The role of propeptide in the refolding of human group IB phospholipase A2

Phospholipase A2 (PLA2, EC 3.1.1.4) catalyzes the hy-drolysis of the 2-acyl ester bond of sn-3-phosphoglycerides,producing a free fatty acid and a lysophospholipid [1].Mammalian secretory phospholipase A2 is tentatively sug-gested to be involved in the in…     

The expression of brain cyclooxygenase-2 is down-regulated in the cytosolic phospholipase A2 knockout mouse

We examined brain phospholipase A2 (PLA2) activity and the expression of enzymes metabolizing arachidonic acid (AA) in cytosolic PLA2 knockout () mice to see if other brain PLA2 can compensate for the absence of cPLA2 alpha and if cPLA2 couples with specific downstream enzymes in the eicosanoid biosynthetic pathway. We found that the rate of formation of prostaglandin E2 (PGE2), an index of net cyclooxygenase (COX) activity, was decreased by 62% in the compared with the control mouse brain. The decrease was accompanied by a 50-60% decrease in mRNA and protein levels of COX-2, but no change in these levels in COX-1 or in PGE synthase. Brain 5-lipoxygenase (5-LO) and cytochrome P450 epoxygenase (cyp2C11) protein levels were also unaltered. Total and Ca2+-dependent PLA2 activities did not differ significantly between and control mice, and protein levels of type VI iPLA2 and type V sPLA2, normalized to actin, were unchanged. These results show that type V sPLA2 and type VI iPLA2 do not compensate for the loss of brain cPLA2 alpha, and that this loss has significant downstream effects on COX-2 expression and PGE2 formation, sparing other AA oxidative enzymes. This suggests that cPLA2 is critical for COX-2-derived eicosanoid production in mouse brain.     

Expression and Induction of Secretory Phospholipase A Group IB in Brain

Summary Secretory phospholipases A₂ (sPLA₂) form a diverse family of enzymes involved in physiologicand pathologic processes. Common among all sPLA₂ is the ability to cleave acyl groups of phospholipids at 2_C of the glycerol backbone, thereby releasingfatty acid and a lysophospholipid. Several sPLA₂ have been cloned and characterized in various tissues.Furthermore, receptors have been identified. In the nervous system sPLA₂ groups IIA, IIE, IIF, V, and XII have been identified, and binding sites for sPLA₂ group IB (sPLA₂-IB) have been found. Here, we report sPLA₂-IB in rat and human brain as well as in neurons in primary culture. The distribution of sPLA₂-IB seems to be mainly neuronal, with the highest abundance occurring in the cerebral cortex and hippocampus. We also find that genes encoding sPLA₂-IB are induced by kainic acid and by electroshock-induced convulsions.Based on the present results we suggest that sPLA₂-IB may be a neuronal intercellular signalling modulator.     

Human group IIA secretory phospholipase A2 potentiates Ca2+ influx through L-type voltage-sensitive Ca2+ channels in cultured rat cortical neurons

Mammalian group IIA secretory phospholipase A2 (sPLA2-IIA) generates prostaglandin D2 (PGD2) and triggers apoptosis in cortical neurons. However, mechanisms of PGD2 generation and apoptosis have not yet been established. Therefore, we examined how second messengers are involved in the sPLA2-IIA-induced neuronal apoptosis in primary cultures of rat cortical neurons. sPLA2-IIA potentiated a marked influx of Ca2+ into neurons before apoptosis. A calcium chelator and a blocker of the L-type voltage-sensitive Ca2+ channel (L-VSCC) prevented neurons from sPLA2-IIA-induced neuronal cell death in a concentration-dependent manner. Furthermore, the L-VSCC blocker ameliorated sPLA2-IIA-induced morphologic alterations and apoptotic features such as condensed chromatin and fragmented DNA. Other blockers of VSCCs such as N type and P/Q types did not affect the neurotoxicity of sPLA2-IIA. Blockers of L-VSCC significantly suppressed sPLA2-IIA-enhanced Ca2+ influx into neurons. Moreover, reactive oxygen species (ROS) were generated prior to apoptosis. Radical scavengers reduced not only ROS generation, but also the sPLA2-IIA-induced Ca2+ influx and apoptosis. In conclusion, we demonstrated that sPLA2-IIA potentiates the influx of Ca2+ into neurons via L-VSCC. Furthermore, the present study suggested that eicosanoids and ROS generated during arachidonic acid oxidative metabolism are involved in sPLA2-IIA-induced apoptosis in cooperation with Ca2+.     

Induction of secretory phospholipase A2 in reactive astrocytes in response to transient focal cerebral ischemia in the rat brain

Although mRNA expression of group IIA secretory phospholipase A2 (sPLA2-IIA) has been implicated in responses to injury in the CNS, information on protein expression remains unclear. In this study, we investigated temporal and spatial expression of sPLA2-IIA mRNA and immunoreactivity in transient focal cerebral ischemia induced in rats by occlusion of the middle cerebral artery. Northern blot analysis showed a biphasic increase in sPLA2-IIA mRNA expression following 60-min of ischemia-reperfusion: an early phase at 30 min and a second increase at a late phase ranging from 12 h to 14 days. In situ hybridization localized the early-phase increase in sPLA2-IIA mRNA to the affected ischemic cortex and the late-phase increase to the penumbral area. Besides sPLA2-IIA mRNA, glial fibrillary acidic protein (GFAP) and cyclo-oxygenase-2 mRNAs, but not cytosolic PLA2, also showed an increase in the penumbral area at 3 days after ischemia-reperfusion. Immunohistochemistry of sPLA2-IIA indicated positive cells in the penumbral area similar to the GFAP-positive astrocytes but different from the isolectin B4-positive microglial cells. Confocal microscopy further confirmed immunoreactivity of sPLA2-IIA in reactive astrocytes but not in microglial cells. Taken together, these results demonstrate for the first time an up-regulation of the inflammatory sPLA2-IIA in reactive astrocytes in response to cerebral ischemia-reperfusion.     

Microglial secreted cathepsin B induces neuronal apoptosis

Activated microglia release a number of substances that can influence neuronal signalling and survival. Here we report that microglia stimulated with the peptide chromogranin A (CGA), secreted the cysteine protease, cathepsin B. Conditioned medium from CGA exposed microglia was neurotoxic to the HT22 hippocampal cell line and to primary cultures of cerebellar granule neurones. In both neuronal cell types, the neurotoxicity could be significantly attenuated with z-FA-fmk or by depletion of microglial conditioned medium with cathepsin B antibody. Conditioned medium from activated microglia or cathepsin B alone induced neuronal apoptosis and caspase 3 activation. Our data indicate that CGA-activated microglia can trigger neuronal apoptosis and that this may be mediated through the secretion of cathepsin B. Since cathepsins may also play a role in the amyloidogenic processing of amyloid precursor protein, these results may have significance for tissue damage and neuronal loss in the neuropathology of Alzheimer's disease.     

Bimodal regulatory effect of melittin and phospholipase A2-activating protein on human type II secretory phospholipase A2

Melittin and phospholipase A2-activating protein (PLAP) are known as efficient activators of secretory phospholipase A2(sPLA2) types I, II, and III when phospholipid liposomes are used as substrate. The present study demonstrates that both peptides can either inhibit or activate sPLA2 depending on the peptide/phospholipid ratio when erythrocyte membranes serve as a biologically relevant substrate. Low concentrations of melittin and PLAP were observed to inhibit sPLA2-triggered release of fatty acids from erythrocyte membranes. The inhibition was reversed at melittin concentrations above 1 microM. PLAP-induced inhibition of sPLA2 persisted steadily throughout the used concentration range (0-150 nM). The two peptides induced a dose-dependent activation of sPLA2 at low concentrations, followed by inhibition when model membranes were used as substrate. This opposite modulatory effect on biological membranes and model membranes is discussed with respect to different mechanisms the interaction of the regulatory peptides with the enzyme molecules and the substrate vesicles.     

Arachidonic acid metabolism in brain physiology and pathology: lessons from genetically altered mouse models

The arachidonic acid (AA) cascade involves the release of AA from the membrane phospholipids by a phospholipase A(2), followed by its subsequent metabolism to bioactive prostanoids by cyclooxygenases coupled with terminal synthases. Altered brain AA metabolism has been implicated in neurological, neurodegenerative, and psychiatric disorders. The development of genetically altered mice lacking specific enzymes of the AA cascade has helped to elucidate the individual roles of these enzymes in brain physiology and pathology. The roles of AA and its metabolites in brain physiology, with a particular emphasis on the phospholipase A(2)/cyclooxygenases pathway, are summarized, and the specific phenotypes of genetically altered mice relevant to brain physiology and neurotoxic models are discussed.     

Secretory phospholipase A2 promotes MMP‐9‐mediated cell death by degrading type I collagen via the ERK pathway at an early stage of chondrogenesis

Background information. sPLA₂ (secretory phospholipase A₂) has been implicated in a wide range of cellular responses, including cell proliferation and ECM (extracellular matrix) remodelling. Even though ECM remodelling is an essential step for chondrogenesis, the expression and functions of sPLA₂ during chondrogenesis have not been studied. Results. In the present study, for the first time, we detect the secretion of sPLA₂ during limb development and suggest that sPLA₂ influences the proliferation and/or survival of limb mesenchymal cells. Treatment of wing bud mesenchymal cells with exogenous sPLA₂ promoted cell death by activating MMP‐9 (matrix metalloproteinase‐9) and increasing type I collagen degradation. The additive chondro‐inhibitory actions were induced by co‐treatment of mp‐BSA (p‐aminophenyl‐mannopyranoside‐BSA), a known ligand of the mannose receptor. Chondro‐inhibitory actions by sPLA₂ were prevented by functional blocking of FcRY (chicken yolk sac IgY receptor), a mannose receptor family member that is the orthologue of the mammalian PLA₂ (phospholipase A₂) receptor and by inhibition of ERK (extracellular‐signal‐regulated kinase) activity. Conclusions. Taken together, our results suggest that elevated levels of sPLA₂ secreted by wing bud mesenchymal cells promote type I collagen degradation by MMP‐9 in a manner typical of receptor‐mediated signalling and that these events lead to cell death.     

25-hydroxycholesterol provokes oligodendrocyte cell line apoptosis and stimulates the secreted phospholipase A2 type IIA via LXR beta and PXR

In several neurodegenerative diseases of the CNS, oligodendrocytes are implicated in an inflammatory process associated with altered levels of oxysterols and inflammatory enzymes such as secreted phospholipase A2 (sPLA2). In view of the scarce literature related to this topic, we investigated oxysterol effects on these myelinating glial cells. Natural oxysterol 25-hydroxycholesterol (25-OH; 1 and 10 μM) altered oligodendrocyte cell line (158N) morphology and triggered apoptosis (75% of apoptosis after 72 h). These effects were mimicked by 22( S )-OH (1 and 10 μM) which does not activate liver X receptor (LXR) but not by a synthetic LXR ligand (T0901317). Therefore, oxysterol-induced apoptosis appears to be independent of LXR. Interestingly, sPLA2 type IIA (sPLA2-IIA) over-expression partially rescued 158N cells from oxysterol-induced apoptosis. In fact, 25-OH, 24( S )-OH, and T0901317 stimulated sPLA2-IIA promoter and sPLA2 activity in oligodendrocyte cell line. Accordingly, administration of T0901317 to mice enhanced sPLA2 activity in brain extracts by twofold. Short interfering RNA strategy allowed to establish that stimulation of sPLA2-IIA is mediated by pregnane X receptor (PXR) at high oxysterol concentration (10 μM) and by LXR β at basal oxysterol concentration. Finally, GC coupled to mass spectrometry established that oligodendrocytes contain oxysterols and express their biosynthetic enzymes, suggesting that they may act through autocrine/paracrine mechanism. Our results show the diversity of oxysterol signalling in the CNS and highlight the positive effects of the LXR/PXR pathway which may open new perspectives in the treatment of demyelinating and neurodegenerative diseases.     

The catalytically active secretory phospholipase A2 type IIA is involved in restenosis development after PTCA in human coronary arteries and generation of atherogenic LDL

Secretory phospholipase A₂ type IIA (sPLA₂) may actively contribute to atherogenesis, acting either within the arterial wall or in plasma. Proinflammatory eicosanoids and lysophospholipids, generated through hydrolysis of cell membrane phospholipids by sPLA₂, initiate and prolong the inflammatory process. In the present study we examined the possible involvement of sPLA₂ in development of restenosis in patients undergoing percutaneous transluminal coronary angioplasty (PTCA). We also investigated whether serum sPLA₂ could catalyze accumulation of lysophosphatidylcholine (LPC) in LDL. Concentrations and catalytic activities of sPLA₂ were measured in blood serum of 49 consenting patients immediately before, 1–7 and 180 days after PTCA. All patients had repeat angiograms at 180-day follow-up. Restenosis was registered in 19 patients. Accumulation of LPC in LDL was evaluated by thin-layer chromatography after incubation of blood serum with LDL. Serum sPLA₂ concentrations increased in all study patients by day 1 post-PTCA, but the increase was significantly greater and more protracted in patients who developed restenosis. Catalytic activities increased significantly 6 days post-PTCA in patients who developed restenosis, whereas for patients without restenosis there was no change in serum sPLA₂ activity throughout the study period in spite of the sPLA2 presence in blood. Incubation of blood serum (6 days post-PTCA) with LDL resulted in accumulation of LPC only for those patients who subsequently developed restenosis. Manoalide, a specific inhibitor of sPLA₂, completely blocked the LPC accumulation. The data indicate that elevated serum sPLA₂ activity after PTCA is associated with restenosis development and may be involved in atherogenic modification of LDL in blood serum. (Mol Cell Biochem 270: 107–113, 2005)     

Role of phospholipase D2 in anti-apoptotic signaling through increased expressions of Bcl-2 and Bcl-xL

We have previously reported that Fas-resistant A20 cells (FasR) have phospholipase D (PLD) activity upregulated by endogenous PLD2 overexpression. In the present study, we investigated how overexpressed PLD2 in FasR could generate survival signals by regulating the protein levels of anti-apoptotic Bcl-2 and Bcl-xL. To confirm the effect of PLD2 on Bcl-2 protein levels, we transfected PLD2 into wild-type murine B lymphoma A20 cells. The transfected cells showed markedly the increases in Bcl-2 and Bcl-xL protein levels, and became resistant to Fas-induced apoptosis, similar to FasR. Treatment of wild-type A20 cells with phosphatidic acid (PA), the metabolic end product of PLD2 derived from phosphatidylcholin, markedly increased levels of anti-apoptotic Bcl-2 and Bcl-xL proteins. Moreover, PA-induced expressions of Bcl-2 and Bcl-xL were enhanced by propranolol, an inhibitor of PA phospholydrolase (PAP), whereas completely blocked by mepacrine, an inhibitor of phospholipase A(2) (PLA(2)), suggesting that PLA(2) metabolite of PA is responsible for the increases in Bcl-2 and Bcl-xL protein levels. We further confirmed the involvement of arachidonic acid (AA) in PA-induced survival signals by showing that 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), PA without AA, was unable to increase Bcl-2 and Bcl-xL proteins. Moreover, PA notably increased cyclooxygenase (COX)-2 protein expression, and PA-induced expression of both Bcl-2 and Bcl-xL was inhibited by NS-398, a specific inhibitor of COX-2. Taken together, these findings demonstrate that PA generated by PLD2 plays an important role in cell survival during Fas-mediated apoptosis through the increased Bcl-2 and Bcl-xL protein levels which resulted from PLA(2) and AA-COX2 pathway.     

Genipin induces cell death via intrinsic apoptosis pathways in human glioblastoma cells

Genipin, a compound derived from Gardenis jasminoides Ellis fruits, was demonstrated to be the specific uncoupling protein 2 (UCP2) inhibitor. UCP2 is a mitochondrial carrier protein that creates proton leaks across the inner mitochondrial membrane, thus uncoupling oxidative phosphorylation from adenosine triphosphate (ATP) synthesis. Several studies revealed that UCP2 is broadly over-expressed in leukemia, colorectal, lung, ovarian, prostate, testicular, and bladder cancers. However, the effect of genipin still needs to be elucidated in neurological malignancies. In this study, we investigated the anticancer effect of genipin in U87MG and A172 cell lines. The anticancer effect of genipin on these cell lines was measured by microculture tetrazoliumtest (MTT), Trypan blue exclusion, and colony formation assays, in the presence of various concentrations of genipin at different time intervals. We assessed apoptosis and measure intracellular reactive oxygen species (ROS) by flow cytometry. Expression of UCP2 and some of the genes involved in apoptosis was analyzed by real-time quantitative polymerase chain reaction (PCR). Results of the MTT assay showed that genipin moderately reduced metabolic activity of both cell lines in dose- and time-dependent manner. Result of Trypan blue exclusion test indicated that the viable cell count decreased in the treated group in a concentration-dependent manner. Genipin also significantly decreased colony formation ability of these cells in a concentration-dependent manner. Result of morphological changes showed that there were significant differences in cell number and morphology in treated groups as compared with the untreated groups. Flow cytometric analysis of U87MG and A172 cells with annexin V/propidium iodide staining, 48 hours after treatment with genipin, displays 22.4% and 26.1% apoptotic population, respectively, in treated cells, in comparison to 7.42% and 9.31% apoptotic cells of untreated cells. After treatment, UCP2 and B-cell lymphoma 2 (BCL ₂) genes are downregulated, and BCL ₂ associated X protein, BCL ₂ antagonist/killer, BCL ₂ interacting killer, and Cytochrome c genes are upregulated. Genipin treatment increased mitochondrial ROS levels and also induced apoptosis through caspase-3 upregulation. In conclusion, the antiproliferative effects of genipin on the growth of both glioblastoma cell lines have been shown in all of these assays, and genipin profoundly induced apoptosis in both cell lines via the UCP2-related mitochondrial pathway through the induction of intracellular ROS.     

Cytosolic phospholipase A2 alpha inhibition prevents neuronal NMDA receptor-stimulated arachidonic acid mobilization and prostaglandin production but not subsequent cell death

Phospholipase A(2) (PLA(2)) enzymes encompass a superfamily of at least 13 extracellular and intracellular esterases that hydrolyze the sn-2 fatty acyl bonds of phospholipids to yield fatty acids and lysophospholipids. The purpose of this study was to characterize which phospholipase paralog regulates NMDA receptor-mediated arachidonic acid (AA) release. Using mixed cortical cell cultures containing both neurons and astrocytes, we found that [(3)H]-AA released into the extracellular medium following NMDA receptor stimulation (100 microM) increased with time and was completely prevented by the addition of the NMDA receptor antagonist MK-801 (10 microM) or by removal of extracellular Ca(2+). Neither diacylglycerol lipase inhibition (RHC-80267; 10 microM) nor selective inhibition of Ca(2+)-independent PLA(2) [bromoenol lactone (BEL); 10 microM] alone had an effect on NMDA receptor-stimulated release of [(3)H]-AA. Release was prevented by methyl arachidonyl fluorophosphonate (MAFP) (5 microM) and AACOCF(3) (1 microM), inhibitors of both cytosolic PLA(2) (cPLA(2)) and Ca(2+)-independent PLA(2) isozymes. This inhibition effectively translated to block of NMDA-induced prostaglandin (PG) production. An inhibitor of p38MAPK, SB 203580 (7.5 microM), also significantly reduced NMDA-induced PG production providing suggestive evidence for the role of cPLA(2)alpha. Its involvement in release was confirmed using cultures derived from mice deficient in cPLA(2)alpha, which failed to produce PGs in response to NMDA receptor stimulation. Interestingly, neither MAFP, AACOCF(3) nor cultures derived from cPLA(2)alpha null mutant animals showed any protection against NMDA-mediated neurotoxicity, indicating that inhibition of this enzyme may not be a viable protective strategy in disorders of the cortex involving over-activation of the NMDA receptor.     

Group IB secretory phospholipase A2 promotes matrix metalloproteinase-2-mediated cell migration via the phosphatidylinositol 3-kinase and Akt pathway

Secretory phospholipase A(2) (sPLA(2)), abundantly expressed in various cells including fibroblasts, is able to promote proliferation and migration. Degradation of collagenous extracellular matrix by matrix metalloproteinase (MMP) plays a role in the pathogenesis of various destructive disorders, such as rheumatoid arthritis, tumor invasion, and metastasis. Here we show that group IB PLA(2) increased pro-MMP-2 activation in NIH3T3 fibroblasts. MMP-2 activity was stimulated by group IB PLA(2) in a dose- and time-dependent manner. Consistent with MMP-2 activation, sPLA(2) decreased expression of type IV collagen. These effects are due to the reduction of tissue inhibitor of metalloproteinase-2 (TIMP-2) and the activation of the membrane type1-MMP (MT1-MMP). The decrease of TIMP-2 levels in conditioned media and the increase of MT1-MMP levels in plasma membrane were observed. In addition, treatment of cells with decanoyl Arg-Val-Lys-Arg-chloromethyl ketone, an inhibitor of pro-MT1-MMP, suppressed sPLA(2)-mediated MMP-2 activation, whereas treatment with bafilomycin A1, an inhibitor of H(+)-ATPase, sustained MMP-2 activation by sPLA(2). The involvement of phosphatidylinositol 3-kinase (PI3K) and Akt in the regulation of MMP-2 activity was further suggested by the findings that PI3K and Akt were phosphorylated by sPLA(2). Expression of p85alpha and Akt mutants, or pretreatment of cells with LY294002, a PI3K inhibitor, attenuated sPLA(2)-induced MMP-2 activation and migration. Taken together, these results suggest that sPLA(2) increases the pro-MMP-2 activation and migration of fibroblasts via the PI3K and Akt-dependent pathway. Because MMP-2 is an important factor directly involved in the control of cell migration and the turnover of extracellular matrix, our study may provide a mechanism for sPLA(2)-promoted fibroblasts migration.     

Secretory phospholipase A2 in SARS-CoV-2 infection and multisystem inflammatory syndrome in children (MIS-C)

Secretory phospholipase 2 (sPLA2) acts as a mediator between proximal and distal events of the inflammatory cascade. Its role in SARS-CoV-2 infection is unknown, but could contribute to COVID-19 inflammasome activation and cellular damage. We present the first report of plasma sPLA2 levels in adults and children with COVID-19 compared with controls. Currently asymptomatic adults with a history of recent COVID-19 infection (≥4 weeks before) identified by SARS-CoV-2 IgG antibodies had sPLA2 levels similar to those who were seronegative (9 ± 6 vs.17 ± 28 ng/mL, P = 0.26). In contrast, children hospitalized with severe COVID-19 had significantly elevated sPLA2 compared with those with mild or asymptomatic SARS-CoV-2 infection (269 ± 137 vs. 2 ± 3 ng/mL, P = 0.01). Among children hospitalized with multisystem inflammatory syndrome in children (MIS-C), all had severe disease requiring pediatric intensive care unit (PICU) admission. sPLA2 levels were significantly higher in those with acute illness <10 days versus convalescent disease ≥10 days (540 ± 510 vs. 2 ± 1, P = 0.04). Thus, sPLA2 levels correlated with COVID-19 severity and acute MIS-C in children, implicating a role in inflammasome activation and disease pathogenesis. sPLA2 may be a useful biomarker to stratify risk and guide patient management for children with acute COVID-19 and MIS-C. Therapeutic compounds targeting sPLA2 and inflammasome activation warrant consideration.     

Serotonin 5-HT(2A) receptor activation induces 2-arachidonoylglycerol release through a phospholipase c-dependent mechanism

To date, several studies have demonstrated that phospholipase C-coupled receptors stimulate the production of endocannabinoids, particularly 2-arachidonoylglycerol. There is now evidence that endocannabinoids are involved in phospholipase C-coupled serotonin 5-HT(2A) receptor-mediated behavioral effects in both rats and mice. The main objective of this study was to determine whether activation of the 5-HT(2A) receptor leads to the production and release of the endocannabinoid 2-arachidonoylglycerol. NIH3T3 cells stably expressing the rat 5-HT(2A) receptor were first incubated with [(3)H]-arachidonic acid for 24 h. Following stimulation with 10 mum serotonin, lipids were extracted from the assay medium, separated by thin layer chromatography, and analyzed by liquid scintillation counting. Our results indicate that 5-HT(2A) receptor activation stimulates the formation and release of 2-arachidonoylglycerol. The 5-HT(2A) receptor-dependent release of 2-arachidonoylglycerol was partially dependent on phosphatidylinositol-specific phospholipase C activation. Diacylglycerol produced downstream of 5-HT(2A) receptor-mediated phospholipase D or phosphatidylcholine-specific phospholipase C activation did not appear to contribute to 2-arachidonoylglycerol formation in NIH3T3-5HT(2A) cells. In conclusion, our results support a functional model where neuromodulatory neurotransmitters such as serotonin may act as regulators of endocannabinoid tone at excitatory synapses through the activation of phospholipase C-coupled G-protein coupled receptors.     

Group IB phospholipase A2 from Pseudonaja textilis

Pseudonaja textilis, an Australian Elapid, is known to produce a highly toxic venom. Both protein profiling and N-terminal sequence analysis showed the presence of four new phospholipases A(2) in this venom. Besides being non-lethal, the phospholipase A(2) proteins were found to be moderately active enzymes and they showed procoagulant property. cDNA cloning and characterization indicated the presence of two isoforms of PLA(2) proteins in a single snake, each containing the "pancreatic loop," characteristic of group IB phospholipase A(2). The genomic cloning also confirmed the presence of two genes each containing four exons that are interrupted by three introns. Phylogenetic analysis showed that the venom group IB PLA(2) gene is primitive and could have evolved from the same ancestor as the mammalian and venom group IA PLA(2) genes. In the present study, we report that the Pt-PLA2 gene could be responsible for the production of PL1, 2, and 3 possibly via RNA editing process.     

Inhibition of secretory phospholipase A2 enzyme by bilirubin: A new role as endogenous anti-inflammatory molecule

Bilirubin is a powerful antioxidant that suppresses the inflammatory process. However its interaction with proinflammatory PLA₂ enzyme is not known. Inhibition of several secretory phospholipase A₂ (sPLA₂) enzyme activities by bilirubin was studied using ¹⁴C-oleate labeled Escherichia coli as substrate. Bilirubin inhibits purified sPLA₂ enzyme from Vipera russellii and Naja naja venom and partially purified sPLA₂ enzymes from human ascitic fluid, pleural fluid and normal serum in a dose dependent manner. IC₅₀ values calculated for these enzymes ranges from 1.75 to 10.5 μM. Inflammatory human sPLA₂ enzymes are more sensitive to inhibition by bilirubin than snake venom sPLA₂s. Inhibition of sPLA₂ activity by bilirubin is independent of calcium concentration. Increasing substrate concentration (upto 180 nmol) did not relieve the inhibition of sPLA₂ by bilirubin and it is irreversible. Bilirubin quenched the relative fluorescence intensity of sPLA₂ in a dose dependent manner in the same concentration range at which in vitro sPLA₂ inhibition was observed. In the presence of bilirubin, apparent shift in the far UV-CD spectra of sPLA₂ was observed, indicating a direct interaction with the enzyme. Inhibition of sPLA₂ induced mouse paw edema by bilirubin confirms its sPLA₂ inhibitory activity in vivo also. These findings indicate that inhibition of sPLA₂ by bilirubin is mediated by direct interaction with the enzyme and bilirubin may act as an endogenous regulator of sPLA₂ enzyme activity.