + cpm formic acid). Percent inhibition of DLODP activity is defined as 100 − (100 × DLODP activity+inhibitor/DLODP activitycontrol). Where indicated, after desalting on Biogel P2 columns, charged oligosaccharides were fractionated on quaternary aminoethyl (QAE)-Sephadex columns (40Varki A. Kornfeld S. The spectrum of anionic oligosaccharides released by endo-beta-N-acetylglucosaminidase H from glycoproteins. Structural studies and interactions with the phosphomannosyl receptor.J. Biol. Chem. 1983; 258: 2808-2818Abstract Full Text PDF PubMed Google Scholar) as previously described (11Peric D. Durrant-Arico C. Delenda C. Dupre T. De Lonlay P. de Baulny H.O. Pelatan C. Bader-Meunier B. Danos O. Chantret I. et al.The compartmentalisation of phosphorylated free oligosaccharides in cells from a CDG Ig patient reveals a novel ER-to-cytosol translocation process.PLoS One. 2010; 5: e11675Crossref PubMed Scopus (21) Google Scholar). DLOs were chromatographed on 4 ml DE-52 cellulose (acetate form) columns (41Spiro M.J. Spiro R.G. Bhoyroo V.D. Glycosylation of proteins by oligosaccharide-lipids. Studies on a thyroid enzyme involved in oligosaccharide transfer and the role of glucose in this reaction.J. Biol. Chem. 1979; 254: 7668-7674Abstract Full Text PDF PubMed Google Scholar) and eluted with 6 vol of CHCl3/methanol/water (10:10:3), 6 vol of CHCl3/methanol/5 mM ammonium acetate (10:10:3), and finally, 6 vol of CHCl3/methanol/100 mM ammonium acetate (10:10:3). Neutral oligosaccharides were separated on silica-coated plastic sheets (0.2 mm thickness) developed in n-propanol-acetic acid-water (3:3:2) for 16–24 h (TLC system 1) (41Spiro M.J. Spiro R.G. Bhoyroo V.D. Glycosylation of proteins by oligosaccharide-lipids. Studies on a thyroid enzyme involved in oligosaccharide transfer and the role of glucose in this reaction.J. Biol. Chem. 1979; 254: 7668-7674Abstract Full Text PDF PubMed Google Scholar). Dolichol and DolP were resolved on prerun silica-coated aluminum sheets (0.2 mm thickness) developed in CHCl3/methanol/NH4OH (65:35:5) for 1 h (TLC system 2). DLOs were separated on prerun silica-coated aluminum sheets (0.2 mm thickness) in CHCl3/methanol/water (10:10:3) for 5 h (TLC system 3) (42Kelleher D.J. Karaoglu D. Gilmore R. Large-scale isolation of dolichol-linked oligosaccharides with homogeneous oligosaccharide structures: determination of steady-state dolichol-linked oligosaccharide compositions.Glycobiology. 2001; 11: 321-333Crossref PubMed Scopus (40) Google Scholar). Mono-, di-, tri-, and tetra-saccharides were separated on cellulose-coated plastic sheets developed in pyridine-ethyl acetate-water-acetic acid (5:5:3:1) for 16 h (TLC system 4). Radioactive components were detected on X-OMAT AR film by fluorography after spraying the dried TLC plates with En3hance and were quantitated by scintillation counting after their elution with water from the silica. Compounds containing terminal nonreducing GlcNAc residues were quantitated by transferring [3H]galactose from UDP-[3H]galactose using bovine milk galactosyltransferase as previously described (43Chantret I. Dancourt J. Barbat A. Moore S.E. Two proteins homologous to the N- and C-terminal domains of the bacterial glycosyltransferase Murg are required for the second step of dolichyl-linked oligosaccharide synthesis in Saccharomyces cerevisiae.J. Biol. Chem. 2005; 280: 9236-9242Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Cells from various mammalian sources contain a detergent- and Mn2+-requiring activity that generates OSP from DLO (12Vleugels W. Duvet S. Peanne R. Mir A.M. Cacan R. Michalski J.C. Matthijs G. Foulquier F. Identification of phosphorylated oligosaccharides in cells of patients with a congenital disorders of glycosylation (CDG-I).Biochimie. 2011; 93: 823-833Crossref PubMed Scopus (21) Google Scholar, 26Anumula K.R. Spiro R.G. Release of glucose-containing polymannose oligosaccharides during glycoprotein biosynthesis. Studies with thyroid microsomal enzymes and slices.J. Biol. Chem. 1983; 258: 15274-15282Abstract Full Text PDF PubMed Google Scholar, 44Baynes J.W. Hsu A.F. Heath E.C. The role of mannosyl-phosphoryl-dihydropolyisoprenol in the synthesis of mammalian glycoproteins.J. Biol. Chem. 1973; 248: 5693-5704Abstract Full Text PDF PubMed Google Scholar, 45Hsu A.F. Baynes J.W. Heath E.C. The role of a dolichol-oligosaccharide as an intermediate in glycoprotein biosynthesis.Proc. Natl. Acad. Sci. USA. 1974; 71: 2391-2395Crossref PubMed Scopus (103) Google Scholar). In preliminary experiments, we confirmed the presence of this activity in human hepatocellular carcinoma HepG2 cell total membrane preparations. In incubation mixtures containing 100 mM Tris HCl, pH 7.5, 0.5% w/v Triton X-100, and 1 mM MnCl2, this activity hydrolyzes both truncated (Man7GlcNAc2-PP-dolichol) and mature (Glc3-0[2-3H]Man9GlcNAc2-PP-dolichol) DLO (A. Massarweh and S.E.H. Moore, unpublished observations). DLO hydrolysis conditions were reevaluated because DLO hydrolysis required large amounts of protein and long incubation times. To do this, HepG2 membranes were incubated with Glc3-0[2-3H]Man9GlcNAc2-PP-dolichol in a reaction mixture containing 100 mM Tris HCl, pH 7.5, 0.5% w\v Triton X-100 and various cations at a final concentration of 10 mM. After stopping the reactions, solvent extractions were performed and radioactivity associated with DLO, protein, OSP, and nfOS plus other neutral sugar (nfOS/S) was quantitated (see Fig. 1A for workup scheme). When reaction mixtures are supplemented with Co2+, Mn2+, or Ni2+, DLO is lost with a concomitant increase in OSP (Fig. 1B). To optimize detergent and pH conditions for OSP generation, Co2+ was used to promote DLO cleavage. OSP generation requires detergent, and the three neutral detergent preparations Triton X-100, NP-40, and IGEPAL have similar capacities to promote this process (Fig. 1C). Further studies were carried out using 0.1% NP-40. The reaction has a pH optimum of 5.5 and is more efficient in HEPES, MOPS, and MES buffers than in Tris, pyridine, or propionate buffers (Fig. 1D). Finally, the divalent cation requirement for the reaction was reevaluated in the presence of 0.1% NP-40 and 100 mM MES buffer at both pH 5.5 and pH 7.5 (Fig. 1E). When the concentrations of Co2+ and Mn2+ were varied at pH 5.5, it was noted that even 10 mM Mn2+ possessed little capacity to provoke OSP generation, whereas 1 mM Co2+ was sufficient to elicit maximal activity. At pH 7.5, the maximal activation seen with Co2+ was lower than that noted at pH 5.5, but this cation appeared to be able to promote DLO cleavage at lower concentrations than at pH 5.5. As indicated in the legend to Fig. 1, the modifications made to the incubation conditions, from Fig. 1B to Fig. 1E, allowed reductions of both the amount of protein required and the assay incubation time. The standard conditions for further exploration of DLO cleavage were 0.1% NP-40, 100 mM MES, pH 5.5, and 1 mM Co2+. As shown in Fig. 2A, a time-dependent decrease in Glc3-0[2-3H]Man9GlcNAc2-PP-dolichol is accompanied by an increase of OSP. It was noted, though, that total recovery of radioactivity from the different fractions was low at the zero and early time points (Fig. 2A). Experiments revealed that this was
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