Bulat and Garrett Final Revision - Journal of Biological Chemistry

JBC Papers in Press. Published on July 29, 2011 as Manuscript M111.269779 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M111.269779

THE PUTATIVE N-ACYLPHOSPHATIDYLETHANOLAMINE SYNTHASE FROM ARABIDOPSIS THALIANA IS A LYSOGLYCEROPHOSPHOLIPID ACYLTRANSFERASE* Evgeny Bulat and Teresa A. Garrett From Department of Chemistry, Vassar College, Box 580, 124 Raymond Avenue, Poughkeepsie, NY 12604, US Running title: Lysoglycerophospholipid acyltransferase from A. thaliana Address correspondence to: Teresa A. Garrett, Ph. D., Box 580 124 Raymond Avenue, Poughkeepsie, NY 12604. Phone: 845-437-5738; Fax: 845-437-5732; Email: [email protected]

N-acyl phosphatidylethanolamine (N-acyl PE) is an anionic minor membrane glycerophospholipid (GPL) that is abundant in animals, higher plants, and certain microorganisms such as yeast and Escherichia coli (1-7). This GPL is of particular interest because of its well-

documented role as the precursor to Nacylethanolamines (NAEs), a class of bioactive lipids that are involved in a variety of physiological processes such as responses to pathogens, plant development and germination in plants (8-10), as well as control of appetite, inflammation, and apoptosis (11-13) in animals. In animals, N-acyl PE synthesis is catalyzed by two distinct biochemical activities. A Ca2+-dependent membrane-associated protein has been characterized in brain, testis, and heart (14). This enzyme has been shown to catalyze the transfer of an acyl chain from the sn-1 position of a di-acylated GPL, functions most efficiently at alkaline pH and requires divalent cations (13, 15, 16). The gene encoding this enzyme has yet to be identified. A lecithin retinol acyltransferase-like protein from rat, RLP-1, has been shown to encode a Ca2+-independent N-acyltransferase (NAT) (17). It is predominantly expressed in testis and is, therefore, thought to be distinct from the Ca2+dependent NAT. Ca2+-independent NAT activity is detected in both the soluble and membrane fractions when over-expressed in COS-7 cells and does not show preference for transfer of the sn-1 or sn-2 acyl chain to the amine of PE (17). Human and mouse homologs have been cloned and shown to possess similar NAT activity (18). In plants N-acyl PE is proposed to be synthesized from PE and un-esterified fatty acids by a membrane-bound N-acyl PE synthase (3, 19, 9). Recently, Faure and colleagues identified a gene from Arabidopsis thaliana, At1g78690p, as encoding an NAT (20) that catalyzes the transfer of an acyl chain from acyl-CoA to the amine of PE. They reported that over-expression of At1g78690p in E. coli led to the accumulation of N-acyl PE in cells and the presence of acyl-CoAdependent N-acyl PE synthase activity in membranes. At1g78690p has homology to

 

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At1g78690p, a gene found in Arabidopsis thaliana, has been reported to encode a Nacyltransferase (NAT)1 that transfers an acyl chain from acyl-CoA to the head-group of phosphatidylethanolamine (PE) to form N-acyl phosphatidylethanolamine (N-acyl PE). Our investigation suggests that At1g78690p is not a PE-dependent NAT, but is instead a lysoglycerophospholipid O-acyltransferase. We over-expressed At1g78690p in Escherichia coli, extracted the cellular lipids, and identified the accumulating glycerophospholipid as acylphosphatidylglycerol (acyl PG). Electrospray ionization quadrupole time-offlight mass spectrometry (ESI-MS) analysis yielded [M-H]- ions corresponding by exact mass to acyl PG rather than N-acyl PE. Collision-induced dissociation mass spectrometry (MS/MS) yielded product ions consistent with acyl PG. In addition, in vitro enzyme assays using both 32P and 14C radiolabeled substrates showed that At1g78690p acylates 1-acyl lysophosphatidylethanolamine (1-acyl lyso PE) and 1-acyl lysophosphatidylglycerol (1-acyl lyso PG), but not PE or phosphatidylglycerol (PG), to form a di-acylated product that co-migrates with PE and PG respectively. We analyzed the di-acylated product formed by At1g78690p using a combination of base hydrolysis, phospholipase D treatment, ESI-MS and MS/MS to show that At1g78690p acylates the sn-2 position of 1-acyl lyso PE and 1-acyl lyso PG.

lysoglycerophospholipid (lyso GPL) acyltransferases such as the lysophosphatidic acid acyltransferase Slc1 from A. thaliana and the lysophosphatidylcholine acyltransferase YPR140wp from Saccharomyces cerevisiae (20, 21). Multiple attempts to detect N-acyl PE synthase activity from cell-free extracts prepared from E. coli induced to over-express At1g78690p were unsuccessful. Therefore, we re-investigated the biochemical activity of At1g78690p in E. coli. In this work, we show that this enzyme is a lyso GPL acyltransferase and that its over-expression in E. coli leads to the accumulation of acylphosphatidylglycerol (acyl PG), not N-acyl PE.

 

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Experimental Procedures Materials: Tryptone and yeast extract were from Fisher Biotech (Fairlawn, New Jersey). Glass-backed Silica Gel 60 thin layer chromatography plates (0.25-mm) and high performance thin layer chromatography (HPTLC) plates were from E. Merck. Solvents were reagent grade from Mallinckrodt. Other chemicals were purchased from VWR or Sigma-Aldrich. GPLs, 1-palmitoyl-2-oleoyl-sn-glycero-3phosphoethanolamine (16:0, 18-1 PE), 1-oleoyl-2hydroxy-sn-glycero-3-phosphoethanolamine (1acyl lyso PE), 1-oleoyl-2-hydroxy-sn-glycero-3phospho-rac-(1-glycerol) (1-acyl lyso PG), 1,2dioleoyl-sn-glycero-3-phosphoethanolamine-Narachidonyl (N-acyl PE), sn-(3-myristoyl-2hydroxyl)-glycerol-1-phospho-sn-3’-(1’, 2’myristoyl)-glycerol (acyl PG), 1-palmitoyl-2oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (16:0, 18:1 PG), and sn-(3-oleoyl-2-hydroxy)glycerol-1-phospho-sn-3'-(1'-oleoyl-2'-hydroxy)glycerol (3, 1’ BMP), were from Avanti Polar Lipids, Alabaster, AL. 32P-PO43- (8500 Ci/mmol, 10 mCi/ml) and palmitoyl-1-14C-CoA (60 mCi/mmol, 0.02 mCi/ml) was from Perkin-Elmer. Palmitoyl-CoA and arachidonyl-CoA, porcine pancreas phospholipase A2 and Streptomyces chromofuscus phospholipase D were from Sigma. Growth of E. coli: pET 15b and pAt1g78690p (a kind gift of D. Coulon, Laboratoire de Biogenèse Membranaire, Université Victor Segalen Bordeaux, France) (20) were transformed into chemically competent E. coli BLR(DE3)pLysS. Transformants were

selected on LB agar containing 10 g of NaCl, 5 g of yeast extract, 10 g of tryptone, 15 g of agar per liter (22) and 100 µg/ml ampicillin (LB-amp) and grown overnight at 37 °C. Single colonies were re-streaked on LB-amp and grown at 37 °C overnight. Overnight cultures were inoculated from a single colony into liquid LB-amp containing 100 µg/ml ampicillin, grown shaking at 225 rpm at 37 °C overnight and then diluted into LB-amp medium to an A600 of 0.01. The culture was grown at 37 °C, shaking at 225 rpm until the A600 was about 0.4-0.5. Isopropyl β -D-1thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM and cells were cultured an additional three hours. Cells were harvested by centrifugation for 20 minutes at 2600 x g and washed with phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) when cells were used for lipid extraction, or with 15 mM Tris, pH 7.4 when cells were used for preparation of protein extracts. Cell pellets were frozen at -80 °C until further use. Extraction of E. coli total lipids: The final cell pellet from a 50 ml growth of E. coli was resuspended in 0.8 ml of PBS and transferred to a disposable glass centrifuge tube equipped with Teflon-lined lids. The cellular lipids were extracted using the method of Bligh and Dyer (23) as described previously (24). Briefly, 1 ml CHCl3 and 2 ml CH3OH were added to the cell suspension to generate a single phase extraction mixture of CHCl3:CH3OH:PBS (1:2:0.8, v/v/v) . After incubation at room temperature for 20 minutes, the mixture was centrifuged at 2600 x g for 15 minutes. The supernatant was transferred to a clean tube and converted to a two-phase BlighDyer extraction mixture (CHCl3:CH3OH:PBS, 2:2:1.8, v/v/v) by the addition of 1 ml of chloroform and 1 ml of PBS. The extraction mixture was centrifuged as above to resolve the phases. The upper phase was removed and the lower phase washed with 2 ml of pre-equilibrated neutral upper phase. The resulting two-phase extraction mixture was centrifuged as described above. The upper phase was discarded and the resulting lower phase dried under N2 gas. Dried lipid films were stored at -20 °C. Thin layer chromatography: Two solvent systems were used to display lipids on silica gel 60 thin layer chromatography (TLC) plates. Solvent system A was CHCl3:CH3OH:H2O (65:25:4, v/v/v)

 

the electrospray ionization source operating at the following settings: nebulizer gas, 21 kPa, curtain gas, 27 kPa, ion-spray voltage, -4500 V, declustering potential, -55 V, focusing potential, 265 V, declustering potential 2, -15 V. The instrument was calibrated using polypropylene glycol (PPG, Applied Biosystems). Under normal operating conditions the resolution of the instrument was 10,000 to 15,000. The mass accuracy of the instrument was between 5 and 20 ppm, and therefore measured masses are given to three decimal places. Collision-induced dissociation mass spectrometry (MS/MS) was performed with a collision energy of -50.0 V (laboratory frame of reference) and N2 as the collision gas. Data acquisition and analysis were performed using the Analyst QS 1.1 software. Exact masses of lipid species and product ions were obtained using CS Chem Draw Pro, version 11.0. In vivo 32P-labeling of lipids: A culture of BLR(DE3)pLysS/pET15b and BLR(DE3)pLysS/pAt1g78690p was grown overnight at 37 °C in LB-amp. This overnight was used to start a 10 ml culture in LB-amp containing 100 µCi of 32P-PO4. This culture, as well as an unlabeled culture for monitoring growth, was incubated at 37 °C. When the A600 reached 0.4-0.5, IPTG was added to a final concentration of 1 mM IPTG. The culture was grown for 3 hours and cells were harvested, extracted and dried as described above. Base-deacylation of 32P labeled lipids: In vivo labeled lipids from BLR(DE3)pLysS/pET15b and BLR(DE3)pLysS/pAt1g78690p (1000 dpm) were re-dissolved in CHCl3:CH3OH (2:1, v/v), treated with NaOH (final concentration 0.2 M) and incubated at room temperature for 60 minutes. The reaction was spotted to TLC with an equivalent amount of untreated samples, developed in solvent system A and phosphorimaged as described above. Preparation of cell-free extracts and membranes: E. coli BLR(DE3)pLysS/pET15b and BLR(DE3)pLysS/pAt1g78690p were grown and induced as described above. The cell pellet from a 50 ml culture was re-suspended in 2 ml of 15 mM Tris, pH 7.4 and cells lysed in a French Pressure cell at 18000 psi. The lysate was centrifuged at 2600 x g to pellet un-lysed cells. The resulting cell-free extract was transferred to a 3 


and was used to display mono-, di-, and triacylated lipids. Solvent system B was 50:40:10:1, C6H12:CH3CH2OCH2CH3:(CH3)2CO:CH3COOH, v/v/v/v and was used to distinguish monoacylglycerol from N-acylethanolamine (9). Lipids were visualized by exposure to iodine vapors, charring with 10% H2SO4 in CH3CH2OH, or by phosphorimaging using a BioRad Personal Molecular Imager. Prep-TLC of lipids: Total lipid extracts were re-dissolved in 2:1 CHCl3:CH3OH, v/v and spotted continuously along the bottom of a 10 cm x 10 cm HPTLC plate. The plate was developed in solvent A and lipids were visualized by exposure to iodine vapors. The region of the TLC plate containing the lipid of interest was wet with water, the silica chips scraped from the plate and placed in a glass centrifuge tube. 2 ml of CHCl3:CH3OH (2:1, v/v) were added to the chips and the silica chips dispersed using a bath sonicator (Laboratory Supplies Company, Hicksville, NY). The dispersed silica chips were pelleted by centrifugation for 10 minutes in a clinical centrifuge. The supernatant was filtered through glass wool and transferred to a glass tube. The pelleted silica chips were re-extracted two additional times to maximize yield of the lipid of interest. All three supernatants were combined and dried under N2. Dried lipid films were stored at -20 °C. Phospholipase D treatment of in vivo accumulating lipid: S. chromofuscus phospholipase D was used following the methods of Imamura and Horiuti (25). Each reaction contained 0.08 units/µl S. chromofuscus phospholipase D re-suspended in 0.05 % BSA and 10mM Tris-HCl pH 8.0, 4 mM CaCl2, 50 mM Tris-HCl pH 8.0, 2.5 mg/ml of Triton X-100 detergent, and 12.5 µg/µl of lipid substrate. Reactions were incubated for 45 minutes at 37 °C, and terminated by plating onto a TLC plate. Mass spectrometry of total lipid extracts and purified accumulating lipid: The dried lipid film was re-dissolved in 100 µl of CHCl3:CH3OH (2:1, v/v) to a concentration of ~ 5mg/ml. This solution was directly infused into the turbo electrospray ionization source of a QSTAR XL quadrupole time-of-flight tandem mass spectrometer (ABI/MDS-Sciex, Toronto, Canada) at 6 µl/min. Mass spectra were obtained scanning from 200 to 2000 Da in negative-ion mode with

 

to an acidic two phase Bligh Dyer by the addition of 3 µl of 6N HCl, 200 µl of CHCl3 and 200 µl of CH3OH (final proportions CHCl3:CH3OH:0.1N HCl, 2:2:1.8, v/v/v). The phases were resolved by centrifugation in a microcentrifuge for 1 min. The lower phase was removed to a fresh tube and the upper phase washed two times with 300 µl of preequilibrated acidic lower phase. The lower phases were combined and dried under N2. The dried lipids were stored at -20°C. In vitro enzyme reactions: 32P-GPLs or 32 P-lyso GPLs were tested as substrates for the At1g78690p enzyme. Reactions contained 100 µM acyl acceptor (GPL or lyso GPL), ~1000 cpm/µl 32P-GPL or 32P-lyso GPL, 100 µM palmitoyl-CoA, 15 mM Tris, pH 7.4, 0.05% Triton X-100, and 0.001-1 mg/ml protein. Reactions were incubated at 37 °C for times indicated and the reaction stopped by spotting a 5 µl portion onto a silica gel-60 TLC plate. TLC plates were developed in solvent A and imaged using a phosphorimager. Reactions were also carried out with 14Cpalmitoyl-CoA (60.0 mCi/mmol) as the labeled substrate. Reactions contained 100 µM acceptor GPL, 100 µM palmitoyl-CoA, ~2000 dpm/µl 14Cpalmitoyl-CoA, 15 mM Tris, pH 7.4, 0.05% Triton X-100, and 0.001-1 mg/ml protein. Reactions were incubated at 37 °C for times indicated and the reaction stopped by spotting a 5 µl portion onto a TLC plate. TLC plates were developed in solvent A and imaged using a phosphorimager. Preparation of in vitro enzyme products: In vitro products were generated in a 2 ml reaction that contained 117 µM arachidonyl-CoA, 2 mM 1oleoyl-lyso PE or 1-oleoyl-lyso PG, 15 mM Tris, pH 7.4, 0.05 % Triton X-100 and 0.46 mg/ml solubilized membranes from BLR(DE3)pLysS/pET15b or BLR(DE3)pLysS/pAt1g78690p. The reaction was incubated for 160 minutes at 37 °C and terminated by Bligh-Dyer extraction as described above. The lower phase was dried under N2 gas and stored at 20 °C until further analysis. Mass spectrometry of in vitro products: The products of the in vitro reaction was resuspended in 100 µl of CHCl3:CH3OH (2:1, v/v) and analyzed using normal phase liquid chromatography electrospray ionization quadrupole time of flight mass spectrometer. The normal phase chromatography was performed 4 


fresh tube and stored at -80 °C. Washed-membranes were prepared by centrifuging the cell-free extract at 4 °C, 100000 x g for 1 hour. The membrane pellet was resuspended by homogenization in 2 ml of 15 mM Tris, pH 7.4 and centrifuged again as described above. The final washed membrane pellet was resuspended by homogenization in 2.0 ml of 15 mM Tris, pH 7.4. For membrane solubilization, Triton X100, Tris-HCl, and ddH2O were added to washed membranes to yield a final concentration of 1% Triton X-100, 15mM Tris pH 7.4, and 1 mg/ml protein. The samples were incubated on ice with periodic inversion for 30 minutes and then spun at 100,000 x g, 4°C for 20 minutes. The supernatant was transferred to a fresh tube to yield solubilized membranes. The concentration of all protein samples was determined using the bicinchoninic acid reagent (Thermo Scientific) with bovine serum albumin as the standard. All protein samples were stored at -80 °C. Generation of 32P-GPLs substrates: E. coli MG1655 were cultured as described above. A 10 ml culture was inoculated to A600 of 0.01 and 1 mCi of 32P-PO43- was added to the culture. Cells were grown to an A600 of ~1.0 and harvested by centrifugation for 15 minutes in a clinical centrifuge. The resulting cell pellet re-suspended in 0.4 ml of PBS and extracted as described above. All extraction volumes were adjusted to maintain the proper Bligh-Dyer extraction mixture ratios (23). The total lipid extract, which contains primarily 32P-PE and 32P-PG, was spotted along the bottom of a 20 cm x 20 cm TLC plate and developed in solvent A. 32P-PE and 32P-PG were identified by phosphorimaging. The silica chips were scraped and extracted as described above. The dried lipid films were re-suspended by sonication in 15 mM Tris, pH 7.4 for use in aqueous reactions. Following this procedure the 32 P-PE and 32P-PG was approximately 95% pure as evaluated by phosphorimager analysis. 32 P-1-acyl lysoPG and 32P-1-acyl lyso PE were generated enzymatically using porcine pancreas phospholipase A2 (PLA2) (26). The reaction mixture (100 µl) containing 32P-PE or 32PPG, 15 mM Tris, pH 8.0, 20 mM CaCl2, 150 mM NaCl, and 0.1 mg/ml of PLA2 was incubated at 37 °C for 1 hour. The reaction mixture was converted

Results Over-expression of At1g78690p in E. coli leads to accumulation of acylphosphatidylglycerol. BLR(DE3)pLysS/pET and BLR(DE3)pLysS/pAt1g78690p were grown and induced with IPTG to express proteins under control of the T7 RNA polymerase promoter (27). Lipids were extracted and displayed on HPTLC plates (Figure 1). A prominent band with an Rf higher than the major di-acylated lipids, PE and PG, accumulates in cells induced to express At1g78690p as compared to the lipid extract from the un-induced and vector control cells. This result is consistent with previous reports from Faure et al from which they concluded that this accumulating lipid was N-acyl PE (20). As shown in Figure 1 (lanes 2 and 3), the accumulating lipid  

nearly co-migrates with an authentic N-acyl PE standard but also with an authentic acyl PG standard (Figure 1, lanes 4 and 5). Negative-ion electrospray ionization mass spectrometry (ESI-MS) was performed on the total lipid extracts from BLR(DE3)pLysS/pET15b and BLR(DE3)pLysS /pAt1g78690p (Figure 2, panels A and B). The predominant ions between m/z 650 and 800 represent doubly charged species [M2H]2- of cardiolipin, and singly charged species [M-H]- of PE and PG by exact mass and MS/MS analysis (24, 28-30). In the 900-1200 a.m.u. range, the negative ions at m/z 915.702, 943.736, 953.743, 955.757, 971.768, 983.769, 985.771, 995.769, 997.780, 1011.780, 1013.799, 1023.786, and 1025.787 are enriched in total lipid extracts from cells over-expressing At1g78690p. These ions correspond by exact mass and MS/MS to acyl PG (see Table 1). The accumulating lipid was purified from lipid extracts of BLR(DE3)pLysS/pAt1g78690p by prep-TLC and analyzed by negative-ion ESI-MS (Figure 2, panel C). The predominant [M-H]- ions in the 870-1060 a.m.u. range correspond by exact mass to the m/z expected for acyl PGs with acyl chains shown in Table 1. MS/MS verified that these ions corresponded to acyl PG. Figure 2, panel D shows the results for the ion at m/z 983.5. The product ions are consistent with a GPL as indicted by the product ion at m/z 152.996 that corresponds to C3H6O5P- (29, 31, 32). Acyl chains corresponding to palmitate (16:0, m/z 255.233) and 12-methylene-palmitoleate (17cp, m/z 267.234) are also seen (33). The product ion at m/z 733.501 corresponds to the loss of one of the 17cp acyl chains. The ions at m/z 391.225 and 403.228 correspond to the loss of the acylated head-group and the 17cp acyl chain or the 16:0 acyl chain as a fatty acid (RCO2H) respectively. The MS/MS spectrum of a synthetic 54:3 (total number of carbons in the acyl chains:total number of unsaturations) acyl PG standard yields analogous product ions (Supplementary Figure 1). In addition, the exact mass of the major [M-H]ions do not match the exact mass predicted for Nacyl PE molecular species nor are the product-ions observed consistent with those of N-acyl PE (4). To further verify that acyl PG is the lipid accumulating in cells over-expressing At1g78690p, the product released by phospholipase D treatment of the isolated lipid 5 


using a Zorbax Rx-SIL (5 µm, 4.6mm x 250 mm) column on an Agilent 1100 HPLC system as described previously (24). The HPLC effluent (0.5 ml/min) was analyzed using an Agilent 6520 quadrupole time-of-flight mass spectrometer. Samples were ionized by electrospray ionization in the negative or positive mode as indicated. Mass spectra were obtained scanning from 100 to 2000 at 1 spectra per second with the following instrument parameters: fragmentor voltage 175V, drying gas temperature - 325 °C, drying gas flow - 11 l/min, nebulizer pressure - 45 psig, capillary voltage - 4000V. Data were collected with the instrument set to 3200 mass range under high resolution conditions at 4 GHz data acquisition rate. Data were collected in profile mode. For MS/MS analysis spectra were obtained scanning from 100 to 2000 at 1 spectra per second with an isolation width of ~4 m/z. The collision energy was -40.0 V (laboratory frame of reference) and N2 was the collision gas. The instrument was calibrated using Agilent ESI-L low concentration tuning mix and under normal operating conditions the resolution of the instrument was ~15,000. The mass accuracy of the instrument was between 1 and 5 ppm, and, therefore measured masses are given to three decimal places. Data acquisition and analysis was performed using Agilent MassHunter Workstation Acquisition Software and Agilent MassHunter Workstation Qualitative Analysis Software (Agilent Technologies, Santa Clara, CA), respectively.

 

0.5 respectively. The 32P-labeled in vivo accumulating lipid was purified by prep-TLC and similarly subjected to base hydrolysis. No product corresponding to Nacylglycerophosphoethanolamine was detected (Supplementary Figure 2). Taken together, this strongly suggests that At1g78690p is not promoting the accumulation of N-acyl PE (or other N-acylated GPLs such as N-acyl lyso PE) in vivo, and is consistent with our identification of the accumulating lipid as acyl PG. Identification of the enzymatic activity of At1g78690p: With the confirmation of acyl PG accumulation in cells over-expressing At1g78690p, we set out to determine the enzymatic activity of this protein. Despite 32 repeated attempts using P-labeled PE and PG as substrates, we were unable to detect the formation of a tri-acylated lipid product from either substrate in an At1g78690p-dependent manner (Supplementary Figure 4). Similar attempts to detect acylation of PE or PG using 14C-labeled acyl-CoA also were unsuccessful (Supplementary Figure 5). In the course of testing for the acylation of 32 P-PE we observed that low levels of 32P-lyso PE, a contaminant in the 32P-PE substrate, decreased in the presence of cell-free extracts prepared from induced BLR(DE3)pLysS/pAt1g78690p (data not shown). This suggested that At1g78690p might acylate lyso GPLs. To test this 32P-1-acyl lyso PE was generated by PLA2 treatment of 32P-PE and tested as a substrate for At1g78690p. When 32P-1acyl lyso PE is used as a substrate, the major product formed when cell-free extracts containing At1g78690p were used as the protein source comigrates with PE (Figure 5). Very little product was formed when cell-free extracts prepared from BLR(DE3)pLysS/pET15b were utilized. No triacylated lipid such as N-acyl PE was detected. 32 P-1-acyl lyso PG was similarly generated by PLA2 treatment of 32P-PG and tested as a substrate for At1g78690p. When 32P-1-acyl lyso PG was tested as a substrate, the major product formed co-migrates with PG (Figure 5). This in vitro assay result was confirmed 14 using C-palmitoyl-CoA as the acyl donor with unlabeled 1-acyl lyso PE and 1-acyl lyso PG as acyl acceptors (Figure 6). When no acyl acceptor is included in the assay, a small amount of product that co-migrates with PE is formed in the presence 6 


was analyzed. If the lipid were predominantly Nacyl PE, then NAE would be released as shown by the treatment of the N-acyl PE standard with PLD (Figure 3, lane 4). If the lipid were acyl PG, then monoacylglycerol (MAG) would be released by PLD treatment as shown by treatment of the acyl PG standard (Figure 3, lane 3). Treatment of the isolated lipid yields a product that corresponds by TLC to MAG (Figure 3, lane 2) consistent with the identification of the accumulating lipid as acyl PG. In TLC solvent B there is a clear difference in the migration of MAG from NAE unlike other solvents in which these fatty acyl derivatives migrate near the solvent front. This allowed us to establish that the accumulating lipid releases MAG as opposed to NAE, consistent with it being acyl PG and not N-acyl PE. Interestingly, an [M-H]- ion at m/z 952.8 detected in the purified accumulating lipid corresponds to N-acyl PE with 50:2 acyl chains (Figure 2) (4). MS/MS analysis of this ion confirmed that it is indeed N-acyl PE (4). While this is likely N-acyl PE that is endogenously produced in E. coli (4) the possibility remained that over-expression of At1g78690p leads to the accumulation of both acyl PG and N-acyl PE. Because N-acyl PE ions are nearly isobaric with acyl PG it is difficult to detect endogenous N-acyl PE with MS without prior separation. To determine if N-acylated lipids were accumulating in cells over-expressing At1g78690p we took advantage of the fact that ester linked, but not amide linked acyl chains, can be removed by treatment with base (34). BLR(DE3)pLysS/pET15b and BLR(DE3)pLysS/pAt1g78690p were continuously labeled with 32P-PO43- and induced with IPTG to promote the accumulation of the lipid in vivo. Cells were extracted and equal counts were subjected to base hydrolysis. As shown in Figure 4 panel A, lipids from cells over-expressing At1g78690p accumulate a lipid with an Rf consistent with N-acyl PE/acyl-PG as compared to cells containing pET15b. When the total lipid extract is treated with base and subject to TLC, only N-acylated lipids migrate off the origin. As can be seen in Figure 4, panel B the amount of Nacylated lipids is not altered when At1g78690p is over-expressed. Quantification of the amount of N-acylated lipid in BLR(DE3)pLysS/pET15b and pAt1g78690p was 12.0% +/- 0.8 and 12.2% +/-

 

oleoyl lyso PE or 1-oleoyl lyso PG as the acyl acceptor, arachidonyl-CoA as the acyl-donor and solubilized membranes generated from induced BLR(DE3)pLysS/pET15b or BLR(DE3)pLysS/At1g78690p. The use of arachidonyl-CoA as the acyl-donor allowed us to generate an in vitro product with a mass distinct from the endogenous PE and PG molecular species present in the solubilized membranes used as the enzyme source. Figure 8 shows the MS analysis of the in vitro product produced by At1g78690p from 1oleoyl lyso PE and arachidonoyl-CoA. Figure 8, panels A and panel B, show the negative-ion LC/ESI-MS in the m/z 660 – 780 of the extracted lipids from the in vitro reaction mixture that utilized solubilized membranes from BLR(DE3)pLysS/pET15b (panel A) or BLR(DE3)pLysS/pAt1g78690p (panel B). The predicted product (m/z 764.5236) is generated only when solubilized membranes containing At1g78690p were used as the enzyme source. The other major ions correspond to [M-H]- ions of endogenous PE molecular species that co-elute during normal phase chromatography. Ions corresponding to the transfer of arachidonate to endogenous molecular species of PE or PG (to generate N-acyl PE or acyl PG) were not detected, corroborating the results obtained using radiolabeled substrates. Negative-ion MS/MS of the ion at m/z 764.5 is shown in Figure 8, panel C. The product ions generated are consistent with the acylation of the sn-2 position of 1-oleoyl-lyso PE. The product ions at m/z 196.038 and m/z 140.011 are characteristic of PE and correspond to glycerophosphoethanolamine and phosphoethanolamine, respectively (29, 30). The product ions corresponding to oleate (18:1, m/z 281.248) and arachidonate (20:4, m/z 303.233) are clearly observed. The presence of the arachidonate product ion strongly indicates that this fatty acyl chain was attached to the precursor ion via an ester linkage to the sn-2 position, the only available site of O-acylation. The arachidonate product ion is not detected in MS/MS analysis of N-arachidonyl lyso PE (Figure 8, panel F) where the arachidonyl chain is attached via an amide linkage to the terminal amine. In Figure 8, panel C the product ions at m/z 478.294 and m/z 460.283 correspond to the loss of the arachidonate 7 


of At1g78690p likely from endogenous lyso PE present in the membranes. When 1-acyl lyso PE is used as the acyl acceptor a product that comigrates is formed only in the presence of At1g78690p. No product that co-migrates with Nacyl PE is detected. When 1-acyl lyso PG is used as the acyl acceptor a product that co-migrates with PG is formed. Interestingly, a product that migrates lower than PG on TLC is also formed. This may be the formation of 14C-1-acyl lyso PG or 14C-2acyl lyso PG from low levels of transacylase or phospholipase activity associated with At1g78690p. In addition, a low amount of product that co-migrates with acyl PG is formed. As shown in Figure 7, the conversion of 1acyl PE to PE (panel A) or 1-acyl PG to PG (panel B) was detected in solubilized membranes prepared from induced BLR(DE3)pLysS/pAt1g78690p (Figure 7, solid circles and squares) but not BLR(DE3)pLysS/pET15b (Figure 7, open circles and square). The conversion is linear with time for approximately ~30 minutes, and is dependent on the inclusion of acyl acceptor (Figure 7, squares versus circles). As mentioned above, low levels of PE and PG are formed in the absence of added 1-acyl lyso PE and 1-acyl lyso PG, presumably from low levels of lyso GPLs present in the solubilized membranes. Under the in vitro assay conditions reported here the specific activity of solubilized membranes for the formation of PE and PG from the respective 1-acyl lyso GPL is 2.3 +/- 0.1 nmol/min/mg and 2.0 +/- 0.1 nmol/min/mg respectively. Determining the site of acylation by At1g78690p: Having established that At1g78690p acylates 1-acyl lyso PE and 1-acyl lyso PG, the site at which the acyl chain is placed was determined. 1-acyl lyso PE could be acylated at the sn-2 hydroxyl or the terminal amine of the head-group. Despite the evidence presented above that N-acylated lipids do not accumulate in cells over-expressing At1g78690p low levels of Nacylation may be possible in vitro. 1-acyl lyso PG has three potential sites of acylation, the sn-2, sn2’ and sn-3’ positions. The site of acylation was determined using normal-phase liquid chromatography-electrospray ionization quadrupole time of flight MS (LC/ESIMS). An in vitro product was generated using 1-

 

reaction mixture that utilized 1-oleoyl lyso PG, arachidonoyl-CoA and solubilized membranes from BLR(DE3)pLysS/pET15b (panel A) or BLR(DE3)pLysS/pAt1g78690p (panel B). The predicted product (m/z 795.5182) is generated only when solubilized membranes containing At1g78690p were used as the enzyme source. The other major ions correspond to [M-H]- ions of endogenous PG molecular species (29, 31) that coelute during normal phase chromatography. Negative-ion MS/MS of the ion at m/z 795.518 is shown in Figure 9, panel C. The product ions generated are consistent with the acylation of the sn-2 position of oleoyl-lyso PG. The product ion at m/z 721.482 corresponds to the loss of the glycerol head-group and is highly suggestive of O-acylation of the sn-2 position and not acylation of the sn-2’ or sn-3’ positions. The product ions at m/z 509.289 and m/z 491.278 correspond to the loss of the arachidonate as a ketene (RCH=O) and the free fatty acid (RCOOH) respectively. The product ion at m/z 531.273 corresponds to the loss of the 18:1 acyl chain. The product ion at m/z 417.241 and m/z 439.226 correspond to loss of the glycerol head-group and the 18:1 and 20:4, respectively as free fatty acids. Positive-ion MS/MS analysis of the in vitro product (m/z 797.519, Figure 9, panel D) shows the formation of a prominent product ion at m/z 625.501 that corresponds to the loss of the glycerol head-group. As with the PE in vitro product, this product ion is consistent with acylation of the sn-2 position by At1g78690p. Figure 9, panel E shows that the corresponding product ion (m/z 577.518) is the major product generated from positive-ion MS/MS analysis of 16:0, 18:1 PG standard. The corresponding product ion is not detected in positive-ion MS/MS analysis of bis(monoacylglycero)phosphate (BMP) (Figure 9, panel F) strongly suggesting that At1g78690p acylates the sn-2 position of 1-oleoyllyso PG to form PG. Discussion In this work we investigated the biochemical function of the enzyme encoded by the A. thaliana gene At1g78690p when it is ectopically expressed in E. coli. We have shown that the A. thaliana enzyme At1g78690p, when over-expressed in E. coli, promotes the accumulation of acyl PG, not N-acyl PE (20), in 8 


as a ketene (RCH=O) and a free acid (RCOOH), respectively (31). The product ion at m/z 500.278 corresponds to the loss of the 18:1 acyl chain as a ketene (RCH=O). The minor product ion at m/z 426.242 corresponds to Narachidonylphosphoethanolamine (see inset of Figure 8 panel F) (4). This may indicate that the in vitro product is N-acylated; however, negativeion MS/MS analysis of 16:0, 18:1 PE standard also yielded a similar minor product ion (Supplementary Figure 6, panels A and B) indicating that the formation of the product ion at m/z 426.242 is formed during the gas phase collision-induced dissociation of PE acylated at the sn-1 and sn-2 positions. Positive-ion MS/MS analysis of the in vitro product (m/z 766.5, Figure 8, panel D) shows the formation of a prominent product ion at m/z 625.516 that corresponds to the loss of phosphoethanolamine (inset, Figure 8, panel D). This product ion is consistent with the arachidonate added to the sn-2 position of 1-oleoyl lyso PE. The corresponding product ion (m/z 577.518) is generated from positive-ion MS/MS analysis of standard 16:0, 18:1 PE (Figure 8, panel E). The corresponding product ion is not detected in positive-ion MS/MS analysis of lyso N-acyl PE generated by PLA2 treatment of standard Narachidonyl di-oleoyl PE (Supplementary Figure 6, panel C). The product ion at m/z 339.286 and m/z 361.270 (Figure 8, panel D) corresponds to loss of arachidonate and oleate, respectively, as a fatty acid from the product ion at m/z 625. The product ion at m/z 330.281 and m/z 308.291 correspond to [arachidonyl-ethanolamineH2O+H]+ and [oleoyl-ethanolamine-H2O+H]+. The corresponding product ion, [oleoylethanolamine-H2O+H]+ at m/z 308.294 is also detected for the standard 16:0, 18:1 PE (Figure 8E). This indicates that these N-acyl product ions are formed during gas phase collision induced dissociation of sn-1, sn-2 acylated PE as seen previously in the negative-ion MS/MS analysis. Taken together, this data suggests that the 20:4 acyl chain is transferred to the sn-2 position and not the terminal amine of the head-group. A similar approach was used to determine the site At1g78690p acylates when 1-oleoyl-lyso PG is used as a substrate. Figure 9, panels A and panel B show the negative-ion ESI-MS in the m/z 700–800 of the extracted lipids from the in vitro

 

growth conditions to optimize the levels of N-acyl PE (39). In our hands a three hour induction of BLR(DE3)pLysS/pAt1g78690p with 0.5 mM IPTG at 37 °C consistently yielded lipid extracts with increased levels of acyl PG and protein extracts with over-expressed lyso GPL transferase activity. Though not investigated, perhaps the extended induction used by Guo et al leads to increased levels of N-acyl PE either as a long-term consequence of increased levels of acyl PG or the presence of this lyso GPL acyltransferase activity in vivo. We have not been able to detect At1g78690p-catalyzed acylation of PE using either 32P labeled PE or with 14C-palmitoyl CoA as was previously reported (20). Careful following of the growth conditions, membrane preparation, and in vitro assay conditions reported in Faure et al did not yield any detectable N-acyl PE synthase activity in our hands. To determine the enzymatic function of At1g78690p we utilized growth conditions, protein extract preparation techniques and in vitro assay conditions previously shown to successfully express and detect the activity of membrane associated lipid metabolic enzymes (40, 41). In particular, the in vitro assay conditions that we used are based on inclusion of non-ionic detergents commonly used in assays for surface dilution kinetic analysis of enzymes that utilize mixed micellar substrates (42). We have clear evidence that At1g78690p efficiently acylates lyso GPLs such as 1-acyl lyso PE and 1-acyl lyso PG. The specific activity of At1g78690p for the acylation of 1-acyl lyso PE and 1-acyl lyso PG in solubilized membranes is approximately 1000 fold higher than that reported previously for the acylation of PE (20). This activity is clearly dependent on both substrates. The formation of di-acylated product is formed when the labeled substrate is the lyso GPL (acyl acceptor) or the acyl-CoA (acyl donor). Preliminary data suggests that At1g78690p does not have a stringent preference for the head-group 1-acyl lyso GPL substrate. 1acyl lyso PE and 1-acyl lyso PG work equally well as substrates. 2-acyl lyso GPLs are also potential substrates that need to be tested. In E. coli, 2-acyl lyso GPLs are generated when the sn-1 acyl chain of PE or PG is transferred to lipoproteins (43) or lipid A (44) making these relevant substrates for the At1g78690p in E. coli. 9 


lipid extracts. Ions corresponding to acyl PG clearly accumulate in lipid extracts prepared from cells over-expressing At1g78690p. The odd integer mass of those [M-H] ions is inconsistent with their previous identification as [M-H]- ions of N-acyl PE (20). In addition, the MS/MS of the major species does not yield acyl chains or product ions that are consistent with N-acyl PE. MS and MS/MS analysis of the purified accumulating lipid corroborates this finding. Previous work has suggested that Nacylated lipids accumulate when At1g78690p is over-expressed. Faure et al suggested N-acyl PE accumulation based on MS analysis and phospholipase D treatment of the accumulating lipid. In our study, the major product formed by the phospholipase D treatment of the isolated accumulating lipid co-migrates with MAG in a TLC system where MAG and NAE have different Rf values. Base hydrolysis of in vivo 32P-labeled lipids strongly suggests that there is little to no accumulation of N-acylated lipids when At1g78690p is over-expressed in E. coli in our hands. Recently, lipid extracts from E. coli overexpressing At1g78690p were shown by Guo et al (39) to have increased levels of N-acyl PE as assessed by LC-MS based detection of N-acyl glycerophosphates following base de-acylation with methyl amine. Triple quadrupole mass spectrometry in multiple reaction-monitoring (MRM) mode was used to detect transitions specific for the N-acyl glycerophosphate products of base hydrolysis of N-acyl PE and thereby quantify N-acyl PE in lipid extracts. Our base hydrolysis data of in vivo 32P-labeled cells indicates that N-acylated lipids do not accumulate in E. coli expressing At1g78690p; however MRM mass spectrometry system used by Guo et al is likely more sensitive. An ion consistent with Nacyl PE (m/z 952.8) is detected in our ESI-MS analysis of the accumulating lipid. This may indicate that N-acyl PE (or other N-acylated lipid) is somewhat increased in cells over-expressing At1g78690p but may not be detected in our analyses. The method of Guo et al would not detect the increases in acyl PG (40) that are seen in our data. The growth conditions we used to induce expression of At1g78690p differ from those used previously (20, 39). Guo et al used modified

While our data strongly suggests that the major site of acylation is the sn-2 hydroxyl of 1acyl lyso PE, we cannot completely rule out that At1g78690p possesses some low level of NAT activity. Negative-ion MS/MS analysis of the in vitro product generated from 1-acyl lyso PE yields a low level of a product ion (m/z 426) that is consistent with N-acylation. While the corresponding product ion (m/z 404) is also observed in MS/MS analysis of the standard PE compound the product ion at m/z 426 could also be the result of low levels of N-acylated 1-acyl lyso PE. Our data also strongly suggests that At1g78690p catalyzes the acylation of the sn-2 hydroxyl of 1-acyl lyso PG. The positive ion MS/MS analysis of the in vitro product generated from 1-acyl lyso PG yields a product ion possible only when the sn-2 position is acylated. However, as with 1-acyl lyso PE, with our current data we cannot completely rule out acylation at the sn-2’ or sn-3’ positions at some level. Lyso GPL acyltransferases play an important role in the Land’s cycle of acyl chain remodeling (47-49). With the identification of At1g78690p as a lyso GPL acyltransferase, its role in relation to the other lyso GPL acyltransferases of A. thaliana (49) needs to be determined. Monoacylated GPLs are acylated by At1g78690p to form di-acylated products in vitro, yet a tri-acylated lipid, acyl PG, accumulates in vivo in E. coli over-expressing At1g78690p. Careful determination of the substrate specificity of this acyltransferase may illuminate the mechanism by which acyl PG accumulates in cells over-expressing At1g78690p and shed light on the complex interplay between lyso GPL metabolism and head-group acylated GPL metabolism in E. coli.

References 1. Merkel, O., Schmid, P. C., Paltauf, F., Schmid, H. H. (2006) Biochim. Biophys. Acta. 1734, 215-219 2. Astarita, G., Ahmed, F., and Piomelli, D. (2008) J. Lipid Res. 49, 48-57 3. Chapman, K. D., and Moore, T. S. J. (1993) Plant Physiol. 102, 761-769 4. Mileykovskaya, E., Ryan, A. C., Mo, X., Lin, C., Khalaf, K. K., Dowhan, W., and Garrett, T. A. (2009) J. Biol. Chem. 284, 2990-3000 5. Rawyler, A. J., and Braendle, R. A. (2001) Plant Physiol. 127, 240-251 6. Holmbäck, J., Karlsson, A., and Arnoldsson, K. C. (2001) Lipids 36, 153-165 7. Coulon, D., Faure, L., Salmon, M., Wattelet, V., and Bessoule, J. Biochimie. In Press, Uncorrected Proof  

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At1g78690p may utilize other acyl donors, acyl acceptors or possess additional enzymatic activities than defined in this work. For example, evidence of transacylation activity is suggested when 1-acyl lyso PG is used as a substrate. With higher concentrations of enzyme, a labeled product is generated that co-migrates with lyso PG. At1g78690p may be able to transfer the sn-2 acyl chain to sn-glycero-3-phospho-3’-glycerol. Alternatively At1g78690p might be able to hydrolyze the sn-1 acyl chain to generate 14Clabeled 2-acyl lyso PG. At1g78690p may also hydrolyze acyl-CoA to yield a free fatty acid and CoA. Free fatty acids are generated in in vitro reactions which we attribute to the two known thioesterases of E. coli, tesA and tesB, soluble enzymes present in E. coli protein extracts (45). Highly purified At1g78690p will be required to identify its enzymatic activities and determine the substrate specificity. Faure et al first investigated At1g78690p because of its homology to YPR140wp, the yeast homolog of tafazzin (20, 21). YPR140wp has been reported to be a membrane associated acylCoA-independent lysophosphatidylcholine acyltransferase (21) consistent, in part, with our report of lyso GPL acyltransferase activity for At1g78690p. Drosophila tafazzin has been shown to be GPL-lyso GPL transacylase with specificity for transferring a linoleoyl residue from PC to monolysocardiolipin (46). Despite the fact that Drosophila tafazzin is ~50% homologous to At1g78690p, tafazzin does not utilize acyl-CoA as an acyl donor. Future research may yield insights into the structural motifs that distinguish between acyl transfer from GPL (or lyso GPL) acyl donors and phosphopantetheine-linked acyl donors (acylCoA or acyl-acyl carrier protein) in acyltransferases.

 

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45. Naggert, J., Narasimhan, M. L., DeVeaux, L., Cho, H., Randhawa, Z. I., Cronan Jr., J. E., Green, B. N., and Smith, S. (1991) J. Biol. Chem. 266, 11044-11050 46. Xu, Y., Malhotra, A., Ren, M., and Schlame, M. (2006) J. Biol. Chem. 281, 39217-39224 47. Shindou, H., and Shimizu, T. (2009) J. Biol. Chem. 284, 1-5 48. Lands, W. E. (2000) Biochim. Biophys. Acta 1483, 1-14 49. Stalber, K., Stahl, U., Stymne, S., and Ohlrogge, J. (2009) BMC Plant Biol. 9, 60-68 Footnotes *

This work was supported in part by LIPID MAPS Large Scale Collaborative Grant Number GM-069338, National Science Foundation Major Research Instrumentation Award #1039659 and a Research Corporation for Science Advancement Cottrell College Science Single Investigator Award #7914.

1

Acknowledgements The authors thank Denis Coulon of the Laboratoire de Biogenèse Membranaire, Université Victor Segalen Bordeaux, France for supplying the plasmid containing At1g78690p. Christian Raetz provided helpful discussions and use of the LIPID MAPS mass spectrometry facility at Duke University Medical Center. Figure Legends Figure 1: Expression of At1g78690p in E. coli leads to the accumulation of a lipid that co-migrates with a tri-acylated lipid. E. coli BLR(DE3)pLysS/pET15b and BLR(DE3)pLysS/pAt1g78690p were grown and induced with IPTG. Lipids were extracted and the accumulating lipid was purified using preparative TLC as described in Experimental Procedures. All lipids were displayed on HPTLC by development in solvent A and visualized by charring with sulfuric acid. Lane 1: Lipid extract from induced BLR(DE3)pLysS/pET15b, Lane 2: Lipid extract from induced BLR(DE3)pLysS/ pAt1g78690p, Lane 3: Purified accumulating lipid, Lane 4: N-acyl PE standard, Lane 5: acyl PG standard. The migration of the major GPLs, PE and PG, are indicated. The asterisk indicates the lipid that accumulates in lipid extracts prepared from cells induced to over-express At1g78690p. Figure 2: Negative-ion ESI-MS of lipid extracts from E. coli BLR(DE3)pLysS/pET15b and BLR(DE3)pLysS/pAt1g78690p. Panel A: Negative-ion ESI-MS from m/z 500 to 1100 of lipid extract prepared from BLR(DE3)pLysS/pET15b. Panel B: Negative-ion ESI-MS from m/z 500 to 1100 of lipid extract prepared from BLR(DE3)pLysS/pAt1g78690p. In Panels A and B the major ions in the m/z 650 to m/z 800 correspond to [M-2H]-2 ions of CL, and [M-H]- ions of PE and PG. Panel C: Negative-ion ESIMS from m/z 900 to 1050 of purified accumulating lipid. The major [M-H]- ions correspond to acyl PG molecular species as shown in Table 1. Panel D: Negative-ion MS/MS of m/z 983.7. The inset shows  

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Abbreviations: PE, phosphatidylethanolamine; N-acyl PE, N-acylphosphatidylethanolamine; PG, phosphatidylglycerol; acyl PG, acylphosphatidylglycerol; ESI-MS, electrospray ionization quadrupole time-of-flight mass spectrometry; a.m.u., atomic mass unit; MS/MS, collision-induced dissociation mass spectrometry; lyso PE, lysophosphatidylethanolamine; lyso PG, lysophosphatidylglycerol; NAE, Nacylethanolamines; GPL, glycerophospholipid; N-acyltransferase, NAT; HPTLC, high performance thin layer chromatography; 3, 1’ BMP, sn-(3-oleoyl-2-hydroxy)-glycerol-1-phospho-sn-3'-(1'-oleoyl-2'hydroxy)-glycerol; LB, Luria-Bertani; IPTG, Isopropyl β -D-1-thiogalactopyranoside; PBS, phosphate buffered saline; PPG, polypropylene glycol; PLA2, phospholipase A2; lyso GPL, lysoglycerophospholipid; TLC, thin layer chromatography; MAG, monoacylglycerol; LC/ESI-MS, liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry; MRM, multiple reaction-monitoring

the major product ions from a predicted precursor ion. At the given molecular mass of 983.7 several distinct molecular species of acyl PG are possible. The inset shows one possible molecular species consistent with the product ion spectra. The MS/MS technique we employed does not allow for definitive assignment of the acyl chains to the sn-1, sn-2, sn-2’ or sn-3’ position. Figure 3: Phospholipase D treatment of the lipid that accumulates upon over-expression of At1g78690p in E. coli. Samples were treated with phospholipase D, displayed by TLC using solvent B and visualized by exposure to iodine vapor. Lane 1: Total lipid extracts from BLR(DE3)pLysS/pET15b, Lane 2: Purified accumulating lipid, Lane 3: Acyl PG standard, Lane 4: N-acyl PE standard.

Figure 5: Acylation of 32P-labeled 1-acyl PE and 1-acyl PG by At1g78690p. Crude extracts (0.05 mg/ml) prepared from induced BLR(DE3)pLysS/pET15b or BLR(DE3)pLysS/At1g78690p were tested for the ability to acylate 32P-labeled 1-acyl lyso PE, or 1-acyl lyso PG using palmitoyl-CoA as the acyl donor in a 10 minute reaction at 37 °C. The E. coli lysophospholipase L2, PldB, (36-38) is known to catalyze the synthesis of acyl PG from PG and lyso PE or lyso PG and may be responsible for the acyl PG formed in these assays. Figure 6: Acylation of 1-acyl PE and 1-acyl PG using 14C-labeled palmitoyl-CoA. Solubilized membranes (0.05 mg/ml) prepared from induced BLR(DE3)pLysS/pET15b or BLR(DE3)pLysS/At1g78690p were tested for acyltransferase activity using un-labeled 1-acyl PE, 1-acyl PG or no acyl acceptor with 14C-labeled palmitoyl-CoA as the acyl donor in a 10 minute reaction at 37 °C. 14C-palmitate released during the reaction by the action of thioesterase (47) present in the solubilized membranes is detected near the solvent front. A small amount of PE is formed in the absence of acyl acceptor presumably from lyso PE present in the solubilized membranes. The product migrating below the PG in the presence of 1-acyl lyso PG may be lyso PG formed by transacylation activity of At1g78690p. Figure 7: Time-dependence of conversion of 1-acyl lyso PE and 1-acyl lyso PG to di-acylated products. Solubilized membranes (0.05 mg/ml) prepared from induced BLR(DE3)pLysS/pET15b (open circles and squares) or BLR(DE3)pLysS/At1g78690p (closed circles and squares) were tested for acyltransferase activity using 1-acyl PE (Panel A), 1-acyl PG (Panel B) (closed circles and squares) or no acyl acceptor (open circles and squares) with 14C-labeled palmitoyl-CoA as the acyl donor. Figure 8: Determination of the site of acylation of 1-acyl lyso PE. Solubilized membranes prepared from induced BLR(DE3)pLysS/pET15b or BLR(DE3)pLysS/At1g78690p were incubated in an in vitro assay mixture using 1-oleoyl lyso PE as the acyl acceptor and arachidonyl-CoA as the acyl donor as described in Experimental Procedures. The lipids from the reaction mixture were extracted and analyzed using LC/ESI-MS and MS/MS. Panel A: Negative-ion LC/ESI-MS from m/z 660 to 780 of lipids extracted from the reaction of solubilized membranes from BLR(DE3)pLysS/pET15b. Panel B: Negative-ion ESI-MS from m/z 660 to 780 of lipids extracted from the reaction of solubilized membranes from BLR(DE3)pLysS/pAt1g78690p. The mass spectra in panel A and B are of the material eluting between minutes 22.0 and 25.0 of the normal phase chromatography. Panel C: Negative-ion MS/MS of the in vitro product at m/z 764.5. Panel D: Positive-ion MS/MS of the in vitro product at m/z 766.5.  

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Figure 4: Base hydrolysis of 32P-labeled lipids from BLR(DE3)pLysS/pET15b and BLR(DE3)pLysS/pAt1g78690p. GPLs were in vivo labeled by culturing in the presence of 32P-PO43and extracted as described in Experimental Procedures. 1000 dpm of each sample was treated with base, spotted to TLC and developed in solvent A. As indicated, lipid extracts from BLR(DE3)pLysS/pAt1g78690p or BLR(DE3)pLysS/pET15b were treated or not treated with 0.2M NaOH. The predicted products and the migration of the base hydrolysis products of acyl-PG and N-acyl PE are shown. Base treatment of the standards yields similar products (Supplementary Figure 3).

Panel E: Positive-ion MS/MS of 16:0, 18:1 PE standard. Panel F: Positive-ion MS/MS of N-arachidonyl 18:1 lyso PE. The insets show the major product ions from the predicted from each precursor ion. Figure 9: Determination of the site of acylation of 1-acyl lyso PG. Solubilized membranes prepared from induced BLR(DE3)pLysS/pET15b or BLR(DE3)pLysS/At1g78690p were incubated in an in vitro assay using 1-oleoyl-lyso PG as the acyl acceptor and arachidonyl-CoA as the acyl donor as described in Experimental Procedures. The lipids from the reaction mixture were extracted and analyzed using ESIMS. Panel A: Negative-ion ESI-MS from m/z 700 to 800 of lipids extracted from the reaction of solubilized membranes from BLR(DE3)pLysS/pET15b. Panel B: Negative-ion ESI-MS from m/z 700 to 800 of lipids extracted from the reaction of solubilized membranes from BLR(DE3)pLysS/pAt1g78690p. The mass spectra in panel A and B are of the material eluting between minutes 16.5 and 19.5 of the normal phase chromatography. Panel C: Negative-ion MS/MS of the in vitro product at m/z 795.5. Panel D: Positive-ion MS/MS of the in vitro product at m/z 797.5. Panel E: Positive-ion MS/MS of 16:0, 18:1 PG standard. Panel F: Positive-ion MS/MS of 3, 1’ BMP. The insets show the major product ions from the predicted from each predicted precursor ion. Downloaded from http://www.jbc.org/ by guest on December 13, 2017

 

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Tables

 


Table 1: Acyl PG species identified in BLR(DE3)pLysS/pAt1g78690p Acyl PG Molecular Molecular Formula Exact Mass Observed Mass Species* [M-H][M-H][M-H]42:0 C48H92O11P875.6383 875.636 42:1 C48H90O11P873.6226 873.623 43:1 C49H92O11P887.6383 887.637 43:2 C49H90O11P 885.6226 885.629 44:1 C50H94O11P901.6539 901.661 44:2 C50H92O11P899.6383 889.660 45:1 C51H96O11P915.6696 915.670 45:2 C51H94O11P 913.6539 913.680 46:1 C52H98O11P929.6852 929.692 46:2 C52H96O11P927.6696 927.698 47:1 C53H100O11P 943.7009 943.701 47:2 C53H98O11P941.6852 941.715 48:1 C54H102O11P957.7165 957.717 48:2 C54H100O11P 955.7009 955.714 49:1 C55H104O11P971.7322 971.730 49:2 C55H102O11P969.7165 969.719 50:2 C56H104O11P983.7322 983.731 50:3 C56H102O11P 981.7165 981.737 51:2 C57H106O11P997.7478 997.747 51:3 C57H104O11P995.7322 995.736 52:2 C58H108O11P 1011.7635 1011.761 52:3 C58H110O11P1009.7478 1009.748 53:2 C59H110O11P1025.7791 1025.773 53:3 C58H108O11P1023.7635 1023.763 54:3 C60H110O11P 1037.7791 1037.779 55:3 C61H112O11P1051.7948 1051.786 *Acyl PG species are denoted by the total number of carbons in the acyl chains:total number of unsaturations in the acyl chains

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The putative N-acylphosphatidylethanolamine synthase from Arabidopsis thaliana is a lysoglycerophospholipid acyltransferase Evgeny Bulat and Teresa A. Garrett J. Biol. Chem. published online July 29, 2011

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