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.

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.

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.

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.

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).

Insights into the subunit arrangement and diversity of paradoxin and taipoxin.

Paradoxin and taipoxin are neurotoxic phospholipases from the inland and coastal species of Australian taipan. Despite their relatively high sequence homology of 70% and 84% for the acidic and basic chains respectively, they differ substantially in reported assays of neurotoxicity. This study provides the first characterisation of paradoxin, which like taipoxin, is a trimer at physiological pH. More broadly, these toxins were found to be composed of a more diverse range of subunits than previously recognised, including newly discovered γTPx isoforms, which give rise to an additional, major conformation of TPx.

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.

Effect of chlorogenic acid (5-Caffeoylquinic Acid) isolated from Baccharis oxyodonta on the structure and pharmacological activities of secretory phospholipase A2 from Crotalus durissus terrificus.

The aim of this paper was to investigate the effect of chlorogenic acid (5-caffeoylquinic acid, 5CQA), isolated from Baccharis oxyodonta, on the structure and pharmacological effect of secretory phospholipase A2 (sPLA2) from Crotalus durissus terrificus. All in vitro and in vivo experiments were conducted using a purified sPLA2 compared under the same experimental conditions with sPLA2 : 5CQA. 5CQA induced several discrete modifications in the secondary structure and the hydrophobic characteristics of native sPLA2 that induced slight changes in the α-helical content, increase in the random coil structure, and decrease of fluorescence of native sPLA2. Moreover, 5CQA significantly decreased the enzymatic activity and the oedema and myonecrosis induced by native sPLA2. As the catalytic activity of sPLA2 plays an important role in several of its biological and pharmacological properties, antibacterial activity was used to confirm the decrease in its enzymatic activity by 5CQA, which induced massive bacterial cell destruction. We found that 5CQA specifically abolished the enzymatic activity of sPLA2 and induced discrete protein unfolding that mainly involved the pharmacological site of sPLA2. These results showed the potential application of 5CQA in the snake poisoning treatment and modulation of the pathological effect of inflammation induced by secretory PLA2.

An evaluation of 3-rhamnosylquercetin, a glycosylated form of quercetin, against the myotoxic and edematogenic effects of sPLA 2 from Crotalus durissus terrificus.

This paper shows the results of quercitrin effects on the structure and biological activity of secretory phospholipase (sPLA2) from Crotalus durissus terrificus, which is the main toxin involved in the pharmacological effects of this snake venom. According to our mass spectrometry and circular dichroism results, quercetin was able to promote a chemical modification of some amino acid residues and modify the secondary structure of C. d. terrificus sPLA2. Moreover, molecular docking studies showed that quercitrin can establish chemical interactions with some of the crucial amino acid residues involved in the enzymatic activity of the sPLA2, indicating that this flavonoid could also physically impair substrate molecule access to the catalytic site of the toxin. Additionally, in vitro and in vivo assays showed that the quercitrin strongly diminished the catalytic activity of the protein, altered its Vmax and Km values, and presented a more potent inhibition of essential pharmacological activities in the C. d. terrificus sPLA2, such as its myotoxicity and edematogenic effect, in comparison to quercetin. Thus, we concluded that the rhamnose group found in quercitrin is most likely essential to the antivenom activities of this flavonoid against C. d. terrificus sPLA2.

Unmasking snake venom of Bothrops leucurus: purification and pharmacological and structural characterization of new PLA2 Bleu TX-III.

Bleu TX-III was isolated from Bothrops leucurus snake venom on one-step analytical chromatography reverse phase HPLC, was homogeneous on SDS-PAGE, and was confirmed by Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry in 14243.8 Da. Multiple alignments of Bleu TX-III show high degree of homology with basic PLA2 myotoxins from other Bothrops venoms. Our studies on local and systemic myotoxicity "in vivo" reveal that Bleu TX-III is myotoxin with local but not systemic action due to the decrease in the plasmatic CK levels when Bleu TX-III is administrated by intravenous route in mice (dose 1 and 5 µg). And at a dose of 20 µg myotoxin behaves like a local and systemic action. Bleu TX-III induced moderate marked paw edema, evidencing the local increase in vascular permeability. The inflammatory events induced in the mice (I.M.) were investigated. The increase in the levels of IL-1, IL-6, and TNF-α was observed in the plasma. It is concluded that Bleu TX-III induces inflammatory events in this model. The enzymatic phospholipid hydrolysis may be relevant to these phenomena. Bothrops leucurus venom is still not extensively explored, and the knowledge of its toxins separately through the study of structure/function will contribute for a better understanding of its action mechanism.

Phospholipase A2 purification and characterization: a case study.

We compared here the purification procedures, the pH, the calcium, the bile salts, and the temperature dependencies as well as the catalytic activities on phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of two purified secreted PLA2 from chicken pancreatic (ChPLA2-IB) and chicken intestinal (ChPLA2-IIA) origins. Interestingly, ChPLA2-IB hydrolyzes efficiently both purified PC and PE, whereas ChPLA2-IIA hydrolyzes only PE and not PC, even after a long incubation period. These analytical results clearly indicate that the catalytic activity of ChPLA2-IIA, measured with the pH-stat and using egg yolk as substrate, is mainly due to the hydrolysis of the PE fraction present in egg yolk.

A catalytically inactive Lys49 PLA2 isoform from Bothrops jararacussu venom that stimulates insulin secretion in pancreatic beta cells.

A new secretory phospholipase A2 (sPLA2) isoform from Bothrops jararacussu venom (BjVIII) has been characterized by causing platelet aggregation, an absent activity in BthTx-I, Prtx-I and PrTx-II sPLA2s. According to our results, BjVIII also enhances insulin release by the pancreatic beta cells. The complete amino acid sequence of the new isoform was determined by Edman degradation and de novo peptide sequencing. These analyses showed a G35K amino acid modification for BjVIII in comparison with BthTx-I, PrTx-I and Prtx-II, a structural difference that has been related to the conflicting biological activities among BjVIII and other Lys49 sPLA2s. The whole set of evidences collected in this work indicates that, besides the C-terminal region and B-wing of PLA2, the calcium binding loop in BjVIII should be considered as an important region, involved in the pharmacological effects of Lys49-sPLA2 isoforms from the Bothrops genus.

Chemical modification of ascorbic acid and evaluation of its lipophilic derivatives as inhibitors of secretory phospholipase A(2) with anti-inflammatory activity.

The halo 6-fatty acid esters of L-ascorbic acid 3a, 3b and 6-fatty acid esters of L-ascorbic acid 5a-g were achieved from L-ascorbic acid 1. Compounds 3a, 3b and 5a-g were evaluated for anti-oxidant, anti-lipid peroxidation, and secretory phospholipase A(2) (sPLA(2)) inhibition in vitro, and sPLA(2) induced mouse paw edema. All the derivatives retained their anti-oxidant property compared to ascorbic acid at 6 × 10(-4)M and are good inhibitors of lipid peroxidation at 1 mg ml(-1) as evaluated by 2, 2-Diphenyl-1-picrylhydrazyl radical and thio-barbituric acid methods, respectively. Compounds 5e and 5f significantly inhibited purified group I sPLA(2) from Naja naja and group II sPLA(2) from Vipera russelli, human synovial fluid and human pleural fluid with IC(50) value ranging from 64 ± 1.95 to 82 ± 1.3 and 48 ± 2.27 to 61 ± 2.23 µM, respectively. The compounds 5e and 5f also showed varying degree of potency in neutralizing indirect hemolytic activity of sPLA(2) at 50 µM concentration, and sPLA(2) induced mouse paw edema at the dose 3 mg/kg. Further docking studies also confirmed that compounds 5e and 5f have maximum interaction with increasing negative energy value. Single molecule possessing both anti-oxidant and anti-inflammatory activities is of great therapeutic significance in inflammatory disorders.

Reductions of phospholipase A(2) inhibition of pulmonary surfactant with hyaluronan.

In acute lung injuries, secretory phospholipase A(2) (sPLA(2)) inhibits surfactants by hydrolyzing phospholipids. Because hyaluronan (HA) reduces hydrolysis of phospholipids by sPLA(2), and because sPLA(2) inhibits surfactant in vitro, the authors hypothesized HA would reduce sPLA(2) inhibition. Surfactants were used alone or mixed with HA and/or sPLA(2) then tested for surface activity in 2 separate assays, or for sPLA(2) activity. Equilibrium surface pressures were identical for surfactant with or without HA. sPLA(2) inhibited surface activity but this inhibitory effect was reduced with HA by 14% in the spreading trough and by 63% in a modified bubble surfactometer. Hyaluronan caused a modest reduction (39%) of sPLA(2) breakdown of labeled phospholipid. Therefore hyaluronan reduces inhibition of surfactants by sPLA(2) in vitro, and reduces the activity of the enzyme.

Snake venoms as a source of compounds modulating sperm physiology: Secreted phospholipases A2 from Oxyuranus scutellatus scutellatus impact sperm motility, acrosome reaction and in vitro fertilization in mice.

The goal of this study was to identify new compounds from venoms able to modulate sperm physiology and more particularly sperm motility. For this purpose, we screened the effects of 16 snake venoms cleared of molecules higher than 15 kDa on sperm motility. Venoms rich in neurotoxins like those from Oxyuranus scutellatus scutellatus or Daboia russelii, were highly potent inhibitors of sperm motility. In contrast, venoms rich in myotoxins like those from Echis carinatus, Bothrops alternatus and Macrovipera lebetina, were inactive. From the main pharmacologically-active fraction of the Taipan snake O. scutellatus s., a proteomic approach allowed us to identify 16 different proteins, among which OS1 and OS2, two secreted phospholipases A2 (sPLA(2)). Purified OS1 and OS2 mimicked the inhibitory effect on sperm motility and were likely responsible for the inhibitory effect of the active fraction. OS1 and OS2 triggered sperm acrosome reaction and induced lipid rearrangements of the plasma membrane. The catalytic activity of OS2 was required to modulate sperm physiology since catalytically inactive mutants had no effect. Finally, sperm treated with OS2 were less competent than control sperm to initiate in vitro normal embryo development. This is the first report characterizing sPLA(2) toxins that modulate in vitro sperm physiology.

Neurotoxic, myotoxic and cytolytic activities of the new basic PLA(2) isoforms BmjeTX-I and BmjeTX-II isolated from the Bothrops marajoensis (Marajó Lancehead) snake venom.

The BmjeTX-I and BmjeTX-II isoforms of PLA(2) were purified from Bothrops marajoensis venom by ion-exchange chromatography and reverse phase HPLC. Both isoforms showed a molecular mass of 13808.89 Da (BmjeTX-I) and 13863.97 Da (BmjeTX-II) determined by based on the determined primary structures and SDS-PAGE and confirmed experimentally by MALDI-TOF mass spectrometry. Multiple alignment of BmjeTX-I and BmjeTX-II isoforms of PLA(2) show high degree of homology with basic PLA(2) myotoxins from other Bothrops venoms. Ex vivo, both isoforms caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other Bothrops species. In chick preparations, contractures to exogenous acetylcholine (55 and 110 microM) or KCl (13.4 mM) were unaltered after complete blockade for the both isoforms BmjeTX-I and BmjeTX-II of PLA(2). These results, which strongly suggested a presynaptic mechanism of action for these toxins. In mice, both isoforms induced myonecrosis and a systemic interleukin-6 response upon intramuscular injection. Both isoforms BmjeTX-I and BmjeTX-II of PLA(2) also induced moderate marked paw edema, evidencing the local increase in vascular permeability. Since both isoforms of PLA(2) exert a strong proinflammatory effect, the enzymatic hydrolysis of phospholipids might be relevant for this phenomenon and produced cytotoxicity in murine skeletal muscle C2C12 myoblasts and myotubes.

Absence of phospholipase A(2) in most Crotalus horridus venom due to translation blockage: comparison with Crotalus horridus atricaudatus venom.

To investigate the peculiar absence of phospholipases A(2) (PLA(2)s) in most Crotalus horridus (CH) venom, we cloned and sequenced the venom PLA(2)s of three CH specimens from different regions. The results revealed that all the venom glands contained mRNAs that encoded an acidic PLA(2) (designated as either CH-E6 or CH-E6'). The predicted CH-E6 from the Iowan CH and CH-E6' from the South Carolinian CH differed by only one amino acid residue, while the PLA(2) cDNA cloned from the Kentuckian CH contained an early stop codon instead of a Tyr(22) codon. Only the individual South Carolinian CH venom was found to contain the CH-E6' protein whose mass was confirmed by MALDI-TOF analysis. Our results suggest that low PLA(2) expression levels in most CH venom can be attributed to translation blockage. We also purified two acidic PLA(2)s and canebrake toxin from the pooled venom of Crotalus horridus atricaudatus (neurotoxic CH subspecies). One of the acidic PLA(2)s was identical to CH-E6 and showed high lipolytic activity and weak anti-platelet activities. The possibility that C. h. atricaudatus could be a hybrid between CH and Crotalus scutulatus is discussed.