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.

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.

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.

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.

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.

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.

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.

A new era of secreted phospholipase A2.

Among more than 30 members of the phospholipase A2 (PLA2) superfamily, secreted PLA2 (sPLA2) enzymes represent the largest family, being Ca(2+)-dependent low-molecular-weight enzymes with a His-Asp catalytic dyad. Individual sPLA2s exhibit unique tissue and cellular distributions and enzymatic properties, suggesting their distinct biological roles. Recent studies using transgenic and knockout mice for nearly a full set of sPLA2 subtypes, in combination with sophisticated lipidomics as well as biochemical and cell biological studies, have revealed distinct contributions of individual sPLA2s to various pathophysiological events, including production of pro- and anti-inflammatory lipid mediators, regulation of membrane remodeling, degradation of foreign phospholipids in microbes or food, or modification of extracellular noncellular lipid components. In this review, we highlight the current understanding of the in vivo functions of sPLA2s and the underlying lipid pathways as revealed by a series of studies over the last decade.

Expression, purification, refolding, and enzymatic characterization of two secretory phospholipases A2 from Neurospora crassa.

Secretory phospholipase A2 (sPLA2) catalyzes the hydrolysis of sn-2 linkage in the glycerophospholipid, thereby releasing fatty acid and 1-acyl lysophospholipid. Among sPLA2s from various organisms and tissues, group XIV fungal/bacterial sPLA2s are relatively less characterized compared to their mammalian counterparts. Here we report cloning, recombinant expression, refolding, and enzymatic characterization of two sPLA2s, NCU06650 and NCU09423, from the filamentous fungus Neurospora crassa. The hexahistidine-tagged putative mature region of both proteins was expressed in Escherichia coli. Inclusion bodies were solubilized using a high hydrostatic pressure refolding technique. NCU06650 was solubilized without any additives at alkaline pH, and the addition of arginine or non-detergent sulfobetain (NDSB) significantly improved the process at acidic pH. In contrast, NCU09423 was solubilized only when NDSB was added at alkaline pH. Both enzymes displayed a Ca(2+)-dependent lipolytic activity toward E. coli membrane. Mass spectrometry analysis using the synthetic phospholipids as substrates demonstrated that both enzymes preferentially cleaved the sn-2 ester linkage of substrates and generated 1-acyl lysophospholipids, demonstrating that they are bona fide PLA2.

Single lipid vesicle assay for characterizing single-enzyme kinetics of phospholipid hydrolysis in a complex biological fluid.

Imaging of individual lipid vesicles is used to track single-enzyme kinetics of phospholipid hydrolysis. The method is employed to quantify the catalytic activity of phospholipase A2 (PLA2) in both pure and complex biological fluids. The measurements are demonstrated to offer a subpicomolar limit of detection (LOD) of human secretory PLA2 (sPLA2) in up to 1000-fold-diluted cerebrospinal fluid (CSF). An additional new feature provided by the single-enzyme sensitivity is that information about both relative concentration variations of active sPLA2 in CSF and the specific enzymatic activity can be simultaneously obtained. When CSF samples from healthy controls and individuals diagnosed with Alzheimer's disease (AD) are analyzed, the specific enzymatic activity is found to be preserved within 7% in the different CSF samples whereas the enzyme concentration differs by up to 56%. This suggests that the previously reported difference in PLA2 activity in CSF samples from healthy and AD individuals originates from differences in the PLA2 expression level rather than from the enzyme activity. Conventional ensemble averaging methods used to probe sPLA2 activity do not allow one to obtain such information. Together with an improvement in the LOD of at least 1 order of magnitude compared to that of conventional assays, this suggests that the method will become useful in furthering our understanding of the role of PLA2 in health and disease and in detecting the pharmacodynamic effects of PLA2-targeting drug candidates.

Class specific peptide inhibitors for secretory phospholipases A2.

Phospholipases A2 (PLA2) catalyze the hydrolytic cleavage of free fatty acids from the sn-2 OH-moiety of glycerophospholipids. These enzymes have a number of functions, from digestion to signaling and toxicity of several venoms. They have also been implicated in inflammation and are connected to diverse diseases, such as cancer, ischemia, atherosclerosis, and schizophrenia. Accordingly, there is a keen interest to develop selective inhibitors for therapeutic use. We recently proposed a novel mechanism for the control of PLA2 activity with highly active protofibrils of PLA2 existing transiently before conversion to inactive amyloid fibrils [19]. In keeping with the above mechanism several algorithms identified (85)KMYFNLI(91) and (17)AALSYGFYG(25) in bee venom (bv) and human lacrimal fluid (Lf) PLA2, respectively, as a regions potentially forming amyloid type aggregates. Interestingly, in keeping with the proposed role of these sequences in the control of the activity of these enzymes, preincubation of 2nM bvPLA2 with (85)KMYFNLI(91) caused complete inhibition of PLA2 activity while the scrambled control peptide YNFLIMK had no effect. Approximately 36% attenuation of the hydrolytic activity of LfPLA2 present in human lacrimal fluid was observed in the presence of 80nM (17)AALSYGFYG(25).

Phospholipase A2 enzymes and the risk of atherosclerosis.

Certain members of the phospholipase A(2) superfamily of enzymes have established causal involvement in atherosclerosis, thus at least two groups of this family of enzymes have been considered potential candidates for the prevention of cardiovascular events. Recently completed experimental animal studies, human biomarker data, vascular imaging studies, and genome-wide atherosclerosis studies provide the rationale for proceeding with clinical outcome trials directed at inhibition of secretory phospholipase A(2) and lipoprotein-associated phospholipase A(2). A clinical trial with the sPLA(2) inhibitor varespladib methyl was recently terminated, while clinical trials with the Lp-PLA(2) inhibitor darapladib are being conducted in coronary heart disease patients. This article reviews the available experimental animal and human trial evidence that serve as the basis for the development of these two classes of phospholipase A(2) inhibitors.

Cloning and functional expression of secreted phospholipases A(2) from Bothrops diporus (Yarará Chica).

Bothrops diporus is a very common viper in Argentina. At present, no complete sequence of secreted phospholipase A(2) (sPLA(2)) from this snake has been reported. We have cloned two sPLA(2) isoenzymes as well as a putative sPLA(2)-like myotoxin from venom gland. The two sPLA(2) were expressed as inclusion bodies in Escherichia coli with an N-terminal tag of ubiquitin. After in vitro renaturation and cleavage step, using an ubiquitin specific peptidase, the recombinants exhibited sPLA(2) activity when analyzed by means of Langmuir dilauroylphosphatidylcholine monolayers as substrate. Both enzymes have a similar surface pressure-activity profile when compared with non-recombinant purified isoforms. To our knowledge, this is the first time that analysis of optimal lateral pressure of substrate monolayers by using the surface barostat technique is performed on recombinant sPLA(2)s.

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.

T-cell and antibody responses to phospholipase A2 from different species show distinct cross-reactivity patterns.

BACKGROUND: Secreted phospholipases A2 (sPLA2) represent antigens to which humans may be rarely or frequently exposed. Thus, the investigation of humoral and cellular immune responses to sPLA2s from different species can provide a suitable model in the study of antibody and T-cell cross-reactivity. METHODS: Specific IgE, IgG1, IgG4, and IgA antibodies were analyzed by ELISA against sPLA2s from pancreas of Bos taurus (BT), Apis mellifera (AM) bee venom, Daboia russellii (DR) and Naja mossambica (NM) snake venoms, and human group III (hGIII) sPLA2 using sera of nonallergic beekeepers, AM-allergic patients, and healthy controls. T-cell cross-reactivity was investigated in PBMC, and T-cell clones (TCC) are generated against AM sPLA2. RESULTS: Hyperimmune and allergic individuals showed high levels of sPLA2-specific IgG4 and significant IgG4 cross-reactivity between BT, DR, and NM sPLA2s. Furthermore, IgE, IgA, and IgG1 cross-reactivities against BT, DR, and NM sPLA2s were also detectable in the range of 22.2-44.8%. Allergic patients showed significant T-cell proliferative response to NM sPLA2 together with increased IFN-γ and IL-13 production even though they had never been exposed to cobra venom. Although nonallergic healthy controls show no cross-reactivity at T-cell level, they did have low levels of IgG4 and IgA against BT, DR, and NM sPLA2s. Human TCC spanning three major T-cell epitopes of AM sPLA2 showed minor proliferative response to NM and hGIII sPLA2s. CONCLUSIONS: This study shows that T cells and antibodies may show cross-reactivity between different species without being naturally exposed to sPLA2s.

Surface pressure-dependent interactions of secretory phospholipase A2 with zwitterionic phospholipid membranes.

The hydrolytic activity of secretory phospholipase A(2) (PLA(2)) is regulated by many factors, including the physical state of substrate aggregates and the chemical nature of phospholipid molecules. In order to achieve strong binding of PLA(2) on its substrates, many previous works have used anionic lipid dispersion to characterize the orientation and penetration depth of PLA(2) molecules on membrane surfaces. In this study, we applied monolayer technique with controllable surface area to investigate the PLA(2)s of Taiwan cobra venom and bee venom on zwitterionic phophatidylcholine monolayers and demonstrated an optimum hydrolytic activity at a surface pressure of 18 and 24 mN/m, respectively. By combining polarized attenuated total reflection Fourier-transform infrared spectroscopy and monolayer-binding experiments, we found that the amount of membrane-bound PLA(2) decreased markedly as the surface pressure of the monolayer was increased. Interestingly, the insertion area of the PLA(2)s decreased to near zero as the surface pressure increased to the optimum pressure for hydrolytic activity. On the basis of the measured infrared dichroic ratio, the orientation of the PLA(2)s bound to zwitterionic membranes was similar to that observed on a negatively charged membrane and was independent of the surface pressure. Our findings suggest that both PLA(2)s were located on the membrane surface rather than penetrating the membrane bilayer and that the deeply inserted mode is not a favorable condition for the hydrolysis of phospholipids in zwitterionic phospholipid membranes. The results are discussed in terms of the easy access of catalytic water for the PLA(2) activity and the mobilization of its substrate and product to facilitate the catalytic process.

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.

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.