Rigidifying Flexible Sites: A Promising Strategy to Improve Thermostability of Lysophospholipase From Pyrococcus abyssi.

High thermostability of the enzymes is one of the distinguishing characteristics that increase their industrial utility. In the current research work, rigidifying the flexible amino acid residues of a lysophospholipase (Pa-LPL) from Pyrococcus abyssi was used as a protein engineering approach to improve its thermostability. A truncated variant of Pa-LPL (t-LPL∆12) was constructed by trimming its 12 amino acid residues (50-61) through overlap extension PCR. The truncated enzyme worked optimally at 65°C and pH 6.5 with remarkable thermostability at 65°C-85°C. In comparison to wild-type Pa-LPL, 5.8 and 1.2-fold increase in half-life (t1/2) of t-LPL∆12 was observed at 65 (optimum temperature) and 95°C, respectively. The activity of t-LPL∆12 was stimulated by 1 mM Cu2+ followed by Ca2+, Ni2+, Co2+, and Mg2+. Both substrate docking and experimental results indicated that the truncated enzyme could hydrolyze a variety of p-nitrophenyl esters. Km, Vmax, and Kcat for enzymatic hydrolysis of p-nitrophenyl butyrate were calculated to be 1 ± 0.087 mM, 1456 ± 36.474 U/mg, and 1.397 × 1011 min-1, respectively. In short, broad substrate specificity and thermostability of t-LPL∆12 are some of the distinctive features that make it an ideal candidate for degumming of vegetable oils.

Lysophospholipase D activity on oral mucosa cells in whole mixed human saliva involves in production of bioactive lysophosphatidic acid from lysophosphatidylcholine.

We reported that lysophosphatidic acid (LPA) is present at 0.8 µM in mixed human saliva (MS). In this study, we examined the distribution, origin, and enzymatic generation pathways of LPA in MS. LPA was distributed in the medium and cell pellet fraction; a true level of soluble LPA in MS was about 150 nM. The soluble LPA was assumed to be generated by ecto-type lysophospholipase D on exfoliated cells in MS from LPC that originated mainly from the major salivary gland saliva. Our results with the albumin-back extraction procedures suggest that a significant pool of LPA is kept in the outer layer of the plasma membranes of detached oral mucosal cells. Such pool of LPA may contribute to wound healing in upper digestive organs including oral cavity. We obtained evidence that the choline-producing activity in MS was mainly due to Ca2+-activated lysophospholipase D activity of glycerophosphodiesterase 7.

Safety evaluation of the food enzyme lysophospholipase from the genetically modified Trichoderma reesei strain DP-Nyc81.

The food enzyme lysophospholipase (2-lysophosphatidylcholine acylhydrolase, EC 3.1.1.5) is produced with the genetically modified Trichoderma reesei strain DP-Nyc81 by Genencor International B.V. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in the processing of cereals and other grains for the production of glucose syrups and other starch hydrolysates. Since residual amounts of food enzyme-total organic solids are removed during these food manufacturing processes, dietary exposure was not calculated and toxicological studies were considered unnecessary. A search for the identity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Autotaxin-Lysophosphatidate Axis: Promoter of Cancer Development and Possible Therapeutic Implications.

Autotaxin (ATX) is a member of the ectonucleotide pyrophosphate/phosphodiesterase (ENPP) family; it is encoded by the ENPP2 gene. ATX is a secreted glycoprotein and catalyzes the hydrolysis of lysophosphatidylcholine to lysophosphatidic acid (LPA). LPA is responsible for the transduction of various signal pathways through the interaction with at least six G protein-coupled receptors, LPA Receptors 1 to 6 (LPAR1-6). The ATX-LPA axis is involved in various physiological and pathological processes, such as angiogenesis, embryonic development, inflammation, fibrosis, and obesity. However, significant research also reported its connection to carcinogenesis, immune escape, metastasis, tumor microenvironment, cancer stem cells, and therapeutic resistance. Moreover, several studies suggested ATX and LPA as relevant biomarkers and/or therapeutic targets. In this review of the literature, we aimed to deepen knowledge about the role of the ATX-LPA axis as a promoter of cancer development, progression and invasion, and therapeutic resistance. Finally, we explored its potential application as a prognostic/predictive biomarker and therapeutic target for tumor treatment.

Safety evaluation of a food enzyme with phospholipase A1 and lysophospholipase activities from the genetically modified Aspergillus niger strain PLN.

The food enzyme with phospholipase A1 (phosphatidycholine 1-acylhydrolase, EC 3.1.1.32) and lysophospholipase (2-lysophosphatidylcholine acylhydrolase, EC 3.1.1.5) activities is produced with the genetically modified Aspergillus niger strain PLN by DSM. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used for the production of refined edible fats and oils by degumming. Since residual amounts of total organic solids are removed during this process, dietary exposure was not calculated and toxicological studies were considered unnecessary for the assessment of this food enzyme. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no matches were found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Application of SUMO fusion technology for the enhancement of stability and activity of lysophospholipase from Pyrococcus abyssi.

Heterologous production of proteins in Escherichia coli has raised several challenges including soluble production of target proteins, high levels of expression and purification. Fusion tags can serve as the important tools to overcome these challenges. SUMO (small ubiquitin-related modifier) is one of these tags whose fusion to native protein sequence can enhance its solubility and stability. In current research, a simple, efficient and cost-effective method is being discussed for the construction of pET28a-SUMO vector. In order to improve the stability and activity of lysophospholipase from Pyrococcus abyssi (Pa-LPL), a 6xHis-SUMO tag was fused to N-terminal of Pa-LPL by using pET28a-SUMO vector. Recombinant SUMO-fused enzyme (6 H-S-PaLPL) works optimally at 35 °C and pH 6.5 with remarkable thermostability at 35-95 °C. Thermo-inactivation kinetics of 6 H-S-PaLPL were also studied at 35-95 °C with first order rate constant (kIN) of 5.58 × 10- 2 h-1 and half-life of 12 ± 0 h at 95 °C. Km and Vmax for the hydrolysis of 4-nitrophenyl butyrate were calculated to be 2 ± 0.015 mM and 3882 ± 22.368 U/mg, respectively. 2.4-fold increase in Vmax of Pa-LPL was observed after fusion of 6xHis-SUMO tag to its N-terminal. It is the first report on the utilization of SUMO fusion tag to enhance the overall stability and activity of Pa-LPL. Fusion of 6xHis-SUMO tag not only aided in the purification process but also played a crucial role in increasing the thermostability and activity of the enzyme. SUMO-fused enzyme, thus generated, can serve as an important candidate for degumming of vegetable oils at industrial scale.

Altered ovarian tissue level of lysophosphatidic acid and mRNA expressions of its metabolic enzymes and receptors in rats received gonadotropin-hyperstimulation.

Lysophosphatidic acid (LPA), a well-studied member of the lysophospholipid family, is known to exert an important bio-effect on oocyte maturation and ovulation in mammals. We attempted to determine how follicle maturation in the rat ovary affects the levels of LPA and its precursor lysophospholipids, as well as mRNA levels of LPA-producing and -degrading enzymes and LPA receptors in rats that received gonadotropin-hyper-stimulation. Tissue levels of lysophospholipids were quantified by LC-MS/MS, and relative mRNA expression levels of LPA-producing and -degrading enzymes, and LPA receptors were measured by RT-PCR. Tissue levels of n-6 polyunsaturated LPAs and LPCs were higher in the ovaries of rats after receiving human chorionic gonadotropin, unlike the distinct profiles of n-3 polyunsaturated LPAs, which had lower levels, and LPCs which had higher levels, after the gonadotropin treatment. The effects of different levels of other polyunsaturated lysophospholipids were variable: decreased levels of lysophosphatidylglycerol, and unaltered levels of lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylserine. The results indicate that expression of mRNA levels of autotaxin and acylglycerol kinase were reduced and expression of lipid phosphate phosphatase 3 was elevated, whereas expressions of two membrane phosphatidic acid phosphatases (A1α and A1ß) and lipid phosphate phosphatase 1 were essentially unaltered in rat ovary at several stages after ovary hyperstimulation. After the gonadotropin treatment, the expression levels of all LPA receptors except LPA3 were decreased at various times. These results are discussed with respect to the physiological processes of the ovarian environment and development in rats.

Metabolism of host lysophosphatidylcholine in Plasmodium falciparum-infected erythrocytes.

The human malaria parasite Plasmodium falciparum requires exogenous fatty acids to support its growth during the pathogenic, asexual erythrocytic stage. Host serum lysophosphatidylcholine (LPC) is a significant fatty acid source, yet the metabolic processes responsible for the liberation of free fatty acids from exogenous LPC are unknown. Using an assay for LPC hydrolysis in P. falciparum-infected erythrocytes, we have identified small-molecule inhibitors of key in situ lysophospholipase activities. Competitive activity-based profiling and generation of a panel of single-to-quadruple knockout parasite lines revealed that two enzymes of the serine hydrolase superfamily, termed exported lipase (XL) 2 and exported lipase homolog (XLH) 4, constitute the dominant lysophospholipase activities in parasite-infected erythrocytes. The parasite ensures efficient exogenous LPC hydrolysis by directing these two enzymes to distinct locations: XL2 is exported to the erythrocyte, while XLH4 is retained within the parasite. While XL2 and XLH4 were individually dispensable with little effect on LPC hydrolysis in situ, loss of both enzymes resulted in a strong reduction in fatty acid scavenging from LPC, hyperproduction of phosphatidylcholine, and an enhanced sensitivity to LPC toxicity. Notably, growth of XL/XLH-deficient parasites was severely impaired when cultured in media containing LPC as the sole exogenous fatty acid source. Furthermore, when XL2 and XLH4 activities were ablated by genetic or pharmacologic means, parasites were unable to proliferate in human serum, a physiologically relevant fatty acid source, revealing the essentiality of LPC hydrolysis in the host environment and its potential as a target for anti-malarial therapy.

Recombinant production and characterization of a metal ion-independent Lysophospholipase from a hyperthermophilic archaeon Pyrococcus abyssi DSM25543.

Genome sequence of Pyrococcus abyssi DSM25543 contains a coding sequence (PAB_RS01410) for α/ß hydrolase (WP_010867387.1). Structural analysis revealed the presence of a consensus motif GXSXG and a highly conserved catalytic triad in the amino acid sequence of α/ß hydrolase that were characteristic features of lysophospholipases. A putative lysophospholipase from P. abyssi with its potential applications in oil degumming and starch processing was heterologously produced in E. coli Rosetta (DE3) pLysS in soluble form followed by its purification and characterization. The recombinant enzyme was found to be active at temperature of 40-90 °C and pH 5.5-7.0. However, the enzyme exhibited its optimum activity at 65 °C and pH 6.5. None of the metal ions (Mn2+, Mg2+, Ni2+, Cu2+, Fe2+, Co2+, Zn2+ and Ca2+) being tested had stimulatory effect on lysophospholipase activity. Km and Vmax for hydrolysis of 4-nitrophenyl butyrate were calculated to be 1 ± 0.089 mM and 1637 ± 24.434 U/mg, respectively. It is the first report on the soluble production and characterization of recombinant lysophospholipase from P. abyssi which exhibits its lipolytic activity in the absence of divalent metal ions. Broad substrate specificity, activity and stability at elevated temperatures make recombinant lysophospholipase an ideal candidate for potential industrial applications.

Lysophospholipase D from Thermocrispum limits psoriatic inflammation by hydrolyzing epidermal lysoplasmalogen produced by group IIF secreted phospholipase A2.

Epidermal lipids play important roles in skin homeostasis and diseases. Psoriasis is an inflammatory disease characterized by keratinocyte hyperproliferation and Th17 immune responses. We previously reported that ethanolamine-type lysoplasmalogen (P-LPE), preferentially produced by group IIF secreted PLA2 (sPLA2-IIF/PLA2G2F) that is expressed in the suprabasal epidermis, promotes epidermal hyperplasia in psoriatic inflammation. Herein, we show that forcible degradation of epidermal P-LPE by topical application of recombinant lysophospholipase D (LyPls-PLD) from Thermocrispum, a lysoplasmalogen-specific hydrolase, attenuated epidermal hyperplasia and inflammation in imiquimod-induced and K5.Stat3C-transgenic mouse psoriasis models. In humans, P-LPE levels were elevated in the tape-stripped stratum corneum of patients with psoriasis. Moreover, in primary cultured human epidermal keratinocytes, aberrant cell proliferation and activation by psoriatic cytokines were sPLA2-IIF/P-LPE-dependent and were suppressed by the addition of LyPls-PLD with a decrease in P-LPE. These findings confirm that the sPLA2-IIF/P-LPE axis in the epidermis indeed regulates psoriasis, that P-LPE is a lipid biomarker that predicts the severity of psoriasis, and that pharmacological removal of this bioactive lipid is useful to prevent the disease. Thus, our study may lead to the development of drug discovery and diagnostic techniques based on this pathway.

COVID-19, Blood Lipid Changes, and Thrombosis.

Although there is increasing evidence that oxidative stress and inflammation induced by COVID-19 may contribute to increased risk and severity of thromboses, the underlying mechanism(s) remain to be understood. The purpose of this review is to highlight the role of blood lipids in association with thrombosis events observed in COVID-19 patients. Among different types of phospholipases A2 that target cell membrane phospholipids, there is increasing focus on the inflammatory secretory phospholipase A2 IIA (sPLA2-IIA), which is associated with the severity of COVID-19. Analysis indicates increased sPLA2-IIA levels together with eicosanoids in the sera of COVID patients. sPLA2 could metabolise phospholipids in platelets, erythrocytes, and endothelial cells to produce arachidonic acid (ARA) and lysophospholipids. Arachidonic acid in platelets is metabolised to prostaglandin H2 and thromboxane A2, known for their pro-coagulation and vasoconstrictive properties. Lysophospholipids, such as lysophosphatidylcholine, could be metabolised by autotaxin (ATX) and further converted to lysophosphatidic acid (LPA). Increased ATX has been found in the serum of patients with COVID-19, and LPA has recently been found to induce NETosis, a clotting mechanism triggered by the release of extracellular fibres from neutrophils and a key feature of the COVID-19 hypercoagulable state. PLA2 could also catalyse the formation of platelet activating factor (PAF) from membrane ether phospholipids. Many of the above lipid mediators are increased in the blood of patients with COVID-19. Together, findings from analyses of blood lipids in COVID-19 patients suggest an important role for metabolites of sPLA2-IIA in COVID-19-associated coagulopathy (CAC).

Role of enterocyte Enpp2 and autotaxin in regulating lipopolysaccharide levels, systemic inflammation, and atherosclerosis.

Conversion of lysophosphatidylcholine to lysophosphatidic acid (LPA) by autotaxin, a secreted phospholipase D, is a major pathway for producing LPA. We previously reported that feeding Ldlr-/- mice standard mouse chow supplemented with unsaturated LPA or lysophosphatidylcholine qualitatively mimicked the dyslipidemia and atherosclerosis induced by feeding a Western diet (WD). Here, we report that adding unsaturated LPA to standard mouse chow also increased the content of reactive oxygen species and oxidized phospholipids (OxPLs) in jejunum mucus. To determine the role of intestinal autotaxin, enterocyte-specific Ldlr-/-/Enpp2 KO (intestinal KO) mice were generated. In control mice, the WD increased enterocyte Enpp2 expression and raised autotaxin levels. Ex vivo, addition of OxPL to jejunum from Ldlr-/- mice on a chow diet induced expression of Enpp2. In control mice, the WD raised OxPL levels in jejunum mucus and decreased gene expression in enterocytes for a number of peptides and proteins that affect antimicrobial activity. On the WD, the control mice developed elevated levels of lipopolysaccharide in jejunum mucus and plasma, with increased dyslipidemia and increased atherosclerosis. All these changes were reduced in the intestinal KO mice. We conclude that the WD increases the formation of intestinal OxPL, which i) induce enterocyte Enpp2 and autotaxin resulting in higher enterocyte LPA levels; that ii) contribute to the formation of reactive oxygen species that help to maintain the high OxPL levels; iii) decrease intestinal antimicrobial activity; and iv) raise plasma lipopolysaccharide levels that promote systemic inflammation and enhance atherosclerosis.

A Plasmodium falciparum lysophospholipase regulates host fatty acid flux via parasite lipid storage to enable controlled asexual schizogony.

Phospholipid metabolism is crucial for membrane biogenesis and homeostasis of Plasmodium falciparum. To generate such phospholipids, the parasite extensively scavenges, recycles, and reassembles host lipids. P. falciparum possesses an unusually large number of lysophospholipases, whose roles and importance remain to be elucidated. Here, we functionally characterize one P. falciparum lysophospholipase, PfLPL3, to reveal its key role in parasite propagation during asexual blood stages. PfLPL3 displays a dynamic localization throughout asexual stages, mainly localizing in the host-parasite interface. Inducible knockdown of PfLPL3 disrupts parasite development from trophozoites to schizont, inducing a drastic reduction in merozoite progenies. Detailed lipidomic analyses show that PfLPL3 generates fatty acids from scavenged host lipids to generate neutral lipids. These are then timely mobilized to allow schizogony and merozoite formation. We then identify inhibitors of PfLPL3 from Medicine for Malaria Venture (MMV) with potent antimalarial activity, which could also serve as pertinent chemical tools to study parasite lipid synthesis.

Cotton peroxisome-localized lysophospholipase counteracts the toxic effects of Verticillium dahliae NLP1 and confers wilt resistance.

Plasma membrane represents a critical battleground between plants and attacking microbes. Necrosis-and-ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs), cytolytic toxins produced by some bacterial, fungal and oomycete species, are able to target on lipid membranes by binding eudicot plant-specific sphingolipids (glycosylinositol phosphorylceramide) and form transient small pores, causing membrane leakage and subsequent cell death. NLP-producing phytopathogens are a big threat to agriculture worldwide. However, whether there are R proteins/enzymes that counteract the toxicity of NLPs in plants remains largely unknown. Here we show that cotton produces a peroxisome-localized enzyme lysophospholipase, GhLPL2. Upon Verticillium dahliae attack, GhLPL2 accumulates on the membrane and binds to V. dahliae secreted NLP, VdNLP1, to block its contribution to virulence. A higher level of lysophospholipase in cells is required to neutralize VdNLP1 toxicity and induce immunity-related genes expression, meanwhile maintaining normal growth of cotton plants, revealing the role of GhLPL2 protein in balancing resistance to V. dahliae and growth. Intriguingly, GhLPL2 silencing cotton plants also display high resistance to V. dahliae, but show severe dwarfing phenotype and developmental defects, suggesting GhLPL2 is an essential gene in cotton. GhLPL2 silencing results in lysophosphatidylinositol over-accumulation and decreased glycometabolism, leading to a lack of carbon sources required for plants and pathogens to survive. Furthermore, lysophospholipases from several other crops also interact with VdNLP1, implying that blocking NLP virulence by lysophospholipase may be a common strategy in plants. Our work demonstrates that overexpressing lysophospholipase encoding genes have great potential for breeding crops with high resistance against NLP-producing microbial pathogens.

The Lysophospholipase PNPLA7 Controls Hepatic Choline and Methionine Metabolism.

The in vivo roles of lysophospholipase, which cleaves a fatty acyl ester of lysophospholipid, remained unclear. Recently, we have unraveled a previously unrecognized physiological role of the lysophospholipase PNPLA7, a member of the Ca2+-independent phospholipase A2 (iPLA2) family, as a key regulator of the production of glycerophosphocholine (GPC), a precursor of endogenous choline, whose methyl groups are preferentially fluxed into the methionine cycle in the liver. PNPLA7 deficiency in mice markedly decreases hepatic GPC, choline, and several metabolites related to choline/methionine metabolism, leading to various symptoms reminiscent of methionine shortage. Overall metabolic alterations in the liver of Pnpla7-null mice in vivo largely recapitulate those in methionine-deprived hepatocytes in vitro. Reduction of the methyl donor S-adenosylmethionine (SAM) after methionine deprivation decreases the methylation of the PNPLA7 gene promoter, relieves PNPLA7 expression, and thereby increases GPC and choline levels, likely as a compensatory adaptation. In line with the view that SAM prevents the development of liver cancer, the expression of PNPLA7, as well as several enzymes in the choline/methionine metabolism, is reduced in human hepatocellular carcinoma. These findings uncover an unexplored role of a lysophospholipase in hepatic phospholipid catabolism coupled with choline/methionine metabolism.

Hepatic phosphatidylcholine catabolism driven by PNPLA7 and PNPLA8 supplies endogenous choline to replenish the methionine cycle with methyl groups.

Choline supplies methyl groups for regeneration of methionine and the methyl donor S-adenosylmethionine in the liver. Here, we report that the catabolism of membrane phosphatidylcholine (PC) into water-soluble glycerophosphocholine (GPC) by the phospholipase/lysophospholipase PNPLA8-PNPLA7 axis enables endogenous choline stored in hepatic PC to be utilized in methyl metabolism. PNPLA7-deficient mice show marked decreases in hepatic GPC, choline, and several metabolites related to the methionine cycle, accompanied by various signs of methionine insufficiency, including growth retardation, hypoglycemia, hypolipidemia, increased energy consumption, reduced adiposity, increased fibroblast growth factor 21 (FGF21), and an altered histone/DNA methylation landscape. Moreover, PNPLA8-deficient mice recapitulate most of these phenotypes. In contrast to wild-type mice fed a methionine/choline-deficient diet, both knockout strains display decreased hepatic triglyceride, likely via reductions of lipogenesis and GPC-derived glycerol flux. Collectively, our findings highlight the biological importance of phospholipid catabolism driven by PNPLA8/PNPLA7 in methyl group flux and triglyceride synthesis in the liver.

The alteration of the expression level of neuropathy target esterase in human neuroblastoma SK-N-SH cells disrupts cellular phospholipids homeostasis.

Neuropathy target esterase (NTE) has been proven to act as a lysophospholipase (LysoPLA) and phospholipase B (PLB) in mammalian cells. In this study, we took human neuroblastoma SK-N-SH cells as the research object and explored the effect of NTE on phospholipid homeostasis. The results showed that phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) levels significantly increased (> 40%), while glycerophosphocholine (GPC) decreased (below 60%) after NTE gene was knockdown in the cells (NTE < 30% of control), which were prepared by gene silencing with dsRNA-NTE. However, in the NTE-overexpressed cells (NTE > 50% of control), which were prepared by expressing recombinant catalytic domain of NTE, LPC remarkably decreased (below 80%) and GPC enhanced (> 40%). Mipafox, a neuropathic organophosphorus compound (OP), significantly inhibited NTE-LysoPLA and NTE-PLB activities (> 95-99% inhibition at 50 µM), which was accompanied with a decreased GPC level (below 40%) although no change of the PC and LPC levels was observed; while paraoxon, a non-neuropathic OP, suppresses neither the activities of NTE-phospholipases nor the levels of PC, LPC, and GPC. Thus, we concluded that both the stable up- or down-regulated expression of NTE gene and the loss of NTE-LysoPLA/PLB activities disrupts phospholipid homeostasis in the cells although the inhibition of NTE activity only decreased GPC content without altering PC and LPC levels.

Monoacylglycerol lipase from marine Geobacillus sp. showing lysophospholipase activity and its application in efficient soybean oil degumming.

Enzymatic degumming is an essential refining process to improve oil quality. In this study, a monoacylglycerol lipase GMGL was derived from marine Geobacillus sp., and was found that not only took monoacylglycerol (MAG) as substrate, but also had activity toward lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE) and glycerolphosphatidylcholine (GPC). Binding free energy showed LPC and LPE could bind with enzyme stably as MAG. It presented great potential in the field of enzymatic degumming. The phosphorus content in crude soybean oil decreased from 680.50 to 2.01 mg/kg and the yield of oil reached to 98.80 % after treating with phospholipase A1 (Lecitase Ultra) combined with lipase GMGL. An ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was developed to identify 21 differential phospholipids between crude soybean oil and enzymatic treatment. This work might shed some light on understanding the catalytic mechanism of monoacylglycerol lipase and provide an effective strategy for enzymatic degumming.

Characterization of recombinant murine GDE4 and GDE7, enzymes producing lysophosphatidic acid and/or cyclic phosphatidic acid.

GDE4 and GDE7 are membrane-bound enzymes that exhibit lysophospholipase D activities. We found that GDE7 produced not only lysophosphatidic acid (LPA) but also cyclic phosphatidic acid (cPA) from lysophospholipids by a transphosphatidylation reaction. In contrast, GDE4 produced only LPA. The analysis of substrate specificity showed that 1-alkyl-lysophosphospholipids were preferred substrates for both enzymes rather than 1-alkyl-lysophospholipids and 1-alkenyl-lysophospholipids. Among the various lysophospholipids with different polar head groups that were tested, lysophosphatidylglycerol and lysophosphatidylserine were preferred substrates for GDE4 and GDE7, respectively. The detailed analysis of the dependency of the enzyme activities of GDE4 and GDE7 on divalent cations suggested multiple divalent cations were bound in the active sites of both enzymes. Taken together, these results suggest the possibility that GDE7 functions as a cPA-producing enzyme in the body.

Gene Expression Data Mining Reveals the Involvement of GPR55 and Its Endogenous Ligands in Immune Response, Cancer, and Differentiation.

G protein-coupled receptor 55 (GPR55) is a recently deorphanized lipid- and peptide-sensing receptor. Its lipidic endogenous agonists belong to lysoglycerophospholipids, with lysophosphatidylinositol (LPI) being the most studied. Peptide agonists derive from fragmentation of pituitary adenylate cyclase-activating polypeptide (PACAP). Although GPR55 and its ligands were implicated in several physiological and pathological conditions, their biological function remains unclear. Thus, the aim of the study was to conduct a large-scale re-analysis of publicly available gene expression datasets to identify physiological and pathological conditions affecting the expression of GPR55 and the production of its ligands. The study revealed that regulation of GPR55 occurs predominantly in the context of immune activation pointing towards the role of the receptor in response to pathogens and in immune cell lineage determination. Additionally, it was revealed that there is almost no overlap between the experimental conditions affecting the expression of GPR55 and those modulating agonist production. The capacity to synthesize LPI was enhanced in various types of tumors, indicating that cancer cells can hijack the motility-related activity of GPR55 to increase aggressiveness. Conditions favoring accumulation of PACAP-derived peptides were different than those for LPI and were mainly related to differentiation. This indicates a different function of the two agonist classes and possibly the existence of a signaling bias.