Characterization of mouse lysophosphatidic acid acyltransferase 3: an enzyme with dual functions in the testis
2008; Elsevier BV; Volume: 50; Issue: 5 Linguagem: Inglês
10.1194/jlr.m800468-jlr200
ISSN1539-7262
AutoresKoichi Yuki, Hideo Shindou, Daisuke Hishikawa, Takao Shimizu,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoGlycerophospholipids are structural and functional components of cellular membranes as well as precursors of various lipid mediators. Using acyl-CoAs as donors, glycerophospholipids are formed by the de novo pathway (Kennedy pathway) and modified in the remodeling pathway (Lands' cycle). Various acyltransferases, including two lysophosphatidic acid acyltransferases (LPAATs), have been discovered from a 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family. Proteins of this family contain putative acyltransferase motifs, but their biochemical properties and physiological roles are not completely understood. Here, we demonstrated that mouse LPAAT3, previously known as mouse AGPAT3, possesses strong LPAAT activity and modest lysophosphatidylinositol acyltransferase activity with a clear preference for arachidonoyl-CoA as a donor. This enzyme is highly expressed in the testis, where CDP-diacylglycerol synthase 1 preferring 1-stearoyl-2-arachidonoyl-phosphatidic acid as a substrate is also highly expressed. Since 1-stearoyl-2-arachidonoyl species are the main components of phosphatidylinositol, mouse LPAAT3 may function in both the de novo and remodeling pathways and contribute to effective biogenesis of 1-stearoyl-2-arachidonoyl-phosphatidylinositol in the testis. Additionally, the expression of this enzyme in the testis increases significantly in an age-dependent manner, and β-estradiol may be an important regulator of this enzyme's induction. Our findings identify this acyltransferase as an alternative important enzyme to produce phosphatidylinositol in the testis. Glycerophospholipids are structural and functional components of cellular membranes as well as precursors of various lipid mediators. Using acyl-CoAs as donors, glycerophospholipids are formed by the de novo pathway (Kennedy pathway) and modified in the remodeling pathway (Lands' cycle). Various acyltransferases, including two lysophosphatidic acid acyltransferases (LPAATs), have been discovered from a 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family. Proteins of this family contain putative acyltransferase motifs, but their biochemical properties and physiological roles are not completely understood. Here, we demonstrated that mouse LPAAT3, previously known as mouse AGPAT3, possesses strong LPAAT activity and modest lysophosphatidylinositol acyltransferase activity with a clear preference for arachidonoyl-CoA as a donor. This enzyme is highly expressed in the testis, where CDP-diacylglycerol synthase 1 preferring 1-stearoyl-2-arachidonoyl-phosphatidic acid as a substrate is also highly expressed. Since 1-stearoyl-2-arachidonoyl species are the main components of phosphatidylinositol, mouse LPAAT3 may function in both the de novo and remodeling pathways and contribute to effective biogenesis of 1-stearoyl-2-arachidonoyl-phosphatidylinositol in the testis. Additionally, the expression of this enzyme in the testis increases significantly in an age-dependent manner, and β-estradiol may be an important regulator of this enzyme's induction. Our findings identify this acyltransferase as an alternative important enzyme to produce phosphatidylinositol in the testis. Tissues maintain distinct content and composition of various glycerophospholipids, such as phosphatidic acid (PA), phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol (PI), phosphatidylserine, and cardiolipin (1MacDonald J.I. Sprecher H. Phospholipid fatty acids remodeling in mammalian cells.Biochim. Biophys. Acta. 1991; 1084: 105-121Crossref PubMed Scopus (248) Google Scholar, 2Wood R. 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At least seven AGPAT family members have been identified in mouse (21Kume K. Shimizu T. cDNA cloning and expression of murine 1-acyl-sn-glycerol-3-phosphate acyltransferase.Biochem. Biophys. Res. Commun. 1997; 237: 663-666Crossref PubMed Scopus (50) Google Scholar, 36Dircks L. Sul H.S. Acyltransferases of de novo glycerophospholipid biosynthesis.Prog. Lipid Res. 1999; 38: 461-479Crossref PubMed Scopus (59) Google Scholar, 37Lu B. Jiang Y.J. Zhou Y. Xu F.Y. Hatch G.M. Choy P.C. Cloning and characterization of murine 1-acyl-sn-glycerol 3-phosphate acyltransferases and their regulation by PPARalpha in murine heart.Biochem. J. 2005; 385: 469-477Crossref PubMed Scopus (92) Google Scholar), and each of them contains a highly conserved putative catalytic motif (NHX4D) and putative substrate binding motif (EGTR) (38Lewin T.M. Wang P. Coleman R.A. 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Identification of a novel sn-glycerol-3-phosphate acyltransferase isoform, GPAT4, as the enzyme deficient in Agpat6−/− mice.J. Lipid Res. 2008; 49: 823-831Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Mouse AGPAT3 has been investigated in the past, but the characterization was far from being done (37Lu B. Jiang Y.J. Zhou Y. Xu F.Y. Hatch G.M. Choy P.C. Cloning and characterization of murine 1-acyl-sn-glycerol 3-phosphate acyltransferases and their regulation by PPARalpha in murine heart.Biochem. J. 2005; 385: 469-477Crossref PubMed Scopus (92) Google Scholar). The existence of at least seven members in AGPAT family raises questions as to the specific role of each member. Therefore, investigation of their tissue distributions or biochemical properties will be important in understanding their biological roles. We present here, to our knowledge, the first detailed biochemical and biological characterization of mouse AGPAT3 (mAGPAT3). Surprisingly, mAGPAT3 possesses both LPAAT and lysophosphatidylinositol acyltransferase (LPIAT) activities and prefers arachidonoyl-CoA as a donor, indicating its dual roles in the de novo and remodeling pathways. Point mutations in highly conserved motifs NHX4D or EGTR completely suppressed both LPAAT and LPIAT activities. The enzyme was localized in the endoplasmic reticulum (ER) and expressed in the liver, kidney, and testis. In the testis, cytidine diphosphodiacylglycerol (CDP-diacylglycerol) synthase 1 is highly expressed and particularly converts 1-stearoyl-2-arachidonoyl-PA to CDP-diacylglycerol, a phospholipid precursor (42Saito S. Goto K. Tonosaki A. Kondo H. Gene cloning and characterization of CDP-diaylglycerol synthase from rat brain.J. Biol. Chem. 1997; 272: 9503-9509Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 43Inglis-Broadgate S.L. Ocaka L. Banerjee R. Gaasenbeek M. Chapple J.P. Cheetham M.E. Clark B.J. Hunt D.M. Halford S. Isolation and characterization of murine Cds (CDP-diacylglycerol synthase) 1 and 2.Gene. 2005; 356: 19-31Crossref PubMed Scopus (40) Google Scholar). This might suggest that mLPAAT3 produces PI effectively. Additionally, mAGPAT3 expression in the testis increases significantly in an age-dependent manner. Since β-estradiol induced this enzyme in testicular cell line, mAGPAT3 may play an important role in the testis coupled with sex hormone. We renamed this enzyme as LPAAT3 according to a proposal for the standardization of LPLAT nomenclature by Shindou and Shimizu (6Shindou, H., and T. Shimizu. Acyl-CoA:lysophospholipid acyltransferases. J. Biol. Chem. In press.Google Scholar). DMEM, 12F-HAM, and RPMI1640 were obtained from Sigma-Aldrich (St. Louis, MO). TLC silica gel plates (type 5721) were purchased from Merck (Darmstadt, Germany). Various lysophospholipids and acyl-CoAs were from Avanti Polar Lipids (Alabaster, AL). [1-14C]Oleoyl-CoA (1.924 GBq/mmol), [1-14C]Linoleoyl-CoA (2.035 GBq/mmol), and [1-14C]Arachidonoyl-CoA (2.035 GBq/mmol) were purchased from Moravec Biochemicals (Mercury Lane, CA). [1-14C]Palmitoyl-CoA (2.22 GBq/mmol) and [3H]acetyl-CoA (185 GBq/mmol) were obtained from GE Healthcare (Buckinghamshire, UK). The entire coding region of mLPAAT3 [DNA Data Bank of Japan (DDBJ) accession number AB377215] was identified in the National Center for Biotechnology Information (NCBI) database. A 1.1 kb cDNA clone encoding the full-length mLPAAT3 was obtained by PCR amplification using a forward primer designed to encode FLAG epitope (DYKDDDDK) in frame with the start codon of target DNA coding region (5′-CTAGCTAGCCACCATGGATTACAAGGATGACGATGACAAGGGCCTGCTTGCCTACCTGAAGACCC), and a reverse primer (CCGCTCGAGTTATTCCTTTTTCTTAAGCTCTTGGTTGCC-3′). Mouse heart cDNA was used as a template. Amplified PCR products were cloned into the pCXN2.1 vector (44Niwa H. Yamamura K. Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector.Gene. 1991; 108: 193-199Crossref PubMed Scopus (4613) Google Scholar) and sequenced. Similarly, mLPAAT1 (DDBJ accession number NM_018862) was identified in NCBI, and full-length mLPAAT1 was obtained by PCR amplification using a forward primer designed to encode FLAG epitope in frame (CTAGCTAGCCACCATGGATTACAAGGATGACGATGACAAGGAGCTGTGGCCCGGGGCCTGG) and a reverse primer (CCGCTCGAGTCAGAGCCGGGCTTCGCCCGCTCCCCC). mLPAAT3 constructs with single mutations in the highly conserved motif NHX4D (His→Ala or Asp→Ala) or EGTR (Glu→Ala) were made using cloned mLPAAT3 cDNA as a template. Amplified PCR products were cloned into the pCXN2.1 vector and sequenced. Details of the method have been described previously (39Harayama T. Shindou H. Ogasawara R. Suwabe A. Shimizu T. Identification of a novel noninflammatory biosynthetic pathway of platelet-activating factor.J. Biol. Chem. 2008; 283: 11097-11106Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Chinese hamster ovary (CHO)-K1 cells were seeded onto 10 cm dishes, at a density of 3 × 106 cells /dish 1 day before transfection. Twelve micrograms each of pCXN2.1 vector or FLAG-mLPAAT3-pCXN2.1 was transfected using Lipofectamine 2000 (Invitrogen). At 48 h after transfection, transfected cells were scraped into 1 ml of ice-cold buffer containing 20 mM Tris-HCl (pH 7.4), 300 mM sucrose, and a proteinase inhibitor cocktail Complete (Roche Applied Science) and sonicated three times on ice for 30 s each time. Cell lysates were centrifuged at 9,000 g for 15 min. Supernatants were then centrifuged at 100,000 g for 1 h. Pellets were suspended in buffer containing 20 mM Tris-HCl (pH 7.4), 300 mM sucrose, and 1 mM EDTA. The protein concentration was measured by the method of Bradford (45Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217389) Google Scholar) using a commercially prepared protein assay solution (Bio-Rad) and BSA (fraction V, fatty acid-free; Sigma-Aldrich) as a standard. Antiserum was generated at SCRUM (Tokyo, Japan). C-terminal peptides were used for immunization of rabbits (EKGSSYGNQELK and FPGEQFKPARRPWT). Specificity of the antiserum was examined by Western blot analysis using mircosomes from vector- and mLPAAT3-transfected cells. Microsomal fractions from mAGPAT4- or AGPAT5-transfected cells were also used as negative controls. Cell lysates were centrifuged at 800 g for 10 min. Supernatants were centrifuged at 9,000 g for 15 min. Supernatants were then centrifuged at 100,000 g for 1 h. Initial 9,000 g pellets were homogenized again and centrifuged at 9,000 g. The pellets were used as 9,000 g pellets. Two micrograms each of 9,000 g pellets, 100,000 g pellets, and 100,000 g supernatants were resolved by 10% SDS-PAGE and transferred to a Hybond ECL nitrocellulose membrane (GE Healthcare UK). The membrane was blocked with 5% skim milk, incubated with anti-FLAG M2 mAb (IBI/Kodak) or anticytochrome c oxidase antibody (Invitrogen), and washed and incubated with horseradish-peroxidase-labeled anti-mouse IgG (GE Healthcare UK). After washing, the membrane was exposed to ECL reagents (GE Healthcare UK) and X-ray film (GE Healthcare UK) to visualize immunoreactive proteins. Expression of the FLAG-tagged target protein was confirmed. For the examination of endogenous mLPAAT3 subcellular localization, rabbit anti-mLPAAT3 antiserum and horseradish-peroxidase-labeled anti-rabbit IgG (GE Healthcare UK) were used as primary and secondary antibodies, respectively. CHO-K1 cells transfected with pCXN2.1 vector or FLAG-mLPAAT3-pCXN2.1 cells were fixed with 4% paraformaldehyde and permeabilized with methanol/acetone solution (1:1, v/v). Cells were incubated with 10 μg/ml primary antibody for 30 min. As a marker, M5 anti-FLAG mouse mAb or anti-FLAG rabbit antibody (Sigma-Aldrich) for FLAG epitope, anti-calnexin antibody (BD Biosciences) for ER, anticytochrome c oxidase antibody (Invitrogen) for mitochondria, and anti-GM130 (Invitrogen) for Golgi were used. Cells were blocked with 1% BSA and then incubated with 10 μg/ml Alexa Fluor 546 goat anti-rabbit IgG (Eugene, OR) and Alexa Fluor 488 goat anti-mouse IgG (Eugene, OR). Confocal microscopy was performed using an LSM510 laser scanning microscope (Carl Zeiss, Germany) equipped with a ×63 water immersion objective lens (numerical aperture = 1.2). Cells were observed by excitation at 543 nm with a He/Ne laser and emission through a 585-nm long-pass filter for the detection of red fluorescence. For the detection of green fluorescence, the excitation was at 488 nm with an argon laser, and emissions were collected using a 505 to 550 nm band-pass filter. Acyltransferase activity was measured by the transfer of [1-14C]acyl-CoAs or [3H]acetyl-CoA to lysophospholipids to form phospholipids. Reaction mixtures contained 100 mM Tris-HCl (pH 7.4), 1 mM EDTA, and indicated concentrations of acyl-CoA, lysophospholipids, and enzyme (100,000 g pellets) in a total volume of 100 μl. The amount of total protein and concentrations of acceptors and donors are described in corresponding figure legends. After incubation at 37°C for 10 min, reactions were stopped by the addition of 300 μl of chloroform:methanol (1:2, v/v). The reaction progressed linearly at least for 10 min. Total lipids were extracted using the Bligh-Dyer method (46Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43088) Google Scholar) and subsequently analyzed by TLC in chloroform:methanol:acetic acid:water (50:25:8:4, v/v/v/v). Bands at positions corresponding to the expected products were visualized with I2 vapor, cut off the plate, placed in Microscinti-O (Perkin-Elmer Life Sciences), and analyzed in an LS6500 liquid scintillation counter (Beckman). Total RNA was prepared using RNeasy Mini Kit (Qiagen). First-strand cDNAs were synthesized using Superscript II (Invitrogen). PCR was conducted in microcapillary tubes, in 20 μl reaction volumes consisting of 2 μl of cDNA solution, 1× FastStart DNA Master SYBR Green I (Roche Applied Science), and 0.5 μM each of the forward and reverse primers. Sequences of primers used in PCR are shown in supplementary Table I. Testicular cell line TM4 cells were cultured in F12-HAM:DMEM (1:1,v/v) containing 5% horse serum (Gibco) and 2.5% FBS. Cells (1 × 105) were incubated with either mock, 100 nM β-estradiol (Sigma-Aldrich), dihydrotestosterone (Sigma-Aldrich), or testosterone (Sigma-Aldrich) for 24 h and collected. C57BL/6J mice were obtained from Clea Japan (Tokyo, Japan). Mice were maintained at 21°C in a light-dark cycle with light from 08:00 to 20:00. Mice were fed with a standard laboratory diet and water ad libitum. All animal studies were conducted in accordance with the guidelines for Animal Research at the University of Tokyo and were approved by the University of Tokyo Ethics Committee for Animal Experiments. Data are presented as mean + SD. P values < 0.05 were considered statistically significant. All statistical calculations were performed using Prism 4 (GraphPad Software). To identify novel LPLATs, we focused on AGPAT family proteins. A phylogenetic tree was drawn by pairwise comparisons of the amino acid sequences of LPAAT family members using ClustalW, DDBJ (http://www.clustalw.ddbj.nig.ac.jp/top-j.html) (Fig. 1A) (47Thompson J.D. Higgins D.G. Gibson T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55973) Google Scholar). The amino acid sequences of mLPAAT1, mLPAAT2, and mLPAAT3 are shown in Fig. 1B. A 1.1 kb cDNA clone encoding the full-length mLPAAT3 enzyme was obtained by PCR amplification. mLPAAT3 encodes a 376 amino acid protein of 43.3 kDa, containing four transmembrane domains, predicted by ConPred II (48Arai M. Mitsuke H. Ikeda M. Xia J-X. Kikuchi T. Satake M. Shimizu T. ConPred II: a consensus prediction method for obtaining transmembrane topology models with high reliability.Nucleic Acids Res. 2004; 32: W390-W393Crossref PubMed Scopus (189) Google Scholar), and the conserved motifs (NHX4D and EGTR) found in members of AGPAT family (Fig. 1B). The protein also possesses the C-terminal sequence motif KKXX (49Shikano S. Li M. Membrane receptor trafficking: evidence of proximal and distal zones conferred by two independent endoplasmic reticulum localized signals.Proc. Natl. Acad. Sci. USA. 2003; 100: 5783-5788Crossref PubMed Scopus (90) Google Scholar), suggesting that mLPAAT3 localizes to the ER, similar to LPCAT1 and LysoPAFAT/LPCAT2 (15Shindou 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 (187) Google Scholar,
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