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.

Isolation and Characterization of A2-EPTX-Nsm1a, a Secretory Phospholipase A2 from Malaysian Spitting Cobra (Naja sumatrana) Venom.

Phospholipase A2 (PLA2) toxins are one of the main toxin families found in snake venom. PLA2 toxins are associated with various detrimental effects, including neurotoxicity, myotoxicity, hemostatic disturbances, nephrotoxicity, edema, and inflammation. Although Naja sumatrana venom contains substantial quantities of PLA2 components, there is limited information on the function and activities of PLA2 toxins from the venom. In this study, a secretory PLA2 from the venom of Malaysian N. sumatrana, subsequently named A2-EPTX-Nsm1a, was isolated, purified, and characterized. A2-EPTX-Nsm1a was purified using a mass spectrometry-guided approach and multiple chromatography steps. Based on LC-MSMS, A2-EPTX-Nsm1a was found to show high sequence similarity with PLA2 from venoms of other Naja species. The PLA2 activity of A2-EPTX-Nsm1 was inhibited by 4-BPB and EDTA. A2-EPTX-Nsm1a was significantly less cytotoxic in a neuroblastoma cell line (SH-SY5Y) compared to crude venom and did not show a concentration-dependent cytotoxic activity. To our knowledge, this is the first study that characterizes and investigates the cytotoxicity of an Asp49 PLA2 isolated from Malaysian N. sumatrana venom in a human neuroblastoma cell line.

Dynamic Role of Phospholipases A2 in Health and Diseases in the Central Nervous System.

Phospholipids are major components in the lipid bilayer of cell membranes. These molecules are comprised of two acyl or alkyl groups and different phospho-base groups linked to the glycerol backbone. Over the years, substantial interest has focused on metabolism of phospholipids by phospholipases and the role of their metabolic products in mediating cell functions. The high levels of polyunsaturated fatty acids (PUFA) in the central nervous system (CNS) have led to studies centered on phospholipases A2 (PLA2s), enzymes responsible for cleaving the acyl groups at the sn-2 position of the phospholipids and resulting in production of PUFA and lysophospholipids. Among the many subtypes of PLA2s, studies have centered on three major types of PLA2s, namely, the calcium-dependent cytosolic cPLA2, the calcium-independent iPLA2 and the secretory sPLA2. These PLA2s are different in their molecular structures, cellular localization and, thus, production of lipid mediators with diverse functions. In the past, studies on specific role of PLA2 on cells in the CNS are limited, partly because of the complex cellular make-up of the nervous tissue. However, understanding of the molecular actions of these PLA2s have improved with recent advances in techniques for separation and isolation of specific cell types in the brain tissue as well as development of sensitive molecular tools for analyses of proteins and lipids. A major goal here is to summarize recent studies on the characteristics and dynamic roles of the three major types of PLA2s and their oxidative products towards brain health and neurological disorders.

Structural, enzymatic and pharmacological profiles of AplTX-II - A basic sPLA2 (D49) isolated from the Agkistrodon piscivorus leucostoma snake venom.

A basic sPLA2 (D49) from the venom of snake Agkistrodon piscivorus leucostoma (AplTX-II) was isolated, purified and characterized. We determined the enzymatic and pharmacological profiles of this toxin. AplTX-II was isolated with a high level of purity through reverse phase chromatography and molecular exclusion. The enzyme showed pI 9.48 and molecular weight of 14,003 Da. The enzymatic activity of the AplTX-II depended on Ca2+ pH and temperature. The comparison of the primary structure with other sPLA2s revealed that AplTX-II presented all the structural reasons expected for a basic sPLA2s. Additionally, we have resolved its structure with the docked synthetic substrate NOBA (4-nitro-3-octanoyloxy benzoic acid) by homology modeling, and performed MD simulations with explicit solvent. Structural similarities were found between the enzyme's modeled structure and other snake sPLA2 X-Ray structures, available in the PDB database. NOBA and active-site water molecules spontaneously adopted stable positions and established interactions in full agreement with the reaction mechanism, proposed for the physiological substrate, suggesting that NOBA hydrolysis is an excellent model to study phospholipid hydrolysis.

Structural and Functional Aspects of Targeting the Secreted Human Group IIA Phospholipase A2.

Human group IIA secretory phospholipase A2 (hGIIA) promotes the proliferation of cancer cells, making it a compelling therapeutic target, but it is also significant in other inflammatory conditions. Consequently, suitable inhibitors of hGIIA have always been sought. The activation of phospholipases A2 and the catalysis of glycerophospholipid substrates generally leads to the release of fatty acids such as arachidonic acid (AA) and lysophospholipid, which are then converted to mediator compounds, including prostaglandins, leukotrienes, and the platelet-activating factor. However, this ability of hGIIA to provide AA is not a complete explanation of its biological role in inflammation, as it has now been shown that it also exerts proinflammatory effects by a catalysis-independent mechanism. This mechanism is likely to be highly dependent on key specific molecular interactions, and the full mechanistic descriptions of this remain elusive. The current candidates for the protein partners that may mediate this catalysis-independent mechanism are also introduced in this review. A key discovery has been that selective inhibition of the catalysis-independent activity of hGIIA is achieved with cyclised derivatives of a pentapeptide, FLSYK, derived from the primary sequence of hGIIA. The effects of hGIIA on cell function appear to vary depending on the pathology studied, and so its mechanism of action is complex and context-dependent. This review is comprehensive and covers the most recent developments in the understanding of the many facets of hGIIA function and inhibition and the insight they provide into their clinical application for disease treatment. A cyclic analogue of FLSYK, c2, the most potent analogue known, has now been taken into clinical trials targeting advanced prostate cancer.

Structural aspects and activation mechanism of human secretory group IIA phospholipase.

Phospholipases are important probes for understanding structure-function relationships of membrane proteins. Many neurotoxins have phospholipase activity, and they have been recognized to be potential therapeutic agents for biological warfare. Understanding the modes of action of these enzymes is important for the development of effective therapeutic strategies. Human secretory phospholipases A2 (sPLA2) interact with cellular membranes and catalyze the hydrolysis of phosphate ester bonds of phospholipids. The activity of these enzymes increases tremendously upon binding to a hydrophobic interface. Using molecular dynamics (MD) simulations in implicit solvent and membrane environments, we investigated alterations in structure and conformation of human sPLA2 upon its interaction with a membrane that may be associated with the activation of the enzyme. In 50 ns MD simulations, starting from six different initial orientations of the protein relative to the membrane surface, the enzyme consistently adopted a membrane-bound configuration in close agreement with the known experimental data. The simulations also reproduced the experimentally determined distribution of hydrophobic and polar side chains on the interfacial binding surface. Differences in the dynamic behavior of the enzyme between the solvent and membrane-bound states were observed. In nonpolar media, the enzyme underwent major conformational rearrangements, which exposed the active site to the membrane. The increased mobility of the surface loop and the ß-wing regions is required for the conformational change, which is essentially induced by the movement of N-terminal helix. Several active site residues underwent structural changes that reorganize the binding site for substrate catalysis. Overall, the results provided a valuable insight into the interfacial behavior of sPLA2 enzyme and suggested that membrane binding is essential but insufficient for sPLA2 activation.

sPLA2 behaves like a prophylactic agent and mediates cellular and humoral immune responses in Plutella xylostella.

Most immune effectors are inducible to microbial pathogen infection while some are already present to act as prophylactic immunity against as yet unseen infection. This study identified secretory phospholipase A2 (sPLA2 ) as a prophylactic factor in diamondback moth, Plutella xylostella. Western blotting using a polyclonal antibody raised against other lepidopteran sPLA2 reacted specifically with ∼25 kDa protein, which was present at approximately 0.4 mM in the plasma of naïve larvae. Interrogation of P. xylostella transcriptomes revealed an open-reading frame for sPLA2 (Px-sPLA2 ), exhibiting high homology with other Group III sPLA2 s. Px-sPLA2 was expressed in all developmental stages. In the larval stage, bacterial challenge induced its expression in hemocytes and fat body but not in gut or epidermis. RNA interference (RNAi) suppressed Px-sPLA2 messenger RNA level and sPLA2 activity in plasma. An inhibition zone assay showed that Px-sPLA2 exhibited antibacterial activities against different species, because specific RNAi knockdown impaired the activity. The RNAi treatment also suppressed the cellular immune response assessed by hemocyte nodule formation and humoral immune response assessed by antimicrobial peptide gene expression. Finally, benzylideneacetone (BZA, a specific sPLA2 inhibitor) treatment inhibited plasma sPLA2 activity of naive larvae in a dose-dependent manner. An addition of BZA significantly increased the bacterial virulence of an entomopathogen, Bacillus thuringiensis. These results suggest that Px-sPLA2 is an immune-associated factor of P. xylostella and its relatively high level of concentration in the plasma of naive larvae strongly suggests its role as a prophylactic factor in defending against pathogens at early infection stages.

Bioaffinity Fishing Procedure Using Secretory Phospholipase A2 for Screening for Bioactive Components: Modulation of Pharmacological Effect Induced by sPLA2 from Crotalus durissus terrificus by Hispidulin from Moquiniastrum floribundum.

Bioaffinity capturing of molecules allows the discovery of bioactive compounds and decreases the need for various stages in the natural compound isolation process. Despite the high selectivity of this technique, the screening and identification methodology depends on the presence of a protein to capture potential ligands. However, some proteins, such as snake secretory phospholipase A2 (sPLA2), have never been investigated using this approach. The purpose of this study was to evaluate the use of a new method for screening natural compounds using a bioaffinity-guided ultrafiltration method on Crotalus durissus terrificus sPLA2 followed by HPLC-MS to identify the compounds, and this method could be used to discover new anti-inflammatory compounds from the various organisms originating from biodiversity. Different extracts were selected to evaluate their ability to inhibit sPLA2 activity. The extracts were incubated with sPLA2 and the resulting mixture was ultrafiltrated to elute unbound components. The resulting compounds were identified by HPLC-MS. We identified hispidulin as one of the components present in the Moquiniastrum floribundum leaf and evaluated the ability of this isolated compound to neutralize the inflammatory activity of sPLA2 from Crotalus durissus terrificus.

Studies of crab digestive phospholipase acting on phospholipid monolayers: Activation by temperature.

Secreted phospholipases A2 (sPLA2) are water-soluble lipolytic enzymes that act at the interface of organized lipid substrates, where the catalytic step is coupled to various interfacial phenomena as enzyme penetration, solubilisation of reaction products, lateral packing and loss of mechanical stability of organized assemblies of phospholipid molecule, among others. Using the monomolecular film technique, we compared the interfacial properties of crab digestive sPLA2 (CDPL) with those of the porcine pancreatic one (PPPL). A kinetic study on the surface pressure dependency of the two sPLA2 was performed using monomolecular films of three different substrates: di C12-PC (1.2-dilauroyl-sn-glycerol-3-phosphocholine); di C12-PG (1.2-dilauroyl-sn-glycerol-3-phosphoglycerol) and di C12-PE (1.2-dilauroyl-sn-glycerol-3-phosphoethanolamine). The use of a substrate in monolayer state, during the catalytic reactions, allows us to monitor the effect of several physicochemical parameters by altering the "quality of interface". The effect of temperature on the hydrolysis rate of these substrates was also checked. Our results show that activities of both phospholipases were affected by the variation of the subphase temperature. CDPL was irreversibly inactivated by p-bromo-phenacyl bromide, the specific inhibitor of sPLA2. The hyperbolic catalytic behaviour observed was coherent with hopping mode of action, one of the two characteristic mechanisms of interfacial catalysis of sPLA2.

Intestinal phospholipase A2 from Sparidae species: Functional properties and cytotoxic potential evaluation.

Marine species have gained significant attention as potential source for a broad spectrum of bioactive proteins. Fish phospholipases A2 (PLA2) have attracted renewed interest due to their excellent properties in lipid digestion. Herein, we report for the first time the catalytic properties of two intestinal secreted PLA2 (sPLA2) identified from Diplodus sargus (IDsPLA2) and Sparus aurata (ISaPLA2). The highest sequence identity was obtained with recently isolated Sparidae digestive PLA2 (45%) and Human pancreatic PLA2 (42%). IDsPLA2 and ISaPLA2 were overexpressed in E. coli as inclusion bodies, refolded and purified. Both enzymes have improved thermostability compared to mammalian pancreatic sPLA2 since they are active and stable at 55 °C, with specific activities of 320 and 190 U mg-1 measured on phosphatidylcholine, respectively. Interestingly, IDsPLA2, but not ISaPLA2, revealed weak toxicity towards macrophages and suggests its involvement in cell membrane degradation. ISaPLA2 was found to be more active than IDsPLA2 when using the monolayer technique at 20 mN m-1. Structural models of both enzymes revealed their differences. In silico docking of phospholipids with both models allowed proposing key amino-acids in substrate binding and selectivity. Overall, these results provide insight into the enzymatic and structural properties of two novel sPLA2 with potential for future applications.

Secretion of secretory phospholipase A2 into Spodoptera exigua larval midgut lumen and its role in lipid digestion.

In insects, lipid digestion is controversial because insects have no bile salts to solubilize dietary lipids. One hypothesis is that a secretory type of phospholipase A2 (sPLA2 ) provides lysophospholipid (LPL) from dietary phospholipids (PLs). We identified a sPLA2 , Se-sPLA2 , in beet armyworm, Spodoptera exigua, that hydrolyses PLs at sn-2. Our goal was to investigate its role in lipid digestion. Se-sPLA2 was expressed in the entire alimentary canal. Incubating the isolated midgut in a cell culture medium led to secretion of Se-sPLA2 and other proteins. Ex vivo RNA interference (RNAi) of Se-sPLA2 expression in isolated midgut culture led to significantly decreased Se-sPLA2 secretion into the medium. Feeding double-stranded RNA specific to Se-sPLA2 to larvae suppressed sPLA2 activity in gut contents. A recombinant Se-sPLA2 was susceptible to benzylideneacetone (BZA), a specific PLA2 inhibitor. After feeding BZA to larvae, we recorded significant decreases in gut content sPLA2 activity, body growth and total haemolymph lipid contents. RNAi against Se-sPLA2 resulted in reduced digestibility. Addition of a specific LPL, 1-palmitoyl-sn-glycero-3-phosphocholine, to BZA-treated larvae rescued digestibility and larval growth. These results strongly bolster our hypothesis that Se-sPLA2 secreted from the midgut acts in lipid digestion by providing necessary LPL to solubilize dietary neutral lipids.

Molecular interactions of phospholipid monolayers with a model phospholipase.

The intrinsic overexpression of secretory phospholipase A2 (sPLA2) in various pro-inflammatory diseases and cancers has the potential to be exploited as a therapeutic strategy for diagnostics and treatment. To explore this potential and advance our knowledge of the role of sPLA2 in related diseases, it is necessary to systematically investigate the molecular interaction of the enzyme with lipids. By employing a Langmuir trough integrated with X-ray reflectivity and grazing incidence X-ray diffraction techniques, this study examined the molecular packing structure of 1,2-palmitoyl-sn-glycero-3-phosphocholine (DPPC) films before and after enzyme adsorption and enzyme-catalyzed degradation. Molecular interaction of sPLA2 (from bee venom) with the DPPC monolayer exhibited Ca2+ dependence. DPPC molecules at the interface without Ca2+ retained a monolayer organization; upon adsorption of sPLA2 to the monolayer the packing became tighter. In contrast, sPLA2-catalyzed degradation of DPPC occurred in the presence of Ca2+, leading to disruption of the ordered monolayer structure of DPPC. The interfacial film became a mixture of highly ordered multilayer domains of palmitic acid (PA) and loosely packed monolayer phase of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (lysoPC) that potentially contained the remaining un-degraded DPPC. The redistribution of lipid degradation products into the third dimension, which produced multilayer PA domains, damaged the structural integrity of the original lipid layer and may explain the bursting of liposomes observed in other studies after a latency period of mixing liposomes with sPLA2. A quantitative understanding of the lipid packing and lipid-enzyme interaction provides an intuitive means of designing and optimizing lipid-related drug delivery systems.

Targeted Lignan Profiling and Anti-Inflammatory Properties of Schisandra rubriflora and Schisandra chinensis Extracts.

Schisandra rubriflora is a dioecious plant of increasing importance due to its lignan composition, and therefore, possible therapeutic properties. The aim of the work was lignan profiling of fruits, leaves and shoots of female (F) and male (M) plants using UHPLC-MS/MS. Additionally, the anti-inflammatory activity of plant extracts and individual lignans was tested in vitro for the inhibition of 15-lipooxygenase (15-LOX), phospholipases A2 (sPLA2), cyclooxygenase 1 and 2 (COX-1; COX-2) enzyme activities. The extracts of fruits, leaves and shoots of the pharmacopoeial species, S. chinensis, were tested for comparison. Twenty-four lignans were monitored. Lignan contents in S. rubriflora fruit extracts amounted to 1055.65 mg/100 g DW and the dominant compounds included schisanhenol, aneloylgomisin H, schisantherin B, schisandrin A, gomisin O, angeloylgomisin O and gomisin G. The content of lignan in leaf extracts was 853.33 (F) and 1106.80 (M) mg/100 g DW. Shoot extracts were poorer in lignans-559.97 (F) and 384.80 (M) mg/100 g DW. Schisantherin B, schisantherin A, 6-O-benzoylgomisin O and angeloylgomisin H were the dominant compounds in leaf and shoot extracts. The total content of detected lignans in S. chinensis fruit, leaf and shoot extracts was: 1686.95, 433.59 and 313.83 mg/100 g DW, respectively. Gomisin N, schisandrin A, schisandrin, gomisin D, schisantherin B, gomisin A, angeloylgomisin H and gomisin J were the dominant lignans in S. chinensis fruit extracts were. The results of anti-inflammatory assays revealed higher activity of S. rubriflora extracts. Individual lignans showed significant inhibitory activity against 15-LOX, COX-1 and COX-2 enzymes.

In silico investigation of the molecular effects caused by R123H variant in secretory phospholipase A2-IIA associated with ARDS.

Phospholipase A2-IIA catalyzes the hydrolysis of the sn-2 ester of glycerophospholipids. A rare c.428G > A (p.Arg143His) variant in PLA2G2A gene was found in two infants affected by acute respiratory distress syndrome (ARDS) by whole coding region and exon/intron boundaries sequencing. To obtain insights into the possible molecular effects of the rare R123H mutation in secretory PLA2-IIA (sPLA2-IIA), molecular modelling, molecular dynamics (MD) using principal component analysis (PCA) and continuum electrostatic calculations were conducted on the crystal structure of the wild type protein and on a generated model structure of the R123H mutant. Analysis of MD trajectories indicate that the overall stability of the protein is not affected by this mutation but nevertheless the catalytically crucial H-bond between Tyr51 and Asp91 as well as main electrostatic interactions in the region close to the mutation site are altered. PCA results indicate that the R123H replacement alter the internal molecular motions of the enzyme and that collective motions are increased. Electrostatic surface potential studies suggest that after mutation the interfacial binding to anionic phospholipid membranes and anionic proteins may be changed. The strengthening of electrostatic interactions may be propagated into the active site region thus potentially affecting the substrate recognition and enzymatic activity. Our findings provide the basis for further investigation and advances our understanding of the effects of mutations on sPLA2 structure and function.

Membrane Allostery and Unique Hydrophobic Sites Promote Enzyme Substrate Specificity.

We demonstrate that lipidomics coupled with molecular dynamics reveal unique phospholipase A2 specificity toward membrane phospholipid substrates. We discovered unexpected headgroup and acyl-chain specificity for three major human phospholipases A2. The differences between each enzyme's specificity, coupled with molecular dynamics-based structural and binding studies, revealed unique binding sites and interfacial surface binding moieties for each enzyme that explain the observed specificity at a hitherto inaccessible structural level. Surprisingly, we discovered that a unique hydrophobic binding site for the cleaved fatty acid dominates each enzyme's specificity rather than its catalytic residues and polar headgroup binding site. Molecular dynamics simulations revealed the optimal phospholipid binding mode leading to a detailed understanding of the preference of cytosolic phospholipase A2 for cleavage of proinflammatory arachidonic acid, calcium-independent phospholipase A2, which is involved in membrane remodeling for cleavage of linoleic acid and for antibacterial secreted phospholipase A2 favoring linoleic acid, saturated fatty acids, and phosphatidylglycerol.

Exploring the binding mechanism and kinetics of Piperine with snake venom secretory Phospholipase A2.

Secreted venom Phospholipase A2 is highly responsible for pharmacological effects like neurotoxicity, myotoxicity, hemolytic, anti-coagulation, and platelet aggregation. Neutralization of these pharmacological behaviors is one of the challenges existing for many decades and a potent drug compound for this is very much needed to control local effects of venom sPLA2. In this study, we investigated binding mechanism and kinetics of inhibition of Piperine (major constitute of Piper nigrum) with sPLA2 using DFT, MD simulation, MM-PBSA, and SPR method. Frontier MO properties were suggested that it procured better chemical reactivity and druglikeness and binding mode of Piperine with EcPLA2 defined that it occupied well in N-terminal hydrophobic cleft. The persistence of Piperine interactions with and without calcium ion was analyzed and confirmed by MD simulation analysis. The dPCA-based FEL shows the nature of apo- and Piperine-bound conformational behavior of EcPLA2 including intermediate forms. Further, binding energy of Piperine was calculated by high-throughput MM-PBSA which states that calcium ion presence enhances the Piperine binding by additional electrostatic interactions. Finally, kinetics of inhibition between Piperine and EcPLA2 implied that it secured better binding affinity (KD: as 1.708 pM) and the result gives clear evidence for the binding mechanism and binding energy calculated. In conclusion, Piperine was authenticated with better drug ability, entrenched binding interaction, and robust kinetics of inhibition with EcPLA2 through which it can become an exceeding drug candidate for pharmacological as well as catalytic activity of sPLA2.

Molecular details of secretory phospholipase A2 from flax (Linum usitatissimum L.) provide insight into its structure and function.

Secretory phospholipase A2 (sPLA2) are low molecular weight proteins (12-18 kDa) involved in a suite of plant cellular processes imparting growth and development. With myriad roles in physiological and biochemical processes in plants, detailed analysis of sPLA2 in flax/linseed is meagre. The present work, first in flax, embodies cloning, expression, purification and molecular characterisation of two distinct sPLA2s (I and II) from flax. PLA2 activity of the cloned sPLA2s were biochemically assayed authenticating them as bona fide phospholipase A2. Physiochemical properties of both the sPLA2s revealed they are thermostable proteins requiring di-valent cations for optimum activity.While, structural analysis of both the proteins revealed deviations in the amino acid sequence at C- & N-terminal regions; hydropathic study revealed LusPLA2I as a hydrophobic protein and LusPLA2II as a hydrophilic protein. Structural analysis of flax sPLA2s revealed that secondary structure of both the proteins are dominated by α-helix followed by random coils. Modular superimposition of LusPLA2 isoforms with rice sPLA2 confirmed monomeric structural preservation among plant phospholipase A2 and provided insight into structure of folded flax sPLA2s.

Evaluation of Rhamnetin as an Inhibitor of the Pharmacological Effect of Secretory Phospholipase A2.

Rhamnetin (Rhm), 3-O-methylquercetin (3MQ), and Rhamnazin (Rhz) are methylated derivatives of quercetin commonly found in fruits and vegetables that possess antioxidant and anti-inflammatory properties. Phospholipase A2 (PLA2) displays several important roles during acute inflammation; therefore, this study aimed at investigating new compounds able to inhibit this enzyme, besides evaluating creatine kinase (CK) levels and citotoxicity. Methylated quercetins were compared with quercetin (Q) and were incubated with secretory PLA2 (sPLA2) from Bothrops jararacussu to determine their inhibitory activity. Cytotoxic studies were performed by using the J774 cell lineage incubated with quercertins. In vivo tests were performed with Swiss female mice to evaluate decreasing paw edema potential and compounds' CK levels. Structural modifications on sPLA2 were made with circular dichroism (CD). Despite Q and Rhz showing greater enzymatic inhibitory potential, high CK was observed. Rhm exhibited sPLA2 inhibitory potential, no toxicity and, remarkably, it decreased CK levels. The presence of 3OH on the C-ring of Rhm may contribute to both its anti-inflammatory and enzymatic inhibition of sPLA2, and the methylation of ring A may provide the increase in cell viability and low CK level induced by sPLA2. These results showed that Rhm can be a candidate as a natural compound for the development of new anti-inflammatory drugs.

Structural basis for functional selectivity and ligand recognition revealed by crystal structures of human secreted phospholipase A2 group IIE.

Secreted phospholipases A2s (sPLA2s) are involved in various pathological conditions such as rheumatoid arthritis and cardiovascular disease. Many inhibitors were developed and studied in clinical trials, but none have reached the market yet. This failure may be attributed to the lack of subtype selectivity for these inhibitors. Therefore, more structural information for subtype sPLA2 is needed to guide the selective inhibitor development. In this study, the crystal structure of human sPLA2 Group IIE (hGIIE), coupled with mutagenesis experiments, proved that the flexible second calcium binding site and residue Asn21 in hGIIE are essential to its enzymatic activity. Five inhibitor bound hGIIE complex structures revealed the key residues (Asn21 and Gly6) of hGIIE that are responsible for interacting with inhibitors, and illustrated the difference in the inhibitor binding pocket with other sPLA2s. This will facilitate the structure-based design of sPLA2's selective inhibitors.

Distinguishing septic from normal donors by detection of sPLA2-IIA from human plasma using a microsieve-based immunoassay.

Bloodstream infections that progress to septic shock are responsible for hundreds of thousands of deaths each year, and are associated with significant healthcare costs. Recent studies have shown that a member of the secreted phospholipase protein family, termed sPLA2-IIA, may play a role during the innate immune response to bacterial infections, and is elevated in the plasma of septic patients. In this report, the feasibility of a simple microsieve-based sPLA2-IIA detection immunoassay was explored. Microsieves containing 5µm pores were covalently coupled with a sPLA2-IIA-specific monoclonal antibody at 0.1, 1.0, and 10µg/mL and then assayed with plasma-based positive and negative controls to determine the optimal coating concentration. Recombinant sPLA2-IIA was then serially diluted to a final concentration of 200, 100, 50, 25, 12.5, and 6.25ng/mL and tested alongside a non-spiked sample to estimate the detection limit of the prototype assay. Recombinant sPLA2-IIA was also spiked into serum, EDTA-plasma, and Lithium-Heparin plasma, in an effort to evaluate assay performance when analyzing these sample matrices. The preliminary limit of detection studies suggests that the microsieve assay is able to distinguish approximately 6-12ng/mL of sPLA2-IIA from a non-spiked sample. When compared to an immunoassay diluent, the microsieve assay also yielded acceptable percent recoveries for each of the three sample matrices spiked with clinically significant levels of sPLA2-IIA. The sPLA2-IIA microsieve assay prototype also clearly distinguished five samples from septic patients from five normal donor samples, and the results were in good agreement with a comparator ELISA test system (R2=0.9347).