FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2016757701> ?p ?o ?g. }

• W2016757701 endingPage "877" @default.
• W2016757701 startingPage "866" @default.
• W2016757701 abstract "Glycerolipids are structural components for membranes and serve in energy storage. We describe here the use of a photodynamic selection technique to generate a population of Chinese hamster ovary cells that display a global deficiency in glycerolipid biosynthesis. One isolate from this population, GroD1, displayed a profound reduction in the synthesis of phosphatidylcholine, phosphatidylethanolamine, and triglycerides but presented high levels of phosphatidic acid and normal levels of phosphatidylinositol synthesis. This was accompanied by a reduction in phosphatidate phosphatase 1 (PAP1) activity. Expression cloning and sequencing of the cDNA obtained from GroD1 revealed a point mutation, Gly-189 → Glu, in glucose-6-phosphate isomerase (GPI), a glycolytic enzyme involved in an inherited disorder that results in anemia and neuromuscular symptoms in humans. GPI activity was reduced by 87% in GroD1. No significant differences were found in DNA synthesis, protein synthesis, and ATP levels, whereas glycerol 3-phosphate levels were increased in the mutant. Expression of wild-type hamster GPI restored GPI activity, glycerolipid biosynthesis, and PAP1 activity in GroD1. Two additional, independently isolated GPI-deficient mutants displayed similar phenotypes with respect to PAP1 activity and glycerolipid biosynthesis. These findings uncover a novel relationship between GPI, involved in carbohydrate metabolism, and PAP1, a lipogenic enzyme. These results may also help to explain neuromuscular symptoms associated with inherited GPI deficiency. Glycerolipids are structural components for membranes and serve in energy storage. We describe here the use of a photodynamic selection technique to generate a population of Chinese hamster ovary cells that display a global deficiency in glycerolipid biosynthesis. One isolate from this population, GroD1, displayed a profound reduction in the synthesis of phosphatidylcholine, phosphatidylethanolamine, and triglycerides but presented high levels of phosphatidic acid and normal levels of phosphatidylinositol synthesis. This was accompanied by a reduction in phosphatidate phosphatase 1 (PAP1) activity. Expression cloning and sequencing of the cDNA obtained from GroD1 revealed a point mutation, Gly-189 → Glu, in glucose-6-phosphate isomerase (GPI), a glycolytic enzyme involved in an inherited disorder that results in anemia and neuromuscular symptoms in humans. GPI activity was reduced by 87% in GroD1. No significant differences were found in DNA synthesis, protein synthesis, and ATP levels, whereas glycerol 3-phosphate levels were increased in the mutant. Expression of wild-type hamster GPI restored GPI activity, glycerolipid biosynthesis, and PAP1 activity in GroD1. Two additional, independently isolated GPI-deficient mutants displayed similar phenotypes with respect to PAP1 activity and glycerolipid biosynthesis. These findings uncover a novel relationship between GPI, involved in carbohydrate metabolism, and PAP1, a lipogenic enzyme. These results may also help to explain neuromuscular symptoms associated with inherited GPI deficiency. The production of glycerolipids, including glycerophospholipids and triacylglycerols, is important for the supply of structural components for membrane production and the storage of excess calories in animal cells. This process must be regulated to adjust to the demands of the cell and the environmental conditions. For example, dividing cells require a constant supply of glycerophospholipids for membrane production, whereas diets rich in carbohydrates result in increased production of fatty acids, which are stored as triglycerides. Dysregulation of glycerolipid biosynthesis results in a number of pathologies, including diabetes and obesity (1Mittra S. Bansal V.S. Bhatnagar P.K. Drug Discov. Today. 2008; 13: 211-218Crossref PubMed Scopus (33) Google Scholar, 2Cheung O. Sanyal A.J. Semin. Liver Dis. 2008; 28: 351-359Crossref PubMed Scopus (85) Google Scholar). Metabolic syndromes have become a prominent health issue in the United States as well as in other developed countries (3Ford E.S. Giles W.H. Dietz W.H. JAMA. 2002; 287: 356-359Crossref PubMed Scopus (5684) Google Scholar, 4Grundy S.M. Arterioscler. Thromb. Vasc. Biol. 2008; 28: 629-636Crossref PubMed Scopus (1047) Google Scholar). The mechanism by which glycerolipid biosynthesis is regulated is still not well defined. This is due, in part, to the fact that, until recently, few of the biosynthetic enzymes had been purified to homogeneity and many of the genes that code for these proteins had yet to be identified. Besides the enzymes directly involved in assembling glycerolipids, there are undoubtedly many genes and gene products involved in controlling glycerolipid biosynthesis. Forward genetics (mutant isolation) is used to identify genes required for a certain phenotype. This way, one can identify factors important for that phenotype, which may not have been predicted given the existing knowledge about the biochemistry in that area. A number of animal cell mutants have been isolated using screening or selection techniques that target cell lines deficient in the synthesis of specific phospholipid species such as phosphatidylcholine (PC), 2The abbreviations used are: PCphosphatidylcholineP121-O-[12-(1′pyrene)]dodecanoic acidCHOChinese hamster ovaryPAP1phosphatidate phosphatase 1GPIglucose-6-phosphate isomerasePEphosphatidylethanolaminePIphosphatidylinositolTGtriglycerideSMsphingomyelinCLcardiolipinPGphosphatidylglycerolDAGdiacylglycerol32Pi[32P]orthophosphateG3Pglycerol 3-phosphatePAphosphatidateMTT3-(4,5-dimethylthiazolyl-2)2,5-diphenyl tetrazolium bromide. sphingomyelin (SM), or phosphatidylserine (5Esko J.D. Raetz C.R. Proc. Natl. Acad. Sci. U.S.A. 1980; 77: 5192-5196Crossref PubMed Scopus (53) Google Scholar, 6Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 7Voelker D.R. Frazier J.L. J. Biol. Chem. 1986; 261: 1002-1008Abstract Full Text PDF PubMed Google Scholar, 8Kuge O. Nishijima M. Akamatsu Y. Proc. Natl. Acad. Sci. U.S.A. 1985; 82: 1926-1930Crossref PubMed Scopus (46) Google Scholar). These mutants have helped in our understanding of the synthesis and importance of these phospholipids. In an effort to identify genes important for regulation of glycerolipid biosynthesis in general, we wanted to generate and select for mutants from an established animal cell line that would display global defects in glycerolipid biosynthesis. phosphatidylcholine 1-O-[12-(1′pyrene)]dodecanoic acid Chinese hamster ovary phosphatidate phosphatase 1 glucose-6-phosphate isomerase phosphatidylethanolamine phosphatidylinositol triglyceride sphingomyelin cardiolipin phosphatidylglycerol diacylglycerol [32P]orthophosphate glycerol 3-phosphate phosphatidate 3-(4,5-dimethylthiazolyl-2)2,5-diphenyl tetrazolium bromide. Using a photodynamic selection technique we have generated a population of CHO cells that display a decreased ability to incorporate fatty acids into complex lipids. Isolates from this population displayed a reduction in glycerolipid biosynthesis accompanied by a reduction in a phosphatidate phosphatase activity (PAP1, sn-3-phosphatidate phosphohydrolase, EC 3.1.3.4). However, expression cloning and sequencing revealed the primary defect to be a point mutation in glucose-6-phosphate isomerase (GPI, d-glucose-6-phosphate aldoseketose isomerase, EC 5.3.1.9), a glycolytic enzyme with moonlighting cytokine functions (9Watanabe H. Takehana K. Date M. Shinozaki T. Raz A. Cancer Res. 1996; 56: 2960-2963PubMed Google Scholar). Mutations in GPI are the second most frequent cause of inherited glycolytic erythroenzymopathy in humans (10Kugler W. Lakomek M. Baillieres Best Pract. Res. Clin. Haematol. 2000; 13: 89-101Crossref PubMed Scopus (57) Google Scholar). This autosomal recessive disorder is characterized by a non-spherocytic hemolytic anemia of variable severity, which can present with neurological dysfunctions (10Kugler W. Lakomek M. Baillieres Best Pract. Res. Clin. Haematol. 2000; 13: 89-101Crossref PubMed Scopus (57) Google Scholar). Our findings presented here reveal an unexpected relationship between GPI and PAP1. They may also yield insight into the nature of communication between carbohydrate metabolism and glycerolipid biosynthesis and may help explain the neurological dysfunctions associated with the human GPI disorders. 1-O-[12-(1′-Pyrene)]dodecanoic acid (P12) was obtained from Invitrogen (Carlsbad, CA). All radioactive compounds were obtained from PerkinElmer Life Sciences. Lipids were purchased from Avanti Polar Lipids (Alabaster, AL). Silica gel 60 TLC plates (EMD Biosciences), Ham's F-12 medium (Cellgro), fetal bovine serum (HyClone), and tissue culture dishes (Corning) were obtained from Fisher Scientific (Pittsburgh, PA). All other reagents, unless otherwise specified, were purchased from Sigma-Aldrich. Cells were maintained in Ham's F-12 medium supplemented with 10% fetal bovine serum, 1 mm glutamine, penicillin G (100 units/ml), and streptomycin (75 units/ml). This growth medium is designated “F12c” throughout the text. Cells were cultured at 33 °C or 40 °C at 5% CO2. The initial parent cell line, ZR-82, is a peroxisome/plasmalogen-deficient strain derived from the CHO-K1 cell line (11Zoeller R.A. Raetz C.R. Proc. Natl. Acad. Sci. U.S.A. 1986; 83: 5170-5174Crossref PubMed Scopus (111) Google Scholar). Other parent cell lines, used to confirm our initial findings, were ZR-87, another CHO-K1 derived peroxisome/plasmalogen-deficient strain (11Zoeller R.A. Raetz C.R. Proc. Natl. Acad. Sci. U.S.A. 1986; 83: 5170-5174Crossref PubMed Scopus (111) Google Scholar), and wild-type CHO-K1. Cells were plated into 100-mm diameter tissue culture dishes at 5 × 105 cells/dish and allowed to attach overnight. The cells were exposed to 200 μg/ml ethylmethane sulfonate for 20 h at 37 °C, the ethylmethane sulfonate-containing medium was removed, and the cells were allowed to grow to confluence at 33 °C (the permissive temperature) to allow establishment of phenotypes prior to selection. Mutagenized cells (107 cells total) were plated into 100-mm diameter tissue culture dishes (106 cells/dish) and allowed to attach overnight at 33 °C. The following day, the cells were shifted to 40 °C. After 2 h at 40 °C, medium containing P12 was added to a final concentration of 5 μm. After 3 h at 40 °C, medium was removed, regular growth medium was added, and the cells were incubated for another hour at 40 °C to remove any P12 fatty acid not assimilated into complex lipids. The cells were then suspended on a 1.5-mm-thick glass plate over a long wavelength (>300 nm) UV source (Black-Ray UV lamp, Model XX-15L, UVP, Inc., San Gabriel, CA) and irradiated from underneath. The distance from the UV source and the duration of the UV irradiation was predetermined to result in >95% cell death. The dishes were then returned to 33 °C to allow growth of the survivors. The surviving cells were pooled and subjected to the same selection process twice more. In the third round of selection, the majority of the cells survived. In all cases, the cells were grown at 33 °C, and the selections were performed at 40 °C. Clonal isolates were generated from the surviving population using limiting dilution at 33 °C. Each isolate was tested for phospholipid biosynthesis as described below. For phospholipid biosynthesis, short term labeling with 32Pi was used. Cells were plated into sterile glass scintillation vials (2.5 × 105 cells/vial) and allowed to attach overnight at 33 °C. The next day, some vials were placed at 40 °C for 2 h while others were left at 33 °C. An aliquot of medium containing 32Pi (25 μCi/ml or 50 μCi/ml) was added, and cells were incubated for 3 h at the appropriate temperature. Medium was removed, and lipids were extracted by the method of Bligh and Dyer (12Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (41814) Google Scholar) in the presence of 300 μg of carrier lipid (total bovine heart extract). An aliquot was taken to determine total chloroform-soluble radioactivity. Phospholipids were separated by two-dimensional TLC using Silica Gel 60 plates, developing in the first dimension with chloroform:methanol:29% ammonium hydroxide (60:30:5, v/v) followed by development in the second dimension using chloroform:methanol:acetic acid:H2O (50:30:6:1, v/v). The labeled species were localized by autoradiography, the labeled bands were scraped into scintillation vials, and radioactivity was quantitated by liquid scintillation spectrometry. Identification of the labeled species was determined by co-migration with authentic standards. Parallel, unlabeled vials were used for protein determination. For phospholipid composition, steady-state labeling with 32Pi was used (13James P.F. Lake A.C. Hajra A.K. Larkins L.K. Robinson M. Buchanan F.G. Zoeller R.A. J. Biol. Chem. 1997; 272: 23540-23546Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Cells were plated into sterile glass scintillation vials (2.5 × 104 cells/vial) at 33 °C and allowed to attach overnight. An aliquot of medium containing 32Pi was then added to achieve a final concentration of 4 μCi/ml. Cells were incubated for 96 h at 33 °C prior to analysis as described above. Cells were labeled with 32Pi (5 μCi/ml) for 4 days (several generations) to uniformly label the phospholipids. Cells were then harvested and plated into unlabeled medium in 6-well dishes at 2 × 105 cells/well. At various times, medium was removed, the cell monolayer was washed once with 3 ml of phosphate-buffered saline, and phospholipids were extracted into 2 ml of methanol containing 300 μg of carrier lipid. After recovery using the method of Bligh & Dyer (12Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (41814) Google Scholar), samples were blown to dryness, resuspended in 1 ml of chloroform, and an aliquot was taken for determination of total phospholipid label using liquid scintillation spectrometry. The individual phospholipids were isolated, and radioactivity was quantitated as described above. Cells were plated into sterile glass scintillation vials (2.5 × 105 cells/vial) and allowed to attach overnight at 33 °C. The next day, vials were shifted to 40 °C for 2 h. An aliquot of medium containing [9,10-3H]oleic acid or [9,10-3H]palmitic acid was added to achieve a final concentration of 2 μm at 2 μCi/ml, and the cells were incubated for 3 h at 40 °C. Labeling medium was removed, and the cell monolayer was incubated for 30 min in F12c medium prior to extraction of cellular lipids as described above. Labeled lipids were separated using single dimension TLC on Silica Gel 60 plates using hexane:ethyl ether:acetic acid (70:30:1; v/v) as the development system. The plates were sprayed with EN3HANCE (PerkinElmer Life Sciences) prior to exposure to x-ray film at −80 °C to localize labeled species. Identification of labeled species was determined by co-migration with authentic standards. Labeled bands were scraped into scintillation vials, and radioactivity was quantified as described above. Parallel, unlabeled vials were used for protein determinations. Cells were plated into 100-mm diameter tissue culture dishes at 106 cells/dish and allowed to attach overnight at 33 °C. The next morning, the medium was changed to F12c containing 100 μm oleic acid, and cells were grown at 33 °C for 48 h after which they were harvested with trypsin and resuspended in 1 ml of phosphate-buffered saline. An aliquot of 50 μl was used for protein determination, and 0.75 ml of this cell suspension was transferred to a glass tube containing 3 ml of methanol: chloroform (2:1, v/v). Lipids were extracted and separated on a Silica Gel G plates using the same single dimension system describe for fatty acid labeling. Plates were charred on a hot plate after spraying the plate with 50% sulfuric acid. Charred plates were scanned, and the densities of the bands of interest were determined using the National Institutes of Health ImageJ program. 3W. S. Rasband (1997–2008) ImageJ, rsb.info.nih.go/ij/, National Institutes of Health. Quantitation was performed by comparison to a standard curve for each neutral lipid class, run in adjacent lanes on the same TLC plate. Cells were plated in 60-mm diameter tissue culture dishes at 105 cells/dish and allowed to attach overnight at 33 °C. The next day, dishes were transferred to 33 °C or 40 °C and allowed to grow. Cells were harvested each day with trypsin and counted using a hemocytometer. Cell growth was also visualized by staining cell colonies grown on tissue culture plates with 0.5% Coomassie Blue in methanol:water:acetic acid (45:45:10, v/v) for 1 h, followed by three washes with methanol:water:acetic acid (45:45:10, v/v). DNA and protein synthesis was determined by measuring the incorporation of [methyl-3H]thymidine and [35S]methionine, respectively, into trichloroacetic acid-insoluble material. Cells were plated into 24-well dishes (2 × 104 cells/well) and allowed to attach overnight at 33 °C. One dish was shifted the next morning to 40 °C 3 h prior to the labeling. [methyl-3H]Thymidine or [35S]methionine was added to yield final radioactive concentrations of 2.5 μCi/ml and 1 μCi/ml, respectively. After 2 h at either 33 °C or 40 °C medium was removed, and 0.5 ml of ice-cold 10% trichloroacetic acid was added to each well. The cell monolayers were washed 5 times with 1 ml of 10% trichloroacetic acid and twice using ice-cold ether:ethanol (1:3, v/v). After drying, cellular material was solubilized in 0.3 ml of 0.5 n NaOH and incubated for 2 h at 37 °C. Aliquots were counted by liquid scintillation spectrometry following neutralization with HCl. Cellular ATP levels were measured using the ENLITEN® luciferase bioluminescent kit assay (Promega, Madison, WI) following the manufacturer's protocol. ATP was extracted in 0.5% trichloroacetic acid, and bioluminescence was measured in a TD 20/20 single tube luminometer (Turner Biosystems, Sunnyvale, CA). Glycerol 3-phosphate (G3P) was extracted in 0.75 ml of 0.1 n NaOH from confluent 100-mm diameter tissue culture dishes. The alkaline extracts were heated for 45 min at 80 °C to selectively destroy NADH, then neutralized with 0.2 n HCl in 0.1 m Tris-HCl (pH 6.8). G3P levels were measured spectrophotometrically by following the reduction of 3-(4,5-dimethylthiazolyl-2)2,5-diphenyl tetrazolium bromide (MTT) (15Larsson K. Ansell R. Eriksson P. Adler L. Mol. Microbiol. 1993; 10: 1101-1111Crossref PubMed Scopus (157) Google Scholar) at 536 nm, in the presence of 0.2 m Tris-HCl (pH 9), 4 mm NAD+, 0.25 mm MTT, 0.25 mm phenazine methosulfate and 10 units/ml glycerol-3-phosphate dehydrogenase. Cells were grown at 33 °C prior to harvest without exposure to 40 °C. Cell monolayers were washed twice using Tris-buffered saline and then scraped into Tris-buffered saline using a rubber policeman. Cells were pelleted and resuspended in ice-cold homogenization buffer (50 mm Tris-HCl, pH 7.2, 1 mm EDTA, 1 mm dithiothreitol) and disrupted by sonication, using a Branson cell disrupter (Branson Sonifier Co.), with two 10-s bursts at a power setting of 30% using a microtip. The sonicated cell suspension was centrifuged for 30 min at 13,000 × g. The resulting supernatant was centrifuged for 90 min at 100,000 × g to pellet membranes and membrane-associated proteins. The supernatant (“soluble fraction”) was collected, and the pellet was resuspended in 0.5 m NaCl in homogenization buffer and centrifuged again for 90 min at 100,000 × g to extract any remnants of soluble, membrane-associated proteins from the membranes. The final pellet was resuspended in homogenization buffer (“membrane fraction”). All fractions were aliquoted and stored at −80 °C prior to assay. [32P] PA was synthesized enzymatically, using diacylglycerol kinase, diolein, and [γ-32P]ATP as described previously (16Carman G.M. Lin Y.P. Methods Enzymol. 1991; 197: 548-553Crossref PubMed Scopus (60) Google Scholar) with the exception that TLC purification was omitted for some PAP assays after observing no significant difference in PAP activity compared with the non-TLC-purified substrate. PAP activity was measured by following the release of water-soluble 32Pi from chloroform-soluble [32P]PA for 20 min at 37 °C. The reaction mixture contained 50 mm Tris-maleate buffer (pH 7.0), 2 mm MgCl2, 0.1 mm [32P]PA (10,000 cpm/nmol), 0.5 mm Triton X-100, and cellular protein (0.1 mg) in a total volume of 0.1 ml. Mg2+-dependent PAP activity (PAP1) represents the difference in activity obtained with or without the addition of 5 mm EDTA to the assay mixture. All enzyme assays were conducted in triplicate and were linear with time and protein concentration. A retroviral cDNA library from human substantia nigra (ViraPort®, Stratagene, La Jolla, CA) was used to infect a GroD1 population. 10 separate pools of 106 cells were infected at a 1:5 (virus:cell) ratio to optimize chances of single gene insertion. After 2 days at 33 °C cells were shifted to 40 °C to obtain temperature-resistant colonies. Genomic DNA was isolated from temperature-resistant clones using the Genomic tip 100/G kit (Qiagen, Valencia, CA), and human cDNA inserts were amplified by PCR using primers against the flanking regions of the cDNA insert in the vector. PCR amplification of genomic DNA was performed by cycling 1 min at 95 °C, 1 min at 64 °C, and 4 min at 72 °C for 40 cycles. Primers were as follows: 5′ Retro, 5′-GGC TGC CGA CCC CGG GGG TGG-3′; 3′pBF, 5′-CGA ACC CCA GAG TCC CGC TCA-3′. PCR products were purified using the QIAquick Spin® kit (Qiagen) and sequenced at Tufts University Core Facility (Boston, MA). GPI activity was measured at room temperature in whole cell homogenates, as described previously (17Gracy R.W. Tilley B.E. Methods Enzymol. 1975; 41: 392-400Crossref PubMed Scopus (64) Google Scholar) by monitoring the production of NADPH from fructose 6-phosphate at 340 nm. Thermal inactivation of the residual GPI activity was performed similarly as described (18Maitra P.K. J. Bacteriol. 1971; 107: 759-769Crossref PubMed Google Scholar); aliquots of whole cell lysate were incubated in a 60 °C water bath for different periods of times and chilled immediately on ice, then assayed for GPI activity. For zymograms, a polyacrylamide native PhastagelTM (Amersham Biosciences) with a gradient of 8–25% was used to separate proteins obtained from soluble fractions of ZR-82 and GroD1, prepared as described above. The gel was stained by immersion in a GPI-specific staining solution (19Rogers T.J. O'Day K. Methods Enzymol. 1983; 92: 237-242Crossref PubMed Scopus (2) Google Scholar) for 30 min at 37 °C. The staining solution was removed by rinsing the gel with deionized water and was fixed with 7% (v/v) acetic acid. Total cellular RNA was isolated from cells with the RNeasy kit (Qiagen), and first strand synthesis of the RNA was performed with SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA). The cDNA coding region for GPI was amplified using the GoTag® Green master mix (Promega) and primers 5′-TCG ACC GTC CGG CTC CGT GT-3′ and 5′-TTT GGC TGG CTC GGC TGA CTT TAT TC-3′. The PCR product was purified and sequenced as described above. The entire coding region of GPI cDNA was amplified from cDNA of CHO-K1 cells using Phusion DNA polymerase (New England BioLabs Inc., Ipswich, MA) and the primers 5′-GTT GTT GAA TTC TCT CCG CTC CCG CCA TGA-3′ and 5′-GTT GTT GAA TTC TTA TTC TAT TCT GAT GTC CCG C-3′, and cloned as an EcoRI fragment into the pBABEpuro vector (20Morgenstern J.P. Land H. Nucleic Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1886) Google Scholar) to generate pBABE(GPI)puro. Insert directionality was confirmed by PCR and sequencing using pBABE 5′ and pBABE 3′ primers (20Morgenstern J.P. Land H. Nucleic Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1886) Google Scholar). HEK 293T cells were co-transfected, using FuGENE 6, with helper virus DNA pCL-10A1 (Imgenex, San Diego, CA) and either equal amounts of pBABE(GPI)puro or pBABEpuro. Supernatants containing retrovirus were harvested 48 h after transfection, filtered, and stored at −80 °C. Cells were infected for 3 h with virus-containing supernatant containing 10 μg/ml Polybrene. Medium was changed to F12c containing 6 μg/ml puromycin 24 h after infection. Protein determinations were performed using the BCA kit and Coomassie protein assay reagent (Pierce). Two-tailed Student's t tests were performed, and values of p ≤ 0.05 were considered significant differences. The ZR-82 cell line is a peroxisome/plasmalogen-deficient variant of the CHO-K1 cell line. This cell line was chosen as the parent strain due to the fact that it accumulates the fluorescent fatty acid P12 almost 3-fold compared with CHO-K1 and is hypersensitive to P12/UV treatment (21Zoeller R.A. Morand O.H. Raetz C.R. J. Biol. Chem. 1988; 263: 11590-11596Abstract Full Text PDF PubMed Google Scholar). ZR-82 cells were mutagenized, and a P12/UV-resistant population of cells was generated as described under “Experimental Procedures.” We assumed that major deficiencies in glycerolipid biosynthesis would be lethal, similar to previously reported cell lines with severe phospholipid biosynthesis deficiencies (5Esko J.D. Raetz C.R. Proc. Natl. Acad. Sci. U.S.A. 1980; 77: 5192-5196Crossref PubMed Scopus (53) Google Scholar). To obtain conditionally lethal (temperature-sensitive) mutants, once mutagenized, the cells were grown at 33 °C (the permissive temperature). One isolate, GroD1, displayed reduced phospholipid biosynthesis; incorporation of 32Pi into phospholipids was reduced by 60% at 33 °C and reduced by 68% at 40 °C in comparison to the parent cell line (Fig. 1A). Separation of the labeled phospholipid classes by two-dimensional TLC revealed that, when compared with the parent strain, the most affected classes in GroD1 were PC and phosphatidylethanolamine (PE) (Fig. 1, B and C), reduced by 85 and 73%, respectively, when measured at 33 °C. The differences were more pronounced at 40 °C. Both of these species are synthesized in mammalian cells using diacylglycerol (DAG) as an intermediate (Fig. 2) (22Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar). The labeling of SM, which depends on PC for donation of the phosphocholine head group (6Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 23Ullman M.D. Radin N.S. J. Biol. Chem. 1974; 249: 1506-1512Abstract Full Text PDF PubMed Google Scholar), was also severely reduced in GroD1 (Fig. 1C). In contrast, phosphatidylinositol (PI), which does not require DAG formation for its synthesis (24Agranoff B.W. Bradley R.M. Brady R.O. J. Biol. Chem. 1958; 233: 1077-1083Abstract Full Text PDF PubMed Google Scholar), did not show a significant reduction over ZR-82 at 33 °C and was only reduced by 22% at 40 °C. Labeling of cardiolipin (CL) and phosphatidylglycerol (PG) was also severely reduced in GroD1 compared with ZR-82. Interestingly, although there appeared to be a severe decrease in the synthesis of phospholipids in GroD1, the labeling of phosphatidic acid (PA), an intermediate for the synthesis of all glycerolipids (25Smith S.W. Weiss S.B. Kennedy E.P. J. Biol. Chem. 1957; 228: 915-922Abstract Full Text PDF PubMed Google Scholar, 26Carman G.M. Han G.S. Trends Biochem. Sci. 2006; 31: 694-699Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), was increased 80% over the parent strain.FIGURE 2Metabolic pathways in the synthesis of the major glycerolipid in animal cells. PAP1 (phosphatidic acid phosphatase 1) catalyzes the dephosphorylation of phosphatidic acid supplying diacylglycerol for the synthesis of PC, PE, and TG. If not dephosphorylated, PA can be converted to CDP-diacylglycerol, which is a precursor of phosphatidylinositol. CDP-eth, CDP-ethanolamine; CDP-cho, CDP-choline; CDP-DAG, CDP-diacylglycerol; and Ino, inositol.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Labeling of cellular lipids with either [9,10-3H]oleate or [9,10-3H]palmitate was consistent with a decrease in glycerolipid biosynthesis (Fig. 3A). Oleate labeling of the phospholipids was reduced by 55% in GroD1, whereas palmitate labeling was reduced by only 25%. This decrease is less severe than that obtained using 32Pi. This is likely due to the fact that fatty acids can be incorporated into pre-existing phospholipids through deacylation/reacylation reactions (27Shindou H. Shimizu T. J. Biol. Chem. 2009; 284: 1-5Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar) (supplemental Fig. S1). The labeling of triglycerides was dramatically reduced in GroD1 using either oleate or palmitate (∼11% of ZR-82 when using either fatty acid). In contrast to triglycerides and phospholipids, the labeling of cholesterol esters was increased in the mutant strain. To confirm the fatty acid labeling data for TG and cholesterol ester, we also measured the cellular accumulation of neutral lipids after 48 h of incubation with 100 μm oleate (Fig. 3, B and C). During this period GroD1 was able to accumulate only 20% of the TG found in ZR-82. However, the mutant cell line was capable of depositing fatty acids in the form of cholesterol esters, displaying increased levels (70% greater than ZR-82). Free cholesterol levels were not significantly different between the two strains under these conditions. We could not detect the accumulation of DAG in either cell line under t" @default.
• W2016757701 created "2016-06-24" @default.
• W2016757701 creator A5013936797 @default.
• W2016757701 creator A5015880535 @default.
• W2016757701 creator A5032155879 @default.
• W2016757701 creator A5041764157 @default.
• W2016757701 creator A5047376925 @default.
• W2016757701 creator A5054019269 @default.
• W2016757701 creator A5069421291 @default.
• W2016757701 creator A5082021389 @default.
• W2016757701 date "2010-01-01" @default.
• W2016757701 modified "2023-10-15" @default.
• W2016757701 title "Isolation of Novel Animal Cell Lines Defective in Glycerolipid Biosynthesis Reveals Mutations in Glucose-6-phosphate Isomerase" @default.
• W2016757701 cites W1431372690 @default.
• W2016757701 cites W1436318950 @default.
• W2016757701 cites W1498869258 @default.
• W2016757701 cites W1510150165 @default.
• W2016757701 cites W1518603252 @default.
• W2016757701 cites W1549255782 @default.
• W2016757701 cites W1565332914 @default.
• W2016757701 cites W1574033922 @default.
• W2016757701 cites W1575864823 @default.
• W2016757701 cites W1577386926 @default.
• W2016757701 cites W1590034396 @default.
• W2016757701 cites W1597780633 @default.
• W2016757701 cites W1676701054 @default.
• W2016757701 cites W1965952267 @default.
• W2016757701 cites W1973942466 @default.
• W2016757701 cites W1982118513 @default.
• W2016757701 cites W1990469104 @default.
• W2016757701 cites W1992625839 @default.
• W2016757701 cites W1997527312 @default.
• W2016757701 cites W1998384937 @default.
• W2016757701 cites W2006659499 @default.
• W2016757701 cites W2010238890 @default.
• W2016757701 cites W2016341707 @default.
• W2016757701 cites W2028709228 @default.
• W2016757701 cites W2029883703 @default.
• W2016757701 cites W2035844149 @default.
• W2016757701 cites W2038060844 @default.
• W2016757701 cites W2051222964 @default.
• W2016757701 cites W2057068035 @default.
• W2016757701 cites W2057680304 @default.
• W2016757701 cites W2059157388 @default.
• W2016757701 cites W2067469548 @default.
• W2016757701 cites W2067522487 @default.
• W2016757701 cites W2069694136 @default.
• W2016757701 cites W2071117792 @default.
• W2016757701 cites W2071810294 @default.
• W2016757701 cites W2073667659 @default.
• W2016757701 cites W2075418587 @default.
• W2016757701 cites W2076822925 @default.
• W2016757701 cites W2078807483 @default.
• W2016757701 cites W2081359387 @default.
• W2016757701 cites W2087576138 @default.
• W2016757701 cites W2102651270 @default.
• W2016757701 cites W2110966981 @default.
• W2016757701 cites W2112668580 @default.
• W2016757701 cites W2128099976 @default.
• W2016757701 cites W2147013322 @default.
• W2016757701 cites W2152311269 @default.
• W2016757701 cites W2156571746 @default.
• W2016757701 cites W2160992291 @default.
• W2016757701 cites W2163560348 @default.
• W2016757701 cites W2164165466 @default.
• W2016757701 cites W2324527909 @default.
• W2016757701 cites W2403030213 @default.
• W2016757701 cites W2417532218 @default.
• W2016757701 cites W2418651706 @default.
• W2016757701 cites W4242561105 @default.
• W2016757701 cites W61835041 @default.
• W2016757701 doi "https://doi.org/10.1074/jbc.m109.068213" @default.
• W2016757701 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2801288" @default.
• W2016757701 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19903819" @default.
• W2016757701 hasPublicationYear "2010" @default.
• W2016757701 type Work @default.
• W2016757701 sameAs 2016757701 @default.
• W2016757701 citedByCount "7" @default.
• W2016757701 countsByYear W20167577012012 @default.
• W2016757701 countsByYear W20167577012019 @default.
• W2016757701 countsByYear W20167577012021 @default.
• W2016757701 countsByYear W20167577012022 @default.
• W2016757701 crossrefType "journal-article" @default.
• W2016757701 hasAuthorship W2016757701A5013936797 @default.
• W2016757701 hasAuthorship W2016757701A5015880535 @default.
• W2016757701 hasAuthorship W2016757701A5032155879 @default.
• W2016757701 hasAuthorship W2016757701A5041764157 @default.
• W2016757701 hasAuthorship W2016757701A5047376925 @default.
• W2016757701 hasAuthorship W2016757701A5054019269 @default.
• W2016757701 hasAuthorship W2016757701A5069421291 @default.
• W2016757701 hasAuthorship W2016757701A5082021389 @default.
• W2016757701 hasBestOaLocation W20167577011 @default.
• W2016757701 hasConcept C181199279 @default.
• W2016757701 hasConcept C185592680 @default.
• W2016757701 hasConcept C2775941552 @default.
• W2016757701 hasConcept C2777790778 @default.
• W2016757701 hasConcept C2781264208 @default.
• W2016757701 hasConcept C54355233 @default.

• next