Identification and Characterization of a Gene Encoding Human LPGAT1, an Endoplasmic Reticulum-associated Lysophosphatidylglycerol Acyltransferase
2004; Elsevier BV; Volume: 279; Issue: 53 Linguagem: Inglês
10.1074/jbc.m406710200
ISSN1083-351X
AutoresYanzhu Yang, Jingsong Cao, Yuguang Shi,
Tópico(s)Pancreatic function and diabetes
ResumoPhosphatidylglycerol (PG) is an important membrane polyglycerolphospholipid required for the activity of a variety of enzymes and is a precursor for synthesis of cardiolipin and bis(monoacylglycerol) phosphate. PG is subjected to remodeling subsequent to its de novo biosynthesis to incorporate appropriate acyl content for its biological functions and to prevent the harmful effect of lysophosphatidylglycerol (LPG) accumulation. The enzymes involved in the remodeling process have not yet been identified. We report here the identification and characterization of a human gene encoding an acyl-CoA: lysophosphatidylglycerol acyltransferase (LPGAT1). Expression of the LPGAT1 cDNA in Sf9 insect and COS-7 cells led to a significant increase in LPG acyltransferase activity. In contrast, no significant acyltransferase activities were detected against glycerol 3-phosphate or a variety of lysophospholipids, including lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylserine. The recombinant human LPGAT1 enzyme recognized various acyl-CoAs and LPGs as substrates but demonstrated clear preference to long chain saturated fatty acyl-CoAs and oleoyl-CoA as acyl donors, which is consistent with the lipid composition of endogenous PGs identified from different tissues. Kinetic analyses of LPGAT1 expressed in COS-7 cells showed that oleoyl-LPG was preferred over palmitoyl-LPG as an acyl receptor, whereas oleoyl-CoA was preferred over lauroyl-CoA as an acyl donor. Consistent with its proposed microsomal origin, LPGAT1 was localized to the endoplasmic reticulum by subcellular fractionation and immunohistochemical analyses. Northern blot analysis indicated that the human LPGAT1 was widely distributed, suggesting a dynamic functional role of the enzyme in different tissues. Phosphatidylglycerol (PG) is an important membrane polyglycerolphospholipid required for the activity of a variety of enzymes and is a precursor for synthesis of cardiolipin and bis(monoacylglycerol) phosphate. PG is subjected to remodeling subsequent to its de novo biosynthesis to incorporate appropriate acyl content for its biological functions and to prevent the harmful effect of lysophosphatidylglycerol (LPG) accumulation. The enzymes involved in the remodeling process have not yet been identified. We report here the identification and characterization of a human gene encoding an acyl-CoA: lysophosphatidylglycerol acyltransferase (LPGAT1). Expression of the LPGAT1 cDNA in Sf9 insect and COS-7 cells led to a significant increase in LPG acyltransferase activity. In contrast, no significant acyltransferase activities were detected against glycerol 3-phosphate or a variety of lysophospholipids, including lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylserine. The recombinant human LPGAT1 enzyme recognized various acyl-CoAs and LPGs as substrates but demonstrated clear preference to long chain saturated fatty acyl-CoAs and oleoyl-CoA as acyl donors, which is consistent with the lipid composition of endogenous PGs identified from different tissues. Kinetic analyses of LPGAT1 expressed in COS-7 cells showed that oleoyl-LPG was preferred over palmitoyl-LPG as an acyl receptor, whereas oleoyl-CoA was preferred over lauroyl-CoA as an acyl donor. Consistent with its proposed microsomal origin, LPGAT1 was localized to the endoplasmic reticulum by subcellular fractionation and immunohistochemical analyses. Northern blot analysis indicated that the human LPGAT1 was widely distributed, suggesting a dynamic functional role of the enzyme in different tissues. The anionic phospholipid phosphatidylglycerol (PG) 1The abbreviations used are: PG, phosphatidylglycerol; GPAT, glycerol-3-phosphate acyltransferase; LPG, lysophosphatidylglycerol; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; LPA, lysophosphatidic acid; LPE, lysophosphatidylethanolamine; LPC, lysophosphatidylcholine; LPI, lysophosphatidylinositol; LPS, lysophosphatidylserine; DAPI, 4,6-diamidino-2-phenylindole; LPGAT1, acyl-CoA:lysophosphatidylglycerol acyltransferase; CDP-DAG, cytidine diphosphate-diacylglycerol; PGP, phosphatidylglycerol phosphate.1The abbreviations used are: PG, phosphatidylglycerol; GPAT, glycerol-3-phosphate acyltransferase; LPG, lysophosphatidylglycerol; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; LPA, lysophosphatidic acid; LPE, lysophosphatidylethanolamine; LPC, lysophosphatidylcholine; LPI, lysophosphatidylinositol; LPS, lysophosphatidylserine; DAPI, 4,6-diamidino-2-phenylindole; LPGAT1, acyl-CoA:lysophosphatidylglycerol acyltransferase; CDP-DAG, cytidine diphosphate-diacylglycerol; PGP, phosphatidylglycerol phosphate. is a common polyglycerolphospholipid required for many cellular functions (1Dowhan W. Annu. Rev. Biochem. 1997; 66: 199-232Crossref PubMed Scopus (773) Google Scholar). PG represents ∼1% of total phospholipids in most mammalian tissues, except for lung, and is found in many subcellular locations, such as microsomal, mitochondrial, and nuclear membranes (2Hostetler K.Y. Hawthorne J.N. Ansell G.B. Phospholipids. Elsevier/North Holland Biomedical Press, Amsterdam1982: 215-261Google Scholar, 3Hallman M. Gluck L. Biochim. Biophys. Acta. 1975; 409: 172-191Crossref PubMed Scopus (92) Google Scholar). In lung, PG represents ∼5% of total phospholipids and is a major component of lung surfactant (2Hostetler K.Y. Hawthorne J.N. Ansell G.B. Phospholipids. Elsevier/North Holland Biomedical Press, Amsterdam1982: 215-261Google Scholar, 3Hallman M. Gluck L. Biochim. Biophys. Acta. 1975; 409: 172-191Crossref PubMed Scopus (92) Google Scholar). PG plays an important role in maintaining the normal function of lung, and absence of PG during fetal development is associated with a high risk of neonatal respiratory distress syndrome (4Hallman M. Kulovich M. Kirkpatrick E. Sugarman R.G. Gluck L. Am. J. Obstet. Gynecol. 1976; 125: 613-617Abstract Full Text PDF PubMed Scopus (231) Google Scholar, 5Greenough A. Eur. J. Pediatr. 1998; 157: 16-18Crossref PubMed Google Scholar). Biochemical evidence indicates that PG is a potential activator of members of the protein kinase C family, including protein kinase C-θ (6Schagger H. Biochim. Biophys. Acta. 2002; 1555: 154-159Crossref PubMed Scopus (302) Google Scholar) and nuclear protein kinase C-βII (7Murray N.R. Fields A.P. J. Biol. Chem. 1998; 273: 11514-11520Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). PG is also a precursor for the biosynthesis of bis(monoacylglycerol) phosphate that plays an important role in liposome formation and endosome organization (8Kobayashi T. Stang E. Fang K.S. Demoerloose P. Parton R.G. Gruenberg J. Nature. 1998; 392: 193-197Crossref PubMed Scopus (647) Google Scholar, 9Matsuo H. Chevallier J. Mayran N. Le Blanc I. Ferguson C. Faure J. Blanc N.S. Matile S. Dubochet J. Sadoul M. Parton R.G. Vilbois F. Gruenberg J. Science. 2004; 303: 531-534Crossref PubMed Scopus (522) Google Scholar). Bis(monoacylglycerol) phosphate is also a predominant phospholipid of macrophages (10Thornburg T. Miller C. Thuren T. King L. Waite M. J. Biol. Chem. 1991; 266: 6834-6840Abstract Full Text PDF PubMed Google Scholar, 11Amidon B. Schmitt J.D. Thuren T. King L. Waite M. Biochemistry. 1995; 34: 5554-5560Crossref PubMed Scopus (32) Google Scholar, 12Heravi J. Waite M. Biochim. Biophys. Acta. 1999; 1437: 277-286Crossref PubMed Scopus (19) Google Scholar) and a target antigen for human antibodies associated with antiphospholipid syndrome characterized by increased risk of thrombosis, recurrent fetal loss, and thrombocytopenia (13Rand J.H. Annu. Rev. Med. 2003; 54: 409-424Crossref PubMed Scopus (76) Google Scholar).The de novo biosynthesis of PG occurs via the cytidine diphosphate-diacylglycerol (CDP-DAG) pathway that begins with formation of CDP-DAG from phosphatidic acid catalyzed by phosphatidic acid:CTP cytidylyltransferase. CDP-DAG is then converted to PG by sequential action of phosphatidylglycerol phosphate (PGP) synthase and PGP phosphatase (Fig. 1) (14Kawasaki K. Kuge O. Chang S.C. Heacock P.N. Rho M. Suzuki K. Nishijima M. Dowhan W. J. Biol. Chem. 1999; 274: 1828-1834Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). PG is an important precursor for the synthesis of cardiolipin, a polyglycerolphospholipid that is required for the activity of a large number of mitochondrial enzymes and carrier proteins (15Schlame M. Rua D. Greenberg M.L. Prog. Lipid Res. 2000; 39: 257-288Crossref PubMed Scopus (656) Google Scholar). Consequently, disruption of phosphatidylglycerophosphate synthase (PGS) gene in yeast causes PG and cardiolipin deficiency and inhibition of growth on nonfermentable carbon sources (16Chang S.C. Heacock P.N. Clancey C.J. Dowhan W. J. Biol. Chem. 1998; 273: 9829-9836Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). In yeast cells, PG appears to substitute for cardiolipin functions (17Chang S.C. Heacock P.N. Mileykovskaya E. Voelker D.R. Dowhan W. J. Biol. Chem. 1998; 273: 14933-14941Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), as evidenced by a significant increase in PG content resulted from disruption of the CRD1 gene that encodes cardiolipin synthase (15Schlame M. Rua D. Greenberg M.L. Prog. Lipid Res. 2000; 39: 257-288Crossref PubMed Scopus (656) Google Scholar, 18Jiang F. Ryan M.T. Schlame M. Zhao M. Gu Z.M. Klingenberg M. Pfanner N. Greenberg M.L. J. Biol. Chem. 2000; 275: 22387-22394Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). PG and cardiolipin deficiency in Chinese hamster ovary cells caused by a mutation in the PGS gene results in mitochondrial morphological and functional abnormalities manifested by increased glycolysis, reduced oxygen consumption, stringent temperature sensitivity for cell growth in glucose-deficient medium, and reduced ATP production. In support for a possible role of PG in oxidative phosphorylation, decreased cardiac PG levels were reported in streptozotocin-induced diabetic rats (19Makino N. Dhalla K.S. Elimban V. Dhalla N.S. Am. J. Physiol. 1987; 253: E202-E207PubMed Google Scholar, 20Hatch G.M. Cao S.G. Angel A. Biochem. J. 1995; 306: 759-764Crossref PubMed Scopus (45) Google Scholar).Phospholipids are known to undergo a rapid deacylationreacylation recycling process to incorporate appropriate fatty acyl composition back to their parent molecules. The re-acylation process is also believed to be important in preventing the accumulation of harmful lysophospholipids. For example, analysis of lipid composition of the rat liver phosphatidylethanolamine after injection of [14C]linoleic acid indicates that 95% of the linoleic acid in 1-stearoyl-2-linoleoylphosphatidylethanolamine was incorporated by means of deacylation and reacylation (21Akesson B. Biochim. Biophys. Acta. 1970; 218: 57-70Crossref PubMed Scopus (58) Google Scholar). The deacylation-reacylation cycle for the remodeling of phosphatidylcholine in mammals has been extensively investigated (22Choy P.C. Skrzypczak M. Lee D. Jay F.T. Biochim. Biophys. Acta. 1997; 1348: 124-133Crossref PubMed Scopus (26) Google Scholar, 23MacDonald J.I. Sprecher H. Biochim. Biophys. Acta. 1991; 1084: 105-121Crossref PubMed Scopus (246) Google Scholar). Likewise, cardiolipin is remodeled by the process subsequent to its de novo synthesis to attain the unique composition of monounsaturated and diunsaturated chains of C18 at the four acyl positions. Defective remodeling of PG and cardiolipin are part of the pathophysiology associated with Barth syndrome (24Vreken P. Valianpour F. Nijtmans L.G. Grivell L.A. Plecko B. Wanders R.J. Barth P.G. Biochem. Biophys. Res. Commun. 2000; 279: 378-382Crossref PubMed Scopus (301) Google Scholar, 25Valianpour F. Wanders R.J. Overmars H. Vreken P. Van Gennip A.H. Baas F. Plecko B. Santer R. Becker K. Barth P.G. J. Pediatr. 2002; 141: 729-733Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 26Schlame M. Kelley R.I. Feigenbaum A. Towbin J.A. Heerdt P.M. Schieble T. Wanders R.J. DiMauro S. Blanck T.J. J. Am. Coll. Cardiol. 2003; 42: 1994-1999Crossref PubMed Scopus (156) Google Scholar), an X-linked cardioskeletal myopathy and neutropenia caused by mutations of an acyltransferase gene (27Bione S. D'Adamo P. Maestrini E. Gedeon A.K. Bolhuis P.A. Toniolo D. Nat. Genet. 1996; 12: 385-389Crossref PubMed Scopus (599) Google Scholar). An enzyme responsible for the acylation of monolysocardiolipin to cardiolipin, monolysocardiolipin acyltransferase, has been characterized in the rat liver and heart (28Schlame M. Rustow B. Biochem. J. 1990; 272: 589-595Crossref PubMed Scopus (104) Google Scholar, 29Ma B.J. Taylor W.A. Dolinsky V.W. Hatch G.M. J. Lipid Res. 1999; 40: 1837-1845Abstract Full Text Full Text PDF PubMed Google Scholar, 30Taylor W.A. Hatch G.M. J. Biol. Chem. 2003; 278: 12716-12721Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and a murine gene encoding a acyl-CoA:lysocardiolipin acyltransferase has recently been cloned and characterized (Fig. 1) (31Cao J. Liu Y. Lockwood J. Burn P. Shi Y. J. Biol. Chem. 2004; 279: 31727-31734Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar).Although lysophosphatidylglycerol acyltransferase (LPGAT) activity has been reported from microsomes of various tissues, including liver (32Wittels B. J. Biol. Chem. 1973; 248: 2906-2911Abstract Full Text PDF PubMed Google Scholar), lung (33Funkhouser J.D. Batenburg J.J. Van Golde L.M. Biochim. Biophys. Acta. 1981; 666: 1-6Crossref PubMed Scopus (20) Google Scholar, 34Sanford G.L. Frosolono M.F. Biochem. Biophys. Res. Commun. 1983; 116: 23-29Crossref PubMed Scopus (6) Google Scholar), and heart (35Cheng P. Dolinsky V. Hatch G.M. Biochim. Biophys. Acta. 1996; 1302: 61-68Crossref PubMed Scopus (11) Google Scholar), the molecular and biochemical nature of the enzyme has not been characterized. In the present study, we report the identification and characterization of a human gene encoding polypeptide possessing activities of LPGAT, designated as LPGAT1. The recombinant LPGAT1 expressed in both Sf9 insect and COS-7 cells catalyzed efficiently the reacylation of LPG to PG with various LPGs and acyl-CoAs as substrates but preferred long saturated fatty acyl-CoAs as acyl donors. Subcellular localization analyses indicated that LPGAT1 was localized in the ER, which is consistent with its proposed microsomal origin.MATERIALS AND METHODSCloning of the Full-length Lysophosphatidylglycerol Acyltransferase cDNA—A human cDNA clone (NCBI accession number BC034621) was identified in NCBI data bases based on its conserved sequence motifs with the human glycerol-3-phosphate acyltransferase (GPAT) (36Lewin T.M. Wang P. Coleman R.A. Biochemistry. 1999; 38: 5764-5771Crossref PubMed Scopus (229) Google Scholar) as a candidate gene for a novel GPAT. This GPAT candidate gene was initially named hGPAT4, and subsequently changed its name to acyl-CoA:lysophosphatidylglycerol acyltransferase (LPGAT1) after functional characterization. A PCR primer pair (forward, 5′-CGGAGTCCAGTGTGAGAATGGCTA-3′, and reverse, 5′-ACGGTGACCTTGACAAGTCCACGT-3′) was used to amplify the full-length coding region of the human LPGAT1 gene from Marathon-Ready cDNA prepared from the human liver (Clontech). Amplification was performed by PCR using Pfx DNA polymerase (Invitrogen) and a thermal cycling condition of 35 cycles (94 °C for 30 s, 62 °C for 30 s, and 68 °C for 2 min), resulting in a 1.2-kb cDNA product that was cloned into the SrfI site of pPCR-script Amp SK(+) vector (Stratagene, La Jolla, CA) and sequenced.Northern Blot Analysis—To analyze the tissue distribution pattern of human LPGAT1 mRNA, multiple human tissue poly(A)+ RNA Northern blots (Clontech) were hybridized with [α-32P]dCTP (3000 Ci/mmol, ICN Radiochemicals)-labeled probes prepared from full-length cDNA of human LPGAT1 gene using a Prime-It RmT Random Primer Labeling Kit (Stratagene, La Jolla, CA). Hybridization was carried out in ULTRAHyb (Ambion, Austin, TX) at 55 °C overnight, followed by three washes at 55 °C in 2× SSC buffer containing 0.1% SDS and 1 mm EDTA. Blots were stripped with boiling 1% SDS to remove radiolabeled probe and re-probed with human radiolabeled G3PDH cDNA as an internal control. The blots were exposed to a PhosphorImager Screen to visualize signals and were quantified by ImageQuant (Amersham Biosciences).Expression of LPGAT1 in Insect Cells—Expression of LPGAT1 in insect cells was performed by using a Bac-to-Bac Baculovirus Expression System (Invitrogen). The human LPGAT1 cDNA that carries the entire coding region was subcloned into EcoRI and NotI sites of the pFastBac vector (Invitrogen), which was subsequently transformed into DH10Bac™ Escherichia coli cells to generate a recombinant bacmid that carries the insertion of the LPGAT1 cDNA. High titer recombinant baculovirus was generated by transfecting the bacmid DNA into Spodoptera frugiperda 9 (Sf9) insect cells followed by several rounds of amplification to increase viral titer. After infection with recombinant baculoviruses for 65 h, Sf9 cells were harvested in ice-cold phosphate-buffered saline (PBS), pelleted by centrifugation, lysed, and assayed immediately for enzyme activity or frozen in liquid N2 for later use. Cell pellets were homogenized in 20 mm NaCl with 20 up-and-down strokes in a motor-driven Dounce homogenizer (Heidolph, Germany) followed by three passages through a 27-gauge needle. The protein concentration in homogenate was determined by a BCA Protein Assay Kit (Pierce) according to the manufacturer's instructions.Expression of LPGAT1 and FLAG-LPGAT1 in Mammalian Cells—A mammalian expression vector for full-length human LPGAT was engineered by subcloning the 1.2-kb cDNA fragment from the pPCR-script Amp SK(+) vector described above into the HindIII and NotI sites of the pcDNA3.1(+)/Hygro mammalian expression vector (Invitrogen). A FLAG-tagged version of LPGAT1 was engineered by PCR amplification with Pfx DNA polymerase using a primer pair (forward, 5′-GCCACCATGGATTACAAGGATGACGACGATAAGGCTATAACTTTGGAAGAAGCT-3′, and reverse, 5′-ACGGTGACCTTGACAAGTCCACGT-3′) designed to add a FLAG tag to the N terminus of LPGAT1 and a thermal cycling condition of 35 cycles (94 °C for 30 s, 53 °C for 30 s, and 68 °C for 2 min). The amplified DNA fragment was cloned into the pPCR-script Amp SK(+) vector (Stratagene, La Jolla, CA) and verified by sequencing. The insert was then subcloned into the HindIII and NotI sites of pcDNA3.1(+)/Hygro vector for transient expression in COS-7 cells. COS-7 cells were maintained under the conditions recommended by American Tissue Culture Collection (ATCC, Manassas, VA). A day before transfection, two million cells were subcultured onto a 100 × 20-mm plate resulting in ∼70% confluence. Cells were transfected with 10 μg of DNA premixed with FuGENE 6 (Roche Diagnostics) according to the manufacturer's instructions. Forty four hours after the transfection, cells were harvested in ice-cold PBS, pelleted by centrifugation, lysed, and assayed immediately or frozen in liquid N2 for later use.In Vitro Assays for GPAT Activity—GPAT activity was determined by conversion of glycerol 3-phosphate to 1-acyl-sn-glycerol 3-phosphate in the presence of acyl-CoA. The reaction was conducted at room temperature in a final volume of 200 μl as described previously (37Haldar D. Vancura A. Methods Enzymol. 1992; 209: 64-72Crossref PubMed Scopus (22) Google Scholar, 38Yet S.F. Moon Y.K. Sul H.S. Biochemistry. 1995; 34: 7303-7310Crossref PubMed Scopus (52) Google Scholar). The reaction mixture contained 75 mm Tris/HCl, pH 7.4, 4 mm MgCl2, 1 mg/ml bovine serum albumin free fatty acids, 8 mm NaF, 50 μm palmitoyl-CoA, 3 mm glycerol 3-phosphate, 1-2.5 μCi of [3H]glycerol 3-phosphate (20 Ci/mmol, American Radiolabeled Chemicals Inc, St. Louis, MO), and 50 μg of cellular homogenate. The reaction was initiated by adding protein homogenate, incubated at room temperature for 10 min, and terminated by adding 0.5 ml of water-saturated 1-butanol. The product from the GPAT enzyme assay was extracted and quantified as described previously (37Haldar D. Vancura A. Methods Enzymol. 1992; 209: 64-72Crossref PubMed Scopus (22) Google Scholar).In Vitro Assays for Lysophospholipid Acyltransferase Activities—The human LPGAT1 was assayed for lysophospholipid acyltransferase activities by measuring the incorporation of radiolabeled acyl moieties of acyl-CoAs (acyl donors) into phospholipids in the presence of relevant lysophospholipids (acyl acceptors). The lysophospholipids used in the experiment include lysophosphatidic acid (LPA), lysophosphatidylethanolamine (LPE), lysophosphatidylcholine (LPC), lysophosphatidylinositol (LPI), lysophosphatidylserine (LPS) (Sigma), and lysophosphatidylglycerol (LPG) (Avanti Polar Lipids, Inc., Alabaster, AL). The reaction mixture contained 80 mm Tris/HCl, pH 7.4, 200 μm lysophospholipid, 20 μm [14C]acyl-CoA (50 mCi/mmol, American Radiolabeled Chemicals Inc), and cell homogenate (50 μg) that contained recombinant LPGAT1 expressed in Sf9 or COS-7 cells, in a total volume of 200 μl. The reaction was initiated by addition of the protein homogenate and was terminated by adding 1 ml of chloroform/methanol (2:1, v/v), after 10 min of incubation at room temperature. The phospholipid product was extracted and separated on TLC plate as described previously (39Choy P.C. Tardi P.G. Mukherjee J.J. Methods Enzymol. 1992; 209: 80-86Crossref PubMed Scopus (14) Google Scholar).In Vitro Assays for LPGAT Activity—Enzymatic reaction was initiated by addition of 50 μg of cell homogenates that contained the recombinant LPGAT1 expressed in COS-7 cells and incubated for 10 min at room temperature in a 200-μl reaction mixture that contained 80 mm Tris/HCl, pH 7.0, 20 μm [14C]acyl-CoA (50 mCi/mmol), and 200 μm of various LPGs, including 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)](sodium salt), 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)](sodium salt), 1-stearoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)](sodium salt), and 1-oleoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)](sodium salt) (Avanti Polar Lipids, Inc., Alabaster, AL). Radiolabeled acyl-CoAs used in the studies were n-octanoyl-CoA, lauroyl-CoA, palmitoyl-CoA, stearoyl-CoA, oleoyl-CoA, linoleoyl-CoA, and arachidonyl-CoA (American Radiolabeled Chemicals Inc., St. Louis, MO). The reaction was terminated by adding 1 ml of chloroform/methanol (2:1, v/v), followed by addition of 0.4 ml of 0.9% KCl to facilitate phase separation. After vigorous vortex for 10 s to extract lipids, phase separation was performed by a brief centrifugation. Aliquots of the organic phase containing phospholipids were dried under a N2 stream and separated by the Linear-K Preadsorbent TLC Plate (Waterman Inc., Clifton, NJ) with the chloroform/methanol/water (65:25:4, v/v). Individual lipid moieties were identified by standards with exposure to I2 vapor.Statistical Analysis—Differences in enzyme activities between different substrates were analyzed by one-way analysis of variance and Student t test.Western Blot Analysis—COS-7 cells transfected with FLAG-tagged LPGAT1 expression vector or empty vector were harvested in Bug Buster HT buffer (Novagen) in the presence of 1× Complete protease inhibitors (Roche Diagnostics) and incubated for 10 min at room temperature with 1% SDS, followed by centrifugation at 20,000 × g for 5 min. The supernatant was used to detect the expression of LPGAT1 by Western analyses. The protein lysate was denatured by boiling for 3 min in 1× loading buffer containing NuPAGE reducing agent, resolved on 4-20% NOVEX Tris-glycerin SDS-PAGE (Invitrogen), and transferred to a nitrocellulose membrane. The membrane was incubated for 2 h at room temperature in washing buffer (0.9% NaCl, 20 mm Tris/HCl, pH 7.5, 0.1% Tween 20) containing 5% nonfat milk to block nonspecific binding. The blots were then incubated with mouse monoclonal anti-FLAG M2 antibody (1.0 μg/ml, Sigma) overnight at 4 °C in the washing buffer. After four washes (5 min each), the membrane was incubated with anti-mouse IgG horseradish peroxidase conjugate (1:5000, Amersham Biosciences) for 1 h at room temperature. The blots were washed four times (5 min each) and visualized with ECL Plus (Amersham Biosciences). The signal was scanned by a PhosphorImager (Amersham Biosciences) and quantified by ImageQuant software.Subcellular Fractionation—Subcellular fractionation analysis was carried out to localize human LPGAT1 transiently expressed in COS-7 cells. Cell pellets were homogenized with a Dounce homogenizer in 10 volumes (w/v) of solution consisting of 0.25 m sucrose, 0.01 m Tris/HCl, pH 7.4, and 1 mm EDTA. The homogenate was first centrifuged at 800 × g for 10 min to remove cell debris and nuclear fractions. The mitochondrial fraction was obtained by centrifuging the supernatant at 8,000 × g for 10 min. Microsomal fraction was prepared from the postmitochondrial supernatant by sedimentation at 100,000 × g for 60 min. The mitochondrial and microsomal fraction was suspended in PBS buffer and stored in aliquots at -80 °C.Immunocytohistochemistry—Cells were grown and transfected on a coverslip (BD Biosciences). Forty eight hours after transfection, cells were first incubated in the growth medium with 100 nm MitoTracker Red CMXRos for 10 min at 37 °C to achieve the specific staining for mitochondria. The cells were then washed two times with PBS (2 min each) and fixed with freshly prepared 4.0% paraformaldehyde pre-warmed at 37 °C. The samples were rinsed twice with PBS (5 min each) and permeabilized with 0.2% Triton X-100 in PBS, followed by incubation for 1 h in 5% normal donkey serum to block nonspecific binding. The samples were then incubated for 2 h at room temperature with mouse monoclonal anti-FLAG M2 antibody (5.0 μg/ml, Sigma) or rabbit anti-calnexin N-terminal polyclonal antibody (1.0 μg/ml, StressGen Biotechnologies Corp, Victoria, Canada). After brief wash with PBS three times, the samples were incubated for 1 h at room temperature with Cy2-conjugated donkey anti-mouse IgG or Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). The cells were also counterstained with DAPI (Molecular Probes, Eugene, OR) to visualize nuclei. The samples were washed four times with PBS and analyzed with a confocal fluorescence microscope (Olympus BX61, Nashua, NH).RESULTSIdentification and Cloning of the Human LPGAT1 Gene—A human LPGAT1 cDNA clone (NCBI accession number BC034621) was initially identified in NCBI data bases based on its conserved sequence motifs with the human GPAT (36Lewin T.M. Wang P. Coleman R.A. Biochemistry. 1999; 38: 5764-5771Crossref PubMed Scopus (229) Google Scholar) as a candidate gene for a novel GPAT. A 1.2-kb cDNA fragment encoding the full-length human LPGAT1 enzyme was cloned by PCR amplification using a cDNA library from human liver and a primer pair designed from the 5′- and 3′-untranslated regions, respectively, of the predicted human LPGAT1 open reading frame. Sequence analysis of the 1.2-kb cDNA suggests a full-length coding region for the enzyme as evidenced by an in-frame stop codon located 110-bp 5′-upstream of the predicted AUG start site embedded within a Kozak consensus sequence for translation initiation. The human LPGAT1 gene predicts a 370-amino acid protein of 43 kDa that carries a 23-amino acid signal-anchor domain at the N terminus and a potential N-linked glycosylation site at position 209 (Fig. 2). The predicted LPGAT1 peptide sequence carries motifs that match all the four conserved domains among members of the GPAT family (36Lewin T.M. Wang P. Coleman R.A. Biochemistry. 1999; 38: 5764-5771Crossref PubMed Scopus (229) Google Scholar), suggesting a functional role as a potential GPAT enzyme. The human LPGAT1 gene is localized on the human chromosome 1 at 1p36.13-q42.3. Results from blast analyses of the public genomic and EST data bases suggest that the LPGAT1 gene is highly conserved throughout eukaryotic species, from fungi to mammals. An ortholog from Caenorhabditis elegans (NCBI accession number NM_073010) shares 38.4% identity and 54.4% similarity, respectively, with the predicted human LPGAT1 peptide sequence.Fig. 2Features of the predicated LPGAT1 peptide sequence. A, the predicted peptide sequence of the human LPGAT1 enzyme. Underlined is a signal-anchor domain located at the N terminus. The conserved sequence motifs for glycerophospholipid acyltransferases are highlighted by red, and the only predicted N-glycosylation site is indicated by blue boldface. B, the hydrophobicity profile of the human LPGAT1 protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Tissue Distribution of the Human LPGAT1 mRNA—The tissue distribution of the human LPGAT1 mRNA was analyzed by Northern blot analysis. Two mRNA splice isoforms of 7.5 and 9.5 kb in length, respectively, were detected in most human tissues examined including peripheral blood, liver, lung, placenta, kidney, and brain (Fig. 3). When normalized with the mRNA level of G3PDH, the expression level was the highest in liver and placenta. In contrast, very low level of expression was detected in human colon.Fig. 3Tissue distribution of the human LPGAT1 mRNAs as examined by Northern blot analyses. A human multiple tissue blot with 2 μg of poly(A)+ RNA from each tissue was hybridized with radiolabeled probe prepared from the full-length cDNA clone of the human LPGAT1 gene (upper panel). The same blots were stripped off residu
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