Phospholipase A2 Production Research Articles

Enhanced baculoviral virulence by suppressing the degradation of an insect immune resolvin, epoxyoctadecamonoenoic acid, in three lepidopteran insects

Epoxyoctadecamonoenoic acids (EpOMEs) are produced from linoleic acid by a cytochrome P450 monooxygenase (CYP) and play a crucial role in terminating excessive and unnecessary immune responses during the late infection stage in insects. This suggests that an increase in the EpOME level may enhance the virulence of insect pathogens against pests. This study tested this hypothesis using a specific inhibitor against soluble epoxide hydrolase (sEH) to degrade EpOMEs, which leads to elevated endogenous EpOME levels. A baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), was used to infect three different lepidopteran insects (Spodoptera exigua, Maruca vitrata, and Plutella xylostella) by oral feeding or hemocoelic injection treatments. Within one hour, the viral infection induced the expression of three different phospholipase A2 (PLA2) genes and, after 12 h, up-regulated the expressions of CYP and sEH genes in Spodopera exigua. As expected, AcMNPV virulence was suppressed by the addition of arachidonic acid (a catalytic product of PLA2) but was enhanced by the addition of either of the EpOME regioisomers. In addition, treatment with a specific sEH inhibitor (AUDA) increased AcMNPV virulence against three different lepidopteran insects, presumably by increasing endogenous EpOME levels. This enhanced effect of EpOMEs on virulence was further supported by specific RNA interference (RNAi), in which RNAi specific to CYP expression decreased AcMNPV virulence while a specific RNAi against sEH expression significantly enhanced virulence. In response to AcMNPV infection, TUNEL assay results showed that S. exigua larvae exhibited apoptosis in the midgut, fat body, and epidermis. Inhibition of apoptosis by a pan-caspase inhibitor, Z-VAD-FMK, significantly increased virulence. Similarly, the addition of AUDA to the viral treatment suppressed the gene expression of five inducible caspases and cytochrome C to suppress apoptosis, which led to a significant increase in the tissue viral titers. These results indicate that EpOMEs play a role in terminating excessive and unnecessary immune responses against viral infection during the late stage by down-regulating antiviral apoptosis in lepidopteran insects.

Quercetin Influences Phospholipase A2 Products in A549 Adenocarcinoma Cells

The effect of quercetin on phospholipase A2 activity and its products has been investigated in A549 adenocarcinoma cells. Quercetin is a polyphenol of medico-biological significance, which has been reported to show antioxidant and anti-tumour beneficial effects. Phospholipids are functionally active molecules, which participate in various cellular processes, acting as second messengers, enzyme activators, membrane transporters, etc. Although there are many studies dealing with the influence of quercetin on the lipid metabolism of different types of cancer cells, the mechanism of this effect
 remains largely unclear. The present study showed that quercetin treatment of A549 cells induced activation of phospholipase A2 (PLA2) activity, the latter being known as a source of pro-inflammatory intermediate metabolites. The phospholipid analysis of membranes from A549 cells revealed reduction of
 phosphatidylcholine (PC) and an increase of the level of lysophosphatidylcholine (LPC). In addition, acyl chain analysis showed an elevation of some polyunsaturated fatty acids, such as arachidonic acid (AA), the latter being a major product of PLA2. The content of lipid peroxides was also elevated in membranes from quercetin-treated A549 cells, which we presume is related to the higher content of polyunsaturated fatty acids, which are an excellent target of oxidative destruction.
 In conclusion, the present results show that treatment of A549 adenocarcinoma cells with the polyphenol quercetin induces activation of membrane-bound PLA2, which is a source polyunsaturated fatty acids, the
 latter producing lipid peroxides. We presume that this mechanism underlies the reported by other authors elevation of oxidative stress, which is induced by quercetin treatment of cancerous cells.

TCA cycle remodeling drives proinflammatory signaling in humans with pulmonary tuberculosis.

The metabolic signaling pathways that drive pathologic tissue inflammation and damage in humans with pulmonary tuberculosis (TB) are not well understood. Using combined methods in plasma high-resolution metabolomics, lipidomics and cytokine profiling from a multicohort study of humans with pulmonary TB disease, we discovered that IL-1β-mediated inflammatory signaling was closely associated with TCA cycle remodeling, characterized by accumulation of the proinflammatory metabolite succinate and decreased concentrations of the anti-inflammatory metabolite itaconate. This inflammatory metabolic response was particularly active in persons with multidrug-resistant (MDR)-TB that received at least 2 months of ineffective treatment and was only reversed after 1 year of appropriate anti-TB chemotherapy. Both succinate and IL-1β were significantly associated with proinflammatory lipid signaling, including increases in the products of phospholipase A2, increased arachidonic acid formation, and metabolism of arachidonic acid to proinflammatory eicosanoids. Together, these results indicate that decreased itaconate and accumulation of succinate and other TCA cycle intermediates is associated with IL-1β-mediated proinflammatory eicosanoid signaling in pulmonary TB disease. These findings support host metabolic remodeling as a key driver of pathologic inflammation in human TB disease.

Comparative expression of recombinant phospholipase A2 in Komagataella phaffii depending on the modification of the alpha-factor signaling peptide

Currently, the Russian market of phospholipase A2 enzyme preparations is represented by commercial preparations of foreign manufacturers: Nagase (Japan) and Maxapal (the Netherlands). However, the growing demand and the need to reduce the cost of production of phospholipase A2 require the development of new super-producers of phospholipase A2. In this connection, the aim of the work is to compare the expression of recombinant phospholipase A2 in Komagataella phaffii depending on the modification of the alpha-factor signaling peptide. The object of the study is the recipient yeast strain Komagataella phaffii X-33. The studies were conducted in accordance with generally accepted norms and approaches. Phospholipase A2 genes from Streptomyces violaceoruber were used for this worK. The target sequences were synthesized in the company "Eurogen" (Russia) and cloned as part of the TE vector pUC57. In the course of the work, the genetic constructs pPICZaA-Pla2 and PPICZmf4iA-Pla2 containing the Streptomyces violaceoruber phospholipase A2 gene were assembled under the native signal a-MF and its modification mf4i. The transformation of the yeast Komagataella phaffii X-33 with the obtained genetic constructs was also carried out. As a result of the conducted studies, it was shown that on average, there were no significant differences in the level of expression and specific activity of recombinant phospholipase A2 in methylotrophic yeast K. Phaffii X-33 when using the native a-MF secretion signal and its modified version mf4i. However, the use of the secretion factor mf4i allows for higher production of phospholipase A2 in individual clones. The obtained data indicate the prospects of using the secretion factor mf4i to create super-producers of enzymes based on yeast K. Phaffii X-33.

Repat33 Acts as a Downstream Component of Eicosanoid Signaling Pathway Mediating Immune Responses of Spodoptera exigua, a Lepidopteran Insect

Simple SummaryEicosanoids are oxygenated polyunsaturated fatty acids containing 20 carbons and subdivided into prostaglandin, leukotriene, and epoxyeicosatrienoic acid. They mediate immune responses in insects as well as mammals. Excessive eicosanoids cause severe inflammatory diseases in humans. However, a lack of eicosanoids induces immunosuppressive conditions in insects, which are highly susceptible to various entomopathogens. Despite the physiological significance of eicosanoids, their molecular action was not clearly understood in insects. This study was focused on Repat (Response to Pathogen) gene family, which might be regulated by eicosanoids. Among 44 members of Repat family genes, Repat33 was highly inducible to Gram-negative bacteria. Its expression was dependent on eicosanoids. Loss of function of Repat33 by RNA interference (RNAi) caused significant immunosuppression of a lepidopteran insect, Spodoptera exigua. Larvae of S. exigua treated by RNAi did not exhibit efficient cellular immune responses. They also failed to express antimicrobial peptide genes at high inducible levels in response to bacterial challenges. These results suggest that Repat33 is a downstream component of eicosanoid immune mediation.Repat (=response to pathogen) is proposed for an immune-associated gene family from Spodoptera exigua, a lepidopteran insect. In this gene family, 46 members (Repat1–Repat46) have been identified. They show marked variations in their inducible expression patterns in response to infections by different microbial pathogens. However, their physiological functions in specific immune responses and their interactions with other immune signaling pathways remain unclear. Repat33 is a gene highly inducible by bacterial infections. The objective of this study was to analyze the physiological functions of Repat33 in mediating cellular and humoral immune responses. Results showed that Repat33 was expressed in all developmental stages and induced in immune-associated tissues such as hemocytes and the fat body. RNA interference (RNAi) of Repat33 expression inhibited the hemocyte-spreading behavior which impaired nodule formation of hemocytes against bacterial infections. Such RNAi treatment also down-regulated expression levels of some antimicrobial genes. Interestingly, Repat33 expression was controlled by eicosanoids. Inhibition of eicosanoid biosynthesis by RNAi against a phospholipase A2 (PLA2) gene suppressed Repat33 expression while an addition of arachidonic acid (a catalytic product of PLA2) to RNAi treatment recovered such suppression of Repat33 expression. These results suggest that Repat33 is a downstream component of eicosanoids in mediating immune responses of S. exigua.

Immunosuppressive Activities of Novel PLA2 Inhibitors from Xenorhabdus hominickii, an Entomopathogenic Bacterium.

Simple SummaryInsect immune responses defend fatal attacks from entomopathogens. A Gram-negative Xenorhabdus hominickii exhibits high entomopathogenicity against lepidopteran insects. During the pathogenic processes, the bacteria suppress host insect immune responses by inhibiting phospholipase A2 (PLA2) enzyme activity with the bacterial secondary metabolites. PLA2 catalyzes eicosanoid biosynthesis. Eicosanoids mediate both cellular and humoral immune responses against various insect pathogens. This study identified eight different PLA2 inhibitors from the bacterial culture broth. Butanol extract of the culture broth possessed high potency to inhibit PLA2 activity. Subsequent fractionations led to identification of eight different compounds. The synthetic compounds also showed PLA2 inhibition and insecticidal activities. Furthermore, the addition of the bacterial PLA2 inhibitors significantly enhance other bacterial pathogenicity, suggesting its potential to be applied for developing novel insecticides. Eicosanoids mediate both cellular and humoral immune responses in insects. Phospholipase A2 (PLA2) catalyzes the first committed step in eicosanoid biosynthesis. It is a common pathogenic target of two entomopathogenic bacteria, Xenorhabdus and Photorhabdus. The objective of this study was to identify novel PLA2 inhibitors from X. hominickii and determine their immunosuppressive activities. To identify novel PLA2 inhibitors, stepwise fractionation of X. hominickii culture broth and subsequent enzyme assays were performed. Eight purified fractions of bacterial metabolites were obtained. Gas chromatography and mass spectrometry (GC-MS) analysis predicted that the main components in these eight fractions were 2-cyanobenzoic acid, dibutylamine, 2-ethyl 1-hexanol, phthalimide (PM), dioctyl terephthalate, docosane, bis (2-ethylhexyl) phthalate, and 3-ethoxy-4-methoxyphenol (EMP). Their synthetic compounds inhibited the activity of PLA2 in hemocytes of a lepidopteran insect, Spodoptera exigua, in a dose-dependent manner. They also showed significant inhibitory activities against immune responses such as prophenoloxidase activation and hemocytic nodulation of S. exigua larvae, with PM and EMP exhibiting the most potent inhibitory activities. These immunosuppressive activities were specific through PLA2 inhibition because an addition of arachidonic acid, a catalytic product of PLA2, significantly rescued such suppressed immune responses. The two most potent compounds (PM and EMP) showed significant insecticidal activities after oral administration. When the compounds were mixed with Bacillus thuringiensis (Bt), they markedly increased Bt pathogenicity. This study identified eight PLA2 inhibitors from bacterial metabolites of X. hominickii and demonstrated their potential as novel insecticides.

Expression of Highly Active Bacterial Phospholipase A2 in Yeast Using Intein-Mediated Delayed Protein Autoactivation.

Phospholipase A2 (PLA2) has found extensive use in industry. However, recombinant PLA2 production in different expression systems is a difficult task because of its toxicity to cell membranes. We report here the development of an effective method for production of highly active PLA2 from Streptomyces violaceoruber strain A-2688 in the yeast Saccharomyces cerevisiae. The method is based on the use of the PRP8 mini-intein (from Penicillium chrysogenum) inserted into the phospholipase sequence with the purpose of temporal inactivation of the enzyme and its subsequent delayed autoactivation. We demonstrate that the most effective site for intein insertion is Ser76 of the mature phospholipase. As a result of intein-containing precursor secretion from yeast cells and its subsequent autocatalytic splicing, highly active enzyme accumulated in the yeast culture fluid. The properties of the obtained recombinant phospholipase A2 protein were similar to those of the native Streptomyces violaceoruber PLA2 protein. A possible evolutionary role of delayed autoactivation of intein-containing proteins is also discussed.

Engineering of a Microbial Cell Factory for the Extracellular Production of Catalytically Active Phospholipase A2 of Streptomyces violaceoruber.

Phospholipase A2 (PLA2) from Streptomyces violaceoruber is a lipolytic enzyme used in a wide range of industrial applications including production of lysolecithins and enzymatic degumming of edible oils. We have therefore investigated expression and secretion of PLA2 in two workhorse microbes, Pichia pastoris and Escherichia coli. The PLA2 was produced to an activity of 0.517 ± 0.012 U/ml in the culture broth of the recombinant P. pastoris. On the other hand, recombinant E. coli BL21 star (DE3), overexpressing the authentic PLA2 (P-PLA2), showed activity of 17.0 ± 1.3 U/ml in the intracellular fraction and 21.7 ± 0.7 U/ml in the culture broth. The extracellular PLA2 activity obtained with the recombinant E. coli system was 3.2-fold higher than the corresponding value reached in a previous study, which employed recombinant E. coli BL21 (DE3) overexpressing codon-optimized PLA2. Finally, we observed that the extracellular PLA2 from the recombinant E. coli P-PLA2 culture was able to hydrolyze 31.1 g/l of crude soybean lecithin, an industrial substrate, to a conversion yield of approximately 95%. The newly developed E. coli-based PLA2 expression system led to extracellular production of PLA2 to a productivity of 678 U/l·h, corresponding to 157-fold higher than that obtained with the P. pastoris-based system. This study will contribute to the extracellular production of a catalytically active PLA2.

Host Immunosuppression Induced by Steinernema feltiae, an Entomopathogenic Nematode, through Inhibition of Eicosanoid Biosynthesis.

Steinernema feltiae K1 (Filipjev) (Nematode: Steinernematidae), an entomopathogenic nematode, was isolated and identified based on its morphological and molecular diagnostic characteristics. Its infective juveniles (IJs) were highly pathogenic to three lepidopteran (LC50 = 23.7–25.0 IJs/larva) and one coleopteran (LC50 = 39.3 IJs/larva) insect species. Infected larvae of the diamondback moth, Plutella xylostella (L.) (Insecta: Lepidoptera), exhibited significant reduction in phospholipase A2 (PLA2) activity in their plasma. The decrease of PLA2 activity was followed by significant septicemia of the larvae infected with S. feltiae. Insecticidal activity induced by S. feltiae was explained by significant immunosuppression in cellular immune responses measured by hemocyte nodule formation and total hemocyte count (THC). Although S. feltiae infection suppressed nodule formation and THC in the larvae, an addition of arachidonic acid (AA, a catalytic product of PLA2) rescued these larvae from fatal immunosuppression. In contrast, an addition of dexamethasone (a specific PLA2 inhibitor) enhanced the nematode’s pathogenicity in a dose-dependent manner. To discriminate the immunosuppressive activity of a symbiotic bacterium (Xenorhabdus bovienii (Proteobacteria: Enterobacterales)) from the nematode, kanamycin was applied to after nematode infection. It significantly inhibited the bacterial growth in the hemolymph. Compared to nematode treatment alone, the addition of antibiotics to nematode infection partially rescued the immunosuppression measured by phenol oxidase activity. Consequently, treatment with antibiotics significantly rescued the larvae from the insecticidal activity of S. feltiae. These results suggest that immunosuppression induced by infection of S. feltiae depends on its symbiotic bacteria by inhibiting eicosanoid biosynthesis, resulting in significant insect mortality. However, the addition of antibiotics or AA could not completely rescue the virulence of the nematode, suggesting that the nematode itself also plays a role in its insecticidal activity.

Variation in pathogenicity of different strains of Xenorhabdus nematophila; Differential immunosuppressive activities and secondary metabolite production

Xenorhabdus nematophila, an entomopathogenic bacterium, is mutualistic with the nematode Steinernema carpocapsae. The bacterium produces secondary metabolites to inhibit target insect phospholipase A2 (PLA2) and induce immunosuppression, which is required for the pathogenicity of this bacterium-nematode complex. However, it was unclear if immunosuppressive intensity of the bacteria was correlated with their insecticidal potency. We compared six different X. nematophila strains inhibiting the immune responses of the beet armyworm (Spodoptera exigua) to explain their virulence variations. In addition to four known strains obtained from the Korean Agricultural Culture Collection, we identified two new strains (SK1 and SK2) of X. nematophila from two different isolates of S. carpocapsae. Although all six strains were virulent, they showed significant variation in median lethal bacterial dosage (LD50). The LD50 of most strains was 15–30 CFU/larva, however, the LD50 of the SK1 strain was more than two-fold higher against S. exigua larvae. Immunosuppressive activities of the six strains were measured by comparing hemocyte-spreading behavior and nodule formation; the SK1 strain was significantly less potent than other bacterial strains. These suppressed hemocyte behaviors were recovered by adding arachidonic acid (a catalytic product of PLA2) into all six strains. Bacterial culture broth was fractionated with different organic solvents and the ability to inhibit immune response and PLA2 activity were assessed. All organic extracts had immunosuppressive activities and PLA2-inhibitory activities. GC-MS analysis showed that these organic extracts possessed a total of 87 different compounds. There were variations in chemical components among the six bacterial strains. Organic extracts of SK1 strain, which exhibited the lowest virulence, contained the least number of secondary metabolites.

Toll/IMD signal pathways mediate cellular immune responses via induction of intracellular PLA 2 expression.

Phospholipase A2 (PLA2 ) hydrolyzes fatty acids from phospholipids at the sn-2 position. Two intracellular PLA2 s, iPLA2 A and iPLA2 B, have been found in Spodoptera exigua. Both are calcium-independent cellular PLA2 . Their orthologs have been found in other insects. These two iPLA2 s are different in ankyrin motif of N terminal region. The objective of this study was to determine whether Toll/immune deficiency (IMD) signal pathways could mediate cellular immune responses via induction of iPLA2 expression. Both iPLA 2 s were expressed in all developmental stages of S. exigua, showing the highest expression in the adult stage. During larval stage, hemocyte is the main tissue showing expression of these iPLA2 s. Both iPLA2 s exhibited similar expression patterns after immune challenge with different microbial pathogens such as virus, bacteria, and fungi. Promoter component analysis of orthologs encoded in S. frugiperda indicated nuclear factor-κB- and Relish-responsible elements on their promoters, suggesting their expression in S. exigua under Toll/IMD immune signaling pathways. RNA interference (RNAi) of MyD88 or Pelle under Toll pathway suppressed inducible expression levels of both iPLA2 s in response to Gram-positive bacteria containing Lys-type peptidoglycan or fungal infection. In contrast, RNAi against Relish under IMD pathway suppressed both iPLA2 s in response to infection with Gram-negative bacteria. Under RNAi conditions, hemocytes significantly lost cellular immune response measured by nodule formation. However, addition of arachidonic acid (a catalytic product of PLA2 ) rescued such immunosuppression. These results suggest that Toll/IMD signal pathways can mediate cellular immune responses via eicosanoid signaling by inducing iPLA2 expression.

Unique enzyme specificity of three human phospholipases A2 toward phospholipids containing sn‐2 omega‐3 and omega‐6 fatty acids

Phospholipase A2 (PLA2) constitutes a superfamily of enzymes that hydrolyzes the sn‐2 acyl‐chain of membrane phospholipids producing lyso‐phospholipids and free fatty acids. The products of PLA2 have various functions, especially omega‐3 and omega‐6 fatty acids which are precursor of various eicosanoids and related metabolites involved in numerous pro‐inflammatory and pro‐resolving cellular responses. The PLA2 superfamily is composed of six different types of enzymes, each with distinct structural features. Among them, the cytosolic PLA2 (cPLA2), calcium‐independent PLA2 (iPLA2), and secreted PLA2 (sPLA2) are well studied. cPLA2 is the main provider of free arachidonic acid (AA), an omega‐6 fatty acid involved in the inflammatory cascade. Several knockout mice studies demonstrated the contribution of iPLA2 to the metabolism of the omega‐3 fatty acid docosahexaenoic acid (DHA) in brain (J. Lipid Res. 2010, 51, 3166–3173). sPLA2 is known for its antibacterial action and involvement in atherosclerosis. Previous studies in our laboratory determined the substrate specificity of all three PLA2s toward a variety of membrane phospholipids (J. Am. Chem. Society 2018 140, 3285–3291). However, the specificity of these enzymes toward phospholipids containing omega‐3 and omega‐6 fatty acids at the sn‐2 position has been unclear. In this study, we have employed a lipidomics‐based LC‐MS/MS assay, which measures the lysophospholipid product to measure the activity of PLA2, and to investigate the sn‐2 acyl‐chain specificity in various phospholipids. We have also developed a lipidomics‐based GC‐MS assay which allow us to measure free fatty acids in mixtures of phospholipid substrates. Although cPLA2 showed relatively high activity toward the omega‐3 eicosapentaenoic acid (EPA), it prefers AA. iPLA2 showed a distinct preference for EPA over AA and a much lesser activity toward phospholipids containing DHA. Unlike the other two PLA2s, sPLA2 showed a remarkable preference for DHA among the omega‐3 and omega‐6 fatty acids. Importantly, similar sn‐2 acyl‐chain specificity was obtained in mixtures of phospholipids. This study investigated the preference of three human PLA2 enzymes for the sn‐2 omega‐3 and omega‐6 fatty acids for the first time. Our findings will help us to understand the difference of function of PLA2 enzymes at the cellular level and their implications in inflammatory diseases.Support or Funding InformationSupported by NIH grant GM20501This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phospholipase A2 products predict the hematopoietic support capacity of horse serum

Horse serum is commonly used as an additive to support the maintenance of hematopoietic progenitor cells in culture. However, the wide variability in the performance of different lots calls for parallel testing of multiple batches over extended periods of culture. Identification of the serum components that determine hematopoietic support would therefore save considerable time and effort and would help to standardize culture procedures. We report here that the ability of horse serum to support the self-renewal of multipotent murine hematopoietic progenitor FDCP-Mix cells is correlated to the concentration of specific fatty acid products of phospholipase A2 and more closely to the spectrum of eicosanoids generated by their further processing through cyclooxygenase and lipoxygenase pathways. Supportive sera have low levels of lysophosphatidylcholine and inflammatory eicosanoids. This links known markers of inflammation, infection and platelet activation to the ability of serum to maintain progenitor cells in an undifferentiated state, providing a means for prospective identification of suitable sera as well as quality control of the production process.

Structural and Aggregation Properties of Alpha-Synuclein Linked to Phospholipase A2 Action.

Alpha-synuclein is a protein involved in the pathogenesis of Parkinson's disease. In vitro observations have shown that specific brain-enriched polyunsaturated fatty acids, such as arachidonic acid, can give rise to a conformational change in alpha-synuclein and ultimately induce its fibrillation. Arachidonic acid is released by phospholipase A2 activity and clinical observations have shown a link between mutations in PLA2G6, the gene responsible for the production of phospholipase A2, and early-onset types of parkinsonism. It is unknown how phospholipase A2-driven release of arachidonic acid can affect the conformation of alphasynuclein. The main objective of this study was to investigate if phospholipase A2-induced release of arachidonic acid can induce changes in conformation and aggregation state of alpha-synuclein. Recombinant human alpha-synuclein was expressed and isolated and incubated in the presence of phosphatidylcholine and phosphatidylserine (PC/PS) containing liposomes. The release of free fatty acids from PC/PS liposomes by bee venom phospholipase A2 was measured with the fluorescent probe acrylodated intestinal fatty acid-binding protein (ADIFAB) and radioactive labelling by preparing liposomes in the presence of L- 3-phosphatidylcholine, 1-stearyl-2[1-14C] arachidonoyl. The effect of free fatty acid release on the conformation of alpha-synuclein was assayed by far-UV circular dichroism and resistance against V8 protease-induced limited proteolysis. Aggregation of alpha-synuclein upon exposure to phospholipase A2-induced action on PC/PS liposomes was measured using thioflavin T fluorescence, SDS-PAGE, gel filtration chromatography, and transmission electron microscopy. RAW264.7 cells were transiently transfected with human alpha-synuclein and release of arachidonic acid was quantified using radiolabeling and liquid scintillation counting. Phospholipase A2 is capable of releasing arachidonic acid from biomimetic phospholipid membranes. Exposure of alpha-synuclein to phospholipase A2-induced release of arachidonic acid from PC/PS liposomes induces a conformational transition of the protein and leads to partial resistance against proteolytic cleavage by V8 protease. Prolonged incubation of alpha-synuclein with arachidonic acid, derived from PC/PS liposomes by phospholipase A2 leads to aggregate formation. In line with this, transiently transfected RAW264.7 cells with alpha-synuclein showed arachidonic acid release and punctate alpha-synuclein staining upon phospholipase A2 activation. The ability of arachidonic acid to drive alpha-synuclein to aggregate was independent of its oxidation state. We present data that suggest a biological context for the previously reported clinical observation that linked mutations in PLA2G6, the gene responsible for the production of phospholipase A2, and early-onset types of parkinsonism. Release of arachidonic acid, independent of its oxidation state, through activation of phospholipase A2-driven hydrolysis of phospholipid membranes, leads to the structural transition and aggregation of alpha-synuclein.

Differential immunosuppression by inhibiting PLA2 affects virulence of Xenorhabdus hominickii and Photorhabdus temperata temperata.

Immunity negatively influences bacterial pathogenicity. Eicosanoids mediate both cellular and humoral immune responses in insects. This study tested a hypothesis that differential bacterial virulence of Xenorhabdus/Photorhabdus is dependent on their inhibitory activity against phospholipase A2 (PLA2) activity. P. temperata subsp. temperata ('Ptt') was more than 40 times more potent than X. hominickii ('Xh'). Although both bacteria suppressed cellular immune responses, Ptt infection suppressed hemocyte nodule formation much more than Xh infection. Their differential immunosuppression appeared to be induced by their secondary metabolites because organic extracts of Ptt-cultured broth exhibited higher inhibitory activities against cellular immune responses than Xn-cultured broth extracts. Humoral immune responses were analyzed by measuring expression levels of 11 antimicrobial peptide (AMP) genes. Among inducible AMPs in hemocytes and fat body, higher number and more kinds of AMPs exhibited lower expression levels in Ptt infection than those in Xh infection. Suppressed immune responses induced by Ptt or Xh infection were significantly rescued by the addition of a catalytic product of PLA2, suggesting that PLA2 was a common inhibitory target. In fact, Ptt infection inhibited PLA2 activity more strongly than Xh infection. RNA interference of a PLA2 gene decreased its expression and significantly increased bacterial virulence. Moreover, addition of PLA2 inhibitor to Xh infection enhanced its virulence, similar to virulence level of Ptt infection. These results suggest that variation in Xenorhabdus/Photorhabdus bacterial virulence can be explained by their differential inhibitory activities against host insect PLA2.

Recombinant-phospholipase A2 production and architecture of inclusion bodies are affected by pH in Escherichia coli

Aggregation of recombinant proteins into inclusion bodies (IBs) is the major drawback of heterologous expression in Escherichia coli. Here, we evaluated the effects of a pH shift after expression induction on recombinant phospholipase A2 production and its aggregation in IBs in E. coli Origami™, as compared to cultures with pH maintained at 7.5 or uncontrolled pH. Cultures shifted from 7.5 to pH 6.5 or 8.5 produced ∼15–25% less biomass as compared with those kept at 7.5 or without pH control. The cultures shifted to pH 8.5 showed a ∼50% higher yield of acetate per biomass, and the rPLA2 yield was improved 2.4-fold. Purified IBs formed at pH 8.5 containing ∼50% of rPLA2, were more susceptible to proteinase-K cleavage and bound less thioflavin-T, indicating lower amyloid content, with the concomitant enrichment of α-helical and random-coil secondary structures, as demonstrated by FTIR. Moreover, only one IB per cell was formed at pH 8.5; instead, more than two were observed under the other culture pH conditions. Nevertheless, under uncontrolled pH conditions, ∼300nm larger IBs were observed. Our work presents evidence of the usefulness of recombinant protein expression cultivated at pH 8.5 allowing the reduction of amyloid content in IBs.

Origin and evolution of group XI secretory phospholipase A2 from flax (Linum usitatissimum) based on phylogenetic analysis of conserved domains.

Phospholipase A2 (PLA2) belongs to class of lipolytic enzymes (EC 3.1.1.4). Lysophosphatidic acid (LPA) and free fatty acids (FFAs) are the products of PLA2 catalyzed hydrolysis of phosphoglycerides at sn-2 position. LPA and FFA that act as second mediators involved in the development and maturation of plants and animals. Mining of flax genome identified two phospholipase A2 encoding genes, viz., LusPLA 2 I and LusPLA 2 II (Linum usitatissimum secretory phospholipase A2). Molecular simulation of LusPLA2s with already characterized plant sPLA2s revealed the presence of conserved motifs and signature domains necessary to classify them as secretory phospholipase A2. Phylogenetic analysis of flax sPLA2 with representative sPLA2s from other organisms revealed that they evolved rapidly via gene duplication/deletion events and shares a common ancestor. Our study is the first report of detailed phylogenetic analysis for secretory phospholipase A2 in flax. Comparative genomic analysis of two LusPLA2s with earlier reported plant sPLA2s, based on their gene architectures, sequence similarities, and domain structures are presented elucidating the uniqueness of flax sPLA2.

Effect of products of PLA2 catalyzed hydrolysis of DLPC on motion of rising bubbles

Local velocities of rising bubbles decrease with the increasing concentration in solution of surface-active, water-soluble species. Therefore, it is possible to use this phenomenon to monitor products of enzymatic reactions, which meet such criteria. In this study, hydrolysis of 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC) catalyzed by calcium-dependent phospholipase A2 (PLA2) (EC3.1.1.4) from porcine pancreas was used as model reaction. The products of this reaction are lauric acid (LA) and 1-lauroyl-2-hydroxy-sn-glycero-3-phosphatidylcholine (Lyso-PC). DLPC was dispersed in a chloroform/methanol mixture that was spread on a free PLA2 solution surface. Air bubbles were then formed at a capillary orifice and the local velocity of rising bubbles as a function of the distance from the capillary tip was monitored. Local velocity profiles were compared with profiles recorded for solutions of pure enzymatic reaction products and their mixtures. Our experiments showed that the product, which had a dominating effect on bubble motion retardation, was lyso-phosphatidylcholine. This can be explained by differences in the kinetics of lauric acid and lyso-phosphatidylcholine transfer from the spread layer to the solution.

Gossypol Increases Expression of the Pro-apoptotic BH3-only Protein NOXA through a Novel Mechanism Involving Phospholipase A2, Cytoplasmic Calcium, and Endoplasmic Reticulum Stress

Gossypol is a putative BH3 mimetic proposed to inhibit BCL2 and BCLXL based on cell-free assays. We demonstrated previously that gossypol failed to directly inhibit BCL2 in cells or induce apoptosis in chronic lymphocytic leukemia (CLL) cells or platelets, which require BCL2 or BCLXL, respectively, for survival. Here, we demonstrate that gossypol rapidly increased activity of phospholipase A2 (PLA2), which led to an increase in cytoplasmic calcium, endoplasmic reticulum (ER) stress, and up-regulation of the BH3-only protein NOXA. Pretreatment with the PLA2 inhibitor, aristolochic acid, abrogated the increase in calcium, ER stress, and NOXA. Calcium chelation also abrogated the gossypol-induced increase in calcium, ER stress, and NOXA, but not the increase in PLA2 activity, indicating that PLA2 is upstream of these events. In addition, incubating cells with the two products of PLA2 (lysophosphatidic acid and arachidonic acid) mimicked treatment with gossypol. NOXA is a pro-apoptotic protein that functions by binding the BCL2 family proteins MCL1 and BFL1. The BCL2 inhibitor ABT-199 is currently in clinical trials for CLL. Resistance to ABT-199 can occur from up-regulation of other BCL2 family proteins, and this resistance can be mimicked by culturing CLL cells on CD154(+) stroma cells. We report here that AT-101, a derivative of gossypol in clinical trials, overcomes stroma-mediated resistance to ABT-199 in primary CLL cells, suggesting that a combination of these drugs may be efficacious in the clinic.

Genotyping of Malassezia pachydermatis isolates from canine healthy skin and atopic dermatitis by internal spacer 1 (IGS1) region analysis

Isolates of Malassezia pachydermatis from healthy dog skin and from dogs with atopic dermatitis were molecularly characterized using internal spacer 1 (IGS1) region analyses, and their phospholipase A2 activity and pH growth profiles were then characterized in vitro. The percentage of isolates from healthy dogs that had the following IGS1 subtypes (isotype, %) were as follows: 1A, 6%; 1B, 27%; 1C, 11%; 2A, 6%; 2B, 6%; 3A, 11%; 3C, 3%; and 3D, 24%. In contrast, 9% of isolates from dogs with atopic dermatitis were isotype IB and 91% were isotype 3D, indicating that isolates of subtype 3D were the most prevalent in dogs with atopic dermatitis. Production of phospholipase A2 was statistically higher in isolates of subtype 3D than in the other subtypes. The subtype 3D isolates showed enhanced growth on alkaline medium compared with non-3D subtype isolates. The main clinical sign of canine Malassezia dermatitis is waxy exudates on the skin, which predispose the patient to development of a yeast overgrowth of the subtype 3D. Increased phospholipase A2 production may be involved in the inflammatory process associated with Malassezia dermatitis.