Identification of a Novel Noninflammatory Biosynthetic Pathway of Platelet-activating Factor*
2008; Elsevier BV; Volume: 283; Issue: 17 Linguagem: Inglês
10.1074/jbc.m708909200
ISSN1083-351X
AutoresT. HARAYAMA, Hideo Shindou, Rie Ogasawara, Akira Suwabe, Takao Shimizu,
Tópico(s)Adipokines, Inflammation, and Metabolic Diseases
ResumoPlatelet-activating factor (PAF) is a potent lipid mediator playing various inflammatory and physiological roles. PAF is biosynthesized through two independent pathways called the de novo and remodeling pathways. Lyso-PAF acetyltransferase (lyso-PAF AT) was believed to biosynthesize PAF under inflammatory conditions, through the remodeling pathway. The first isolated lyso-PAF AT (LysoPAFAT/LPCAT2) had consistent properties. However, we show in this study the finding of a second lyso-PAF AT working under noninflammatory conditions. We partially purified a Ca2+-independent lyso-PAF AT from mouse lung. Immunoreactivity for lysophosphatidylcholine acyltransferase 1 (LPCAT1) was detected in the active fraction. Lpcat1-transfected Chinese hamster ovary cells exhibited both LPCAT and lyso-PAF AT activities. We confirmed that LPCAT1 transfers acetate from acetyl-CoA to lyso-PAF by the identification of an acetyl-CoA (and other acyl-CoAs) interacting site in LPCAT1. We further showed that LPCAT1 activity and expression are independent of inflammatory signals. Therefore, these results suggest the molecular diversity of lyso-PAF ATs is as follows: one (LysoPAFAT/LPCAT2) is inducible and activated by inflammatory stimulation, and the other (LPCAT1) is constitutively expressed. Each lyso-PAF AT biosynthesizes inflammatory and physiological amounts of PAF, depending on the cell type. These findings provide important knowledge for the understanding of the diverse pathological and physiological roles of PAF. Platelet-activating factor (PAF) is a potent lipid mediator playing various inflammatory and physiological roles. PAF is biosynthesized through two independent pathways called the de novo and remodeling pathways. Lyso-PAF acetyltransferase (lyso-PAF AT) was believed to biosynthesize PAF under inflammatory conditions, through the remodeling pathway. The first isolated lyso-PAF AT (LysoPAFAT/LPCAT2) had consistent properties. However, we show in this study the finding of a second lyso-PAF AT working under noninflammatory conditions. We partially purified a Ca2+-independent lyso-PAF AT from mouse lung. Immunoreactivity for lysophosphatidylcholine acyltransferase 1 (LPCAT1) was detected in the active fraction. Lpcat1-transfected Chinese hamster ovary cells exhibited both LPCAT and lyso-PAF AT activities. We confirmed that LPCAT1 transfers acetate from acetyl-CoA to lyso-PAF by the identification of an acetyl-CoA (and other acyl-CoAs) interacting site in LPCAT1. We further showed that LPCAT1 activity and expression are independent of inflammatory signals. Therefore, these results suggest the molecular diversity of lyso-PAF ATs is as follows: one (LysoPAFAT/LPCAT2) is inducible and activated by inflammatory stimulation, and the other (LPCAT1) is constitutively expressed. Each lyso-PAF AT biosynthesizes inflammatory and physiological amounts of PAF, depending on the cell type. These findings provide important knowledge for the understanding of the diverse pathological and physiological roles of PAF. Phosphatidylcholine (PC) 4The abbreviations used are: PC, phosphatidylcholine; PLA2, phospholipase A2; LPC, lysophosphatidylcholine; LPCAT, LPC acyltransferase; PAF, platelet-activating factor; cPLA2α, cytosolic phospholipase A2α; lyso-PAF AT, lyso-PAF acetyltransferase; COX, cyclooxygenase; LPS, lipopolysaccharides; APMSF, 4-amidinophenylmethanesulfonyl fluoride; ODN, CpG oligonucleotide; poly(I:C), polyinosine-polycytidylic acid; CHO, Chinese hamster ovary; WT, wild type; TLR, Toll-like receptor; MAPK, mitogen-activated protein kinase; ANOVA, analysis of variance. is the most abundant glycerophospholipid in eukaryotes and exerts various biological functions. PC is biosynthesized through a de novo pathway (1Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar), and the sn-2 acyl group is subsequently modified through a remodeling pathway (2Lands W.E. J. Biol. Chem. 1958; 231: 883-888Abstract Full Text PDF PubMed Google Scholar). In the remodeling pathway, PC is cleaved at its sn-2 position by phospholipase A2 (PLA2) (EC 3.1.1.4), generating lysophosphatidylcholine (LPC). Next, an acyl group is transferred to LPC by LPC acyltransferase (LPCAT) (EC 2.3.1.23), generating PC with various acyl compositions. Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is an ether analogue of PC and contains an acetyl group at its sn-2 position. PAF is a potent lipid mediator that has many inflammatory and noninflammatory roles (3Ishii S. Nagase T. Tashiro F. Ikuta K. Sato S. Waga I. Kume K. Miyazaki J. Shimizu T. EMBO J. 1997; 16: 133-142Crossref PubMed Scopus (130) Google Scholar, 4Prescott S.M. Zimmerman G.A. Stafforini D.M. McIntyre T.M. Annu. Rev. Biochem. 2000; 69: 419-445Crossref PubMed Scopus (588) Google Scholar, 5Ishii S. Shimizu T. Prog. Lipid Res. 2000; 39: 41-82Crossref PubMed Scopus (329) Google Scholar). PAF has been implicated in many inflammatory diseases, such as thrombosis (6Zimmerman G.A. McIntyre T.M. Prescott S.M. Stafforini D.M. Crit. Care Med. 2002; 30: S294-S301Crossref PubMed Scopus (325) Google Scholar), multiple sclerosis (7Kihara Y. Ishii S. Kita Y. Toda A. Shimada A. Shimizu T. J. Exp. Med. 2005; 202: 853-863Crossref PubMed Scopus (65) Google Scholar), acute lung injury (8Nagase T. Ishii S. Kume K. Uozumi N. Izumi T. Ouchi Y. Shimizu T. J. Clin. Investig. 1999; 104: 1071-1076Crossref PubMed Scopus (105) Google Scholar), asthma (9Ishii S. Nagase T. Shindou H. Takizawa H. Ouchi Y. Shimizu T. J. Immunol. 2004; 172: 7095-7102Crossref PubMed Scopus (42) Google Scholar), and anaphylaxis (10Ishii S. Kuwaki T. Nagase T. Maki K. Tashiro F. Sunaga S. Cao W.H. Kume K. Fukuchi Y. Ikuta K. Miyazaki J. Kumada M. Shimizu T. J. Exp. Med. 1998; 187: 1779-1788Crossref PubMed Scopus (235) Google Scholar). Under noninflammatory circumstances, PAF plays roles in several physiological processes such as glycogen degradation of fetal lung (11Hoffman D.R. White R.G. Angle M.J. Maki N. Johnston J.M. J. Biol. Chem. 1988; 263: 9316-9319Abstract Full Text PDF PubMed Google Scholar), fertility (12Krausz C. Gervasi G. Forti G. Baldi E. Hum. Reprod. 1994; 9: 471-476Crossref PubMed Scopus (65) Google Scholar), and long term potentiation of neurons (13Chen C. Magee J.C. Marcheselli V. Hardy M. Bazan N.G. J. Neurophysiol. 2001; 85: 384-390Crossref PubMed Scopus (60) Google Scholar). Similarly to PC, PAF is biosynthesized by the de novo and remodeling pathways. In the remodeling pathway, alkyl-PC is cleaved at its sn-2 position by PLA2, generating lyso-PAF (alkyl-LPC). Among PLA2s, cytosolic PLA2α (cPLA2α) plays a major role in PAF production of inflammatory cells (14Shindou H. Ishii S. Uozumi N. Shimizu T. Biochem. Biophys. Res. Commun. 2000; 271: 812-817Crossref PubMed Scopus (58) Google Scholar). PAF is then biosynthesized from lyso-PAF by lyso-PAF acetyltransferase (lyso-PAF AT) (EC 2.3.1.67) (15Wykle R.L. Malone B. Snyder F. J. Biol. Chem. 1980; 255: 10256-10260Abstract Full Text PDF PubMed Google Scholar). It was long thought that PAF production occurs through the remodeling pathway under inflammatory conditions, whereas the de novo pathway produces physiological (noninflammatory) PAF (as summarized in Fig. 5E, upper panel) (4Prescott S.M. Zimmerman G.A. Stafforini D.M. McIntyre T.M. Annu. Rev. Biochem. 2000; 69: 419-445Crossref PubMed Scopus (588) Google Scholar, 16Snyder F. Biochim. Biophys. Acta. 1995; 1254: 231-249Crossref PubMed Scopus (184) Google Scholar). It has been demonstrated that endogenous lyso-PAF AT activity is enhanced by several inflammatory stimuli, probably both by post-translational modifications and by mRNA induction (17Shindou H. Ishii S. Yamamoto M. Takeda K. Akira S. Shimizu T. J. Immunol. 2005; 175: 1177-1183Crossref PubMed Scopus (39) Google Scholar). This activity was shown to be Ca2+-dependent (18Domenech C. Machado-De Domenech E. Söling H.D. J. Biol. Chem. 1987; 262: 5671-5676Abstract Full Text PDF PubMed Google Scholar, 19Gómez-Cambronero J. Nieto M.L. Mato J.M. Sánchez-Crespo M. Biochim. Biophys. Acta. 1985; 845: 511-515Crossref PubMed Scopus (21) Google Scholar). We recently reported the cDNA cloning of a lyso-PAF AT, LysoPAFAT/LPCAT2. This enzyme has both lyso-PAF AT and LPCAT activities in the presence of Ca2+ (20Shindou H. Hishikawa D. Nakanishi H. Harayama T. Ishii S. Taguchi R. Shimizu T. J. Biol. Chem. 2007; 282: 6532-6539Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). The lyso-PAF AT activity and the mRNA level of LysoPAFAT/LPCAT2 are enhanced by various stimuli, consistently with the endogenous lyso-PAF AT activity in inflammatory cells. However, it was unknown whether additional lyso-PAF AT(s) exist. The gene with the highest homology to Lysopafat/Lpcat2 is Lpcat1 (21Nakanishi H. Shindou H. Hishikawa D. Harayama T. Ogasawara R. Suwabe A. Taguchi R. Shimizu T. J. Biol. Chem. 2006; 281: 20140-20147Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). LPCAT1 is thought to be involved in the biosynthesis of pulmonary surfactant lipids based on several experimental observations as follows: substrate preference for medium-chain saturated acyl-CoAs to produce disaturated PC, high expression in alveolar type II cells, and a robust induction at the perinatal period (21Nakanishi H. Shindou H. Hishikawa D. Harayama T. Ogasawara R. Suwabe A. Taguchi R. Shimizu T. J. Biol. Chem. 2006; 281: 20140-20147Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 22Chen X. Hyatt B.A. Mucenski M.L. Mason R.J. Shannon J.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11724-11729Crossref PubMed Scopus (151) Google Scholar). Both LPCAT1 and LysoPAFAT/LPCAT2 are members of the lysophospholipid acyltransferase family. The members of this family contain four conserved acyltransferase motifs (motifs 1–4) (23Lewin T.M. Wang P. Coleman R.A. Biochemistry. 1999; 38: 5764-5771Crossref PubMed Scopus (233) Google Scholar). Although several studies were performed based on site-directed mutagenesis using other lysophospholipid acyltransferases, the precise role of each motif is not yet clear (23Lewin T.M. Wang P. Coleman R.A. Biochemistry. 1999; 38: 5764-5771Crossref PubMed Scopus (233) Google Scholar, 24Dircks L.K. Ke J. Sul H.S. J. Biol. Chem. 1999; 274: 34728-34734Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 25Yamashita A. Nakanishi H. Suzuki H. Kamata R. Tanaka K. Waku K. Sugiura T. Biochim. Biophys. Acta. 2007; 1771: 1202-1215Crossref PubMed Scopus (68) Google Scholar). Here we show the finding of a novel Ca2+-independent lyso-PAF AT activity in the murine lung, mediated by LPCAT1. Depending on the substrate concentration, LPCAT1 recognizes a wide range of acyl-CoAs as substrates, ranging from acetyl-CoA to palmitoyl-CoA. We further characterized the nature of this substrate specificity by site-directed mutagenesis and identified several amino acid residues responsible for acyl-CoA (including acetyl-CoA) binding. We investigated the role of LPCAT1 in PAF biosynthesis and showed that LPCAT1 is neither activated nor up-regulated at the mRNA level by inflammatory stimuli, in contrast to LysoPAFAT/LPCAT2. Thus, LPCAT1 may be involved in the noninflammatory PAF production. This relationship is similar to that of the two cyclooxygenases (COXs). COX-1 is constitutive and exerts protective functions, whereas COX-2 is induced in inflammation and plays many pathological roles (26Smith W.L. Langenbach R. J. Clin. Investig. 2001; 107: 1491-1495Crossref PubMed Scopus (533) Google Scholar). Our findings show the existence of a noninflammatory remodeling pathway involved in PAF biosynthesis. The enzymes involved in each remodeling pathway were expressed differently depending on the cell type. We propose that PAF is mainly biosynthesized by cPLA2α and LysoPAFAT/LPCAT2 in the inflammatory remodeling pathway, whereas PLA2s and LPCAT1 produce PAF in the noninflammatory remodeling pathway. Thus, the classical hypothesis needs some revision, and physiological levels of PAF may be produced not only through the de novo pathway but also through the noninflammatory remodeling pathway. Materials—[3H]Acetyl-CoA, 1-[1-14C]palmitoyl-LPC, [1-14C] palmitoyl-CoA, a HiTrap DEAE-Sepharose Fast Flow column (5:50) (DEAE-Sepharose column), andÄKTAExplorer 10S were obtained from GE Healthcare. Lyso-PAF was purchased from Cayman Chemical Co. (Ann Arbor, MI). 1-O-Alkenyl-LPC was from Doosan (Toronto, Canada). All species of 1-acyl-LPC and acyl-CoA ranging from butanoyl-CoA to arachidoyl-CoA were obtained from Avanti Polar Lipids (Alabaster, AL). Phosphatidylcholine (PC) from frozen egg yolk and lipopolysaccharides (LPS) from Salmonella minnesota were purchased from Sigma. BIGCHAP was from DOJINDO Laboratories (Kumamoto, Japan). QuikChange site-directed mutagenesis kit was from Stratagene (La Jolla, CA). Animals—C57BL/6J mice and Sprague-Dawley rats were obtained from Clea Japan, Inc. (Tokyo, Japan). Mice were maintained in a light-dark cycle with light from 8:00 to 20:00 at 21 °C. 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. Preparation of Microsomes from Mouse Tissues—Mouse lung, spleen, brain, liver, and kidney were homogenized with a Polytron homogenizer in 3× volume of cold buffer containing 100 mm Tris-HCl (pH 7.4), 300 mm sucrose, 5 mm 2-mercaptoethanol, 20 μm 4-amidinophenylmethanesulfonyl fluoride (APMSF), and a protease inhibitor mixture Complete (1×). After centrifugation for 10 min at 9,000 × g, the supernatant was collected and centrifuged for 1 h at 100,000 × g. The pellet was resuspended in a buffer containing 20 mm Tris-HCl (pH 7.4), 300 mm sucrose, 5 mm 2-mercaptoethanol, 20 μm APMSF and 1× Complete. Partial Purification of Murine Ca2+-independent Lyso-PAF AT—The microsomal fraction of lung was solubilized with 0.5% BIGCHAP (w/v) for 40 min and centrifuged at 100,000 × g for 1 h. The supernatant of this centrifugation was designated solubilized microsomes. Solubilized microsomes were applied on a DEAE-Sepharose column equilibrated with Buffer A containing 20 mm Tris-HCl (pH 7.4), 5 mm 2-mercaptoethanol, 20 μm APMSF, and 0.1% BIGCHAP usingÄKTAExplorer 10S. Proteins were eluted by a 160–500 mm linear gradient of NaCl. Measurement of Lyso-PAF AT and LPCAT Activities—Lyso-PAF AT and LPCAT activities were measured as described previously, using microplate chromatography or thin layer chromatography (20Shindou H. Hishikawa D. Nakanishi H. Harayama T. Ishii S. Taguchi R. Shimizu T. J. Biol. Chem. 2007; 282: 6532-6539Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 21Nakanishi H. Shindou H. Hishikawa D. Harayama T. Ogasawara R. Suwabe A. Taguchi R. Shimizu T. J. Biol. Chem. 2006; 281: 20140-20147Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 27Kume K. Waga I. Shimizu T. Anal. Biochem. 1997; 246: 118-122Crossref PubMed Scopus (7) Google Scholar). For the measurement of lyso-PAF AT activity, the protein was incubated with 0–200 μm [3H]acetyl-CoA (1.11 GBq/mmol) and 20 μm lyso-PAF in a buffer containing 20 mm Tris-HCl (pH 7.4), 5 mm 2-mercaptoethanol, 2 mm CaCl2, 20 μm APMSF, 1× Complete, and 1 mg/ml PC. Eventually, EDTA, CoCl2, CuCl2, FeCl2, MgCl2, or MnCl2 was used instead of CaCl2 in some experiments. For the measurement of LPCAT activity, protein was incubated with 0–200 μm acyl-CoA and 50 μm LPC in a buffer containing 100 mm Tris-HCl (pH 7.4), 1 mm EDTA, and 1 mg/ml PC. As radiolabeled substrate, [14C]palmitoyl-CoA or [14C]palmitoyl-LPC was utilized. Production of Anti-LysoPAFAT/LPCAT2 and Anti-LPCAT1 Antisera—Antisera were generated at Immuno-Biological Laboratories (Gunma, Japan). C-terminal peptides were used for immunization of rabbits (Lyso-PAFAT/LPCAT2, SNKVSPESQEEGTSDKKVD, and LPCAT1, EMYPDYAEDYLYPDQTHFDS). Specificity of the antisera was examined by Western blot using microsomes from vector-, Lysopafat/Lpcat2-, or Lpcat1-transfected cells. Western Blot Analysis—Western blot analyses were performed as described previously (21Nakanishi H. Shindou H. Hishikawa D. Harayama T. Ogasawara R. Suwabe A. Taguchi R. Shimizu T. J. Biol. Chem. 2006; 281: 20140-20147Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Antisera were used in a dilution factor of 1:1000. Site-directed Mutagenesis of LPCAT1—Mutants of LPCAT1 were constructed using QuikChange site-directed mutagenesis kit or by overlap extension PCR (28Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6833) Google Scholar). The primer sets utilized are listed in the supplemental material. Identification of PAF by PAF Receptor Binding Assay and Mass Spectrometry—PAF receptor binding assay was done as described previously (14Shindou H. Ishii S. Uozumi N. Shimizu T. Biochem. Biophys. Res. Commun. 2000; 271: 812-817Crossref PubMed Scopus (58) Google Scholar). Mass spectrometry was performed as described previously (20Shindou H. Hishikawa D. Nakanishi H. Harayama T. Ishii S. Taguchi R. Shimizu T. J. Biol. Chem. 2007; 282: 6532-6539Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 29Harrison K.A. Clay K.L. Murphy R.C. J. Mass Spectrom. 1999; 34: 330-335Crossref PubMed Google Scholar) and as precisely described in supplemental Fig. 5. Isolation and Stimulation of Mouse Peritoneal Cells—Mouse peritoneal macrophages induced by thioglycolate were prepared as described previously (17Shindou H. Ishii S. Yamamoto M. Takeda K. Akira S. Shimizu T. J. Immunol. 2005; 175: 1177-1183Crossref PubMed Scopus (39) Google Scholar). The cells were treated with 100 ng/ml LPS, 0.8 μm CpG oligonucleotide (ODN) 1826, or 1 μg/ml polyinosine-polycytidylic acid (poly(I:C)) for 16 h. Cells were then washed with ice-cold buffer containing 20 mm Tris-HCl (pH 7.4) and 300 mm sucrose. Total RNAs were then extracted and subsequently used for the synthesis of first-strand cDNA. Isolation of Rat Alveolar Type II Cells and Macrophages—Alveolar type II cells were isolated by the method of Dobbs and Mason (30Dobbs L.G. Mason R.J. J. Clin. Investig. 1979; 63: 378-387Crossref PubMed Scopus (279) Google Scholar), as described previously (21Nakanishi H. Shindou H. Hishikawa D. Harayama T. Ogasawara R. Suwabe A. Taguchi R. Shimizu T. J. Biol. Chem. 2006; 281: 20140-20147Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Alveolar macrophages for RNA extraction were recovered from bronchoalveolar lavage fluid of 7-week-old Sprague-Dawley rats. Rats were anesthetized and euthanized, and bronchoalveolar lavage fluid was obtained by four times lavage using phosphate-buffered saline. A part of the cells was cytocentrifuged with Cytospin 3 at 200 rpm for 2 min and stained with Diff-Quick confirming that >90% of the cells were macrophages. Quantitative PCR—Quantitative PCR experiments were performed as described previously (21Nakanishi H. Shindou H. Hishikawa D. Harayama T. Ogasawara R. Suwabe A. Taguchi R. Shimizu T. J. Biol. Chem. 2006; 281: 20140-20147Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) using LightCycler 1.5 (Roche Diagnostics). The primers used are indicated in the supplemental material. Software—All statistical calculations were performed using Prism 4 (GraphPad Software). Edmundson wheel analysis was performed using GENETYX-MAC version 13.0.6 (Genetyx Corp.). Identification of a Ca2+-independent Lyso-PAF AT Activity—To characterize PAF biosynthesis in mice, we measured lyso-PAF acetyltransferase activities in lung, spleen, brain, liver, and kidney microsomes in the presence of EDTA or Ca2+. Consistently with the expression pattern of Lysopafat/Lpcat2 (20Shindou H. Hishikawa D. Nakanishi H. Harayama T. Ishii S. Taguchi R. Shimizu T. J. Biol. Chem. 2007; 282: 6532-6539Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), high lyso-PAF AT activity was observed in spleen microsomes in the presence of Ca2+, which was inhibited by EDTA (Ca2+-dependent lyso-PAF AT activity) (Fig. 1A). The Ca2+-dependent lyso-PAF AT activity was weaker in brain and liver microsomes and not detectable in kidney microsomes. Surprisingly, EDTA did not inhibit lyso-PAF AT activity in lung microsomes, indicating the presence of a Ca2+-independent lyso-PAF AT (Fig. 1A). Partial Purification of LPCAT1 as a Lyso-PAF AT—We next tried to identify the Ca2+-independent lyso-PAF AT by partial purification. After solubilization (with 0.5% (w/v) BIGCHAP), lung microsomes retained the Ca2+-independent lyso-PAF AT activity (Fig. 1C). Solubilized microsomes were applied on a DEAE-Sepharose column and fractionated (Fig. 1B). For each fraction, Ca2+-independent lyso-PAF AT activity was measured. The activity was detected from fractions 10 to 13 (Fig. 1C). We performed Western blot using anti-LysoPAFAT/LPCAT2 antiserum. Two major bands appeared in the spleen microsomes and the solubilized lung microsomes, the lower one having the size expected from the primary sequence of LysoPAFAT/LPCAT2 (60 kDa). However, the 60-kDa band was barely detected after fractionation (Fig. 1D). Thus, the proteins exhibiting immunoreactivity against LysoPAFAT/LPCAT2 were different from that with Ca2+-independent lyso-PAF AT activity (Fig. 1, C and D). We performed similar experiments using anti-LPCAT1 antiserum, because LPCAT1 possesses the highest homology (48.2%) to LysoPAFAT/LPCAT2. The immunoreactivity was present in fractions 10–14, with similarity to the Ca2+-independent lyso-PAF AT activity (Fig. 1, C and E), suggesting that LPCAT1 might be the Ca2+-independent lyso-PAF AT in lung. To obtain more direct evidence for the lyso-PAF AT activity in LPCAT1, microsomes were prepared from Lpcat1- or vector-transfected Chinese hamster ovary (CHO)-K1 cells, and lyso-PAF AT activity was measured. Lpcat1-transfected cells had a higher lyso-PAF AT activity than the control cells, indicating that LPCAT1 is the Ca2+-independent lyso-PAF AT partially purified from murine lung (Fig. 2A). Characterization of LPCAT1 as a Lyso-PAF AT—We characterized kinetic properties of LPCAT1. The apparent Km values for acetyl-CoA and lyso-PAF were 82.4–128.2 and 7.9–18.4 μm, respectively (supplemental Fig. 1, A and B). Lyso-PAF AT activity of LPCAT1 was similar with or without Ca2+ (Fig. 2A). On the other hand, the activity of LysoPAFAT/LPCAT2 was detected in the presence of Ca2+ but not EDTA (Fig. 2A). The activity of LPCAT1 was not influenced by the presence of Co2+, Mg2+, or Mn2+, whereas Cu2+ and Fe2+ inhibited it. The activity of LysoPAFAT/LPCAT2 was detected in the presence of Co2+, Mg2+, Fe2+, or Mn2+ but not Cu2+ (Fig. 2A). The activity of LPCAT1 was unaltered by the presence of dithiothreitol, but the activity of LysoPAFAT/LPCAT2 was 2-fold increased (supplemental Fig. 1C). The pH optimum for LPCAT1 activity was around pH 7.5 (supplemental Fig. 1D). We next investigated the lysophospholipid preference of LPCAT1. LPCAT1 showed high activity toward alkyl-LPC (C16), acyl-LPC (C16), and alkenyl-LPC (mixture). The activity was lower for acyl-LPC (C18) and not significant for alkyl-LPC (C18) (Fig. 2B). Thus, LPCAT1 can synthesize PAF (C16) much more efficiently than PAF (C18). Acetyltransferase activity of LPCAT1 had not been reported previously, probably because of the concentration of acetyl-CoA used (21Nakanishi H. Shindou H. Hishikawa D. Harayama T. Ogasawara R. Suwabe A. Taguchi R. Shimizu T. J. Biol. Chem. 2006; 281: 20140-20147Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 22Chen X. Hyatt B.A. Mucenski M.L. Mason R.J. Shannon J.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11724-11729Crossref PubMed Scopus (151) Google Scholar). Thus, we measured acyltransferase activities of LPCAT1 with two different concentrations (10 and 200 μm) of various acyl-CoAs (C2 to C20) as acyl donors. At 10 μm acyl-CoA, LPCAT1 utilized all saturated acyl-CoAs ranging from hexanoyl-CoA (C6) to palmitoyl-CoA (C16) as substrates, and the maximal activity was observed for decanoyl-CoA (C10) (Fig. 2C). On the other hand, at 200 μm acyl-CoA, the substrate preference of LPCAT1 shifted to shorter chain acyl-CoAs (Fig. 2D). Similarly to our previous report, LPCAT1 showed no acetyltransferase activity, at the substrate concentration of 10 μm (Fig. 2C). However, we could detect the activity for acetyl-CoA (C2) at 200 μm, as expected from the result shown in supplemental Fig. 1A. Activity for butanoyl-CoA (C4) was also seen, but it was not significantly different from that in vector-transfected cells. Thus, LPCAT1 recognizes a wide range of acyl-CoAs (from C2 to C16) and exerts acetyltransferase activity at a high concentration of acetyl-CoA. Analysis of Conserved Motifs in LPCAT1—To prove that the wide range of acyltransferase activity seen in Lpcat1-transfected cells is directly exerted by LPCAT1, we tried to characterize the recognition sites of acyl-CoAs in LPCAT1. Because acyl-CoA recognition may be a common characteristic of acyltransferases, we hypothesized that some of the acyltransferase motifs may play roles in acyl-CoA recognition. We compared the sequences of the following lysophospholipid acyltransferases: glycerol-3-phosphate acyltransferase 1, lysophosphatidic acid acyltransferase α and β, lysophosphatidylglycerol acyltransferase 1, and lysocardiolipin acyltransferase 1. From the aligned sequences of these enzymes, conserved His135 and Asp140 in motif 1 and Glu208 and Gly209 in motif 3 were found in LPCAT1. The amino acids in motif 2 were less conserved, and the following three regions were candidates for motif 2 in LPCAT1: from Gly164 to Arg168, from Gly164 to Arg171, and from Phe174 to Arg177. Motif 4 consists of a conserved Pro surrounded by hydrophobic amino acids. Three Pro residues in LPCAT1, namely Pro227, Pro230 and Pro233, fulfilled this criterion (Fig. 3A). We next investigated the effects of the deletion of each motif on the activity of LPCAT1. Each motif region (from His135 to Asp140, from Gly164 to Arg177, from Ile203 to Gly209, and from Pro227 to Pro233) was deleted from LPCAT1, generating mutants del1, del2, del3, and del4. Each mutant was transfected into CHO-K1 cells, and the expression was examined by Western blot. Each mutant was expressed but was weaker than wild type LPCAT1 (WT) (supplemental Fig. 2A). Lyso-PAF AT and LPCAT activities were then measured by thin layer chromatography analyses. Lyso-PAF AT and LPCAT activities were abolished in these deletion mutants (supplemental Fig. 2B). We then examined the effect of point mutations in each motif. We generated mutants H135A and D140A in motif 1; G164A, R168A, R171A, F174A, and R177A in motif 2; E208A and G209A in motif 3; and P227A, P230A, and P233A in motif 4. Each mutant was transfected into CHO-K1 cells, and the expression was detected by Western blot. The expression levels of D140A, R171A, F174A, E208A, and P233A decreased, whereas those of other mutants were similar to that of WT (supplemental Fig. 2, C–F). For each mutant, we measured lyso-PAF AT (1-hexadecyl-lyso-PAF and acetyl-CoA as substrates) and LPCAT (1-myristoyl-LPC and palmitoyl-CoA as substrates) activities relative to WT (hereafter, we will refer these relative activities as “remaining activity”). H135A, D140A, and E208A had no remaining activity, whereas R168A, R171A, G209A, P227A, P230A, and P233A had partially remaining lyso-PAF AT and LPCAT activities (Fig. 3, B–E). Notably, in G164A, lyso-PAF AT activity was slightly increased (∼150%), but LPCAT activity was similar to WT (∼100%). F174A and R177A had no detectable lyso-PAF AT activity (<10%), whereas LPCAT activity was only partially diminished (∼40%) (Fig. 3C). Analysis of Motif 2 in LPCAT1—We investigated further lyso-PAF AT and LPCAT activities in F174A and R177A. The relative lyso-PAF AT and LPCAT activities were not affected by reaction buffers, lysophospholipid species (lyso-PAF and LPC), or acyl-chain length at the sn-1 position of LPC (C14 and C16) (data not shown). However, the relative activity for acetyl-CoA was different from that for palmitoyl-CoA (Fig. 3C and Fig. 4A). We measured LPCAT activities of F174A and R177A for various chain length acyl-CoAs (C2 to C16). The substrate concentrations were 200 μm for short-chain acyl-CoAs (C2 to C8), and 10 μm for medium-chain acyl-CoAs (C10 to C16). The remaining LPCAT activities in F174A and R177A were higher for longer acyl-CoAs as substrates (Fig. 4A). We confirmed that such relationships were found in several concentrations of acyl-CoAs (supplemental Fig. 3, A–C). Thus, the preference of F174A and R177A for longer chain acyl-CoA was not because of substrate concentration but because of acyl chain length. These results suggest that mutations in motif 2 modulate acyl-CoA selectivity. Motif 2 of LPCAT1 contains many hydrophobic amino acids conserved among species, thus these residues may interact with acyl-CoA (Fig. 4B and supplemental Fig. 4A). The hydrophobic region (from Trp163 to Tyr169) was predicted to form an α-helix using the PredictProtein Server (31Rost B. Yachdav G. Liu J. Nucleic Acids Res. 2004; 32: W321-W326Crossref PubMed Scopus (1183) Google Scholar). When this hydrophobic region (in this example, Ile162 to Arg171) is illustrated by an Edmundson wheel representation (32Schiffer M. Edmundson A.B. Biophys. J. 1967; 7: 121-135Abstract Full Text PDF PubMed Scopus (901) Google Scholar), all hydrophobic residues are located at one side (Fig. 4B). This suggests that this region forms an α-helix, and the hydrophobic residues are located favorably to interact with the acyl chain. To investigate their roles in acyl-CoA interaction, we constructed mutants of each hydrophobic residue (I160A, I162A, W163A, L166A, I167
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