Purification, characterization, and cDNA cloning of acidic platelet aggregation inhibiting phospholipases A2 from the snake venom of Vipera lebetina (Levantine viper)

Two novel acidic phospholipase A₂s (PLA₂) were isolated by size exclusion chromatography and reversed-phase chromatography from the crude Vipera lebetina venom. The molecular masses of VLPLA₂-1 (13,704 Da) and VLPLA₂-2 (13,683 Da) and their internal tryptic peptides were determined by MALDI-TOF mass-spectrometry. When tested in human platelet-rich plasma, both enzymes showed a potent inhibitory effect on aggregation induced by ADP and collagen. Chemical modification with p-bromophenacylbromide abolished the enzymatic activity of PLA₂; its anti-platelet activity was fully inhibited in case of collagen as inducer and partially inhibited in case of ADP as inducer. The complete cDNAs encoding PLA₂ were cloned from a single venom gland cDNA library. Complete amino acid sequences of the VLPLA₂ were deduced from the cDNA sequences. The full-length cDNA sequences of the VLPLA₂ possess 615 bp and encode an open reading frame of 138 amino acids that include signal peptide (16 amino acids) and mature enzyme (122 amino acids). The VLPLA₂s have significant sequence similarity to many other phospholipase A₂s from snake venoms. The phylogenetic analysis on the basis of the amino acid sequence homology demonstrates that VLPLA₂s grouped with other Asp49 PLA₂s and they appear to share a close evolutionary relationship with the European vipers.     

Chemical modifications of phospholipases A2 from snake venoms: effects on catalytic and pharmacological properties

Phospholipases A₂ (PLA₂s) constitute major components of snake venoms and have been extensively investigated not only because they are very abundant in these venoms but mainly because they display a wide range of biological effects, including neurotoxic, myotoxic, cytotoxic, edema-inducing, artificial membrane disrupting, anti-coagulant, platelet aggregation inhibiting, hypotensive, bactericidal, anti-HIV, anti-tumoral, anti-malarial and anti-parasitic. Due to this functional diversity, these structurally similar proteins aroused the interest of many researchers as molecular models for study of structure–function relationships. One of the main experimental strategies used for the study of myotoxic PLA₂s is the traditional chemical modification of specific amino acid residues (His, Met, Lys, Tyr, Trp and others) and examination of the consequent effects upon the enzymatic, toxic and pharmacological activities. This line of research has provided useful insights into the structural determinants of the action of these enzymes and, together with additional strategies, supports the concept of the presence of ‘pharmacological sites’ distinct from the catalytic site in snake venom myotoxic PLA₂s.     

Understanding the in vitro neuromuscular activity of snake venom Lys49 phospholipase A2 homologues

Phospholipases A₂ (PLA₂s) with a lysine substituting for the highly conserved aspartate 49, Lys49 PLA₂ homologues, are important myotoxic components in venoms from snakes of Viperidae family. These proteins induce conspicuous myonecrosis by a catalytically-independent mechanism. Traditionally, the Lys49 PLA₂ homologues are classified as non-neurotoxic myotoxins given their inability to cause lethality or paralytic effects when injected in vivo, even at relatively high doses. However, a series of in vitro studies has shown that several Lys49 PLA₂ homologues from Bothrops snake venoms induce neuromuscular blocking activity on nerve-muscle preparations in vitro. The interpretation of these findings has created some confusion in the literature, raising the question whether the Lys49 PLA₂ homologues present some neurotoxic activity. The present article reviews the in vitro neuromuscular effects of Lys49 PLA₂ homologues and discusses their possible mechanisms of action. It was concluded that the neuromuscular blockade induced by Lys49 PLA₂ homologues in isolated preparations is mainly a consequence of the general membrane-destabilizing effect of these toxins.     

Mapping the structural determinants of presynaptic neurotoxicity of snake venom phospholipases A2

The structural features of presynaptically neurotoxic secretory phospholipases A(2) (sPLA(2)s) that are responsible for their potent and specific action are still a matter of debate. To identify the residues that distinguish a highly neurotoxic sPLA(2), ammodytoxin A (AtxA), from a structurally similar but more than two orders of magnitude less toxic Russell's viper sPLA(2), VIIIa, we prepared a range of mutants and compared their properties. The results show that the structural features that confer high neurotoxicity to AtxA extend from its C-terminal part, with a central role of the residues Y115, I116, R118, N119 (the YIRN cluster) and F124, across the interfacial binding surface (IBS) in the vicinity of F24, to the N-terminal helix whose residues M7 and G11 are located on the edges of the IBS. Competition binding studies indicate that the surface of interaction with the neuronal M-type sPLA(2) receptor R180 extends over a similar region of the molecule. In addition, the YIRN cluster of AtxA is crucial for the high-affinity interaction with two intracellular binding proteins, calmodulin and R25. The concept of a single "presynaptic neurotoxic site" on the surface of snake venom sPLA(2)s is not consistent with these results which suggest that different parts of the toxin molecule are involved in distinct steps of presynaptic neurotoxicity.     

Inflammatory effects of snake venom myotoxic phospholipases A2

Snake venom phospholipases A₂ (PLA₂) show a remarkable functional diversity. Among their toxic activities, some display the ability to cause rapid necrosis of skeletal muscle fibers, thus being myotoxic PLA₂s. Besides myotoxicity, these enzymes evoke conspicuous inflammatory and nociceptive events in experimental models. Local inflammation and pain are important characteristics of snakebite envenomations inflicted by viperid and crotalid species, whose venoms are rich sources of myotoxic PLA₂s. Since the discovery that mammalian PLA₂ is a key enzyme in the release of arachidonic acid, the substrate for the synthesis of several lipid inflammatory mediators, much interest has been focused on this enzyme in the context of inflammation. The mechanisms involved in the proinflammatory action of secretory PLA₂s are being actively investigated, and part of the knowledge on secretory PLA₂ effects has been gained by using snake venom PLA₂s as tools, due to their high structural homology with human secretory PLA₂s. The inflammatory events evoked by PLA₂s are primarily associated with enzymatic activity and to the release of arachidonic acid metabolites. However, catalytically inactive Lys49 PLA₂s trigger inflammatory and nociceptive responses comparable to those of their catalytically active counterparts, thereby evidencing that these proteins promote inflammation and pain by mechanisms not related to phospholipid hydrolysis nor to mobilization of arachidonic acid. These studies have provided a boost to the research in this field and various approaches have been used to identify the amino acid residues and the specific sites of interaction of myotoxic PLA₂s with cell membranes potentially involved in the PLA₂-induced inflammatory and nociceptive effects. This work reviews the proinflammatory and nociceptive effects evoked by myotoxic PLA₂s and their mechanisms of action.     

A phospholipase A2 isolated from Lachesis muta snake venom increases the survival of retinal ganglion cells in vitro

We have previously showed that a phospholipase A₂ isolated from Lachesis muta snake venom and named LM-PLA₂-I displayed particular biological activities, as hemolysis, inhibition on platelet aggregation, edema induction and myotoxicity. In the present work, we evaluated the effect of LM-PLA₂-I on the survival of axotomized rat retinal ganglion cells kept in vitro, as well as its mechanism of action. Our results clearly showed that treatment with LM-PLA₂-I increased the survival of ganglion cells (100% when compared to control cultures) and the treatment of LM-PLA₂-I with p-bromophenacyl bromide abolished this effect. This result indicates that the effect of LM-PLA₂-I on ganglion cell survival is entirely dependent on its enzymatic activity and the generation of lysophosphatidylcholine (LPC) may be a prerequisite to the observed survival. In fact, commercial LPC mimicked the effect of LM-PLA₂-I upon ganglion cell survival. To investigate the mechanism of action of LM-PLA₂-I, cultures were treated with chelerythrine chloride, BAPTA-AM, rottlerin and also with an inhibitor of c-junc kinase (JNKi). Our results showed that rottlerin and JNK inhibitor abolished the LM-PLA₂-I on ganglion cell survival. Taken together, our results showed that LM-PLA₂-I and its enzymatic product, LPC promoted survival of retinal ganglion cells through the protein kinase C pathway and strongly suggest a possible role of the PLA₂ enzyme and LPC in controlling the survival of axotomized neuronal cells.     

Inhibitory effect of phospholipase A2 isolated from Crotalus durissus terrificus venom on macrophage function

Recent work demonstrated that crotoxin, the main toxin of Crotalus durissus terrificus venom, inhibits macrophage spreading and phagocytic activities. The crotoxin molecule is composed of two subunits, an acidic non-toxic and non-enzymatic polypeptide named crotapotin and a weakly toxic basic phospholipase A₂ (PLA₂). In the present work, the active subunit responsible for the inhibitory effect of crotoxin on macrophage function was investigated. Peritoneal macrophages harvested from naive rats were used. Crotapotin (2.12, 3.75, or 8.37 nM/ml), added for 2 h to the medium of peritoneal cell incubation, did not modify the spreading and phagocytic activities of these cells. On the other hand, the PLA₂ (1.43, 2.86, or 6.43 nM/ml) subunit caused a significant reduction (30, 33, and 35%, respectively) of the spreading activity. The PLA₂ also inhibited the phagocytosis of opsonised zymosan, opsonised sheep erythrocytes, and Candida albicans, indicating that this inhibitory effect is not dependent on the type of receptor involved in the phagocytosis process. The inhibitory effect of PLA₂ was not due to loss of cell membrane integrity, since macrophage viability was higher than 95%. These findings indicate that the inhibitory effect of crotoxin on macrophage spreading and phagocytic activities is caused by the phospholipase A₂ subunit.     

Renal and cardiovascular effects of Bothrops marajoensis venom and phospholipase A2

Bothrops marajoensis is found in the savannah of Marajó Island in the State of Pará and regions of Amapá State, Brazil. The aim of the work was to study the renal and cardiovascular effects of the B. marajoensis venom and phospholipase A₂ (PLA₂). The venom was fractionated by Protein Pack 5PW. N-terminal amino acid sequencing of sPLA₂ showed amino acid identity with other lysine K49 sPLA₂s of snake venom. B. marajoensis venom (30 μg/mL) decreased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate and sodium tubular transport. PLA₂ 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 PLA₂ 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 PLA₂ 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.     

Snake venom Lys49 myotoxins: From phospholipases A2 to non-enzymatic membrane disruptors

Snake venoms often contain toxins that cause a rapid necrosis of skeletal muscle fibers, referred to as myotoxins. The most common among them are phospholipases A₂ (PLA₂s), enzymes that have two independent evolutionary origins in snake venoms. Within the group II PLA₂s found in viperid venoms, a particular subgroup emerged, in which the otherwise conserved Asp49 of their catalytic center is replaced by Lys49. These intriguing proteins, referred to as Lys49 myotoxins, lost the ability to catalyze phospholipid hydrolysis, but still induce myonecrosis by a non-enzymatic mechanism based on membrane permeabilization as the critical event. Such mechanism is only partially understood. This review briefly describes the general structural and functional characteristics of the Lys49 myotoxins, and summarizes four proposed models of their functional “toxic site”. Finally, it discusses some novel insights into their mode of action, in particular examining arguments and experimental observations that could shed light on the possible nature of their membrane target on skeletal muscle cells, which remains elusive.     

Identification of linear B-cell epitopes on myotoxin II, a Lys49 phospholipase A2 homologue from Bothrops asper snake venom

Knowledge on toxin immunogenicity at the molecular level can provide valuable information for the improvement of antivenoms, as well as for understanding toxin structure-function relationships. The aims of this study are two-fold: first, to identify the linear B-cell epitopes of myotoxin II from Bothrops asper snake venom, a Lys49 phospholipase A₂ homologue; and second, to use antibodies specifically directed against an epitope having functional relevance in its toxicity, to probe the dimeric assembly mode of this protein in solution. Linear B-cell epitopes were identified using a library of overlapping synthetic peptides spanning its complete sequence. Epitopes recognized by a rabbit antiserum to purified myotoxin II, and by three batches of a polyvalent (Crotalidae) therapeutic antivenom (prepared in horses immunized with a mixture of B. asper, Crotalus simus, and Lachesis stenophrys venoms) were mapped using an enzyme-immunoassay based on the capture of biotinylated peptides by immobilized streptavidin. Some of the epitopes identified were shared between the two species, whereas others were unique. Differences in epitope recognition were observed not only between the two species, but also within the three batches of equine antivenom. Epitope V, located at the C-terminal region of this protein, is known to be relevant for toxicity and neutralization. Affinity-purified rabbit antibodies specific for this site were able to immunoprecipitate myotoxin II, suggesting that the two copies of epitope V are simultaneously available to antibody binding, which would be compatible with the mode of dimerization known as “conventional” dimer.     

cDNA cloning, structural, and functional analyses of venom phospholipases A2 and a Kunitz-type protease inhibitor from steppe viper Vipera ursinii renardi

Snake venom phospholipases A₂ (PLA₂s) display a wide array of biological activities and are each characteristic to the venom. Here, we report on the cDNA cloning and characterization of PLA₂s from the steppe viper Vipera ursinii renardi venom glands. Among the five distinct PLA₂ cDNAs cloned and sequenced, the most common were the clones encoding a basic Ser-49 containing PLA₂ (Vur-S49). Other clones encoded either ammodytin analogs I1, I2d and I2a (designated as Vur-PL1, Vur-PL2 and Vur-PL3, respectively) or an ammodytoxin-like PLA₂ (Vurtoxin). Additionally, a novel Kunitz-type trypsin inhibitor for this venom species was cloned and sequenced. Comparison of these PLA₂ and Kunitz inhibitor sequences with those in the sequence data banks suggests that the viper V. u. renardi is closely related to Vipera ammodytes and Vipera aspis. Separation of V. u. renardi venom components by gel-filtration and ion-exchange chromatography showed the presence of many PLA₂ isoforms. Remarkably, the most abundant PLA₂ isolated was Vur-PL2 while Vur-S49 analog was in very low yield. There are great differences between the proportion of cDNA clones and that of the proteins isolated. Two Vur-PL2 isoforms (designated as Vur-PL2A and Vur-PL2B) indistinguishable by masses, peptide mass fingerprinting, N-terminal sequences and CD spectroscopy were purified from the pooled venom. However, when rechromatographed on cation-exchanger, Vur-PL2A showed only one peak corresponding to Vur-PL2B, suggesting the existence of conformers for Vur-PL2. Vur-PL2B was weakly cytotoxic to rat pheochromocytoma PC12 cells and showed both strong anticoagulant and anti-platelet activities. This is the first case of a strong anticoagulating ammodytin I analog in Vipera venom.     

Isolation of a snake venom phospholipase A2 (PLA2) inhibitor (AIPLAI) from leaves of Azadirachta indica (Neem): Mechanism of PLA2 inhibition by AIPLAI in vitro condition

A compound (AIPLAI (Azadirachta indica PLA(2) inhibitor)) purified from the methanolic leaf extract of A. indica (Neem) inhibits the cobra and Russell's viper venoms (RVVs) phospholipase A(2) enzymes in a dose-dependent manner. Inhibition of catalytic and tested pharmacological properties of cobra venom (Naja naja and Naja kaouthia) PLA(2) enzymes by AIPLAI is significantly higher (P<0.05) compared to the inhibition of PLA(2) enzymes of crude RVV (Daboia russelli) when tested under the same condition. Kinetic study reveals that in in vitro condition, AIPLAI inhibits the purified N. kaouthia PLA(2) enzymes in a non-competitive manner. The AIPLAI is quite stable at room temperature. The present study shows that AIPLAI holds good promise for the development of novel anti-snake venom drug in future.     

Identification and characterization of phospholipase A2 inhibitors from the serum of the Japanese rat snake, Elaphe climacophora

Two distinct phospholipase A₂ (PLA₂) inhibitory proteins (PLIs) were purified from the serum of the Japanese rat snake, Elaphe climacophora. The 150-kDa inhibitor, a trimer of a 50-kDa subunit, specifically inhibited the basic PLA₂ purified from the venom of Gloydius brevicaudus, whereas the 120-kDa one composed of two distinct 25-kDa subunits, A and B, inhibited both the acidic and basic PLA₂s of G. brevicaudus. On the basis of their amino acid sequences, these inhibitors were assigned as PLIβ and PLIγ, respectively. A PLIα homolog (PLIα-like protein; PLIα-LP) having an apparent molecular weight of 50-kDa and composed of 15-kDa subunits was also purified from the E. climacophora serum. This homolog was immunoreactive with antibody raised against the G. brevicaudus PLIα, but lacked in the inhibitory activity toward the acidic and basic PLA₂s. The cDNAs encoding PLIα-LP, PLIβ, PLIγ-A, and PLIγ-B were cloned from liver RNA, and their nucleotide sequences were compared with those of other venomous and non-venomous snakes.     

Phospholipase A2 inhibitors (βPLIs) are encoded in the venom glands of Lachesis muta (Crotalinae, Viperidae) snakes

Phospholipase A₂ inhibitors (PLIs) are glycoproteins secreted by snake liver into the circulating blood aiming the self-protection against toxic venom phospholipases A₂. In the present study, we describe the first complete nucleotide sequence of a βPLI from venom glands of a New World snake, Lachesis muta. The deduced primary structure was compared to other known βPLIs and recent literature findings of other possible roles of PLIs in snakes are discussed.     

Molecular evolution of myotoxic phospholipases A2 from snake venom

After two decades of study, we draw the conclusion that venom-gland phospholipase A₂ (PLA₂) isozymes, including PLA₂ myotoxins of Crotalinae snakes, have evolved in an accelerated manner to acquire their diverse physiological activities. In this review, we describe how accelerated evolution of venom PLA₂ isozymes was discovered. This type of evolution is fundamental for other venom isozyme systems. Accelerated evolution of venom PLA₂ isozyme genes is due to rapid change in exons, but not in introns and the flanking regions, being completely opposite to the case of the ordinary isozyme genes. The molecular mechanism by which proper base substitutions had occurred in the particular sites of venom isozyme genes is a puzzle to be solved in future studies. It should be noted that accelerated evolution occurred until the isozymes had acquired their particular function and, since then, they have evolved with less frequent mutation, possibly for functional conservation. We also found that interisland mutations occurred in venom PLA₂ isozymes. The relationships between mutation and its driving force are speculative and the real mechanism remains a mystery.     

Leucocyte recruitment induced by type II phospholipases A2 into the rat pleural cavity

Bothropstoxin-I (BthTX-I) and bothropstoxin-II (BthTX-II) are Lys-49 and Asp-49 phospholipases A₂ (PLA₂s), respectively, isolated from Bothrops jararacussu venom. Piratoxin-I (PrTX-I) is a Lys-49 PLA₂ isolated from Bothrops pirajai venom. In this study, the ability of BthTX-I, BthTX-II and PrTX-I to recruit leucocytes into the rat pleural cavity and potential mechanisms underlying this effect were investigated. Intrapleural injection of either BthTX-I or PrTX-I (10–100 μg/cavity each) caused a significant leucocyte infiltration at 12 h after injection. The maximal cell migration was observed with the dose of 30 μg/cavity (14.9±15.5 and 17.6±1.6×10⁶ cells/cavity, respectively). Leucocyte counts consisted mainly of mononuclear cells, but significant amounts of neutrophils and eosinophils were also observed. Intrapleural injection of BthTX-II (10–100 μg/cavity) caused a marked leucocyte infiltration at 6 and 12 h after injection. The maximal response was observed with the dose of 100 μg/cavity (57.3±3.4×10⁶ cells/cavity, 6 h). The leucocyte counts were mainly composed of neutrophils and mononuclear cells. The treatment of either BthTX-I (30 μg/cavity, 12 h) or BthTX-II (30 μg/cavity, 6 h) with the PLA₂ inhibitor p-bromophenacyl bromide (p-BPB) had no effect on the total and differential leucocyte counts induced by these proteins. The same treatment partially reduced the PrTX-I-induced pleural leucocyte infiltration. In rats depleted of the histamine and 5-hydroxytryptamine (5-HT) stores by chronic treatment with compound 48/80, the total leucocyte counts in response to BthTX-I, BthTX-II and PrTX-I was not significantly affected compared to control animals. In addition, BthTX-I, BthTX-II and PrTX-I (100 μg/ml each) significantly degranulated pleural mast cells in vitro leading to the release of [¹⁴C]5-hydroxytryptamine ([¹⁴C]5-HT). p-BPB and heparin (50 IU/ml) significantly reduced the [¹⁴C]5-HT release induced by these PLA₂s. Our results demonstrate that BthTX-I, BthTX-II and PrTX-I recruit leucocyte into the pleural cavity of the rat by mechanisms unrelated to enzymatic activity and pleural mast cell degranulation.     

Natural phospholipase A2 myotoxin inhibitor proteins from snakes, mammals and plants

A renewed interest in the phenomenon of inter- and intra-species resistance towards the toxicity of snake venoms, coupled with the search for new strategies for treatment of snake envenomations, has prompted the discovery of proteins which neutralize the major toxic components of these venoms. Among these emerging groups of proteins are inhibitors of toxic phospholipases A₂ (PLA₂s), many of which exhibit a wide range of toxic effects including muscle-tissue damage, neurotoxicity, and inflammation. These proteins have been isolated from both venomous and non-venomous snakes, mammals, and most recently from medicinal plant extracts. The snake blood-derived inhibitors have been grouped into three major classes, α, β, and γ, based on common structural motifs found in other proteins with diverse physiological properties. In mammals, DM64, an anti-myotoxic protein isolated from opossum serum, belongs to the immunoglobulin super gene family and is homologous to human α₁B-glycoprotein and DM43, a metalloproteinase inhibitor from the same organism. In plants, a short note is made of WSG, a newly described anti-toxic-PLA₂ glycoprotein isolated from Withania somnifera (Ashwaganda), a medicinal plant whose aqueous extracts neutralize the PLA₂ activity of the Naja naja venom. The implications of these new groups of PLA₂ toxin inhibitors in the context of our current understanding of snake biology as well as in the development of novel therapeutic reagents in the treatment of snake envenomations worldwide are discussed.     

Comparison of the enzymatic and edema-produscing activities of two venom phospholipase A2 enzymes

The edema-producing activity of NNAVPLA₂, an acidic phospholipase A₂ (PLA₂) enzyme from Naja naja atra venom (NNAV), was less potent than that of TMVPLA₂ II, a basic PLA; from Trimeresurus mucrosquamatus venom (TMV). These edema-forming effects were greatly suppressed by pretreatment of rats with diphenhydramine/ methysergide or compound 48/80, which reduced the tissue content of histamine and serotonin. Heparin abolished and suppressed the paw edema caused by protamine and TMVPLA₂ II, respectively, but had no effect on the NNAVPLA₂-induced response. In isolated rat peritoneal mast cells, both PLA₂ concentration dependently induced the release of histamine and β-glueuronidase. Again, TMVPLA₂ II was more potent than NNAVPLA₂. This degranulation effect of mast cells caused by TMVPLA₂ II and protamine was inhibited by heparin, while that caused by NNAVPLA₂ was unaffected. The edema-forming and mast cell degranulation effects were greatly decreased in both PBPB-modified NNAVPLA₂ and PBPB-modified TMVPLA₂ II, in which the catalytic activity of the enzymes was completely lost. PBPB-modified TMVPLA₂ II-induced paw edema was also suppressed by heparin. Furthermore, this edematous response was totally reversed in rat pretreated with aspirin in combination with diphenhydramine and methysergide. These results suggest that the edema-forming effect of PLA₂ is probably dependent on the presence of catalytic, positive charge and pharmacological sites on its molecule.     

Purification and characterization of the biological effects of phospholipase A2 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 PLA₂ 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 São 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 PLA₂ proteins (named BcPLA₂1, BcPLA₂2 and BcPLA₂3) found to be active in B. caissarum extracts. MALDI-TOF mass spectrometry of BcPLA₂1 showed one main peak at 14.7 kDa. The N-terminal amino acid sequence of BcPLA₂1 showed high amino acid sequence identity with PLA₂ group III protein isolated from the Mexican lizard (PA23 HELSU, HELSU, PA22 HELSU) and with the honey bee Apis mellifera (PLA₂ and 1POC_A). In addition, BcPLA₂1 also showed significant overall homology to bee PLA₂. The enzymatic activity induced by native BcPLA₂1 (20 μg/well) was reduced by chemical treatment with p-bromophenacyl bromide (p-BPB) and with morin. BcPLA₂1 strongly induced insulin secretion in presence of high glucose concentration. In isolated kidney, the PLA₂ from B. caissarum increased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, and sodium, potassium and chloride levels of excretion. BcPLA₂1, however, did not increase the perfusion pressure on the mesenteric vascular bed. In conclusion, PLA₂, 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.     

Lipid domain formation modulates activities of snake venom phospholipase A2 enzymes

The goal of the present study is to elucidate the effect of lipid domain formation on activities of Naja naja atra and Bungarus multicinctus phospholipase A₂ (PLA₂) enzymes. Sphingomyelin inhibited enzymatic activity and membrane-damaging activity of PLA₂ against egg yolk phosphatidylcholine (EYPC), while cholesterol and cholesterol sulfate abrogated the inhibitory effect of sphingomyelin. The ability of cholesterol and cholesterol sulfate to abolish the inhibitory effect of sphingomyelin was closely related to their capacity to induce domain formation in EYPC/sphingomyelin vesicles. Laurdan fluorescence measurement revealed that membrane packing of EYPC/sphingomyelin vesicles was differently affected by cholesterol and cholesterol sulfate. Unlike cholesterol, cholesterol sulfate was unable to promote domain formation in dipalmitoylphosphatidylcholine (DPPC) vesicles. Cholesterol increased but cholesterol sulfate reduced PLA₂ activity against DPPC. Self-quenching studies and trinitrophenylation of Lys residues revealed that PLA₂ enzymes adopted different membrane-bound mode upon absorption onto the membrane bilayers comprised of different lipid compositions. Collectively, our data indicate that lipid domain formation regulates PLA₂ activity, and suggest that the physical state of membrane bilayers changes the interactive mode of PLA₂ with phospholipids.