Peeking into the Stingers: A Comprehensive SWATH-MS Study of the European Hornet Vespa crabro (Linnaeus, 1758) (Hymenoptera: Vespidae) Venom Sac Extracts.

This study aimed to investigate the venom sac extracts (VSEs) of the European hornet (EH) Vespa crabro (Linnaeus, 1758) (Hymenoptera: Vespidae), focusing on the differences between stinging females, gynes (G), and workers (W), at the protein level. Using a quantitative "Sequential Window Acquisition of all Theoretical Fragment Ion Mass Spectra" (SWATH-MS) analysis, we identified and quantified a total of 240 proteins. Notably, within the group, 45.8% (n = 110) showed significant differential expression between VSE-G and VSE-W. In this set, 57.3% (n = 63) were upregulated and 42.7% (n = 47) downregulated in the G. Additionally, the two-hundred quantified proteins from the class Insecta belong to sixteen different species, six of them to the Hymenoptera/Apidae lineage, comprising seven proteins with known potential allergenicity. Thus, phospholipase A1 (Vesp v 1), phospholipase A1 verutoxin 2b (VT-2b), hyaluronidase A (Vesp v 2A), hyaluronidase B (Vesp v 2B), and venom allergen 5 (Vesp v 5) were significantly downregulated in the G, and vitellogenin (Vesp v 6) was upregulated. Overall, 46% of the VSE proteins showed differential expression, with a majority being upregulated in G. Data are available via ProteomeXchange with identifier PXD047955. These findings shed light on the proteomic differences in VSE between EH castes, potentially contributing to our understanding of their behavior and offering insights for allergy research.

Case study of chemical and enzymatic degumming processes in soybean oil production at an industrial plant.

The vegetable oil degumming process plays a critical role in refining edible oil. Phospholipids (PL) removal from crude extracted soybean oil (SBO) by the enzymatic degumming process has been investigated in this work. Enzymatic degumming of extracted SBO with microbial phospholipase A1 PLA-1 Quara LowP and Lecitase Ultra enzymes have also been studied comparatively. The main novelty of our work is the use of the enzymatic degumming process on an industrial scale (600 tons a day). Many parameters have been discussed to understand in detail the factors affecting oil losses during the degumming process. The factors such as chemical conditioning (CC) by phosphoric acid 85%, the enzyme dosage mg/kg (feedstock dependent), the enzymatic degumming reaction time, and the characteristics of the plant-processed SBO have been discussed in detail. As a main point, the degummed oil with a phosphorus content of < 10 mg/kg increases yield. Quara LowP and Lecitase Ultra enzymes are not specific for certain phospholipids PL; however, the conversion rate depends on the SBO phospholipid composition. After 4 h, over 99% of Phospholipids were degraded to their lysophospholipid LPL (lysolecithin). The results showed a significant effect of operating parameters and characteristics of different origins of SBO, fatty acids FFA content, Phosphorus content and total divalent metals (Calcium Ca, Magnesium Mg and Iron Fe mg/kg) content on the oil loss. The benefit of using enzymatic degumming of vegetable oils rather than traditional chemical refining is that the enzymatic degumming process reduces total oil loss. This decrease is known as enzymatic yield. The enzymatic degumming also decreases wastewater and used chemicals and running costs; moreover, it enables physical refining by lowering the residue phosphorus to < 10 mg/kg.

Interfacial adsorption and activity of pancreatic lipase-related protein 2 onto heterogeneous plant lipid model membranes.

Pancreatic lipase related-protein 2 (PLRP2) exhibits remarkable galactolipase and phospholipase A1 activities, which depend greatly on the supramolecular organization of the substrates and the presence of surfactant molecules such as bile salts. The objective of the study was to understand the modulation of the adsorption mechanisms and enzymatic activity of Guinea pig PLRP2 (gPLRP2), by the physical environment of the enzyme and the physical state of its substrate. Langmuir monolayers were used to reproduce homogeneous and heterogeneous photosynthetic model membranes containing galactolipids (GL), and/or phospholipids (PL), and/or phytosterols (pS), presenting uncharged or charged interfaces. The same lipid mixtures were also used to form micrometric liposomes, and their gPLRP2 catalyzed digestion kinetics were investigated in presence or in absence of bile salts (NaTDC) during static in vitro, so called "bulk", digestion. The enzymatic activity of gPLRP2 onto the galactolipid-based monolayers was characterized with an optimum activity at 15 mN/m, in the absence of bile salts. gPLRP2 showed enhanced adsorption onto biomimetic model monolayer containing negatively charged lipids. However, the compositional complexity in the heterogeneous uncharged model systems induced a lag phase before the initiation of lipolysis. In bulk, no enzymatic activity could be demonstrated on GL-based liposomes in the absence of bile salts, probably due to the high lateral pressure of the lipid bilayers. In the presence of NaTDC (4 mM), however, gPLRP2 showed both high galactolipase and moderate phospholipase A1 activities on liposomes, probably due to a decrease in packing and lateral pressure upon NaTDC adsorption, and subsequent disruption of liposomes.

The Phospholipase A1 Activity of Glycerol Ester Hydrolase (Geh) Is Responsible for Extracellular 2-12(S)-Methyltetradecanoyl-Lysophosphatidylglycerol Production in Staphylococcus aureus.

Phosphatidylglycerol (PG) is the major membrane phospholipid of Staphylococcus aureus and predominately consists of molecular species with ≥16-carbon acyl chains in the 1-position and anteiso 12(S)-methyltetradecaonate (a15) esterified at the 2-position. The analysis of the growth media for PG-derived products shows S. aureus releases essentially pure 2-12(S)-methyltetradecanoyl-sn-glycero-3-phospho-1'-sn-glycerol (a15:0-LPG) derived from the hydrolysis of the 1-position of PG into the environment. The cellular lysophosphatidylglycerol (LPG) pool is dominated by a15-LPG but also consists of ≥16-LPG species arising from the removal of the 2-position. Mass tracing experiments confirmed a15-LPG was derived from isoleucine metabolism. A screen of candidate secreted lipase knockout strains pinpointed glycerol ester hydrolase (geh) as the gene required for generating extracellular a15-LPG, and complementation of a Δgeh strain with a Geh expression plasmid restored extracellular a15-LPG formation. Orlistat, a covalent inhibitor of Geh, also attenuated extracellular a15-LPG accumulation. Purified Geh hydrolyzed the 1-position acyl chain of PG and generated only a15-LPG from a S. aureus lipid mixture. The Geh product was 2-a15-LPG, which spontaneously isomerizes with time to a mixture of 1- and 2-a15-LPG. Docking PG in the Geh active site provides a structural rationale for the positional specificity of Geh. These data demonstrate a physiological role for Geh phospholipase A1 activity in S. aureus membrane phospholipid turnover. IMPORTANCE Glycerol ester hydrolase, Geh, is an abundant secreted lipase whose expression is controlled by the accessory gene regulator (Agr) quorum-sensing signal transduction pathway. Geh is thought to have a role in virulence based on its ability to hydrolyze host lipids at the infection site to provide fatty acids for membrane biogenesis and substrates for oleate hydratase, and Geh inhibits immune cell activation by hydrolyzing lipoprotein glycerol esters. The discovery that Geh is the major contributor to the formation and release of a15-LPG reveals an unappreciated physiological role for Geh acting as a phospholipase A1 in the degradation of S. aureus membrane phosphatidylglycerol. The role(s) for extracellular a15-LPG in S. aureus biology remain to be elucidated.

Molecular insights on PS-PLA1 lipase activity of human ABHD16B.

The human alpha beta hydrolase domain (ABHD) proteins are ubiquitous and regulate the cellular lipids' anabolic and catabolic processes. The structural aspects for specific biochemical function of many ABHD proteins related to physiological disorders and its link to pathological conditions remain unknown. Here putative human ABHD16B protein was overexpressed in Saccharomyces cerevisiae for its biological activity. In-vitro enzymatic assay of the recombinant ABHD16B protein with fluorescently tagged glycerophospholipids revealed that the PLA1 activity is observed with phosphatidylserine (PS). In addition, it efficiently hydrolyzed monoacylglycerol over triacylglycerols. Further, molecular dynamic simulations and per residue binding free energy decomposition analysis revealed that the origin of PS-specific PLA1 activity of ABHD16B is due to the electrostatic interaction of the PS head group with K8, R319, and E178, which led to having the hydrogen bond interaction of sn-1 acyl chain ester to the catalytic site residues. Site-directed mutagenesis of the 245GXSXG249 motif of ABHD16B reduced the maximal lipase activity of PS and MAG. In summary, these results revealed that ABHD16B plays a vital role in PS selectivity that in turn, controls the specific subcellular pools of 2-LPS metabolism in the tissues at low pH.

Phosphorylation and subcellular localization of human phospholipase A1, DDHD1/PA-PLA1.

Protein phosphorylation is the most common post-translational modification of proteins and functions as a molecular switch for their regulation. This modification is reversibly regulated by protein kinases and phosphatases. In most cases, the phosphorylation of enzymes positively or negatively regulates enzyme activity. However, we found that the phosphorylation of DDHD1 phospholipase A1 (PLA1) did not affect PLA1 activity. Integrated analyses, including phospho-proteomics, Phos-tag SDS-PAGE, PLA1 enzyme assays, and immunofluorescent microscopy, revealed the subcellular localization of DDHD1 without greatly affecting its PLA1 activity. Our findings may contribute to understanding rare clinical cases that concern the implications of protein phosphorylation.

Phospholipase A1 Member A Deficiency Alleviates Mannan-Induced Psoriatic Arthritis in Mice Model.

Synovial fluids from rheumatoid and psoriatic arthritis patients have high levels of PLA1A. The current study was to understand PLA1A functions in the pathophysiology of rheumatic diseases. We generated Pla1a−/− mice to assess their phenotype and the impact of PLA1A deficiency on the development of mannan-induced psoriatic arthritis (MIP). Mice were evaluated routinely for the induced symptoms. On the day of sacrifice, blood samples were collected for hematology analysis and prepared for plasma. Livers were collected. Lymph node immune cells were analyzed using flow cytometry. We performed µCT scans of hind paws from naïve and mannan-induced female mice. Cytokines/chemokines were quantified using Luminex in hind paw tissues and plasma of female mice. Pla1a−/− mice showed a slight increase in circulating and lymph node lymphocytes. CD4+ T cells contributed most to this increase in lymph nodes (p = 0.023). In the MIP model, the lymph node ratios of CD3+ to CD19+ and CD4+ to CD8+ were higher in Pla1a−/− mice. Pla1a−/− mice were less susceptible to MIP (p < 0.001) and showed reduced bone erosions. Pla1a−/− mice also showed reduced IL-17, KC, IP-10, MIP-1ß, LIF, and VEGF in hind paw tissues as compared to WT mice (p < 0.05). These findings indicated that PLA1A deficiency protected from the development of the MIP disease. The data suggested that PLA1A could contribute to MIP through increased activation of lymphocytes, possibly those producing IL-17.

Phosphatidylserine-Specific Phospholipase A1 Alleviates Lipopolysaccharide-Induced Macrophage Inflammation by Inhibiting MAPKs Activation.

Macrophages are a key in innate immune responses and play vital roles in homeostasis and inflammatory diseases. Phosphatidylserine-specific phospholipase A1 (PS-PLA1) is a specific phospholipase which hydrolyzes fatty acid from the sn-1 position of phosphatidylserine (PS) to produce lysophosphatidylserine (lysoPS). Both PS and lysoPS are associated with activation of immune cells including macrophages. However, the effect of PS-PLA1 on macrophage inflammation remains unclear. The purpose of this study is to evaluate the role of PS-PLA1 in lipopolysaccharide (LPS)-induced macrophage inflammation. Alterations of PS-PLA1 expression in LPS-stimulated RAW264.7 macrophages were investigated via Western blot. PS-PLA1 stable knockdown and overexpression RAW264.7 cell lines were generated by infecting cells with appropriate lentiviral vectors, respectively. PS-PLA1 expression was found to be dramatically upregulated in RAW264.7 macrophages after LPS stimulation. PS-PLA1 knockdown promotes while PS-PLA1 overexpression ameliorates the release of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and nitric oxide from RAW264.7 cells and M1 macrophage polarization. Additionally, PS-PLA1 knockdown facilitates phosphorylation of p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), while PS-PLA1 overexpression attenuates their phosphorylation. Moreover, mitogen-activated protein kinase (MAPK) inhibitors block the release of TNF-α and IL-1ß in PS-PLA1 knockdown RAW264.7 cells after LPS stimulation. These findings suggest PS-PLA1 ameliorates LPS-induced macrophage inflammation by inhibiting MAPKs activation, and PS-PLA1 might be considered as a target for modulating macrophage inflammation.

Exploring Interfacial Hydrolysis of Artificial Neutral Lipid Monolayer and Bilayer Catalyzed by Phospholipase C.

Phospholipase C (PLC) represents an important type of enzymes with the feature of hydrolyzing phospholipids at the position of the glycerophosphate bond, among which PLC extracted from Bacillus cereus (BC-PLC) has been extensively studied owing to its similarity to hitherto poorly characterized mammalian analogues. This study focuses on investigating the interfacial hydrolysis mechanism of phosphatidylcholine (PC) monolayer and bilayer membranes catalyzed by BC-PLC using sum frequency generation vibrational spectroscopy (SFG-VS) and laser scanning confocal microscopy (LSCM). We found that, upon interfacial hydrolysis, BC-PLC was adsorbed onto the lipid interface and catalyzed the lipolysis with no net orientation, as evidenced by the silent amide I band, indicating that ordered PLC alignment was not a prerequisite for the enzyme activity, which is very different from what we have reported for phospholipase A1 (PLA1) and phospholipase A2 (PLA2) [Kai, S. Phys. Chem. Chem. Phys. 2018, 20(1), 63-67; Wang, F. Langmuir 2019, 35(39), 12831-12838; Zhang, F. Langmuir 2020, 36(11), 2946-2953]. For the PC monolayer, one of the two hydrolysates, phosphocholine, desorbed from the interface into the aqueous phase, while the other one, diacylglycerol (DG), stayed well packed with high order at the interface. For the PC bilayer, phosphocholine dispersed into the aqueous phase too, similar to the monolayer case; however, DG, presumably formed clusters with the unreacted lipid substrates and desorbed from the interface. With respect to both the monolayer and bilayer cases, mechanistic schematics were presented to illustrate the different interfacial hydrolysis processes. Therefore, this model experimental study in vitro provides significant molecular-level insights and contributes necessary knowledge to reveal the lipolysis kinetics with respect to PLC and lipid membranes with monolayer and bilayer structures.

An adaptive teosinte mexicana introgression modulates phosphatidylcholine levels and is associated with maize flowering time.

Native Americans domesticated maize (Zea mays ssp. mays) from lowland teosinte parviglumis (Zea mays ssp. parviglumis) in the warm Mexican southwest and brought it to the highlands of Mexico and South America where it was exposed to lower temperatures that imposed strong selection on flowering time. Phospholipids are important metabolites in plant responses to low-temperature and phosphorus availability and have been suggested to influence flowering time. Here, we combined linkage mapping with genome scans to identify High PhosphatidylCholine 1 (HPC1), a gene that encodes a phospholipase A1 enzyme, as a major driver of phospholipid variation in highland maize. Common garden experiments demonstrated strong genotype-by-environment interactions associated with variation at HPC1, with the highland HPC1 allele leading to higher fitness in highlands, possibly by hastening flowering. The highland maize HPC1 variant resulted in impaired function of the encoded protein due to a polymorphism in a highly conserved sequence. A meta-analysis across HPC1 orthologs indicated a strong association between the identity of the amino acid at this position and optimal growth in prokaryotes. Mutagenesis of HPC1 via genome editing validated its role in regulating phospholipid metabolism. Finally, we showed that the highland HPC1 allele entered cultivated maize by introgression from the wild highland teosinte Zea mays ssp. mexicana and has been maintained in maize breeding lines from the Northern United States, Canada, and Europe. Thus, HPC1 introgressed from teosinte mexicana underlies a large metabolic QTL that modulates phosphatidylcholine levels and has an adaptive effect at least in part via induction of early flowering time.

Current Knowledge on Mammalian Phospholipase A1, Brief History, Structures, Biochemical and Pathophysiological Roles.

Phospholipase A1 (PLA1) is an enzyme that cleaves an ester bond at the sn-1 position of glycerophospholipids, producing a free fatty acid and a lysophospholipid. PLA1 activities have been detected both extracellularly and intracellularly, which are well conserved in higher eukaryotes, including fish and mammals. All extracellular PLA1s belong to the lipase family. In addition to PLA1 activity, most mammalian extracellular PLA1s exhibit lipase activity to hydrolyze triacylglycerol, cleaving the fatty acid and contributing to its absorption into the intestinal tract and tissues. Some extracellular PLA1s exhibit PLA1 activities specific to phosphatidic acid (PA) or phosphatidylserine (PS) and serve to produce lysophospholipid mediators such as lysophosphatidic acid (LPA) and lysophosphatidylserine (LysoPS). A high level of PLA1 activity has been detected in the cytosol fractions, where PA-PLA1/DDHD1/iPLA1 was responsible for the activity. Many homologs of PA-PLA1 and PLA2 have been shown to exhibit PLA1 activity. Although much has been learned about the pathophysiological roles of PLA1 molecules through studies of knockout mice and human genetic diseases, many questions regarding their biochemical properties, including their genuine in vivo substrate, remain elusive.

Efficient immobilized phospholipase A1 on Mo-basing nanomaterials for enzymatic degumming.

Six kinds of Mo-basing nanomaterials (MoO3 , MoO3 @Ru, Mo-PDA, MoPC , MoP, CNT@MoS2 ) were successfully synthesized, which were employed as carriers to immobilize phospholipase A1 (PLA1) for the hydrolysis of phospholipids (PLs). PLA1 was immobilized by a simple adsorption-precipitation-cross-linking to form an "enzyme net" covering on nanoparticles. The greatest advantage of these nanoparticles was their strong hydrophilic surface. It not only permitted their dispersion in the aqueous phase, but also showed the strong affinity for PLs in the organic phase, because amphiphilic PLs had the polar head group and higher hydrophilicity than other oils components. Michaelis-Menten analysis revealed that higher catalytic activity and enzyme-substrate affinity were observed in several immobilized PLA1 than its free form. MoO3 was confirmed to be the best candidate for carrier. The highest specific activity of MoO3 -immobilized PLA1 reached 43.1 U/mg, which was about 1.8 times higher than that of free PLA1 (24.4 U/mg). In addition, the stability and recycling were also enhanced. The robust immobilized PLA1 was prepared in this work, showing great potential for the enzymatic degumming.

Crystal Structures of the Plant Phospholipase A1 Proteins Reveal a Unique Dimerization Domain.

Phospholipase is an enzyme that hydrolyzes various phospholipid substrates at specific ester bonds and plays important roles such as membrane remodeling, as digestive enzymes, and the regulation of cellular mechanism. Phospholipase proteins are divided into following the four major groups according to the ester bonds they cleave off: phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD). Among the four phospholipase groups, PLA1 has been less studied than the other phospholipases. Here, we report the first molecular structures of plant PLA1s: AtDSEL and CaPLA1 derived from Arabidopsis thaliana and Capsicum annuum, respectively. AtDSEL and CaPLA1 are novel PLA1s in that they form homodimers since PLAs are generally in the form of a monomer. The dimerization domain at the C-terminal of the AtDSEL and CaPLA1 makes hydrophobic interactions between each monomer, respectively. The C-terminal domain is also present in PLA1s of other plants, but not in PLAs of mammals and fungi. An activity assay of AtDSEL toward various lipid substrates demonstrates that AtDSEL is specialized for the cleavage of sn-1 acyl chains. This report reveals a new domain that exists only in plant PLA1s and suggests that the domain is essential for homodimerization.

Urine autotaxin levels reflect the disease activity of sarcoidosis.

Since the clinical outcome of patients with sarcoidosis is still unpredictable, a good prognostic biomarker is necessary. Autotaxin (ATX) and phosphatidylserine-specific phospholipase A1 (PS-PLA1) function as main enzymes to produce lysophospholipids (LPLs), and these enzymes are attracting attention as useful biomarkers for several chronic inflammatory diseases. Here, we investigated the relationships between LPLs-producing enzymes and the disease activity of sarcoidosis. In total, 157 patients with sarcoidosis (active state, 51%) were consecutively enrolled. Using plasma or urine specimens, we measured the values of LPLs-producing enzymes. Urine ATX (U-ATX) levels were significantly lower in the active state compared to those in the inactive state, while the plasma ATX (P-ATX) and PS-PLA1 levels showed no significant difference between these two states. Concerning the comparison with existing clinical biomarkers for sarcoidosis, U-ATX showed a weak negative correlation to ACE, P-ATX a weak positive correlation to both ACE and sIL-2R, and PS-PLA1 a weak positive one to sIL-2R. Notably, only the U-ATX levels inversely fluctuated depending on the status of disease activity whether OCS had been used or not. These findings suggest that U-ATX is likely to be a novel and useful molecule for assessing the disease activity of sarcoidosis.

Phosphatidylserine-Specific Phospholipase A1 Limits Aggressiveness of Lung Adenocarcinoma by Lysophosphatidylserine and Protein Kinase A-Dependent Pathway.

Lipid metabolic abnormalities in cancer cells are increasingly being studied. Several studies have reported that phosphatidylserine-specific phospholipase A1 (PLA1A) might be involved in the pathogenesis of cancers. Nevertheless, the function and mechanistic details of PLA1A in lung adenocarcinoma (LUAD) progression remain largely undefined. In the present study, low PLA1A expression was correlated with poor prognosis in patients with LUAD. Results from in vitro and in vivo animal studies showed that overexpressed PLA1A suppressed the proliferation of LUAD cells in vitro and tumor growth in vivo through regulation of cyclin abundance, thereby inducing S-phase arrest. Meanwhile, PLA1A overexpression attenuated migration and invasion of LUAD cells, including by inhibiting the epithelial-mesenchymal transition. Mechanistically, PLA1A overexpression inhibited aggressiveness of LUAD cells through elevated lysophosphatidylserine, which acts via G-protein-coupled receptor 174, further activating cAMP/protein kinase A pathway. Activating G-protein-coupled receptor 174/protein kinase A pathway may involve effects on cell cycle regulators and transcription factors-regulated epithelial-mesenchymal transition. The study uncovered the mechanism through which PLA1A regulates LUAD proliferation, invasion, and migration. These results demonstrate the potential use of PLA1A as a biomarker for diagnosing LUAD, which may therefore potentially serve as a therapeutic target for LUAD.

A defective lysophosphatidic acid-autophagy axis increases miscarriage risk by restricting decidual macrophage residence.

Massive infiltrated and enriched decidual macrophages (dMφ) have been widely regarded as important regulators of maternal-fetal immune tolerance and trophoblast invasion, contributing to normal pregnancy. However, the characteristics of metabolic profile and the underlying mechanism of dMφ residence remain largely unknown. Here, we observe that dMφ display an active glycerophospholipid metabolism. The activation of ENPP2-lysophosphatidic acid (LPA) facilitates the adhesion and retention, and M2 differentiation of dMφ during normal pregnancy. Mechanistically, this process is mediated through activation of the LPA receptors (LPAR1 and PPARG/PPARγ)-DDIT4-macroautophagy/autophagy axis, and further upregulation of multiple adhesion factors (e.g., cadherins and selectins) in a CLDN7 (claudin 7)-dependent manner. Additionally, poor trophoblast invasion and placenta development, and a high ratio of embryo loss are observed in Enpp2±, lpar1-/- or PPARG-blocked pregnant mice. Patients with unexplained spontaneous abortion display insufficient autophagy and cell residence of dMφ. In therapeutic studies, supplementation with LPA or the autophagy inducer rapamycin significantly promotes dMφ autophagy and cell residence, and improves embryo resorption in Enpp2± and spontaneous abortion mouse models, which should be dependent on the activation of DDIT4-autophagy-CLDN7-adhesion molecules axis. This observation reveals that inactivation of ENPP2-LPA metabolism and insufficient autophagy of dMφ result in resident obstacle of dMφ and further increase the risk of spontaneous abortion, and provides potential therapeutic strategies to prevent spontaneous abortion.Abbreviations: ACTB: actin beta; ADGRE1/F4/80: adhesion G protein-coupled receptor E1; Atg5: autophagy related 5; ATG13: autophagy related 13; BECN1: beclin 1; CDH1/E-cadherin: cadherin 1; CDH5/VE-cadherin: cadherin 5; CFSE: carboxyfluorescein succinimidyl ester; CLDN7: claudin 7; CSF1/M-CSF: colony stimulating factor 1; CSF2/GM-CSF: colony stimulating factor 2; Ctrl: control; CXCL10/IP-10: chemokine (C-X-C) ligand 10; DDIT4: DNA damage inducible transcript 4; dMφ: decidual macrophage; DSC: decidual stromal cells; ENPP2/ATX: ectonucleotide pyrophosphatase/phosphodiesterase 2; Enpp2±: Enpp2 heterozygous knockout mouse; ENPP2i/PF-8380: ENPP2 inhibitor; EPCAM: epithelial cell adhesion molecule; ESC: endometrial stromal cells; FGF2/b-FGF: fibroblast growth factor 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GPCPD1: glycerophosphocholine phosphodiesterase 1; HE: heterozygote; HIF1A: hypoxia inducible factor 1 subunit alpha; HNF4A: hepatocyte nuclear factor 4 alpha; HO: homozygote; ICAM2: intercellular adhesion molecule 2; IL: interleukin; ITGAV/CD51: integrin subunit alpha V; ITGAM/CD11b: integrin subunit alpha M; ITGAX/CD11b: integrin subunit alpha X; ITGB3/CD61: integrin subunit beta 3; KLRB1/NK1.1: killer cell lectin like receptor B1; KRT7/cytokeratin 7: keratin 7; LPA: lysophosphatidic acid; LPAR: lysophosphatidic acid receptor; lpar1-/-: lpar1 homozygous knockout mouse; LPAR1i/AM966: LPAR1 inhibitor; LY6C: lymphocyte antigen 6 complex, locus C1; LYPLA1: lysophospholipase 1; LYPLA2: lysophospholipase 2; Lyz2: lysozyme 2; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MARVELD2: MARVEL domain containing 2; 3-MA: 3-methyladenine; MBOAT2: membrane bound O-acyltransferase domain containing 2; MGLL: monoglyceride lipase; MRC1/CD206: mannose receptor C-type 1; MTOR: mechanistic target of rapamycin kinase; NP: normal pregnancy; PDGF: platelet derived growth factor; PLA1A: phospholipase A1 member A; PLA2G4A: phospholipase A2 group IVA; PLPP1: phospholipid phosphatase 1; pMo: peripheral blood monocytes; p-MTOR: phosphorylated MTOR; PPAR: peroxisome proliferator activated receptor; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; PPARGi/GW9662: PPARG inhibitor; PTPRC/CD45: protein tyrosine phosphatase receptor type, C; Rapa: rapamycin; RHEB: Ras homolog, mTORC1 binding; SA: spontaneous abortion; SELE: selectin E; SELL: selectin L; siCLDN7: CLDN7-silenced; STAT: signal transducer and activator of transcription; SQSTM1: sequestosome 1; TJP1: tight junction protein 1; VCAM1: vascular cell adhesion molecule 1; WT: wild type.

Phospholipase A1 Member A Activates Fibroblast-like Synoviocytes through the Autotaxin-Lysophosphatidic Acid Receptor Axis.

Lysophosphatidylserine (lysoPS) is known to regulate immune cell functions. Phospholipase A1 member A (PLA1A) can generate this bioactive lipid through hydrolysis of sn-1 fatty acids on phosphatidylserine (PS). PLA1A has been associated with cancer metastasis, asthma, as well as acute coronary syndrome. However, the functions of PLA1A in the development of systemic autoimmune rheumatic diseases remain elusive. To investigate the possible implication of PLA1A during rheumatic diseases, we monitored PLA1A in synovial fluids from patients with rheumatoid arthritis and plasma of early-diagnosed arthritis (EA) patients and clinically stable systemic lupus erythematosus (SLE) patients. We used human primary fibroblast-like synoviocytes (FLSs) to evaluate the PLA1A-induced biological responses. Our results highlighted that the plasma concentrations of PLA1A in EA and SLE patients were elevated compared to healthy donors. High concentrations of PLA1A were also detected in synovial fluids from rheumatoid arthritis patients compared to those from osteoarthritis (OA) and gout patients. The origin of PLA1A in FLSs and the arthritic joints remained unknown, as healthy human primary FLSs does not express the PLA1A transcript. Besides, the addition of recombinant PLA1A stimulated cultured human primary FLSs to secrete IL-8. Preincubation with heparin, autotaxin (ATX) inhibitor HA130 or lysophosphatidic acid (LPA) receptor antagonist Ki16425 reduced PLA1A-induced-secretion of IL-8. Our data suggested that FLS-associated PLA1A cleaves membrane-exposed PS into lysoPS, which is subsequently converted to LPA by ATX. Since primary FLSs do not express any lysoPS receptors, the data suggested PLA1A-mediated pro-inflammatory responses through the ATX-LPA receptor signaling axis.

Genome-wide expression analysis of phospholipase A1 (PLA1) gene family suggests phospholipase A1-32 gene responding to abiotic stresses in cotton.

Cotton is the most important crop for the production of natural fibres used in the textile industry. High salinity, drought, cold and high temperature represent serious abiotic stresses, which seriously threaten cotton production. Phospholipase AS has an irreplaceable role in lipid signal transmission, growth and development and stress events. Phospholipase A can be divided into three families: PLA1, PLA2 and pPLA. Among them, the PLA1 family is rarely studied in plants. In order to study the potential functions of the PLA1 family in cotton, the bioinformatics analysis of the PLA1 family was correlated with cotton adversity, and tissue-specific analysis was performed. Explore the structure-function relationship of PLA1 members. It is found that the expression of GbPLA1-32 gene is affected by a variety of environmental stimuli, indicating that it plays a very important role in stress and hormone response, and closely associates the cotton adversity with this family. Through further functional verification, we found that virus-induced GbPLA1-32 gene silencing (VIGS) caused Gossypium barbadense to be sensitive to salt stress. This research provides an important basis for further research on the molecular mechanism of cotton resistance to abiotic stress.

Studies on the Structure and Properties of Membrane Phospholipase A1 Inclusion Bodies Formed at Low Growth Temperatures Using GFP Fusion Strategy.

The effect of cultivation temperatures (37, 26, and 18 °C) on the conformational quality of Yersinia pseudotuberculosis phospholipase A1 (PldA) in inclusion bodies (IBs) was studied using green fluorescent protein (GFP) as a folding reporter. GFP was fused to the C-terminus of PldA to form the PldA-GFP chimeric protein. It was found that the maximum level of fluorescence and expression of the chimeric protein is observed in cells grown at 18 °C, while at 37 °C no formation of fluorescently active forms of PldA-GFP occurs. The size, stability in denaturant solutions, and enzymatic and biological activity of PldA-GFP IBs expressed at 18 °C, as well as the secondary structure and arrangement of protein molecules inside the IBs, were studied. Solubilization of the chimeric protein from IBs in urea and SDS is accompanied by its denaturation. The obtained data show the structural heterogeneity of PldA-GFP IBs. It can be assumed that compactly packed, properly folded, proteolytic resistant, and structurally less organized, susceptible to proteolysis polypeptides can coexist in PldA-GFP IBs. The use of GFP as a fusion partner improves the conformational quality of PldA, but negatively affects its enzymatic activity. The PldA-GFP IBs are not toxic to eukaryotic cells and have the property to penetrate neuroblastoma cells. Data presented in the work show that the GFP-marker can be useful not only as target protein folding indicator, but also as a tool for studying the molecular organization of IBs, their morphology, and localization in E. coli, as well as for visualization of IBs interactions with eukaryotic cells.