Phospholipase A2 sensitive liposomes for delivery of small interfering RNA (siRNA)

Small interfering RNA (siRNA) is potent and highly specific for gene silencing and there is currently a lot of enthusiasm for developing siRNA into a drug. However, for most therapeutic applications of siRNA, delivery systems are needed. These delivery systems have multiple requirements and should on one hand ideally be stable carriers protecting the siRNA from degradation and on the other hand assist the siRNA in overcoming membrane barriers for intracellular delivery to the cytosol. Long-circulating liposomes, which are sensitive to secretory phospholipase A(2) (sPLA(2)) are feasible delivery systems for systemic administration of drugs due to their passive targeting to pathological tissue via the enhanced permeability and retention (EPR) effect and their site-specific, enzyme-triggered release of encapsulated drug in response to sPLA(2) which exists locally at elevated levels at, e.g,. sites of inflammation. However, recent data suggest that endosomal membrane destabilizing approaches could be addressed to design sPLA(2)-sensitive liposomes as successful delivery systems for siRNA to the RNA interference pathway in the cytoplasm upon systemic administration.     

Liposomes containing alkylated methotrexate analogues for phospholipase A2 mediated tumor targeted drug delivery

Two lipophilic methotrexate analogues have been synthesized and evaluated for cytotoxicity against KATO III and HT-29 human colon cancer cells. Both analogues contained a C₁₆-alkyl chain attached to the γ-carboxylic acid and one of the analogues had an additional benzyl group attached to the α-carboxylic acid. The cytotoxicity of the γ-alkylated compound towards KATO III (IC₅₀ = 55 nM) and HT-29 (IC₅₀ = 400 nM) cell lines, was unaffected by the alkylation, whereas the additional benzyl group on the α-carboxyl group made the compound nontoxic. The γ-derivative with promising cytotoxicity was incorporated into liposomes that were designed to be particularly susceptible to a liposome degrading enzyme, secretory phospholipase A₂ (sPLA₂), which is found in high concentrations in tumors of several different cancer types. Liposome incorporation was investigated by differential scanning calorimetry (DSC), and sPLA₂ hydrolysis was examined by fluorescence spectroscopy and high performance liquid chromatography (HPLC). The results showed that the methotrexate (MTX)-analogue could be incorporated into liposomes that were degradable by sPLA₂. However, the in vitro cytotoxicity of the MTX-liposomes against KATO III and HT-29 cancer cells was found to be independent of sPLA₂ hydrolysis, indicating that the alkylated MTX-analogue was available for cancer cell uptake even in the absence of liposome hydrolysis. Using a DSC based method for assessing the anchoring stability of alkylated compounds in liposomes, it was demonstrated that the MTX-analogue partitioned into the water phase and thereby became available for cell uptake. It was concluded that liposomes containing alkylated MTX-analogues show promise as a drug delivery system, although the MTX-analogue needs to be more tightly anchored to the liposomal carrier. Also, the developed DSC-assay for studying the anchoring stability of alkylated drugs will be a useful tool in the development of liposomal drug delivery systems.     

Secreted Phospholipases A2 - not just Enzymes: Revisited

Secreted phospholipases A₂ (sPLA₂s) participate in a very broad spectrum of biological processes through their enzymatic activity and as ligands for membrane and soluble receptors. The physiological roles of sPLA₂s as enzymes have been very well described, while their functions as ligands are still poorly known. Since the last overview of sPLA₂-binding proteins (sPLA₂-BPs) 10 years ago, several important discoveries have occurred in this area. New and more sensitive analytical tools have enabled the discovery of additional sPLA₂-BPs, which are presented and critically discussed here. The structural diversity of sPLA₂-BPs reveals sPLA₂s as very promiscuous proteins, and we offer some structural explanations for this nature that makes these proteins evolutionarily highly advantageous. Three areas of physiological engagement of sPLA₂-BPs have appeared most clearly: cellular transport and signalling, and regulation of the enzymatic activity of sPLA₂s. Due to the multifunctionality of sPLA₂s, they appear to be exceptional pharmacological targets. We reveal the potential to exploit interactions of sPLA₂s with other proteins in medical terms, for the development of original diagnostic and therapeutic procedures. We conclude this survey by suggesting the priority questions that need to be answered.     

Leukotriene receptor antagonists,LY293111 and ONO-1078, protect neurons from the sPLA2-IB-induced neuronal cell death independently of blocking their receptors

In the ischemic brain, leukotrienes (LTs) are increased and their receptor antagonists protect neurons. However, it has not yet been sufficiently clarified how antagonists for LT receptors exhibit neuroprotective effects. In the present study, we evaluated protective effects of receptor antagonists for LTB₄ (LY293111) and cysteinyl LTs (ONO-1078) in the primary culture of rat cortical neurons. The group IB secretory phospholipase A₂ (sPLA₂-IB)-induced neuronal cell death had been established as the in vitro model for cerebral ischemia. sPLA₂-IB triggered the influx of Ca²⁺ into neurons via L-type voltage-dependent calcium channel (L-VDCC). Subsequently, the enzyme produced eicosanoids including LTB₄ before neuronal cell death. Neither administration of LTB₄ nor cysteinyl LTs such as LTC₄, LTD₄ and LTE₄ killed neurons. However, both LY293111 and ONO-1078 significantly prevented neurons from the neurotoxicity of sPLA₂-IB, suggesting that the two LT receptor blockers protected neurons through alternative pathways beside LT receptors. An L-VDCC blocker does not only inhibit the influx of Ca²⁺ into neurons but also rescues neurons from the sPLA₂-IB-induced neuronal cell death. The two LT receptor antagonists also blocked the sPLA₂-IB-induced Ca²⁺ influx significantly. Thus, LTs exhibited no neurotoxicity, but their receptor antagonists protected neurons directly in the in vitro ischemic model. Furthermore, the suppression of L-VDCC appeared to be involved in the neuroprotective effects of LY293111 and ONO-1078 independent of blocking their receptors.     

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.     

Emerging roles of secreted phospholipase A2 enzymes: An update

Phospholipase A₂ (PLA₂) enzymes catalyze the hydrolysis of the sn-2 position of glycerophospholipids to produce free fatty acids and lysophospholipids. More than one third of the mammalian PLA₂ enzymes belong to the secreted PLA₂ (sPLA₂) family, which consists of low molecular mass, Ca²⁺-requiring enzymes with a His–Asp catalytic dyad. Individual sPLA₂ enzymes exhibit unique tissue and cellular localizations and specific enzymatic properties, suggesting their distinct biological roles. The past decade has met a new era of the sPLA₂ research field toward deciphering their in vivo functions by developing several specific tools and methods. These include i) the production of transgenic and knockout mouse lines for several sPLA₂s, ii) the development of specific analytical tools including the production of large amounts of recombinant sPLA₂ proteins, and iii) applying mass spectrometry lipidomics to unveil their specific enzymatic properties occurring in vivo. It is now obvious that individual sPLA₂s are involved in diverse biological events through lipid mediator-dependent and -independent processes, act redundantly or non-redundantly in the context of physiology and pathophysiology, and may represent potential drug targets or novel bioactive molecules in certain situations. In this review, we will highlight the newest understanding of the biological roles of sPLA₂s in the past few years.     

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.     

Activities and interactions among phospholipases A2 during thapsigargin-induced S49 cell death

The purpose of this study was to determine the roles of calcium-dependent phospholipase A₂ (cPLA₂) and calcium-independent phospholipase A₂ (iPLA₂) in thapsigargin-induced membrane susceptibility to secretory phospholipase A₂ (sPLA₂) and programmed cell death. ³H-arachidonic acid release was observed in the presence of thapsigargin. This release was inhibited partially by an inhibitor of iPLA₂ (BEL) and completely by an inhibitor of both cPLA₂ and iPLA₂ (MAFP) suggesting that these enzymes were active during apoptosis. The process of cell death did not require the activity of either enzyme since neither inhibitor impeded the progression of apoptosis. However, both inhibitors increased the susceptibility of the membrane to sPLA₂ in the presence of thapsigargin. In the case of BEL, this effect appeared to involve direct induction of apoptosis in a sub-population of the cells independent of the action of iPLA₂. In conclusion, the results suggested that cPLA₂ and iPLA₂ are active during thapsigargin-induced apoptosis in S49 cells and that cPLA₂ tempers the tendency of the cells to become susceptible to sPLA₂ during apoptosis.     

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.     

Type-IIA secreted phospholipase A2 is an endogenous antibiotic-like protein of the host

Type-IIA secreted phospholipase A₂ (sPLA₂-IIA) has been proposed to play a role in the development of inflammatory diseases. It has been shown to release arachidonic acid, the precursor of proinflammatory eicosanoids, to hydrolyze phospholipids of pulmonary surfactant, and to bind to specific receptors located on cell surface membranes. However, the most established biological role of sPLA₂-IIA is related to its potent bactericidal property in particular toward Gram-positive bacteria. This enzyme is present in animal and human biological fluids at concentrations sufficient to kill bacteria. Human recombinant sPLA₂-IIA is able to kill Gram-positive bacteria at concentrations as low as 1.1 ng/ml. This remarkable property is due to the unique preference of sPLA₂-IIA for anionic phospholipids such as phosphatidylglycerol, the main phospholipid component of bacterial membranes. Much higher concentrations of sPLA₂-IIA are required for its action on host cell membranes and surfactant both of which are mainly composed by phosphatidylcholine, a poor substrate for sPLA₂-IIA. Transgenic mice over-expressing human sPLA₂-IIA are resistant to infection by Staphylococcus aureus, Escherichia coli, and Bacillus anthracis, the etiological agent of anthrax. Conversely, certain bacteria, such as B. anthracis, E. coli and Bordetella pertussis are able to inhibit sPLA₂-IIA expression by host cells, thus highlighting a mechanism by which these bacteria can subvert the host immune system. Intranasal instillation of recombinant sPLA₂-IIA protects mice from mortality caused by pulmonary anthrax. Interestingly, this protective effect was obtained even with B. anthracis strains that down-regulate the expression of endogenous sPLA₂-IIA, indicating that instilled sPLA₂-IIA can overcome the subversive action of B. anthracis. We conclude that sPLA₂-IIA is an efficient endogenous antibiotic of the host and can play a role in host defense against pathogenic bacteria. It can be used as a therapeutic agent in adjunct with current therapy to treat bacteria resistant to multiple antibiotics.     

Emerging roles of secreted phospholipase A2 enzymes: Lessons from transgenic and knockout mice

Among the emerging phospholipase A₂ (PLA₂) superfamily, the secreted PLA₂ (sPLA₂) family consists of low-molecular-mass, Ca²⁺-requiring extracellular enzymes with a His-Asp catalytic dyad. To date, more than 10 sPLA₂ enzymes have been identified in mammals. Individual sPLA₂s exhibit unique tissue and cellular localizations and enzymatic properties, suggesting their distinct pathophysiological roles. Despite numerous enzymatic and cell biological studies on this enzyme family in the past two decades, their precise in vivo functions still remain largely obscure. Recent studies using transgenic and knockout mice for several sPLA₂ enzymes, in combination with lipidomics approaches, have opened new insights into their distinct contributions to various biological events such as food digestion, host defense, inflammation, asthma and atherosclerosis. In this article, we overview the latest understanding of the pathophysiological functions of individual sPLA₂ isoforms fueled by studies employing transgenic and knockout mice for several sPLA₂s.     

Development of a Cell-Based Bioassay for Phospholipase A2-Triggered Liposomal Drug Release

The feasibility of exploiting secretory phospholipase A₂ (sPLA₂) enzymes, which are overexpressed in tumors, to activate drug release from liposomes precisely at the tumor site has been demonstrated before. Although the efficacy of the developed formulations was evaluated using in vitro and in vivo models, the pattern of sPLA₂-assisted drug release is unknown due to the lack of a suitable bio-relevant model. We report here on the development of a novel bioluminescence living-cell-based luciferase assay for the monitoring of sPLA₂-triggered release of luciferin from liposomes. To this end, we engineered breast cancer cells to produce both luciferase and sPLA₂ enzymes, where the latter is secreted to the extracellular medium. We report on setting up a robust and reproducible bioassay for testing sPLA₂-sensitive, luciferin remote-loaded liposomal formulations, using 1,2-distearoyl-sn-glycero-3-phosphatidylcholine/1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPC/DSPG) 7:3 and DSPC/DSPG/cholesterol 4:3:3 as initial test systems. Upon their addition to the cells, the liposomes were degraded almost instantaneously by sPLA₂ releasing the encapsulated luciferin, which provided readout from the luciferase-expressing cells. Cholesterol enhanced the integrity of the formulation without affecting its susceptibility to sPLA₂. PEGylation of the liposomes only moderately broadened the release profile of luciferin. The provided bioassay represents a useful tool for monitoring active drug release in situ in real time as well as for testing and optimizing of sPLA₂-sensitive lipid formulations. In addition, the bioassay will pave the way for future in-depth in vitro and in vivo studies.     

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.     

Differential contributions of protein kinase C isoforms in the regulation of group IIA secreted phospholipase A2 expression in cytokine-stimulated rat fibroblasts

Protein kinase C (PKC) is a family of serine/threonine kinases involved in various signal transduction pathways. We investigated the roles of PKC in the regulation of group IIA secreted phospholipase A₂ (sPLA₂-IIA) expression in cytokine-stimulated rat fibroblastic 3Y1 cells. Here we show that the induction of sPLA₂-IIA by proinflammatory cytokines was under the control of both classical cPKCα and atypical aPKCλ/ι pathways by using PKC inhibitors, a PKC activator, and PKC knockdowns. Treatment of 3Y1 cells with PKC selective inhibitors having broad specificity, such as chelerythrine chloride and GF109203X, blocked IL-1β/TNFα-dependent induction of sPLA₂-IIA protein in a dose-dependent manner. Treatment with the PKC activator phorbol 12-myristate 13-acetate (PMA), which activates cPKC and novel nPKC isoforms, markedly attenuated the cytokine-dependent induction of sPLA₂-IIA expression. In comparison, 24-h pretreatment with PMA, which down-regulates these PKC isoforms, markedly enhanced sPLA₂-IIA expression. Results with short hairpin RNA (shRNA)-mediated knockdown of PKC isoforms revealed that the cytokine-induced sPLA₂-IIA expression was markedly enhanced in cPKCα knockdown cells compared to those in replicate control cells. In contrast, knockdown of the aPKCλ/ι isoform reduced the cytokine-induced expression of sPLA₂-IIA. These results suggest that the aPKCλ/ι pathway is required for the induction of sPLA₂-IIA expression and that the cPKCα pathway acts as a negative regulator of sPLA₂-IIA expression in cytokine-stimulated rat fibroblasts.     

Phospholipase A2-like activity of human bocavirus VP1 unique region

Human bocavirus (HBoV) is a new parvovirus first discovered in 2005, which is associated with acute respiratory infection. Analysis of sequence homology has revealed that a putative phospholipase A₂ (PLA₂) motif exists in the VP1 unique region of HBoV. However, little is known about whether the VP1 unique region of HBoV has PLA₂ enzymatic activity and how these critical residues contribute to its PLA₂ activity. To address these issues, the VP1 unique region protein and four of its mutants, were expressed in Eschericha coli. The purified VP1 unique protein (VP1U) showed a typical Ca²⁺-dependent secreted PLA₂-like (sPLA₂) activity, which was inhibited by sPLA₂-specific inhibitors in a time-dependent manner. Mutation of one of the amino acids (21Pro, 41His, 42Asp or 63Asp) in VP1U almost eliminated the sPLA₂ activity of HBoV VP1U. These data indicate that VP1U of HBoV has sPLA₂-like enzymatic activity, and these residues are crucial for its sPLA₂-like activity. Potentially, VP1U may be a target for the development of anti-viral drugs for HBoV.     

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.     

Systemic siRNA delivery to leukocyte-implicated diseases

Short interfering RNA (siRNA), a small duplex of RNA fragment, has proved as an extremely useful research tool to interrogate gene functions in test tubes. However, the transformation of siRNAs from a functional genomic tool into a new therapeutic modality has been hindered by ineffective delivery methods for systemic administration. In this review, we will discuss the recent advances in formulating new delivery strategies that target siRNAs to specific cells following systemic administration. Special emphasis will be given to leukocytes, since siRNA delivery remains exceptionally challenging here due to the unavailability of effective delivery technologies. We will not only detail new platforms that utilize leukocyte integrins as receptor targets for siRNAs delivery, but also show how one of these strategies has been utilized for in vivo drug target validation of a novel anti-inflammatory target, cyclin D1, for inflammatory bowel diseases.     

In silico and in vitro characterization of phospholipase A2 isoforms from soybean (Glycine max)

At the present, no secreted phospholipase A₂ (sPLA₂) from soybean (Glycine max) was investigated in detail. In this work we identified five sequences of putative secreted sPLA₂ from soybean after a BLAST search in G. max database. Sequence analysis showed a conserved PA2c domain bearing the Ca²⁺ binding loop and the active site motif. All the five mature proteins contain 12 cysteine residues, which are commonly conserved in plant sPLA₂s. We propose a phylogenetic tree based on sequence alignment of reported plant sPLA₂s including the novel enzymes from G. max. According to PLA₂ superfamily, two of G. max sPLA₂s are grouped as XIA and the rest of sequences as XIB, on the basis of differences found in their molecular weights and deviating sequences especially in the N- and C-terminal regions of the isoenzymes. Furthermore, we report the cloning, expression and purification of one of the putative isoenzyme denoted as GmsPLA₂-XIA-1. We demonstrate that this mature sPLA₂ of 114 residues had PLA₂ activity on Triton:phospholipid mixed micelles and determine the kinetic parameters for this system. We generate a model based on the known crystal structure of sPLA₂ from rice (isoform II), giving first insights into the three-dimensional structure of folded GmsPLA₂-XIA-1. Besides describing the spatial arrangement of highly conserved pair HIS-49/ASP-50 and the Ca⁺² loop domains, we propose the putative amino acids involved in the interfacial recognition surface. Additionally, molecular dynamics simulations indicate that calcium ion, besides its key function in the catalytic cycle, plays an important role in the overall stability of GmsPLA₂-XIA-1 structure.     

Genes encoding phospholipases A2 mediate insect nodulation reactions to bacterial challenge

We propose that expression of four genes encoding secretory phospholipases A₂ (sPLA₂) mediates insect nodulation responses to bacterial infection. Nodulation is the quantitatively predominant cellular defense reaction to bacterial infection. This reaction is mediated by eicosanoids, the biosynthesis of which depends on PLA₂-catalyzed hydrolysis of arachidonic acid (AA) from cellular phospholipids. Injecting late instar larvae of the red flour beetle, Tribolium castaneum, with the bacterium, Escherichia coli, stimulated nodulation reactions and sPLA₂ activity in time- and dose-related manners. Nodulation was inhibited by pharmaceutical inhibitors of enzymes involved in eicosanoid biosynthesis, and the inhibition was rescued by AA. We cloned five genes encoding sPLA₂ and expressed them in E. coli cells to demonstrate these genes encode catalytically active sPLA₂s. The recombinant sPLA₂s were inhibited by sPLA₂ inhibitors. Injecting larvae with double-stranded RNAs specific to each of the five genes led to reduced expression of the corresponding sPLA₂ genes and to reduced nodulation reactions to bacterial infections for four of the five genes. The reduced nodulation was rescued by AA, indicating that expression of four genes encoding sPLA₂s mediates nodulation reactions. A polyclonal antibody that reacted with all five sPLA₂s showed the presence of the sPLA₂ enzymes in hemocytes and revealed that the enzymes were more closely associated with hemocyte plasma membranes following infection. Identifying specific sPLA₂ genes that mediate nodulation reactions strongly supports our hypothesis that sPLA₂s are central enzymes in insect cellular immune reactions.     

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.