Putative N-acylphosphatidylethanolamine synthase from Arabidopsis thaliana is a lysoglycerophospholipid acyltransferase

Abstract

AT1G78690, a gene found in Arabidopsis thaliana, has been reported to encode a N-acyltransferase that transfers an acyl chain from acyl-CoA to the headgroup of phosphatidylethanolamine (PE) to form N-acylphosphatidylethanolamine (N-acyl-PE). Our investigation suggests that At1g78690p is not a PE-dependent N-acyltransferase but is instead a lysoglycerophospholipid O-acyltransferase. We overexpressed AT1G78690 in Escherichia coli, extracted the cellular lipids, and identified the accumulating glycerophospholipid as acylphosphatidylglycerol (acyl-PG). Electrospray ionization quadrupole time-of-flight 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 (32)P- and (14)C-radiolabeled substrates showed that AT1G78690 acylates 1-acyllysophosphatidylethanolamine (1-acyllyso-PE) and 1-acyllysophosphatidylglycerol (1-acyllyso-PG), but not PE or phosphatidylglycerol (PG), to form a diacylated product that co-migrates with PE and PG, respectively. We analyzed the diacylated product formed by AT1G78690 using a combination of base hydrolysis, phospholipase D treatment, ESI-MS, and MS/MS to show that AT1G78690 acylates the sn-2-position of 1-acyllyso-PE and 1-acyllyso-PG.

FIGURE 1.

Expression of AT1G78690 in E. coli leads to the accumulation of a lipid that co-migrates with a triacylated 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 under “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 overexpress At1g78690p.

FIGURE 2.

Negative ion ESI-MS of lipid extracts from E. coli BLR(DE3)pLysS/pET15b and BLR(DE3)pLysS/pAt1g78690p. A, negative ion ESI-MS from m/z 500 to 1100 of lipid extract prepared from BLR(DE3)pLysS/pET15b. B, negative ion ESI-MS from m/z 500 to 1100 of lipid extract prepared from BLR(DE3)pLysS/pAt1g78690p. In A and B, the major ions in m/z 650–800 correspond to [M − 2H]²⁻ ions of CL and [M − H]⁻ ions of PE and PG. C, negative ion ESI-MS 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. D, negative ion MS/MS of m/z 983.7. The inset shows 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 overexpression of AT1G78690 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 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]PO₄³⁻ and extracted as described under “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 0.2 m 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 (supplemental Fig. 3).

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-acyllyso-PE, or 1-acyllyso-PG using palmitoyl-CoA as the acyl donor in a 10-min reaction at 37 °C. The E. coli lysophospholipase L2, PldB (–37), 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 unlabeled 1-acyl-PE, 1-acyl-PG, or no acyl acceptor with ¹⁴C-labeled palmitoyl-CoA as the acyl donor in a 10-min 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-acyllyso-PG may be lyso-PG formed by transacylation activity of AT1G78690.

FIGURE 7.

Time dependence of conversion of 1-acyllyso-PE and 1-acyllyso-PG to diacylated 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-acyllyso-PE (A), 1-acyllyso-PG (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-acyllyso-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-oleoyllyso-PE as the acyl acceptor and arachidonyl-CoA as the acyl donor as described under “Experimental Procedures.” The lipids from the reaction mixture were extracted and analyzed using LC/ESI-MS and MS/MS. 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. 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 A and B are of the material eluting between minutes 22.0 and 25.0 of the normal phase chromatography. C, negative ion MS/MS of the in vitro product at m/z 764.5. D, positive ion MS/MS of the in vitro product at m/z 766.5. E, positive ion MS/MS of 16:0, 18:1 PE standard. F, positive ion MS/MS of N-arachidonyl 18:1 lyso-PE. The insets show the major product ions predicted from each precursor ion.

FIGURE 9.

Determination of the site of acylation of 1-acyllyso-PG. Solubilized membranes prepared from induced BLR(DE3)pLysS/pET15b or BLR(DE3)pLysS/At1g78690p were incubated in an in vitro assay using 1-oleoyllyso-PG as the acyl acceptor and arachidonyl-CoA as the acyl donor as described under “Experimental Procedures.” The lipids from the reaction mixture were extracted and analyzed using ESI-MS. 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. 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 A and B are of the material eluting between minutes 16.5 and 19.5 of the normal phase chromatography. C, negative ion MS/MS of the in vitro product at m/z 795.5. D, positive ion MS/MS of the in vitro product at m/z 797.5. E, positive ion MS/MS of 16:0, 18:1 PG standard. F, positive ion MS/MS of 3,1′-BMP. The insets show the major product ions predicted from each predicted precursor ion.