Viscous macromolecules inhibit erythrocyte hemolysis induced by snake venom phospholipase A2

The effect of medium viscosity on lysis of red blood cells (RBC) induced by snake venom phospholipase A2 (PLA2) was examined. The medium viscosity was modified by the addition of various macromolecules which differ in their chemical nature and in their capacity to increase fluid viscosity. PLA2 and Ca++ were applied to cells suspended in viscous medium to induce hemolysis. It was found that the hemolysis is inhibited in direct proportion to increasing viscosity of the extracellular fluid. This phenomenon was observed with aggregated as well as disaggregated RBC. To examine whether the viscosity interferes with the accessibility of the enzyme to the cell, the medium viscosity was modified after binding of the enzyme to the cells; PLA2 was added to a RBC suspension in the presence of Ba++ which binds the enzyme to the cell membrane but does not activate it. The cell-enzyme complex was separated by gel filtration and suspended in viscous medium in the presence of Ca++ which activates the reaction. Also in this case RBC lysis was inhibited as the medium viscosity was increased. It is proposed that the action of PLA2 on RBC membrane is regulated by the viscosity of the cell surface aqueous environment.     

Dexamethasone directly inhibits snake venom phospholipase A

Dexamethasone was found to directly inhibit snake venom phospholipase A2 within 3 to 10 minutes. To detect this effect, the incubation time seems to be critical. Moreover, the inhibitory effect of dexamethasone and mepacrine were additive with each other. We speculate that this direct inhibitory effect on phospholipase A2 plays a part in its strong biological activity.     

Toxicity domain in presynaptically toxic phospholipase A2 of snake venom

About 42 complete amino-acid sequences of phospholipases A2 (phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) are known, including those of 13 presynaptically toxic enzymes, but the structural features responsible for the neurotoxicity and distinguishing the toxins from the non-neurotoxic enzymes are far from being clear. In this study, we examined the charged-residue distributions and hydrophobic characteristics based on the sequence data and the predicted tertiary structure and proposed a possible toxicity domain. We found that the presynaptically toxic enzymes have three or four more basic amino-acid residues than the non-neurotoxic enzymes at positions 59, 60, 65, 70-73 and 97 or 98. These residues appear to cluster near the surface region at the N-terminal side. The cationic nature of this basic cluster in the toxin is enhanced by the alpha-amino group of the N-terminus and the dipole moment of helices 96-110 and 1-10. Moreover, these toxic-site residues are usually associated with hydrophobic regions at 1-7, 64-81 and 97-109.     

Quercetin as an inhibitor of snake venom secretory phospholipase A2

As polyphenolic compounds isolated from plants extracts, flavonoids have been applied to various pharmaceutical uses in recent decades due to their anti-inflammatory, cancer preventive, and cardiovascular protective activities. In this study, we evaluated the effects of the flavonoid quercetin on Crotalus durissus terrificus secretory phospholipase A2 (sPLA2), an important protein involved in the release of arachidonic acid from phospholipid membranes. The protein was chemically modified by treatment with quercetin, which resulted in modifications in the secondary structure as evidenced through circular dichroism. In addition, quercetin was able to inhibit the enzymatic activity and some pharmacological activities of sPLA2, including its antibacterial activity, its ability to induce platelet aggregation, and its myotoxicity by approximately 40%, but was not able to reduce the inflammatory and neurotoxic activities of sPLA2. These results suggest the existence of two pharmacological sites in the protein, one that is correlated with the enzymatic site and another that is distinct from it. We also performed molecular docking to better understand the possible interactions between quercetin and sPLA2. Our docking data showed the existence of hydrogen-bonded, polar interactions and hydrophobic interactions, suggesting that other flavonoids with similar structures could bind to sPLA2. Further research is warranted to investigate the potential use of flavonoids as sPLA2 inhibitors.     

N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2

A novel phospholipase A2 (PLA2) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C18 chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A2s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A2s are distinct from the other subgroups (D49 PLA2, S49 PLA2 and K49 PLA2) and represent a unique subgroup of snake venom group II PLA2, named N49 PLA2 subgroup.     

Therapeutic application of natural inhibitors against snake venom phospholipase A(2)

Natural inhibitors occupy an important place in the potential to neutralize the toxic effects caused by snake venom proteins and enzymes. It has been well recognized for several years that animal sera, some of the plant and marine extracts are the most potent in neutralizing snake venom phospholipase A(2) (svPLA(2)). The implication of this review to update the latest research work which has been accomplished with svPLA(2) inhibitors from various natural sources like animal, marine organisms presents a compilation of research in this field over the past decade and revisiting the previous research report including those found in plants. In addition to that the bioactive compounds/inhibitor molecules from diverse sources like aristolochic alkaloid, flavonoids and neoflavonoids from plants, hydrocarbones -2, 4 dimethyl hexane, 2 methylnonane, and 2, 6 dimethyl heptane obtained from traditional medicinal plants Tragia involucrata (Euphorbiaceae) member of natural products involved for the inhibitory potential of phospholipase A(2) (PLA(2)) enzymes in vitro and also decrease both oedema induced by snake venom as well as human synovial fluid PLA(2). Besides marine natural products that inhibit PLA(2) are manoalide and its derivatives such as scalaradial and related compounds, pseudopterosins and vidalols, tetracylne from synthetic chemicals etc. There is an overview of the role of PLA(2) in inflammation that provides a rationale for seeking inhibitors of PLA(2) as anti-inflammatory agents. However, more studies should be considered to evaluate antivenom efficiency of sera and other agents against a variety of snake venoms found in various parts of the world. The implications of these new groups of svPLA(2) toxin inhibitors in the context of our current understanding of snake biology as well as in the development of new novel antivenoms therapeutics agents in the efficient treatment of snake envenomations are discussed.     

Structure and function comparison of Micropechis ikaheka snake venom phospholipase A2 isoenzymes

Comparison of the crystal structures of three Micropechis ikaheka phospholipase A2 isoenzymes (MiPLA2, MiPLA3 and MiPLA4, which exhibit different levels of pharmacological effects) shows that their C-terminus (residues 110-124) is the most variable. M-Type receptor binding affinity of the isoenzymes has also been investigated and MiPLA4 binds to the rabbit M-type receptor with high affinity. Examination of surface charges of the isoenzymes reveals a trend of increase in positive charges with potency. The isoenzymes are shown to oligomerize in a concentration-dependent manner in a semi-denaturing gel. The C-termini of the medium (MiPLA4) and highly potent (MiPLA2) isoenzyme molecules cluster together, forming a highly exposed area. A BLAST search using the sequence of the most potent MiPLA2 results in high similarity to Staphylococcus aureus clotting factor A and cadherin 11. This might explain the myotoxicity, anticoagulant and hemoglobinuria effects of MiPLA2s.     

Mechanisms by which intracellular calcium induces susceptibility to secretory phospholipase A2 in human erythrocytes

Exposure of human erythrocytes to the calcium ionophore ionomycin rendered them susceptible to the action of secretory phospholipase A(2) (sPLA(2)). Analysis of erythrocyte phospholipid metabolism by thin-layer chromatography revealed significant hydrolysis of both phosphatidylcholine and phosphatidylethanolamine during incubation with ionomycin and sPLA(2). Several possible mechanisms for the effect of ionomycin were considered. Involvement of intracellular phospholipases A(2) was excluded since inhibitors of these enzymes had no effect. Assessment of membrane oxidation by cis-parinaric acid fluorescence and comparison to the oxidants diamide and phenylhydrazine revealed that oxidation does not participate in the effect of ionomycin. Incubation with ionomycin caused classical physical changes to the erythrocyte membrane such as morphological alterations (spherocytosis), translocation of aminophospholipids to the outer leaflet of the membrane, and release of microvesicles. Experiments with phenylhydrazine, KCl, quinine, merocyanine 540, the calpain inhibitor E-64d, and the scramblase inhibitor R5421 revealed that neither phospholipid translocation nor vesicle release was required to induce susceptibility. Results from fluorescence spectroscopy and two-photon excitation scanning microscopy using the membrane probe laurdan argued that susceptibility to sPLA(2) is a consequence of increased order of membrane lipids.     

Cobra venom phospholipase A2 inhibition by manoalide. A novel type of phospholipase inhibitor

Manoalide, an unusual nonsteroidal sesterterpenoid recently isolated from sponge, antagonizes phorbol-induced inflammation but not that induced by arachidonic acid, suggesting that manoalide acts prior to the cyclooxygenase step in prostaglandin synthesis, possibly by inhibiting phospholipase A2. We have now studied the inhibitory effect of manoalide on a homogeneous preparation of phospholipase A2 from cobra venom. For a given concentration of manoalide, the inhibition of phospholipase A2 activity toward dipalmitoylphosphatidylcholine/Triton X-100 mixed micelles is time-dependent and plateaus at about 85% inhibition of the initial velocity even after extensive preincubation. Metal ions (Ca2+, Ba2+, Mn2+) increase the inhibition, while lysophosphatidylcholine and substrate micelles protect. Increasing manoalide concentration shows increasing inhibition of the initial velocity until a plateau is reached, giving a typical saturation curve with a linear double-reciprocal plot. Under typical conditions (20-min preincubation, 40 degrees C, pH 7.1), 50% inhibition is achieved at a manoalide concentration of about 2 X 10(-6) M. The data indicate that manoalide is a potent inhibitor of the cobra venom phospholipase A2. Manoalide is now shown to react irreversibly with lysine residues in the enzyme. Surprisingly, the cobra venom phospholipase normally acts poorly on phosphatidylethanolamine as substrate, but after reaction with manoalide, the enzyme is somewhat more active toward this substrate rather than being inhibited. This suggests that a lysine residue may be important in understanding the substrate specificity of phospholipase A2.     

The molecular cloning of a phospholipase A2 from Bothrops jararacussu snake venom: Evolution of venom group II phospholipase A2's may imply gene duplications

The sequence coding for a snake venom phospholipase A₂ (PLA₂), BJUPLA₂, has been cloned from a Bothrops jararacussu venom gland cDNA library. The cDNA sequence predicts a precursor containing a 16-residue signal peptide followed by a molecule of 122 amino acid residues with a strong sequence similarity to group II snake venom PLA₂'s. A striking feature of the cDNA is the high sequence conservation of the 5 and 3 untranslated regions in cDNAs coding for PLA₂'s from a number of viper species. The greatest sequence variation was observed between the regions coding for the mature proteins, with most substitutions occurring in nonsynonymous sites. The phylogenetic tree constructed by alignment of the amino acid sequence of BJUPLA₂ with group II PLA₂'s in general groups them according to current taxonomical divisions and/or functional activity. It also suggests that gene duplications may have occurred at a number of different points during the evolution of snake venom group II PLA₂'s.The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number X76289.Correspondence to: A.M. Moura-da-Silva     

Isolation and functional characterization of proinflammatory acidic phospholipase A2 from Bothrops leucurus snake venom

In the present study, an acidic PLA(2), designated Bl-PLA(2), was isolated from Bothrops leucurus snake venom through two chromatographic steps: ion-exchange on CM-Sepharose and hydrophobic chromatography on Phenyl-Sepharose. Bl-PLA(2) was homogeneous on SDS-PAGE and when submitted to 2D electrophoresis the molecular mass was 15,000Da and pI was 5.4. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA(2)s from snake venoms. Its specific activity was 159.9U/mg and the indirect hemolytic activity was also higher than that of the crude venom. Bl-PLA(2) induced low myotoxic and edema activities as compared to those of the crude venom. Moreover, the enzyme was able to induce increments in IL-12p40, TNF-α, IL-1β and IL-6 levels and no variation of IL-8 and IL-10 in human PBMC stimulated in vitro, suggesting that Bl-PLA(2) induces proinflammatory cytokine production by human mononuclear cells. Bothrops leucurus venom is still not extensively explored and knowledge of its components will contribute for a better understanding of its action mechanism.     

Isolation and enzymatic characterization of a basic phospholipase A2 from Bothrops jararacussu snake venom

A novel basic phospholipase A₂ (PLA2) isoform was isolated from Bothrops jararacussu snake venom and partially characterized. The venom was fractionated by HPLC ion-exchange chromatography in ammonium bicarbonate buffer, followed by reverse-phase HPLC to yield the protein Bj IV. Tricine SDS-PAGE in the presence or absence of dithiothreitol showed that Bj IV had a molecular mass of 15 and 30 kDa, respectively. This enzyme was able to form multimeric complexes (30, 45, and 60 kDa). Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWSWGQMIQETGLLPSYTTY . . .) showed a high degree of homology with basic D49 PLA₂ myotoxins from other Bothrops venoms. Bj IV had high PLA₂ activity and produced moderate myonecrosis in skeletal muscle, but showed no neuromuscular activity in mouse phrenic nerve-diaphragm preparations. Bj IV showed allosteric enzymatic behavior, with maximal activity at pH 8.2 and 35-45°C. Full PLA₂ activity required Ca²⁺ but was inhibited by Cu²⁺ and Zn²⁺, and by Cu²⁺ and Mg²⁺ in the presence and absence of Ca²⁺, respectively. Crotapotins from Crotalus durissus terrificus rattlesnake venom significantly inhibited the enzymatic activity of Bj IV. The latter observation suggested that the binding site for crotapotin in this PLA₂ was similar to that in the basic PLA₂ of the crotoxin complex from C. d. terrificus venom. The presence of crotapotin-like proteins capable of inhibiting the catalytic activity of D49 PLA₂ could partly explain the low PLA₂ activity of Bothrops venoms.     

Cobra venom phospholipase A2 immobilized to porocus glass beads.

Phospholipase A₂ (EC 3.1.1.4) from cobra venom (Naja naja naja) has been covalently immobilized to aryl amine porous glass beads by diazo coupling. The attachment of the enzyme to the glass beads is apparently through tyrosine. The activity of the immobilized enzyme toward phospholipid substrate has been monitored using the Triton X-100/phospholipid mixed micelle assay system. The activity of the immobilized phospholipase A₂ toward phosphatidylcholine is about 160 μmol min^?1 ml^?1 of glass beads, and the specific activity is about 13 μmol min^?1 mg^?1 of protein in this assay system. The pH rate profile and apparent pK_a in 10 mm Ca²⁺ of the immobilized enzyme parallels that of the soluble enzyme. The substrate specificity of the immobilized enzyme toward individual phospholipid species in mixed micelles is phosphatidylcholine ? phosphatidylethanolamine. In binary lipid mixtures in mixed micelles containing phosphatidylcholine and phosphatidylethanolamine together, a reversal in specificity is observed, and phosphatidylethanolamine is the preferred substrate. This unusual specificity reversal in binary mixtures is also observed for the soluble enzyme. The activity of the immobilized enzyme toward phospholipid inserted in mixed micelles is the same as toward a synthetic phospholipid which forms monomers, while a 20-fold decrease in activity toward monomeric substrate is observed for the soluble enzyme. The immobilized enzyme is stable at temperatures of 90 °C as is the soluble enzyme. However, p-bromphenacyl bromide, a reagent which inactivates the soluble enzyme, does not inactivate the immobilized enzyme. The immobilized enzyme can be stored frozen for several months and is reusable. The mechanism of action of immobilized phospholipase A₂ from cobra venom and the potential usefullness of the bound enzyme as a probe for phospholipids in surfaces of membranes is considered.     

Evolutionary relationships and implications for the regulation of phospholipase A2 from snake venom to human secreted forms

Summary The amino acid sequences of 40 secreted phospholipase A₂'s (PLA₂) were aligned and a phylogenetic tree derived that has three main branches corresponding to elapid (group I), viperid (group II), and insect venom types of PLA₂. The human pancreatic and recently determined nonpancreatic sequences in the comparison align with the elapid and viperid categories, repectively, indicating that at least two PLA₂ genes existed in the vertebrate line before the divergence of reptiles and mammals about 200–300 million years ago. This allows resolution for the first time of major genetic events in the evolution of current PLA₂'s and the relationship of human PLA₂'s to those of snake venom, many of which are potent toxins. Implications for possible mechanisms of regulation of mammalian intra- and extracellular PLA₂'s are discussed, as well as issues relating to the search for the controlling enzymes in arachidonic acid release, prostaglandin generation, and signal transduction.     

Assay for phospholipase A activity of snake venom with asolectin as substrate

A reliable, rapid, and relatively inexpensive assay for phospholipase A activity in Naja naj venom is described. Asolectin, a mixture of soybean phospholipids, is used as the substrate instead of the more expensive, natural substrate l-α-lecithin. Phospholipase A activity of snake venom, monitored by hydroxamate formation, obeys Michaelis-Menten kinetics and is proportional to time and to venom concentration. The pH profile of activity shows a broad plateau from pH 7.0 to 9.5 and a shoulder between pH 5 and 6.