The phospholipase A2 superfamily and its group numbering system.

The superfamily of phospholipase A(2) (PLA(2)) enzymes currently consists of 15 Groups and many subgroups and includes five distinct types of enzymes, namely the secreted PLA(2)s (sPLA(2)), the cytosolic PLA(2)s (cPLA(2)), the Ca(2+) independent PLA(2)s (iPLA(2)), the platelet-activating factor acetylhydrolases (PAF-AH), and the lysosomal PLA(2)s. In 1994, we established the systematic Group numbering system for these enzymes. Since then, the PLA(2) superfamily has grown continuously and over the intervening years has required several updates of this Group numbering system. Since our last update, a number of new PLA(2)s have been discovered and are now included. Additionally, tools for the investigation of PLA(2)s and approaches for distinguishing between the different Groups are described.

Cytokine-stimulated secretion of group II phospholipase A2 by rat mesangial cells. Its contribution to arachidonic acid release and prostaglandin synthesis by cultured rat glomerular cells.

Potent pro-inflammatory cytokines, such as interleukin 1 (IL-1) or tumor necrosis factor (TNF) alpha have been found to increase group II phospholipase A2 (PLA2) synthesis and secretion by mesangial cells. In all cases 85-90% of the enzyme is secreted from the cells and a parallel increase in prostaglandin (PG)E2 synthesis is observed. We report here that co-incubation with a monoclonal antibody that specifically binds and neutralizes rat group II PLA2 attenuates IL-1 beta and TNF alpha-stimulated PGE2 production by 45% and 52%, respectively. CGP43182, a specific inhibitor of group II PLA2, potently blocks mesangial cell group II PLA2 in vitro with a half-maximal inhibitory concentration (IC50) of 1.5 microM, while only slightly affecting mesangial cell high molecular weight PLA2. CGP 43182 markedly attenuates IL-1 beta- and TNF alpha-stimulated PGE2 synthesis in intact mesangial cells with IC50's of 1.3 and 1.0 microM, respectively. PLA2 secreted from cytokine-stimulated mesangial cells was purified to homogeneity. Addition of the purified enzyme to unstimulated mesangial cells causes a marked release of arachidonic acid and a subsequent increased synthesis of PGE2. Moreover, addition of purified PLA2 to a cloned rat glomerular epithelial cell line and cultured bovine glomerular endothelial cells augmented both arachidonic acid release and PGE2 synthesis, with the endothelial cells being especially sensitive. Thus, cytokine-triggered synthesis and secretion of group II PLA2 by mesangial cells contributes, at least in part, to the observed synthesis of PGE2 that occurs in parallel to the enzyme secretion. Furthermore, extracellular PLA2 secreted by mesangial cells is able to stimulate arachidonic acid release and PGE2 synthesis by the adjacent endothelial and epithelial cells. These data suggest that expression and secretion of group II PLA2 triggered by pro-inflammatory cytokines may crucially participate in the pathogenesis of inflammatory processes within the glomerulus.

Cell-specific regulation of type II phospholipase A2 expression in rat mesangial cells.

IL-1 stimulates mesangial cells to synthesize specific proteins, including a non-pancreatic (Type II) phospholipase A2 (PLA2). We have studied the regulation of PLA2 by proinflammatory mediators, implicated in the pathogenesis of glomerulonephritis, and have assessed whether the activation of second messenger systems modulates or mimics PLA2 gene expression by cytokines. IL-1 alpha and beta, TNF alpha, and LPS, but not serum, IL-2, or PDGF, potently induce PLA2 mRNA, and enzyme expression. IL-1-stimulated mesangial cells express a 1.0 kB PLA2 mRNA transcript that is induced in a dose- and time-dependent manner. IL-1-stimulated increases in steady-state PLA2 mRNA abundance result from a moderate increase in PLA2 transcription rate that is amplified by the prolonged persistence of the transcript. Forskolin and dibutyryl cAMP potentiate IL-1-induced PLA2 mRNA and enzyme expression, but have no effect in the absence of cytokine. 12-tetradecanoyl phorbol 13-acetate, sn-1, 2-dioctanoyl glycerol or 1-oleoyl-2-acetyl-sn-glycerol fail to induce PLA2 expression or to alter the effect of IL-1 when coincubated with the cytokine. In contrast, serum deprivation for 24 h specifically enhances IL-1-stimulated PLA2. Genistein potentiates PLA2 mRNA expression in cells exposed to both IL-1 and serum. The inhibitory effect of serum on IL-1-induced PLA2 mRNA abundance is reproduced by PDGF but not dexamethasone. These data demonstrate that the signaling pathways directly engaged by IL-1 to induce PLA2 expression in mesangial cells interact with several second messenger systems in a cell-specific manner. We speculate that IL-1 induces specialized changes in mesangial cell structure and function through direct activation of a transcription factor(s), that result in induction of a specific gene set.

Posttranscriptional effect of insulin-like growth factor-I on interleukin-1beta-induced type II-secreted phospholipase A2 gene expression in rabbit articular chondrocytes.

Large amounts of type II-secreted phospholipase A2 (type II sPLA2) are secreted into inflammatory synovial fluid and they are believed to induce the synthesis of lipid mediators by articular chondrocytes. Preliminary experiments showed that insulin-like growth factor-I, which counteracts cartilage degradation in arthritis, inhibits interleukin-1beta-induced type II sPLA2 gene expression in rabbit articular chondrocytes (Berenbaum, F., G. Thomas, S. Poiraudeau, G. Bereziat, M.T. Corvol, and J. Masliah. 1994. FEBS Lett. 340: 51-55). The present study showed that IL-1beta induced the sustained synthesis of prostaglandin E2 and a parallel increase in type II sPLA2 gene expression (assessed by enzymatic activity and Northern blot analysis), but no increase in cytosolic PLA2 gene expression (assessed by Northern and Western blot analysis) or cytosolic PLA2 activity in rabbit articular chondrocytes. IGF-I inhibited both IL-1beta-stimulated PGE2 synthesis and type II sPLA2 gene expression, but had no effect on cytosolic PLA2 gene expression. Nuclear run-on experiments revealed that IL-1beta stimulated the transcription rate of type II sPLA2 gene, giving rise to long-lived mRNA in cells treated with actinomycin D. IGF-I did not affect transcription rate, suggesting that it acts as a post-transcriptional step. Sucrose density gradient analysis of the translation step showed no effect of IGF-I on the entry of type II sPLA2 mRNA into the polysomal pool or on its distribution into the various polysomal complexes, suggesting that IGF-I does not act on the translation of the mRNA. Lastly, IGF-I strongly decreased the half-life of IL-1beta-induced type II sPLA2 mRNA (from 92 to 12 h), suggesting that IGF-I destabilizes mRNA. These data demonstrate that IL-1beta stimulates the transcription rate of the type II sPLA2 gene and gives rise to a very stable mRNA. In contrast, IGF-I decreases the half-life of the type II sPLA2 message.

Phospholipase A2 in chamber angle of normal eyes and patients with primary open angle glaucoma and exfoliation glaucoma.

PURPOSE: Phospholipase A2 (PLA2) is a growing family of lipolytic enzymes that play a key role in various biological processes including general lipid metabolism, membrane homeostasis, and in diseases such as atherosclerosis, arthritis, and acute pancreatitis. Oxidative stress as well as inflammation may be associated with glaucoma pathogenesis. Therefore, our aim was to examine the expression of group IIA secretory PLA2 (sPLA2-IIA), group V secretory PLA2 (sPLA2-V), calcium-independent PLA2 (iPLA2), and cytosolic PLA2 (cPLA2) type in the trabecular meshwork (TM) and the canal of Schlemm in normal eyes and in juxtacanalicular tissue samples from patients with primary open angle glaucoma (POAG) or exfoliation glaucoma (ExG). METHODS: TM tissues were isolated from healthy donor eyes for corneal transplantation. Specimens of inner wall of the Schlemm's canal and the juxtacanalicular tissue were collected during deep sclerectomy from the eyes of patients who had POAG or ExG. Antibodies against PLA2s (sPLA2-IIA, sPLA2-V, iPLA2, and cPLA2) and a standard immunohistochemical procedure were used for the analysis. Quantification of immunoreactions was provided using a Photoshop-based image analysis. Double-staining immunofluorescence of macrophages and sPLA2-IIA was performed by using confocal microscopy. RESULTS: sPLA2-IIA was not present in normal TM. In contrast, sPLA2-IIA levels were significantly higher in glaucoma patients than in controls. Furthermore, sPLA2-IIA expression was much higher in POAG when compared to ExG. iPLA2 was found to predominate in normal human TM, and it demonstrated strong labeling in the uveal and corneoscleral meshwork. The staining of juxtacanalicular meshwork was only moderate in density. In contrast, expression of the enzyme was significantly decreased in glaucoma patients, especially in ExG, when compared to normal controls or to POAG. In addition, strong regional differences were detected in sPLA2-IIA and iPLA2 levels in POAG, whereas immunostaining of these enzymes was much lower and rather uniform throughout ExG sample. In POAG, sPLA2-IIA staining was restricted to certain parts of the trabecular samples where sPLA2-IIA positive macrophages were also present. Immunostaining of sPLA2-V or cPLA2 was low, and no significant changes were found in levels of these enzymes between normal and glaucomatous samples. CONCLUSIONS: sPLA2-IIA, an oxidative stress marker in atherosclerosis, is overexpressed especially in POAG. This result supports the hypothesis that oxidative stress may play a significant role in the pathogenesis of POAG. In ExG, a dramatic decrease in the expression level of iPLA2, a housekeeping enzyme in phospholipid remodeling, may indicate imbalance in phospholipid turnover and also inhibition of normal physiological functions in the TM. These findings may contribute to understanding the pathogenesis of POAG and ExG and may be important for the development of novel therapeutic strategies to different glaucomas.

Multiple forms of secretory phospholipase A2 in plants.

Multiple secretory phospholipase A2 (sPLA2) genes have been identified in plants and encode isoforms with distinct regulatory and catalytic properties. Elucidation of this genetic and biochemical heterogeneity has provided important clues to the regulation and function of the individual enzymes. An increasing body of evidence shows that their lipid products, lysophospholipids and free fatty acids, mediate a variety of cellular responses, including plant growth, development, and responses to stress and defense. This review discusses the newly-acquired information on plant sPLA2s including the molecular and biochemical characteristics, and signaling functions of each isoform.

Differential inhibition of group IVA and group VIA phospholipases A2 by 2-oxoamides.

Inhibitors of the Group IVA phospholipase A(2) (GIVA cPLA(2)) and GVIA iPLA(2) are useful tools for defining the roles of these enzymes in cellular signaling and inflammation. We have developed inhibitors of GVIA iPLA(2) building upon the 2-oxoamide backbone that are uncharged, containing ester groups. Although the most potent inhibitors of GVIA iPLA(2) also inhibited GIVA cPLA(2), there were three 2-oxoamide compounds that selectively and weakly inhibited GVIA iPLA(2). We further show that several potent 2-oxoamide inhibitors of GIVA cPLA(2) containing free carboxylic groups (Kokotos et al. J. Med. Chem. 2002, 45, 2891-2893) do not inhibit GVIA iPLA(2) and are, therefore, selective GIVA cPLA(2) inhibitors.

Discovery of novel [Arg49]phospholipase A2 isozymes from Protobothrops elegans venom and regional evolution of Crotalinae snake venom phospholipase A2 isozymes in the southwestern islands of Japan and Taiwan.

Protobothrops (formerly Trimeresurus) elegans, a Crotalinae snake, inhabits Ishigaki and Iriomote islands of the Sakishima Islands of Japan which are located between Okinawa island of Japan and Taiwan. Two phospholipase A(2) (PLA(2)) isozymes were purified to homogeneity from P. elegans venom and sequenced. This led to a discovery of novel PLA(2) isozymes with Arg at position 49, that is, [Arg(49)]PLA(2) forms, named PeBP(R)-I and PeBP(R)-II. They are polymorphic at position 3, Val for PeBP(R)-I and Ile for PeBP(R)-II. The cDNAs encoding PeBP(R)-I and PeBP(R)-II were cloned. The cDNA encoding an [Asp(49)]PLA(2) named PePLA(2) was also obtained. In contrast to PLA(2) isozymes from Protobothrops genus with 122 amino acid residues, PeBP(R)-I and PeBP(R)-II are composed of 121 amino acid residues due to lack of Pro at position 90. They exhibited necrotic and edema-inducing activities but no hemorrhagic activity was detected. A phylogenetic tree constructed for venom PLA(2) isozymes of Protobothrops genus and of related genera in the southwestern islands of Japan and Taiwan revealed that PeBP(R)-I and PeBP(R)-II of P. elegans are evolutionarily much closer to PmK49PLA(2), a [Lys(49)]PLA(2), from P. mucrosquamatus (Taiwan) than BPI and BPII, both [Lys(49)]PLA(2) forms, from P. flavoviridis (Amami-Oshima and Tokunoshima islands of Japan). Such evolutionary relationships are also seen in neutral [Asp(49)]PLA(2) isozymes from the three Protobothrops species. Thus, P. elegans is the species much closer to P. mucrosquamatus than P. flavoviridis. Their evolutionary distances seem to be well related to geological history of the islands where they have lived. In addition, it was clearly noted that Ovophis okinavensis (Amami-Oshima), which had formerly belonged to the Trimeresurus genus, and Trimeresurus stejnegeri (Taiwan) are the species fairly distant from Protobothrops genus.

The molecular biology of the group VIA Ca2+-independent phospholipase A2.

The group VIA PLA2 is a member of the PLA2 superfamily. This enzyme, which is cytosolic and Ca2+-independent, has been designated iPLA2beta to distinguish it from another recently cloned Ca2+-independent PLA2. Features of iPLA2beta molecular structure offer some insight into possible cellular functions of the enzyme. At least two catalytically active iPLA2beta isoforms and additionalsplicing variants are derived from a single gene that consists of at least 17 exons located on human chromosome 22q13.1. Potential tumor suppressor genes also reside at or near this locus. Structural analyses reveal that iPLA2beta contains unique structural features that include a serine lipase consensus motif (GXSXG), a putative ATP-binding domain, an ankyrin-repeat domain, a caspase-3 cleavage motif DVTD138Y/N, a bipartite nuclear localization signal sequence, and a proline-rich region in the human long isoform. iPLA2beta is widely expressed among mammalian tissues, with highest expression in testis and brain. iPLA2beta prefers to hydrolyze fatty acid at the sn-2 fatty acid substituent but also exhibits phospholipase A1, lysophospholipase, PAF acetylhydrolase, and transacylase activities. iPLA2beta may participate in signaling, apoptosis, membrane phospholipid remodeling, membrane homeostasis, arachidonate release, and exocytotic membrane fusion. Structural features and the existence of multiple splicing variants of iPLA2beta suggest that iPLA2beta may be subject to complex regulatory mechanisms that differ among cell types. Further study of its regulation and interaction with other proteins may yield insight into how its structural features are related to its function.

Phospolipase A2 and apoptosis.

Phospolipase A(2) (PLA(2)) is the esterase activity that cleaves the sn-2 ester bond in glycerophospholipids, releasing free fatty acids and lysophospholipids. The PLA(2) activity is found in a variety of enzymes which can be divided in several types based on their Ca(2+) dependence for their activity; Ca(2+)-dependent secretory phosholipases (sPLA(2)s) and cytosolic phospholipases (cPLA(2)s), and Ca(2+)-independent phospholipase A(2)s (iPLA(2)s). These enzymes also show diverse size and substrate specificity (i.e., in the fatty acid chain length and extent of saturation). Among the fatty acids released by PLA(2), arachidonic acid (AA) is of particular biological importance, because it is subsequently converted to prostanoids and leukotrienes by cyclooxygenases (COX) and lipoxygenases (LOX), respectively. Free AA may also stimulate apoptosis through activation of sphingomyelinase. Alternatively, it is suggested that oxidized metabolites generated from AA by LOX induce apoptosis. Although the precise mechanisms remain to be elucidated, changes are observed in glycerolipid metabolism during apoptotic processes. In some cells induced to undergo apoptosis, AA is released concomitant with loss of cell viability, caspase activation and DNA fragmentation. Such AA releases appear to be mediated by activation of cPLA(2) and/or iPLA(2). For example, tumor necrosis factor-alpha (TNF-alpha)-induced cell death is mediated by cPLA(2), whereas Fas-induced apoptosis appears to be mediated by iPLA(2). Some discrepancies among early experimental results were probably caused by differences in the experimental conditions such as the serum concentration, inhibitors used that are not necessarily specific to a single-type enzyme, or differential expression of each PLA(2) in cells employed in the experiments. Recent studies eliminated such problems, by carefully defining the experimental conditions, and using multiple inhibitors that show different specificities. Accordingly, more convincing data are available that demonstrate involvement of some PLA(2)s in the apoptotic processes. In addition to cPLA(2) and iPLA(2), sPLA(2)s were recently found to play roles in apoptosis. Moreover, new proteins that appear to control PLA(2)s are being discovered. Here, the roles of PLA(2)s in apoptosis are discussed by reviewing recent reports.

Trifluoromethyl ketones and methyl fluorophosphonates as inhibitors of group IV and VI phospholipases A(2): structure-function studies with vesicle, micelle, and membrane assays.

A series of fatty alkyl trifluoromethyl ketones and methyl fluorophosphonates have been prepared and tested as inhibitors and inactivators of human groups IV and VI phospholipases A(2) (cPLA(2) and iPLA(2)). Compounds were analyzed with phospholipid vesicle-, detergent-phospholipid mixed-micelle-, and natural membrane-based assays, and, with few exceptions, the relative inhibitor potencies measured with the three assays were similar. Ph(CH(2))(4)COCF(3) and Ph(CH(2))(4)PO(OMe)F emerged as a potent inhibitor and inactivator, respectively, of iPLA(2), and both are poorly effective against cPLA(2). Of all 13 fatty alkyl trifluoromethyl ketones tested, the trifluoromethyl ketone analog of arachidonic acid is the most potent cPLA(2) inhibitor, and structurally similar compounds including the trifluoromethyl ketone analog of docosahexenoic acid are much poorer cPLA(2) inhibitors. Inactivation of cPLA(2) by fatty alkyl fluoromethylphosphonates is greatly promoted by binding of enzyme to the interface. The use of both vesicles and mixed micelles to assay phospholipase A(2) inhibitors and inactivators present at low mol fraction in the interface provides reliable rank order potencies of a series of compounds that correlate with their behavior in a natural membrane assay.

The expanding superfamily of phospholipase A(2) enzymes: classification and characterization.

The phospholipase A(2) (PLA(2)) superfamily consists of a broad range of enzymes defined by their ability to catalyze the hydrolysis of the middle (sn-2) ester bond of substrate phospholipids. The hydrolysis products of this reaction, free fatty acid and lysophospholipid, have many important downstream roles, and are derived from the activity of a diverse and growing superfamily of PLA(2) enzymes. This review updates the classification of the various PLA(2)'s now described in the literature. Four criteria have been employed to classify these proteins into one of the 11 Groups (I-XI) of PLA(2)'s. First, the enzyme must catalyze the hydrolysis of the sn-2 ester bond of a natural phospholipid substrate, such as long fatty acid chain phospholipids, platelet activating factor, or short fatty acid chain oxidized phospholipids. Second, the complete amino acid sequence of the mature protein must be known. Third, each PLA(2) Group should include all of those enzymes that have readily identifiable sequence homology. If more than one homologous PLA(2) gene exists within a species, then each paralog should be assigned a Subgroup letter, as in the case of Groups IVA, IVB, and IVC PLA(2). Homologs from different species should be classified within the same Subgroup wherever such assignments are possible as is the case with zebra fish and human Group IVA PLA(2) orthologs. The current classification scheme does allow for historical exceptions of the highly homologous Groups I, II, V, and X PLA(2)'s. Fourth, catalytically active splice variants of the same gene are classified as the same Group and Subgroup, but distinguished using Arabic numbers, such as for Group VIA-1 PLA(2) and VIA-2 PLA(2)'s. These four criteria have led to the expansion or realignment of Groups VI, VII and VIII, as well as the addition of Group XI PLA(2) from plants.

Calcium-independent phospholipase A(2): structure and function.

The classical Ca(2+)-independent phospholipase A(2) enzyme, now known as Group VIA PLA(2), was initially purified and characterized from the P388D(1) macrophage-like cell line. The corresponding cDNA was subsequently cloned from a variety of sources, and it is now known that multiple splice variants of the enzyme are expressed, some of which may act as negative regulators of the active enzyme. Group VIA PLA(2) has a consensus lipase motif (GTSTG) containing the catalytic serine, is 85-88 kDa, and exists in an aggregated form. The enzyme contains multiple ankyrin repeats, which may play a role in oligomerization. The Group VIA enzyme exhibits lysophospholipase activity as well as phospholipase A(2) activity, and it is capable of hydrolyzing a wide variety of phospholipid substrates. A major function of Group VIA PLA(2) is to mediate phospholipid remodeling, but the enzyme may play other roles as well. Other Ca(2+)-independent PLA(2) enzymes have more recently been identified, and it may be possible to discriminate between the various Ca(2+)-independent PLA(2) enzymes based on sequence or inhibitor-sensitivity. However, the physiological functions of the newly identified enzymes have yet to be elucidated.

Increasing molecular diversity of secreted phospholipases A(2) and their receptors and binding proteins.

Secreted phospholipases A(2) (sPLA(2)s) form a large family of structurally related enzymes which are widespread in nature. Snake venoms are known for decades to contain a tremendous molecular diversity of sPLA(2)s which can exert a myriad of toxic and pharmacological effects. Recent studies indicate that mammalian cells also express a variety of sPLA(2)s with ten distinct members identified so far, in addition to the various other intracellular PLA(2)s. Furthermore, scanning of nucleic acid databases fueled by the different genome projects indicates that several sPLA(2)s are also present in invertebrate animals like Drosophila melanogaster as well as in plants. All of these sPLA(2)s catalyze the hydrolysis of glycerophospholipids at the sn-2 position to release free fatty acids and lysophospholipids, and thus could be important for the biosynthesis of biologically active lipid mediators. However, the recent identification of a variety of membrane and soluble proteins that bind to sPLA(2)s suggests that the sPLA(2) enzymes could also function as high affinity ligands. So far, most of the binding data have been accumulated with venom sPLA(2)s and group IB and IIA mammalian sPLA(2)s. Collectively, venom sPLA(2)s have been shown to bind to membrane and soluble mammalian proteins of the C-type lectin superfamily (M-type sPLA(2) receptor and lung surfactant proteins), to pentraxin and reticulocalbin proteins, to factor Xa and to N-type receptors. Venom sPLA(2)s also associate with three distinct types of sPLA(2) inhibitors purified from snake serum that belong to the C-type lectin superfamily, to the three-finger protein superfamily and to proteins containing leucine-rich repeats. On the other hand, mammalian group IB and IIA sPLA(2)s can bind to the M-type receptor, and group IIA sPLA(2)s can associate with lung surfactant proteins, factor Xa and proteoglycans including glypican and decorin, a mammalian protein containing a leucine-rich repeat.

Roles of secretory phospholipases A(2) in inflammatory diseases and trauma.

Six distinct secretory small molecular weight phospholipases A(2) (PLA(2)) have been cloned and characterized from human tissues. Two of them, pancreatic group IB PLA(2) (PLA(2)-IB) and synovial-type group IIA PLA(2) (PLA(2)-IIA) have been studied as to their association to various inflammatory diseases. PLA(2)-IB is a digestive enzyme synthesized by pancreatic acinar cells. In acute pancreatitis, which is characterized by destruction of pancreatic tissue, PLA(2)-IB is released into the circulation, but its role in pancreatic and other tissue damage is still hypothetical. The concentration of PLA(2)-IIA increases in blood plasma in generalized inflammatory response resulting from infections, chronic inflammatory diseases, acute pancreatitis, trauma and surgical operations. PLA(2)-IIA is synthesized in a number of gland cells and is present in cellular secretions on mucosal surfaces including Paneth cells of intestinal mucosa, prostatic gland cells and seminal plasma, and lacrimal glands and tears. PLA(2)-IIA is expressed in hepatoma-derived cells in vitro and hepatocytes in vivo. PLA(2)-IIA is regarded as an acute phase protein and seems to function as an antibacterial agent especially effective against Gram-positive bacteria. Other putative functions in the inflammatory reaction include hydrolysis of cell membrane phospholipids and release of arachidonic acid for prostanoid synthesis.

Sera of patients suffering from inflammatory diseases contain group IIA but not group V phospholipase A(2).

During recent years, the high phospholipase A(2) (PLA(2)) concentrations at sites of inflammation and in circulation in several life-threatening diseases, such as sepsis, multi-organ dysfunction and acute respiratory distress syndrome, has generally been ascribed to the non-pancreatic group IIA PLA(2). Recently the family of secreted low molecular mass PLA(2) enzymes has rapidly expanded. In some cases, a newly described enzyme appeared to be cross-reactive with antibodies against the group IIA enzyme. For this reason, reports describing the expression of group IIA PLA(2) during inflammatory conditions need to be reevaluated. Here we describe the identification of the PLA(2) activity in sera of acute chest syndrome patients and in sera of trauma victims. In both cases, the PLA(2) activity was identified as group IIA. This classification was based upon cross-reactivity with monoclonal antibodies against group IIA PLA(2) which do not recognize the recombinant human group V enzyme. Moreover, purification of the enzymatic activity from the two sera followed by N-terminal amino acid sequence analyses revealed only the presence of group IIA enzyme.

Regulation of the expression of group IIA and group V secretory phospholipases A(2) in rat mesangial cells.

Rat mesangial cells synthesize and secrete a secretory phospholipase A(2) upon stimulation of the cells with cytokines, like IL-1beta and TNF and with cAMP elevating agents like forskolin. This enzyme was previously characterized to belong to group IIA sPLA(2). The discovery of several other low molecular weight phospholipases, like group IIC in murine testis and group V in human and rat heart, prompted investigations on the presence of group IIC and group V sPLA(2) in rat mesangial cells. This was done by isolating the RNA from stimulated cells and performing RT-PCR, using primers specific for group IIC and V sPLA(2). The results indicate that rat mesangial cells upon stimulation express next to group IIA also group V sPLA(2). No indications were obtained for the expression of group IIC sPLA(2). The regulation of the expression of group V sPLA(2) at the mRNA level was further investigated by examining the time-dependent expression, the influence of dexamethasone and the signaling route of the IL-1beta stimulation. The results show that the IL-1beta induced expression of group V sPLA(2) mRNA was time dependent and, similar to that of group IIA sPLA(2) mRNA, involves activation of NF-kappaB. However, in contrast to the group IIA sPLA(2), the expression of group V sPLA(2) was not influenced by the presence of dexamethasone. The expression of both phospholipases was also examined at the protein level in stimulated mesangial cells. Western blot analysis shows that stimulated mesangial cells synthesize both group IIA and group V sPLA(2) protein but the expression of group V is lower compared to that of group IIA sPLA(2). In addition, the extent of secretion into the medium appears to be considerably higher for group IIA than for group V sPLA(2).

Comparative proteomics and subtyping of venom phospholipases A2 and disintegrins of Protobothrops pit vipers.

To explore the venom diversity and systematics of pit vipers under the genus Protobothrops, the venom phospholipases A2 (PLA2s) of P. mangshanensis, P. elegans and P. tokarensis were purified and characterized for the first time. The results were compared with the corresponding venom data of other co-generic species including P. mucrosquamatus, P. flavoviridis and P. jerdonii. Based on sequence features at the N-terminal regions, we identified five PLA2 subtypes, i.e., the Asp49-PLA2s with N6, E6 or R6 substitution and the Lys49-PLA2. However, not all subtypes were expressed in each of the species. Venom N6-PLA2s from P. mangshanensis and P. tokarensis venom were weakly neurotoxic toward chick biventer cervicis tissue preparations. The venoms of P. tokarensis and P. flavoviridis contained identical PLA2 isoforms. In most Protobothrop disintegrins, sequences flanking the RGD-motif are conserved. Phylogenetic analyses based on amino acid sequences of both families of the acidic PLA2s and the disintegrins clarify that these species could belong to a monophyletic group.

Platelet-derived growth factor and fibroblast growth factor differentially regulate interleukin 1beta- and cAMP-induced group II phospholipase A2 expression in rat renal mesangial cells.

Expression of group II phospholipase A2 (PLA2; EC 3.1.1.4) in rat renal mesangial cells is triggered in response to two principal classes of activating signals. These two groups of activators comprise inflammatory cytokines such as interleukin 1beta (IL-1beta) or tumor necrosis factor alpha and agents that elevate cellular levels of cyclic AMP (cAMP) such as forskolin, an activator of adenylate cyclase. Treatment of mesangial cells with IL-1beta or forskolin for 24 h induces group II PLA2 activity secreted into cell culture supernatants by about 15-fold and 11-fold, respectively. Platelet-derived growth factor (PDGF)-BB potently inhibits secretion of IL-1beta- and forskolin-induced group II PLA2 activity. By Western and Northern blot analyses, we demonstrate that this is due to a reduction of PLA2 protein levels and the corresponding PLA2 mRNA steady-state levels. Basic fibroblast growth factor (bFGF) virtually does not inhibit IL-1beta-stimulated group II PLA2 activity, but markedly inhibits forskolin-induced expression of group II PLA2 activity. These effects are caused by changes in the corresponding PLA2 protein and PLA2 mRNA steady-state levels. Inhibition of protein kinase C (PKC) by the potent and selective PKC inhibitor calphostin C converted the inhibitory action of PDGF into a bFGF-type of response thus suggesting that PKC is a major effector in PDGF-induced inhibition of IL-1beta-stimulated group II sPLA2 expression. In summary, our data suggest that PDGF and bFGF differentially modulate in a stimulus-specific manner the expression of group II PLA2 in mesangial cells.

Structure, function, and regulation of group V phospholipase A(2).

The hydrolysis of membrane phospholipid by phospholipase A(2) (PLA(2)) is a key step in the production of inflammatory eicosanoids. Recent cell studies have shown that secretory group V PLA(2) (gVPLA(2)) is involved in agonist-induced eicosanoid biosynthesis in mouse P388D1 cell line, mast cells, and transfected HEK 293 cells. gVPLA(2) is homologous to other group II PLA(2) family members but has distinctive enzymatic properties, including its activity to effectively hydrolyze phosphatidylcholine (PC) vesicles and the outer plasma membrane of mammalian cells. Mutational studies showed that gVPLA(2) has a unique structure that allows effective binding to PC membranes and efficient catalysis of an active-site-bound PC substrate. Thanks to this unique structure and activity, exogenously added gVPLA(2) can induce the eicosanoid biosynthesis in unstimulated inflammatory cells, including human neutrophils and eosinophils, suggesting that it might be able to trigger inflammatory responses under certain physiological conditions. Extensive structure-function and cell studies showed that gVPLA(2) could act directly on the outer plasma membranes of neutrophils and eosinophils. The release of fatty acids and lysophospholipids from the cell surfaces induces the translocation and activation of cytosolic PLA(2) and 5-lipoxygenase, resulting in the leukotriene synthesis. In case of neutrophils, induction of leukotriene B(4) synthesis by gVPLA(2) leads to the phosphorylation of cytosolic PLA(2) by a leukotriene B(4) receptor and MAP kinase-mediated mechanism. Finally, heparan sulfate proteoglycans in neutrophils appear to play a role of internalizing and degrading the cell surface-bound gVPLA(2) to protect the cells from extensive lipolytic damage.