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An accurate and versatile method for determining the acyl group-introducing position of lysophospholipid acyltransferases
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An accurate and versatile method for determining the acyl groupintroducing position of lysophospholipid acyltransferases Hiroki Kawanaa,b, Kuniyuki Kanoa,b, Hideo Shindouc,d, Asuka Inouea,b, Takao Shimizud,e, ⁎ Junken Aokia,b, a Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-Ku, Sendai 980-8578, Japan b AMED-LEAP, Chiyoda-ku, Tokyo 100-0004, Japan c Department of Lipid Signaling, National Center for Global Health and Medicine, Shinjuku-ku, Tokyo 162-8655, Japan d AMED-CREST, Chiyoda-ku, Tokyo 100-0004, Japan e Departments of Lipidomics, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Phospholipid sn-2-Acyl lysophospholipid sn-1-Acyl lysophospholipid Lysophospholipid acyltransferase LPCAT1 LYCAT LPCAT3
Lysophospholipid acyltransferases (LPLATs) incorporate a fatty acid into the hydroxyl group of lysophospholipids (LPLs) and are critical for determining the fatty acid composition of phospholipids. Previous studies have focused mainly on their molecular identification and their substrate specificity regarding the polar head groups and acyl-CoAs. However, little is known about the positional specificity of the hydroxyl group of the glycerol backbone (sn-2 or sn-1) at which LPLATs introduce a fatty acid. This is mainly due to the instability of LPLs used as an acceptor, especially for LPLs with a fatty acid at the sn-2 position of the glycerol backbone (sn-2LPLs), which are essential for the enzymatic assay to determine the positional specificity. In this study, we established a method to determine the positional specificity of LPLAT by preparing stable sn-2-LPLs in combination with PLA2 digestion, and applied the method for determining the positional specificity of several LPLATs including LPCAT1, LYCAT and LPCAT3. We found that LPCAT1 introduced palmitic acid both at the sn-1 and sn2 positions of palmitoyl-LPC, while LYCAT and LPCAT3 specifically introduced stearic acid at the sn-1 position of LPG and arachidonic acid at the sn-2 position of LPC, respectively. The present method for evaluating the positional specificity could also be used for biochemical characterization of other LPLATs.
1. Introduction Lysophospholipid acyltransferases (LPLATs) introduce an acyl group into lysophospholipids (LPLs) at either the sn-1 or sn-2 position of the glycerol backbone and are crucial for determining the fatty acid composition of phospholipids. Recent studies have identified several LPLAT isozymes that showed preference for the polar heads of LPLs and acyl-CoAs [1,2]. However, for most of the LPLAT isozymes, the glycerol positions (sn-1 or sn-2) at which they introduce fatty acids have not been determined. It is generally accepted that an acyl chain linked either at the sn-1 or sn-2 position of LPLs spontaneously moves to another position, yielding a mixture of LPLs with the acyl chain at either the sn-1 or sn-2 position. For example, 1-palmitoyl-lysophosphatidylcholine (1-palmitoyl-sn-glycero-3-phosphorylcholine), which is converted from the corresponding
phosphatidylcholine (PC) by phospholipase A2 (PLA2) reaction, is actually an equilibrium mixture consisting of approximately 90% 1-palmitoyl-2-lyso- and 10% 2-palmitoyl-1-lyso-isomers [3]. The acyl chain movement, which is known as an intramolecular acyl migration, occurs especially fast in LPLs with a fatty acid moiety at the sn-2 position (sn-2LPLs) [3,4]. Thus sn-2-LPLs are unstable and are quickly converted to the corresponding sn-1-LPLs, especially under neutral or alkaline conditions. This has hampered the LPLAT enzyme assay, especially the assay in which an acyl chain is introduced at the sn-1 position of sn-2acyl LPLs. We and others have shown that the intramolecular acyl migration reaction does not proceed under acidic conditions [3,4]. Thus, it is reasonable to assume that sn-2-LPLs can be prepared more efficiently by hydrolyzing PLs with phospholipase A1 (PLA1) and then lowering the pH of the reactant. In this study, we first tried to prepare a high-grade
⁎ Corresponding author at: Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-Ku, Sendai 980-8578, Japan. E-mail address: [email protected] (J. Aoki).
https://doi.org/10.1016/j.bbalip.2019.02.008 Received 1 October 2018; Received in revised form 2 February 2019; Accepted 24 February 2019 1388-1981/ © 2019 Published by Elsevier B.V.
Please cite this article as: Hiroki Kawana, et al., BBA - Molecular and Cell Biology of Lipids, https://doi.org/10.1016/j.bbalip.2019.02.008
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Fig. 1. Preparation of sn-1- and sn-2-palmitoyl dominant LPCs. (A–C) Ratio of 2-palmitoyl-1-lyso-PC (sn-2-palmitoyl LPC: filled) and 1-palmitoyl-2-lyso-PC (sn-1-palmitoyl LPC: dashed) in the present LPC preparations used for LPCAT1 assay. “sn-2-pal (palmitoyl) dominant LPC” was prepared from DPPC by Rhizomucor miehei lipase (PLA1), and “sn-1-pal (palmitoyl) dominant LPC” was prepared from “sn-2-pal (palmitoyl) dominant LPC” by using a spontaneous acylmigration reaction. (A and B) Ratio of sn-1 and sn-2palmitoyl LPC before (A) and after (B) 37 °C 10 minute incubation. (C) Ratio of sn-1 and sn-2palmitoyl LPC preparations in the deuterium-labeled palmitoyl LPC. The data are representative of three independent experiments with similar results.
2. Material and methods
sn-2-LPL preparation. We then used it as well as its sn-1 isomer to determine the specificity of LPLATs for the sn-1 or sn-2 position. We first selected LPCAT1 as a model of LPLAT molecules since it is involved in the synthetic pathway of a symmetric phospholipid, dipalmitoyl PC [5–7], possibly by introducing palmitic acid to either the sn-1 position of 2-palmitoyl-1-lysophosphatidylcholine (LPC) or to the sn-2 position of 1-palmitoyl-2-LPC. Thus, we assumed that LPCAT1 is an ideal LPLAT to determine its positional specificity. We also selected two other LPLATs, LYCAT and LPCAT3, which have been suggested to introduce an acyl chain in position-specific manner [8,9]. Our present results indicated that LPCAT1 introduced a fatty acid to both the sn-1 and sn-2 positions, while LYCAT and LPCAT3 selectively introduced a fatty acid to the sn-1 and sn-2 positions, respectively, demonstrating the present method is suitable for determining the positional specificity of LPLATs.
2.1. Materials Dulbecco's modified Eagle's medium (DMEM) was purchased from Nissui Pharmaceutical (Tokyo, Japan). Fetal bovine serum (FBS) and Opti-MEM were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Honey bee venom PLA2, lipase from Rhizomucor miehei which has intrinsic PLA1 activity and palmitoyl- [13C16] coenzyme A were obtained from Sigma-Aldrich (St. Louis, MO). All glycerophospholipids and lysophospholipids including palmitoyl-d62 PC (1,2-dipalmitoyld62-sn-glycero-3-phosphocholine) were purchased from Avanti Polar Lipids (Alabaster, AL). LC-MS grade methanol and acetonitrile were purchased from Kanto Chemical (Tokyo, Japan). Chloroform, formic acid and ammonium formate were purchased from Fujifilm-Wako
2
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labeled, [13C]-labeled, deuterium-labeled palmitoyl LPC and stearoyl LPC are 496.3, 512.3, 526.3 and 524.4 respectively. The m/z values for non-labeled DPPC, DPPC with a non-labeled palmitoyl and a [13C]-labeled palmitoyl ([13C16]DPPC), and DPPC with a [13C]-labeled palmitoyl and deuterium-labeled palmitoyl ([13C16]-[d31] DPPC), and 38:4 (stearoyl-arachidonoyl) PC are 734.6, 750.6, 780.6 and 810.6 respectively. At MS3, phosphocholine fragments (m/z = 184.1) were detected. To measure phosphatidylglycerol (PG), at MS1 the m/z values of [M + NH4]+ ion for 36:1 (stearoyl-oleoyl) PG is 794.6. At MS3, corresponding diacylglycerol fragment (m/z = 605.5) was detected in positive ion mode. For measurement of lysophosphatidylglycerol (LPG), at MS1 the m/z values of [M-H]− ion for oleoyl LPG is 509.3. At MS3, corresponding fatty acid fragment (m/z = 281.2) was detected in negative ion mode. To confirm incorporated fatty acid composition of the assay reactants, the product ions were scanned in negative mode to detect [M + HCOO]− ions at MS1.
(Osaka, Japan). 2.2. Preparation of sn-1-acyl dominant LPLs and sn-2-acyl dominant LPLs To obtain palmitoyl lysophosphatidylcholine (LPC) with palmitic acid at the sn-2 position of the glycerol backbone (sn-2-palmitoyl LPC), dipalmitoyl PC (DPPC) was subjected to an in vitro PLA1 reaction using the method by M. Okudaira et al. [4] with some modifications. Nonlabeled or deuterium-labeled (d62) DPPC (1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine) (1 μmol each) was incubated with reaction mixture containing 78 mM phosphate, 10% diethyl ether, 2 mM CaCl2, 0.05% TritonX-100 and 4.5 μg/ml Rhizomucor miehei lipase (pH 7.4). The mixture was incubated at 37 °C for 80 min. Then the reaction was stopped by adding 9 volumes of acidic methanol (pH 4.0) previously described [4]. After centrifugation (21,500 ×g, 5 min), the supernatant was applied to a Bond Elut C18 solid phase cartridge column (Agilent Technologies) to remove the remaining DPPC and free palmitic acid, the byproduct of the PLA1 reaction. The extract was dried by evaporation and dissolved in acidic methanol. The sn-1/sn-2 ratio and the concentration of sn-2-palmitoyl LPC preparation were determined by LC-MS/MS as previously described [4] with slight modifications (see below). The sn-2-palmitoyl LPC preparation predominantly contained LPC with palmitic acid at the sn-2 position (> 95%) and the level of remaining PC was very low (< 0.001% of LPC). The preparation, which we called “stock sn-2-palmitoyl dominant LPC”, was stored at −80 °C. The sn-1 LPC isomer was prepared from the sn-2 LPC isomer by using the spontaneous acyl-migration reaction. Since the acyl-migration reaction is dependent on the pH of the solution and the incubation time, the “stock sn-2-palmitoyl dominant LPC” was dissolved in weak alkaline solution (100 mM Tis-HCl (pH 8.9)). To prepare the sn-2 LPC isomer, the reaction was stopped immediately by adding acidic methanol. To prepare the sn-1 LPC isomer, the reaction mixture was incubated for 2 h at 37 °C before stopping the reaction. The new preparations were called “sn-2-palmitoyl dominant LPC” and “sn-1-palmitoyl dominant LPC”, respectively. We confirmed that the prepared “sn-1-palmitoyl dominant LPC” and “sn-2-palmitoyl dominant LPC” predominantly contained LPC with palmitic acid at the sn-1 position (> 90%) and sn-2 position (> 90%), respectively (Fig. 1). We also prepared sn-1and sn-2-oleoyl dominant LPG and sn-1and sn-2-stearoyl dominant LPC using the same procedure as stated above, except that dioleoyl PG (DOPG) and distearoyl PC (DSPC) were used instead of DPPC.
2.4. Plasmids and vectors cDNA for human LPCAT1 (hLPCAT1; NCBI accession number NM_ 024830) was amplified by PCR using PrimeSTAR HS Polymerase (TAKARA BIO Inc.) and HEK293A cell cDNA as a template. The PCR primers used were as follows. Forward 5′-CGCCACCATGAGGCTGCGGGGATGCGG-3′ Reverse 5′-GAGCTCGAGCTAATCCAGCTTCTTGCGAAC-3′ The amplified cDNA fragments were inserted into the pCAGGS vector (a gift from J. Miyazaki, Osaka University) with or without an Nterminal FLAG-tag. The nucleotide sequences of all the plasmids prepared were checked by DNA sequencing. Mouse LPCAT1, LYCAT and LPCAT3 clone in pCXN2.1 vector were previously described [5,8,9,11]. 2.5. Cell culture and transfection of plasmids HEK293A cells (Thermo Fisher Scientific) were maintained in DMEM supplemented with 10% FBS, 2 mM L-Glutamine (Thermo Fisher Scientific), 100 U/ml penicillin (Sigma-Aldrich), and 100 μg/ml streptomycin (Thermo Fisher Scientific) at 37 °C in the presence of 5% CO2 gas. HEK293A cells were transfected with cDNAs encoding several LPLATs using Opti-MEM and Lipofectamine 2000 (Thermo Fisher Scientific).
2.3. LC-MS/MS analyses
2.6. Preparation of membrane fractions
Lysophospholipids (LPLs) were measured as previously described [4] with minor modifications. The LC-MS/MS system consisted of an UltiMate 3000 HPLC system and TSQ Quantiva Triple-Stage Quadrupole mass spectrometer (Thermo Fisher Scientific) equipped with a heated-electrospray ionization-II (HESI-II) source. For HPLC, we used a reverse-phase column (Capcell Pak C18 ACR (250 mm × 1.5 mm,3 μm particle size; Osaka Soda) with a gradient elution of solvent A (5 mM ammonium formate in water, pH 4.0) and solvent B (5 mM ammonium formate in 95% (v/v) acetonitrile, pH 4.0) at 200 μl/min. This method enabled us to separate 1-acyl-2-lyso-PL (sn-1-acyl LPL) and 2-acyl-1lyso-PL (sn-2-acyl LPL). Diacyl-phospholipids (PLs) including PC and PG were quantified by a similar method [10] with slight modifications. The LC-MS/MS system consisted of a NANOSPACE SI-II HPLC system (Osaka Soda) and TSQ Quantum Ultra triple quadrupole mass spectrometer (Thermo Fisher Scientific) equipped with a heated-electrospray ionization-II (HESI-II) source. LC separation was performed using a reverse-phase column (Capcell Pak C8 UG120, 150 mm × 1.5 mm, 5 μm particle size; Osaka soda) with a gradient elution of solvent A and solvent B (described above) at 400 μl/min. LPC and PC were detected by selected reaction monitoring (SRM) in positive ion mode. At MS1, the m/z values of [M + H]+ ions for non-
HEK293A cells were harvested and transferred to ice-cold TSC buffer (20 mM Tris-HCl, pH 7.4, 300 mM sucrose and cOmplete™ Protease Inhibitor Cocktail (Roche, Mannheim, Germany)), and were sonicated ten times for 10 s using a probe-type sonicator (MICROTEC). The cell homogenates were centrifuged at 800 ×g for 10 min, and the supernatants were further centrifuged at 100,000 ×g for 1 h. The resultant pellets were re-suspended in TSE buffer (20 mM Tris-HCl, pH 7.4, 300 mM sucrose and 1 mM EDTA). Protein concentrations were determined with a BCA protein assay kit (Thermo Fisher Scientific). The membrane fractions were used as an enzyme source. 2.7. LPL acyltransferase assays LPC acyltransferase (LPCAT) activities that introduce palmitic acid into palmitoyl LPC were determined by measuring the product of the LPCAT reaction, i.e. DPPC with a slight modification of T. Harayama et al. [7] In the LPCAT reaction, membrane fractions (containing LPCAT1 recombinant protein), LPC (“sn-1-palmitoyl dominant LPC” or “sn-2-palmitoyl dominant LPC”) and [13C16]palmitoyl-CoA were mixed and incubated at 37 °C for 10 min. The final reaction mixture contained 110 mM Tris-HCl (pH 7.4), 150 mM sucrose, 0.5 mM EDTA, 10 μM LPC, 2 μM Acyl-CoA, 0.015% Tween-20 and 0.05 μg of membrane protein. 3
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products were subjected to the PLA2 reaction and the position of the incorporated palmitic acid in the resulting palmitoyl-LPC was determined. Since palmitoyl-LPC can be produced from palmitoyl-containing PC in the membrane fraction that was used as an enzyme source, which may disturb measurement of the palmitoyl LPC concentration, a deuterium-labeled [d31]palmitoyl LPC (both “sn-1-palmitoyl dominant LPC” and “sn-2-palmitoyl dominant LPC”) was prepared from [d62] DPPC (possessing two [d31]-labeled palmitic acids) and used instead of non-labeled palmitoyl LPC as an acceptor. [13C16][d31] DPPC produced by the LPCAT1 assay was reacted with PLA2. The position of [13C16]palmitic acid and [d31]palmitic acid in the resulting palmitoyl LPC was then determined. To separate [13C16]-[d31] DPPC from [d31]palmitoyl LPC (because it was used an acceptor, the reaction mixture had excess [d31]palmitoyl LPC), the assay products were applied to a Bond Elut C8 solid phase cartridge column (Agilent Technologies) and eluted with 5 mM ammonium formate in 99.5% (v/ v) methanol. The eluted fraction containing [13C16]-[d31] DPPC was evaporated and dissolved in 100 mM Tris-HCl buffer containing 0.1% Triton X-100. The PLA2 reaction was started by adding 0.1unit bee venom PLA2, allowed to continue at 37 °C for 10 min and then stopped by acidic methanol containing 1 μM 17:0 LPC and 100 nM 17:0 LPA as an internal standard. After centrifugation (21,500 ×g, 5 min), the supernatant was subjected to LC-MS/MS analysis to determine the concentrations of sn-1 [13C16]palmitoyl LPC and sn-1 [d31]palmitoyl LPC. The LYCAT and LPCAT3 assay products were also digested with PLA2, and the resulting sn-1-acyl-LPG and sn-1-acyl-LPC were analyzed by LCMS/MS. 2.9. Data analysis The apparent Km and Vmax values were calculated from the Michaelis-Menten equation using GraphPad Prism 7.0 software (GraphPad). Fig. 2. LPCAT1 activities toward sn-1 and sn-2-pal dominant LPCs. LPCAT assays were performed using “sn-1-pal dominant LPC” or “sn-2-pal dominant LPC” as an acceptor, and a membrane fraction of LPCAT1-overexpressing HEK293A cells as an enzyme source. The results using recombinant mouse LPCAT1 (A) and human LPCAT1 (B) are shown on the right and the results using control membrane fraction (mock) were also shown on the left. Data are shown as the mean + SD of three data points. Three experiments using independently-prepared membrane fractions were performed with similar results.
3. Results 3.1. Preparation of LPC with palmitic acid predominantly at the sn-1 or sn2 position To determine at which position of the LPC glycerol backbone LPCAT1 introduces palmitic acid, we first prepared two types of palmitoyl-LPC that have palmitic acid at either the sn-2 or sn-1 position from dipalmitoyl PC (DPPC). As described in Material and methods section, the two LPC isomers were separated and quantified by LC-MS/ MS to determine their purity. The analysis revealed that the present sn2 LPC isomer preparation was composed of > 90% of 2-palmitoyl-1lyso-PC and < 10% of 1-palmitoyl-2-lyso-PC, while the sn-1 LPC isomer preparation was composed of > 90% of 1-palmitoyl-2-lyso-PC and < 10% of 2-palmitoyl-1-lyso-PC (Fig. 1A). After incubating the lipids for 10 min in enzymatic assay buffer, the assay condition for the preset LPCAT1 assay, most of the 2-palmitoyl-1-lyso-PC remained intact (Fig. 1B). In the following studies, we call the sn-1- and sn-2-palmitoyl LPC isomer preparations as the sn-1- and sn-2-palmitoyl dominant LPCs, respectively. We also produced deuterium labeled sn-1-palmitoyl dominant LPC and sn-2-palmitoyl dominant LPC from deuterium labeled DPPC (Fig. 1C).
The reaction was stopped by adding chloroform/methanol (1:2). Dilauryl-PC (DLPC) or dilauryl-PG (DLPG) was added as an internal standard and lipids were extracted by the Bligh & Dyer method. The organic (lower) layer was evaporated, dissolved in methanol, and the amounts of DPPC containing [13C16]-labeled palmitic acid were quantified by LC–MS/MS. To quantify the amount of labeled-DPPC, commercially available non-labeled DPPC was used as a calibration standard. For kinetics analysis, various concentrations of LPC (“sn-1palmitoyl dominant LPC” or “sn-2-palmitoyl dominant LPC”) and [13C16]palmitoyl-CoA were subjected to the LPCAT reaction using fixed concentrations of [13C16]palmitoyl-CoA (2 μM) and LPC (10 μM), respectively. Similar assay conditions were employed for LPG acyltransferase (LPGAT) activities for LYCAT and LPC acyltransferase (LPCAT) activities for LPCAT3. LPGAT activities for LYCAT were determined in the presence of LYCAT-overexpressed membrane fraction (0.05 μg) and stearoyl-CoA using sn-2 or sn-1-oleoyl dominant LPG. LPCAT activities for LPCAT3 were determined in the presence of LPCAT3-overexpressed membrane fraction (0.005 μg) and arachidonoyl-CoA using sn-2 or sn-1-stearoyl dominant LPC.
3.1.1. LPCAT1 introduces palmitic acid at both the sn-1 and sn-2 positions of LPC In order to determine the position of the LPC glycerol backbone at which LPCAT1 introduces palmitic acid, we performed an LPCAT assay using sn-1-palmitoyl dominant LPC or sn-2-palmitoyl dominant LPC as the acceptor, [13C16]palmitoyl-CoA as the acyl donor, and the membrane fraction of LPCAT1-overexpressed cells as the enzyme source. We evaluated the LPCAT1 activity by measuring the [13C16] DPPC formed. Using the membrane fraction from HEK293A cells expressing mouse
2.8. Confirmation of the position of incorporated fatty acid To determine position of incorporated palmitic acid, LPCAT1 assay 4
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Fig. 3. Confirmation of the position of incorporated palmitic acid. (A) Fragment spectrum of [13C16]palmitoyl-palmitoyl-PC produced by LPCAT1 reaction. LPCAT1 assay products containing [13C16]palmitoyl-palmitoyl-PC were subjected to mass spectrometry (MS) analysis. In the negative ion mode, PC [M + HCOO]− ions were fragmented using a collision energy of 45 eV and two fatty acid (FA) fragments (palmitic acid m/z = 255.2 and [13C16]palmitic acid m/z = 271.2) were detected. Left and right panels show representative fragment patterns of assay products using “sn-1-pal dominant LPC” and “sn-2-pal dominant LPC” as an acceptor, respectively. (B and C) Determination of the position of incorporated [13C16]palmitic acid by using PLA2 reaction and LC-MS/ MS. (B) LPCAT1 activities using sn-1- or sn-2-[d31]pal dominant LPC as an acceptor and recombinant human LPCAT1. Similar results were obtained when nonlabeled sn-1 or sn-2 pal dominant LPC was used (Fig. 2). Data are shown as the mean + SD of three data points. Three experiments using independently-prepared membrane fractions were performed with similar results. (C) Ratio of sn-1-[13C16]palmitoyl-LPC (dot) and sn1-[d31]palmitoyl-LPC (shadowed) in PLA2-digested assay mixtures. LPCAT1 assay products [13C16]palmitoyl-[d31] palmitoyl-PC were subjected to bee venom PLA2 treatment. The resulting LPC species were analyzed by LC-MS/ MS. The data are representative of three independent experiments with similar results.
LPCAT1, significant amounts of [13C16]palmitic acid were incorporated in both sn-1-palmitoyl dominant LPC and sn-2-palmitoyl dominant LPC although the amount incorporated in the latter was almost twice the amount incorporated in the former (Fig. 2A). Interestingly, the membrane fraction prepared from mock-transfected HEK293A cells also had similar [13C16]palmitic acid-incorporating activity (Fig. 2A), indicating that LPCAT1 is endogenously expressed in HEK293 cells. A similar result was obtained when human LPCAT1 expressed in HEK293 cells (Fig. 2B). The difference in activity could not have been caused by the substrate preparation method because the activity for sn-1-palmitoyl dominant LPC prepared from the sn-2-palmitoyl dominant LPC was
almost the same as the activity for commercially available palmitoylLPC which has a similar content of 1-palmitoyl-2-lyso-PC (data not shown). [13C16]DPPC formed by the LPCAT1 reaction would be either 1[13C16]palmitoyl-2-palmitoyl-PC or 1-palmitoyl-2-[13C16]palmitoyl -PC. To confirm which isomers were produced, we subjected the reactants to MS/MS analysis. In negative mode, formate adducts ([M-HCOO]-) of reactants PC were fragmented by collision induced dissociation (CID). The resulting fragments obtained from both the sn-2-palmitoyl dominant LPC and the sn-1-palmitoyl dominant LPC reactants were 255.2 and 271.2, which correspond to unlabeled and [13C16]-labeled palmitic 5
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concentration of 2 μM and a varying the concentration of LPC (Fig. 4B). The apparent Km value toward [13C16]palmitoyl-CoA was 0.23 ± 0.12 μM for sn-1-palmitoyl dominant LPC and 0.64 ± 0.16 μM for sn-2-palmitoyl dominant LPC. The Km value toward LPC was 2.08 ± 0.12 μM for sn-1-palmitoyl dominant LPC and 3.67 ± 0.08 μM for sn-2-palmitoyl dominant LPC. In both cases, the Vmax values toward sn-2-palmitoyl dominant LPC were found to be about 2 times higher than those toward sn-1-palmitoyl dominant LPC. Therefore, sn-1-palmitoyl LPC appears to have a slightly higher affinity for LPCAT1 than sn-2-palmitoyl LPC, while sn-2-palmitoyl LPC has a higher turnover rate. 3.2. LYCAT and LPCAT3 introduce stearic acid at the sn-1 position of LPG and arachidonic acid at the sn-2 positions of LPC, respectively We next applied the method to determine the acyl position of other LPLATs. We chose LYCAT and LPCAT3. Preceding studies suggested that LYCAT introduced stearic acid (18:0) selectively at the sn-1 position of lysophosphatidylinositol (LPI) [8,15,16], while LPCAT3 was critical for introduction of polyunsaturated fatty acids (PUFAs), especially arachidonic acid (20:4) at the sn-2 position of various LPLs, generating various types of arachidonoyl phospholipids [17,18]. To examine the activity of LYCAT, we used stearoyl-CoA as a donor and lysophosphatidylglycerol (LPG) as an acceptor instead of LPI, since LYCAT was able to introduce stearic acid into LPG with a similar manner to LPI [19,20]. We prepared an sn-2-oleoyl dominant LPG substrate from di-oleoyl phosphatidylglycerol (PG) by PLA1 reaction and then converted the sn-2-oleoyl dominant LPG to an sn-1-oleoyl dominant LPG in alkaline condition as we did for palmitoyl LPC. We confirmed the present sn-2 LPG isomer preparation (sn-2-acyl dominant LPG) was composed of > 90% of 2-oleoyl-1-lyso-PG and < 10% of 1oleoyl-2-lyso-PG, while the sn-1 LPG isomer preparation (sn-1-acyl dominant LPG) was composed of > 90% of 1-oleoyl-2-lyso-PG and < 10% of 2-oleoyl-1-lyso-PG (Fig. 5A left). Unlike LPCAT1 which introduced palmitic acid both at the sn-1 and sn-2 position of LPC, LYCAT introduced stearic acid predominantly at the sn-1 position of LPG (Fig. 5B left and Fig. 5C left). Similarly, we prepared an sn-2-stearoyl dominant LPC and an sn-1stearoyl dominant LPC substrates from di-stearoyl phosphatidylcholine (PC) and used them for evaluation of LPCAT3 activity using arachidonoyl-CoA as an acyl donor. In contrast to LPCAT1 and LYCAT, LPCAT3 was found to introduce arachidonic acid predominantly at the sn-2 position of LPC (Fig. 5A right, Fig. 5B right and Fig. 5C right).
Fig. 4. Kinetic analyses of LPCAT1 activities toward sn-1- and sn-2-pal dominant LPCs. (A and B) The kinetic analyses to determine the Km and Vmax values for human LPCAT1 were performed using varying concentration of [13C16]palmitoyl-CoA (A) and palmitoyl-LPC (B). Closed circles, sn-2-pal dominant LPC; open circles, sn-1-pal dominant LPC. Results obtained using mock-transfected membrane fraction as an enzyme source were also shown. Closed triangles, sn-2-pal dominant LPC; open triangles, sn-1-pal dominant LPC. Data are shown as the mean ± SD of three data points. The results are representative of three independent experiments with similar results. The Km and Vmax values calculated from three independent experiments are shown as the mean ± SD in the text.
acid, respectively. Interestingly, the intensities of the two fragments were different among the two reactants (Fig. 3A). Since the fatty acid moiety is more easily liberated from the sn-2 position than from the sn-1 position [12–14], 1-palmitoyl-2-[13C16]palmitoyl-PC was abundant in the reactants produced from sn-1-palmitoyl dominant LPC, while 1[13C16]palmitoyl-2-palmitoyl-PC was abundant in the reactants produced from sn-2-palmitoyl dominant LPC. To perform more quantitative analysis, we used sn-2-[d31]palmitoyl dominant LPC and sn-1-[d31]palmitoyl dominant LPC as the acceptors in the LPCAT1 reaction. A similar result was obtained when unlabeled palmitoyl LPC was used as the acceptor (Fig. 3B). Double-labeled [13C16]-[d31]DPPC formed by the LPCAT1 reaction would be either 1[13C16]palmitoyl-2-[d31]palmitoyl-PC or 1-[d31]palmitoyl-2-[13C16] palmitoyl-PC. The reactants were digested with PLA2 and analyzed by LC-MS/MS. When sn-2-[d31]palmitoyl dominant LPC was used as the acceptor, the major LPC detected in PLA2 reactant was 1-[13C16]palmitoyl-LPC, while when sn-1-[d31]palmitoyl dominant LPC was used as the acceptor, the major LPC detected was 1-[d31]palmitoyl-LPC (Fig. 3C). The above two results clearly show that LPCAT1 incorporated palmitic acid at both the sn-1 position and sn-2 position of LPC.
4. Discussion In this study, we established a method to determine the acyl-introducing position of LPLATs, which was found to be applicable at least three LPLATs. The present method utilized sn-2 dominant LPLs, which have a fatty acid mainly at the sn-2 position of glycerol backbone, and sn-1 dominant LPLs, which have a fatty acid mainly at the sn-1 position of glycerol backbone, in combination with corresponding acyl-CoA species. We first demonstrated that LPCAT1, which was shown to have a role in synthesizing DPPC in the lung surfactant, actually has an activity to introduce palmitic acid at both the sn-1 and the sn-2 position of LPC. Interestingly, when ether-linked LPLs, i.e., sn-1-O-(9-cis-octadecenyl)-sn-glycero-3-phosphocholine (1-OGPC) and sn-2-O-(9-cis-octadecenyl)-sn-glycero-3-phosphocholine (2-OGPC), were used as acceptors, plant LPCATs had an ability to introduce fatty acids into both sn-1 and sn-2 position of LPC [21]. However, these acceptors (especially 2OGPC) are artificial substrates because they are hardly detected in biological samples. Thus, it remains unclear whether the artificial substrates reflect the behavior of natural substrates. In this study, we evaluated the mammalian LPCAT1 activity using natural substrates as acceptors, i.e. sn-1-palmitoyl dominant and sn-2-palmitoyl dominant LPC. Therefore, our present assay system has an advantage over the
3.1.2. Kinetic parameters of sn-2 and sn-1-palmitoyl dominant LPCs To determine the kinetic parameters, we used human LPCAT1 and either [1] an LPC concentration of 10 μM with varying concentrations of [13C16]palmitoyl-CoA (Fig. 4A) or [2] a [13C16]palmitoyl-CoA 6
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Fig. 5. Positions of glycerol backbone at which LYCAT and LPCAT3 introduce a fatty acid. (A) Ratio of 2-oleoyl-1-lyso-PG (sn-2-oleoyl LPG: filled) and 1-oleoyl-2-lyso-PG (sn-1-oleoyl LPG: dashed) in the present LPG preparations used for LYCAT assay (left). Ratio of 2-stearoyl-1-lyso-PC (sn-2-stearoyl LPC: filled) and 1-stearoyl-2-lyso-PC (sn-1-stearoyl LPC: dashed) in the present LPC preparations used for LPCAT3 assay (right). (B) LYCAT (left) and LPCAT3 (right) activities toward “sn-1acyl dominant LPLs” and “sn-2-acyl dominant LPLs”. LYCAT activity was determined using “sn-1oleoyl dominant LPG” or “sn-2-oleoyl dominant LPG” as an acceptor, stearoyl-CoA as a donor and a membrane fraction of mouse LYCAT-overexpressing HEK293A cells as an enzyme source (left). LPCAT3 activity was determined using “sn-1-stearoyl dominant LPC” or “sn-2-stearoyl dominant LPC” as an acceptor, arachidonoyl-CoA as a donor and a membrane fraction of mouse LPCAT3-overexpressing HEK293A cells as an enzyme source (right). The results using control membrane fraction (mock) were also shown. Data are shown as the mean + SD of three independent experiments. (C) Ratio of sn-1-acyl LPLs in PLA2-digested assay products. LYCAT assay products (36:1 (stearoyl-oleoyl) PG) or LPCAT3 assay products (38:4 (stearoyl-arachidonoyl) PC) were subjected to bee venom PLA2 digestion. The resulting LPL species were analyzed by LC-MS/MS. Ratio of sn-1-stearoyl LPG (closed) and sn-1-oleoyl LPG (open) in PLA2-digested LYCAT products (left) and ratio of sn-1-arachidonoyl LPC (closed) and sn-1-stearoyl LPC (open) in PLA2-digested LPCAT3 products (right) were shown. The data are representative of three independent experiments with similar results.
We also determined the acyl-introducing position of LYCAT and LPCAT3. As previously suggested, we confirmed that LYCAT introduced stearic acid predominantly at the sn-1 position and LPCAT3 introduced arachidonic acid predominantly at the sn-2 position. The present study revealed that there are at least three types of LPLATs: (1) LPLATs which introduced fatty acid to both the sn-1 and the sn-2 positions (e.g. LPCAT1), (2) LPLATs which introduced fatty acid specifically to the sn1position (e.g. LYCAT) and (3) LPLATs which introduced fatty acid specifically to the sn-2 position (e.g. LPCAT3). Since various types of pure synthetic phospholipids are now commercial available, the present method can be applied essentially for all types of LPLs and LPLATs. At present, the positional specificities of most LPLATs remain unclear. Thus, the present method will definitely become the first choice one which helps to determine the positional specificities of other LPLATs.
previous methods in that it has a more biological context. In the preceding studies performing acyltransferase assay toward the sn-1 position, using freshly prepared sn-2-acyl LPI produced by PLA1, yeast PSI1, C. elegans, acl-8, 9 and 10, and mammalian LYCAT were able to introduce stearic acid (18:0) at the sn-1 position of sn-2acyl LPI [8,15,16]. However, these studies did not determine the precise concentration of sn-1- and sn-2-acyl LPLs used for the acyltransferase assay. In our experiment, we quantified the amount of sn-2palmitoyl LPC before and after the acyltransferase assay, and confirmed that the assay could be performed in the presence of sufficient sn-2palmitoyl LPC (Fig. 1). In addition, to confirm the positional specificity of the assay products, we utilized both [13C16]palmitoyl CoA and [d31] palmitoyl LPC in combination with the PLA2 reaction. Importantly, our method makes it possible to quantify the introduction of acyl chains at both the sn-1 and the sn-2 positions. 7
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Transparency document [9]
The Transparency document associated with this article can be found, in online version.
[10]
Acknowledgements [11]
This work was supported by the Leading Advanced Projects for medical innovation (AMED-LEAP) Grant Number JP18gm0010004h0002 (to J.A.) from the Japan Agency for Medical Research and Development (AMED) and KAKENHI Grant Numbers JP16K15113 and JP15H05899 (to J.A.) from the Japan Society for the Promotion of Science (JSPS).
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[13]
[14]
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BBA - Molecular and Cell Biology of Lipids journal homepage: www.elsevier.com/locate/bbalip
An accurate and versatile method for determining the acyl groupintroducing position of lysophospholipid acyltransferases Hiroki Kawanaa,b, Kuniyuki Kanoa,b, Hideo Shindouc,d, Asuka Inouea,b, Takao Shimizud,e, ⁎ Junken Aokia,b, a Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-Ku, Sendai 980-8578, Japan b AMED-LEAP, Chiyoda-ku, Tokyo 100-0004, Japan c Department of Lipid Signaling, National Center for Global Health and Medicine, Shinjuku-ku, Tokyo 162-8655, Japan d AMED-CREST, Chiyoda-ku, Tokyo 100-0004, Japan e Departments of Lipidomics, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Phospholipid sn-2-Acyl lysophospholipid sn-1-Acyl lysophospholipid Lysophospholipid acyltransferase LPCAT1 LYCAT LPCAT3
Lysophospholipid acyltransferases (LPLATs) incorporate a fatty acid into the hydroxyl group of lysophospholipids (LPLs) and are critical for determining the fatty acid composition of phospholipids. Previous studies have focused mainly on their molecular identification and their substrate specificity regarding the polar head groups and acyl-CoAs. However, little is known about the positional specificity of the hydroxyl group of the glycerol backbone (sn-2 or sn-1) at which LPLATs introduce a fatty acid. This is mainly due to the instability of LPLs used as an acceptor, especially for LPLs with a fatty acid at the sn-2 position of the glycerol backbone (sn-2LPLs), which are essential for the enzymatic assay to determine the positional specificity. In this study, we established a method to determine the positional specificity of LPLAT by preparing stable sn-2-LPLs in combination with PLA2 digestion, and applied the method for determining the positional specificity of several LPLATs including LPCAT1, LYCAT and LPCAT3. We found that LPCAT1 introduced palmitic acid both at the sn-1 and sn2 positions of palmitoyl-LPC, while LYCAT and LPCAT3 specifically introduced stearic acid at the sn-1 position of LPG and arachidonic acid at the sn-2 position of LPC, respectively. The present method for evaluating the positional specificity could also be used for biochemical characterization of other LPLATs.
1. Introduction Lysophospholipid acyltransferases (LPLATs) introduce an acyl group into lysophospholipids (LPLs) at either the sn-1 or sn-2 position of the glycerol backbone and are crucial for determining the fatty acid composition of phospholipids. Recent studies have identified several LPLAT isozymes that showed preference for the polar heads of LPLs and acyl-CoAs [1,2]. However, for most of the LPLAT isozymes, the glycerol positions (sn-1 or sn-2) at which they introduce fatty acids have not been determined. It is generally accepted that an acyl chain linked either at the sn-1 or sn-2 position of LPLs spontaneously moves to another position, yielding a mixture of LPLs with the acyl chain at either the sn-1 or sn-2 position. For example, 1-palmitoyl-lysophosphatidylcholine (1-palmitoyl-sn-glycero-3-phosphorylcholine), which is converted from the corresponding
phosphatidylcholine (PC) by phospholipase A2 (PLA2) reaction, is actually an equilibrium mixture consisting of approximately 90% 1-palmitoyl-2-lyso- and 10% 2-palmitoyl-1-lyso-isomers [3]. The acyl chain movement, which is known as an intramolecular acyl migration, occurs especially fast in LPLs with a fatty acid moiety at the sn-2 position (sn-2LPLs) [3,4]. Thus sn-2-LPLs are unstable and are quickly converted to the corresponding sn-1-LPLs, especially under neutral or alkaline conditions. This has hampered the LPLAT enzyme assay, especially the assay in which an acyl chain is introduced at the sn-1 position of sn-2acyl LPLs. We and others have shown that the intramolecular acyl migration reaction does not proceed under acidic conditions [3,4]. Thus, it is reasonable to assume that sn-2-LPLs can be prepared more efficiently by hydrolyzing PLs with phospholipase A1 (PLA1) and then lowering the pH of the reactant. In this study, we first tried to prepare a high-grade
⁎ Corresponding author at: Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-Ku, Sendai 980-8578, Japan. E-mail address: [email protected] (J. Aoki).
https://doi.org/10.1016/j.bbalip.2019.02.008 Received 1 October 2018; Received in revised form 2 February 2019; Accepted 24 February 2019 1388-1981/ © 2019 Published by Elsevier B.V.
Please cite this article as: Hiroki Kawana, et al., BBA - Molecular and Cell Biology of Lipids, https://doi.org/10.1016/j.bbalip.2019.02.008
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Fig. 1. Preparation of sn-1- and sn-2-palmitoyl dominant LPCs. (A–C) Ratio of 2-palmitoyl-1-lyso-PC (sn-2-palmitoyl LPC: filled) and 1-palmitoyl-2-lyso-PC (sn-1-palmitoyl LPC: dashed) in the present LPC preparations used for LPCAT1 assay. “sn-2-pal (palmitoyl) dominant LPC” was prepared from DPPC by Rhizomucor miehei lipase (PLA1), and “sn-1-pal (palmitoyl) dominant LPC” was prepared from “sn-2-pal (palmitoyl) dominant LPC” by using a spontaneous acylmigration reaction. (A and B) Ratio of sn-1 and sn-2palmitoyl LPC before (A) and after (B) 37 °C 10 minute incubation. (C) Ratio of sn-1 and sn-2palmitoyl LPC preparations in the deuterium-labeled palmitoyl LPC. The data are representative of three independent experiments with similar results.
2. Material and methods
sn-2-LPL preparation. We then used it as well as its sn-1 isomer to determine the specificity of LPLATs for the sn-1 or sn-2 position. We first selected LPCAT1 as a model of LPLAT molecules since it is involved in the synthetic pathway of a symmetric phospholipid, dipalmitoyl PC [5–7], possibly by introducing palmitic acid to either the sn-1 position of 2-palmitoyl-1-lysophosphatidylcholine (LPC) or to the sn-2 position of 1-palmitoyl-2-LPC. Thus, we assumed that LPCAT1 is an ideal LPLAT to determine its positional specificity. We also selected two other LPLATs, LYCAT and LPCAT3, which have been suggested to introduce an acyl chain in position-specific manner [8,9]. Our present results indicated that LPCAT1 introduced a fatty acid to both the sn-1 and sn-2 positions, while LYCAT and LPCAT3 selectively introduced a fatty acid to the sn-1 and sn-2 positions, respectively, demonstrating the present method is suitable for determining the positional specificity of LPLATs.
2.1. Materials Dulbecco's modified Eagle's medium (DMEM) was purchased from Nissui Pharmaceutical (Tokyo, Japan). Fetal bovine serum (FBS) and Opti-MEM were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Honey bee venom PLA2, lipase from Rhizomucor miehei which has intrinsic PLA1 activity and palmitoyl- [13C16] coenzyme A were obtained from Sigma-Aldrich (St. Louis, MO). All glycerophospholipids and lysophospholipids including palmitoyl-d62 PC (1,2-dipalmitoyld62-sn-glycero-3-phosphocholine) were purchased from Avanti Polar Lipids (Alabaster, AL). LC-MS grade methanol and acetonitrile were purchased from Kanto Chemical (Tokyo, Japan). Chloroform, formic acid and ammonium formate were purchased from Fujifilm-Wako
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labeled, [13C]-labeled, deuterium-labeled palmitoyl LPC and stearoyl LPC are 496.3, 512.3, 526.3 and 524.4 respectively. The m/z values for non-labeled DPPC, DPPC with a non-labeled palmitoyl and a [13C]-labeled palmitoyl ([13C16]DPPC), and DPPC with a [13C]-labeled palmitoyl and deuterium-labeled palmitoyl ([13C16]-[d31] DPPC), and 38:4 (stearoyl-arachidonoyl) PC are 734.6, 750.6, 780.6 and 810.6 respectively. At MS3, phosphocholine fragments (m/z = 184.1) were detected. To measure phosphatidylglycerol (PG), at MS1 the m/z values of [M + NH4]+ ion for 36:1 (stearoyl-oleoyl) PG is 794.6. At MS3, corresponding diacylglycerol fragment (m/z = 605.5) was detected in positive ion mode. For measurement of lysophosphatidylglycerol (LPG), at MS1 the m/z values of [M-H]− ion for oleoyl LPG is 509.3. At MS3, corresponding fatty acid fragment (m/z = 281.2) was detected in negative ion mode. To confirm incorporated fatty acid composition of the assay reactants, the product ions were scanned in negative mode to detect [M + HCOO]− ions at MS1.
(Osaka, Japan). 2.2. Preparation of sn-1-acyl dominant LPLs and sn-2-acyl dominant LPLs To obtain palmitoyl lysophosphatidylcholine (LPC) with palmitic acid at the sn-2 position of the glycerol backbone (sn-2-palmitoyl LPC), dipalmitoyl PC (DPPC) was subjected to an in vitro PLA1 reaction using the method by M. Okudaira et al. [4] with some modifications. Nonlabeled or deuterium-labeled (d62) DPPC (1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine) (1 μmol each) was incubated with reaction mixture containing 78 mM phosphate, 10% diethyl ether, 2 mM CaCl2, 0.05% TritonX-100 and 4.5 μg/ml Rhizomucor miehei lipase (pH 7.4). The mixture was incubated at 37 °C for 80 min. Then the reaction was stopped by adding 9 volumes of acidic methanol (pH 4.0) previously described [4]. After centrifugation (21,500 ×g, 5 min), the supernatant was applied to a Bond Elut C18 solid phase cartridge column (Agilent Technologies) to remove the remaining DPPC and free palmitic acid, the byproduct of the PLA1 reaction. The extract was dried by evaporation and dissolved in acidic methanol. The sn-1/sn-2 ratio and the concentration of sn-2-palmitoyl LPC preparation were determined by LC-MS/MS as previously described [4] with slight modifications (see below). The sn-2-palmitoyl LPC preparation predominantly contained LPC with palmitic acid at the sn-2 position (> 95%) and the level of remaining PC was very low (< 0.001% of LPC). The preparation, which we called “stock sn-2-palmitoyl dominant LPC”, was stored at −80 °C. The sn-1 LPC isomer was prepared from the sn-2 LPC isomer by using the spontaneous acyl-migration reaction. Since the acyl-migration reaction is dependent on the pH of the solution and the incubation time, the “stock sn-2-palmitoyl dominant LPC” was dissolved in weak alkaline solution (100 mM Tis-HCl (pH 8.9)). To prepare the sn-2 LPC isomer, the reaction was stopped immediately by adding acidic methanol. To prepare the sn-1 LPC isomer, the reaction mixture was incubated for 2 h at 37 °C before stopping the reaction. The new preparations were called “sn-2-palmitoyl dominant LPC” and “sn-1-palmitoyl dominant LPC”, respectively. We confirmed that the prepared “sn-1-palmitoyl dominant LPC” and “sn-2-palmitoyl dominant LPC” predominantly contained LPC with palmitic acid at the sn-1 position (> 90%) and sn-2 position (> 90%), respectively (Fig. 1). We also prepared sn-1and sn-2-oleoyl dominant LPG and sn-1and sn-2-stearoyl dominant LPC using the same procedure as stated above, except that dioleoyl PG (DOPG) and distearoyl PC (DSPC) were used instead of DPPC.
2.4. Plasmids and vectors cDNA for human LPCAT1 (hLPCAT1; NCBI accession number NM_ 024830) was amplified by PCR using PrimeSTAR HS Polymerase (TAKARA BIO Inc.) and HEK293A cell cDNA as a template. The PCR primers used were as follows. Forward 5′-CGCCACCATGAGGCTGCGGGGATGCGG-3′ Reverse 5′-GAGCTCGAGCTAATCCAGCTTCTTGCGAAC-3′ The amplified cDNA fragments were inserted into the pCAGGS vector (a gift from J. Miyazaki, Osaka University) with or without an Nterminal FLAG-tag. The nucleotide sequences of all the plasmids prepared were checked by DNA sequencing. Mouse LPCAT1, LYCAT and LPCAT3 clone in pCXN2.1 vector were previously described [5,8,9,11]. 2.5. Cell culture and transfection of plasmids HEK293A cells (Thermo Fisher Scientific) were maintained in DMEM supplemented with 10% FBS, 2 mM L-Glutamine (Thermo Fisher Scientific), 100 U/ml penicillin (Sigma-Aldrich), and 100 μg/ml streptomycin (Thermo Fisher Scientific) at 37 °C in the presence of 5% CO2 gas. HEK293A cells were transfected with cDNAs encoding several LPLATs using Opti-MEM and Lipofectamine 2000 (Thermo Fisher Scientific).
2.3. LC-MS/MS analyses
2.6. Preparation of membrane fractions
Lysophospholipids (LPLs) were measured as previously described [4] with minor modifications. The LC-MS/MS system consisted of an UltiMate 3000 HPLC system and TSQ Quantiva Triple-Stage Quadrupole mass spectrometer (Thermo Fisher Scientific) equipped with a heated-electrospray ionization-II (HESI-II) source. For HPLC, we used a reverse-phase column (Capcell Pak C18 ACR (250 mm × 1.5 mm,3 μm particle size; Osaka Soda) with a gradient elution of solvent A (5 mM ammonium formate in water, pH 4.0) and solvent B (5 mM ammonium formate in 95% (v/v) acetonitrile, pH 4.0) at 200 μl/min. This method enabled us to separate 1-acyl-2-lyso-PL (sn-1-acyl LPL) and 2-acyl-1lyso-PL (sn-2-acyl LPL). Diacyl-phospholipids (PLs) including PC and PG were quantified by a similar method [10] with slight modifications. The LC-MS/MS system consisted of a NANOSPACE SI-II HPLC system (Osaka Soda) and TSQ Quantum Ultra triple quadrupole mass spectrometer (Thermo Fisher Scientific) equipped with a heated-electrospray ionization-II (HESI-II) source. LC separation was performed using a reverse-phase column (Capcell Pak C8 UG120, 150 mm × 1.5 mm, 5 μm particle size; Osaka soda) with a gradient elution of solvent A and solvent B (described above) at 400 μl/min. LPC and PC were detected by selected reaction monitoring (SRM) in positive ion mode. At MS1, the m/z values of [M + H]+ ions for non-
HEK293A cells were harvested and transferred to ice-cold TSC buffer (20 mM Tris-HCl, pH 7.4, 300 mM sucrose and cOmplete™ Protease Inhibitor Cocktail (Roche, Mannheim, Germany)), and were sonicated ten times for 10 s using a probe-type sonicator (MICROTEC). The cell homogenates were centrifuged at 800 ×g for 10 min, and the supernatants were further centrifuged at 100,000 ×g for 1 h. The resultant pellets were re-suspended in TSE buffer (20 mM Tris-HCl, pH 7.4, 300 mM sucrose and 1 mM EDTA). Protein concentrations were determined with a BCA protein assay kit (Thermo Fisher Scientific). The membrane fractions were used as an enzyme source. 2.7. LPL acyltransferase assays LPC acyltransferase (LPCAT) activities that introduce palmitic acid into palmitoyl LPC were determined by measuring the product of the LPCAT reaction, i.e. DPPC with a slight modification of T. Harayama et al. [7] In the LPCAT reaction, membrane fractions (containing LPCAT1 recombinant protein), LPC (“sn-1-palmitoyl dominant LPC” or “sn-2-palmitoyl dominant LPC”) and [13C16]palmitoyl-CoA were mixed and incubated at 37 °C for 10 min. The final reaction mixture contained 110 mM Tris-HCl (pH 7.4), 150 mM sucrose, 0.5 mM EDTA, 10 μM LPC, 2 μM Acyl-CoA, 0.015% Tween-20 and 0.05 μg of membrane protein. 3
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products were subjected to the PLA2 reaction and the position of the incorporated palmitic acid in the resulting palmitoyl-LPC was determined. Since palmitoyl-LPC can be produced from palmitoyl-containing PC in the membrane fraction that was used as an enzyme source, which may disturb measurement of the palmitoyl LPC concentration, a deuterium-labeled [d31]palmitoyl LPC (both “sn-1-palmitoyl dominant LPC” and “sn-2-palmitoyl dominant LPC”) was prepared from [d62] DPPC (possessing two [d31]-labeled palmitic acids) and used instead of non-labeled palmitoyl LPC as an acceptor. [13C16][d31] DPPC produced by the LPCAT1 assay was reacted with PLA2. The position of [13C16]palmitic acid and [d31]palmitic acid in the resulting palmitoyl LPC was then determined. To separate [13C16]-[d31] DPPC from [d31]palmitoyl LPC (because it was used an acceptor, the reaction mixture had excess [d31]palmitoyl LPC), the assay products were applied to a Bond Elut C8 solid phase cartridge column (Agilent Technologies) and eluted with 5 mM ammonium formate in 99.5% (v/ v) methanol. The eluted fraction containing [13C16]-[d31] DPPC was evaporated and dissolved in 100 mM Tris-HCl buffer containing 0.1% Triton X-100. The PLA2 reaction was started by adding 0.1unit bee venom PLA2, allowed to continue at 37 °C for 10 min and then stopped by acidic methanol containing 1 μM 17:0 LPC and 100 nM 17:0 LPA as an internal standard. After centrifugation (21,500 ×g, 5 min), the supernatant was subjected to LC-MS/MS analysis to determine the concentrations of sn-1 [13C16]palmitoyl LPC and sn-1 [d31]palmitoyl LPC. The LYCAT and LPCAT3 assay products were also digested with PLA2, and the resulting sn-1-acyl-LPG and sn-1-acyl-LPC were analyzed by LCMS/MS. 2.9. Data analysis The apparent Km and Vmax values were calculated from the Michaelis-Menten equation using GraphPad Prism 7.0 software (GraphPad). Fig. 2. LPCAT1 activities toward sn-1 and sn-2-pal dominant LPCs. LPCAT assays were performed using “sn-1-pal dominant LPC” or “sn-2-pal dominant LPC” as an acceptor, and a membrane fraction of LPCAT1-overexpressing HEK293A cells as an enzyme source. The results using recombinant mouse LPCAT1 (A) and human LPCAT1 (B) are shown on the right and the results using control membrane fraction (mock) were also shown on the left. Data are shown as the mean + SD of three data points. Three experiments using independently-prepared membrane fractions were performed with similar results.
3. Results 3.1. Preparation of LPC with palmitic acid predominantly at the sn-1 or sn2 position To determine at which position of the LPC glycerol backbone LPCAT1 introduces palmitic acid, we first prepared two types of palmitoyl-LPC that have palmitic acid at either the sn-2 or sn-1 position from dipalmitoyl PC (DPPC). As described in Material and methods section, the two LPC isomers were separated and quantified by LC-MS/ MS to determine their purity. The analysis revealed that the present sn2 LPC isomer preparation was composed of > 90% of 2-palmitoyl-1lyso-PC and < 10% of 1-palmitoyl-2-lyso-PC, while the sn-1 LPC isomer preparation was composed of > 90% of 1-palmitoyl-2-lyso-PC and < 10% of 2-palmitoyl-1-lyso-PC (Fig. 1A). After incubating the lipids for 10 min in enzymatic assay buffer, the assay condition for the preset LPCAT1 assay, most of the 2-palmitoyl-1-lyso-PC remained intact (Fig. 1B). In the following studies, we call the sn-1- and sn-2-palmitoyl LPC isomer preparations as the sn-1- and sn-2-palmitoyl dominant LPCs, respectively. We also produced deuterium labeled sn-1-palmitoyl dominant LPC and sn-2-palmitoyl dominant LPC from deuterium labeled DPPC (Fig. 1C).
The reaction was stopped by adding chloroform/methanol (1:2). Dilauryl-PC (DLPC) or dilauryl-PG (DLPG) was added as an internal standard and lipids were extracted by the Bligh & Dyer method. The organic (lower) layer was evaporated, dissolved in methanol, and the amounts of DPPC containing [13C16]-labeled palmitic acid were quantified by LC–MS/MS. To quantify the amount of labeled-DPPC, commercially available non-labeled DPPC was used as a calibration standard. For kinetics analysis, various concentrations of LPC (“sn-1palmitoyl dominant LPC” or “sn-2-palmitoyl dominant LPC”) and [13C16]palmitoyl-CoA were subjected to the LPCAT reaction using fixed concentrations of [13C16]palmitoyl-CoA (2 μM) and LPC (10 μM), respectively. Similar assay conditions were employed for LPG acyltransferase (LPGAT) activities for LYCAT and LPC acyltransferase (LPCAT) activities for LPCAT3. LPGAT activities for LYCAT were determined in the presence of LYCAT-overexpressed membrane fraction (0.05 μg) and stearoyl-CoA using sn-2 or sn-1-oleoyl dominant LPG. LPCAT activities for LPCAT3 were determined in the presence of LPCAT3-overexpressed membrane fraction (0.005 μg) and arachidonoyl-CoA using sn-2 or sn-1-stearoyl dominant LPC.
3.1.1. LPCAT1 introduces palmitic acid at both the sn-1 and sn-2 positions of LPC In order to determine the position of the LPC glycerol backbone at which LPCAT1 introduces palmitic acid, we performed an LPCAT assay using sn-1-palmitoyl dominant LPC or sn-2-palmitoyl dominant LPC as the acceptor, [13C16]palmitoyl-CoA as the acyl donor, and the membrane fraction of LPCAT1-overexpressed cells as the enzyme source. We evaluated the LPCAT1 activity by measuring the [13C16] DPPC formed. Using the membrane fraction from HEK293A cells expressing mouse
2.8. Confirmation of the position of incorporated fatty acid To determine position of incorporated palmitic acid, LPCAT1 assay 4
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Fig. 3. Confirmation of the position of incorporated palmitic acid. (A) Fragment spectrum of [13C16]palmitoyl-palmitoyl-PC produced by LPCAT1 reaction. LPCAT1 assay products containing [13C16]palmitoyl-palmitoyl-PC were subjected to mass spectrometry (MS) analysis. In the negative ion mode, PC [M + HCOO]− ions were fragmented using a collision energy of 45 eV and two fatty acid (FA) fragments (palmitic acid m/z = 255.2 and [13C16]palmitic acid m/z = 271.2) were detected. Left and right panels show representative fragment patterns of assay products using “sn-1-pal dominant LPC” and “sn-2-pal dominant LPC” as an acceptor, respectively. (B and C) Determination of the position of incorporated [13C16]palmitic acid by using PLA2 reaction and LC-MS/ MS. (B) LPCAT1 activities using sn-1- or sn-2-[d31]pal dominant LPC as an acceptor and recombinant human LPCAT1. Similar results were obtained when nonlabeled sn-1 or sn-2 pal dominant LPC was used (Fig. 2). Data are shown as the mean + SD of three data points. Three experiments using independently-prepared membrane fractions were performed with similar results. (C) Ratio of sn-1-[13C16]palmitoyl-LPC (dot) and sn1-[d31]palmitoyl-LPC (shadowed) in PLA2-digested assay mixtures. LPCAT1 assay products [13C16]palmitoyl-[d31] palmitoyl-PC were subjected to bee venom PLA2 treatment. The resulting LPC species were analyzed by LC-MS/ MS. The data are representative of three independent experiments with similar results.
LPCAT1, significant amounts of [13C16]palmitic acid were incorporated in both sn-1-palmitoyl dominant LPC and sn-2-palmitoyl dominant LPC although the amount incorporated in the latter was almost twice the amount incorporated in the former (Fig. 2A). Interestingly, the membrane fraction prepared from mock-transfected HEK293A cells also had similar [13C16]palmitic acid-incorporating activity (Fig. 2A), indicating that LPCAT1 is endogenously expressed in HEK293 cells. A similar result was obtained when human LPCAT1 expressed in HEK293 cells (Fig. 2B). The difference in activity could not have been caused by the substrate preparation method because the activity for sn-1-palmitoyl dominant LPC prepared from the sn-2-palmitoyl dominant LPC was
almost the same as the activity for commercially available palmitoylLPC which has a similar content of 1-palmitoyl-2-lyso-PC (data not shown). [13C16]DPPC formed by the LPCAT1 reaction would be either 1[13C16]palmitoyl-2-palmitoyl-PC or 1-palmitoyl-2-[13C16]palmitoyl -PC. To confirm which isomers were produced, we subjected the reactants to MS/MS analysis. In negative mode, formate adducts ([M-HCOO]-) of reactants PC were fragmented by collision induced dissociation (CID). The resulting fragments obtained from both the sn-2-palmitoyl dominant LPC and the sn-1-palmitoyl dominant LPC reactants were 255.2 and 271.2, which correspond to unlabeled and [13C16]-labeled palmitic 5
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concentration of 2 μM and a varying the concentration of LPC (Fig. 4B). The apparent Km value toward [13C16]palmitoyl-CoA was 0.23 ± 0.12 μM for sn-1-palmitoyl dominant LPC and 0.64 ± 0.16 μM for sn-2-palmitoyl dominant LPC. The Km value toward LPC was 2.08 ± 0.12 μM for sn-1-palmitoyl dominant LPC and 3.67 ± 0.08 μM for sn-2-palmitoyl dominant LPC. In both cases, the Vmax values toward sn-2-palmitoyl dominant LPC were found to be about 2 times higher than those toward sn-1-palmitoyl dominant LPC. Therefore, sn-1-palmitoyl LPC appears to have a slightly higher affinity for LPCAT1 than sn-2-palmitoyl LPC, while sn-2-palmitoyl LPC has a higher turnover rate. 3.2. LYCAT and LPCAT3 introduce stearic acid at the sn-1 position of LPG and arachidonic acid at the sn-2 positions of LPC, respectively We next applied the method to determine the acyl position of other LPLATs. We chose LYCAT and LPCAT3. Preceding studies suggested that LYCAT introduced stearic acid (18:0) selectively at the sn-1 position of lysophosphatidylinositol (LPI) [8,15,16], while LPCAT3 was critical for introduction of polyunsaturated fatty acids (PUFAs), especially arachidonic acid (20:4) at the sn-2 position of various LPLs, generating various types of arachidonoyl phospholipids [17,18]. To examine the activity of LYCAT, we used stearoyl-CoA as a donor and lysophosphatidylglycerol (LPG) as an acceptor instead of LPI, since LYCAT was able to introduce stearic acid into LPG with a similar manner to LPI [19,20]. We prepared an sn-2-oleoyl dominant LPG substrate from di-oleoyl phosphatidylglycerol (PG) by PLA1 reaction and then converted the sn-2-oleoyl dominant LPG to an sn-1-oleoyl dominant LPG in alkaline condition as we did for palmitoyl LPC. We confirmed the present sn-2 LPG isomer preparation (sn-2-acyl dominant LPG) was composed of > 90% of 2-oleoyl-1-lyso-PG and < 10% of 1oleoyl-2-lyso-PG, while the sn-1 LPG isomer preparation (sn-1-acyl dominant LPG) was composed of > 90% of 1-oleoyl-2-lyso-PG and < 10% of 2-oleoyl-1-lyso-PG (Fig. 5A left). Unlike LPCAT1 which introduced palmitic acid both at the sn-1 and sn-2 position of LPC, LYCAT introduced stearic acid predominantly at the sn-1 position of LPG (Fig. 5B left and Fig. 5C left). Similarly, we prepared an sn-2-stearoyl dominant LPC and an sn-1stearoyl dominant LPC substrates from di-stearoyl phosphatidylcholine (PC) and used them for evaluation of LPCAT3 activity using arachidonoyl-CoA as an acyl donor. In contrast to LPCAT1 and LYCAT, LPCAT3 was found to introduce arachidonic acid predominantly at the sn-2 position of LPC (Fig. 5A right, Fig. 5B right and Fig. 5C right).
Fig. 4. Kinetic analyses of LPCAT1 activities toward sn-1- and sn-2-pal dominant LPCs. (A and B) The kinetic analyses to determine the Km and Vmax values for human LPCAT1 were performed using varying concentration of [13C16]palmitoyl-CoA (A) and palmitoyl-LPC (B). Closed circles, sn-2-pal dominant LPC; open circles, sn-1-pal dominant LPC. Results obtained using mock-transfected membrane fraction as an enzyme source were also shown. Closed triangles, sn-2-pal dominant LPC; open triangles, sn-1-pal dominant LPC. Data are shown as the mean ± SD of three data points. The results are representative of three independent experiments with similar results. The Km and Vmax values calculated from three independent experiments are shown as the mean ± SD in the text.
acid, respectively. Interestingly, the intensities of the two fragments were different among the two reactants (Fig. 3A). Since the fatty acid moiety is more easily liberated from the sn-2 position than from the sn-1 position [12–14], 1-palmitoyl-2-[13C16]palmitoyl-PC was abundant in the reactants produced from sn-1-palmitoyl dominant LPC, while 1[13C16]palmitoyl-2-palmitoyl-PC was abundant in the reactants produced from sn-2-palmitoyl dominant LPC. To perform more quantitative analysis, we used sn-2-[d31]palmitoyl dominant LPC and sn-1-[d31]palmitoyl dominant LPC as the acceptors in the LPCAT1 reaction. A similar result was obtained when unlabeled palmitoyl LPC was used as the acceptor (Fig. 3B). Double-labeled [13C16]-[d31]DPPC formed by the LPCAT1 reaction would be either 1[13C16]palmitoyl-2-[d31]palmitoyl-PC or 1-[d31]palmitoyl-2-[13C16] palmitoyl-PC. The reactants were digested with PLA2 and analyzed by LC-MS/MS. When sn-2-[d31]palmitoyl dominant LPC was used as the acceptor, the major LPC detected in PLA2 reactant was 1-[13C16]palmitoyl-LPC, while when sn-1-[d31]palmitoyl dominant LPC was used as the acceptor, the major LPC detected was 1-[d31]palmitoyl-LPC (Fig. 3C). The above two results clearly show that LPCAT1 incorporated palmitic acid at both the sn-1 position and sn-2 position of LPC.
4. Discussion In this study, we established a method to determine the acyl-introducing position of LPLATs, which was found to be applicable at least three LPLATs. The present method utilized sn-2 dominant LPLs, which have a fatty acid mainly at the sn-2 position of glycerol backbone, and sn-1 dominant LPLs, which have a fatty acid mainly at the sn-1 position of glycerol backbone, in combination with corresponding acyl-CoA species. We first demonstrated that LPCAT1, which was shown to have a role in synthesizing DPPC in the lung surfactant, actually has an activity to introduce palmitic acid at both the sn-1 and the sn-2 position of LPC. Interestingly, when ether-linked LPLs, i.e., sn-1-O-(9-cis-octadecenyl)-sn-glycero-3-phosphocholine (1-OGPC) and sn-2-O-(9-cis-octadecenyl)-sn-glycero-3-phosphocholine (2-OGPC), were used as acceptors, plant LPCATs had an ability to introduce fatty acids into both sn-1 and sn-2 position of LPC [21]. However, these acceptors (especially 2OGPC) are artificial substrates because they are hardly detected in biological samples. Thus, it remains unclear whether the artificial substrates reflect the behavior of natural substrates. In this study, we evaluated the mammalian LPCAT1 activity using natural substrates as acceptors, i.e. sn-1-palmitoyl dominant and sn-2-palmitoyl dominant LPC. Therefore, our present assay system has an advantage over the
3.1.2. Kinetic parameters of sn-2 and sn-1-palmitoyl dominant LPCs To determine the kinetic parameters, we used human LPCAT1 and either [1] an LPC concentration of 10 μM with varying concentrations of [13C16]palmitoyl-CoA (Fig. 4A) or [2] a [13C16]palmitoyl-CoA 6
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Fig. 5. Positions of glycerol backbone at which LYCAT and LPCAT3 introduce a fatty acid. (A) Ratio of 2-oleoyl-1-lyso-PG (sn-2-oleoyl LPG: filled) and 1-oleoyl-2-lyso-PG (sn-1-oleoyl LPG: dashed) in the present LPG preparations used for LYCAT assay (left). Ratio of 2-stearoyl-1-lyso-PC (sn-2-stearoyl LPC: filled) and 1-stearoyl-2-lyso-PC (sn-1-stearoyl LPC: dashed) in the present LPC preparations used for LPCAT3 assay (right). (B) LYCAT (left) and LPCAT3 (right) activities toward “sn-1acyl dominant LPLs” and “sn-2-acyl dominant LPLs”. LYCAT activity was determined using “sn-1oleoyl dominant LPG” or “sn-2-oleoyl dominant LPG” as an acceptor, stearoyl-CoA as a donor and a membrane fraction of mouse LYCAT-overexpressing HEK293A cells as an enzyme source (left). LPCAT3 activity was determined using “sn-1-stearoyl dominant LPC” or “sn-2-stearoyl dominant LPC” as an acceptor, arachidonoyl-CoA as a donor and a membrane fraction of mouse LPCAT3-overexpressing HEK293A cells as an enzyme source (right). The results using control membrane fraction (mock) were also shown. Data are shown as the mean + SD of three independent experiments. (C) Ratio of sn-1-acyl LPLs in PLA2-digested assay products. LYCAT assay products (36:1 (stearoyl-oleoyl) PG) or LPCAT3 assay products (38:4 (stearoyl-arachidonoyl) PC) were subjected to bee venom PLA2 digestion. The resulting LPL species were analyzed by LC-MS/MS. Ratio of sn-1-stearoyl LPG (closed) and sn-1-oleoyl LPG (open) in PLA2-digested LYCAT products (left) and ratio of sn-1-arachidonoyl LPC (closed) and sn-1-stearoyl LPC (open) in PLA2-digested LPCAT3 products (right) were shown. The data are representative of three independent experiments with similar results.
We also determined the acyl-introducing position of LYCAT and LPCAT3. As previously suggested, we confirmed that LYCAT introduced stearic acid predominantly at the sn-1 position and LPCAT3 introduced arachidonic acid predominantly at the sn-2 position. The present study revealed that there are at least three types of LPLATs: (1) LPLATs which introduced fatty acid to both the sn-1 and the sn-2 positions (e.g. LPCAT1), (2) LPLATs which introduced fatty acid specifically to the sn1position (e.g. LYCAT) and (3) LPLATs which introduced fatty acid specifically to the sn-2 position (e.g. LPCAT3). Since various types of pure synthetic phospholipids are now commercial available, the present method can be applied essentially for all types of LPLs and LPLATs. At present, the positional specificities of most LPLATs remain unclear. Thus, the present method will definitely become the first choice one which helps to determine the positional specificities of other LPLATs.
previous methods in that it has a more biological context. In the preceding studies performing acyltransferase assay toward the sn-1 position, using freshly prepared sn-2-acyl LPI produced by PLA1, yeast PSI1, C. elegans, acl-8, 9 and 10, and mammalian LYCAT were able to introduce stearic acid (18:0) at the sn-1 position of sn-2acyl LPI [8,15,16]. However, these studies did not determine the precise concentration of sn-1- and sn-2-acyl LPLs used for the acyltransferase assay. In our experiment, we quantified the amount of sn-2palmitoyl LPC before and after the acyltransferase assay, and confirmed that the assay could be performed in the presence of sufficient sn-2palmitoyl LPC (Fig. 1). In addition, to confirm the positional specificity of the assay products, we utilized both [13C16]palmitoyl CoA and [d31] palmitoyl LPC in combination with the PLA2 reaction. Importantly, our method makes it possible to quantify the introduction of acyl chains at both the sn-1 and the sn-2 positions. 7
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Transparency document [9]
The Transparency document associated with this article can be found, in online version.
[10]
Acknowledgements [11]
This work was supported by the Leading Advanced Projects for medical innovation (AMED-LEAP) Grant Number JP18gm0010004h0002 (to J.A.) from the Japan Agency for Medical Research and Development (AMED) and KAKENHI Grant Numbers JP16K15113 and JP15H05899 (to J.A.) from the Japan Society for the Promotion of Science (JSPS).
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