The neurotoxic phospholipase A2 associates,through a non-phosphorylated binding motif,with 14-3-3 protein gamma and epsilon isoforms

Two novel acceptors for ammodytoxin C, a presynaptically neurotoxic phospholipase A(2) from snake venom, have been purified from porcine cerebral cortex by a toxin-affinity-based procedure. Using tandem mass spectrometry, the isolated acceptors were identified as 14-3-3 gamma and epsilon isoforms, highly conserved cytoplasmic proteins involved in the regulation of numerous physiological processes. The interaction between ammodytoxin C and 14-3-3 proteins is direct and not mediated by calmodulin, a high-affinity acceptor for both ammodytoxin C and 14-3-3 proteins, as demonstrated in pull-down experiments and by surface plasmon resonance. The latter technique gave an apparent dissociation constant of 1.0+/-0.2 microM for the interaction between chip-immobilized 14-3-3 and ammodytoxin C. 14-3-3 usually interacts with proteins through specific phospho-Ser/Thr motifs. Ammodytoxin C is not a phospho-protein, therefore the interaction must occur through a non-phosphorylated binding site, most probably the KEESEK sequence at its C-terminal end. The interaction we describe suggests an explanation for the pathophysiological effects evoked by some secreted phospholipases A(2), such as the inhibition of protein phosphorylation, of terminal ion currents, and of neurotransmission, as well as the initiation of neuronal cell death, all processes regulated by 14-3-3 proteins.     

Binding to the high-affinity M-type receptor for secreted phospholipases A(2) is not obligatory for the presynaptic neurotoxicity of ammodytoxin A

R180, isolated from porcine brain cortex, is a high-affinity membrane receptor for ammodytoxin A (AtxA), a secreted phospholipase A(2) (sPLA(2)) and presynaptically active neurotoxin from venom of the long-nosed viper (Vipera ammodytes ammodytes). As a member of the M-type sPLA(2) receptors, present on the mammalian plasma membrane, R180 has been proposed to be responsible for one of the first events in the process of presynaptic neurotoxicity, the binding of the toxin to the nerve cell. To test this hypothesis, we prepared and analyzed three N-terminal fusion proteins of AtxA possessing a 12 or 5 amino acid residue peptide. The presence of such an additional "propeptide" prevented interaction of the toxin with the M-type receptor but not its lethality in mouse and neurotoxic effects on a mouse phrenic nerve-hemidiaphragm preparation. In addition, antibodies raised against the sPLA(2)-binding C-type lectin-like domain 5 of the M-type sPLA(2) receptor were unable to abolish the neurotoxic action of AtxA on the neuromuscular preparation. The specific enymatic activities of the fusion AtxAs were two to three orders of magnitude lower from that of the wild type, yet resulting in a similar but less pronounced neurotoxic profile on the neuromuscular junction. This is in accordance with other data showing that a minimal enzymatic activity suffices for presynaptic toxicity of sPLA(2)s to occur. Our results indicate that the interaction of AtxA with the M-type sPLA(2) receptor at the plasma membrane is not essential for presynaptic activity of the toxin. Interaction of AtxA with two intracellular proteins, calmodulin and the R25 receptor, was affected but not prevented by the presence of the N-terminal fusion peptides, implying that these proteins may play a role in the sPLA(2) neurotoxicity.     

Amino acid and cDNA sequences of a neutral phospholipase A2 from the long-nosed viper (Vipera ammodytes ammodytes) venom.

The amino acid sequence of a non-toxic phospholipase A2, ammodytin I2, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) and its cDNA sequence have been determined. The protein sequence was elucidated by sequencing the peptides generated by CNBr cleavage, mild acid hydrolysis and tryptic digestion of maleylated and non-maleylated protein. Sequencing of the cDNA showed that the protein is synthesized as an 137-amino-acid-residue precursor molecule consisting of a 16-residue signal peptide, followed by a 121-residue mature enzyme. Ammodytin I2 cDNA shows 73% nucleotide and 59% amino acid identities in the mature protein region in comparison to that of ammodytoxin A, the most presynaptically neurotoxic phospholipase A2 from the long-nosed viper. Identities in the signal-peptide region are considerably higher, 96% and 100%, respectively.     

Amino-acid sequence of ammodytoxin B partially reveals the location of the site of toxicity of ammodytoxins

The complete amino-acid sequence of ammodytoxin B, a presynaptically toxic phospholipase A2 isolated from Vipera ammodytes ammodytes venom, was determined by manual and automated protein sequencing. Ammodytoxin B (i.v. LD50 = 0.58 mg/kg for white mice) is 30-fold less toxic than ammodytoxin A, the most toxic phospholipase isolated from the same venom. The two proteins (each 122 residues long) differ in only 3 residues located in positions 115, 118 and 119 (numbering according to R. Renetseder et al. (1985) J. Biol. Chem. 260, 11627-11634) suggesting that an exposed hydrophobic residue in position 115 and a basic residue in position 118 may be responsible for the increased toxicity of ammodytoxin A and should form at least one part of the site of toxicity in ammodytoxins.     

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).     

Ammodytoxin A, a highly lethal phospholipase A2 from Vipera ammodytes ammodytes venom

The amino acid sequence of ammodytoxin A, the most toxic presynaptically active phospholipase A2 isolated from Vipera ammodytes ammodytes venom, was determined. The primary structure was deduced from peptides obtained by Staphylococcus aureus proteinase and trypsin digestion of reduced and carboxymethylated protein and from the automated Edman degradation of the N-terminal part of the non-reduced molecule. According to the sequence, the enzyme classifies to the subgroup IIA of the phospholipase A2 family of enzymes. The location of basic residues believed to be responsible for the toxic activity of presynaptically active phospholipases differs substantially from those in the highly toxic enzymes of other subgroups. Comparison of the sequence with sequences of other snake venom enzymes indicates that the toxic site(s) may not be the same in all subgroups of presynaptically active phospholipases.     

Ammodytoxin, a neurotoxic secreted phospholipase A(2), can act in the cytosol of the nerve cell

Recent identification of intracellular proteins that bind ammodytoxin (calmodulin, 14-3-3 proteins, and R25) suggests that this snake venom presynaptically active phospholipase A(2) acts intracellularly. As these ammodytoxin acceptors are cytosolic and mitochondrial proteins, the toxin should be able to enter the cytosol of a target cell and remain stable there to interact with them. Using laser scanning confocal microscopy we show here that Alexa-labelled ammodytoxin entered the cytoplasm of the rat hippocampal neuron and subsequently also its nucleus. The transport of proteins into the nucleus proceeds via the cytosol of a cell, therefore, ammodytoxin passed the cytosol of the neuron on its way to the nucleus. Although it is not yet clear how ammodytoxin is translocated into the cytosol of the neuron, our results demonstrate that its stability in the cytosol is not in question, providing the evidence that the toxin can act in this cellular compartment.     

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.     

Comparative structural studies of two natural isoforms of ammodytoxin,phospholipases A2 from Vipera ammodytes ammodytes which differ in neurotoxicity and anticoagulant activity

Ammodytoxin A (AtxA) and its natural isoform AtxC from the venom of Vipera ammodytes ammodytes belong to group IIA-secreted phospholipases A₂ which catalyze the hydrolysis of glycerophospholipids and exhibit strong neurotoxic and anticoagulant effects. The two isoforms, which differ in sequence by only two amino acid residues (Phe124 > Ile and Lys128 > Glu), display significant differences in toxicity and anticoagulant properties and act on protein targets including neurotoxic proteic receptors and coagulation factor Xa with significantly different strengths of binding.In order to characterize the structural basis of these functional differences, we have determined the crystal structures of the two isoforms. Comparison of the structures shows that the mutation Lys128 > Glu in AtxC could perturb interactions with FXa, resulting in lower anticoagulant activity, since the side chain of Glu128 is partly buried, making a stabilizing hydrogen bond with the main-chain nitrogen atom of residue Thr35. This interaction leads to a displacement of the main polypeptide chain at positions 127 and 128 (identified by mutagenesis as important for interaction with FXa), and a different orientation of the side chain of unmutated Lys127. The mutation Phe124 > Ile in AtxC induces no significant conformational changes, suggesting that the differences in toxicity of the two isoforms are due essentially to differences in surface complementarity in the interaction of the toxin with the neurotoxic protein receptor. The crystal structures also reveal a novel dimeric quaternary association involving significant hydrophobic interactions between the N-terminal α-helices of two molecules of ammodytoxin related by crystallographic symmetry. Interactions at the dimer interface include important contributions from Met7, which is unique to ammodytoxin. Equilibrium sedimentation experiments are consistent with the crystallographic model.Competition experiments using SPR technology show complete inhibition of AtxA binding to FXa by calmodulin (CaM). The crystal structure shows that the C-terminal region, important for binding to FXa and CaM, is fully exposed and accessible for interaction with proteic receptors in both the monomeric and dimeric forms of ammodytoxin described here.     

Neutralization of lethal potency and inhibition of enzymatic activity of a phospholipase A2 neurotoxin, crotoxin, by non-precipitating antibodies (Fab)

Rabbit antibodies were prepared against both purified catalytic (component-B) and purified non-catalytic (component-A) subunits of crotoxin, the major phospholipase A2 neurotoxin from the South American rattlesnake. They cross-react with crotoxin-like toxins from the venom of several Crotalus species as well as with single-chain phospholipase A2 neurotoxins from Crotalid and Viperid venoms (agkistrodontoxin and ammodytoxin A) but not from Elapid venoms (notexin). Immunological cross-reactions of anti-component-A and anti-component-B sera with crotoxin and with its isolated components A and B showed that component-A exposes determinants of low immunogenicity which are present on component-B, whereas the major antigenic determinants of component-B are not present on component-A. Anti-component-B antibodies, but not anti-component-A antibodies, neutralize the lethal potency of crotoxin and inhibit its enzymatic activity. Furthermore, non-precipitating anti-component-B Fab fragments were as potent as antibodies, indicating that crotoxin neutralization results from the binding of the antibodies to the catalytic subunit, rather than the formation of an immunoprecipitate.     

A presynaptically toxic secreted phospholipase A2 is internalized into motoneuron-like cells where it is rapidly translocated into the cytosol

The molecular mechanism of the presynaptic toxicity of secreted phospholipase A2 (sPLA2) neurotoxins, including that of ammodytoxin A (AtxA), has not been resolved. Here we report the action of AtxA on mouse motoneuron-like cells, on which it induced characteristic neurotoxic effects on synaptic vesicles and on the reorganization of F-actin. AtxA also released fatty acids from the plasmalemma. Its significantly less neurotoxic V31W mutant showed similar effects on cells but with a much higher rate of hydrolysis than the wild-type, indicating that high enzymatic activity alone is not sufficient for the observed effects. The neurotoxic action was observed by confocal microscopy of a fluorescently labelled AtxA and by electron microscopy of a nanogold-labelled toxin. The Atx-binding proteins were tagged by a photo-cross-linking reagent conjugated to the toxin. AtxA was taken up rapidly by the cells, where it interacted within minutes with calmodulin and 14-3-3 proteins in the cytosol. These data demonstrate, for the first time, the translocation of an sPLA2 from the extracellular space into the cytosol of a cell. Such an event may thus be important in explaining the action of a range of homologous endogenous sPLA2 enzymes in mammals whose roles in various cellular processes are not yet completely understood.     

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.     

Identification of a novel binding site for calmodulin in ammodytoxin A, a neurotoxic group IIA phospholipase A2.

The molecular mechanism of the presynaptic neurotoxicity of snake venom phospholipases A2 (PLA2s) is not yet fully elucidated. Recently, new high-affinity binding proteins for PLA2 toxins have been discovered, including the important intracellular Ca2+ sensor, calmodulin (CaM). In the present study, the mode of interaction of group IIA PLA2s with the Ca2+-bound form of CaM was investigated by mutational analysis of ammodytoxin A (AtxA) from the long-nosed viper (Vipera ammodytes ammodytes). Several residues in the C-terminal part of AtxA were found to be important in this interaction, particularly those in the region 115-119. In support of this finding, introduction of Y115, I116, R118 and N119, present in AtxA, into a weakly neurotoxic PLA2 from Russell's viper (Daboia russellii russellii) increased by sevenfold its binding affinity for CaM. Furthermore, two out of four peptides deduced from different regions of AtxA were able to compete with the toxin in binding to CaM. The nonapeptide showing the strongest inhibition was that comprising the AtxA region 115-119. This stretch contributes to a distinct hydrophobic patch within the region 107-125 in the C-terminal part of the molecule. This lacks any substantial helical structure and is surrounded by several basic residues, which may form a novel binding motif for CaM on the molecular surface of the PLA2 toxin.     

Phorbol myristate acetate stimulates formation of phosphatidyl inositol 4-phosphate and phosphatidyl inositol 4,5-bisphosphate in human platelets

There are conflicting data in the literature as to whether or not the Ca2+ activation of phospholipase A2 is mediated by the calcium binding protein calmodulin. In the present study the membrane-bound phospholipase A2 enzymes in rat and human platelets were shown to be absolutely Ca2+ dependent but were not stimulated by the addition of calmodulin. A partially purified phospholipase A2 from rat platelet membrane, which contained little endogenous calmodulin, also was not stimulated by calmodulin addition. Both isolated and membrane-bound phospholipase A2 were inhibited by the non-specific calmodulin antagonist trifluoperazine but the inhibition was not overcome by adding calmodulin. There was thus no evidence from these studies that phospholipase A2 is calmodulin regulated.     

The role of aspartic acid-49 in the active site of phospholipase A2. A site-specific mutagenesis study of porcine pancreatic phospholipase A2 and the rationale of the enzymatic activity of [lysine49]phospholipase A2 from Agkistrodon piscivorus piscivorus' venom

In order to probe the role of Asp-49 in the active site of porcine pancreatic phospholipase A2 two mutant proteins were constructed containing either Glu or Lys at position 49. Their enzymatic activities and their affinities for substrate and for Ca2+ ions were examined in comparison with the native enzyme. Enzymatic characterization indicated that the presence of Asp-49 is essential for effective hydrolysis of phospholipids. Conversion of Asp-49 to either Glu or Lys strongly reduces the binding of Ca2+ ions in particular for the lysine mutant but the affinity for substrate analogues is hardly affected. Extensive purification of [Lys49]phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus yielded a protein which was 4000 times less active than the basic [Asp49]phospholipase A2 from this venom. Inhibition studies with p-bromophenacyl bromide showed that this residual activity was due to a small amount of contaminating enzyme and that the Lys-49 homologue itself is inactive. The results obtained both with the porcine pancreatic phospholipase A2 mutants and with the native venom enzymes show that Asp-49 is essential for the catalytic action of phospholipase A2.     

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.     

Studies on phospholipase A inhibitor in blood plasma. II. Interaction of phospholipase A inhibitor with phospholipase A and its specificity

Non-competitive inhibition of snake venom phospholipase A2 which has been exhibited by bovine plasma phospholipase A inhibitor, a kind of lipoprotein, was not observed unless the inhibitor was preincubated with the enzyme. The inhibition seemed to be due to the formation of the enzyme-inhibitor complex, which was identified by immunoelectrophoresis. The enzyme-inhibitor interaction was observed maximally on incubation at physiological pH, but not below pH 5. The inhibitor was inactivated by trypsin digestion and heat treatment. It suppressed the phospholipase A2 activities of rat blood plasma as well as of the snake venom and porcine pancreas, but not the enzyme activities such as those of phospholipase C of Bacillus cereus, lipase of porcine pancreas, trypsin, and papain. The inhibitor also showed the ability to decrease membrane-bound phospholipase A1 and A2 activities in intracellular organelles such as plasma membranes, mitochondria, lysosomes, and microsomes. In view of these facts, it was concluded that the plasma inhibitor is specific for phospholipase A.     

The amino acid sequence of a myotoxic phospholipase from the venom of Bothrops asper

A myotoxic, basic phospholipase A2 (pI greater than 9.5) with anticoagulant activity has been purified from the venom of Bothrops asper, and its amino acid sequence determined by automated Edman degradation. It is distinct from the B. asper phospholipase A2 known as myotoxin I [Lomonte, B. and Gutierrez, J. M., 1989, Toxicon 27, 725] but cross-reacts with myotoxin I rabbit antisera, suggesting that the proteins are closely related isoforms. To our knowledge, this is the first myotoxic phospholipase to be sequenced that lacks presynaptic neurotoxicity (iv LD50 approximately equal to 8 micrograms/g in mice). The protein appears to exist as a monomer, contains 122 amino acids, and fits with subgroup IIA of other sequenced phospholipase A2 molecules. Its primary sequence shows greatest identity with ammodytoxin B (67%), a phospholipase A2 presynaptic neurotoxin from Vipera ammodytes ammodytes venom. Hydropathy profiles of B. asper phospholipase and the ammodytoxins also show great similarities. In contrast, even though the amino acid sequence identities between B. asper phospholipase and the basic subunit of crotoxin remain high (64%), their hydropathy profiles differ substantially. Domains and residues that may be responsible for neurotoxicity are discussed.     

The interaction between the presynaptic phospholipase neurotoxins beta-bungarotoxin and crotoxin and mixed detergent-phosphatidylcholine micelles. A comparison with non-neurotoxic snake venom phospholipases A2

Certain phospholipase A2 enzymes (E.C.3.1.1.4) selectively inhibit neurotransmitter release from cholinergic nerve terminals. Both specific acceptor proteins and the physical state of nerve terminal phospholipids have been implicated in studies of the mechanism of phospholipase neurotoxin action. Here we have examined the effects of charge on a micellar phospholipid substrate by comparing the enzyme activity and binding of two neurotoxic phospholipases (beta-bungarotoxin and crotoxin) with other non-neurotoxic phospholipases. This has been achieved by altering either the phospholipid or the ionic charge of the detergent in the mixed phospholipid micelle. The neurotoxic phospholipases were only active on negatively charged micelles, whereas the non-neurotoxic enzymes were equally active in hydrolyzing neutral micelles. This distinction was also reflected in binding studies; the non-neurotoxic phospholipases bound to both types of substrate, whereas beta-bungarotoxin and crotoxin selectively bound to negatively charged micellar structures. These experiments suggest that, in addition to the existence of any specific acceptor proteins, neurotoxin binding is also governed by the charge on the lipid phase of the nerve terminal membrane.