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Lysophosphatidylserine (LysoPS) has been shown to have lipid mediator-like actions to induce mast cell degranulation and suppress T lymphocyte proliferation. Recently, three G protein-coupled receptors (GPCRs), LPS1/GPR34, LPS2/P2Y10, and LPS3/GPR174, were found to react specifically with LysoPS, raising the possibility that LysoPS exerts its roles through these receptors. In this study, we show that LPS3 is expressed in various T cell subtypes and is involved in suppression of Interleukin-2 (IL-2) production in CD4 T cells. We found that LysoPS suppressed the IL-2 production from activated T cells at the mRNA and protein levels. In addition, LysoPS did not have such an effect on the splenocytes and CD4 T cells isolated from LPS3-deficient mice. In LPS3-deficient splenocytes and CD4 T cells, anti-CD3/anti-CD28-triggered IL-2 production is somewhat increased. Interestingly, LysoPS with various fatty acids was up-regulated upon T cell activation. The present study raised the possibility that LysoPS exerts its immunosuppressive roles by down-regulating IL-2 production through a LysoPS-LPS3 axis in T cells.

The ligands of certain G-protein-coupled receptors (GPCRs) have been identified as endogenous lipids, such as lysophosphatidylserine (LysoPS). Here, we analyzed the molecular basis of the structure–activity relationship of ligands of GPR34, one of the LysoPS receptor subtypes, focusing on recognition of the long-chain fatty acid moiety by the hydrophobic pocket. By introducing benzene ring(s) into the fatty acid moiety of 2-deoxy-LysoPS, we explored the binding site’s preference for the hydrophobic shape. A tribenzene-containing fatty acid surrogate with modifications of the terminal aromatic moiety showed potent agonistic activity toward GPR34. Computational docking of these derivatives with a homology modeling/molecular dynamics-based virtual binding site of GPR34 indicated that a kink in the benzene-based lipid surrogates matches the L-shaped hydrophobic pocket of GPR34. A tetrabenzene-based lipid analogue bearing a bulky tert-butyl group at the 4-position of the terminal benzene ring exhibited potent ...

Food-borne trans-fatty acids (TFAs) are mainly produced as byproducts during food manufacture. Recent epidemiological studies have revealed that TFA consumption is a major risk factor for various disorders, including atherosclerosis. However, the underlying mechanisms in this disease etiology are largely unknown. Here we have shown that TFAs potentiate activation of apoptosis signal-regulating kinase 1 (ASK1) induced by extracellular ATP, a damage-associated molecular pattern leaked from injured cells. Major food-associated TFAs such as elaidic acid (EA), linoelaidic acid, and trans-vaccenic acid, but not their corresponding cis isomers, dramatically enhanced extracellular ATP-induced apoptosis, accompanied by elevated activation of the ASK1-p38 pathway in a macrophage-like cell line, RAW264.7. Moreover, knocking out the ASK1-encoding gene abolished EA-mediated enhancement of apoptosis. We have reported previously that extracellular ATP induces apoptosis through the ASK1-p38 pathway activated by reactive oxygen species generated downstream of the P2X purinoceptor 7 (P2X7). However, here we show that EA did not increase ATP-induced reactive oxygen species generation but, rather, augmented the effects of calcium/calmodulin-dependent kinase II-dependent ASK1 activation. These results demonstrate that TFAs promote extracellular ATP-induced apoptosis by targeting ASK1 and indicate novel TFA-associated pathways leading to inflammatory signal transduction and cell death that underlie the pathogenesis and progression of TFA-induced atherosclerosis. Our study thus provides insight into the pathogenic mechanisms of and proposes potential therapeutic targets for these TFA-related disorders.

Under a quiescent state, microglia exhibit a ramified shape, rather than the amoeboid-like morphology following injury or inflammation. The manipulation of microglial morphology in vitro has not been very successful, which has impeded the progress of microglial studies. We demonstrate that lysophosphatidylserine (LysoPS), a kind of lysophospholipids, rapidly and substantially alters the morphology of primary cultured microglia to an in vivo-like ramified shape in a receptor independent manner. This mechanism is mediated by Cdc42 activity. LysoPS is incorporated into the plasma membrane and converted to phosphatidylserine (PS) via the Lands' cycle. The accumulated PS on the membrane recruits Cdc42. Both Cdc42 and PS colocalize predominantly in primary and secondary processes, but not in peripheral branches or tips of microglia. Along with the morphological changes LysoPS suppresses inflammatory cytokine production and NF-kB activity. The present study provides a tool to manipulate a microglial phenotype from an amoeboid to a fully ramified in vitro, which certainly contributes to studies exploring microglial physiology and pathology.

Lysophosphatidylserine (LysoPS) has been shown to have lipid mediator-like actions to induce mast cell degranulation and suppress T lymphocyte proliferation. Recently, three G protein-coupled receptors (GPCRs), LPS

The ligands of certain G-protein-coupled receptors (GPCRs) have been identified as endogenous lipids, such as lysophosphatidylserine (LysoPS). Here, we analyzed the molecular basis of the structure-activity relationship of ligands of GPR34, one of the LysoPS receptor subtypes, focusing on recognition of the long-chain fatty acid moiety by the hydrophobic pocket. By introducing benzene ring(s) into the fatty acid moiety of 2-deoxy-LysoPS, we explored the binding site's preference for the hydrophobic shape. A tribenzene-containing fatty acid surrogate with modifications of the terminal aromatic moiety showed potent agonistic activity toward GPR34. Computational docking of these derivatives with a homology modeling/molecular dynamics-based virtual binding site of GPR34 indicated that a kink in the benzene-based lipid surrogates matches the L-shaped hydrophobic pocket of GPR34. A tetrabenzene-based lipid analogue bearing a bulky tert-butyl group at the 4-position of the terminal benzene ring exhibited potent GPR34 agonistic activity, validating the present hydrophobic binding pocket model.

Lysophospholipids (LysoGPs) serve as lipid mediators and precursors for synthesis of diacyl phospholipids (GPs). LysoGPs detected in cells have various acyl chains attached at either the sn-1 or sn-2 position of the glycerol backbone. In general, acyl chains at the sn-2 position of 2-acyl-1-LysoGPs readily move to the sn-1 position, generating 1-acyl-2-lyso isomers by a nonenzymatic reaction called intra-molecular acyl migration, which has hampered the detection of 2-acyl-1-LysoGPs in biological samples. In this study, we developed a simple and versatile method to separate and quantify 2-acyl-1- and 1-acyl-2-LysoGPs. The main point of the method was to extract LysoGPs at pH 4 and 4°C, conditions that were found to completely eliminate the intra-molecular acyl migration. Under the present conditions, the relative amounts of 2-acyl-1-LysoGPs and 1-acyl-2-LysoGPs did not change at least for 1 week. Further, in LysoGPs extracted from cells and tissues under the present conditions, most of the saturated fatty acids (16:0 and 18:0) were found in the sn-1 position of LysoGPs, while most of the PUFAs (18:2, 20:4, 22:6) were found in the sn-2 position. Thus the method can be used to elucidate the in vivo role of 2-acyl-1-LysoGPs.

Lysophosphatidylserine (LysoPS) is an endogenous lipid mediator that specifically activates membrane proteins of the P2Y and its related families of G protein-coupled receptors (GPCR), GPR34 (LPS1), P2Y10 (LPS2), and GPR174 (LPS3). Here, in order to increase potency and receptor selectivity, we designed and synthesized LysoPS analogues containing the conformational constraints of the glycerol moiety. These reduced structural flexibility by fixation of the glycerol framework of LysoPS using a 2-hydroxymethyl-3-hydroxytetrahydropyran skeleton, and related structures identified compounds which exhibited high potency and selectivity for activation of GPR34 or P2Y10. Morphing of the structural shape of the 2-hydroxymethyl-3-hydroxytetrahydropyran skeleton into a planar benzene ring enhanced the P2Y10 activation potentcy rather than the GPR34 activation.

Lysophosphatidylserine (LysoPS), a deacylated form of phosphatidylserine (PS), has been assumed to serve as a bioactive lysophospholipid mediator. However, LysoPS has received little attention because its mode of actions as well as its synthetic pathways have been obscure. Recently G protein-coupled receptors (GPCRs) specific for LysoPS, and enzymes responsible for the generation of LysoPS have been identified. These LysoPS-related molecules are mainly expressed on immune cells, which led us to assume that LysoPS may have some roles in inflammation. Here we summarised the newly discovered LysoPS receptors and enzymes including our recent works.

GPR34 is a G protein-coupled receptor belonging to the P2Y family. Here, we attempted to resolve conflicting reports about whether it is a functional lysophosphatidylserine (LysoPS) receptor. In HEK293 cells expressing human, mouse or rat GPR34 and Gα chimera between Gαq and Gαi1(Gq/i1), LysoPS quickly elevated intracellular Ca(2+) ion levels ([Ca(2+)](i)). LysoPS also stimulated alkaline phosphatase (AP)-tagged TGFα (AP-TGFα) release in GPR34-expressing HEK293 cells and induced the migration of CHO-K1 cells expressing GPR34. Other lysophospholipids did not induce these actions. Replacement of the serine residue of LysoPS abolished the reactivity of LysoPS with GPR34, indicating that GPR34 strictly recognizes the serine head group of LysoPS. Recombinant phosphatidylserine-specific phospholipase A(1) (PS-PLA(1)) that deacylates fatty acid at the sn-1 position of PS and produces 2-acyl-LysoPS, but not catalytically inactive mutant PS-PLA(1), stimulated the release of AP-TGFα from GPR34-expressing cells. Consistent with the result, LysoPS was detected in the cells treated with wild-type PS-PLA(1) but not with the mutant PS-PLA(1). PS treated with PLA(1) was much more effective at stimulating AP-TGFα release than PS treated with PLA(2). In addition, migration-resistant 2-acyl-1-deoxy-LysoPS, a 2-acyl-LysoPS analogue, was much more potent than 1-acyl-2-deoxy-LysoPS. The present studies confirm that GPR34 is a cellular receptor for LysoPS, especially with a fatty acid at the sn-2 position.

The expression of phosphatidylserine-specific phospholipase A1 (PS-PLA1) is most upregulated in the genes of peripheral blood cells from chronic rejection model rats bearing long-term surviving cardiac allografts. The expression profile of PS-PLA1 in peripheral blood cells responsible for the immune response may indicate a possible biological marker for rejection episodes. In this study, PS-PLA1 mRNA expression was examined in human THP-1-derived macrophages. The effects of several immunosuppressive agents on this expression were also examined in in vitro experiments. A real-time RT-PCR analysis revealed that PS-PLA1 mRNA expression was found in human THP-1-derived macrophages. This expression was enhanced in the cells stimulated with lipopolysaccharide (LPS), a toll-like receptor (TLR) 4 ligand. Other TLR ligands (TLR2, 3, 5, 7, and 9) did not show a significant induction of PS-PLA1 mRNA. The time course of the mRNA expression profiles was different between PS-PLA1 and tumor necrosis factor-α (TNF-α), which showed a maximal expression at 12 and 1 h after LPS stimulation, respectively. Among the observed immunosuppressive agents, corticosteroids, prednisolone, 6α-methylprednisolone, dexamethasone, and beclomethasone inhibited PS-PLA1 expression with half-maximal inhibitory concentrations less than 3.0 nM, while methotrexate, cyclosporine A, tacrolimus, 6-mercaptopurine, and mycophenoic acid showed either a weak or moderate inhibition. These results suggest that the expression of PS-PLA1 mRNA in THP-1-derived macrophages is activated via TLR4 and it is inhibited by corticosteroids, which are used at high dosages to suppress chronic allograft rejection.

It is now widely accepted that lysophospholipids (LPLs), a product of the phospholipase A reaction, function as mediators through G-protein-coupled receptors. Notably, recent studies of lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) have revealed their essential roles in vivo. On the other hand, other LPLs such as lysophosphatidylserine (LPS), lysophosphatidylthreonine (LPT), lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI) and lysophosphatidylglycerol (LPG) have been reported to show lipid mediator-like responses both in vivo (LPS and LPT) and in vitro (LPS, LPT, LPE and LPG), while very little is known about their receptor, synthetic enzyme and patho-physiological roles. In this review, we summarize the actions of these LPLs as lipid mediators including LPS, LPT, LPE and LPG.

The function of phosphatidylserine-specific phospholipase A1 (PS-PLA1), a phospholipase that acts specifically on phosphatidylserine and produces lysophosphatidylserine, a lysophospholipid mediator, has not been fully elucidated. We evaluated the role of PS-PLA1 in oncogenesis and metastasis of colorectal cancer (CRC).Specimens from 85 patients with CRC were immunostained with a monoclonal antibody against PS-PLA1. The correlation between PS-PLA1 expression and the clinicopathological variables was analyzed.Tumor depth and hematogenous metastasis independently positively correlated with PS-PLA1 expression. High PS-PLA1 expression was associated with shorter disease-free survival, although it was not an independent predictive factor.PS-PLA1 expression in CRC is associated with tumor invasion and metastasis.

It is now widely accepted that lysophospholipids (LPLs), a product of the phospholipase A reaction, function as mediators through G protein-coupled receptors (GPCRs). Notably, recent studies of lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) have revealed their essential roles in vivo. In addition, recent studies have identified several GPCRs for other lysophospholipids such as lysophosphatidylserine (LysoPS) and lysophosphatidylinositol (LPI). In this chapter, we summarize the actions and production of these LPLs as lipid mediators including LysoPS and LPI.

GPR34, a P2Y receptor family member, was identified as a candidate lysophosphatidylserine (LysoPS) receptor in 2006. However, it was controversial whether LysoPS is a real ligand for GPR34. Kitamura et al. (GPR34 is a receptor for lysophosphatidylserine with a fatty acid at the sn-2 position. J. Biochem. 2012;151:511-518), using several methods for detecting activation of G protein-coupled receptor (GPCR) and chemically synthesized LysoPS analogues, concluded that GPR34 is a cellular receptor for LysoPS, especially with a fatty acid at the sn-2 position. Furthermore, three additional GPCRs belonging to the P2Y family were recently identified as GPCRs for LysoPS, supporting the idea that GPR34 is a LysoPS receptor.

Lysophosphatidylserine (1-oleoyl-2 R-lysophosphatidylserine, LysoPS) has been shown to have lipid mediator-like actions such as stimulation of mast cell degranulation and suppression of T lymphocyte proliferation, although the mechanisms of LysoPS actions have been elusive. Recently, three G protein-coupled receptors (LPS1/GPR34, LPS2/P2Y10 and LPS3/GPR174) were found to react specifically with LysoPS, raising the possibility that LysoPS serves as a lipid mediator that exerts its role through these receptors. Previously, we chemically synthesized a number of LysoPS analogues and evaluated them as agonists for mast-cell degranulation. Here, we used a transforming growth factor-α (TGFα) shedding assay to see if these LysoPS analogues activated the three LysoPS receptors. Modification of the serine moiety significantly reduced the ability of the analogues to activate the three LysoPS receptors, whereas modification of other parts resulted in loss of activity in receptor-specific manner. We found that introduction of methyl group to serine moiety (1-oleoyl-lysophosphatidylallothreonine) and removal of sn-2 hydroxyl group (1-oleoyl-2-deoxy-LysoPS) resulted in reduction of reactivity with LPS1 and LPS3, respectively. Accordingly, we synthesized a LysoPS analogue with the two modifications (1-oleoyl-2-deoxy-lysophosphatidylallothreonine) and found it to be an LPS2-selective agonist. These pharmacological tools will definitely help to identify the biological roles of these LysoPS receptors.

A single-format method to detect multiple G protein-coupled receptor (GPCR) signaling, especially Gα(12/13) signaling, presently has limited throughput and sensitivity. Here we report a transforming growth factor-α (TGFα) shedding assay, in which GPCR activation is measured as ectodomain shedding of a membrane-bound proform of alkaline phosphatase-tagged TGFα (AP-TGFα) and its release into conditioned medium. AP-TGFα shedding response occurred almost exclusively downstream of Gα(12/13) and Gα(q) signaling. Relying on chimeric Gα proteins and promiscuous Gα(16) protein, which can couple with Gα(s)- and Gα(i)-coupled GPCRs and induce Gα(q) signaling, we used the TGFα shedding assay to detect 104 GPCRs among 116 human GPCRs. We identified three orphan GPCRs (P2Y10, A630033H20 and GPR174) as Gα(12/13)-coupled lysophosphatidylserine receptors. Thus, the TGFα shedding assay is useful for studies of poorly characterized Gα(12/13)-coupled GPCRs and is a versatile platform for detecting GPCR activation including searching for ligands of orphan GPCRs.

Members of the pancreatic lipase family exhibit both lipase activity toward triacylglycerol and/or phospholipase A(1) (PLA(1)) activity toward certain phospholipids. Some members of the pancreatic lipase family exhibit lysophospholipase activity in addition to their lipase and PLA(1) activities. Two such enzymes, phosphatidylserine (PS)-specific PLA(1) (PS-PLA(1)) and phosphatidic acid (PA)-selective PLA(1)α (PA-PLA(1)α, also known as LIPH) specifically hydrolyze PS and PA, respectively. However, little is known about the mechanisms that determine their substrate specificities. Crystal structures of lipases and mutagenesis studies have suggested that three surface loops, namely, β5, β9, and lid, have roles in determining substrate specificity. To determine roles of these loop structures in the substrate recognition of these PLA(1) enzymes, we constructed a number of PS-PLA(1) mutants in which the three surface loops are replaced with those of PA-PLA(1)α. The results indicate that the surface loops, especially the β5 loop, of PA-PLA(1)α play important roles in the recognition of PA, whereas other structure(s) in PS-PLA(1) is responsible for PS preference. In addition, β5 loop of PS-PLA(1) has a crucial role in lysophospholipase activity toward lysophosphatidylserine. The present study revealed the critical role of lipase surface loops, especially the β5 loop, in determining substrate specificities of PLA(1) enzymes.

In response to various exogenous stimuli, mast cells (MCs) release a wide variety of inflammatory mediators stored in their cytoplasmic granules and this release initiates subsequent allergic reactions. Lysophosphatidylserine (lysoPS) has been known as an exogenous inducer to potentiate histamine release from MCs, though even at submicromolar concentrations. In this study, through SAR studies on lysoPS against MC degranulation, we identified lysoPT, a threonine-containing lysophospholipid and its 2-deoxy derivative as novel strong agonists. LysoPT and its 2-deoxy derivative induced histamine release from MCs both in vitro and in vivo at a concentration less than one-tenth that of lysoPS. Notably, lysoPT did not activate a recently proposed lysoPS receptor on MCs, GPR34, demonstrating the presence of another undefined receptor reactive to both lysoPS and lysoPT that is involved in MC degranulation. Thus, the present strong agonists, lysoPT and its 2-deoxy derivative, will be useful tools to understand the mechanisms of lysoPS-induced activation of degranulation of MCs.

Phospholipase A 1 (PLA 1 ) is an enzyme that hydrolyzes phospholipids and produces 2-acyl-lysophospholipids and fatty acids and is conserved in a wide range of organisms. Mammals have several enzymes that exhibit PLA 1 activity in vitro . The extracellular PLA 1 s include phosphatidylserine (PS)-specific PLA 1 (PS-PLA 1 ), membrane-associated phosphatidic acid (PA)-selective PLA 1 s (mPA-PLA 1 α and mPA-PLA 1 β), hepatic lipase (HL), endothelial lipase (EL) and pancreatic lipase-related protein 2 (PLRP2), all of which belong to the pancreatic lipase gene family. The former three PLA 1 s differ from other members in their substrate specificities, structural features and gene organizations, and form a subfamily in the pancreatic lipase gene family. PS-PLA 1 , mPA-PLA 1 α and mPA-PLA 1 β exhibit only PLA 1 activity, while HL, EL and PLRP2 show triacylglycerol-hydrolyzing activity in addition to PLA 1 activity. The tertiary structures of lipases have two surface loops, the lid and the β9 loop. The lid and the β9 loop cover the active site in its closed conformation. An alignment of amino acid sequences of the pancreatic lipase gene family members revealed two molecular characteristics of PLA 1 s in the two surface loops. First, lipase members exhibiting PLA 1 activity (PS-PLA 1 , mPA-PLA 1 α and mPA-PLA 1 β, EL, guinea pig PLRP2 and PLA 1 from hornet venom (DolmI)) have short lids. Second, PS-PLA 1 , mPA-PLA 1 α, mPA-PLA 1 β and DolmI, which exhibit only PLA 1 activity, have short β9 loops. Thus, the two surface loops appear to be involved in the ligand recognition. PS-PLA 1 and mPA-PLA 1 s specifically hydrolyze PS and PA, respectively, producing their corresponding lysophospholipids. Lysophosphatidylserine and lysophosphatidic acid have been defined as lipid mediators with multiple biological functions. Thus, these PLA 1 s have a role in the production of these lysophospholipid mediators.

Upon various stimulations, mast cells (MCs) release a wide variety of chemical mediators stored in their cytoplasmic granules, which then initiates subsequent allergic reactions. Lysophosphatidylserine (LysoPS), a kind of lysophospholipid, potentiates the histamine release from MCs triggered by antigen stimulation. We previously showed through structure-activity studies of LysoPS analogs that LysoPS with a methyl group at the carbon of the serine residue, i.e., lysophosphatidylthreonine (LysoPT), is extremely potent in stimulating the MC degranulation. In this study, as our continuing study to identify more potent LysoPS analogs, we developed LysoPS analogs with fatty acid surrogates. We found that the substitution of oleic acid to an aromatic fatty acid surrogate (C3-pH-p-O-C11) in 2-deoxy-1-LysoPS resulted in significant increase in the ability to induce MCs degranulation compared with 2-deoxy-1-LysoPS with oleic acid. Conversion of the serine residue into the threonine residue further increased the activity of MC degranulation both in vitro and in vivo. The resulting super agonist, 2-deoxy-LysoPT with C3-pH-p-O-C11, will be a useful tool to elucidate the mechanisms of stimulatory effect of LysoPS on MC degranulation., Highlights • Lysophosphatidylserine (LysoPS) stimulates degranulation of mast cell (MC). • We evaluated various LysoPS analogs for their MC degranulation-stimulating activity. • We identified a threonine containing LysoPS analogs as a super agonist. • The MC LysoPS receptor is different from the known LysoPS receptors. • The super agonist helps to identify the putative MC LysoPS receptor.