Sequence and crystal structure determination of a basic phospholipase A2 from common krait (Bungarus caeruleus) at 2.4 A resolution: identification and characterization of its pharmacological sites

This is the first phospholipase A2 (PLA2) structure from the family of kraits. The protein was isolated from Bungarus caeruleus (common krait) and the primary sequence was determined using cDNA approach. Three-dimensional structure of this presynaptic neurotoxic PLA2 from group I has been determined by molecular replacement method using the model of PLA2 component of beta2-bungarotoxin (Bungarus multicinctus) and refined using CNS package to a final R-factor of 20.1 % for all the data in resolution range 20.0-2.4 A. The final refined model comprises 897 protein atoms and 77 water molecules. The overall framework of krait phospholipase A2 with three long helices and two short antiparallel beta-strands is extremely similar to those observed for other group I PLA2s. However, the critical parts of PLA2 folding are concerned with its various functional loops. The conformations of these loops determine the efficiency of enzyme action and presence/absence of various pharmacological functions. In the present structure calcium-binding loop is occupied by a sodium ion with a 7-fold co-ordination. The conformation of loop 55-75 in krait PLA2 corresponds to a very high activity of the enzyme. A comparison of its sequence with multimeric PLA2s clearly shows the absence of critical residues such as Tyr3, Trp61 and Phe64, which are involved in the multimerization of PLA2 molecules. The protein shows anticoagulant and neurotoxic activities.     

Natural inhibitors of toxic phospholipases A(2)

Endogenous proteins isolated from the serum of snakes have been found to be natural inhibitors displaying anti-hemorrhagic, anti-neurotoxic or anti-myotoxic activity. Some of these proteins inhibit phospholipase A(2) (PLA(2)) activity. We review in brief here the properties, structure and classification of these PLA(2) inhibitors (PLIs), focusing in particular on the mechanism of neutralization of the toxic PLA(2)s by anti-neurotoxic PLIs. We also discuss: 1) the protection provided by these molecules against endogenous snake venom PLA(2)s; 2) their specificity for neurotoxic snake venom PLA(2)s (beta-neurotoxins) and non-toxic mammalian secreted sPLA(2)s; and 3) the domains of the inhibitor and PLA(2) potentially involved in the binding of these two molecules. Purified and characterized natural inhibitors of PLA(2)s may be used to develop more effective therapeutic strategies for dealing with snake envenomation. Moreover, the structural and, in some cases, functional similarity of natural inhibitors to various mammalian proteins suggests that these mammalian proteins may themselves behave as PLA(2) inhibitors. Thus, these proteins may have important physiological functions in regulating the activities of neurotoxic PLA(2) and non-toxic sPLA(2).     

Identification of beta-type phospholipase A(2) inhibitor in a nonvenomous snake,Elaphe quadrivirgata

A novel serum protein inhibiting specifically the enzymatic activity of the basic phospholipase A(2) (PLA(2)) from the venom of the Chinese mamushi snake (Agkistrodon blomhoffii siniticus) was purified from a nonvenomous Colubridae snake, Elaphe quadrivirgata. The purified inhibitor was a 150-kDa glycoprotein having a trimeric structure, composed of two homologous 50-kDa subunits. Their amino acid sequences, containing leucine-rich repeats, were typical of the beta-type PLA(2) inhibitor (PLIbeta), previously identified from the serum of A. blomhoffii siniticus. The inhibitor inhibited exclusively group II basic PLA(2)s and did not inhibit other kinds of PLA(2)s. This is the first paper reporting the existence of PLIbeta in a nonvenomous snake. The existence of PLIbeta in the nonvenomous snake reflects that PLIbetas are widely distributed over the snake species and participate commonly in regulating the physiological activities of the unidentified target PLA(2)s.     

Modeling of acanthoxin A1, a PLA2 enzyme from the venom of the common death adder (Acanthophis antarcticus)

The phospholipase A2 enzyme, acanthoxin, found in the venom of the common death adder (Acanthophis antarcticus) as with other snake PLA2 enzymes displays neurotoxic activity. It is unclear whether this neurotoxic activity particular to some snake PLA2 enzymes is a result of structural differences solely within the catalytic sites or at a distant location upon the molecules. We have predicted the three-dimensional structure of one of the two predominant isoforms of acanthoxin (A1) using comparative protein modeling techniques. Given the high degree of homology and the availability of a high quality crystallographic structure, notexin was used as a molecular template to construct an all atom model of acanthoxin. The model was made using the program MODELLER3 and then refined with X-PLOR. Comparison between the predicted structure of acanthoxin and several X-ray structures of toxic and nontoxic PLA2 enzymes has led to a testable two-step proposal of neurotoxic PLA2 activity; involving the favorable binding to acceptor molecules followed by enzymatic intrusion upon the target membrane. The electrostatic potentials across the molecular surfaces of toxic and nontoxic PLA2 enzymes were calculated (GRASP) and it was found that the toxic PLA2 enzymes possessed a charge distribution on the noncatalytic surface not identified in the nontoxic PLA2 enzymes. Thus we have identified residues potentially involved in the interaction of the PLA2 enzymes with their acceptor molecules. Furthermore, the proposed acceptor molecule recognition site is distant from the catalytic site which upon binding of the PLA2 to the acceptor molecule may enhance the enzymatic ability of the toxic PLA2 enzymes on particular cell types.     

Enzymatic activity and inhibition of the neurotoxic complex vipoxin from the venom of Vipera ammodytes meridionalis

Vipoxin from the venom of Vipera ammodytes meridionalis is an unique neurotoxic complex between a toxic phospholipase A2 and a highly homologous non-toxic protein inhibitor. It is an example of evolution of a catalytic and toxic function into inhibitory and non-toxic one. The activity of the V. ammodytes meridionalis toxin is 1.7 times higher than that of the closely related (92% sequence identity) neurotoxic complex RV4/RV7 from the venom of Vipera russelli formosensis The enhanced enzymatic activity of vipoxin is attributed to limited structural changes, in particular to the substitutions G54R and Q78K in the PLA2 subunit of the complex and to the T54R substitution in the inhibitor. Oleyloxyethylphosphocholine, aristolochic acid and vitamin E suppressed the enzymatic activity of vipoxin and its isolated PLA2 subunit. These compounds influence inflammatory processes in which PLA2 is implicated. The peptide Lys-Ala-Ile-Tyr-Ser, which is an integral part of the PLA2 components of the two neurotoxic complexes from V. ammodytes meridionalis and V. russelli formosensis (sequence 70-74) activated vipoxin increasing its PLA2 activity by 23%. This is in contrast to the inhibitory effect of the respective pentapeptides with 70-74 sequences on other group II PLA2s. Surprisingly, the same peptide inhibited 46% of the V. russelli formosensis PLA2 activity. The limited changes in the structure of the two highly homologous neurotoxins lead to considerable differences in their interaction with native peptides.     

The X-ray structure of a snake venom Gln48 phospholipase A2 at 1.9A resolution reveals anion-binding sites

Phospholipase A2 is an "interfacial" enzyme and its binding to negatively charged surfaces is an important step during catalysis. The Gln48 phospholipase A2 from the venom of Vipera ammodytes meridionalis plays the role of chaperone and directs a toxic His48 PLA2 onto its acceptor. In the venom the two phospholipases A2 exist as a postsynaptic neurotoxic complex, Vipoxin. The X-ray structure of Gln48 PLA2, complexed to sulphate ions, which mimic the negatively charged groups of anionic membranes, has been determined by the molecular replacement method and refined to 1.9A resolution. The protein forms a homodimer stabilized by ionic, hydrophobic, and hydrogen-bond interactions. The structure reveals two anion-binding sites per subunit. These sites are probably involved in interactions with the negatively charged membrane surface and, in this way, in the "targeting" of the toxic component to the receptors of the postsynaptic membranes. In the absence of the chaperone subunit the toxin changes the target of the physiological attack. A comparison of the homodimeric Gln48 PLA2 structure with that of the heterodimeric Vipoxin reveals differences in regions involved in the pharmacological activity of the toxin. This fact, except the active site histidine substitution, can explain the absence of toxicity in the Gln48 protein in comparison to the His48 phospholipase A2.     

Catalytically inactive phospholipase A2 homologue binds to vascular endothelial growth factor receptor-2 via a C-terminal loop region

VEGF (vascular endothelial growth factor) regulates neovascularization through binding to its receptor KDR (kinase insert domain-containing receptor; VEGF receptor-2). We recently identified a catalytically inactive PLA(2) (phospholipase A(2)) homologue (KDR-bp) in the venom of eastern cottonmouth (Agkistrodon piscivorus piscivorus) as a third KDR-binding protein, in addition to VEGF(165) and tissue inhibitor of metalloproteinase-3. KDR-bp binds to the extracellular domain of KDR with a K(d) of 10(-8) M, resulting in specific blockade of endothelial cell growth induced by VEGF(165). Inactive PLA(2) homologues are widely distributed in the venoms of Viperidae snakes and are known to act as myotoxins. In the present study, we demonstrated that KDR-binding ability is a common characteristic for inactive PLA(2) homologues in snake venom, but not for active PLA(2)s such as neurotoxic and platelet aggregation-modulating PLA(2)s. To understand better the KDR and KDR-bp interaction, we resolved the binding region of KDR-bp using eight synthetic peptides designed based on the structure of KDR-bp. A synthetic peptide based on the structure of the C-terminal loop region of KDR-bp showed high affinity for KDR, but other peptides did not, suggesting that the C-terminal loop region of KDR-bp is involved in the interaction with KDR. The results of the present study provide insight into the binding of inactive PLA(2) homologues to KDR, and may also assist in the design of novel anti-KDR molecules for anti-angiogenic therapy.     

Purification of a Class B1 platelet aggregation inhibitor phospholipase A2 from Indian cobra (Naja Naja) venom

A platelet aggregation inhibitor phospholipase A(2) (NND-IV-PLA(2)) was isolated from Naja naja (Eastern India) venom by a combination of cation and anion exchange chromatography. NND-IV-PLA(2) is the most catalytically active enzyme isolated from the Indian cobra venom. The acidic PLA(2) profile of Eastern regional Indian cobra venom is distinctly different from that of the western regional venom. However the acidic PLA(2)s from both the regions follow the pattern of increasing catalytic activity with increase in acidic nature of the PLA(2) isoform. NND-IV-PLA(2) is a Class B1 platelet aggregation inhibitor and inhibits platelet aggregation induced by ADP, collagen and epinephrine. Modification of active site histidine abolishes both catalytic activity and platelet aggregation inhibition activities while aristolochic acid, a phospholipase A(2) inhibitor has only partial effect on the two activities.     

Simultaneous activation of PLA2 and PLC are required to promote acrosomal reaction stimulated by progesterone via G-proteins

The acrosome reaction (AR) is a special exocytotic process promoted by signal transduction pathways studied in many laboratories. Progesterone (P4) is one of the trigger molecules proposed. Upon the binding of P4 to its receptor, several molecules could be activated, including G-proteins, phospholipase A(2) (PLA(2)), and phospholipase C (PLC). The role of these molecules was analyzed in this study using the Chlortetracycline (CTC) protocol to detect and quantify the AR. Incubation of capacitated sperm cells with GTPgammas (GTPgammas, a mimetic of G-protein activation), arachidonic acid (AA, product of PLA(2) action), or phorbol ester (PMA, an activator of PLC) for 15 min increased the AR to a similar percentage as P4. Conversely, a decrease in the AR was detected when sperm cells were incubated with P4 after preincubation with: GDPbetaS (GDP, an inhibitor of G-protein activation), ONO RS-82 (ONO, an inhibitor of PLA(2)), or neomycin (Neo, an inhibitor of PLC) for 15 min. To analyze the activation sequence of G proteins, PLA(2), and PLC combinations of these mimetic/inhibitors were used during successive incubation periods. Inhibition promoted by GDP, ONO, and Neo were overcome by 15-min incubation with GTPgammas, AA, or PMA, respectively. But GTPgammas or P4 did not reverse the inhibition due to incubation with Neo and ONO. Interestingly, this dual inhibition was reverted by another 15-min incubation with AA or PMA. Results presented here could indicate that the AR triggered by P4 is driven by activation of G-proteins, that in turn activate PLA(2) and PLC simultaneously, that finally promote acrosomal exocytosis.     

Interaction of the neurotoxic and nontoxic secretory phospholipases A2 with the crotoxin inhibitor from Crotalus serum.

Crotalus durissus terrificus snakes possess a protein in their blood, named crotoxin inhibitor from Crotalus serum (CICS), which protects them against crotoxin, the main toxin of their venom. CICS neutralizes the lethal potency of crotoxin and inhibits its phospholipase A2 (PLA2) activity. The aim of the present study is to investigate the specificity of CICS towards snake venom neurotoxic PLA2s (beta-neurotoxins) and nontoxic mammalian PLA2s. This investigation shows that CICS does not affect the enzymatic activity of pancreatic and nonpancreatic PLA2s, bee venom PLA2 and Elapidae beta-neurotoxins but strongly inhibits the PLA2 activity of Viperidae beta-neurotoxins. Surface plasmon resonance and PAGE studies further demonstrated that CICS makes complexes with monomeric and multimeric Viperidae beta-neurotoxins but does not interact with nontoxic PLA2s. In the case of dimeric beta-neurotoxins from Viperidae venoms (crotoxin, Mojave toxin and CbICbII), which are made by the noncovalent association of a PLA2 with a nonenzymatic subunit, CICS does not react with the noncatalytic subunit, instead it binds tightly to the PLA2 subunit and induces the dissociation of the heterocomplex. In vitro assays performed with Torpedo synaptosomes showed a protective action of CICS against Viperidae beta-neurotoxins but not against other PLA2 neurotoxins, on primary and evoked liberation of acetylcholine. In conclusion, CICS is a specific PLA2 inhibitor of the beta-neurotoxins from the Viperidae family.     

Three-dimensional structure of a presynaptic neurotoxic phospholipase A2 from Daboia russelli pulchella at 2.4 A resolution

The phospholipase A(2 )from Daboia russelli pulchella (DPLA(2)) is the only known member of subclass II of group IIA. The three-dimensional structure of this presynaptic neurotoxic DPLA(2) enzyme has been determined at 2.4 A resolution. The structure was determined by the molecular replacement method using the model Crotalus atrox, and refined using X-PLOR to a final R-factor of 18.8 % for all data in the resolution range 20.0 A-2.4 A. The final refined model comprises 1888 atoms from two crystallographically independent protein molecules and 160 water oxygen atoms. The overall folding of DPLA(2), with three long helices and two short antiparallel beta-strands is grossly similar to those observed for other PLA(2)s. In the present structure, the calcium binding site is empty but the conformation of the calcium binding loop is similar to those observed in the calcium bound states. Two spatially adjacent regions of residues 55-61 (a typical beta-turn I) and 83-94 (a well defined loop) are remarkably different in conformation, electrostatic characteristics and inter-segmental interactions from those found in non-neurotoxic PLA(2)s. Yet another striking structural feature in DPLA(2 )pertains to the stretch of residues 53-77, which has a series of positively charged residues protruding outwardly. The above segment is presumed to be involved in the anticoagulant activity. A unique hydrophobic patch including residues Leu17, Ala18, Ile19, Pro20, Phe106 and Leu110 is found on the surface together with an equally emphatic region of -OH groups containing residues such as Ser21, Tyr22, Ser23, Ser24, Tyr25 and Tyr28. The interactions between two molecules of DPLA(2) in the asymmetric unit are remarkably different from those observed in the standard dimers and trimers of PLA(2)s, leaving the enzyme's active site fully exposed for enzyme-substrate reactions, it makes this structure one of the most favourable examples for structure-based drug design through soaking experiments.     

Two groups of entomopathogenic bacteria, Photorhabdus and Xenorhabdus, share an inhibitory action against phospholipase A2 to induce host immunodepression

Photorhabdus and Xenorhabdus are two genera of entomopathogenic bacteria having a mutualistic relationship with their respective nematode hosts, Heterorhabditis and Steinernema. One of the pathogenic mechanisms of these bacteria includes host immunodepression, which leads to lethal septicemia. It has been known that X. nematophila inhibits phospholipase A2 (PLA2) to induce host immunodepression. Here, we tested the hypothesis of PLA2 inhibition using another bacterial species involved in other genera. P. temperata subsp. temperata is the intestinal symbiont of an entomopathogenic nematode, H. megidis. The bacteria caused potent pathogenicity in a dose-dependent manner against the fifth instar larvae of a test target insect, Spodoptera exigua, as early as 24 h after the intra-hemocoelic injection. In response to the live bacterial injection, hemocyte nodulation (a cellular immune response) and prophenoloxidase (pPO) activation were inhibited, while the injection of heat-killed bacteria significantly induced both immune reactions. The immunodepression induced by the live bacteria was reversed by the addition of arachidonic acid, the catalytic product of phospholipase A2. In contrast, the addition of dexamethasone, a specific PLA2 inhibitor to the heat-killed bacterial treatment, inhibited both immune capacities. In addition to a previously known PLA2 inhibitory action of X. nematophila, the inhibition of P. temperata temperata on PLA2 suggests that bacteria symbiotic to entomopathogenic nematodes share a common pathogenic target to result in an immunodepressive state of the infected insects. To prove this generalized hypothesis, we used other bacterial species (X. bovienni, X. poinarii, and P. luminescens) involved in these two genera. All our experiments clearly showed that these other bacteria also share their inhibitory action against PLA2 to induce host immunodepression.     

Purification of a post-synaptic neurotoxic phospholipase A2 from Naja naja venom and its inhibition by a glycoprotein from Withania somnifera

A post-synaptic neurotoxic phospholipase A(2) (PLA(2)) has been purified from Indian cobra Naja naja venom. It was associated with a peptide in the venom. The association was disrupted using 8 M urea. It is denoted to be a basic protein by its behavior on both ion exchange chromatography and electrophoresis. It is toxic to mice, LD(50) 1.9 mg/kg body weight (ip). It is proved to be post-synaptic PLA(2) by chymographic experiment using frog nerve-muscle preparation. A glycoprotein, (WSG) was isolated from a folk medicinal plant Withania somnifera. The WSG inhibited the phospholipase A(2) activity of NN-XIa-PLA(2,) isolated from the cobra venom, completely at a mole-to-mole ratio of 1:2 (NN-XIa-PLA(2): WSG) but failed to neutralize the toxicity of the molecule. However, it reduced the toxicity as well as prolonged the death time of the experimental mice approximately 10 times when compared to venom alone. The WSG also inhibited several other PLA(2) isoforms from the venom to varying extent. The interaction of the WSG with the PLA(2) is confirmed by fluorescence quenching and gel-permeation chromatography. Chemical modification of the active histidine residue of PLA(2) using p-brophenacyl bromide resulted in the loss of both catalytic activity as well as neurotoxicity of the molecule. These findings suggest that the venom PLA(2) has multiple sites on it; perhaps some of them are overlapping. Application of the plant extract on snakebite wound confirms the medicinal value associated with the plant.     

A novel prokaryotic phospholipase A2. Characterization, gene cloning, and solution structure

Until now, phospholipase A(2) (PLA(2); EC 3.1.14) has been found only from eukaryotic sources. In the present study, we found a secreted PLA(2), which is produced by a soil bacterium, Streptomyces violaceoruber A-2688, demonstrating that the enzyme is the first phospholipase A(2) identified in prokaryote. After characterization of the novel PLA(2), a gene encoding the enzyme was cloned, sequenced, and overexpressed using a Streptomyces host-vector system. The amino acid sequence showed that the prokaryotic PLA(2) has only four cysteines and less homology to the eukaryotic ones, which have 12-16 cysteines. The solution structures of the prokaryotic PLA(2), bound and unbound with calcium(II) ion, were determined by using the NMR technique and structure calculation. The overall structure of the S. violaceoruber PLA(2), which is composed of only five alpha-helices, is completely different from those of eukaryotic PLA(2)s, which consist of beta-sheets and alpha-helices. The structure of the calcium-binding domain is obviously distinct from that without the ion; the ligands for the calcium(II) ion are the two carboxylates of Asp(43) (monodentate) and Asp(65) (bidentate), the carbonyl oxygen of Leu(44), and three water molecules. A calcium-binding experiment showed that the calcium dissociation constant ( approximately 5 mm) for the prokaryotic PLA(2) is much larger than those of eukaryotic ones.     

Structural analysis of secretory phospholipase A2 from Clonorchis sinensis: therapeutic implications for hepatic fibrosis

Hepatic fibrosis is a common complication of the infection by the parasite, Clonorchis sinensis. There is a high incidence of this disease in the Asian countries with an increased risk of conversion to cancer. A secretory phospholipase A(2) (PLA(2)) enzyme from the parasite is implicated in the pathology. This is an attractive drug target in the light of extensive structural characterization of this class of enzyme. In this study, the structure of the enzyme was modeled based on its sequence homology to the group III bee venom PLA(2). On analysis, the overall structure essentially is comprised of three helices, two sets of β-wings and an elongated C-terminal extension. The structure is stabilized by four disulfide bonds. The structure is comprised of a calcium binding loop, active site and a substrate binding hydrophobic channel. The active site of the enzyme shows the classical features of PLA(2) with the participation of the three residues: histidine-aspartic acid-tyrosine in hydrogen bond formation. This is an interesting variation from the house keeping group III PLA(2) enzyme of human which has a histidine-aspartic acid and phenylalanine arrangement at the active site. This difference is therefore an important structural parameter that can be exploited to design specific inhibitor molecules against the pathogen PLA(2). Likewise, there are certain unique structural features in the hydrophobic channel and the putative membrane binding surface of the PLA(2) from Clonorchis sinensis that not only help understand the mechanism of action but also provide knowledge for a targeted therapy of liver fibrosis caused by the parasite.     

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.     

An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits hemocyte phagocytosis of Spodoptera exigua by inhibiting phospholipase A(2)

Phagocytosis is a hemocytic behavior against bacterial infection. An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits immune responses of target insects and causes hemolymph septicemia. This study analyzed how X. nematophila could inhibit phagocytosis to increase its pathogenicity. Granular cells and plasmatocytes were the main phagocytic hemocytes of Spodoptera exigua determined by observing fluorescence-labeled bacteria in the cytosol. X. nematophila significantly inhibited phagocytosis of both hemocytes, while heat-killed X. nematophila lost its inhibitory potency. However, co-injection of X. nematophila with arachidonic acid did not show any significant inhibition of hemocyte phagocytosis. In fact, hemocytes of S. exigua infected with X. nematophila showed significant reduction in phospholipase A(2) (PLA(2)) activity. Dexamethasone, a specific PLA(2) inhibitor, significantly inhibited phagocytosis of both cell types. However, the inhibitory effect of dexamethasone was recovered by addition of arachidonic acid. Incubation of hemocytes with benzylideneacetone, a metabolite of X. nematophila, inhibited phagocytosis in a dose-dependent manner. These results suggest that X. nematophila produces and secretes PLA(2) inhibitor(s), which in turn inhibit the phagocytic response of hemocytes.     

Crystal structure of a carbohydrate induced homodimer of phospholipase A2 from Bungarus caeruleus at 2.1A resolution

This is the first crystal structure of a carbohydrate induced dimer of phospholipase A(2) (PLA(2)). This is an endogenous complex formed between two PLA(2) molecules and two mannoses. It was isolated from Krait venom (Bungarus caeruleus) and crystallized as such. The complete amino acid sequence of PLA(2) was determined using cDNA method. Three-dimensional structure of the complex has been solved with molecular replacement method and refined to a final R-factor of 0.192 for all the data in the resolution range 20.0-2.1A. The presence of mannose molecules in the protein crystals was confirmed using dinitrosalicylic acid test and the molecular weight of the dimer was verified with MALDI-TOF. As indicated by dynamic light scattering and analytical ultracentrifugation the dimer was also stable in solution. The good quality non-protein electron density at the interface of two PLA(2) molecules enabled us to model two mannoses. The mannoses are involved extensively in interactions with protein atoms of both PLA(2) molecules. Some of the critical amino acid residues such as Asp 49 and Tyr 31, which are part of the substrate-binding site, are found facing the interface and interacting with mannoses. The structure of the complex clearly shows that the dimerization is caused by mannoses and it results in the loss of enzymatic activity.     

Crystallization and preliminary X-ray diffraction studies of a toxic phospholipase A2 from the venom of Vipera ammodytes meridionalis complexed to a synthetic inhibitor

A toxic phospholipase A(2) (PLA(2)) is isolated from the neurotoxic complex Vipoxin, the major lethal component of the venom of Vipera ammodytes meridionalis. The enzyme is complexed to the synthetic inhibitor elaidoylamide and crystallized. The crystals belong to the space group P2(1)2(1)2(1), with unit cell dimensions a=46.57 A, b=82.68 A, c=119.47 A and beta=90 degrees. Initial diffraction data to 3.3 A resolution are collected.     

The first phospholipase inhibitor from the serum of Vipera ammodytes ammodytes

Ammodytoxins are neurotoxic secretory phospholipase A(2) molecules, some of the most toxic components of the long-nosed viper (Vipera ammodytes ammodytes) venom. Envenomation by this and by closely related vipers is quite frequent in southern parts of Europe and serotherapy is used in the most severe cases. Because of occasional complications, alternative medical treatment of envenomation is needed. In the present study, ammodytoxin inhibitor was purified from the serum of V. a. ammodytes using two affinity procedures and a gel exclusion chromatography step. The ammodytoxin inhibitor from V. a. ammodytes serum consists of 23- and 25-kDa glycoproteins that form an oligomer, probably a tetramer, of about 100 kDa. N-terminal sequencing and immunological analysis revealed that both types of subunit are very similar to gamma-type secretory phospholipase A(2) inhibitors. The ammodytoxin inhibitor from V. a. ammodytes serum is a potent inhibitor of phospholipase activity and hence probably also the neurotoxicity of ammodytoxins. Discovery of the novel natural inhibitor of these potent secretory phospholipase A(2) toxins opens up prospects for the development of new types of small peptide inhibitors for use in regulating the physiological and pathological activities of secretory phospholipases A(2).