PLAAT1 expression triggers fragmentation of mitochondria in an enzyme activity-dependent manner.

The phospholipase A and acyltransferase (PLAAT) family is a protein family consisting of five members (PLAAT1-5), which acts as phospholipid-metabolizing enzymes with phospholipase A1/A2 and N-acyltransferase activities. Since we previously reported that the overexpression of PLAAT3 in mammalian cells causes the specific disappearance of peroxisomes, in the present study we examined a possible effect of PLAAT1 on organelles. We prepared HEK293 cells expressing mouse PLAAT1 in a doxycycline-dependent manner and found that the overexpression of PLAAT1 resulted in the transformation of mitochondria from the original long rod shape to a round shape, as well as their fragmentation. In contrast, the overexpression of a catalytically inactive point mutant of PLAAT1 did not generate any morphological change in mitochondria, suggesting the involvement of catalytic activity. PLAAT1 expression also caused the reduction of peroxisomes, while the levels of the marker proteins for ER, Golgi apparatus and lysosomes were almost unchanged. In PLAAT1-expressing cells, the level of dynamin-related protein 1 responsible for mitochondrial fission was increased, whereas those of optic atrophy 1 and mitofusin 2, both of which are responsible for mitochondrial fusion, were reduced. These results suggest a novel role of PLAAT1 in the regulation of mitochondrial biogenesis.

PLAAT1 deficiency alleviates high-fat diet-induced hepatic lipid accumulation in mice.

The phospholipase A and acyltransferase (PLAAT) family is composed of three isoforms in mice (PLAAT1, 3, and 5), all of which function as phospholipid-metabolizing enzymes exhibiting phospholipase A1 /A2 and acyltransferase activities. Plaat3-deficient (Plaat3-/- ) mice were previously reported to show lean phenotype and remarkable hepatic fat accumulation under high-fat diet (HFD) feeding, while Plaat1-/- mice have not been analyzed. In the present study, we generated Plaat1-/- mice and investigated the effects of PLAAT1 deficiency on HFD-induced obesity, hepatic lipid accumulation, and insulin resistance. After HFD treatment, PLAAT1 deficiency caused a lower body weight gain compared to wild-type mice. Plaat1-/- mice also showed reduced liver weight with negligible hepatic lipid accumulation. In accordance with these findings, PLAAT1 deficiency improved HFD-induced hepatic dysfunction and lipid metabolism disorders. Lipidomics analysis in the liver revealed that in Plaat1-/- mice, the levels of various glycerophospholipids tended to increase, while all classes of lysophospholipids examined tended to decrease, suggesting that PLAAT1 functions as phospholipase A1 /A2 in the liver. Interestingly, the HFD treatment of wild-type mice significantly increased the mRNA level of PLAAT1 in the liver. Furthermore, the deficiency did not appear to elevate the risk of insulin resistance in contrast to PLAAT3 deficiency. These results suggested that the suppression of PLAAT1 improves HFD-induced overweight and concomitant hepatic lipid accumulation.

Vasoactive intestinal peptide blockade suppresses tumor growth by regulating macrophage polarization and function in CT26 tumor-bearing mice.

Macrophages are a major population of immune cells in solid cancers, especially colorectal cancers. Tumor-associated macrophages (TAMs) are commonly divided into M1-like (tumor suppression) and M2-like (tumor promotion) phenotypes. Vasoactive intestinal peptide (VIP) is an immunoregulatory neuropeptide with a potent anti-inflammatory function. Inhibition of VIP signaling has been shown to increase CD8+ T cell proliferation and function in viral infection and lymphoma. However, the role of VIP in macrophage polarization and function in solid tumors remains unknown. Here, we demonstrated that conditioned medium from CT26 (CT26-CM) cells enhanced M2-related marker and VIP receptor (VPAC) gene expression in RAW264.7 macrophages. VIP hybrid, a VIP antagonist, enhanced M1-related genes but reduced Mrc1 gene expression and increased phagocytic ability in CT26-CM-treated RAW264.7 cells. In immunodeficient SCID mice, VIP antagonist alone or in combination with anti-PD-1 antibody attenuated CT26 tumor growth compared with the control. Analysis of tumor-infiltrating leukocytes found that VIP antagonist increased M1/M2 ratios and macrophage phagocytosis of CT26-GFP cells. Furthermore, Vipr2 gene silencing or VPAC2 activation affected the polarization of CT26-CM-treated RAW264.7 cells. In conclusion, the inhibition of VIP signaling enhanced M1 macrophage polarization and macrophage phagocytic function, resulting in tumor regression in a CT26 colon cancer model.

ß-1,4-Galactan suppresses lipid synthesis in sebaceous gland cells via TLR4.

Sebum is a lipid mixture secreted from sebaceous glands of the skin. The excessive secretion of sebum causes acne vulgaris and seborrheic dermatitis, while its deficiency causes xerosis. Therefore, the appropriate control of sebum secretion is crucially important to keep the skin healthy. In the present study, we evaluated the effects of naturally occurring polysaccharides on lipid biosynthesis in hamster sebaceous gland cells. Among the tested polysaccharides, ß-1,4-galactan, the main chain of type I arabinogalactan, most potently suppressed lipid synthesis in the sebaceous gland cells as analysed by oil red O staining. Toll-like receptor (TLR)4 inhibitors counteracted this suppressive effect and lipopolysaccharide, a TLR4 ligand, mimicked this effect, suggesting the involvement of the TLR4 signalling pathway. In the cells ß-1,4-galactan significantly decreased mRNA levels of lipogenesis-related transcription factors (peroxisomeGraphical Abstract$\includegraphics{\bwartpath }$ proliferator-activated receptor γ and sterol regulatory element-binding protein 1) and enzymes (acetyl-CoA carboxylase and fatty acid synthase) as well as the glucose transporter GLUT4. Furthermore, ß-1,4-galactan increased the production of lactic acid serving as a natural moisturizing factor and enhanced the proliferation of sebaceous gland cells. These results suggest potential of ß-1,4-galactan as a material with therapeutic and cosmetic values for the skin.

Assay of NAT Activity.

In animal tissues, N-acyltransferase (NAT) catalyzes the first reaction in the biosynthetic pathway of bioactive N-acylethanolamines, in which an acyl chain is transferred from the sn-1 position of the donor phospholipid, such as phosphatidylcholine, to the amino group of phosphatidylethanolamine, resulting in the formation of N-acylphosphatidylethanolamine. NAT has long been known to be stimulated by Ca2+ and hence referred to as Ca2+-dependent NAT. Later, this enzyme was identified as cPLA2ε (also referred to as PLA2G4E). On the other hand, members of the phospholipase A/acyltransferase (PLAAT) family (also known as HRAS-like suppressor family) show Ca2+-independent NAT activity. In this chapter, we describe (1) partial purification of Ca2+-dependent NAT from rat brain, (2) purification of recombinant cPLA2ε and PLAAT-2, and (3) NAT assay using radiolabeled substrate.

Formation of N-acyl-phosphatidylethanolamines by cytosolic phospholipase A2ε in an ex vivo murine model of brain ischemia.

N-Acyl-phosphatidylethanolamines (NAPEs), a minor class of membrane glycerophospholipids, accumulate along with their bioactive metabolites, N-acylethanolamines (NAEs) during ischemia. NAPEs can be formed through N-acylation of phosphatidylethanolamine by cytosolic phospholipase A2ε (cPLA2ε, also known as PLA2G4E) or members of the phospholipase A and acyltransferase (PLAAT) family. However, the enzyme responsible for the NAPE production in brain ischemia has not yet been clarified. Here, we investigated a possible role of cPLA2ε using cPLA2ε-deficient (Pla2g4e-/-) mice. As analyzed with brain homogenates of wild-type mice, the age dependency of Ca2+-dependent NAPE-forming activity showed a bell-shape pattern being the highest at the first week of postnatal life, and the activity was completely abolished in Pla2g4e-/- mice. However, liquid chromatography-tandem mass spectrometry revealed that the NAPE levels of normal brain were similar between wild-type and Pla2g4e-/- mice. In contrast, post-mortal accumulations of NAPEs and most species of NAEs were only observed in decapitated brains of wild-type mice. These results suggested that cPLA2ε is responsible for Ca2+-dependent formation of NAPEs in the brain as well as the accumulation of NAPEs and NAEs during ischemia, while other enzyme(s) appeared to be involved in the maintenance of basal NAPE levels.

Defunctionalizing intracellular organelles such as mitochondria and peroxisomes with engineered phospholipase A/acyltransferases.

Organelles vitally achieve multifaceted functions to maintain cellular homeostasis. Genetic and pharmacological approaches to manipulate individual organelles are powerful in probing their physiological roles. However, many of them are either slow in action, limited to certain organelles, or rely on toxic agents. Here, we design a generalizable molecular tool utilizing phospholipase A/acyltransferases (PLAATs) for rapid defunctionalization of organelles via remodeling of the membrane phospholipids. In particular, we identify catalytically active PLAAT truncates with minimal unfavorable characteristics. Chemically-induced translocation of the optimized PLAAT to the mitochondria surface results in their rapid deformation in a phospholipase activity dependent manner, followed by loss of luminal proteins as well as dissipated membrane potential, thus invalidating the functionality. To demonstrate wide applicability, we then adapt the molecular tool in peroxisomes, and observe leakage of matrix-resident functional proteins. The technique is compatible with optogenetic control, viral delivery and operation in primary neuronal cultures. Due to such versatility, the PLAAT strategy should prove useful in studying organelle biology of diverse contexts.

Involvement of the γ Isoform of cPLA2 in the Biosynthesis of Bioactive N-Acylethanolamines.

Arachidonylethanolamide (anandamide) acts as an endogenous ligand of cannabinoid receptors, while other N-acylethanolamines (NAEs), such as palmitylethanolamide and oleylethanolamide, show analgesic, anti-inflammatory, and appetite-suppressing effects through other receptors. In mammalian tissues, NAEs, including anandamide, are produced from glycerophospholipid via N-acyl-phosphatidylethanolamine (NAPE). The ɛ isoform of cytosolic phospholipase A2 (cPLA2) functions as an N-acyltransferase to form NAPE. Since the cPLA2 family consists of six isoforms (α, ß, γ, δ, ɛ, and ζ), the present study investigated a possible involvement of isoforms other than ɛ in the NAE biosynthesis. Firstly, when the cells overexpressing one of the cPLA2 isoforms were labeled with [14C]ethanolamine, the increase in the production of [14C]NAPE was observed only with the ɛ-expressing cells. Secondly, when the cells co-expressing ɛ and one of the other isoforms were analyzed, the increase in [14C]N-acyl-lysophosphatidylethanolamine (lysoNAPE) and [14C]NAE was seen with the combination of ɛ and γ isoforms. Furthermore, the purified cPLA2γ hydrolyzed not only NAPE to lysoNAPE, but also lysoNAPE to glycerophospho-N-acylethanolamine (GP-NAE). Thus, the produced GP-NAE was further hydrolyzed to NAE by glycerophosphodiesterase 1. These results suggested that cPLA2γ is involved in the biosynthesis of NAE by its phospholipase A1/A2 and lysophospholipase activities.

Roles of Endocannabinoids and Endocannabinoid-Like Molecules in Energy Homeostasis and Metabolic Regulation: A Nutritional Perspective.

The endocannabinoid system is involved in signal transduction in mammals. It comprises principally G protein-coupled cannabinoid receptors and their endogenous agonists, called endocannabinoids, as well as the enzymes and transporters responsible for the metabolism of endocannabinoids. Two arachidonic acid-containing lipid molecules, arachidonoylethanolamide (anandamide) and 2-arachidonoylglycerol, function as endocannabinoids. N-acylethanolamines and monoacylglycerols, in which the arachidonic acid chain is replaced with a saturated or monounsaturated fatty acid, are not directly involved in the endocannabinoid system but exhibit agonistic activities for other receptors. These endocannabinoid-like moleculesinclude palmitoylethanolamide, oleoylethanolamide (OEA), and 2-oleoylglycerol. Endocannabinoids stimulate feeding behavior and the anabolism of lipids and glucose, while OEA suppresses appetite. Both central and peripheral systems are included in these nutritional and metabolic contexts. Therefore, they have potential in the treatment and prevention of obesity. We outline the structure, metabolism, and biological activities of endocannabinoids and related molecules, and focus on their involvement in energy homeostasis and metabolic regulation.

Involvement of acid ceramidase in the degradation of bioactive N-acylethanolamines.

Bioactive N-acylethanolamines (NAEs) include palmitoylethanolamide, oleoylethanolamide, and anandamide, which exert anti-inflammatory, anorexic, and cannabimimetic actions, respectively. The degradation of NAEs has been attributed to two hydrolases, fatty acid amide hydrolase and NAE acid amidase (NAAA). Acid ceramidase (AC) is a lysosomal enzyme that hydrolyzes ceramide (N-acylsphingosine), which resembles NAAA in structure and function. In the present study, we examined the role of AC in the degradation of NAEs. First, we demonstrated that purified recombinant human AC can hydrolyze various NAEs with lauroylethanolamide (C12:0-NAE) as the most reactive NAE substrate. We then used HEK293 cells metabolically labeled with [14C]ethanolamine, and revealed that overexpressed AC lowered the levels of 14C-labeled NAE. As analyzed with liquid chromatography-tandem mass spectrometry, AC overexpression decreased the amounts of different NAE species. Furthermore, suppression of endogenous AC in LNCaP prostate cells by siRNA increased the levels of various NAEs. Lastly, tissue homogenates from mice genetically lacking saposin D, a presumable activator protein of AC, showed much lower hydrolyzing activity for NAE as well as ceramide than the homogenates from wild-type mice. These results demonstrate the ability of AC to hydrolyze NAEs and suggest its physiological role as a third NAE hydrolase.

Intracellular Ca2+-dependent formation of N-acyl-phosphatidylethanolamines by human cytosolic phospholipase A2ε.

N-Acyl-phosphatidylethanolamines (NAPEs) are known to be precursors of bioactive N-acylethanolamines (NAEs), including the endocannabinoid arachidonoylethanolamide (anandamide) and anti-inflammatory palmitoylethanolamide. In mammals, NAPEs are produced by N-acyltransferases, which transfer an acyl chain from the sn-1 position of glycerophospholipid to the amino group of phosphatidylethanolamine (PE). Recently, the ɛ isoform of cytosolic phospholipase A2 (cPLA2ɛ) was found to be Ca2+-dependent N-acyltransferase. However, it was poorly understood which types of phospholipids serve as substrates in living cells. In the present study, we established a human embryonic kidney 293 cell line, in which doxycycline potently induces human cPLA2ɛ, and used these cells to analyze endogenous substrates and products of cPLA2ɛ with liquid chromatography-tandem mass spectrometry. When treated with doxycycline and Ca2+ ionophore, the cells produced various species of diacyl- and alkenylacyl-types of NAPEs as well as NAEs in large quantities. Moreover, the levels of diacyl- and alkenylacyl-types of PEs and diacyl-phosphatidylcholines (PCs) decreased, while those of lysophosphatidylethanolamines and lysophosphatidylcholines increased. These results suggested that cPLA2ɛ Ca2+-dependently produces NAPEs by utilizing endogenous diacyl- and alkenylacyl-types of PEs as acyl acceptors and diacyl-type PCs and diacyl-type PEs as acyl donors.

The role of intracellular anionic phospholipids in the production of N-acyl-phosphatidylethanolamines by cytosolic phospholipase A2ɛ.

N-Acyl-phosphatidylethanolamines (NAPEs) represent a class of glycerophospholipids and serve as the precursors of bioactive N-acylethanolamines, including arachidonoylethanolamide (anandamide), palmitoylethanolamide and oleoylethanolamide. NAPEs are produced in mammals by N-acyltransferases, the enzymes which transfer an acyl chain of glycerophospholipids to the amino group of phosphatidylethanolamine. Recently, the ɛ isoform of cytosolic phospholipase A2 (cPLA2ɛ, also called PLA2G4E) was identified as Ca2+-dependent N-acyltransferase. We showed that the activity is remarkably stimulated by phosphatidylserine (PS) in vitro. In the present study, we investigated whether or not endogenous PS regulates the function of cPLA2ɛ in living cells. When PS synthesis was suppressed by the knockdown of PS synthases in cPLA2ɛ-expressing cells, the cPLA2ɛ level and its N-acyltransferase activity were significantly reduced. Mutagenesis studies revealed that all of C2, lipase and polybasic domains of cPLA2ɛ were required for its proper localization as well as the enzyme activity. Liposome-based assays showed that several anionic glycerophospholipids, including PS, phosphatidic acid and phosphatidylinositol 4,5-bisphosphate, enhance the Ca2+-dependent binding of purified cPLA2ɛ to liposome membrane and stimulate its N-acyltransferase activity. Altogether, these results suggested that endogenous PS and other anionic phospholipids affect the localization and enzyme activity of cPLA2ɛ.

Endocannabinoids and related N-acylethanolamines: biological activities and metabolism.

The plant Cannabis sativa contains cannabinoids represented by Δ9-tetrahydrocannabinol, which exert psychoactivity and immunomodulation through cannabinoid CB1 and CB2 receptors, respectively, in animal tissues. Arachidonoylethanolamide (also referred to as anandamide) and 2-arachidonoylglycerol (2-AG) are well known as two major endogenous agonists of these receptors (termed "endocannabinoids") and show various cannabimimetic bioactivities. However, only 2-AG is a full agonist for CB1 and CB2 and mediates retrograde signals at the synapse, strongly suggesting that 2-AG is physiologically more important than anandamide. The metabolic pathways of these two endocannabinoids are completely different. 2-AG is mostly produced from inositol phospholipids via diacylglycerol by phospholipase C and diacylglycerol lipase and then degraded by monoacylglycerol lipase. On the other hand, anandamide is concomitantly produced with larger amounts of other N-acylethanolamines via N-acyl-phosphatidylethanolamines (NAPEs). Although this pathway consists of calcium-dependent N-acyltransferase and NAPE-hydrolyzing phospholipase D, recent studies revealed the involvement of several new enzymes. Quantitatively major N-acylethanolamines include palmitoylethanolamide and oleoylethanolamide, which do not bind to cannabinoid receptors but exert anti-inflammatory, analgesic, and anorexic effects through receptors such as peroxisome proliferator-activated receptor α. The biosynthesis of these non-endocannabinoid N-acylethanolamines rather than anandamide may be the primary significance of this pathway. Here, we provide an overview of the biological activities and metabolisms of endocannabinoids (2-AG and anandamide) and non-endocannabinoid N-acylethanolamines.

Phosphatidylserine-stimulated production of N-acyl-phosphatidylethanolamines by Ca2+-dependent N-acyltransferase.

N-acyl-phosphatidylethanolamine (NAPE) is known to be a precursor for various bioactive N-acylethanolamines including the endocannabinoid anandamide. NAPE is produced in mammals through the transfer of an acyl chain from certain glycerophospholipids to phosphatidylethanolamine (PE) by Ca2+-dependent or -independent N-acyltransferases. The ε isoform of mouse cytosolic phospholipase A2 (cPLA2ε) was recently identified as a Ca2+-dependent N-acyltransferase (Ca-NAT). In the present study, we first showed that two isoforms of human cPLA2ε function as Ca-NAT. We next purified both mouse recombinant cPLA2ε and its two human orthologues to examine their catalytic properties. The enzyme absolutely required Ca2+ for its activity and the activity was enhanced by phosphatidylserine (PS). PS enhanced the activity 25-fold in the presence of 1 mM CaCl2 and lowered the EC50 value of Ca2+ >8-fold. Using a PS probe, we showed that cPLA2ε largely co-localizes with PS in plasma membrane and organelles involved in the endocytic pathway, further supporting the interaction of cPLA2ε with PS in living cells. Finally, we found that the Ca2+-ionophore ionomycin increased [14C]NAPE levels >10-fold in [14C]ethanolamine-labeled cPLA2ε-expressing cells while phospholipase A/acyltransferase-1, acting as a Ca2+-independent N-acyltransferase, was insensitive to ionomycin for full activity. In conclusion, PS potently stimulated the Ca2+-dependent activity and human cPLA2ε isoforms also functioned as Ca-NAT.

Peripheral tissue levels and molecular species compositions of N-acyl-phosphatidylethanolamine and its metabolites in mice lacking N-acyl-phosphatidylethanolamine-specific phospholipase D.

N-acylethanolamines (NAEs), a class of lipid mediators, are produced from N-acyl-phosphatidylethanolamine (NAPE) by several pathways, including the direct release by NAPE-specific phospholipase D (NAPE-PLD) or the multistep pathway via sn-glycero-3-phospho-N-acylethanolamine (Gp-NAE). Using liquid chromatography-tandem mass spectrometry, we compared peripheral tissue levels of NAPE, Gp-NAE and NAE in NAPE-PLD-deficient (NAPE-PLD-/-) and wild type (WT) mice. NAPE-PLD was suggested to play a major role in the NAPE degradation in heart, kidney, and liver, but not in jejunum, because the NAPE levels except jejunum were significantly higher in NAPE-PLD-/- mice than in WT mice. The deletion of NAPE-PLD failed to alter the NAE levels of these tissues, suggesting its limited role in the NAE production. The enzyme assays with tissue homogenates confirmed the presence of NAPE-PLD-independent pathways in these peripheral tissues. Gp-NAE species having an acyl moiety with 22 carbons and 6 double bonds was enriched in these peripheral tissues. As for sn-2 acyl species of NAPE, 18:2-acyl-containing NAPE species were predominant over 18:1-containing species in heart, liver, and jejunum. Our results show that both molecular species composition of NAPE, NAE and Gp-NAE and their dependencies on Napepld are different among the peripheral tissues, suggesting that each tissue has distinct metabolic pathways and these NAE-containing lipids play tissue-specific roles.

An involvement of phospholipase A/acyltransferase family proteins in peroxisome regulation and plasmalogen metabolism.

The H-Ras-like suppressor (HRASLS) is a protein family consisting of five members in humans. Despite their discovery as tumor suppressors, we demonstrated that all these proteins are phospholipid-metabolizing enzymes, such as phospholipase (PL) A1 /A2 and acyltransferase. We thus proposed to rename HRASLS1-5 as PLA/acyltransferase (PLAAT)-1-5. Notably, PLAATs exhibit N-acyltransferase activity to biosynthesize N-acylated ethanolamine phospholipids, including N-acyl-plasmalogen, which serve as precursors of bioactive N-acylethanolamines. Furthermore, the overexpression of PLAAT-3 in animal cells causes disappearance of peroxisomes and a remarkable reduction in plasmalogen levels. This finding might be related to the inhibitory effect of PLAAT-3 on the chaperone activity of the peroxin PEX19. In this article, we will review our recent findings about PLAAT proteins, with special reference to their roles in peroxisome biogenesis and plasmalogen metabolism.

Mammalian enzymes responsible for the biosynthesis of N-acylethanolamines.

Bioactive N-acylethanolamines (NAEs) are ethanolamides of long-chain fatty acids, including palmitoylethanolamide, oleoylethanolamide and anandamide. In animal tissues, NAEs are biosynthesized from membrane phospholipids. The classical "transacylation-phosphodiesterase" pathway proceeds via N-acyl-phosphatidylethanolamine (NAPE), which involves the actions of two enzymes, NAPE-generating Ca2+-dependent N-acyltransferase (Ca-NAT) and NAPE-hydrolyzing phospholipase D (NAPE-PLD). Recent identification of Ca-NAT as Ɛ isoform of cytosolic phospholipase A2 enabled the further molecular biological approaches toward this enzyme. In addition, Ca2+-independent NAPE formation was shown to occur by N-acyltransferase activity of a group of proteins named phospholipase A/acyltransferases (PLAAT)-1-5. The analysis of NAPE-PLD-deficient mice confirmed that NAEs can be produced through multi-step pathways bypassing NAPE-PLD. The NAPE-PLD-independent pathways involved three members of the glycerophosphodiesterase (GDE) family (GDE1, GDE4 and GDE7) as well as α/ß-hydrolase domain-containing protein (ABHD)4. In this review article, we will focus on recent progress made and latest insights in the enzymes involved in NAE synthesis and their further characterization.

A quantitative study on splice variants of N-acylethanolamine acid amidase in human prostate cancer cells and other cells.

N-Acylethanolamine acid amidase (NAAA) is a lysosomal enzyme, hydrolyzing various bioactive N-acylethanolamines with a preference for palmitoylethanolamide. Human NAAA mRNA was previously reported to consist of multiple 3'-end splice variants. However, their tissue distributions and roles have not been examined yet. In the present study, we first identified four major splice variants (tentatively referred to as a1, a2, b2, and c2) in a human prostate cancer cell line LNCaP, which were composed of exons 1-11, exons 1-10 and 12, exons 1-9 and 12, and exons 1-8 and 12, respectively. We next developed quantitative polymerase chain reaction methods to individually quantify these NAAA variants as well as collectively measure all the variants. Among various human prostate cancer cells, the total levels of NAAA mRNAs in androgen-sensitive cells like LNCaP were higher than those in androgen-insensitive cells. In all of these prostate cells and other human cells, variants a1 and b2 showed the highest and lowest expression levels, respectively, among the four variants. Interestingly, ratios of the four variants were different by cell type. Variants a1 and a2 encoded the same full-length NAAA protein, which was catalytically active, while b2 and c2 were translated to C-terminally truncated proteins. As expressed in HEK293 cells these truncated forms were detected as catalytically inactive precursor proteins, but not as mature forms. These results revealed wide distribution of multiple variants of NAAA mRNA in various human cells and suggested that the proteins from some variants are catalytically inactive.

Comparative analyses of isoforms of the calcium-independent phosphatidylethanolamine N-acyltransferase PLAAT-1 in humans and mice.

N-Acylphosphatidylethanolamines (NAPEs) are a class of glycerophospholipids, which are known as precursors for different bioactive N-acylethanolamines. We previously reported that phospholipase A/acyltransferase-1 (PLAAT-1), which was originally found in mammals as a tumor suppressor, catalyzes N-acylation of phosphatidylethanolamines to form NAPEs. However, recent online database suggested the presence of an uncharacterized isoform of PLAAT-1 with an extra sequence at the N terminus. In the present study, we examined the occurrence, intracellular localization, and catalytic properties of this longer isoform, as well as the original shorter isoform from humans and mice. Our results showed that human tissues express the longer isoform but not the short isoform at all. In contrast, mice expressed both isoforms with different tissue distribution. Unlike the cytoplasmic localization of the shorter isoform, the long isoform was found in both cytoplasm and nucleus, inferring that the extra sequence harbors a nuclear localization signal. As assayed with purified proteins, neither isoform required calcium for full activity. Moreover, the overexpression of each isoform remarkably increased cellular NAPE levels. These results conclude that the new long isoform of PLAAT-1 is a calcium-independent N-acyltransferase existing in both cytoplasm and nucleus and suggest a possible formation of NAPEs in various membrane structures including nuclear membrane. J. Lipid Res 2016. 57: 2051-2060.

Calcium-dependent generation of N-acylethanolamines and lysophosphatidic acids by glycerophosphodiesterase GDE7.

N-Acylethanolamines form a class of lipid mediators and include an endocannabinoid arachidonoylethanolamide (anandamide), analgesic and anti-inflammatory palmitoylethanolamide, and appetite-suppressing oleoylethanolamide. In animal tissues, N-acylethanolamines are synthesized from N-acylated ethanolamine phospholipids directly by N-acylphosphatidylethanolamine-hydrolyzing phospholipase D or through multi-step pathways via N-acylethanolamine lysophospholipids. We previously reported that glycerophosphodiesterase (GDE) 4, a member of the GDE family, has lysophospholipase D (lysoPLD) activity hydrolyzing N-acylethanolamine lysophospholipids to N-acylethanolamines. Recently, GDE7 was shown to have lysoPLD activity toward lysophosphatidylcholine to produce lysophosphatidic acid (LPA). Here, we examined the reactivity of GDE7 with N-acylethanolamine lysophospholipids as well as the requirement of divalent cations for its catalytic activity. When overexpressed in HEK293 cells, recombinant GDE7 proteins of human and mouse showed lysoPLD activity toward N-palmitoyl, N-oleoyl, and N-arachidonoyl-lysophosphatidylethanolamines and N-palmitoyl-lysoplasmenylethanolamine to generate their corresponding N-acylethanolamines and LPAs. However, GDE7 hardly hydrolyzed glycerophospho-N-palmitoylethanolamine. Overexpression of GDE7 in HEK293 cells increased endogenous levels of N-acylethanolamines and LPAs. Interestingly, GDE7 was stimulated by micromolar concentrations of Ca2+ but not by millimolar concentrations of Mg2+, while GDE4 was stimulated by Mg2+ but was insensitive to Ca2+. GDE7 was widely distributed in various tissues of humans and mice with the highest levels in their kidney tissues. These results suggested that GDE7 is a novel Ca2+-dependent lysoPLD, which is involved in the generation of both N-acylethanolamines and LPAs.