Proteolysis sensitizes LDL particles to phospholipolysis by secretory phospholipase A2 group V and secretory sphingomyelinase

LDL particles that enter the arterial intima become exposed to proteolytic and lipolytic modifications. The extracellular hydrolases potentially involved in LDL modification include proteolytic enzymes, such as chymase, cathepsin S, and plasmin, and phospholipolytic enzymes, such as secretory phospholipases A₂ (sPLA₂-IIa and sPLA₂-V) and secretory acid sphingomyelinase (sSMase). Here, LDL was first proteolyzed and then subjected to lipolysis, after which the effects of combined proteolysis and lipolysis on LDL fusion and on binding to human aortic proteoglycans (PG) were studied. Chymase and cathepsin S led to more extensive proteolysis and release of peptide fragments from LDL than did plasmin. sPLA₂-IIa was not able to hydrolyze unmodified LDL, and even preproteolysis of LDL particles failed to enhance lipolysis by this enzyme. However, preproteolysis with chymase and cathepsin S accelerated lipolysis by sPLA₂-V and sSMase, which resulted in enhanced fusion and proteoglycan binding of the preproteolyzed LDL particles. Taken together, the results revealed that proteolysis sensitizes the LDL particles to hydrolysis by sPLA₂-V and sSMase. By promoting fusion and binding of LDL to human aortic proteoglycans, the combination of proteolysis and phospholipolysis of LDL particles potentially enhances extracellular accumulation of LDL-derived lipids during atherogenesis.     

Acidity and lipolysis by group V secreted phospholipase A2 strongly increase the binding of apoB-100-containing lipoproteins to human aortic proteoglycans

Local acidic areas characterize diffuse intimal thickening (DIT) and advanced atherosclerotic lesions. The role of acidity in the modification and extra- and intracellular accumulation of triglyceride-rich VLDL and IDL particles has not been studied before. Here, we examined the effects of acidic pH on the activity of recombinant human group V secreted phospholipase A₂ (sPLA₂-V) toward small VLDL (sVLDL), IDL, and LDL, on the binding of these apoB-100-containing lipoproteins to human aortic proteoglycans, and on their uptake by human monocyte-derived macrophages. At acidic pH, the ability of sPLA₂-V to lipolyze the apoB-100-containing lipoproteins was moderately, but significantly, increased while binding of the lipoproteins to proteoglycans increased > 60-fold and sPLA₂-V-modification further doubled the binding. Moreover, acidic pH more than doubled macrophage uptake of soluble complexes of sPLA₂-V-LDL with aortic proteoglycans. Proteoglycan-affinity chromatography at pH 7.5 and 5.5 revealed that sVLDL, IDL, and LDL consisted of populations with different proteoglycan-binding affinities, and, surprisingly, the sVLDL fractions with the highest proteoglycan-affinity contained only low amounts of apolipoproteins E and C-III. Our results suggest that in atherosclerotic lesions with acidic extracellular pH, sPLA₂-V is able to lipolyze sVLDL, IDL, and LDL, and increase their binding to proteoglycans. This is likely to provoke extracellular accumulation of lipids derived from these atherogenic lipoprotein particles and to increase the progression of the atherosclerotic lesions.     

Oxidized phosphatidylcholine stimulates activity of secretory phospholipase A2 group IIA and abolishes sphingomyelin-induced inhibition of the enzyme

We have shown recently that oxidized but not native lipoproteins stimulate the activity of secretory phospholipase A2 group IIA (sPLA₂(IIA)). Since oxidized lipoproteins potentially contain considerable amounts of oxidized phosphatidylcholine, we examined the effect of oxidized palmitoyl arachidonyl phosphatidylcholine (oxPC) and the competitive effects of oxPC and sphingomyelin (SM) on sPLA₂(IIA) activity.OxPC either added to the assay medium as separated liposomes or incorporated in varied amounts into LDL progressively enhanced the activity of purified human sPLA₂(IIA) and abolished the inhibitory effect of LDL-incorporated SM on the enzyme activity. OxPC completely abolished the inhibitory effect of SM at the oxPC/SM concentration ratio 1/2. On the other hand, SM suppressed the activating effect of oxPC in a dose-dependent manner, abolishing it almost completely at a concentration 8 times as high as that of oxPC.Thus, changes in the oxPC/SM concentration ratio in LDL may affect the regulatory mechanisms of sPLA₂(IIA) activity in human blood, inducing stimulation or inhibition of the enzyme. Influence on regulation of sPLA₂(IIA) activity can be useful in the development of new therapeutic approaches to the treatment of cardiovascular diseases.     

Type II secretory phospholipase A2 and prognosis in patients with stable coronary heart disease: mendelian randomization study

Background

Serum type II secretory phospholipase A₂ (sPLA₂-IIa) has been found to be predictive of adverse outcomes in patients with stable coronary heart disease. Compounds targeting sPLA₂-IIa are already under development. This study investigated if an association of sPLA₂-IIa with secondary cardiovascular disease (CVD) events may be of causal nature or mainly a matter of confounding by correlated cardiovascular risk markers.

Methodology/Principal Findings

Eight-year follow-up data of a prospective cohort study (KAROLA) of patients who underwent in-patient rehabilitation after an acute cardiovascular event were analysed. Associations of polymorphisms (SNP) in the sPLA₂-IIa-coding gene PLA2G2A with serum sPLA₂-IIa and secondary fatal or non-fatal CVD events were examined by multiple regression. Hazard ratios (HR) were compared with those expected if the association between sPLA₂-IIa and CVD were causal. The strongest determinants of sPLA₂-IIa (rs4744 and rs10732279) were associated with an increase of serum concentrations by 81% and 73% per variant allele. HRs (95% confidence intervals) estimating the associations of the SNPs with secondary CVD events were increased, but not statistically significant (1.16 [0.89–1.51] and 1.18 [0.91–1.52] per variant allele, respectively). However, these estimates were very similar to those expected when assuming causality (1.18 and 1.17), based on an association of natural log-transformed sPLA₂-IIa concentration with secondary events with HR = 1.33 per unit.

Conclusion

The present findings regarding genetic polymorphisms, determination of serum sPLA₂-IIa, and prognosis in CVD patients are consistent with a genuine causal relationship and thus might point to a valid drug target for prevention of secondary CVD events.     

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.     

Therapy with interleukin-2 induces the systemic release of phospholipase-A2

Therapy with interleukin-2 (IL-2) induces remissions in some forms of cancer. This treatment however, is accompanied by side-effects which, in part, may be mediated by the formation of eicosanoids and plateletactivating factor. We investigated the systemic release of phospholipase A₂ (PLA₂), a rate-limiting enzyme in the formation of these lipid mediators, in patients receiving IL-2. In a pilot study of 4 patients we observed an increase in PLA₂ activity in serial plasma samples obtained during the first day after a bolus infusion of IL-2, which increase closely correlated with that of antigen levels of secretory phospholipase A₂ (sPLA₂) as measured by enzyme-linked immunosorbent assay (r=0.92;P<0.001). In 20 patients, receiving 12×10⁶–18×10⁶ IU IL-2/m², we then investigated the course of antigenic levels of sPLA₂ in relation to those of the cytokines tumour necrosis factor (TNF) and interleukin-6 (IL-6) (both cytokines may induce sPLA₂ in vivo). From 4 h on, sPLA₂ levels significantly increased, reaching a peak 24 h after the IL-2 infusion. Subsequent IL-2 infusions even induced a further increase of sPLA₂. This increase of sPLA₂ was presumably not due to a direct effect of IL-2 on, for example, hepatocytes, since this cytokine, in contrast to IL-1, IL-6, TNF and interferon , was not able to induce the synthesis of sPLA₂ by Hep G2 cells in vitro. Consistent with this, plasma levels of TNF and IL-6 in the patients rose, reaching peak levels before a zenith of sPLA₂ occurred, i.e at 2 h and 4 h after the start of the IL-2 infusion respectively. sPLA₂ levels significantly correlated with the development of the side-effects increase in body weight (r=0.49;P<0.0001) and decrease in mean arterial blood pressure (r=0.40;P<0.0001). Moreover, maximum sPLA₂ levels induced by IL-2 were higher in patients who had progressive disease after therapy than in patients who had stable disease or a partial response.     

Induction of distinct sets of secretory phospholipase A2 in rodents during inflammation

Although the expression of the prototypic secretory phospholipase A₂ (sPLA₂), group IIA (sPLA₂-IIA), is known to be up-regulated during inflammation, it remains uncertain if other sPLA₂ enzymes display similar or distinct profiles of induction under pathological conditions. In this study, we investigated the expression of several sPLA₂s in rodent inflammation models. In lipopolysaccharide (LPS)-treated mice, the expression of sPLA₂-V, and to a lesser extent that of sPLA₂-IID, -IIE, and -IIF, were increased, whereas that of sPLA₂-X was rather constant, in distinct tissues. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema, in which the expression of sPLA₂-IID, -IIF and -V was increased, was significantly reduced by YM-26734, a competitive sPLA₂-IIA inhibitor that turned out to inhibit sPLA₂-IID, -IIE, -V and -X as well. In contrast, sPLA₂-IIA was dominant in carageenin-induced pleurisy in rats, where the accumulation of exudate fluids and leukocytes was significantly ameliorated by YM-26734. These results indicate that distinct sPLA₂s can participate in inflammatory diseases according to tissues, animal species, and types of inflammation.     

Secretory phospholipase A2 does not appear to be associated with phenotypic variation in familial adenomatous polyposis

Recent studies in mice have provided strong evidence for a modifier gene that is capable of effecting the expression of the mouse equivalent of familial adenomatous polyposis (FAP). A candidate gene has been proposed, namely secretory phospholipase A₂ (sPLA₂). Increased tumor number in mice was correlated with low levels of sPLA₂ expression and the presence of truncating mutations within the sPLA₂ gene. In an attempt to determine whether any genetic alterations in the sPLA₂ gene were associated with the expression of FAP in man, we investigated the genetic structure of sPLA₂ in 97 polyposis coli patients presenting with various disease phenotypes, and its expression in 8 FAP patients displaying markedly different disease characteristics. In the current study no inactivating mutations in the sPLA₂ gene were identified, suggesting that human sPLA₂ is not associated with phenotypic variation in FAP. Received: 25 March 1996 / Revised: 9 April 1996     

The role of mast cell-derived secreted phospholipases A2 in respiratory allergy

Secreted phospholipases A₂ (sPLA₂s) are molecules released in plasma and biological fluids of patients with systemic inflammatory, autoimmune and allergic diseases. These molecules exert proinflammatory effects by either enzymatic-mechanisms or through binding to surface molecules expressed on inflammatory cells. sPLA₂s are released at low levels in the normal airways and tend to increase during respiratory allergies (e.g., rhinitis and bronchial asthma) as the result of local secretion. Several sPLA₂ isoforms are expressed in the human lung and some of them (e.g., group IIA and group X) are released in the airways of patients with rhinitis or asthma. Mast cells play a major role in the pathogenesis of respiratory allergies and other chronic inflammatory lung diseases. Recent evidence indicates that mast cells purified from human lung express most of the sPLA₂ isoforms so far described. IgE-mediated activation of these cells induce the release of sPLA₂s suggesting that mast cells are a main source of extracellular sPLA₂s during allergic reactions. Once released, sPLA₂s may contribute to the generation of eicosanoids (e.g., PGD₂ and LTC₄) and to the release of preformed mediators (e.g., histamine) by an autocrine loop involving the interaction of sPLA₂s with surface molecules such as heparan sulphate proteoglycans or the M-type receptor. Thus, mast cell-derived sPLA₂s may play an important role in the initiation and amplification of the inflammatory reactions in patients with allergic rhinitis and bronchial asthma.     

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.     

Ectopically Expressed Pro-group X Secretory Phospholipase A2 Is Proteolytically Activated in Mouse Adrenal Cells by Furin-like Proprotein Convertases: IMPLICATIONS FOR THE REGULATION OF ADRENAL STEROIDOGENESIS*

Group X secretory phospholipase A₂ (GX sPLA₂) hydrolyzes mammalian cell membranes, liberating free fatty acids and lysophospholipids. GX sPLA₂ is produced as a pro-enzyme (pro-GX sPLA₂) that contains an N-terminal 11-amino acid propeptide ending in a dibasic motif, suggesting cleavage by a furin-like proprotein convertase (PC). Although propeptide cleavage is clearly required for enzymatic activity, the protease(s) responsible for pro-GX sPLA₂ activation have not been identified. We previously reported that GX sPLA₂ negatively regulates adrenal glucocorticoid production, likely by suppressing liver X receptor-mediated activation of steroidogenic acute regulatory protein expression. In this study, using a FLAG epitope-tagged pro-GX sPLA₂ expression construct (FLAG-pro-GX sPLA₂), we determined that adrenocorticotropic hormone (ACTH) enhanced FLAG-pro-GX sPLA₂ processing and phospholipase activity secreted by Y1 adrenal cells. ACTH increased the expression of furin and PCSK6, but not other members of the PC family, in Y1 cells. Overexpression of furin and PCSK6 in HEK 293 cells significantly enhanced FLAG-pro-GX sPLA₂ processing, whereas siRNA-mediated knockdown of both PCs almost completely abolished FLAG-pro-GX sPLA₂ processing in Y1 cells. Expression of either furin or PCSK6 enhanced the ability of GX sPLA₂ to suppress liver X receptor reporter activity. The PC inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone significantly suppressed FLAG-pro-GX sPLA₂ processing and sPLA₂ activity in Y1 cells, and it significantly attenuated GX sPLA₂-dependent inhibition of steroidogenic acute regulatory protein expression and progesterone production. These findings provide strong evidence that pro-GX sPLA₂ is a substrate for furin and PCSK6 proteolytic processing and define a novel mechanism for regulating corticosteroid production in adrenal cells.     

Key Role of Group V Secreted Phospholipase A2 in Th2 Cytokine and Dendritic Cell-Driven Airway Hyperresponsiveness and Remodeling

Background

Previous work has shown that disruption of the gene for group X secreted phospholipase A₂ (sPLA₂-X) markedly diminishes airway hyperresponsiveness and remodeling in a mouse asthma model. With the large number of additional sPLA₂s in the mammalian genome, the involvement of other sPLA₂s in the asthma model is possible – in particular, the group V sPLA₂ (sPLA₂-V) that like sPLA₂-X is highly active at hydrolyzing membranes of mammalian cells.

Methodology and Principal Findings

The allergen-driven asthma phenotype was significantly reduced in sPLA₂-V-deficient mice but to a lesser extent than observed previously in sPLA₂-X-deficient mice. The most striking difference observed between the sPLA₂-V and sPLA₂-X knockouts was the significant impairment of the primary immune response to the allergen ovalbumin (OVA) in the sPLA₂-V^−/− mice. The impairment in eicosanoid generation and dendritic cell activation in sPLA2-V^−/− mice diminishes Th2 cytokine responses in the airways.

Conclusions

This paper illustrates the diverse roles of sPLA₂s in the immunopathogenesis of the asthma phenotype and directs attention to developing specific inhibitors of sPLA₂-V as a potential new therapy to treat asthma and other allergic disorders.     

Group X Secretory Phospholipase A2 Regulates the Expression of Steroidogenic Acute Regulatory Protein (StAR) in Mouse Adrenal Glands

We developed C57BL/6 mice with targeted deletion of group X secretory phospholipase A₂ (GX KO). These mice have ∼80% higher plasma corticosterone concentrations compared with wild-type (WT) mice under both basal and adrenocorticotropic hormone (ACTH)-induced stress conditions. This increased corticosterone level was not associated with increased circulating ACTH or a defect in the hypothalamic-pituitary axis as evidenced by a normal response to dexamethasone challenge. Primary cultures of adrenal cells from GX KO mice exhibited significantly increased corticosteroid secretion compared with WT cells. Conversely, overexpression of GX secretory phospholipase A₂ (sPLA₂), but not a catalytically inactive mutant form of GX sPLA₂, significantly reduced steroid production 30–40% in Y1 mouse adrenal cell line. This effect was reversed by the sPLA₂ inhibitor, indoxam. Silencing of endogenous M-type receptor expression did not restore steroid production in GX sPLA₂-overexpressing Y1 cells, ruling out a role for this sPLA₂ receptor in this regulatory process. Expression of steroidogenic acute regulatory protein (StAR), the rate-limiting protein in corticosteroid production, was ∼2-fold higher in adrenal glands of GX KO mice compared with WT mice, whereas StAR expression was suppressed in Y1 cells overexpressing GX sPLA₂. Results from StAR-promoter luciferase reporter gene assays indicated that GX sPLA₂ antagonizes StAR promoter activity and liver X receptor-mediated StAR promoter activation. In summary, GX sPLA₂ is expressed in mouse adrenal glands and functions to negatively regulate corticosteroid synthesis, most likely by negatively regulating StAR expression.     

Assessing Phospholipase A2 Activity toward Cardiolipin by Mass Spectrometry

Cardiolipin, a major component of mitochondria, is critical for mitochondrial functioning including the regulation of cytochrome c release during apoptosis and proper electron transport. Mitochondrial cardiolipin with its unique bulky amphipathic structure is a potential substrate for phospholipase A₂ (PLA₂) in vivo. We have developed mass spectrometric methodology for analyzing PLA₂ activity toward various cardiolipin forms and demonstrate that cardiolipin is a substrate for sPLA₂, cPLA₂ and iPLA₂, but not for Lp-PLA₂. Our results also show that none of these PLA₂s have significant PLA₁ activities toward dilyso-cardiolipin. To understand the mechanism of cardiolipin hydrolysis by PLA₂, we also quantified the release of monolyso-cardiolipin and dilyso-cardiolipin in the PLA₂ assays. The sPLA₂s caused an accumulation of dilyso-cardiolipin, in contrast to iPLA₂ which caused an accumulation of monolyso-cardiolipin. Moreover, cardiolipin inhibits iPLA₂ and cPLA₂, and activates sPLA₂ at low mol fractions in mixed micelles of Triton X-100 with the substrate 1-palmitoyl-2-arachidonyl-sn-phosphtidylcholine. Thus, cardiolipin functions as both a substrate and a regulator of PLA₂ activity and the ability to assay the various forms of PLA₂ is important in understanding its function.     

Inhibition of type 2A secretory phospholipase A2 reduces death of cardiomyocytes in acute myocardial infarction

During acute myocardial infarction (AMI), ischemia leads to necrotic areas surrounded by border zones of reversibly damaged cardiomyocytes, showing membrane flip-flop. During reperfusion type IIA secretory phopholipase A₂ (sPLA₂-IIA) induces direct cell-toxicity and facilitates binding of other inflammatory mediators on these cardiomyocytes. Therefore, we hypothesized that the specific sPLA₂-IIA-inhibitor PX-18 would reduce cardiomyocyte death and infarct size in vivo. Wistar rats were treated with PX-18 starting minutes after reperfusion, and at day 1 and 2 post AMI. After 28 days hearts were analyzed. Furthermore, the effect of PX-18 on membrane flip-flop and apoptosis was investigated in vitro. PX-18 significantly inhibited sPLA₂-IIA activity and reduced infarct size (reduction 73 ± 9%, P < 0.05), compared to the vehicle-treated group, without impairing wound healing. In vitro, PX-18 significantly reduced reversible membrane flip-flop and apoptosis in cardiomyocytes. However, no sPLA₂-IIA activity could be detected, suggesting that PX-18 also exerted a protective effect independent of sPLA₂-IIA. In conclusion, PX-18 is a potent therapeutic to reduce infarct size by inhibiting sPLA₂-IIA, and possibly also by inhibiting apoptosis of cardiomyocytes in a sPLA₂-IIA independent manner. A. van Dijk and P. A. J. Krijnen have contributed equally to the study.     

The Role of Secretory Phospholipase A2 in the Central Nervous System and Neurological Diseases

Secretory phospholipase A₂ (sPLA₂s) are small secreted proteins (14–18 kDa) and require submillimolar levels of Ca²⁺ for liberating arachidonic acid from cell membrane lipids. In addition to the enzymatic function, sPLA₂ can exert various biological responses by binding to specific receptors. Physiologically, sPLA₂s play important roles on the neurotransmission in the central nervous system and the neuritogenesis in the peripheral nervous system. Pathologically, sPLA₂s are involved in the neurodegenerative diseases (e.g., Alzheimer’s disease) and cerebrovascular diseases (e.g., stoke). The common pathology (e.g., neuronal apoptosis) of Alzheimer’s disease and stroke coexists in the mixed dementia, suggesting common pathogenic mechanisms of the two neurological diseases. Among mammalian sPLA₂s, sPLA₂-IB and sPLA₂-IIA induce neuronal apoptosis in rat cortical neurons. The excess influx of calcium into neurons via l-type voltage-dependent Ca²⁺ channels mediates the two sPLA₂-induced apoptosis. The elevated concentration of intracellular calcium activates PKC, MAPK and cytosolic PLA₂. Moreover, it is linked with the production of reactive oxygen species and apoptosis through activation of the superoxide producing enzyme NADPH oxidase. NADPH oxidase is involved in the neurotoxicity of amyloid β peptide, which impairs synaptic plasticity long before its deposition in the form of amyloid plaques of Alzheimer’s disease. In turn, reactive oxygen species from NADPH oxidase can stimulate ERK1/2 phosphorylation and activation of cPLA₂ and result in a release of arachidonic acid. sPLA₂ is up-regulated in both Alzheimer’s disease and cerebrovascular disease, suggesting the involvement of sPLA₂ in the common pathogenic mechanisms of the two diseases. Thus, our review presents evidences for pathophysiological roles of sPLA₂ in the central nervous system and neurological diseases.     

Liquid-liquid immiscibility in model membranes activates secretory phospholipase A2

Secretory phospholipase A₂ (sPLA₂) hydrolyzes phosphatidylcholines (PC) within lipid bilayers to produce lyso-PC and a fatty acid, which can act as signaling molecule in biological membranes. The activity of sPLA₂ depends on the membrane structure. Bilayer defects, curvature, and gel-fluid micro-heterogeneity are known to activate sPLA₂. Here, we investigate if liquid-liquid immiscibility within model membranes is sufficient for sPLA₂ activation. The onset of the hydrolytic activity of cobra-venom sPLA₂ towards mixed monolayers of dimyristoyl-PC (DMPC)/cholesterol 2:1 (mol/mol) has been determined using infrared reflection-absorption spectroscopy (IRRAS) and polarization-modulated (PM-) IRRAS. The lag phase of sPLA₂ activity increases exponentially with rising surface pressures starting at 12 mN/m. This indicates that enzyme activation is hampered at higher surface pressures. Below 12 mN/m, no lag phase is observed, and sPLA₂ is efficiently activated. The surface pressure that is critical for sPLA₂ activation correlates with the critical miscibility pressure according to the phase diagram of DMPC and cholesterol. Thus, coexisting, liquid-phase domains provide sufficient boundaries to activate sPLA₂. Moreover, liquid-liquid immiscibility is an activating mechanism for sPLA₂ that also applies to biological membranes under physiological conditions because the corresponding bilayer structure is associated with that of membrane rafts.     

Group IID,IIE, IIF and III secreted phospholipase A2s

Among the 11 members of the secreted phospholipase A₂ (sPLA₂) family, group IID, IIE, IIF and III sPLA₂s (sPLA₂-IID, -IIE, -IIF and -III, respectively) are “new” isoforms in the history of sPLA₂ research. Relative to the better characterized sPLA₂s (sPLA₂-IB, -IIA, -V and -X), the enzymatic properties, distributions, and functions of these “new” sPLA₂s have remained obscure until recently. Our current studies using knockout and transgenic mice for a nearly full set of sPLA₂s, in combination with comprehensive lipidomics, have revealed unique and distinct roles of these “new” sPLA₂s in specific biological events. Thus, sPLA₂-IID is involved in immune suppression, sPLA₂-IIE in metabolic regulation and hair follicle homeostasis, sPLA₂-IIF in epidermal hyperplasia, and sPLA₂-III in male reproduction, anaphylaxis, colonic diseases, and possibly atherosclerosis. In this article, we overview current understanding of the properties and functions of these sPLA₂s and their underlying lipid pathways in vivo.     

On the Role of Protein Disulfide Isomerase in the Retrograde Cell Transport of Secreted Phospholipases A2

Following the finding that ammodytoxin (Atx), a neurotoxic secreted phospholipase A₂ (sPLA₂) in snake venom, binds specifically to protein disulfide isomerase (PDI) in vitro we show that these proteins also interact in living rat PC12 cells that are able to internalize this group IIA (GIIA) sPLA₂. Atx and PDI co-localize in both differentiated and non-differentiated PC12 cells, as shown by fluorescence microscopy. Based on a model of the complex between Atx and yeast PDI (yPDI), a three-dimensional model of the complex between Atx and human PDI (hPDI) was constructed. The Atx binding site on hPDI is situated between domains b and b’. Atx interacts hPDI with an extensive area on its interfacial binding surface. The mammalian GIB, GIIA, GV and GX sPLA₂s have the same fold as Atx. The first three sPLA₂s have been detected intracellularly but not the last one. The models of their complexes with hPDI were constructed by replacement of Atx with the respective mammalian sPLA₂ in the Atx—hPDI complex and molecular docking of the structures. According to the generated models, mammalian GIB, GIIA and GV sPLA₂s form complexes with hPDI very similar to that with Atx. The contact area between GX sPLA₂ and hPDI is however different from that of the other sPLA₂s. Heterologous competition of Atx binding to hPDI with GV and GX sPLA₂s confirmed the model-based expectation that GV sPLA₂ was a more effective inhibitor than GX sPLA₂, thus validating our model. The results suggest a role of hPDI in the (patho)physiology of some snake venom and mammalian sPLA₂s by assisting the retrograde transport of these molecules from the cell surface. The sPLA₂–hPDI model constitutes a valuable tool to facilitate further insights into this process and into the (patho)physiology of sPLA₂s in relation to their action intracellularly.     

Group X secretory phospholipase A2 induces potent productions of various lipid mediators in mouse peritoneal macrophages

We have previously shown the expression of group X secretory phospholipase A₂ (sPLA₂-X) in mouse splenic macrophages and its powerful potency for releasing fatty acids from various intact cell membranes. Here, we examined the potency of sPLA₂-X in the production of lipid mediators in murine peritoneal macrophages. Mouse sPLA₂-X was found to induce a marked release of fatty acids including arachidonic acid and linoleic acid, which contrasted with little, if any, release by the action of group IB and IIA sPLA₂s. In resting macrophages, sPLA₂-X elicited a modest production of prostaglandin E₂ and thromboxane A₂. After the induction of cyclooxygenase-2 (COX-2) by pretreatment with lipopolysaccharide, a dramatic increase in the production of these eicosanoids was observed in sPLA₂-X-treated macrophages, which was completely blocked by the addition of either the specific sPLA₂ inhibitor indoxam or the COX inhibitor indomethacin. In accordance with its higher hydrolyzing activity toward phosphatidylcholine, mouse sPLA₂-X induced a potent production of lysophosphatidylcholine. These findings strongly suggest that sPLA₂-X plays a critical role in the production of various lipid mediators from macrophages. These events might be relevant to the progression of various pathological states, including chronic inflammation and atherosclerosis.