Molecular Characterization of Three Novel Phospholipase A2 Proteins from the Venom of Atheris chlorechis, Atheris nitschei and Atheris squamigera

Secretory phospholipase A2 (sPLA2) is known as a major component of snake venoms and displays higher-order catalytic hydrolysis functions as well as a wide range of pathological effects. Atheris is not a notoriously dangerous genus of snakes although there are some reports of fatal cases after envenomation due to the effects of coagulation disturbances and hemorrhaging. Molecular characterization of Atheris venom enzymes is incomplete and there are only a few reports in the literature. Here, we report, for the first time, the cloning and characterization of three novel cDNAs encoding phospholipase A2 precursors (one each) from the venoms of the Western bush viper (Atheris chlorechis), the Great Lakes bush viper (Atheris nitschei) and the Variable bush viper (Atheris squamigera), using a “shotgun cloning” strategy. Open-reading frames of respective cloned cDNAs contained putative 16 residue signal peptides and mature proteins composed of 121 to 123 amino acid residues. Alignment of mature protein sequences revealed high degrees of structural conservation and identity with Group II venom PLA2 proteins from other taxa within the Viperidae. Reverse-phase High Performance Liquid Chromatography (HPLC) profiles of these three snake venoms were obtained separately and chromatographic fractions were assessed for phospholipase activity using an egg yolk suspension assay. The molecular masses of mature proteins were all identified as approximately 14 kDa. Mass spectrometric analyses of the fractionated oligopeptides arising from tryptic digestion of intact venom proteins, was performed for further structural characterization.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) as a therapeutic target to prevent retinal vasopermeability during diabetes

Lipoprotein-associated phospholipase A2 (Lp-PLA2) hydrolyses oxidized low-density lipoproteins into proinflammatory products, which can have detrimental effects on vascular function. As a specific inhibitor of Lp-PLA2, darapladib has been shown to be protective against atherogenesis and vascular leakage in diabetic and hypercholesterolemic animal models. This study has investigated whether Lp-PLA2 and its major enzymatic product, lysophosphatidylcholine (LPC), are involved in blood-retinal barrier (BRB) damage during diabetic retinopathy. We assessed BRB protection in diabetic rats through use of species-specific analogs of darapladib. Systemic Lp-PLA2 inhibition using SB-435495 at 10 mg/kg (i.p.) effectively suppressed BRB breakdown in streptozotocin-diabetic Brown Norway rats. This inhibitory effect was comparable to intravitreal VEGF neutralization, and the protection against BRB dysfunction was additive when both targets were inhibited simultaneously. Mechanistic studies in primary brain and retinal microvascular endothelial cells, as well as occluded rat pial microvessels, showed that luminal but not abluminal LPC potently induced permeability, and that this required signaling by the VEGF receptor 2 (VEGFR2). Taken together, this study demonstrates that Lp-PLA2 inhibition can effectively prevent diabetes-mediated BRB dysfunction and that LPC impacts on the retinal vascular endothelium to induce vasopermeability via VEGFR2. Thus, Lp-PLA2 may be a useful therapeutic target for patients with diabetic macular edema (DME), perhaps in combination with currently administered anti-VEGF agents.

Rare coding variants in the phospholipase D3 gene confer risk for Alzheimer's disease.

Genome-wide association studies (GWAS) have identified several risk variants for late-onset Alzheimer's disease (LOAD)1, 2. These common variants have replicable but small effects on LOAD risk and generally do not have obvious functional effects. Low-frequency coding variants, not detected by GWAS, are predicted to include functional variants with larger effects on risk. To identify low-frequency coding variants with large effects on LOAD risk, we carried out whole-exome sequencing (WES) in 14 large LOAD families and follow-up analyses of the candidate variants in several large LOAD case–control data sets. A rare variant in PLD3 (phospholipase D3; Val232Met) segregated with disease status in two independent families and doubled risk for Alzheimer’s disease in seven independent case–control series with a total of more than 11,000 cases and controls of European descent. Gene-based burden analyses in 4,387 cases and controls of European descent and 302 African American cases and controls, with complete sequence data for PLD3, reveal that several variants in this gene increase risk for Alzheimer’s disease in both populations. PLD3 is highly expressed in brain regions that are vulnerable to Alzheimer’s disease pathology, including hippocampus and cortex, and is expressed at significantly lower levels in neurons from Alzheimer’s disease brains compared to control brains. Overexpression of PLD3 leads to a significant decrease in intracellular amyloid-β precursor protein (APP) and extracellular Aβ42 and Aβ40 (the 42- and 40-residue isoforms of the amyloid-β peptide), and knockdown of PLD3 leads to a significant increase in extracellular Aβ42 and Aβ40. Together, our genetic and functional data indicate that carriers of PLD3 coding variants have a twofold increased risk for LOAD and that PLD3 influences APP processing. This study provides an example of how densely affected families may help to identify rare variants with large effects on risk for disease or other complex traits.

Patatin-like phospholipase domain-containing protein 3 and Liver Disease: Opportunities to Unravel Mechanisms Underlying Statistical Associations

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Effects of morin on snake venom phospholipase A(2) (PLA(2))

Flavonoids are potent anti-inflammatory compounds isolated from several plant extracts, and have been used experimentally against inflammatory processes. In this work, a PLA(2) isolated from the Crotalus durissus cascavella venom and rat paw oedema were used as a model to. study the effect of flavonoids on PLA(2). We observed that a treatment of PLA(2) with morin induces several modifications in the aromatic amino acids, with accompanying changes in its amino acid composition. In addition, results from circular dichroism spectroscopy and UV scanning revealed important structural modifications. Concomitantly, a considerable decrease in the enzymatic and antibacterial activities was observed, even though anti-inflammatory and neurotoxic activities were not affected. These apparent controversial results may be an indication that PLA(2) possess a second pharmacological site which does not affect or depend on the enzymatic activity. (c) 2005 Elsevier Ltd. All rights reserved.

Purification and renal effects of phospholipase A(2) isolated from Bothrops insularis venom

Bothrops insularis venom contains a variety of substances presumably responsible for several pharmacological effects. We investigated the biochemical and biological effects of phospholipase A(2) protein isolated from B. insularis venom and the chromatographic profile showed 7 main fractions and the main phospholipase A(2) (PLA(2)) enzymatic activity was detected in fractions IV and V. Fraction IV was submitted to a new chromatographic procedure on ion exchange chromatography, which allowed the elution of 5 main fractions designated as lV-1 to IV-5, from which lV-4 constituted the main fraction. The molecular homogeneity of this fraction was characterized by high-performance liquid chromatography (HPLC) and demonstrated by mass spectrometry (MS), which showed a molecular mass of 13984.20 Da; its N-terminal sequence presented a high amino acid identity (up to 95%) with the PLA(2) of Bothrops jararaca and Bothrops asper. Phospholipase A(2) isolated from B. insularis (Bi PLA(2)) venom (10 mu g/mL) was also studied as to its effect on the renal function of isolated perfused kidneys of Wistar rats (n = 6). Bi PLA(2) increased perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa+) and chloride tubular reabsorption (%TCl-) decreased at 120 min, without alteration in potassium transport. In conclusion, PLA(2) isolated from B. insularis venom promoted renal alterations in the isolated perfused rat kidney. (c) 2007 Elsevier Ltd. All rights reserved.

Purification and characterization of the biological effects of phospholipase A(2) from sea anemone Bunodosoma caissarum

Sea anemones contain a variety of biologically active substances. Bunodosoma caissarum is a sea anemone from the Cnidaria phylum, found only in Brazilian coastal waters. The aim of the present work was to study the biological effects of PLA(2) isolated from the sea anemone B. caissarum on the isolated perfused kidney, the arteriolar mesenteric bed and on insulin secretion. Specimens of B. caissarum were collected from the Sao Vicente Channel on the southern coast of the State of São Paulo, Brazil. Reverse phase HPLC analysis of the crude extract of B. caissarum detected three PLA(2) proteins (named BcPLA(2)1, BCPLA(2)2 and BcPLA(2)3) found to be active in B. caissarum extracts. MALDI-TOF mass spectrometry of BcPLA(2)1 showed one main peak at 14.7 kDa. The N-terminal amino acid sequence of BcPLA(2)1 showed high amino acid sequence identity with PLA(2) group III protein isolated from the Mexican lizard (PA23 HELSU, HELSU, PA22 HELSU) and with the honey bee Apis mellifera (PLA(2) and 1POC_A). In addition, BcPLA(2)1 also showed significant overall homology to bee PLA(2). The enzymatic activity induced by native BCPLA(2)1 (20 mu g/well) was reduced by chemical treatment with p-bromophenacyl bromide (p-BPB) and with morin. BcPLA(2)1 strongly induced insulin secretion in presence of high glucose concentration. In isolated kidney, the PLA(2) from B. caissarum increased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, and sodium, potassium and chloride levels of excretion. BcPLA(2)1, however, did not increase the perfusion pressure on the mesenteric vascular bed. In conclusion, PLA(2), a group III phospholipase isolated from the sea anemone B. caissarum, exerted effects on renal function and induced insulin secretion in conditions of high glucose concentration. (C) 2009 Elsevier Ltd. All rights reserved.

Abdominal hyperalgesia in secretory phospholipase A(2)-induced rat pancreatitis: Distinct roles of NK1 receptors

We investigated the potential of secretory phospholipase A(2) (sPLA(2))-induced pancreatitis to promote abdominal hyperalgesia, as well as to depolarize sensory fibres in vitro using a grease-gap technique. Pancreatitis was induced by the injection of sPLA(2) from Crotalus durissus terrificus (sPLA(2) Cdt, 300 mu g kg(-1)) venom into the common bile duct of rats. Pancreatic inflammatory signs, serum amylase levels and abdominal hyperalgesia were evaluated in rats treated or not with SR140333, a tachykinin NK1 receptor antagonist. Injection of sPLA(2) Cdt caused pancreatic oedema formation and increased pancreatic neutrophil infiltration and serum amylase at 4 h, which returned to normality by 24 h, except for the neutrophil infiltration, which was still increased at this time point. Animals injected with sPLA(2) exhibited a lower withdrawal threshold to electronic von Frey stimulation in the upper abdominal region at 4 h, but not 24 h, post-injection when compared with saline-injected rats. Pre-treatment of animals with SR140333 significantly reduced the sPLA(2) Cdt-induced abdominal hyperalgesia, without affecting the other parameters. Neither sPLA(2) Cdt nor sPLA(2) from Naja mocambique mocambique venom depolarized capsaicin-sensitive sensory fibres from rat vagus nerve, but they decreased the propagated compound action potentials in both A and C fibres. These data show for the first time that NK1 receptors play an important role in the early abdominal hyperalgesia in a rat model of sPLA(2)-induced pancreatitis, suggesting that these receptors are of importance in the development of pain in the pancreatitis condition. We also provide evidence that sPLA(2)s do not directly depolarize sensory fibres in vitro. (C) 2011 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved.

Role of phospholipase A(2) and tyrosine kinase in Clostridium difficile toxin A-induced disruption of epithelial integrity, histologic inflammatory damage and intestinal secretion

Clostridium difficile-associated disease causes diarrhea to fulminant colitis and death. We investigated the role of phospholipase A(2) (PLA(2)) inhibitors, aristolochic acid (AA), bromophenacyl bromide BPB and quinacrine (QUIN) on the C. difficile toxin A-induced disruption of epithelial integrity, histologic inflammatory damage and intestinal secretion. Toxin A caused severe hemorrhagic and inflammatory fluid secretion at 6-8 h in rabbit ileal segments, an effect that was significantly inhibited by QUIN (71%, P < 0.01), AA (87%, P < 0.0001) or by BPB (51%, P < 0.01). The secretory effect of toxin A was also inhibited in segments adjacent to those with AA (89%, P < 0.01). Furthermore, QUIN or AA substantially reduced the histologic damage seen after 6-8 h in rabbit ileal segments. The cyclooxygenase inhibitor, indomethacin, also significantly inhibited (96%; n = 6) the secretory effects of toxin A in ligated rabbit intestinal segments. The destruction by toxin A of F-actin at the light junctions of T-84 cell monolayers was not inhibited by AA or BPB. AA or QUIN had no effect on the T-84 cell tissue resistance reduction over 8-24 h after toxin A exposure. All the inhibitors were shown to be effective in the doses administered direct in ileal loops to inhibit PLA(2) activity. The data suggest that PLA(2) is involved in the major pathway of toxin A-induced histologic inflammatory damage and hemorrhagic fluid secretion. Cop. right (C) 2008 John Wiley & Sons, Ltd.

Umbelliferone induces changes in the structure and pharmacological activities of Bn IV, a phospholipase A(2) isoform isolated from Bothrops neuwiedi

In this paper was demonstrated that umbelliferone induces changes in structure and pharmacological activities of Bn IV, a lysine 49 secretory phospholipase A(2) (sPLA2) from Both tops neuwiedi. Incubation of Bn IV with umbelliferone virtually abolished platelet aggregation, edema, and myotoxicity induced by native Bn IV. The amino acid sequence of Bn IV showed high sequence similarities with other Lys49 sPLA2s from B. jararacussu (BthTx-I), B. pirajai (PrTx-I), and B. neuwiedi pauloensis (Bn SP6 and Bn SP7). This sPLA2 also has a highly conserved C-terminal amino acid sequence, which has been shown as important for the pharmacological activities of Lys49 sPLA2. Sequencing of Bn IV previously treated with umbelliferone revealed modification of S(1) and S(20). Fluorescent spectral analysis and circular dichroism (CD) studies showed that umbelliferone modified the secondary structure of this protein. Moreover, the pharmacological activity of Bn IV is driven by synergism of the C-terminal region with the a-helix motifs, which are involved in substrate binding of the Asp49 and Lys49 residues of 5PLA2 and have a direct effect on the Ca2+-independent membrane damage of some secretory snake venom PLA2. For Bn IV, these interactions are potentially important for triggering the pharmacological activity of this 5PLA2. (C) 2011 Elsevier Ltd. All rights reserved.

Effect of umbelliferone (7-hydroxycoumarin, 7-HOC) on the enzymatic, edematogenic and necrotic activities of secretory phospholipase A2 (sPLA2) isolated from Crotalus durissus collilineatus venom

Flavonoids, coumarins and other polyphenolic compounds are powerful antioxiants both in hydrophilic and lipophylic environments with diverse pharmacological properties including anti-inflammatory activity. Despite being widely used as powerful therapeutic agents for blood coagulation disorders, more specifically to control some serine protease enzymes, the mechanism of anti-inflammatory activity of coumarins is unknown, unlike that of flavonoids. Although their controlling effect on serine proteases is well acknowledged, their action on secretory phospholipase A2 (sPLA2) remains obscure. The present study describes the interaction between umbelliferone (7-HOC) and the sPLA2 from Crotalus durissus collilineatus venom. In vitro inhibition of sPLA2 enzymatic activity by 7-HOC was estimated using 4N3OBA as substrate, resulting in an irreversible decrease in such activity proportional to 7-HOC concentration. The biophysical interaction between 7-HOC and sPLA2 was examined by fluorescent spectral analysis and circular dichroism studies. Results from both techniques clearly showed that 7-HOC strongly modified the secondary structure of this enzyme and CD spectra revealed that it strongly decreased sPLA2 alphahelical conformation. In addition, two-dimensional electrophoresis indicated an evident difference between HPLC-purified native and 7-HOC-treated sPLA2s, which were used in pharmacological experiments to compare their biological activities. In vivo anti-inflammatory activity was assessed by the sPLA2-induced mouse paw edema model, in which 7-HOC presented an effect similar to those of dexamethasone and cyproheptacline against the pro-inflammatory effect induced by native sPLA2 on the mouse paw edema, mast cell degranulation and skin edema. on the other hand, 7-HOC exhibited a more potent inhibitory effect on sPUL2 than that of p-bromophenacyl bromide (p-BPB). Our data suggest that 7-HOC interacts with sPLA2 and causes some structural modifications that lead to a sharp decrease or inhibition of the edematogenic and myotoxic activities of this enzyme, indicating its potential use to suppress inflammation induced by sPLA2 from the snake venom. (C) 2008 Published by Elsevier Ltd.

The effect of phospholipase A2 from Crotalus durissus collilineatus on Leishmania (Leishmania) amazonensis infection

In this study, the effect of phospholipase A2 (PLA2) derived from Crotalus durissus collilineatus was evaluated in vitro and in vivo on experimental cutaneous leishmaniasis. The promastigote and amastigote forms treated with PLA2 presented increased growth rate. In vivo studies showed that PLA2-treated Leishmania (Leishmania) amazonensis promastigotes increased the size of lesions in BALB/c mice, and histopathological analysis showed numerous necrotic regions presenting a higher density of polymorphonuclear, mononuclear, and amastigote cells. Additionally, infected macrophages treated with PLA2 were able to generate prostaglandin E2 (PGE2). Cytokine quantification showed that the supernatant from infected macrophages presented moderate and high amounts of IL-2 and IL-10, respectively. However, in PLA2-treated infected macrophages, suppression of IL-2 levels occurred, but not of IL-10 levels. Observation also revealed that both the supernatant and lysate of L. (L.) amazonensis promastigotes exhibited PLA2 activity, which, in the presence of dexamethasone, showed no reduction in their activities; while glucocorticoid maintained the ability of promastigote forms to infect macrophages, which presented values similar to controls. In conclusion, the results indicate that PLA2 may be a progression factor for cutaneous leishmaniasis, since the PLA2 effect suppressed IL-2 levels and generated PGE2, an inflammatory lipid mediator.

Renal and cardiovascular effects of Bothrops marajoensis venom and phospholipase A(2)

Bothrops marajoensis is found in the savannah of Marajo Island in the State of Par S and regions of Amapa State, Brazil. The aim of the work was to study the renal and cardiovascular effects of the B. marajoensis venom and phospholipase A(2) (PLA(2)). The venom was fractionated by Protein Pack 5PW. N-terminal amino acid sequencing of sPLA(2) showed amino acid identity with other lysine K49sPLA(2)s of snake venom. B. marajoensis venom (30 mu g/mL) decreased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate and sodium tubular transport. PLA(2) did not change the renal parameters. The perfusion pressure of the mesenteric bed did not change after infusion of venom. In isolated heart, the venom decreased the force of contraction and increased PP but did not change coronary flow. In the arterial pressure, the venom and PLA(2) decreased mean arterial pressure and cardiac frequency. The presence of atrial flutter and late hyperpolarisation reversed, indicating QRS complex arrhythmia and dysfunction in atrial conduction. In conclusion, B. marajoensis venom and PLA(2) induce hypotension and bradycardia while simultaneously blocking electrical conduction in the heart. Moreover, the decrease in glomerular filtration rate, urinary flow and electrolyte transport demonstrates physiological changes to the renal system. (C) 2009 Elsevier Ltd. All rights reserved.