L. pneumophila, an important facultative intracellular bacterium, infects the human lung and environmental protozoa. At least fifteen phospholipases A (PLA) are encoded in its genome. Three of which, namely PlaA, PlaC, and PlaD, belong to the GDSL lipase family abundant in bacteria and higher plants. PlaA is a lysophospholipase A (LPLA) that destabilizes the phagosomal membrane in absence of a protective factor. PlaC shows PLA and glycerophospholipid: cholesterol acyltransferase (GCAT) activities which are activated by zinc metalloproteinase ProA via cleavage of a disulphide loop. In this work, we compared GDSL enzyme activities, their secretion, and activation of PlaA. We found that PlaA majorly contributed to LPLA, PlaC to PLA, and both substrate-dependently to GCAT activity. Western blotting revealed that PlaA and PlaC are type II-secreted and both processed by ProA. Interestingly, ProA steeply increased LPLA but diminished GCAT activity of PlaA. Deletion of 20 amino acids within a predicted disulfide loop of PlaA had the same effect. In summary, we propose a model by which ProA processes PlaA via disulfide loop cleavage leading to a steep increase in LPLA activity. Our results help to further characterize the L. pneumophila GDSL hydrolases, particularly PlaA, an enzyme acting in the Legionella-containing phagosome.

Drosophila melanogaster is one of the most famous insects in biological research. It is widely used to analyse functions of different genes. The phosphatidylcholine lysophospholipase gene swiss cheese was initially shown to be important in the fruit fly nervous system. However, the role of this gene in non-nervous cell types has not been elucidated yet, and the evolutional explanation for the conservation of its function remains elusive. In this study, we analyse expression pattern and some aspects of the role of the swiss cheese gene in the fitness of Drosophila melanogaster. We describe the spatiotemporal expression of swiss cheese throughout the fly development and analyse the survival and productivity of swiss cheese mutants. We found swiss cheese to be expressed in salivary glands, midgut, Malpighian tubes, adipocytes, and male reproductive system. Dysfunction of swiss cheese results in severe pupae and imago lethality and decline of fertility, which is impressive in males. The latter is accompanied with abnormalities of male locomotor activity and courtship behaviour, accumulation of lipid droplets in testis cyst cells and decrease in spermatozoa motility. These results suggest that normal swiss cheese is important for Drosophila melanogaster fitness due to its necessity for both specimen survival and their reproductive success.

Mammalian patatin-like phospholipase domain containing proteins (PNPLAs) play critical roles in triglyceride hydrolysis, phospholipids metabolism, and lipid droplet (LD) homeostasis. PNPLA7 is a lysophosphatidylcholine hydrolase anchored on the endoplasmic reticulum which associates with LDs through its catalytic region (PNPLA7-C) in response to increased cyclic nucleotide levels. However, the interaction of PNPLA7 with LDs through its catalytic region is unknown. Herein, we demonstrate that PNPLA7-C localizes to the mature LDs ex vivo and also colocalizes with pre-existing LDs. Localization of PNPLA7-C with LDs induces LDs clustering via non-enzymatic intermolecular associations, while PNPLA7 alone does not induce LD clustering. Residues 742-1016 contains four putative transmembrane domains which act as a LD targeting motif and are required for the localization of PNPLA7-C to LDs. Furthermore, the N-terminal flanking region of the LD targeting motif, residues 681-741, contributes to the LD targeting, whereas the C-terminal flanking region (1169-1326) has an anti-LD targeting effect. Interestingly, the LD targeting motif does not exhibit lysophosphatidylcholine hydrolase activity even though it associates with LDs phospholipid membranes. These findings characterize the specific functional domains of PNPLA7 mediating subcellular positioning and interactions with LDs, as wells as providing critical insights into the structure of this evolutionarily conserved phospholipid-metabolizing enzyme family.

White matter injury has been frequently reported in HIV+ patients. Previous studies showed that HIV-1 Tat (transactivator of transcription), a viral protein that is produced and secreted by HIV-infected cells, is toxic to young, immature oligodendrocytes (OLGs). Adding Tat to the culture medium reduced the viability of immature OLGs, and the surviving OLGs exhibited reduced process networks. OLGs produce and secrete autotaxin (ATX), an ecto-enzyme containing a lysophospholipase D (lysoPLD) activity that converts lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a lipid signaling molecule that stimulates OLG differentiation. We hypothesized that Tat affects OLG development by interfering with the ATX-LPA signaling pathway. Our data show that Tat treatment leads to changes in the expression of OLG differentiation genes and the area of OLG process networks, both of which can be rescued by LPA. Tat-treated OLGs showed no change in LPA receptor expression but significantly decreased extracellular ATX levels and lysoPLD activity. In Tat transgenic mice, expression of Tat in vivo leads to decreased OLG ATX secretion. Furthermore, co-immunoprecipitation experiments revealed a potential physical interaction between Tat and ATX. Together, these data strongly suggest two functional implications of Tat blocking ATX's lysoPLD activity. On one hand, it attenuates OLG differentiation, and on the other hand it interferes with the protective effects of LPA on OLG process morphology.

TesA from Pseudomonas aeruginosa belongs to the GDSL hydrolase family of serine esterases and lipases that possess a broad substrate- and regiospecificity. It shows high sequence homology to TAP, a multifunctional enzyme from Escherichia coli exhibiting thioesterase, lysophospholipase A, protease and arylesterase activities. Recently, we demonstrated high arylesterase activity for TesA, but only minor thioesterase and no protease activity. Here, we present a comparative analysis of TesA and TAP at the structural, biochemical and physiological levels. The crystal structure of TesA was determined at 1.9 Å and structural differences were identified, providing a possible explanation for the differences in substrate specificities. The comparison of TesA with other GDSL-hydrolase structures revealed that the flexibility of active-site loops significantly affects their substrate specificity. This assumption was tested using a rational approach: we have engineered the putative coenzyme A thioester binding site of E. coli TAP into TesA of P. aeruginosa by introducing mutations D17S and L162R. This TesA variant showed increased thioesterase activity comparable to that of TAP. TesA is the first lysophospholipase A described for the opportunistic human pathogen P. aeruginosa. The enzyme is localized in the periplasm and may exert important functions in the homeostasis of phospholipids or detoxification of lysophospholipids.

Lysophospholipase I (LYPLA1) is an important protein with multiple functions. In this study, the full-length cDNA of the LYPLA1 gene from Ovis aries (OaLypla1) was cloned using primers and rapid amplification of cDNA ends (RACE) technology. The full-length OaLypla1 was 2457 bp with a 5'-untranslated region (UTR) of 24 bp, a 3'-UTR of 1740 bp with a poly (A) tail, and an open reading frame (ORF) of 693 bp encoding a protein of 230 amino acid residues with a predicted molecular weight of 24,625.78 Da. Phylogenetic analysis showed that the OaLypla1 protein shared a high amino acid identity with LYPLA1 of Bos taurus. The recombinant OaLypla1 protein was expressed and purified, and its phospholipase activity was identified. Monoclonal antibodies (mAb) against OaLypla1 that bound native OaLypla1 were generated. Real-time PCR analysis revealed that OaLypla1 was constitutively expressed in the liver, spleen, lung, kidney, and white blood cells of sheep, with the highest level in the kidney. Additionally, the mRNA levels of OaLypla1 in the buffy coats of sheep challenged with virulent or avirulent Brucella strains were down-regulated compared to untreated sheep. The results suggest that OaLypla1 may have an important physiological role in the host response to bacteria. The function of OaLypla1 in the host response to bacterial infection requires further study in the future.

Autotaxin (ATX) is a tumor cell motility-stimulating factor, originally isolated from melanoma cell supernatants. ATX had been proposed to mediate its effects through 5'-nucleotide pyrophosphatase and phosphodiesterase activities. However, the ATX substrate mediating the increase in cellular motility remains to be identified. Here, we demonstrated that lysophospholipase D (lysoPLD) purified from fetal bovine serum, which catalyzes the production of the bioactive phospholipid mediator, lysophosphatidic acid (LPA), from lysophosphatidylcholine (LPC), is identical to ATX. The Km value of ATX for LPC was 25-fold lower than that for the synthetic nucleoside substrate, p-nitrophenyl-tri-monophosphate. LPA mediates multiple biological functions including cytoskeletal reorganization, chemotaxis, and cell growth through activation of specific G protein-coupled receptors. Recombinant ATX, particularly in the presence of LPC, dramatically increased chemotaxis and proliferation of multiple different cell lines. Moreover, we demonstrate that several cancer cell lines release significant amounts of LPC, a substrate for ATX, into the culture medium. The demonstration that ATX and lysoPLD are identical suggests that autocrine or paracrine production of LPA contributes to tumor cell motility, survival, and proliferation. It also provides potential novel targets for therapy of pathophysiological states including cancer.

Our recent study has found that gut bacteria Enterococcus faecalis contributes to hypertension and upregulates lysophospholipase A1 (LYPLA1) in the renal medulla of rats. This work aimed to investigate the role of LYPLA1 in the development of E. faecalis-induced hypertension. Compared to control, E. faecalis treatment increased blood pressure (BP), serum angiotensin II, sodium reabsorption, and expression of αENaC and LYPLA1 in the renal medulla of mice, and these effects were attenuated by knockdown of LYPLA1. Moreover, the intrarenal lypla1 overexpression increased sodium reabsorption and BP. Further studies showed that LYPLA1 promoted the accumulation of renal glycerophosphocholine (GPC), which directly elevated the expression of αENaC and sodium reabsorption. In addition, enriched abundance of LYPLA1 in the renal medulla and urine was also observed in other hypertensive animals. Overall, our results demonstrate that LYPLA1 contributes to E. faecalis-induced hypertension by accumulating GPC and activating ENaC in the renal medulla.

Glycerophosphodiester phosphodiesterases (GDPD) are enzymes which degrade various glycerophosphodiesters to produce glycerol-3-phosphate and the corresponding alcohol moiety. Apart from this, a very interesting finding is that this enzyme could be used in the degradation of toxic organophosphorus esters, which has resulted in much attention on the biochemical and application research of GDPDs. In the present study, a novel GDPD from Pyrococcus furiosus DSM 3638 (pfGDPD) was successfully expressed in Escherichia coli and biochemically characterized. This enzyme hydrolyzed bis(p-nitrophenyl) phosphate, one substrate analogue of organophosphorus diester, with an optimal reaction temperature 55 °C and pH 8.5. The activity of pfGDPD was strongly dependent on existing of bivalent cations. It was strongly stimulated by Mn(2+) ions, next was Co(2+) and Ni(2+) ions. Further investigations were conducted on its substrate selectivity towards different phospholipids. The results indicated that except of glycerophosphorylcholine (GPC), this enzyme also possessed lysophospholipase D activity toward both sn1-lysophosphatidylcholine (1-LPC) and sn2-lysophosphatidylcholine (2-LPC). Higher activity was found for 1-LPC than 2-LPC; however, no hydrolytic activity was found for phosphatidylcholine (PC). Molecular docking based on the 3D-modeled structure of pfGDPD was conducted in order to provide a structural foundation for the substrate selectivity.

Although advanced lipidomics technology facilitates quantitation of intracellular lipid components, little is known about the regulation of lipid metabolism in cancer cells. Here, we show that disruption of the Gdpd3 gene encoding a lysophospholipase D enzyme significantly decreased self-renewal capacity in murine chronic myelogenous leukaemia (CML) stem cells in vivo. Sophisticated lipidomics analyses revealed that Gdpd3 deficiency reduced levels of certain lysophosphatidic acids (LPAs) and lipid mediators in CML cells. Loss of Gdpd3 also activated AKT/mTORC1 signalling and cell cycle progression while suppressing Foxo3a/β-catenin interaction within CML stem cell nuclei. Strikingly, CML stem cells carrying a hypomorphic mutation of Lgr4/Gpr48, which encodes a leucine-rich repeat (LRR)-containing G-protein coupled receptor (GPCR) acting downstream of Gdpd3, displayed inadequate disease-initiating capacity in vivo. Our data showing that lysophospholipid metabolism is required for CML stem cell maintenance in vivo establish a new, biologically significant mechanism of cancer recurrence that is independent of oncogene addiction.

In a previous study we purified a novel lysoPLD (lysophospholipase D) which converts LPC (lysophosphatidylcholine) into a bioactive phospholipid, LPA (lysophosphatidic acid), from the rat brain. In the present study, we identified the purified 42 and 35 kDa proteins as the heterotrimeric G protein subunits Gα(q) and Gβ(1) respectively. When FLAG-tagged Gα(q) or Gβ(1) was expressed in cells and purified, significant lysoPLD activity was observed in the microsomal fractions. Levels of the hydrolysed product choline increased over time, and the Mg(2+) dependency and substrate specificity of Gα(q) were similar to those of lysoPLD purified from the rat brain. Mutation of Gα(q) at amino acids Lys(52), Thr(186) or Asp(205), residues that are predicted to interact with nucleotide phosphates or catalytic Mg(2+), dramatically reduced lysoPLD activity. GTP does not compete with LPC for the lysoPLD activity, indicating that these substrate-binding sites are not identical. Whereas the enzyme activity of highly purified FLAG-tagged Gα(q) overexpressed in COS-7 cells was ~4 nmol/min per mg, the activity from Neuro2A cells was 137.4 nmol/min per mg. The calculated K(m) and V(max) values for lysoPAF (1-O-hexadecyl-sn-glycero-3-phosphocholine) obtained from Neuro2A cells were 21 μM and 0.16 μmol/min per mg respectively, similar to the enzyme purified from the rat brain. These results reveal a new function for Gα(q) and Gβ(1) as an enzyme with lysoPLD activity. Tag-purified Gα(11) also exhibited a high lysoPLD activity, but Gα(i) and Gα(s) did not. The lysoPLD activity of the Gα subunit is strictly dependent on its subfamily and might be important for cellular responses. However, treatment of Hepa-1 cells with Gα(q) and Gα(11) siRNAs (small interfering RNAs) did not change lysoPLD activity in the microsomal fraction. Clarification of the physiological relevance of lysoPLD activity of these proteins will need further studies.

Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by multiorgan hamartomas, including renal angiomyolipomas and pulmonary lymphangioleiomyomatosis (LAM). TSC2 deficiency leads to hyperactivation of mTOR Complex 1 (mTORC1), a master regulator of cell growth and metabolism. Phospholipid metabolism is dysregulated upon TSC2 loss, causing enhanced production of lysophosphatidylcholine (LPC) species by TSC2-deficient tumor cells. LPC is the major substrate of the secreted lysophospholipase D autotaxin (ATX), which generates two bioactive lipids, lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). We report here that ATX expression is upregulated in human renal angiomyolipoma-derived TSC2-deficient cells compared with TSC2 add-back cells. Inhibition of ATX via the clinically developed compound GLPG1690 suppressed TSC2-loss associated oncogenicity in vitro and in vivo and induced apoptosis in TSC2-deficient cells. GLPG1690 suppressed AKT and ERK1/2 signaling and profoundly impacted the transcriptome of these cells while inducing minor gene expression changes in TSC2 add-back cells. RNA-sequencing studies revealed transcriptomic signatures of LPA and S1P, suggesting an LPA/S1P-mediated reprogramming of the TSC lipidome. In addition, supplementation of LPA or S1P rescued proliferation and viability, neutral lipid content, and AKT or ERK1/2 signaling in human TSC2-deficient cells treated with GLPG1690. Importantly, TSC-associated renal angiomyolipomas have higher expression of LPA receptor 1 and S1P receptor 3 compared with normal kidney. These studies increase our understanding of TSC2-deficient cell metabolism, leading to novel potential therapeutic opportunities for TSC and LAM. SIGNIFICANCE: This study identifies activation of the ATX-LPA/S1P pathway as a novel mode of metabolic dysregulation upon TSC2 loss, highlighting critical roles for ATX in TSC2-deficient cell fitness and in TSC tumorigenesis.

A novel poxvirus gene has been characterized within the genome of ectromelia virus. It has significant similarity to a family of lysophospholipases suggesting that it may function in the degradation of lysophospholipids. Since these molecules are active in the stimulation of inflammation, we hypothesize that this gene may play a role in virus virulence. This gene is expressed early in the ectromelia virus replication cycle, before DNA replication. We have also characterized a human cDNA that encodes a protein which is 49.5% identical to the ectromelia virus protein. By its presence in multiple cDNA libraries, this human gene is known to be expressed in a variety of body tissues and is likely to function in the normal regulation of lysophospholipid levels. This family of proteins have conserved blocks of amino acids that are indicative of a serine-aspartic acid-histidine catalytic triad, similar to those used by true lipases and a number of esterases.

Liver fibrosis is an excessive wound-healing reaction that requires the participation of inflammatory cells and hepatic stellate cells (HSCs). The pathogenesis of liver fibrosis caused by viruses and alcohol has been well characterized, but the molecular mechanisms underlying liver fibrosis induced by the liver fluke Clonorchis sinensis are poorly understood. Lysophospholipase A (LysoPLA), which deacylates lysophospholipids, plays a critical role in mediating the virulence and pathogenesis of parasites and fungi; however, the roles of C. sinensis lysophospholipase A (CsLysoPLA) in C. sinensis-induced liver fibrosis remain unknown.

African trypanosomosis, a parasitic disease caused by protozoan parasites transmitted by tsetse flies, affects both humans and animals in sub-Saharan Africa. While the human form (HAT) is now limited to foci, the animal form (AAT) is widespread and affects the majority of sub-Saharan African countries, and constitutes a real obstacle to the development of animal breeding. The control of AAT is hampered by a lack of standardized and easy-to used diagnosis tools. This study aimed to evaluate the diagnostic potential of TbLysoPLA and TbGK proteins from Trypanosoma brucei brucei for AAT serodiagnosis in indirect ELISA using experimental and field sera, individually, in combination, and associated with the BiP C-terminal domain (C25) from T. congolense. These novel proteins were characterized in silico, and their sequence analysis showed strong identities with their orthologs in other trypanosomes (more than 60% for TbLysoPLA and more than 82% for TbGK). TbLysoPLA displays a low homology with cattle (<35%) and Piroplasma (<15%). However, TbGK shares more than 58% with cattle and between 45-55% with Piroplasma. We could identify seven predicted epitopes on TbLysoPLA sequence and 14 potential epitopes on TbGK. Both proteins were recombinantly expressed in Escherichia coli. Their diagnostic potential was evaluated by ELISA with sera from cattle experimentally infected with T. congolense and with T.b. brucei, sera from cattle naturally infected with T. congolense, T. vivax and T.b. brucei. Both proteins used separately had poor diagnostic performance. However, used together with the BiP protein, they showed 60% of sensitivity and between 87-96% of specificity, comparable to reference ELISA tests. In conclusion, we showed that the performance of the protein combinations is much better than the proteins tested individually for the diagnosis of AAT.

We previously detected a submicromolar concentration of lysophosphatidic acid (LPA) in human saliva. Here, we compare LPA concentrations in human gingival crevicular fluid (GCF) from patients with periodontitis and healthy controls, and examine how the local LPA levels are regulated enzymatically. The concentrations of LPA and its precursor lysophospholipids in GCF was measured by liquid chromatography-tandem mass spectrometry. The LPA-producing and LPA-degrading enzymatic activities were measured by quantifying the liberated choline and free fatty acid, respectively. The concentration of LPA in GCF of periodontitis patients was lower than that of healthy controls, due to higher soluble lysophospholipase activity toward LPA. LPA was found to prevent survival of Sa3, a human gingival epithelium-derived tumor cell line, activate Sa3 through Ca2+ mobilization, and release interleukin 6 from Sa3 in vitro. Furthermore, local injection of LPA into the gingiva attenuated ligature-induced experimental alveolar bone loss induced by oral bacteria inoculation in a rat model of periodontitis in vivo. A high concentration of LPA in human GCF is necessary to maintain normal gingival epithelial integrity and function, protecting the progression of periodontitis.

Autotaxin (ATX) is an extracellular lysophospholipase D that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). Both ATX and LPA have been shown to be involved in many cancers. However, the functional role of ATX and the regulation of ATX expression in human hepatocellular carcinoma (HCC) remain elusive.

A family of glycerol-based lysolipid mediators comprises lysophosphatidic acid as a representative phospholipidic member but also a monoacylglycerol as a non-phosphorus-containing member. These critical lysolipid mediators are known to be produced from different lysophospholipids by actions of lysophospholipases C and D in mammals. Some members of the glycerophosphodiesterase (GDE) family have attracted recent attention due to their phospholipid-metabolizing activity. In this study, we found selective depletion of lysophosphatidylinositol among lysophospholipids in the culture medium of COS-7 cells transfected with a vector containing glycerophosphodiester phosphodiesterase 2 (GDPD2, GDE3). Thin-layer chromatography and liquid chromatography-tandem mass spectrometry of lipids extracted from GDE3-transfected COS-7 cells exposed to fluorescent analogs of phosphatidylinositol (PI) revealed that GDE3 acted as an ecto-type lysophospholipase C preferring endogenous lysophosphatidylinositol and PI having a long-chain acyl and a short-chain acyl group rather than endogenous PI and its fluorescent analog having two long chain acyl groups. In MC3T3-E1 cells cultured with an osteogenic or mitogenic medium, mRNA expression of GDE3 was increased by culturing in 10% fetal bovine serum for several days, concomitant with increased activity of ecto-lysophospholipase C, converting arachidonoyl-lysophosphatidylinositol, a physiological agonist of G protein-coupled receptor 55, to arachidonoylglycerol, a physiological agonist of cannabinoid receptors 1 and 2. We suggest that GDE3 acts as an ecto-lysophospholipase C, by switching signaling from lysophosphatidylinositol to that from arachidonoylglycerol in an opposite direction in mouse bone remodeling.

Phospholipases are hydrolytic enzymes that play crucial roles in vivo and also possess immense biotechnological potential. In the present study, the phospholipase B of Trichosporon asahii MSR54 was overexpressed in E. coli and characterized. The 68-kDa enzyme was monomeric in solution and possessed phospholipase, lysophospholipase, esterase and acyltransferase activities. It was maximally active at pH 8.0 and 40 °C. The enzyme retained >50% activity between pH 3.0-8.0 and had a half-life of 30 min at 60 °C. Its activity was not metal dependent and was stable in the presence of most metal ions. Its catalytic efficiency on lysophosphatidyl choline was 1.0 × 103 mM-1 h-1. Site directed mutagenesis revealed R121 (present in the GYRAMV motif), S194 (present in the conserved GLSGG motif) and D420 (present in LVDXGE motif) to be the crucial amino acid residues for esterolytic activity. S194 and D420 were also the catalytic amino acids for lysophospholipase and phospholipase activities of the enzymes, while R121 was not involved in catalysis of phospholipid substrates. Further, it was found that cysteine residues in C61 and C354 were involved in disulphide linkages that imparted the properties of thiol activation and thermostability, respectively.

Previous work from our laboratory showed that the Gram-negative aquatic pathogen Vibrio cholerae can take up a much wider repertoire of fatty acids than other Gram-negative organisms. The current work elaborated on the ability of V. cholerae to exploit an even more diverse pool of lipid nutrients from its environment. We have demonstrated that the bacterium can use lysophosphatidylcholine as a metabolite for growth. Using a combination of thin-layer chromatography and mass spectrometry, we also showed that lysophosphatidylcholine-derived fatty acid moieties can be used for remodeling the V. cholerae membrane architecture. Furthermore, we have identified a lysophospholipase, VolA (Vibrio outer membrane lysophospholipase A), required for these activities. The enzyme is well conserved in Vibrio species, is coexpressed with the outer membrane fatty acid transporter FadL, is one of very few surface-exposed lipoprotein enzymes to be identified in Gram-negative bacteria and the first instance of a surface lipoprotein phospholipase. We propose a model whereby the bacterium efficiently couples the liberation of fatty acid from lysophosphatidylcholine to its subsequent metabolic uptake. An expanded ability to scavenge diverse environmental lipids at the bacterial surface increases overall bacterial fitness and promotes homeoviscous adaptation through membrane remodeling.