Characterization of human lysophospholipid acyltransferase 3
2009; Elsevier BV; Volume: 50; Issue: 8 Linguagem: Inglês
10.1194/jlr.m800398-jlr200
ISSN1539-7262
AutoresShilpa Jain, Xiaoling Zhang, Preeti J. Khandelwal, Aleister J. Saunders, Brian S. Cummings, Peter Oelkers,
Tópico(s)Sphingolipid Metabolism and Signaling
ResumoEsterifying lysophospholipids may serve a variety of functions, including phospholipid remodeling and limiting the abundance of bioactive lipids. Recently, a yeast enzyme, Lpt1p, that esterifies an array of lysophospholipids was identified. Described here is the characterization of a human homolog of LPT1 that we have called lysophosphatidylcholine acyltransferase 3 (LPCAT3). Expression of LPCAT3 in Sf9 insect cells conferred robust esterification of lysophosphatidylcholine in vitro. Kinetic analysis found apparent cooperativity with a saturated acyl-CoA having the lowest K0.5 (5 μM), a monounsaturated acyl-CoA having the highest apparent Vmax (759 nmol/min/mg), and two polyunsaturated acyl-CoAs showing intermediate values. Lysophosphatidylethanolamine and lysophosphatidylserine were also utilized as substrates. Electrospray ionization mass spectrometric analysis of phospholipids in Sf9 cells expressing LPCAT3 showed a relative increase in phosphatidylcholine containing saturated acyl chains and a decrease in phosphatidylcholine containing unsaturated acyl chains. Targeted reduction of LPCAT3 expression in HEK293 cells had essentially an opposite effect, resulting in decreased abundance of saturated phospholipid species and more unsaturated species. Reduced LPCAT3 expression resulted in more apoptosis and distinctly fewer lamellipodia, suggesting a necessary role for lysophospholipid esterification in maintaining cellular function and structure.—Jain, S., X. Zhang, P. J. Khandelwal, A. J. Saunders, B. S. Cummings, and P. Oelkers. Characterization of human lysophospholipid acyltransferase 3. Esterifying lysophospholipids may serve a variety of functions, including phospholipid remodeling and limiting the abundance of bioactive lipids. Recently, a yeast enzyme, Lpt1p, that esterifies an array of lysophospholipids was identified. Described here is the characterization of a human homolog of LPT1 that we have called lysophosphatidylcholine acyltransferase 3 (LPCAT3). Expression of LPCAT3 in Sf9 insect cells conferred robust esterification of lysophosphatidylcholine in vitro. Kinetic analysis found apparent cooperativity with a saturated acyl-CoA having the lowest K0.5 (5 μM), a monounsaturated acyl-CoA having the highest apparent Vmax (759 nmol/min/mg), and two polyunsaturated acyl-CoAs showing intermediate values. Lysophosphatidylethanolamine and lysophosphatidylserine were also utilized as substrates. Electrospray ionization mass spectrometric analysis of phospholipids in Sf9 cells expressing LPCAT3 showed a relative increase in phosphatidylcholine containing saturated acyl chains and a decrease in phosphatidylcholine containing unsaturated acyl chains. Targeted reduction of LPCAT3 expression in HEK293 cells had essentially an opposite effect, resulting in decreased abundance of saturated phospholipid species and more unsaturated species. Reduced LPCAT3 expression resulted in more apoptosis and distinctly fewer lamellipodia, suggesting a necessary role for lysophospholipid esterification in maintaining cellular function and structure.—Jain, S., X. Zhang, P. J. Khandelwal, A. J. Saunders, B. S. Cummings, and P. Oelkers. Characterization of human lysophospholipid acyltransferase 3. Lysophospholipids (lysoPLs2, monoacylglyercophospholipids) may be esterified for several physiological reasons. One is the intracellular remodeling of glycerophospholipids (PL). Pairing phospholipase A2 activity with lysoPL esterification allows for the alteration of PL acyl-chain composition (Lands Cycle) (1Lands W.E. Metabolism of glycerolipids. 2. The enzymatic acylation of lysolecithin.J. Biol. Chem. 1960; 235: 2233-2237Abstract Full Text PDF PubMed Google Scholar). Acyl chain composition of PL may impact cellular membrane dynamics (2Holte L.L. Separovic F. Gawrisch K. Nuclear magnetic resonance investigation of hydrocarbon chain packing in bilayers of polyunsaturated phospholipids.Lipids. 1996; 31: S199-S203Crossref PubMed Google Scholar) and influence cellular processes such as endocytosis (3Mahoney E.M. Scott W.A. Landsberger F.R. Hamill A.L. Cohn Z.A. Influence of fatty acyl substitution on the composition and function of macrophage membranes.J. Biol. Chem. 1980; 255: 4910-4917Abstract Full Text PDF PubMed Google Scholar). The predominance of palmitoyl chains in surfactant phospholipids (4Post M. Schuurmans E.A. Batenburg J.J. Van Golde L.M. Mechanisms involved in the synthesis of disaturated phosphatidylcholine by alveolar type II cells isolated from adult rat lung.Biochim. Biophys. 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Acta. 2002; 1582: 81-88Crossref PubMed Scopus (197) Google Scholar). How cells esterify lysoPL has recently begun to be elucidated. Acyl-CoA independent, transacylation can be mediated by the mitochondrial tafazzin (5Xu Y. Malhotra A. Ren M. Schlame M. The enzymatic function of tafazzin.J. Biol. Chem. 2006; 281: 39217-39224Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Acyl-CoA dependent esterification of lysoPC, lysophosphatidic acid (lysoPA), and lysophosphatidylglycerol (lysoPG) can be mediated in the endoplasmic reticulum (ER) by lysophosphatidylcholine acyltransferase 1 (LPCAT1) (8Nakanishi H. Shindou H. Hishikawa D. Harayama T. Ogasawara R. Suwabe A. Taguchi R. Shimizu T. Cloning and characterization of mouse lung-type acyl-CoA:lysophosphatidylcholine acyltransferase 1 (LPCAT1). Expression in alveolar type II cells and possible involvement in surfactant production.J. Biol. Chem. 2006; 281: 20140-20147Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 9Chen X. Hyatt B.A. Mucenski M.L. Mason R.J. Shannon J.M. Identification and characterization of a lysophosphatidylcholine acyltransferase in alveolar type II cells.Proc. Natl. Acad. Sci. USA. 2006; 103: 11724-11729Crossref PubMed Scopus (151) Google Scholar). Saturated acyl-CoA substrates are used preferentially but not exclusively. LPCAT2 is 48% identical to LPCAT1, localizes to the ER and Golgi, has a broader pattern of tissue expression, and only uses lysoPC as an acyl acceptor (10Shindou H. Hishikawa D. Nakanishi H. Harayama T. Ishii S. Taguchi R. Shimizu T. A single enzyme catalyzes both platelet-activating factor production and membrane biogenesis of inflammatory cells. Cloning and characterization of acetyl-CoA:LYSO-PAF acetyltransferase.J. Biol. Chem. 2007; 282: 6532-6539Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Among the lysoPC species utilized is 1-alkyl lysoPC and 1-alkenyl lysoPC, indicating the likely importance of this enzyme in platelet activating factor (PAF) synthesis. A third mouse paralog can also esterify lysoPC with a preference for palmitoyl-CoA (11Soupene E. Fyrst H. Kuypers F.A. Mammalian acyl-CoA:lysophosphatidylcholine acyltransferase enzymes.Proc. Natl. Acad. Sci. USA. 2008; 105: 88-93Crossref PubMed Scopus (81) Google Scholar). The S. cerevisiae genome contains no gene with distinct sequence identity to these mouse LPCAT. However, an unrelated gene, LPT1 (also called ALE1, LPA1, and SLC4) encodes for a lysoPL acyltransferase (LPLAT) (12Riekhof W.R. Wu J. Gijon M.A. Zarini S. Murphy R.C. Voelker D.R. Lysophosphatidylcholine metabolism in Saccharomyces cerevisiae: the role of P-type ATPases in transport and a broad specificity acyltransferase in acylation.J. Biol. Chem. 2007; 282: 36853-36861Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 16Chen Q. Kazachkov M. Zheng Z. Zou J. The yeast acylglycerol acyltransferase LCA1 is a key component of Lands cycle for phosphatidylcholine turnover.FEBS Lett. 2007; 581: 5511-5516Crossref PubMed Scopus (53) Google Scholar). Lpt1p can esterify all the lyso species of the major phospholipids, has highest capacity for unsaturated acyl-CoA species, and belongs to the membrane bound o-acyltransferase (MBOAT) gene family (17Hofmann K. A superfamily of membrane-bound O-acyltransferases with implications for Wnt signaling.Trends Biochem. Sci. 2000; 25: 111-112Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar) which has many representatives in mammalian genomes. Pioneering work by Hishikawa et al. (18Hishikawa D. Shindou H. Kobayashi S. Nakanishi H. Taguchi R. Shimizu T. Discovery of a lysophospholipid acyltransferase family essential for membrane asymmetry and diversity.Proc. Natl. Acad. Sci. USA. 2008; 105: 2830-2835Crossref PubMed Scopus (210) Google Scholar) identified and characterized three mouse MBOAT paralogs, LPCAT3, LPCAT4, and LPEAT1, that can esterify lysoPC, lysoPE and/or lysoPS. Zhou et al. (19Zhao Y. Chen Y.Q. Bonacci T.M. Bredt D.S. Li S. Bensch W.R. Moller D.E. Kowala M. Konrad R.J. Cao G. Identification and characterization of a major liver lysophosphatidylcholine acyltransferase.J. Biol. Chem. 2008; 283: 8258-8265Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) have shown that human LPCAT3 esterified lysoPC and lysoPS in vitro and that reduction of its expression in Huh7 cells dramatically reduced LPCAT activity. We have performed similar kinetic analyses of human LPCAT3, with some different results. We have used mass spectrometry to analyze the effect of LPCAT3 over-expression and reduced expression on phospholipid composition. Data are also provided that support a role for LPCAT3 in regulating cell growth and apoptosis. Lysophosphatidylethanolamine (lysoPE), lysophosphatidylserine (lysoPS), lysophosphatidylinositol (lysoPI), 1-O-alkyl lysoPC, and lysoPG were from Avanti Polar Lipids (Alabaster, AL). [1-14C] palmitoyl- lysoPC (55mCi/mmol) was from Perkin-Elmer Life. All other chemicals were obtained from Sigma or Fisher. Full-length sequences were aligned using clustalW2 (20Labarga A. Valentin F. Anderson M. Lopez R. Web services at the European Bioinformatics Institute.Nucleic Acids Res. 2007; 35: W6-11Crossref PubMed Scopus (158) Google Scholar). For percent identity, the conserved amino acids were counted and divided by the length of the shorter of the two proteins compared. Transmembrane domains were predicted by the TMHMM2.0 program (21Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes.J. Mol. Biol. 2001; 305: 567-580Crossref PubMed Scopus (9084) Google Scholar). A 4.4 kb human LPCAT3 cDNA clone (GenBank Acc. #BC065194) was obtained from the American Type Culture Collection in pCMVsport6. Restriction digest with SmaI, PvuII released the open reading frame with 45 bp of 5′ UTR and 121 bp of 3′ UTR. This fragment was ligated into StuI digested pFastbac and sequenced. Subsequent baculovirus expression in Sf9 insect cells was performed in the Wistar Institute Protein Expression Core Facility. The pFastbac/LPCAT3 plasmid and a pFastbac/Glutathione S-transferase (GST) control were transposed into DH10Bac cells. Recovered bacmid DNA was screened by PCR for proper transposition of the transfer vector into the baculovirus genome. Positive bacmid DNA was transfected into Sf9 cells, and passage 1 (P1) virus stocks were recovered 96 h. posttransfection. A high-titer P2 virus stock was generated by infecting Sf9 at a multiplicity of infection (MOI) of ∼0.1, followed by incubation for 96–120 h. For production, 1 × 106 Sf9 cells/ml in Sf900-II medium (Invitrogen) at 28°C were infected with viruses at an MOI of 1. Cells were harvested 48 h postinfection. Sf9 cells were homogenized in 1 mM EDTA / 200 mM sucrose / 100 mM Tris-HCl, pH 7.4 by 10–15 passages through a 26.5 gauge needle (22Yen C.L. Stone S.J. Cases S. Zhou P. Farese Jr., R.V. Identification of a gene encoding MGAT1, a monoacylglycerol acyltransferase.Proc. Natl. Acad. Sci. USA. 2002; 99: 8512-8517Crossref PubMed Scopus (151) Google Scholar). Unlysed cells were removed by centrifugation at 1,000 g for 5 min at 4°C. The supernatant was stored frozen (− 70°C) prior to assays. Lysophosphatidylcholine acyltransferase (LPCAT) activity was measured by the incorporation of [1-14C]palmitoyl lysoPC into phosphatidylcholine (PC). The reaction contained 100 mM Tris-HCl, pH 7.4, 50 μM [1-14C]palmitoyl lysoPC (50,000 dpm/nmol), 1–150 μM of the respective acyl-CoA, and 1 μg of cell lysate protein in a total volume of 100 μl. Fixed time assays were performed for 7.5 min at 28°C or 5 min at 37°C. Reactions were stopped by adding chloroform: methanol (2:1) and lipids were extracted, resolved by thin layer chromatography, and quantified as described elsewhere (8Nakanishi H. Shindou H. Hishikawa D. Harayama T. Ogasawara R. Suwabe A. Taguchi R. Shimizu T. Cloning and characterization of mouse lung-type acyl-CoA:lysophosphatidylcholine acyltransferase 1 (LPCAT1). Expression in alveolar type II cells and possible involvement in surfactant production.J. Biol. Chem. 2006; 281: 20140-20147Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). EZ-Fit software was used for Hill-Plots. To calculate Vmax/Km, Vmax was first changed to nM/min/mg to remove volume from the units of the ratio. These assays were essentially performed as described previously (13Jain S. Stanford N. Bhagwat N. Seiler B. Costanzo M. Boone C. Oelkers P. Identification of a novel lysophospholipid acyltransferase in Saccharomyces cerevisiae.J. Biol. Chem. 2007; 282: 30562-30569Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The reaction mixture contained 100 mM Tris-HCl, pH 7.4, 50 μM of the respective lysoPL, 50 μM oleoyl CoA, 1 mM dithionitrobenzoic acid (DTNB), and 2–5 μg cell lysate protein in a total volume of 1 ml. Real time assays were performed for 5 min at room temperature or 3 min at 37°C. Cellular PL was extracted using chloroform and methanol according to the method of Bligh and Dyer (23Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42689) Google Scholar). Following transfection, Sf9 cells were homogenized in 3 ml of methanol: water (2:0.8), transferred to a glass test tube, and then 1.25 ml chloroform was added. Tubes were vortexed for 30 s and allowed to sit for 10 min on ice. After centrifugation at 213 g for 1 min, the chloroform layer was collected. The extraction was repeated and lipid extracts combined, dried under argon, reconstituted with 50 μl of methanol:chloroform (2:1), and stored at −20°C. Lipid phosphorus was quantified using malachite green (22Yen C.L. Stone S.J. Cases S. Zhou P. Farese Jr., R.V. Identification of a gene encoding MGAT1, a monoacylglycerol acyltransferase.Proc. Natl. Acad. Sci. USA. 2002; 99: 8512-8517Crossref PubMed Scopus (151) Google Scholar). 10 μl of lipid extract was dried under argon. 200 μl of perchloric acid was added and heated at 130°C for 2–3 h. To this was added 1 ml of dH2O, 1.5 ml of reagent C (4.2 g ammonium molybdate tetrahydrate in 100 ml 5 N HCl and 0.15 g malachite green oxalate in 300 ml dH20), and 200 μl of 1.5% (v/v) Tween 20. After 25 min at room temperature, the A590 was measured. Lipid extracts (500 pmol/ μl) were prepared by reconstituting in chloroform: methanol (2:1). Mass spectrometry was performed as described previously (24Taguchi R. Hayakawa J. Takeuchi Y. Ishida M. Two-dimensional analysis of phospholipids by capillary liquid chromatography/electrospray ionization mass spectrometry.J. Mass Spectrom. 2000; 35: 953-966Crossref PubMed Scopus (141) Google Scholar). Samples were analyzed using a Trap XCT ion-trap mass spectrometer (Agilent Technologies, Santa Clara, CA) equipped with an ESI source. 5 μl of sample was introduced by means of a flow injector into the ESI source at a rate of 0.2 ml/min. The elution solvent was acetonitrile:methanol:water (2:3:1, v/v/v) containing 0.1% (w/v) ammonium formate (pH 6.4). The mass spectrometer was operated in the positive and negative ionization modes with a nitrogen drying gas flow-rate of 8.0 L/min at 350°C, nebulizer pressure at 30 psi, and capillary voltage at 3 kV. As previously described (24Taguchi R. Hayakawa J. Takeuchi Y. Ishida M. Two-dimensional analysis of phospholipids by capillary liquid chromatography/electrospray ionization mass spectrometry.J. Mass Spectrom. 2000; 35: 953-966Crossref PubMed Scopus (141) Google Scholar), qualitative identification of individual PL molecular species was based on their calculated theoretical monoisotopic mass values and subsequent MSn fragmentation to identify the polar head groups. Relative quantification was done by comparison to the most abundant phospholipid in each sample, which corresponded to m/z 760 or 34:1 (16:0, 18:1) PC. MassLynx 4.0 software was used for data analysis. MSn fragmentation was performed on the same instrument under similar conditions except that the ion source and ion optic parameters were optimized with respect to the positive molecular ion of interest. Data from four separate Sf9 infections underwent ANOVA using SAS software. Individual means were compared using Student's t-test. HEK293 (human embryonic kidney) cells were seeded in 24 well plates at a concentration of 2.5 × 105 cells/well (∼30% confluence) and allowed to grow for 24 h prior to transfection. 1 μg of pSM2 either encoding for a short hairpin RNA (shRNA) specific for LPCAT3 (Open Biosystems clone #V2HS 200012) or for a nonsilencing (NS) control (with a target sequence not complementary to any known human gene) was gently mixed with 1 μl of lipofectamine transfection reagent (InVitrogen Life Sciences) in 100 μl of Opti-MEM I reduced-serum medium (Life Sciences). After 20 min at room temperature, the mixtures were added drop-wise onto the cells. Subsequent experimentation was done after culturing in serum-free DMEM at 37°C for 24 h. Activity of Complex I of the electron transport chain was determined by adding 30 μl of 5 mg/ml MTT (3-(4, dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) to each well. After 2 h incubation at 37°C, the media was replaced with 800 μl of DMSO to dissolve the resulting purple formazan, and the absorbance at 544 nm was measured with a FLUOstar OPTIMA plate reader (BMG Labtechnologies, Inc., Durham, NC). Cell number and viability were assessed using the ViaCount® assay reagent on EasyCyte Mini Guava flow cytometer (Guava Technologies, Hayward, CA) according to the manufacturer's instructions. Briefly, 25 μl cell suspensions were combined with 450 μl of assay reagent containing 7-Amino-actinomycin D (7-AAD) and propidium iodide (PI). Following 5 min incubation, cell number and viability were assessed by alterations in both 7-AAD and PI staining as well as alterations in forward scatter. Apoptosis and necrosis were assessed using annexin V (apoptotic cell marker) and propidium iodide (PI, necrotic cell marker) staining and flow cytometry as previously described (25Cummings B.S. Schnellmann R.G. Cisplatin-induced renal cell apoptosis: caspase 3-dependent and -independent pathways.J. Pharmacol. Exp. Ther. 2002; 302: 8-17Crossref PubMed Scopus (309) Google Scholar) with modifications. Cisplatin (50 μM) and tert-butylhydroperoxide (500 μM) were used as positive controls for apoptosis and necrosis, respectively. Following treatment, media were removed, cells washed twice with PBS and incubated in binding buffer (10 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, pH = 7.4) containing annexin V-FITC (25 μg/ml) and PI (25 μg/ml) for 10 min. Cells were washed three times using binding buffer, released from the monolayers using a rubber policeman and staining quantified using a Becton Dickinson FacsCalibur flow cytometer. For each measurement, 10,000 events were counted. Cell morphology was determined using phase-contrast microscopy as previously described (25Cummings B.S. Schnellmann R.G. Cisplatin-induced renal cell apoptosis: caspase 3-dependent and -independent pathways.J. Pharmacol. Exp. Ther. 2002; 302: 8-17Crossref PubMed Scopus (309) Google Scholar, 26Cummings B.S. Kinsey G.R. Bolchoz L.J. Schnellmann R.G. Identification of caspase-independent apoptosis in epithelial and cancer cells.J. Pharmacol. Exp. Ther. 2004; 310: 126-134Crossref PubMed Scopus (55) Google Scholar) with modifications. Briefly, cells were washed twice with PBS, fixed for 20 min using 10% buffered formalin, 4% formaldehyde, washed with PBS, covered with mounting media (Sigma), and cover slips applied. Visualization was performed using a TE300 Eclipse microscope (Nikon, Melville, NY). Using the yeast Lpt1p amino acid sequence as the query, a blastp search (27Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. Basic local alignment search tool.J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70338) Google Scholar) identified three human proteins with about 25% identity. These likely paralogs were originally identified as MBOAT 1, 2, and 5 with conservation of a putative acyltransferase domain (Pfam PFO3062) (17Hofmann K. A superfamily of membrane-bound O-acyltransferases with implications for Wnt signaling.Trends Biochem. Sci. 2000; 25: 111-112Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar). MBOAT5 was chosen for initial analysis because, like Lpt1, it contains a C-terminal ER retention signal, KKXX (28Nilsson T. Jackson M. Peterson P.A. Short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum.Cell. 1989; 58: 707-718Abstract Full Text PDF PubMed Scopus (367) Google Scholar). Subsequent studies have renamed human MBOAT5 as LPCAT3 (19Zhao Y. Chen Y.Q. Bonacci T.M. Bredt D.S. Li S. Bensch W.R. Moller D.E. Kowala M. Konrad R.J. Cao G. Identification and characterization of a major liver lysophosphatidylcholine acyltransferase.J. Biol. Chem. 2008; 283: 8258-8265Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 29Kazachkov M. Chen Q. Wang L. Zou J. Substrate preferences of a lysophosphatidylcholine acyltransferase highlight its role in phospholipid remodeling.Lipids. 2008; 43: 895-902Crossref PubMed Scopus (41) Google Scholar, 30Matsuda S. Inoue T. Lee H.C. Kono N. Tanaka F. Gengyo-Ando K. Mitani S. Arai H. Member of the membrane-bound O-acyltransferase (MBOAT) family encodes a lysophospholipid acyltransferase with broad substrate specificity.Genes Cells. 2008; 13: 879-888Crossref PubMed Scopus (57) Google Scholar). The predicted protein has 487 amino acids, a molecular weight of 56 kDa, and seven algorithm-identified (21Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes.J. Mol. Biol. 2001; 305: 567-580Crossref PubMed Scopus (9084) Google Scholar) transmembrane domains. A putative full-length cDNA clone for LPCAT3 was found in the mammalian gene collection (31Strausberg R.L. Feingold E.A. Klausner R.D. Collins F.S. The mammalian gene collection.Science. 1999; 286: 455-457Crossref PubMed Scopus (232) Google Scholar) and inserted into baculovirus. This virus was used to drive expression in Sf9 insect cells. Precedents indicated that Sf9 cells harbor low levels of endogenous acyltransferase activity in general (22Yen C.L. Stone S.J. Cases S. Zhou P. Farese Jr., R.V. Identification of a gene encoding MGAT1, a monoacylglycerol acyltransferase.Proc. Natl. Acad. Sci. USA. 2002; 99: 8512-8517Crossref PubMed Scopus (151) Google Scholar, 32Cheng D. Chang C.C. Qu X. Chang T.Y. Activation of acyl-coenzyme A:cholesterol acyltransferase by cholesterol or by oxysterol in a cell-free system.J. Biol. Chem. 1995; 270: 685-695Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Parallel infection with a GST-encoding virus allowed for the measurement of endogenous activity. In vitro LPCAT assays at 28°C found LPCAT3 expression to confer 10-fold higher activity than GST-expression (Fig. 1). This temperature was primarily used for subsequent assays as it yielded 5-fold more activity than parallel assays at the physiological temperature of 37°C (Fig. 1). To kinetically characterize the substrate specificity of LPCAT3, acyl-CoA concentration series assays were performed with four qualitatively different species. These represent the main classes of long chain acyl-CoAs: saturated (stearoyl-CoA); monounsaturated (oleoyl-CoA); polyunsaturated, omega-6 (arachidonic acid); and polyunsaturated, omega-3 (α-linolenoyl-CoA). Since the oleoyl-CoA curve was visibly sigmoidal (Fig. 2), suggesting cooperativity, Hill plots of all kinetic data were generated (see supplementary Figs. I and II). Cooperativity was found in all cases except linolenyl-CoA at 28°C. These same plots were used to determine the K0.5 and apparent Vmax values (Table 1). In terms of affinity (K0.5), the substrate preference was stearoyl-CoA > oleoyl-CoA ≥ arachidonyl-CoA ≥ α-linolenoyl-CoA. Arachidonyl-CoA showed higher affinity at 28°C than at 37°C. In terms of capacity (Vmax), oleoyl-CoA had the highest at 28°C and linolenoyl-CoA at 37°C. Because all of the reactions have the same, although undetermined, enzyme concentration, Vmax/K0.5 should be proportional to the catalytic efficiency (kcat/Km). This value was the highest for oleoyl-CoA. Clearly, the degree and placement of double bonds within the acyl-CoA substrate affects utilization by LPCAT3. α-Linolenoyl-CoA was the only substrate for which concentrations above 110 μM did not inhibit activity.TABLE 1Kinetic parameters of human LPCAT3Acyl-CoATempK0.5Apparent VmaxVmax / K0.5Hill number°CμMnmol/min/mgmin−1 mg −1stearoyl-CoA (18:0)285.1509.82.7oleoyl-CoA (18:1)2846.575916.32.0arachidonyl-CoA (20:4)2855.85299.52.0α-linolenoyl-CoA (18:3)28214.02681.31.0stearoyl-CoA (18:0)372.162.63.7oleoyl-CoA (18:1)3712.2877.11.6arachidonyl-CoA (20:4)37245.0820.32.0α-linolenoyl-CoA (18:3)37232.03231.41.4Kinetic data from Fig. 2 and supplementary Fig. II were used to calculate K0.5 and apparent Vmax using Hill plots (see supplementary Figs. I and III). LPCAT, lysophosphatidylcholine acyltransferase. Open table in a new tab Kinetic data from Fig. 2 and supplementary Fig. II were used to calculate K0.5 and apparent Vmax using Hill plots (see supplementary Figs. I and III). LPCAT, lysophosphatidylcholine acyltransferase. The kinetics of LPCAT3 were further analyzed by performing a lysoPC substrate concentration series with oleoyl-CoA as the acyl donor (Fig. 3). Again, cooperativity was observed with a Hill plot showing a Hill number of 1.7, K0.5 of 13.1 μM, and an apparent Vmax of 486 nmol/min/mg. Since oleoyl-CoA was kept at 50 μM, a value that turned out to be less than saturating, the Vmax was less than that observed for oleoyl-CoA. Further analysis was performed with a single concentration (50 μM) of lysoPC species with varying sn-1 acyl chains (Fig. 4). Neither length nor degree of saturation affected utilization. However, the ether linkage in 1-alkyl lysoPC abrogated activity (data not shown). Varying the head group on the lysoPL also had a distinct effect on activity. Comparing 14:0 lysoPE to 14:0 lysoPC and 18:1 lysoPS to 18:1 lysoPC shows a 7-fold preference for lysoPC (Fig. 4). However, other species of lysoPE and lysoPS may be utilized to a greater degree. Esterification of lysophosphatidic acid (lysoPA), lysoPG, and lysoPI was assayed for but not detected (data not shown). This was also the case for lysoPS at 37°C (see supplementary Fig. IV). The structural features common to the acyl-acceptors utilized by LPCAT3 are an amine in the head group and a sn-1 acyl group attached through an ester linkage.Fig. 4LysoPL substrate specificity of LPCAT3. LPLAT activity was measured using 50 μM oleoyl-CoA and 50 μM of lysoPC (1-myristoyl (C14:0), 1-palmitoyl (C16:0), 1-stearoyl (C18:0), 1-oleoyl (C18:1)), 1-myristoyl (14:0) lysoPE, and 1-oleoyl (18:1) lysoPS at 28°C. The spectrophotometric method was used, as described in Materials and Methods, with lysates prepared from LPCAT3 or GST expressing Sf9 cells. The data represents mean ± SE; n = 4. Data from parallel assays at 37°C are shown in supplementary Fig. IV. GST, glutathione S-transferase; LPCAT, lysophosphatidylcholine acyltransferase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To begin to examine the physiological role of LPCAT3 in PL metabolism, the effect of expressing LPCAT3 on the PL profile in Sf9 cells was measured. ESI-MS was performed on four independently generated cell lysates expressing LPCAT3 and GST. To account for alteration in ionization efficiency, the relative abundance of each PL species was expressed relative to the most abundant species (34:1 PC; m/z 760) within the sample. Normalizing to an added standard (28:0 PC, m/z 678) was also performed and showed similar trends (data not shown). After analyzing the abundance of 73 different m/z values in the positive mode, 10 showed a significant difference between the LPCAT3 and GST expressing cells. Assigning specific PL
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