Deficiency in ethanolamine plasmalogen leads to altered cholesterol transport
2003; Elsevier BV; Volume: 44; Issue: 1 Linguagem: Inglês
10.1194/jlr.m200363-jlr200
ISSN1539-7262
AutoresNatalie J. Munn, Emily Arnio, Dailan Liu, Raphael A. Zoeller, Laura Liscum,
Tópico(s)Cholesterol and Lipid Metabolism
ResumoPlasmalogens are a major sub-class of ethanolamine and choline phospholipids in which the sn-1 position has a long chain fatty alcohol attached through a vinyl ether bond. These phospholipids are proposed to play a role in membrane fusion-mediated events. In this study, we investigated the role of the ethanolamine plasmalogen plasmenylethanolamine (PlsE11167) in intracellular cholesterol transport in Chinese hamster ovary cell mutants NRel-4 and NZel-1, which have single gene defects in PlsEtn biosynthesis. We found that PlsEtn was essential for specific cholesterol transport pathways, those from the cell surface or endocytic compartments to acyl-CoA/cholesterol acyltransferase in the endoplasmic reticulum. The movement of cholesterol from the endoplasmic reticulum or endocytic compartments to the cell surface was normal in PlsEtn-deficient cells. Also, vesicle trafficking was normal in PlsEtn-deficient cells, as measured by fluid phase endocytosis and exocytosis, as was the movement of newly-synthesized proteins to the cell surface. The mutant cholesterol transport phenotype was due to the lack of PlsEtn, since it was corrected when NRel-4 cells were transfected with a cDNA encoding the missing enzyme or supplied with a metabolic intermediate that enters the PlsEtn biosynthetic pathway downstream of the defect.Future work must determine the precise role that plasmalogens have on cholesterol transport to the endoplasmic reticulum. Plasmalogens are a major sub-class of ethanolamine and choline phospholipids in which the sn-1 position has a long chain fatty alcohol attached through a vinyl ether bond. These phospholipids are proposed to play a role in membrane fusion-mediated events. In this study, we investigated the role of the ethanolamine plasmalogen plasmenylethanolamine (PlsE11167) in intracellular cholesterol transport in Chinese hamster ovary cell mutants NRel-4 and NZel-1, which have single gene defects in PlsEtn biosynthesis. We found that PlsEtn was essential for specific cholesterol transport pathways, those from the cell surface or endocytic compartments to acyl-CoA/cholesterol acyltransferase in the endoplasmic reticulum. The movement of cholesterol from the endoplasmic reticulum or endocytic compartments to the cell surface was normal in PlsEtn-deficient cells. Also, vesicle trafficking was normal in PlsEtn-deficient cells, as measured by fluid phase endocytosis and exocytosis, as was the movement of newly-synthesized proteins to the cell surface. The mutant cholesterol transport phenotype was due to the lack of PlsEtn, since it was corrected when NRel-4 cells were transfected with a cDNA encoding the missing enzyme or supplied with a metabolic intermediate that enters the PlsEtn biosynthetic pathway downstream of the defect. Future work must determine the precise role that plasmalogens have on cholesterol transport to the endoplasmic reticulum. Plasmalogens are a major class of ethanolamine and choline phospholipids in which the sn-1 position of the glycerol backbone has a long chain fatty alcohol attached through a vinyl ether bond (1Nagan N. Zoeller R.A. Plasmalogens: biosynthesis and functions.Prog. Lipid Res. 2001; 40: 199-229Google Scholar). They constitute 18% of total phospholipid mass in humans, with the highest amounts in heart, striated muscle, and nervous tissue (2Horrocks L.A. Sharma M. Plasmalogens and O-alkyl glycerophospholipids.in: Hawthorne N. Ansell G.B. Phospholipids J. Elsevier, Amsterdam.1982: 51-92Google Scholar). In the Chinese hamster ovary (CHO) cell line, 11% of the total phospholipids are plasmalogens, primarily ethanolamine plasmalogen, called plasmenylethanolamine (PlsEtn) (3Nagan N. Hajra A.K. Larkins L.K. Lazarow P. Purdue P.E. Rizzo W.B. Zoeller R.A. Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol.Biochem. J. 1998; 332: 273-279Google Scholar). CHO cells do not contain plasmenylcholine (4Zoeller R.A. Raetz C.R. Strategies for isolating somatic cell mutants defective in lipid biosynthesis.Methods Enzymol. 1992; 209: 34-51Google Scholar). The cellular distribution of plasmalogens has not been thoroughly characterized, although they are reportedly present in plasma membrane (5Koizumi K. Shimizu S. Koizumi K.T. Nishida K. Sato C. Ota K. Yamanaka N. Rapid isolation and lipid characterization of plasma membranes from normal and malignant lymphoid cells of mouse.Biochim. Biophys. Acta. 1981; 649: 393-403Google Scholar), synaptic vesicles (6Breckenridge W.C. Morgan I.G. Zanetta J.P. Vincendon G. Adult rat brain synaptic vesicles. II. Lipid composition.Biochim. Biophys. Acta. 1973; 320: 681-686Google Scholar), and secretory granules (7Bjerrum O.W. Nielsen H. Borregaard N. Quantitative analysis of phospholipids and demonstration of plasmalogen in human neutrophil subcellular fractions by high-performance liquid chromatography.Scand. J. Clin. Lab. Invest. 1989; 49: 613-622Google Scholar). In erythrocytes, plasmalogens are found to be oriented with the same asymmetric distribution as their diacyl analogs (8Fellmann P. Herve P. Devaux P.F. Transmembrane distribution and translocation of spin-labeled plasmalogens in human red blood cells.Chem. Phys. Lipids. 1993; 66: 225-230Google Scholar). For such an abundant phospholipid, plasmalogen functions are quite obscure. Current in vitro models suggest that PlsEtn plays a role in membrane fusion-mediated events (9Lohner K. Is the high propensity of ethanolamine plasmalogens to form non-lamellar lipid structures manifested in the properties of biomembranes?.Chem. Phys. Lipids. 1996; 81: 167-184Google Scholar) and protects against reactive oxygen species (10Zoeller R.A. Lake A.C. Nagan N. Gaposchkin D.P. Legner M.A. Lieberthal W. Plasmalogens as endogenous antioxidants: somatic cell mutants reveal the importance of the vinyl ether.Biochem. J. 1999; 338: 769-776Google Scholar). In this study, we investigated the role of PlsEtn in cholesterol metabolism, prompted by the finding that cholesterol efflux to HDL is aberrant in plasmalogen-deficient RAW mutant macrophages (11Mandel H. Sharf R. Berant M. Wanders R.J. Vreken P. Aviram M. Plasmalogen phospholipids are involved in HDL-mediated cholesterol efflux: insights from investigations with plasmalogen-deficient cells.Biochem. Biophys. Res. Commun. 1998; 250: 369-373Google Scholar). The lack of plasmalogens did not alter the rate of cellular cholesterol transfer to HDL; instead, it appeared to reduce the pool of cholesterol available for efflux. Another study showed that levels of PlsEtn are reduced in brain tissue from the Niemann-Pick C (NPC) mouse model (12Schedin S. Sindelar P.J. Pentchev P. Brunk U. Dallner G. Peroxisomal impairment in Niemann-Pick type C disease.J. Biol. Chem. 1997; 272: 6245-6251Google Scholar). NPC cells exhibit lysosomal storage of cholesterol, gangliosides, and other lipids, as well as aberrant cholesterol homeostatic responses (13Liscum L. Niemann-Pick type C mutations cause lipid traffic jam.Traffic. 2000; 1: 218-225Google Scholar). If plasmalogens affect the cellular cholesterol distribution, then reduced plasmalogen levels might exacerbate the cholesterol transport defective phenotype of NPC and contribute to disease progression. Our model systems were CHO cell mutants NRel-4 and NZel-1, which display greatly reduced levels of PlsEtn due to defects in different steps of plasmalogen biosynthesis (3Nagan N. Hajra A.K. Larkins L.K. Lazarow P. Purdue P.E. Rizzo W.B. Zoeller R.A. Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol.Biochem. J. 1998; 332: 273-279Google Scholar, 14Nagan N. Hajra A.K. Das A.K. Moser H.W. Moser A. Lazarow P. Purdue P.E. Zoeller R.A. A fibroblast cell line defective in alkyl-dihydroxyacetone phosphate synthase: a novel defect in plasmalogen biosynthesis.Proc. Natl. Acad. Sci. USA. 1997; 94: 4475-4480Google Scholar). NRel-4 has a defect in dihydroxyacetonephosphate acyltransferase (DHAPAT) (3Nagan N. Hajra A.K. Larkins L.K. Lazarow P. Purdue P.E. Rizzo W.B. Zoeller R.A. Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol.Biochem. J. 1998; 332: 273-279Google Scholar), the enzyme that catalyzes the first step in plasmalogen biosynthesis. NZel-1 has a defect in alkyl-dihydroxyacetone phosphate (DHAP) synthase (14Nagan N. Hajra A.K. Das A.K. Moser H.W. Moser A. Lazarow P. Purdue P.E. Zoeller R.A. A fibroblast cell line defective in alkyl-dihydroxyacetone phosphate synthase: a novel defect in plasmalogen biosynthesis.Proc. Natl. Acad. Sci. USA. 1997; 94: 4475-4480Google Scholar), which catalyzes the second step in plasmalogen biosynthesis. Our analysis revealed that PlsEtn is essential for specific cholesterol transport pathways. In PlsEtn deficient cells, the movement of cholesterol from endocytic compartments or the plasma membrane to ACAT in the endoplasmic reticulum (ER) was deficient. Cholesterol movement to the plasma membrane appeared to be normal, as did vesicular protein trafficking. Defective cholesterol transport pathways were restored in NRel-4 cells transfected with a cDNA encoding the missing enzyme, DHAPAT, or supplied with an intermediate that enters the PlsEtn biosynthetic pathway downstream of the metabolic defect. These data support a role for PlsEtn in specific cholesterol transport pathways, those from the cell surface to the cell interior. [9,10-3H]oleic acid (5 Ci/mmol), [1-14C]oleoyl-CoA (59 mCi/mmol), [1,2-3H]cholesterol (45 Ci/mmol), [1,2,6,7-3H]cholesteryl linoleate (75 Ci/mmol), [1,2,6,7-3H]cholesteryl oleate (78 Ci/mmol), cholesteryl [1-14C]oleate (57 mCi/mmol), [14C(U)]sucrose (600 mCi/mmol), and EasyTag Express 35S Protein Labeling Mix (1175 Ci/mmol) were obtained from NEN Life Science Products. Sodium 125I (103 mCi/ml) was from Amersham Biosciences. Tissue culture reagents were from Life Technologies, Inc. or Sigma. Lipids were obtained from Sigma or Steraloids. Mevinolin was a generous gift of Merck Research Laboratory. Other chemicals were from Sigma unless otherwise indicated. Compounds were dissolved as follows: 25-hydroxycholesterol in ethanol, cholesterol in ethanol, and amphotericin B in dimethyl sulfoxide. LDL was prepared by ultracentrifugation (15Goldstein J.L. Basu S.K. Brown M.S. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells.Methods Enzymol. 1983; 98: 241-260Google Scholar). LDL labeled with [3H]cholesteryl linoleate ([3H]CL-LDL) was prepared with an average specific activity of 21,000 cpm/nmol of total cholesteryl linoleate (16Faust J.R. Goldstein J.L. Brown M.S. Receptor-mediated uptake of low density lipoprotein and utilization of its cholesterol for steroid synthesis in cultured mouse adrenal cells.J. Biol. Chem. 1977; 252: 4861-4871Google Scholar). LDL labeled with [3H]cholesteryl oleate ([3H]CO-LDL) was prepared with an average specific activity of 38,000 dpm/nmol of total cholesteryl ester (16Faust J.R. Goldstein J.L. Brown M.S. Receptor-mediated uptake of low density lipoprotein and utilization of its cholesterol for steroid synthesis in cultured mouse adrenal cells.J. Biol. Chem. 1977; 252: 4861-4871Google Scholar). 125I-LDL was prepared by the iodine monochloride method (15Goldstein J.L. Basu S.K. Brown M.S. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells.Methods Enzymol. 1983; 98: 241-260Google Scholar). Lipoprotein-deficient serum (LPDS) was prepared as described, omitting the thrombin incubation (15Goldstein J.L. Basu S.K. Brown M.S. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells.Methods Enzymol. 1983; 98: 241-260Google Scholar). The following media were prepared: H-5% newborn calf serum (NCS) and H-10% NCS (Ham's F-12 medium containing 5% or 10% (v/v) newborn calf serum, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 20 mM HEPES, pH 7.1); H-5% LPDS and H-1% LPDS [H-5% NCS in which 5% (v/v) newborn calf serum was replaced with 5 or 1% (v/v) lipoprotein-deficient calf serum, respectively]; H-5% LPDS/mev (H-5% LPDS containing 20 μM mevinolin and 0.5 mM mevalonate). The following buffers were prepared: TBS (50 mM Tris-Cl and 155 mM NaCl, pH 7.4) and PBS (1.5 mM KH2HPO4, 2.7 mM KCl, and 137 mM NaCl, pH 7.3). All cells were grown as monolayers in a humidified incubator (5% CO2) at 37°C in H-5% NCS. The mutant cell lines, NRel-4 (3Nagan N. Hajra A.K. Larkins L.K. Lazarow P. Purdue P.E. Rizzo W.B. Zoeller R.A. Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol.Biochem. J. 1998; 332: 273-279Google Scholar) and NZel-1 (14Nagan N. Hajra A.K. Das A.K. Moser H.W. Moser A. Lazarow P. Purdue P.E. Zoeller R.A. A fibroblast cell line defective in alkyl-dihydroxyacetone phosphate synthase: a novel defect in plasmalogen biosynthesis.Proc. Natl. Acad. Sci. USA. 1997; 94: 4475-4480Google Scholar), were isolated as described previously. NRel-4 and NZel-1 cells display deficiencies in DHAPAT and alkyl-DHAP synthase, respectively. Both show a 90% loss in PlsEtn levels compared with wild-type cells. NRel-4.15 is a clonal isolate, stably expressing human DHAPAT cDNA (kindly provided by Wilhelm Just, Univ. Heidelberg, and directionally inserted into the pBK-CMV expression vector; Stratagene) and containing wild-type plasmalogen levels. NRel-4.CMV has been transfected with vector alone and is plasmalogen-deficient (Zoeller et al., unpublished observations). CHO mutant 2-2 was isolated by Dahl et al. (17Dahl N.K. Reed K.L. Daunais M.A. Faust J.R. Liscum L. Isolation and characterization of Chinese hamster ovary cells defective in the intracellular metabolism of LDL-derived cholesterol.J. Biol. Chem. 1992; 267: 4889-4896Google Scholar) and has the NPC phenotype. On day 0 of each experiment, monolayer stock flasks of CHO-K1 and mutant cells were trypsinized, and cells were seeded as indicated in the individual experiment. On day 0, cells were seeded into 6-well plates (20,000 cells/well) in H-5% NCS. On day 2, cells were fed H-5% NCS. On day 3, cells were washed with Hank's balanced salt solution (HBSS) and fed H-5% LPDS. Experiments were conducted on day 4. Following additions of LDL or 25-hydroxycholesterol, monolayers were pulsed with 100 μM [3H]oleate (prepared with a specific activity of 8,700 cpm/nmol) bound to albumin (15Goldstein J.L. Basu S.K. Brown M.S. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells.Methods Enzymol. 1983; 98: 241-260Google Scholar). After 2 h of [3H]oleate incubation, cells were washed with TBS, lipids were extracted with hexane-isopropanol (3:2, v/v), and cholesterol [3H]oleate was isolated and quantified (18Liscum L. Faust J.R. The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in Chinese hamster ovary cells cultured with 3-b-[2-(diethylamino)ethoxy]androst-5-en-17-one.J. Biol. Chem. 1989; 264: 11796-11806Google Scholar). After lipid extraction, the monolayers were dissolved in 0.1 N NaOH and aliquots removed for protein determination (19Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Google Scholar) using BSA as a standard. Cholesterol esterification is defined as nmol [3H]oleate incorporated into cholesteryl [3H]oleate/h/mg protein. On day 0, cells were seeded in 100-mm dishes (300–500,000 cells/dish) in H-5% NCS. Cells were cultured until 80% confluent, with media changes every 2 days. On the day of assay, cells were refed 10 ml/dish H-5% NCS. After 2 h, cells were washed with PBS, subjected to hypotonic shock, and scraped as described (20Chang T.Y. Limanek J.S. Chang C.C. A simple and efficient procedure for the rapid homogenization of cultured animal cells grown in monolayer.Anal. Biochem. 1981; 116: 298-302Google Scholar). Aliquots of 150 μl with ∼200–250 μg cell protein were used per assay reaction. The reaction was initiated by the addition of 20 μl of [14C]oleoyl-CoA (15 nmol; 30,000 dpm/nmol) containing 300 μg of BSA in 125 mM Tris-HCL, pH 7.8. Assays were carried out at 37°C for 3–15 min. Lipids were extracted and analyzed by TLC on silica gel 60 plates as described (21Chang C.C.Y. Doolittle G.M. Chang T.Y. Cycloheximide sensitivity in regulation of acyl coenzyme A:cholesterol acyltransferase activity in chinese hamster ovary cells. 1. Effect of exogenous sterols.Biochemistry. 1986; 25: 1693-1699Google Scholar). On day 0, cells were seeded into 6-well plates (25,000 cells/well) in H-5% NCS. On day 2, cells were fed H-5% NCS. On day 3, cells were washed in HBSS and fed H-5% LPDS. On day 4, cells were incubated for 6 h with either 125I-LDL or [3H]CL-LDL. The uptake and proteolytic degradation of 125I-LDL was measured by incubating cells with 20 μg/ml 125I-LDL in the absence and presence of 500 μg/ml of unlabeled LDL. After 6 h, the release of 125I-monoiodotyrosine was quantified as described (15Goldstein J.L. Basu S.K. Brown M.S. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells.Methods Enzymol. 1983; 98: 241-260Google Scholar). Specific degradation was calculated by subtracting the value obtained in the presence of unlabeled LDL from that obtained in its absence. The uptake and lysosomal hydrolysis of [3H]CL-LDL was measured by quantifying cell-associated [3H]cholesteryl linoleate and [3H]cholesterol as described previously (22Underwood K.W. Jacobs N.L. Howley A. Liscum L. Evidence for a cholesterol transport pathway from lysosomes to endoplasmic reticulum that is independent of the plasma membrane.J. Biol. Chem. 1998; 273: 4266-4274Google Scholar). The amount of LDL-derived [3H]cholesterol that was re-esterified by ACAT to form cholesteryl [3H]oleate was also quantified as described previously (22Underwood K.W. Jacobs N.L. Howley A. Liscum L. Evidence for a cholesterol transport pathway from lysosomes to endoplasmic reticulum that is independent of the plasma membrane.J. Biol. Chem. 1998; 273: 4266-4274Google Scholar). On day 0, cells were seeded in 4-well Falcon 4104 chamber slides (1,500 cells/well) in H-5% NCS. On day 2, cells were refed. On day 3, cells were washed with PBS and processed for filipin fluorescence microscopy as described (23Jacobs N.L. Andemariam B. Underwood K.W. Panchalingam K. Sternberg D. Kielian M. Liscum L. Analysis of a Chinese hamster ovary cell mutant with defective mobilization of cholesterol from the plasma membrane to the endoplasmic reticulum.J. Lipid Res. 1997; 38: 1973-1987Google Scholar). On day 0, cells were seeded into 6-well plates (25,000 cells/well) in H-5% NCS. On day 2, cells were washed in HBSS and fed H-5% LPDS. On day 3, cells were incubated in H-5% LPDS containing 20 μg/ml [3H]CL-LDL. After 2 h, cells were treated with cholesterol oxidase, and the conversion of [3H]cholesterol to [3H]cholestenone was quantified as described (24Porn M.I. Slotte J.P. Reversible effects of sphingomyelin degradation on cholesterol distribution and metabolism in fibroblasts and transformed neuroblastoma cells.Biochem. J. 1990; 271: 121-126Google Scholar). On day 0, cells were seeded in 96-well plates (15,000 cells/well) in H-5% NCS. On day 1, cells were fed H-5% LPDS. On day 2, cells were fed H-5% LPDS/mev. After 8 h, cells were fed H-5% LPDS/mev with indicated additions of LDL. On day 3, cells were incubated 5 h in H-1% LPDS with or without 100 μg/ml amphotericin B. After 5 h, cells were washed with HBSS. Cell viability was assessed using a colorimetric 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as described (22Underwood K.W. Jacobs N.L. Howley A. Liscum L. Evidence for a cholesterol transport pathway from lysosomes to endoplasmic reticulum that is independent of the plasma membrane.J. Biol. Chem. 1998; 273: 4266-4274Google Scholar). Cell survival is defined as the mean MTT cleaved (A560) per well from three wells treated with amphotericin B expressed as a percentage of the mean MTT cleaved (A560) per well from three wells not treated with amphotericin B. On day 0, cells were seeded into 6-well plates (20,000 cells/well) in H-5% NCS. On day 2, cells were washed with HBSS and fed H-5% LPDS. Additions of 1.3 μCi/ml [3H]cholesterol in ethanol were made at staggered times. On day 4, cells were washed with TBS. Lipids were extracted, separated, and quantified as described (22Underwood K.W. Jacobs N.L. Howley A. Liscum L. Evidence for a cholesterol transport pathway from lysosomes to endoplasmic reticulum that is independent of the plasma membrane.J. Biol. Chem. 1998; 273: 4266-4274Google Scholar). On day 0, cells were seeded in 6-well plates (25,000 cells/well) in H-5% NCS. On day 1, cells were washed with HBSS and fed H-5% LPDS. On day 3, cells were fed H-5% LPDS + 2% 2-hydroxypropyl-β-cyclodextrin. Additions of 60 μCi/ml [3H]acetate were made at staggered times. At time of harvest, the media were removed and centrifuged. The supernatant was extracted with petroleum ether, and the upper phase containing the [3H]cholesterol was removed and evaporated to dryness. Cell monolayers were washed once quickly, 2 × 7 min with TBS-BSA, and twice quickly with TBS. Cellular lipids were extracted using hexane-isopropyl alcohol (3:2, v/v). Monolayers were dissolved in 0.1 N NaOH, and aliquots were taken for protein determination. [3H]cholesterol was isolated by TLC using heptane-ethyl ether (90:60, v/v). The endogenously labeled [3H]sterols cochromatograph with authentic cholesterol; however, they can be resolved by HPLC into [3H]cholesterol (70%) and [3H]desmosterol (30%). They are referred to as [3H]cholesterol. On day 0, cells were seeded in 6-well plates (25,000 cells/well) in H-5% NCS. On day 1, cells were washed with HBSS and fed H-5% LPDS. On day 3, cells were fed H-5% LPDS and additions of 60 μCi/ml [3H]acetate were made at staggered times. At time of harvest, cells were treated with cholesterol oxidase, and the conversion of [3H]cholesterol to [3H]cholestenone was quantified as described (24Porn M.I. Slotte J.P. Reversible effects of sphingomyelin degradation on cholesterol distribution and metabolism in fibroblasts and transformed neuroblastoma cells.Biochem. J. 1990; 271: 121-126Google Scholar). On day 0, cells were seeded into 100-mm dishes (100,000 cells/dish) in H-5% NCS. On day 2, cells were washed with HBSS and fed H-5% NCS or H-5% LPDS. On day 4, cells were washed with TBS. Lipids were extracted and quantified as previously described (23Jacobs N.L. Andemariam B. Underwood K.W. Panchalingam K. Sternberg D. Kielian M. Liscum L. Analysis of a Chinese hamster ovary cell mutant with defective mobilization of cholesterol from the plasma membrane to the endoplasmic reticulum.J. Lipid Res. 1997; 38: 1973-1987Google Scholar), except that each sample consisted of one 100-mm dish. Gas chromatography was conducted isothermally using a DB-17 capillary column (15 m × 0.53 mm, Alltech) at 245°C. On day 0, cells were seeded in 6-well plates (50,000 cells/well) in H-5% NCS. On day 1, cells were refed 1 ml of Dulbecco's Modified Eagle Medium (no methionine, no cysteine) containing 10 mM ammonium chloride and 50 μCi EasyTag Express 35S. At staggered times, the media were removed and centrifuged, and subjected to TCA precipitation. The pellets were dissolved in 0.5 N NaOH and subjected to liquid scintillation counting. On day 0, cells were seeded in 24-well plates (12,000 cells/well) in H-5% NCS. On day 1, cells were refed H-10% NCS with and without 20 μM 1-monopalmityl glyceryl ether (Doosan Serdary Research Laboratories). This compound is readily taken into cells, entering the pathway downstream of DHAPAT and alkyl-DHAP synthase. Incubation with this substrate over a period of 2–3 days restores PlsEtn levels to normal in NRel-4 cells (3Nagan N. Hajra A.K. Larkins L.K. Lazarow P. Purdue P.E. Rizzo W.B. Zoeller R.A. Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol.Biochem. J. 1998; 332: 273-279Google Scholar). On day 4, cells were refed 200 μl of Dulbecco's Modified Eagle Medium (no methionine, no cysteine) after which staggered additions of EasyTag Express 35S were made (50 μCi/well). At time of harvest, the media were removed and centrifuged. Aliquots were subjected to 12% SDS-PAGE, after which gels were fixed, impregnated with Autofluor (National Diagnostics), and exposed to film. Densitometric analysis was performed using an AlphaImager 2200. To measure endocytosis, on day 0, cells were seeded into 6-well dishes (50,000 cells/dish) in H-5% NCS. On day 1, cells were refed H-10% NCS with and without 20 μM 1-monopalmityl glyceryl ether. On day 4, [14C]sucrose (260 μCi) was dried under nitrogen at 37°C and dissolved in 260 μl water. Cells were refed 1 ml of H-5% NCS after which staggered additions of 10 μCi [14C]sucrose were made. At time of harvest, cells were washed with TBS containing 2 mg/ml BSA (once quickly, then six times, 3 min each), then with TBS (once quickly, then 5 min). Cells were solubilized with 1 ml 1% SDS for 30 min. Aliquots of the cell extract were subjected to liquid scintillation counting in 10 ml of ReadySafe and protein determination (19Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Google Scholar). To measure clearance of endocytosed [14C]sucrose, on day 0, cells were seeded in 6-well plates (25,000 cells/well) in H-5% NCS. On day 2, cells were refed H-5% NCS. [14C]sucrose was dried under nitrogen at 37°C, dissolved in water, added to H-5% NCS, and stored at 4°C overnight. On day 3, cells were refed 0.5 ml H-5% NCS containing 70–200 μCi [14C]sucrose. After 3 h, cells were washed six times with H-5% NCS and refed 1 ml H-5% NCS. At the indicated time, the media were removed and subjected to centrifugation (15,000 g, 5 min). Cells were washed with TBS containing 2 mg/ml BSA (once quickly, then 5 min), then with TBS (once quickly, then 5 min). Cells were solubilized with 1 ml 1% SDS for 30 min. Aliquots of media and cell extract were subjected to liquid scintillation counting in 10 ml of ReadySafe. Plasmalogen synthesis begins in peroxisomes, where DHAPAT catalyzes the formation of acyl-DHAP (25Lee T.C. Biosynthesis and possible biological functions of plasmalogens.Biochim. Biophys. Acta. 1998; 1394: 129-145Google Scholar) (Fig. 1). Alkyl-DHAP synthase catalyzes the second step in the pathway, forming the ether bond to produce alkyl-DHAP. Plasmalogen synthesis then continues in the ER. NRel-4 and NZel-1 cells are independently isolated CHO cell mutants defective in DHAPAT and alkyl-DHAP synthase, respectively (3Nagan N. Hajra A.K. Larkins L.K. Lazarow P. Purdue P.E. Rizzo W.B. Zoeller R.A. Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol.Biochem. J. 1998; 332: 273-279Google Scholar, 14Nagan N. Hajra A.K. Das A.K. Moser H.W. Moser A. Lazarow P. Purdue P.E. Zoeller R.A. A fibroblast cell line defective in alkyl-dihydroxyacetone phosphate synthase: a novel defect in plasmalogen biosynthesis.Proc. Natl. Acad. Sci. USA. 1997; 94: 4475-4480Google Scholar). Unlike other plasmalogen-deficient mutants (26Zoeller R.A. Raetz C.R. Isolation of animal cell mutants deficient in plasmalogen biosynthesis and peroxisome assembly.Proc. Natl. Acad. Sci. USA. 1986; 83: 5170-5174Google Scholar, 27Fujiki Y. Molecular defects in genetic diseases of peroxisomes.Biochim. Biophys. Acta. 1997; 1361: 235-250Google Scholar), NRel-4 and NZel-1 have single enzyme defects and intact functional peroxisomes (3Nagan N. Hajra A.K. Larkins L.K. Lazarow P. Purdue P.E. Rizzo W.B. Zoeller R.A. Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol.Biochem. J. 1998; 332: 273-279Google Scholar, 14Nagan N. Hajra A.K. Das A.K. Moser H.W. Moser A. Lazarow P. Purdue P.E. Zoeller R.A. A fibroblast cell line defective in alkyl-dihydroxyacetone phosphate synthase: a novel defect in plasmalogen biosynthesis.Proc. Natl. Acad. Sci. USA. 1997; 94: 4475-4480Google Scholar). These cells make excellent model systems in which to study the relationship between plasmalogen and cholesterol metabolism. We were interested in investigating this relationship because of the apparent effect of plasmalogens on the pool of cellular cholesterol available for efflux to HDL (11Mandel H. Sharf R. Berant M. Wanders R.J. Vreken P. Aviram M. Plasmalogen phospholipids are involved in HDL-mediated cholesterol efflux: insights from investigations with plasmalogen-deficient cells.Biochem. Biophys. Res. Commun. 1998; 250: 369-373Google Scholar). If plasmalogens play a role in membrane fission and fusion (9Lohner K. Is the high propensity of ethanolamine plasmalogens to form non-lamellar lipid structures manifested in the properties of biomembranes?.Chem. Phys. Lipids. 1996; 81: 167-184Google Scholar), a PlsEtn deficiency may alter cholesterol transport pathways throughout the cell. ACAT is a resident ER enzyme (28Chang C.C.Y. Chen J. Thomas M.A. Cheng D. Priore V.A.D. Newton R.S. Pape M.E. Chang T-Y. Regulation and immunolocalization of acyl-coenzyme A:cholesterol acyltransferase in mammalian cells as studied with specific antibodies.J. Biol. Chem. 1995; 49: 29532-29540Google Scholar) that catalyzes cholestero
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