Lysophosphatidylethanolamine Acyltransferase Research Articles

Admission lysophosphatidylethanolamine acyltransferase level predicts the severity and prognosis of community-acquired pneumonia.

Early diagnosis and prognosis of patients with community-acquired pneumonia (CAP) are still difficult clinical challenges. This study aimed to investigate the role of lysophosphatidylethanolamine acyltransferase (LPEAT) in CAP and to evaluate the effectiveness of this enzyme as an indicator of disease severity and risk of death in CAP. This retrospective, multi-center study was conducted in 2017. A total of 267 patients with CAP were included. Of these 267 patients, 175 patients had non-severe CAP (non-SCAP) and 92 patients had severe CAP (SCAP). In addition, we recruited 15 healthy volunteers and 42 hospitalized disease controls in our study. The demographic and clinical characteristics were recorded for all participants. Admission levels of LPEAT were determined by quantitative enzyme-linked immunosorbent assay. Admission levels of LPEAT in patients with SCAP were significantly higher, particularly in non-survivors and were not affected by the causative etiology. Furthermore, when the patients were stratified according to PSI and CURB-65 scores, the patients with high severity scores had higher LPEAT levels upon admission than patients with low severity scores. LPEAT also performed well in predicting SCAP in patients with CAP. Moreover, LPEAT could predict the 30-day mortality rate of patients with CAP, and combining LPEAT with the clinical severity score further improved the accuracy of mortality prediction. Elevated LPEAT levels can reliably predict the severity of illness in patients with CAP at the time of admission. Adding LPEAT to clinical scoring methods could improve prognostic accuracy. Trial registration ClinicalTrials.gov, NCT03093220. Registered on March 28th, 2017.

Phospholipid:Diacylglycerol Acyltransferase1 Overexpression Delays Senescence and Enhances Post-heat and Cold Exposure Fitness.

In an alternative pathway to acyl-CoA: diacylglycerol acyltransferase (DGAT)-mediated triacylglycerol (TAG) synthesis from diacylglycerol, phospholipid:diacylglycerol acyltransferase (PDAT) utilizes not acyl-CoA but an acyl group from sn-2 position of a phospholipid, to form TAG. The enzyme’s activity in vitro matches DGAT’s in a number of plant species, however its main function in plants (especially in vegetative tissue) is debatable. In the presented study, we cultivated PDAT1-overexpressing, pdat1 knockout and wild-type lines of Arabidopsis thaliana through their whole lifecycle. PDAT1 overexpression prolonged Arabidopsis lifespan in comparison to wild-type plants, whereas knocking out pdat1 accelerated the plant’s senescence. After subjecting the 3-week old seedlings of the studied lines (grown in vitro) to 2-h heat stress (40°C) and then growing them for one more week in standard conditions, the difference in weight between wild-type and PDAT1-overexpressing lines increased in comparison to the difference between plants grown only in optimal conditions. In another experiment all lines exposed to 2-week cold stress experienced loss of pigment, except for PDAT1-overexpressing lines, which green rosettes additionally weighed 4 times more than wild-type. Our results indicate that plants depleted of PDAT1 are more susceptible to cold exposure, while PDAT1 overexpression grants plants a certain heat and cold resilience. Since it was shown, that lysophospholipids may be intertwined with stress response, we decided to also conduct in vitro assays of acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) and acylCoA:lysophosphatidylethanolamine acyltransferase (LPEAT) activity in microsomal fractions from the PDAT1-overexpressing Arabidopsis lines in standard conditions. The results show significant increase in LPEAT and LPCAT activity in comparison to wild-type plants. PDAT1-overexpressing lines’ rosettes also present twice as high expression of LPCAT2 in comparison to control. The presented study shows how much heightened expression of PDAT1 augments plant condition after stress and extends its lifespan.

Lysophosphatidylethanolamine acyltransferase 2 (LPEAT2) incorporates DHA into phospholipids and has possible functions for fatty acid-induced cell death

Glycerophospholipids, one of the main constituents of biological membranes, are synthesized from glycerol-3-phosphate through the de novo pathway, and are reconstituted through the remodeling pathway. Lysophosphatidylethanolamine acyltransferase 2 (LPEAT2), one of the enzymes that play a role in the remodeling pathway, has been previously reported to have LPEAT, lysophosphatidylcholine acyltransferase (LPCAT) and lysophosphatidylglycerol acyltransferase (LPGAT) activities with 16:0-CoA, 18:0-CoA, and 18:1-CoA as donors. In this study, we found that LPEAT2 is active with 22:6-CoA. Knockdown studies using Neuro 2A cells showed that LPEAT2 has endogenous LPEAT activity with 22:6-CoA, and that LPEAT2 has functions for modulating 22:6/20:4 ratios of phospholipids. In addition, we demonstrated that Neuro 2A cells overexpressing LPEAT2 underwent cell death with necrotic morphology when differentiated into neuron-like cells, with supplementation with 22:6 (DHA). These results suggest that LPEAT2 plays a role in inducing cell death DHA-dependently. This study will lead to better understand how DHA levels are regulated in phospholipids, especially in the brain where LPEAT2 is highly expressed. Our study also provides insight to understand the mechanism of cell death induced by DHA.

Correction: Novel Lysophospholipid Acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b Responsible for Generation of Palmitate-Docosahexaenoate-Phosphatidylcholine and Phosphatidylethanolamine.

[This corrects the article DOI: 10.1371/journal.pone.0102377.].

Cloning and characterization of a gene encoding lysophosphatidylethanolamine acyltransferase (LPEAT) in the green microalga Myrmecia incisa Reisigl

PDF HTML阅读 XML下载 导出引用 引用提醒 缺刻缘绿藻溶血磷脂酰乙醇胺酰基转移酶(LPEAT)的基因克隆与特征分析 DOI: 作者: 作者单位: 1. 水产种质资源发掘与利用教育部重点实验室, 上海海洋大学, 上海 201306;2. 海洋生物科学国际联合研究中心, 上海海洋大学, 上海 201306;3. 水产科学国家级实验教学示范中心, 上海海洋大学, 上海 201306 作者简介: 周志刚(1964-),博士,教授,主要从事藻类学及藻类生物技术的研究.E-mail:zgzhou@shou.edu.cn 通讯作者: 中图分类号: S917 基金项目: 国家自然科学基金项目（31772821，31402274）；国家海洋局可再生能源专项基金项目（SHME2011SW02）；上海高校水产科学高峰学科建设项目. Cloning and characterization of a gene encoding lysophosphatidylethanolamine acyltransferase (LPEAT) in the green microalga Myrmecia incisa Reisigl Author: Affiliation: 1. The Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources Conferred by Ministry of Education, Shanghai Ocean University, Shanghai 201306, China;2. International Research Center for Marine Biosciences Conferred by Ministry of Science and Technology, Shanghai Ocean University, Shanghai 201306, China;3. National Demonstration Center for the Experimental Teaching of Fisheries Science, Shanghai Ocean University, Shanghai 201306, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:缺刻缘绿藻三酰甘油（TAG）中花生四烯酸（ArA）占其总脂肪酸含量的68.0%。为了弄清ArA是如何被优先地用于合成TAG，鉴于Lands循环是通过改变膜脂的脂肪酸组成进而影响TAG的脂肪酸组成，本研究选择在Lands循环中起关键作用的溶血磷脂酰乙醇胺酰基转移酶（LPEAT）作为突破口。运用反转录PCR与3'-及5'-cDNA末端快速扩增技术，自缺刻缘绿藻（；它的cDNA序列全长1303 bp，其中，5'-非翻译区（UTR）长129 bp，3'-UTR长193 bp，开放阅读框长981 bp，编码326个氨基酸残基；以缺刻缘绿藻基因组DNA为模板扩增得到该基因长为1871 bp的DNA序列；这两序列的比对结果显示，含有6个内含子，将其开放阅读框（ORF）分割成7个外显子。通过多序列比对及生物信息学分析，发现MiLPEAT存在一个磷酸酰基转移酶结构域PlsC，并含有LPEAT家族所具有的NH（x）4D、FPEGT等4个特征性模体；Wolfpsort等在线预测结果以及MiLPEAT羧基端存在的双赖氨酸模体"KKxx"，暗示它可能位于内质网并参与分泌途径。基于不同植物LPEAT的氨基酸序列所构建的邻接聚类图表明，MiLPEAT因与2型LPEAT有不同的序列特征而形成不同的分支，但却与1型LPEAT聚类在一起，推测它们的功能可能更近似。通过荧光定量PCR检测，发现MiLPEAT的基因转录量在氮饥饿8 h时显著（<0.01）减少49%，但磷脂酰乙醇胺（PE）相对丰度的增加却不显著；推测在氮饥饿过程中增加的磷脂酰乙醇胺（PE）可能被磷脂：二酰甘油酰基转移酶作用以合成为TAG，致使缺刻缘绿藻TAG含量的增加。 Abstract:Reisigl, a coccoid green microalga, is an oleaginous alga that can accumulate an unprecedentedly high level of arachidonic acid (ArA, 20:4w6)-rich triacylglycerols (TAG) under the growth stress of nitrogen starvation. ArA accounts for 68.0% of total fatty acids in TAG. How is ArA preferentially utilized for the biosynthesis of TAG? The Lands' cycle plays an important role in the composition change of fatty acids of phospholipids, thus altering the composition of fatty acids in TAG. Lysophosphatidylethanolamine acyltransferase (LPEAT), a key enzyme in the Lands' cycle, was the focus of the present study. was cloned using reverse transcription-PCR and 3°-and 5'-cDNA rapid amplification of cDNA ends technique. The full-length cDNA was 1303 bp long, and contained a 129-bp 5'-untranslated region (UTR) and 193-bp 3'-UTR. The length of the open read frame was 981 bp that encoded a 326-amino acid protein. The DNA sequence of with the isolated algal genomic DNA as a template, and it was 1871 bp long. Comparison of the cDNA and DNA sequences showed that possessed 6 introns that separated the coding sequence into 7 exons. Multiple sequence alignment and bioinformatics analysis of LPEATs from different species demonstrated that MiLPEAT possessed a phosphate acyltransferase domain, PlsC, thus suggesting that it was one member of the lysophospholipid acyltransferase (LPLAT) superfamily. MiLPEAT also had the 4 typical motifs, NH(x)4D, GCxYVxR, FPEGT, and PVxPVx, which are characterized in the LPLAT superfamily. Both the prediction, as analyzed online by Wolfsport and Protein Prowler, and the presence of a dilysine motif at the carboxyl terminus of MiLPEAT, implied that MiLPEAT might reside at the algal endoplasmic reticulum and possibly participate in the secretion pathway. A neighbor-joining phylogeny was constructed on the basis of deduced amino acids of LPEATs from different species of plants. It illustrated that MiLPEAT was so different from LPEAT2 that they located at different clades due to their various characteristics of sequences. MiLPEAT was clustered phylogenetically with LPEAT1, suggesting their similar functions in the acylation of phospholipids. Quantitative real-time PCR detection pointed out that <0.05) at 8 h after treatment with nitrogen starvation in . By coincidence, the relative abundance of lysophosphatidylethanolamine (LPE) in the microalgal cells reduced by approximately 49% at an extreme significance level (<0.01) under the nitrogen starvation stress. The corresponding phosphatidylethanolamine (PE) generated from LPE as catalyzed by MiLPEAT, however, did not increase significantly. It is assumed that the net increase of PE under nitrogen starvation in was possibly utilized for TAG biosynthesis by the phospholipid DAG acyltransferase (PDAT), so that the content of TAG has been reported to increase. This research lays a foundation for us to understand the TAG and phospholipid biosynthetic pathway and how to regulate TAG synthesis in . 参考文献 相似文献 引证文献

Phosphatidylethanolamine dynamics are required for osteoclast fusion

Osteoclasts, responsible for bone resorption, are multinucleated cells formed by cell-cell fusion of mononuclear pre-osteoclasts. Although osteoclast fusion is a pivotal step for osteoclastogenesis, little is known about the mechanism involved. To clarify the underlying process, we investigated dynamics of membrane phospholipids during osteoclastogenesis in vitro. We found that the cellular content of phospholipids, phosphatidylethanolamine (PE) in particular, was increased during osteoclast differentiation. Furthermore, PE was greatly increased in the outer leaflet of the plasma membrane bilayer during osteoclastogenesis, being concentrated in filopodia involved in cell-cell fusion. Immobilisation of the cell surface PE blocked osteoclast fusion, revealing the importance of PE abundance and distribution. To identify the molecules responsible for these PE dynamics, we screened a wide array of lipid-related genes by quantitative PCR and shRNA-mediated knockdown. Among them, a PE-biosynthetic enzyme, acyl-CoA:lysophosphatidylethanolamine acyltransferase 2 (LPEAT2), and two ATP-binding cassette (ABC) transporters, ABCB4 and ABCG1, were markedly increased during osteoclastogenesis, and their knockdown in pre-osteoclasts led to reduction in PE exposure on the cell surface and subsequent osteoclast fusion. These findings demonstrate that the PE dynamics play an essential role in osteoclast fusion, in which LPEAT2, ABCB4 and ABCG1 are key players for PE biosynthesis and redistribution.

Acyl-CoA:Lysophosphatidylethanolamine Acyltransferase Activity Regulates Growth of Arabidopsis.

Arabidopsis (Arabidopsis thaliana) contains two enzymes (encoded by the At1g80950 and At2g45670 genes) preferentially acylating lysophosphatidylethanolamine (LPE) with acyl-coenzyme A (CoA), designated LYSOPHOSPHATIDYLETHANOLAMINE ACYLTRANSFERASE1 (LPEAT1) and LPEAT2. The transfer DNA insertion mutant lpeat2 and the double mutant lpeat1 lpeat2 showed impaired growth, smaller leaves, shorter roots, less seed setting, and reduced lipid content per fresh weight in roots and seeds and large increases in LPE and lysophosphatidylcholine (LPC) contents in leaves. Microsomal preparations from leaves of these mutants showed around 70% decrease in acylation activity of LPE with 16:0-CoA compared with wild-type membranes, whereas the acylation with 18:1-CoA was much less affected, demonstrating that other lysophospholipid acyltransferases than the two LPEATs could acylate LPE The above-mentioned effects were less pronounced in the single lpeat1 mutant. Overexpression of either LPEAT1 or LPEAT2 under the control of the 35S promotor led to morphological changes opposite to what was seen in the transfer DNA mutants. Acyl specificity studies showed that LPEAT1 utilized 16:0-CoA at the highest rate of 11 tested acyl-CoAs, whereas LPEAT2 utilized 20:0-CoA as the best acyl donor. Both LPEATs could acylate either sn position of ether analogs of LPC The data show that the activities of LPEAT1 and LPEAT2 are, in a complementary way, involved in growth regulation in Arabidopsis. It is shown that LPEAT activity (especially LPEAT2) is essential for maintaining adequate levels of phosphatidylethanolamine, LPE, and LPC in the cells.

Lysophosphatidylethanolamine acyltransferase 1/membrane-bound O-acyltransferase 1 regulates morphology and function of P19C6 cell-derived neurons.

Glycerophospholipids, which are components of biomembranes, are formed de novo by the Kennedy pathway and subsequently mature through the Lands cycle. Lysophospholipid acyltransferases (LPLATs) are key enzymes in both pathways and influence the fatty acid composition of biomembranes. Neuronal differentiation is characterized by neurite outgrowth, which requires biomembrane biosynthesis. However, the role of LPLATs in neuronal differentiation remains unknown. In this study, we examined whether LPLATs are involved in neuronal differentiation using all-trans-retinoic acid (ATRA)-treated P19C6 cells. In these cells, mRNA levels of lysophosphatidylethanolamine acyltransferase (LPEAT)-1/membrane-bound O-acyltransferase (MBOAT)-1 were higher than those in undifferentiated cells. LPEAT enzymatic activity increased with 16:0- and 18:1-CoA as acyl donors. When LPEAT1/MBOAT1 was knocked down with small interfering RNA (siRNA), outgrowth of neurites and expression of neuronal markers decreased in ATRA-treated P19C6 cells. Voltage-dependent calcium channel activity was also suppressed in these cells transfected with LPEAT1/MBOAT1 siRNA. These results suggest that LPEAT1/MBOAT1 plays an important role in neurite outgrowth and function.-Tabe, S., Hikiji, H., Ariyoshi, W., Hashidate-Yoshida, T., Shindou, H., Okinaga, T., Shimizu, T., Tominaga, K., Nishihara, T. Lysophosphatidylethanolamine acyltransferase 1/membrane-bound O-acyltransferase 1 regulates morphology and function of P19C6 cell-derived neurons.

Possible Role of Different Yeast and Plant Lysophospholipid:Acyl-CoA Acyltransferases (LPLATs) in Acyl Remodelling of Phospholipids.

Recent results have suggested that plant lysophosphatidylcholine:acyl-coenzyme A acyltransferases (LPCATs) can operate in reverse in vivo and thereby catalyse an acyl exchange between the acyl-coenzyme A (CoA) pool and the phosphatidylcholine. We have investigated the abilities of Arabidopsis AtLPCAT2, Arabidopsis lysophosphatidylethanolamine acyltransferase (LPEAT2), S. cerevisiae lysophospholipid acyltransferase (Ale1) and S. cerevisiae lysophosphatidic acid acyltransferase (SLC1) to acylate lysoPtdCho, lysoPtdEtn and lysoPtdOH and act reversibly on the products of the acylation; the PtdCho, PtdEtn and PtdOH. The tested LPLATs were expressed in an S. cervisiaeale1 strain and enzyme activities were assessed in assays using microsomal preparations of the different transformants. The results show that, despite high activity towards lysoPtdCho, lysoPtdEtn and lysoPtdOH by the ALE1, its capacities to operate reversibly on the products of the acylation were very low. Slc1 readily acylated lysoPtdOH, lysoPtdCho and lysoPtdEtn but showed no reversibility towards PtdCho, very little reversibility towards PtdEtn and very high reversibility towards PtdOH. LPEAT2 showed the highest levels of reversibility towards PtdCho and PtdEtn of all LPLATs tested but low ability to operate reversibly on PtdOH. AtLPCAT2 showed good reversible activity towards PtdCho and PtdEtn and very low reversibility towards PtdOH. Thus, it appears that some of the LPLATs have developed properties that, to a much higher degree than other LPLATs, promote the reverse reaction during the same assay conditions and with the same phospholipid. The results also show that the capacity of reversibility can be specific for a particular phospholipid, albeit the lysophospholipid derivatives of other phospholipids serve as good acyl acceptors for the forward reaction of the enzyme.Electronic supplementary materialThe online version of this article (doi:10.1007/s11745-015-4102-0) contains supplementary material, which is available to authorized users.

Novel lysophospholipid acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b responsible for generation of palmitate-docosahexaenoate-phosphatidylcholine and phosphatidylethanolamine.

N-3 polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid (DHA, 22:6n-3), have been reported to play roles in preventing cardiovascular diseases. The major source of DHA is fish oils but a recent increase in the global demand of DHA and decrease in fish stocks require a substitute. Thraustochytrids, unicellular marine protists belonging to the Chromista kingdom, can synthesize large amounts of DHA, and, thus, are expected to be an alternative to fish oils. DHA is found in the acyl chain(s) of phospholipids as well as triacylglycerols in thraustochytrids; however, how thraustochytrids incorporate DHA into phospholipids remains unknown. We report here a novel lysophospholipid acyltransferase (PLAT1), which is responsible for the generation of DHA-containing phosphatidylcholine and phosphatidylethanolamine in thraustochytrids. The PLAT1 gene, which was isolated from the genomic DNA of Aurantiochytrium limacinum F26-b, was expressed in Saccharomyces cerevisiae, and the FLAG-tagged recombinant enzyme was characterized after purification with anti-FLAG affinity gel. PLAT1 shows wide specificity for donor substrates as well as acceptor substrates in vitro, i.e, the enzyme can adopt lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine and lysophosphatidylinositol as acceptor substrates, and 15:0/16:0-CoA and DHA-CoA as donor substrates. In contrast to the in vitro experiment, only lysophosphatidylcholine acyltransferase and lysophosphatidylethanolamine acyltransferase activities were decreased in plat1-knockout mutants, resulting in a decrease of 16:0-DHA-phosphatidylcholine (PC) [PC(38∶6)] and 16:0-DHA-phosphatidylethanolamine (PE) [PE(38∶6)], which are two major DHA-containing phospholipids in A. limacinum F26-b. However, the amounts of other phospholipid species including DHA-DHA-PC [PC(44∶12)] and DHA-DHA-PE [PE(44∶12)] were almost the same in plat-knockout mutants and the wild-type. These results indicate that PLAT1 is the enzyme responsible for the generation of 16:0-DHA-PC and 16:0-DHA-PE in the thraustochytrid.

Lysophosphatidylcholine Acyltransferase 3 Is the Key Enzyme for Incorporating Arachidonic Acid into Glycerophospholipids during Adipocyte Differentiation

Cellular membranes contain glycerophospholipids, which have important structural and functional roles in cells. Glycerophospholipids are first formed in the de novo pathway (Kennedy pathway) and are matured in the remodeling pathway (Lands’ cycle). Recently, lysophospholipid acyltransferases functioning in Lands’ cycle were identified and characterized. Several enzymes involved in glycerophospholipid biosynthesis have been reported to have important roles in adipocytes. However, the role of Lands’ cycle in adipogenesis has not yet been reported. Using C3H10T1/2, a cell line capable of differentiating to adipocyte-like cells in vitro, changes of lysophospholipid acyltransferase activities were investigated. Lysophosphatidylcholine acyltransferase (LPCAT), lysophosphatidylethanolamine acyltransferase (LPEAT) and lysophosphatidylserine acyltransferase (LPSAT) activities were enhanced, especially with 18:2-CoA and 20:4-CoA as donors. Correspondingly, mRNA expression of LPCAT3, which possesses LPCAT, LPEAT and LPSAT activities with high specificity for 18:2- and 20:4-CoA, was upregulated during adipogenesis. Analysis of acyl-chain compositions of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) showed a change in their profiles between preadipocytes and adipocytes, including an increase in the percentage of arachidonic acid-containing phospholipids. These changes are consistent with the activities of LPCAT3. Therefore, it is possible that enhanced phospholipid remodeling by LPCAT3 may be associated with adipocyte differentiation.

Biosynthesis of phosphatidylcholine by human lysophosphatidylcholine acyltransferase 1

Pulmonary surfactant is a complex of phospholipids and proteins lining the alveolar walls of the lung. It reduces surface tension in the alveoli, and is critical for normal respiration. Pulmonary surfactant phospholipids consist mainly of phosphatidylcholine (PC) and phosphatidylglycerol (PG). Although the phospholipid composition of pulmonary surfactant is well known, the enzyme(s) involved in its biosynthesis have remained obscure. We previously reported the cloning of murine lysophosphatidylcholine acyltransferase 1 (mLPCAT1) as a potential biosynthetic enzyme of pulmonary surfactant phospholipids. mLPCAT1 exhibits lysophosphatidylcholine acyltransferase (LPCAT) and lysophosphatidylglycerol acyltransferase (LPGAT) activities, generating PC and PG, respectively. However, the enzymatic activity of human LPCAT1 (hLPCAT1) remains controversial. We report here that hLPCAT1 possesses LPCAT and LPGAT activities. The activity of hLPCAT1 was inhibited by N-ethylmaleimide, indicating the importance of some cysteine residue(s) for the catalysis. We found a conserved cysteine (Cys(211)) in hLPCAT1 that is crucial for its activity. Evolutionary analyses of the close homologs of LPCAT1 suggest that it appeared before the evolution of teleosts and indicate that LPCAT1 may have evolved along with the lung to facilitate respiration. hLPCAT1 mRNA is highly expressed in the human lung. We propose that hLPCAT1 is the biosynthetic enzyme of pulmonary surfactant phospholipids.

Regulation of phospholipid synthesis in yeast

Phospholipid synthesis in the yeast Saccharomyces cerevisiae is a complex process that involves regulation by both genetic and biochemical mechanisms. The activity levels of phospholipid synthesis enzymes are controlled by gene expression (e.g., transcription) and by factors (lipids, water-soluble phospholipid precursors and products, and covalent modification of phosphorylation) that modulate catalysis. Phosphatidic acid, whose levels are controlled by the biochemical regulation of key phospholipid synthesis enzymes, plays a central role in the regulation of phospholipid synthesis gene expression.

Molecular Identification of a Novel Mammalian Brain Isoform of Acyl-CoA:Lysophospholipid Acyltransferase with Prominent Ethanolamine Lysophospholipid Acylating Activity, LPEAT2

Acyl-CoA-dependent lysophospholipid acyltransferases play an important role in attaining the appropriate molecular species of phospholipids. A number of genes encoding these activities were recently identified. It has become clear that multiple genes can encode one enzymatic activity and that a given gene may encode multiple activities. Here we report the identification of a gene encoding a mammalian acyl-CoA-dependent lysophospholipid acyltransferase with prominent activity toward ethanolamine-containing lysophospholipids, which we termed acyl-CoA:lysophosphatidylethanolamine acyltransferase 2, LPEAT2 (previously annotated as AYTL3 or AGPAT7). LPEAT2 is predominantly expressed in brain, coinciding with an enrichment of phosphatidylethanolamine in this tissue. Ectopic expression of LPEAT2 in mammalian HEK293T cells led to a dramatic increase (up to 9-fold) in LPEAT activity when compared with cells transfected with empty vector or an unrelated acyltransferase. LPEAT2 also exhibited significant acyl-CoA-dependent acyltransferase activity toward 1-O-alkenyl-lysophosphatidylethanolamine, lysophosphatidylglycerol, 1-O-alkyl-lysophosphatidylcholine, lysophosphatidylserine, and lysophosphatidylcholine but lacked appreciable acylating activity toward glycerol 3-phosphate, lysophosphatidic acid, lysophosphatidylinositol, and diacylglycerol, demonstrating multiple but selective functions of LPEAT2 as an enzyme involved in phospholipid remodeling. LPEAT2 recognizes a broad range of medium and long chain fatty acyl-CoA, and its activity was not affected by Ca(2+). When overexpressed in mammalian cells, LPEAT2 is localized to the endoplasmic reticulum. siRNA-mediated knockdown of LPEAT2 in HEK293T cells significantly decreased LPEAT and 1-alkenyl-LPEAT activities but did not affect other lysophospholipid acylating activities. These findings identify LPEAT2 as an important enzyme in the biosynthesis of ethanolamine-containing phospholipids, especially in brain.

Identification and Characterization of the Major Lysophosphatidylethanolamine Acyltransferase in Saccharomyces cerevisiae

We recently demonstrated that yeast actively import lysophosphatidylethanolamine (lyso-PtdEtn) through the action of plasma membrane P-type ATPases and rapidly acylate it to form PtdEtn. The predominant lyso-PtdEtn acyltransferase (LPEAT) activity present in cellular extracts is acyl-CoA dependent, but the identity of the gene encoding this activity was unknown. We now demonstrate that a previously uncharacterized open reading frame, YOR175C, encodes the major acyl-CoA-dependent LPEAT activity in yeast and henceforth refer to it as ALE1 (acyltransferase for lyso-PtdEtn). Ale1p is an integral membrane protein and is highly enriched in the mitochondria-associated endoplasmic reticulum membrane. It is a member of the membrane-bound O-acyltransferase family and possesses a dibasic motif at its C terminus that is likely responsible for Golgi retrieval and retention in the endoplasmic reticulum. An ale1Delta strain retains only trace amounts of acyl-CoA-dependent LPEAT activity, and strains lacking the capacity for PtdEtn synthesis via the phosphatidylserine decarboxylase and Kennedy pathways show a stringent requirement for both exogenous lyso-PtdEtn and a functional ALE1 gene for viability. Ale1p catalytic activity has a pH optimum between pH 7 and 7.5 and a strong preference for unsaturated acyl-CoA substrates.

Lysophosphatidylethanolamine acyltransferase activity is elevated during cardiac cell differentiation

We examined if elevation in lysophosphatidylethanolamine acyltransferase activity was associated with elevation in phosphatidylethanolamine content during differentiation of P19 teratocarcinoma cells into cardiac myocytes. P19 cells were induced to undergo differentiation into cardiac myocytes by the addition of 1% dimethylsulfoxide to the medium. Immunofluorescence microscopy revealed the presence of striated myosin at 8 days post-dimethylsulfoxide addition confirming differentiation into cardiac cells. The content of phosphatidylethanolamine was increased 2.1-fold ( P<0.05) in differentiated cells compared to undifferentiated cells, whereas the content of phosphatidylcholine was reduced 29% ( P<0.05). There were no alterations in the pool sizes of other phospholipids, including cardiolipin. The relative abundance of fatty acids in phospholipids of P19 cells was 18:1>18:0>16:1=18:2>16:0=14:0>20:4 and differentiation did not affect the relative amounts of these fatty acids within individual phospholipids. When cells were incubated with [1,3- 3H]glycerol, radioactivity incorporated into phosphatidylethanolamine was elevated 5.8-fold, whereas radioactivity incorporated into phosphatidylcholine was unaltered. Ethanolaminephosphotransferase, cholinephosphotransferase and membrane CTP:phosphocholine cytidylyltransferase activities were elevated in differentiated cells compared to undifferentiated cells, whereas membrane and cytosolic phospholipase A 2 activities were unaltered. Lysophosphatidylethanolamine acyltransferase activities were elevated 2.4-fold ( P<0.05). Lysophosphatidylcholine acyltransferase, monolysocardiolipin acyltransferase, acyl-Coenzyme A synthetase and acyl-Coenzyme A hydrolase activities were unaltered in differentiated cells compared to undifferentiated cells. We postulate that during cardiac cell differentiation, the observed elevation in lysophosphatidylethanolamine acyltransferase activity accompanies the elevation in phosphatidylethanolamine mass, possibly to maintain the fatty acyl composition of this phospholipid within the membrane.

Coenzyme A-dependent and -independent acyl transfer between dog heart microsomal phospholipids

We have recently shown that dog heart microsomes catalyze the transfer of acyl groups from the sn-2 position of exogenous phosphatidylcholine to lysophosphatidylethanolamine with strong preference for arachidonate over linoleate (Biochem. Biophys. Res. Commun. 129, 381–388 (1985)). We now report that the addition of 0.5 mM CoA enhances the acyl transfer activity 3–4-fold but reduces the selectivity for arachidonate. Acyl transfer in the absence of CoA exhibits a pH optimum of 7.5–8.5, whereas two pH optima (7.5 and 4.5) are observed in the presence of CoA with transfer activity at pH 4.5 exceeding that of pH 7.5 by 4–5-fold. The plasmalogen (alkenyl) analog of lysophosphatidylethanolamine is an equally effective acyl acceptor in the absence of CoA but less effective in its presence. The microsomal acyl-CoA/ lysophosphatidylethanolamine acyltransferase does not favor arachidonate over linoleate. Therefore, transacylation from phosphatidylcholine may account for the high arachidonate content of dog heart microsomal phosphatidylethanolamine and its plasmalogen analog. In fact, acyl transfer from endogenous lipids to 1-[1′- 14C] palmitoyl-2- lyso-sn- glycerophosphoethanolamine results in the generation of mostly (over 80%) tetraunsaturated phosphatidylethanolamine. This proportion is reduced by the addition of CoA and, even more, by CoA plus acyl-CoA-generating cofactors. We conclude that in dog heart microsomes, lysophosphatidylethanolamine can be acylated by different mechanisms, of which the CoA-independent transacylase exhibits the greatest acyl selectivity.

Acyltransferase activities in adult rat type II pneumocyte-derived subcellular fractions

1. 1. Acyl-CoA: lysophosphatidylcholine, acyl-CoA: lysophosphatidylethanolamine, and lysophosphatidylcholine: lysophosphatidylcholine acyltransferases were investigated using subcellular fractions derived from adult rat type II pneumocytes in primary culture. Acyl-CoA: lysophospholipid acyltransferase activities were determined to be microsomal, while lysophosphatidylcholine: lysophosphatidylcholine acyltransferase activity was found to be cytosolic. Total palmitoyl CoA: lysophosphatidylcholine acyltransferase activity was 30-fold greater than lysophosphatidylcholine: lysophosphatidylcholine acyltransferase activity, indicating that the former enzyme is more important in the synthesis of dipalmitoyl phosphatidylcholine. 2. 2. Palmitoyl-CoA and oleoyl-CoA lysophosphatidylcholine acyltransferase activities were approximately equal under optimal substrate conditions. Specific activities of the enzyme using arachidoyl-CoA and arachidonoyl-CoA were 46% and 18%, respectively, of those with palmitoyl-CoA. Acyl-CoA: lysophosphatidylethanolamine acyltransferase showed a preference for palmitoyl-CoA as opposed to oleoyl-CoA under optimal conditions. However, when equimolar concentrations of either palmitoyl-CoA and oleoyl-CoA or palmitoyl-CoA and arachidoyl-CoA were assayed together, the relative utilization of the two substrates was found to be dependent on total acyl-CoA concentration. At higher concentrations, the incorporation of palmitoyl-CoA into phosphatidylcholine was less than other acyl-CoAs. However, at lower concentrations palmitoyl-CoA was utilized quite selectively. Whole lung microsomes did not show as marked a preference for palmitoyl-CoA as did type II pneumocyte microsomes under these same conditions. In similar experiments, low total acyl-CoA concentrations produced greater incorporation of oleoyl-CoA into phosphatidylethanolamine. For both enzymes total activity at the lowest concentrations used was at least 45% that at optimal conditions. This demonstrates that the type II pneumocyte acyltransferase system(s) can selectively utilize palmitoyl-CoA. 3. 3. No evidence for direct exchange of palmitoyl-CoA with 1-saturated-2-unsaturated phosphatidylcholine in subcellular fractions from type II pneumocytes was found.