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• W2000664329 abstract "Sequence analysis of cDNA clones corresponding to a number of genes located in the class III region of the human major histocompatibility complex (MHC), in the chromosome band 6p21.3, has shown that the G15 gene encodes a 283-amino acid polypeptide with significant homology over the entire polypeptide with the enzyme lysophosphatidic acid acyltransferase (LPAAT) from different yeast, plant, and bacterial species. The amino acid sequence of the MHC-encoded human LPAAT (hLPAATα) is 48% identical to the recently described hLPAAT (Eberhardt, C., Gray, P. W., and Tjoelker, L. W. (1997) J. Biol. Chem.272, 20299–20305), which is encoded by a gene located on chromosome 9p34.3. LPAAT is the enzyme that in lipid metabolism converts lysophosphatidic acid (LPA) into phosphatidic acid (PA). The expression of the hLPAATα polypeptide in the baculovirus system and in mammalian cells has shown that it is an intracellular protein that contains LPAAT activity. Cell extracts from insect cells overexpressing hLPAATα were analyzed in different LPAAT enzymatic assays using, as substrates, different acyl acceptors and acyl donors. These cell extracts were found to contain up to 5-fold more LPAAT activity compared with control cell extracts, indicating that the hLPAATα specifically converts LPA into PA, incorporating different acyl-CoAs with different affinities. The hLPAATα polypeptide expressed in the mammalian Chinese hamster ovary cell line was found, by confocal immunofluorescence, to be localized in the endoplasmic reticulum. Due to the known role of LPA and PA in intracellular signaling and inflammation, the hLPAATα gene represents a candidate gene for some MHC-associated diseases. Sequence analysis of cDNA clones corresponding to a number of genes located in the class III region of the human major histocompatibility complex (MHC), in the chromosome band 6p21.3, has shown that the G15 gene encodes a 283-amino acid polypeptide with significant homology over the entire polypeptide with the enzyme lysophosphatidic acid acyltransferase (LPAAT) from different yeast, plant, and bacterial species. The amino acid sequence of the MHC-encoded human LPAAT (hLPAATα) is 48% identical to the recently described hLPAAT (Eberhardt, C., Gray, P. W., and Tjoelker, L. W. (1997) J. Biol. Chem.272, 20299–20305), which is encoded by a gene located on chromosome 9p34.3. LPAAT is the enzyme that in lipid metabolism converts lysophosphatidic acid (LPA) into phosphatidic acid (PA). The expression of the hLPAATα polypeptide in the baculovirus system and in mammalian cells has shown that it is an intracellular protein that contains LPAAT activity. Cell extracts from insect cells overexpressing hLPAATα were analyzed in different LPAAT enzymatic assays using, as substrates, different acyl acceptors and acyl donors. These cell extracts were found to contain up to 5-fold more LPAAT activity compared with control cell extracts, indicating that the hLPAATα specifically converts LPA into PA, incorporating different acyl-CoAs with different affinities. The hLPAATα polypeptide expressed in the mammalian Chinese hamster ovary cell line was found, by confocal immunofluorescence, to be localized in the endoplasmic reticulum. Due to the known role of LPA and PA in intracellular signaling and inflammation, the hLPAATα gene represents a candidate gene for some MHC-associated diseases. Lysophosphatidic acid acyltransferase (LPAAT), 1The abbreviations used are: LPAAT, lysophosphatidic acid acyltransferase; hLPAAT, human LPAAT; MHC, major histocompatibility complex; LPA, lysophosphatidic acid; PA, phosphatidic acid; ER, endoplasmic reticulum; DAG, diacylglycerol; PC, phosphatidylcholine; IL, interleukin; mAb, monoclonal antibody; LPE, lysophosphatidylethanolamine; LPC, lysophosphatidylcholine; bp, base pair(s); PCR, polymerase chain reaction; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid). also known as 1-acyl-sn-glycerol-3-phosphate acyltransferase (EC2.3.1.51), is the enzyme that converts lysophosphatidic acid (LPA) into phosphatidic acid (PA) in the lipid metabolism. LPA or 1-acyl-sn-glycerol 3-phosphate consists of a glycerol backbone with a fatty acyl chain at the sn-1 position, a hydroxyl group at the sn-2 position, and a phosphate group at the sn-3 position. In the endoplasmic reticulum (ER) membrane, LPA is formed from glycerol 3-phosphate through the action of glycerol-3-phosphate acyltransferase. LPA is then further acylated in the ER by LPAAT to yield PA, the precursor of all glycerolipids. The rate of acylation of LPA to PA is very high, and consequently, there is little accumulation of LPA at the site of biosynthesis. PA can either be hydrolyzed by phosphatidic acid phosphohydrolase to yield diacylglycerol (DAG) or, alternatively, can be converted to CDP-DAG for the synthesis of more complex phospholipids in the ER, from which they are transported to different subcellular compartments. PA can be produced by this de novo synthesis or, alternatively, by phospholipase D hydrolysis of phospholipids, such as phospha- tidylcholine (PC) and phosphatidylethanolamine, or through phosphorylation of DAG by DAG kinase (for a general description, see Refs. 1Voet D. Voet J.G. Biochemistry. 2nd Ed. John Wiley & Sons, Inc., New York1995Google Scholar and 2Lodish H. Baltimore D. Berk A. Zipursky S.L. Matsudaira P. Darnell J. Molecular Cell Biology. 3rd Ed. Scientific American Books, New York1995Google Scholar). Naturally occurring glycerolipids generally exhibit a nonrandom distribution of the acyl constituents; saturated fatty acids are esterified predominantly at the C-1 position and unsaturated fatty acids at the C-2 position (for a review see Refs. 3Yamashita S. Hosaka K. Miki Y. Numa S. Methods Enzymol. 1981; 71: 528-536Crossref PubMed Scopus (26) Google Scholar and 4Bell R.M. Coleman R.A. Boyer P.D. 3rd Ed. The Enzymes. XVI. Academic Press, Inc., New York1983: 87-111Google Scholar). Previous studies carried out using a semipurified LPAAT from rat liver microsomes indicated that LPAAT exhibited a significant acyl donor specificity for monoeic and dienoeic acyl-CoA thioesters (5Yamashita S. Hosaka K. Numa S. Eur. J. Biochem. 1973; 38: 25-31Crossref PubMed Scopus (68) Google Scholar). However, the activity of the enzyme was essentially not affected by the fatty acid constituent of the acyl acceptor (LPA), except that the 1-stearoyl and 1-arachidonoyl LPAs were somewhat less effective acyl acceptors (6Miki Y. Hosaka K. Yamashita S. Handa H. Numa S. Eur. J. Biochem. 1977; 81: 433-441Crossref PubMed Scopus (26) Google Scholar). This acyl acceptor specificity was also found for lyso-PC acyltransferase when using different lysophosphatidylcholine (LPC) as substrates. The acyl acceptor specificity of LPAAT, however, does depend on the polar head group, and LPAAT is highly specific for LPA. In contrast, lyso-PC acyltransferase utilizes several acyl acceptors differing in the polar head group, except that LPA and lysophosphatidylethanol are ineffective substrates (6Miki Y. Hosaka K. Yamashita S. Handa H. Numa S. Eur. J. Biochem. 1977; 81: 433-441Crossref PubMed Scopus (26) Google Scholar). The human major histocompatibility complex (MHC) spans ∼4 megabase pairs in the chromosome band 6p21.3 and is divided into three regions (7Campbell R.D. Trowsdale J. Immunol. Today. 1997; 18 (centerfold)Google Scholar). The class I and II regions contain the classical MHC genes, which encode cell-surface glycoproteins involved in the presentation of antigenic peptides to T cells during an immune response. These are interspersed with a large number of other genes, some of which encode proteins involved in antigen processing. The class I and class II regions are separated by the central class III region that spans 1100 kilobase pairs of DNA (7Campbell R.D. Trowsdale J. Immunol. Today. 1997; 18 (centerfold)Google Scholar, 8Aguado B. Milner C.M. Campbell R.D. Browning M. McMichael A. HLA/MHC: Genes, Molecules, and Function. Bios Scientific Publishers Ltd, Oxford, UK1996: 39-75Google Scholar). Characterization of a 220-kilobase pair segment of DNA located between the class II region and the complementC4 genes in the class III region of the human MHC has revealed that the region contains at least nine genes (9Kendall E. Sargent C.A. Campbell R.D. Nucleic Acids Res. 1990; 18: 7251-7257Crossref PubMed Scopus (77) Google Scholar, 10Sugaya K. Fukagawa T. Matsumoto K. Mita K. Takahashi T.-I. Ando A. Inoko H. Ikemura T. Genomics. 1994; 23: 408-419Crossref PubMed Scopus (209) Google Scholar, 11Bristow J. Tee M.K. Gitelman S.E. Mellon S.H. Miller W.L. J. Cell. Biol. 1993; 122: 265-278Crossref PubMed Scopus (256) Google Scholar, 12Aguado B. Campbell R.D. Genomics. 1995; 25: 650-659Crossref PubMed Scopus (18) Google Scholar, 13Neeper M. Schmidt A.M. Brett J. Yan S.D. Wang F. Pan Y.C. Elliston K. Stern D. Shaw A. J. Biol. Chem. 1992; 267: 14998-15004Abstract Full Text PDF PubMed Google Scholar, 14Khanna A. Campbell R.D. Biochem. J. 1996; 319: 81-89Crossref PubMed Scopus (15) Google Scholar, 15Min J. Shukla H. Kozono H. Bronson S.K. Weissman S.M. Chaplin D.D. Genomics. 1995; 30: 149-156Crossref PubMed Scopus (23) Google Scholar), NOTCH-4, G18, PBX-2 (G17), RAGE, G16, G15, G14, G13 (Creb-rp), and TN-X (tenascin-X), of which only five (NOTCH-4, PBX-2, RAGE, Creb-rp, and TN-X), at present, encode proteins of known or putative function (for a review, see Ref.8Aguado B. Milner C.M. Campbell R.D. Browning M. McMichael A. HLA/MHC: Genes, Molecules, and Function. Bios Scientific Publishers Ltd, Oxford, UK1996: 39-75Google Scholar). We have found that one of the uncharacterized genes (G15) encodes a protein that has significant homology with LPAAT from bacteria, plant, and yeast species, suggesting that it could be the human homologue. During the preparation of this manuscript, two other papers have been published describing the cloning and expression of human LPAAT (hLPAAT) (16West J. Tompkins C.K. Balantac N. Nudelman E. Meengs B. White T. Bursten S. Coleman J. Kumar A. Singer J.W. Leung D.W. DNA Cell Biol. 1997; 16: 691-701Crossref PubMed Scopus (112) Google Scholar, 17Stamps A.C. Elmore M.A. Hill M.E. Kelly K. Makda A.A. Finnen M.J. Biochem. J. 1997; 326: 455-461Crossref PubMed Scopus (43) Google Scholar). In addition, another hLPAAT has been described (16West J. Tompkins C.K. Balantac N. Nudelman E. Meengs B. White T. Bursten S. Coleman J. Kumar A. Singer J.W. Leung D.W. DNA Cell Biol. 1997; 16: 691-701Crossref PubMed Scopus (112) Google Scholar, 18Eberhardt C. Gray P.W. Tjoelker L.W. J. Biol. Chem. 1997; 272: 20299-20305Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), which is encoded by a gene located in chromosome 9 (18Eberhardt C. Gray P.W. Tjoelker L.W. J. Biol. Chem. 1997; 272: 20299-20305Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). For convenience, West et al. (16West J. Tompkins C.K. Balantac N. Nudelman E. Meengs B. White T. Bursten S. Coleman J. Kumar A. Singer J.W. Leung D.W. DNA Cell Biol. 1997; 16: 691-701Crossref PubMed Scopus (112) Google Scholar) named the two hLPAATs as LPAATα and LPAATβ. In this report, we describe the finding, through cDNA sequence analysis and expression of the encoded polypeptide in insect and mammalian cell lines, that the MHC class III region geneG15 codes for a hLPAAT (hLPAATα). We also report a detailed characterization of the enzymatic activity of hLPAATα and the localization of the enzyme in the ER. Screening of a U937 cDNA library, using two overlapping cosmids (D3A and E91) from the MHC class III region as probes, resulted in the isolation of 22 cDNA clones (9Kendall E. Sargent C.A. Campbell R.D. Nucleic Acids Res. 1990; 18: 7251-7257Crossref PubMed Scopus (77) Google Scholar). Characterization of these clones by restriction enzyme mapping revealed that pG15–3B contained a full-length cDNA insert of ∼2.1 kilobase pairs. Both strands of this cDNA were sequenced by the dideoxy chain termination method after random sonicated fragments were cloned in the size range 300–1000 bp by blunt end ligation intoSmaI-cut M13mp18. The sequence was assembled using the Staden (19Staden R. Bishop M.J. Rawlings C.J. Nucleic Acid and Protein Sequence Analysis: A Practical Approach. IRL Press, Oxford, UK1987: 173-217Google Scholar) programs and determined with a degeneracy of 9.0. Computer analyses were performed using the software package of the University of Wisconsin Genetics Computer Group (GCG) (20Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448Crossref PubMed Scopus (9381) Google Scholar). To remove the 3′- and 5′-flanking sequences of the G15 cDNA (hLPAATα), to express only the coding sequence under the control of the polyhedrin promoter, a PCR copy of the open reading frame was generated using oligonucleotide primers that also created XbaI sites adjacent to the initiating AUG codon and the stop codon. The oligonucleotide primers used were as follows: sense (5′-GCGGTCTAGA ATGGATTTGTGGCCAGG-3′) and antisense (5′-CCGCTCTAGAGCCAGGGTTCACCCACC-3′), in which the XbaI sites are in italics, and the initiating and stop codons are in boldface type. This PCR copy was gel-isolated and ligated into the XbaI-digested plasmid pBluescript KS+ (pBlsc), and several clones were sequenced. The insert of one clone (pG15Bls) that did not include any PCR errors was excised and ligated to the XbaI-cut baculovirus transfer vector pAcCL29.1 (21Livingstone C. Jones I. Nucleic Acids Res. 1989; 17: 2366Crossref PubMed Scopus (59) Google Scholar), kindly donated by Dr. I. Jones (NERC Institute of Virology and Environmental Microbiology, Oxford), to yield pG15Bac (hLPAATα). Spodoptera frugiperda (Sf21) insect cells and wild type baculovirus (Autographa californica nuclear polyhedrosis virus) were kindly donated by Dr A. Alcamı́ (Sir William Dunn School of Pathology, Oxford). Sf21 insect cells were grown in TC100 medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal calf serum. Sf21 cells were cotransfected with BacPAK6 DNA (CLONTECH) and pG15Bac (hLPAATα), using Lipofectin (Life Technologies, Inc.), to generate hLPAATα recombinants. Ten clones were isolated and plaque-purified four times. The expression of the recombinant hLPAATα was confirmed by metabolic labeling, and one of these viruses, vG15Bac (hLPAATα), was selected for further experiments. The recombinant AcB15R has been described previously (22Alcamı́ A. Smith G.L. Cell. 1992; 71: 153-167Abstract Full Text PDF PubMed Scopus (410) Google Scholar). Sf21 cells were infected at 10 plaque forming units/cell and pulse-labeled from 24 to 27 h postinfection with 200 μCi/ml Tran35S-label (ICN Biomedicals; a mixture of ∼80% [35S]methionine and ∼20% [35S]cysteine, 1200 Ci/mmol) in methionine-free TC100 medium in the absence of serum. Cells and extracellular media were analyzed by SDS-polyacrylamide gel electrophoresis in 12% acrylamide gels. Radioactive bands were detected by fluorography with Amplify (Amersham Corp.). To express the hLPAATα fused at the C terminus to a T7.Tag sequence (MASMTGGQQMGRDP), for which specific monoclonal antibodies are commercially available (T7.TagmAb) (Novagen), the last 270 bp encoding the C terminus of the coding sequence of hLPAATα (from pG15Bls) were PCR-amplified. In this amplification, a NcoI site at the 3′-end of the cDNA was created to remove the stop codon and to fuse hLPAATα in frame to the T7.Tag sequence, located in T7.TagpBlsc (kindly donated by C. Winchester, from this laboratory). The oligonucleotide primers used for the PCR were as follows: sense (5′-CCATCTTGCAGTGCAGGCC-3′) and antisense (5′-TAGCCATGG CACCGCCCCCAGGCTTCTTC-3′), in which theNcoI site is indicated in italics and the disrupted stop codon is shown in boldface type. The PCR product was isolated and cloned into pBlsc, the DNA sequence was confirmed, and one clone (pG15COOH-Blsc) was digested with AvaI–NcoI to isolate the insert. To obtain the 5′-end of the coding sequence, pG15Bls was digested with XbaI, end-filled, and then digested with AvaI, and the 0.68-kilobase pair blunt end/AvaI fragment was isolated and ligated together with theAvaI–NcoI fragment encoding the C-terminal region into EcoRV/NcoI-digested T7.TagpBlsc. The proper ligation of the two fragments into the vector was confirmed by sequence analysis. A HindIII–XbaI insert, containing the hLPAATα cDNA fused to the T7.Tag sequence, was isolated and cloned into pcDNA3 (Invitrogen) cut with HindIII–XbaI to generate pcDNA3G15Tag. CHO cells were electroporated with linearized (SspI) pcDNA3G15Tag or pcDNA3 and incubated for 2 days with GMEMS (for CHO cells, Advanced Protein Products Ltd.), supplemented with 10% (v/v) fetal calf serum. After that time, cells were subcultured at different densities containing different concentrations of geneticin sulfate (G418). Expression of the protein was confirmed after 13 days of transfection by Western blot analysis (ECL method, Amersham) using the T7.TagmAb, and those groups of cells expressing the hLPAATα recombinant protein were diluted to obtain single clones expressing hLPAATα. Expression of the protein in the single clones was confirmed by Western blotting, and three of them were analyzed by immunofluorescence and for LPAAT activity. One of the clones, CHOG15 (hLPAATα), was chosen for subsequent experiments. Single clones containing only the vector were created in parallel, and the presence of the vector was confirmed by PCR screening using specific pcDNA3 primers. Three of these clones were used in immunofluorescence and enzyme activity assays as negative controls for the hLPAATα transfectants, and one of these, CHOV, was chosen for further experiments. Sf21 cells were infected with the wild type (A. californica nuclear polyhedrosis virus) or vG15Bac (hLPAATα) baculovirus at low multiplicity of infection (2 plaque-forming units/cell). Cells were harvested 72 h postinfection (or when the cytopathic effect was total) and resuspended in 50 mm Tris-HCl, pH 8, followed by four cycles of freeze-thaw and then Dounce homogenization. The homogenates were spun down for 10 min at 2000 rpm, and the supernatant was aliquoted and stored at −70 °C. Miki et al. (6Miki Y. Hosaka K. Yamashita S. Handa H. Numa S. Eur. J. Biochem. 1977; 81: 433-441Crossref PubMed Scopus (26) Google Scholar) showed that a semipurified LPAAT, obtained from rat liver microsomes, contained similar activity for 1-myristoyl, 1-palmitoyl, 1-oleoyl, and 1-linoleoyl LPAs. For convenience, we have used in our assays 1-oleoyl LPA, since it was possible to obtain this compound also in a radiolabeled form. The spectrophotometric analysis for measurement of LPAAT activity was performed at room temperature essentially as described by Yamashita et al. (3Yamashita S. Hosaka K. Miki Y. Numa S. Methods Enzymol. 1981; 71: 528-536Crossref PubMed Scopus (26) Google Scholar) with minor modifications. The enzyme activity was assayed by measuring the reaction of the thiol group of the released CoA with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) spectrophotometrically giving an increase in absorbance at 413 nm. A typical incubation mixture consisted of 100 mmTris-HCl, pH 7.4, 1 mm DTNB, 50 μm LPA (oleoyl-sn-glycerol 3-phosphate) (Sigma), 10–45 μm acyl-CoA (Sigma), and 50–150 μg of cell homogenate in a total volume of 1 ml. DTNB was added as a 0.01 msolution in 0.1 m potassium phosphate buffer, pH 7.0. The reaction was initiated by the addition of acyl-CoA after preincubation of the enzyme with all of the other components for 2 min. A molar absorbance of 13,600 m−1 was used to calculate the activity. To assay the enzyme activity by TLC, the assay procedure was essentially as described above except that 4 μg of enzyme homogenate in a total volume of 10 μl was used, and instead of adding DTNB, 0.02 μCi/μl 3H-LPA (1-oleoyl) (NEN Life Science Products) was used. The assay was also performed using 50 μmunlabeled LPA and 0.005 μCi/μl 14C-oleoyl-CoA (Amersham). The reaction was terminated by directly spotting it onto a silica gel 60 TLC plate (Merck) and developed in chloroform/methanol/acetic acid/water (25:10:3:1). To determine the position of substrate and products, LPA and PA standards were loaded and visualized by exposure to iodine vapors. When 3H-LPA was used, fluorography was done by immersion of the TLC plate in chloroform containing 10% 2,5-diphenyloxazole for 10 s and then dried without exposure to iodine vapors. The labeled products were detected by autoradiography. Cells were grown on glass cover slides, fixed in 4% paraformaldehyde in 250 mm Hepes, pH 7.4, for 30 min, and quenched in 50 mm NH4Cl in PBS for 15 min at room temperature. The primary and secondary antibodies were added in 0.2% gelatin, 0.05% saponin in PBS for 45 min. The secondary antibody was fluorescein isothiocyanate-conjugated anti-mouse-IgG (Sigma Immunochemicals), while the 1D3 primary monoclonal antibody (mAb), was kindly donated by Dr D. Vaux (Sir William Dunn School of Pathology, Oxford). Nonpermeabilized conditions were without detergent and involved incubation of the primary mAb at 4 °C to avoid permeabilization and internalization of the mAbs and membrane proteins. Immunofluorescence was observed using a Bio-Rad MRC 1024 confocal microscope. A nearly full-length cDNA clone (pG15–3B) corresponding to the single copy gene G15, located in the MHC class III region, was isolated from a U937 cDNA library (9Kendall E. Sargent C.A. Campbell R.D. Nucleic Acids Res. 1990; 18: 7251-7257Crossref PubMed Scopus (77) Google Scholar) and sequenced. The 2045-bp cDNA insert, with a poly(A) signal AATAAA 17 bp upstream of a 21-bp poly(A) tail at one end (Fig.1 a), contained a single long open reading frame, which was predicted to encode a 31.7-kDa protein. The hydrophobicity plot (Fig. 1 b) of the 283-amino acid predicted polypeptide (Fig. 1 a) revealed the presence of seven potential hydrophobic regions, suggesting that the G15gene product could be a membrane-spanning protein. In addition, at the N terminus there are two putative signal cleavage sites after amino acids 22 and 58, respectively. The FastA and BestFit programs from the GCG package revealed significant sequence homology between the entire sequence of theG15 gene product and LPAAT from different organisms (Fig.2) such as bacteria (Hemophilus influenzae (30.4% identity) (23Fleischmann R.D. Adams M.D. White O. Clayton R.A. Kirkness E.F. Kerlavage A.R. Bult C.J. Tomb J.F. Dougherty B.A. Merrick J.M. McKenney K. Sutton G. Fitzhugh W. Fields C. Gocayne J.D. Scott J. Shirley R. Liu L.I. Glodek A. Kelley J.M. Weidman J.F. Phillips C.A. Spriggs T. Hedblom E. Cotton M.D. Utterback T.R. Hanna M.C. Nguyen D.T. Saudek D.M. Brandon R.C. Fine L.D. Fritchman J.L. Fuhrmann J.L. Geoghagen N.S.M. Gnehm C.L. McDonald L.A. Small K.V. Fraser C.M. Smith H.O. Venter J.C. Science. 1995; 269: 496-512Crossref PubMed Scopus (4673) Google Scholar), Salmonella typhimurium (28.1% identity) (24Luttinger A.L. Springer A.L. Schmid M.B. New Biol. 1991; 3: 687-697PubMed Google Scholar), Escherichia coli(27.7% identity) (25Coleman J. Mol. Gen. Genet. 1992; 232: 295-303Crossref PubMed Scopus (104) Google Scholar), Neisseria gonorrhoeae (24.9% identity), and Neisseria meningitidis (24.0% identity) (26Swartley J.S. Balthazar J.T. Coleman J. Shafer W.M. Stephens D.S. Mol. Microbiol. 1995; 18: 401-412Crossref PubMed Scopus (19) Google Scholar)); yeast (Saccharomyces cerevisiae (30.9% identity) (27Nagiec M.M. Wells G.B. Lester R.L. Dickson R.C. J. Biol. Chem. 1993; 268: 22156-22163Abstract Full Text PDF PubMed Google Scholar)); and plant (Limnantes alba (28.4% identity) (28Lassner M.W. Levering C.K. Davies H.M. Knutzon D.S. Plant Physiol. 1995; 109: 1389-1394Crossref PubMed Scopus (113) Google Scholar) and Cocos nucifera (23.6% identity) (29Knutzon D.S. Lardizabal K.D. Nelsen J.S. Bleibaum J.L. Davies H.M. Metz J.G. Plant Physiol. 1995; 109: 999-1006Crossref PubMed Scopus (104) Google Scholar) (Fig. 2 a)). Multiple sequence alignment of these sequences, using the Pile-up program from the GCG package, shows that they are highly related (Fig.2 b), suggesting that the MHC-linked G15 gene encodes a human LPAAT. Comparison of the G15 gene product (hLPAATα) with the hLPAAT (hLPAATβ) encoded by a gene located in chromosome 9 (16West J. Tompkins C.K. Balantac N. Nudelman E. Meengs B. White T. Bursten S. Coleman J. Kumar A. Singer J.W. Leung D.W. DNA Cell Biol. 1997; 16: 691-701Crossref PubMed Scopus (112) Google Scholar, 18Eberhardt C. Gray P.W. Tjoelker L.W. J. Biol. Chem. 1997; 272: 20299-20305Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), using the BestFit program, shows that they are 47.6% identical and 70.5% similar to each other (Fig. 2 a) over the entire amino acid sequences (Fig. 2 c).Figure 2Percentage of amino acid identities (ID) or similarities (SIM) (a) and amino acid sequence alignment (b) of the complete amino acid sequence of the G15 gene product and LPAATs from different organisms. Human (G15 (hLPAATα) and hLPAATβ), S. cerevisiae (Scerevisiae), H. influenzae (Hemoph), S. typhimurium(Salmonella), E. coli (Ecoli), L. alba (Lalba), C. nucifera(Cnucifera), N. gonorrhoeae (Gonocc), and N. meningitidis (Meningo) are shown.a, the BestFit scores are shown. b, the conserved potential transmembrane sequences are underlined, and the potential active center is double underlined. c, BestFit analysis of hLPAATα (top line) and hLPAATβ (bottom line).View Large Image Figure ViewerDownload (PPT) To characterize the G15 gene product (hLPAATα), the protein was expressed in insect cells using the baculovirus system. Radiolabeling of Sf21 insect cells infected with vG15Bac (hLPAATα) showed, in cell extracts, a major specific polypeptide of 26 kDa and a minor specific polypeptide of 28 kDa that were not observed in infections with a control virus expressing the secreted vaccinia virus interleukin 1β (IL-1β) receptor (AcB15R) (22Alcamı́ A. Smith G.L. Cell. 1992; 71: 153-167Abstract Full Text PDF PubMed Scopus (410) Google Scholar) or with the wild type virus (Fig.3 a). hLPAATα was not detected in the medium (Fig. 3 a), in contrast to the secreted control AcB15R. When the radiolabeling was performed in the presence of tunicamycin, no difference in the hLPAATα size was observed (data not shown), indicating that the single potentialN-glycosylation site situated at amino acids 184–186 (Fig.1 a) is not glycosylated, consistent with its predicted cytosolic localization (see below). For expression in CHO cells, a 14-amino acid T7 epitope tag (T7.Tag) was fused to the C terminus of hLPAATα, and stable cell lines expressing hLPAATα (CHOG15) or containing only the transfer vector pcDNA3 (CHOV) were produced. Western blot analysis of cell extracts from the cell lines CHO, CHOG15 (hLPAATα), or CHOV using a T7.TagmAb showed a specific tagged hLPAATα band of 27 kDa only in the cell line CHOG15 (hLPAATα) (Fig. 3 b). The SigCleave program in the GCG package predicts two signal peptides, one that would be cleaved after amino acid 22 and the second after amino acid 58 (Fig. 1). Since the expected molecular mass for hLPAATα is ∼31.7 kDa, including the signal peptide, the ∼28- and ∼26-kDa forms found in insect cells could be explained by the processing of hLPAATα after each of the potential cleavage sites. However, in CHOG15 (hLPAATα) cells, only a single band of ∼27 kDa was seen. When the molecular weight of the 14-amino acid T7.Tag is taken into account, the size of the hLPAATα polypeptide will be ∼26 kDa, and this is similar in size to the major band found for hLPAATα expressed in insect cells using baculovirus (Fig. 3). This suggests that the major cleavage site for the signal peptide is at amino acid 58 and that the cleavage at amino acid 22 observed in insect cells could be due to aberrant processing. Since it is also possible that anomalous migration of the protein (due to its hydrophobic nature) during SDS-polyacrylamide gel electrophoresis is taking place, further experiments will be required to characterize the amino terminus of the mature protein. The Predict-protein program (EMBL, Heidelberg) (30Rost B. Casadio R. Fariselli P. Sander C. Protein Sci. 1995; 4: 521-533Crossref PubMed Scopus (643) Google Scholar), using the alignment of the different LPAATs and assuming cleavage of the hLPAATα after amino acid 58, predicts only two transmembrane domains, one from amino acid 130 to 147 and one from amino acid 195 to 211 (Fig.1 and 2 b). The predicted topology of the protein would be as illustrated in Fig. 4, with the amino terminus in the ER lumen, followed by a transmembrane domain, a cytoplasmic loop, another transmembrane domain, and the C terminus in the ER lumen. A highly conserved region, distinct from the transmembrane regions, between amino acids 176 and 196, would be located on the cytosolic side of the ER membrane, where phospholipid synthesis occurs (31Coleman R.A. Bell R.M. Boyer P.D. 3rd Ed. The Enzymes. XVI. Academic Press, Inc., New York1983: 605-625Google Scholar), indicating a potential active center of the enzyme (Fig. 2 b and 4). Comparative sequence analysis of the different LPAATs with the available glycerol-3-phosphate acyltransferase sequences revealed that they show a higher sequence similarity in this region, supporting the hypothesis of this being the active center (data not shown). The two conserved transmembrane regions could also be part of the active center and select for the length and degree of saturation of the acyl chains (Figs. 2 b and 4). In this model, the potential glycosylation site would be located on the cytosolic side of the ER and for this reason would be unavailable for glycosylation. The model would still be valid for the localization of the active center and carboxyl terminus even if" @default.
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• W2000664329 title "Characterization of a Human Lysophosphatidic Acid Acyltransferase That Is Encoded by a Gene Located in the Class III Region of the Human Major Histocompatibility Complex" @default.
• W2000664329 cites W1503258737 @default.
• W2000664329 cites W1520271242 @default.
• W2000664329 cites W1590099198 @default.
• W2000664329 cites W1598208661 @default.
• W2000664329 cites W1605154807 @default.
• W2000664329 cites W1832073808 @default.
• W2000664329 cites W1967303459 @default.
• W2000664329 cites W1971403296 @default.
• W2000664329 cites W1983964526 @default.
• W2000664329 cites W1990229869 @default.
• W2000664329 cites W2005581282 @default.
• W2000664329 cites W2013300862 @default.
• W2000664329 cites W2015292449 @default.
• W2000664329 cites W2029236303 @default.
• W2000664329 cites W2033936924 @default.
• W2000664329 cites W2047786555 @default.
• W2000664329 cites W2051412610 @default.
• W2000664329 cites W2054090667 @default.
• W2000664329 cites W2060055554 @default.
• W2000664329 cites W2077294307 @default.
• W2000664329 cites W2083077798 @default.
• W2000664329 cites W2091974362 @default.
• W2000664329 cites W2092869660 @default.
• W2000664329 cites W2093529055 @default.
• W2000664329 cites W2142146144 @default.
• W2000664329 cites W2142872187 @default.
• W2000664329 cites W2146396614 @default.
• W2000664329 cites W2156994055 @default.
• W2000664329 cites W2336675162 @default.
• W2000664329 cites W2397113429 @default.
• W2000664329 cites W2402973226 @default.
• W2000664329 cites W4229834841 @default.
• W2000664329 cites W4247231961 @default.
• W2000664329 cites W87923103 @default.
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