Mass spectrometry analysis of the phospholipase A(2) activity of snake pre-synaptic neurotoxins in cultured neurons.

Snake pre-synaptic phospholipase A(2) neurotoxins paralyse the neuromuscular junction by releasing phospholipid hydrolysis products that alter curvature and permeability of the pre-synaptic membrane. Here, we report results deriving from the first chemical analysis of the action of these neurotoxic phospholipases in neurons, made possible by the use of high sensitivity mass spectrometry. The time-course of the phospholipase A(2) activity (PLA(2)) hydrolysis of notexin, beta-bungarotoxin, taipoxin and textilotoxin acting in cultured neurons was determined. At variance from their enzymatic activities in vitro, these neurotoxins display comparable kinetics of lysophospholipid release in neurons, reconciling the large discrepancy between their in vivo toxicities and their in vitro enzymatic activities. The ratios of the lyso derivatives of phosphatidyl choline, ethanolamine and serine obtained here together with the known distribution of these phospholipids among cell membranes, suggest that most PLA(2) hydrolysis takes place on the cell surface. Although these toxins were recently shown to enter neurons, their intracellular hydrolytic action and the activation of intracellular PLA(2)s appear to contribute little, if any, to the phospholipid hydrolysis measured here.

Phospholipases A2 isolated from Micrurus lemniscatus coral snake venom: behavioral, electroencephalographic, and neuropathological aspects.

The present study evaluated four phospholipase A2 (PLA2) (Mlx-8, Mlx-9, Mlx-11 and Mlx-12) isolated from Micrurus lemniscatus snake venom (Elapidae). The effects of intrahippocampal administration of these toxins have been determined on behavior, electroencephalography, and neuronal degeneration in rats. These four PLA2 toxins induced motor and EEG alterations in a dose-dependent manner. Behavioral convulsions were characterized by clonic movements and were often accompanied by EEG alterations. Mlx toxins were convulsive but weakly epileptogenic, since low rates of seizure discharges were observed in EEG records. Neuronal injury seemed to depend on the dose of the toxin used. The highest doses of toxins caused severe intoxication and death of some animals. The injury of hippocampal cells was characterized by massive neuronal loss and hippocampal gliosis. A high occurrence of compulsive scratching was observed. Moreover, the onset of seizures induced by Mlx toxins was markedly delayed. The similarities between the effects of Mlx PLA2s and those isolated from other Elapidae snakes venoms suggest that their toxicity are probably due to their specific binding to neuronal membranes and to the catalysis of phospholipid hydrolysis, producing lysophospholipids and fatty acids. These compounds likely disturb the membrane conformation, causing a marked increase in the release of neurotransmitters and concurrent inhibition of vesicle fission and recycling. These toxins can be a useful tool to investigate properties of endogenous secretory PLA2s and therefore may be important both to study mechanisms involved in neurotransmitter release at nerve terminals and to explain the convulsive properties of PLA2s toxins.

Novel genes encoding six kinds of three-finger toxins in Ophiophagus hannah (king cobra) and function characterization of two recombinant long-chain neurotoxins.

Three-finger toxins are a family of low-molecular-mass toxins (<10 kDa) having very similar three-dimensional structures. In the present study, 19 novel cDNAs coding three-finger toxins were cloned from the venom gland of Ophiophagus hannah (king cobra). Alignment analysis showed that the putative peptides could be divided into six kinds of three-finger toxins: LNTXs (long-chain neurotoxins), short-chain neurotoxins, cardiotoxins (CTXs), weak neurotoxins, muscarinic toxins and a toxin with a free SH group. Furthermore, a phylogenetic tree was established on the basis of the toxin cDNAs and the previously reported similar nucleotide sequences from the same source venom. It indicated that three-finger-toxin genes in O. hannah diverged early in the course of evolution by long- and short-type pathways. Two LNTXs, namely rLNTX1 (recombinant LNTX1) and rLNTX3, were expressed and showed cytolytic activity in addition to their neurotoxic function. By comparing the functional residues, we offer some possible explanations for the differences in their neurotoxic function. Moreover, a plausible elucidation of the additonal cytolytic activity was achieved by hydropathy-profile analysis. This, to our knowledge, is the first observation that recombinant long chain alpha-neurotoxins have a CTX-like cytolytic activity.

Lachesis muta (Viperidae) cDNAs reveal diverging pit viper molecules and scaffolds typical of cobra (Elapidae) venoms: implications for snake toxin repertoire evolution.

Efforts to describe toxins from the two major families of venomous snakes (Viperidae and Elapidae) usually reveal proteins belonging to few structural types, particular of each family. Here we carried on an effort to determine uncommon cDNAs that represent possible new toxins from Lachesis muta (Viperidae). In addition to nine classes of typical toxins, atypical molecules never observed in the hundreds of Viperidae snakes studied so far are highly expressed: a diverging C-type lectin that is related to Viperidae toxins but appears to be independently originated; an ohanin-like toxin, which would be the third member of the most recently described class of Elapidae toxins, related to human butyrophilin and B30.2 proteins; and a 3FTx-like toxin, a new member of the widely studied three-finger family of proteins, which includes major Elapidae neurotoxins and CD59 antigen. The presence of these common and uncommon molecules suggests that the repertoire of toxins could be more conserved between families than has been considered, and their features indicate a dynamic process of venom evolution through molecular mechanisms, such as multiple recruitments of important scaffolds and domain exchange between paralogs, always keeping a minimalist nature in most toxin structures in opposition to their nontoxin counterparts.

Structural difference between group I and group II cobra cardiotoxins: X-ray, NMR, and CD analysis of the effect of cis-proline conformation on three-fingered toxins.

Natural homologues of cobra cardiotoxins (CTXs) were classified into two structural subclasses of group I and II based on the amino acid sequence and circular dichroism analysis, but the exact differences in their three-dimensional structures and biological significance remain elusive. We show by circular dichroism, NMR spectroscopic, and X-ray crystallographic analyses of a newly purified group I CTX A6 from eastern Taiwan cobra (Naja atra) venoms that its loop I conformation adopts a type VIa turn with a cis peptide bond located between two proline residues of PPxY. A similar "banana-twisted" conformation can be observed in other group I CTXs and also in cyclolinopeptide A and its analogues. By binding to the membrane environment, group I CTX undergoes a conformational change to adopt a more extended hydrophobic domain with beta-sheet twisting closer to the one adopted by group II CTX. This result resolves a discrepancy in the CTX structural difference reported previously between solution as well as crystal state and shows that, in addition to the hydrophobicity, the exact loop I conformation also plays an important role in CTX-membrane interaction. Potential protein targets of group I CTXs after cell internalization are also discussed on the basis of the determined loop I conformation.

A novel phospholipase A(2) from the venom glands of Bungarus candidus: cloning and sequence-comparison.

The presence of phospholipase A(2) (PLA(2)) in the venom of Malayan krait (Bungarus candidus) and its structure were studied. The PLA(2) cDNAs from the venom gland of B. candidus (Indonesia origin) were amplified by the polymerase chain reactions (PCR) and cloned. The primers used were based on the cDNA sequences of several homologous B. multicinctus venom PLA(2)s. In addition to the A-chains of beta-bungarotoxins, a novel B. candidus PLA(2) was cloned and its full amino acid sequence deduced. Having totally 125 amino acid residues, the PLA(2) contains a pancreatic loop and is 61% identical to the acidic PLA(2) of king cobra venom. However, the enzyme was not detected from the venom sample. Its structural relationships to other elapid venom PLA(2)s were analyzed with a phylogenetic tree and discussed.

Electrospray liquid chromatography/mass spectrometry fingerprinting of Acanthophis (death adder) venoms: taxonomic and toxinological implications.

Death adders (genus Acanthophis) are unique among elapid snakes in both morphology and venom composition. Despite this genus being among the most divergent of all elapids, the venom has been historically regarded as relatively quite simple. In this study, liquid chromatography/mass spectrometry (LC/MS) analysis has revealed a much greater diversity in venom composition, including the presence of molecules of novel molecular weights that may represent a new class of venom component. Furthermore, significant variation exists between species and populations, which allow for the LC/MS fingerprinting of each species. Mass profiling of Acanthophis venoms clearly demonstrates the effectiveness of this technique which underpins fundamental studies ranging from chemotaxonomy to drug design.

Comparison of three classes of snake neurotoxins by homology modeling and computer simulation graphics.

We present a systematic structure comparison of three major classes of postsynaptic snake toxins, which include short and long chain alpha-type neurotoxins plus one angusticeps-type toxin of black mamba snake family. Two novel alpha-type neurotoxins isolated from Taiwan cobra (Naja naja atra) possessing distinct primary sequences and different postsynaptic neurotoxicities were taken as exemplars for short and long chain neurotoxins and compared with the major lethal short-chain neurotoxin in the same venom, i.e., cobrotoxin, based on the derived three-dimensional structure of this toxin in solution by NMR spectroscopy. A structure comparison among these two alpha-neurotoxins and angusticeps-type toxin (denoted as FS2) was carried out by the secondary-structure prediction together with computer homology-modeling based on multiple sequence alignment of their primary sequences and established NMR structures of cobrotoxin and FS2. It is of interest to find that upon pairwise superpositions of these modeled three-dimensional polypeptide chains, distinct differences in the overall peptide flexibility and interior microenvironment between these toxins can be detected along the three constituting polypeptide loops, which may reflect some intrinsic differences in the surface hydrophobicity of several hydrophobic peptide segments present on the surface loops of these toxin molecules as revealed by hydropathy profiles. Construction of a phylogenetic tree for these structurally related and functionally distinct toxins corroborates that all long and short toxins present in diverse snake families are evolutionarily related to each other, supposedly derived from an ancestral polypeptide by gene duplication and subsequent mutational substitutions leading to divergence of multiple three-loop toxin peptides.

Elapid venom toxins: multiple recruitments of ancient scaffolds.

Nigroxins A and B, two myotoxic phospholipases A2 (PLA2s) from the venom of the American elapid Micrurus nigrocinctus, belong to a new PLA2 subclass. Their primary structures were established and compared with those of PLA2s that have already been studied with respect to myotoxic activity. The combination of amino acid residues Arg15, Ala100, Asn108 and a hydrophobic residue at position 109 is present exclusively in class I PLA2s that display myotoxic activity. These residues cluster within a surface region rich in positive charges and are suggested to play a role in the interaction with the target membrane of the muscle fibers. It is concluded that the myotoxic PLA2s resulted from recruitment of an ancient scaffold. Dendrotoxins and alpha-neurotoxins are similarly derived from other old structures, which are, however, now also present in nontoxic proteins that are widely distributed throughout the animal kingdom. The evolutionary pathways by which elapid PLA2s acquired myotoxicity and dendrotoxins acquired K+-channel blocker activity are traced. They demonstrate how existing scaffolds were adapted stepwise to serve toxic functions by exchange of a few surface-exposed residues.

Four new postsynaptic neurotoxins from Naja naja sputatrix venom: cDNA cloning, protein expression, and phylogenetic analysis.

cDNAs encoding 4 short chain alpha-neurotoxins from Malayan spitting cobra (Naja naja sputatrix) venom were cloned and expressed in Escherichia coli. The recombinant toxins possessed identical amino acid sequences to alpha-neurotoxins. This is the first report on cloning and expression of isoforms of neurotoxins from a species of spitting cobra. Two of these isoforms were also identified in the crude venom by reverse phase-high performance liquid chromatography (RP-HPLC), capillary electrophoresis followed by mass spectrometry and characterized by protein sequencing. Based on the variable amino acid residues, the neurotoxins in N. n. sputatrix could be assigned to 2 major groups, 10E11T and 10Q11A, which could be further subdivided into 10E11T28S: 10E11T28G and 10Q11A28S; 10Q11A28G respectively. These substitutions were also found to be unique to N. n. sputatrix neurotoxins. Phylogenetic analysis based on molecular properties of the toxins provided further support for the classification of N. n. sputatrix neurotoxin into 2 fundamental groups.

[Genetic origin of venom variability: impact on the preparation of antivenin serums]. / L'origine génétique de la variabilité des venins: impact sur la préparation des sérums antivenimeux.

One of the main possible origin of the biochemical variations of venoms could be genetic. We studied the venom of members of litters born in a snake farm (12 Crotalus atrox and 21 Naja haje). We first used the electrophoresis in cellulose acetate (AE). Then, variations were confirmed by immunoelectrophoresis (AIE) using an antivenom (IPSER Africa, Pasteur Mérieux Sérums & Vaccines) and immunsera prepared on rabbit from i) venom presenting the maximum of bands in electrophoresis (complete venom) and ii) pure toxins (neurotoxin-alpha and cardiotoxin-gamma). At last, the toxicity of some samples was measured and the ability of SAV to neutralise the corresponding sample was measured. The AE of C. atrox venoms showed a good homogeneity, probably due to a good genetic stability of the investigated group. On the other hand, N. haje venoms have revealed a great heterogeneity. The 13 samples were allocated to five groups according to the absence of some fractions compared to the complete venom. The AIE showed that the neurotoxin-alpha is present in every sample, but variable in quantity, even when it did not appear on AE. We suggest that these pattern variations are due either to relative variations of protein fractions in samples or to modifications of the chemical composition of the neurotoxin-alpha. However, the variation of toxicity between the different samples questioned the neutralisation ability of antivenoms. We propose that venom sample choice for SAV production should be based on biochemical criteria and toxicity of samples rather than random pooling.

Development of immunoassays for determination of circulating venom antigens during envenomations by coral snakes (Micrurus species).

A reverse agglutination assay and two capture enzyme-linked immunoassays (ELISAs) for the quantitative determination of Micrurus nigrocinctus nigrocinctus venom antigens in fluids were developed using affinity-purified polyclonal antibodies and a cocktail of three monoclonal antibodies. The lower detection limit was 0.3 mg/ml for the reverse agglutination assay and 4 ng/ml for the capture ELISAs. The optical densities of both ELISAs correlated very well with venom concentrations in the range 4-333 ng/ml (r = 0.99). The ability of these assays to detect venoms of several medically important Micrurus species was studied. Besides detecting homologous venom, both ELISAs were also useful to quantitate venom from M. fulvius, M. dumerilii carinicauda and M. alleni. Using biotinylated polyclonal antibodies, M. n. nigrocinctus venom antigens were detected in sera or plasma from rabbits and mice during experimental envenomations with lethal and sublethal venom doses. The assays described in this work are promising tests to estimate the severity of poisoning in envenomations by the most important coral snakes of North and Central America.

Taxonomic changes and toxinology: systematic revisions of the Asiatic cobras (Naja naja species complex)

Until recently, all Asiatic cobra populations were regarded as belonging to one single species, Naja naja. Recent revisions have shown that there are in fact at least 10 full species of Asiatic Naja. In order to allow the existing literature to be reconciled with these recent discoveries, an interpretation of the older nomenclature is provided. Problematic areas, especially concerning the species N. sumatrana and N. siamensis, are highlighted.

Ectopic and increased expression of Fasciclin II alters motoneuron growth cone guidance.

We used the enhancer detection/GAL4 system in Drosophila to direct increased levels of Fasciclin II (Fas II) expression on motoneuron growth cones and axons and to direct ectopic Fas II expression on other cells they encounter. Four classes of abnormal phenotypes are observed: "bypass" phenotypes, in which axons fail to defasciculate at the choice point where they would normally enter their muscle target region and instead extend past their target; "detour" phenotypes, in which these bypass growth cones enter their muscle target region at a different location; "stall" phenotypes, in which axons that enter their muscle target region fail to defasciculate from one another to probe their muscle targets; and "misroute" phenotypes, in which growth cones are diverted onto abnormal pathways by contact with Fas II-positive cells. These phenotypes show that changes in the pattern and level of Fas II expression can alter growth cone guidance, apparently in part by modulating the ability of these growth cones to respond to other guidance cues.

A comparative study on the electrophoretic patterns of snake venoms.

1. Examination of the polyacrylamide gel electrophoretic (PAGE) and SDS-PAGE patterns of snake venoms shows that these patterns are useful for species differentiation (and hence identification) for snakes of certain genera but have only limited application for snakes from some other genera, due either to the marked individual variations in the venoms or the lack of marked interspecific differences within the same genus. 2. There is no substantial intersubspecific difference in the electrophoretic patterns of the venoms. 3. In general there are no common characteristics in the electrophoretic patterns of the venom at the generic level because of the wide variations in the electrophoretic patterns of venoms of snakes within the same genus. 4. At the familial level, the venoms of Elapidae exhibited SDS-PAGE patterns distinct from those of Crotalidae.

Differing stabilities of snake venom cardiotoxins in acidic aqueous acetonitrile.

1. Although snake venom cardiotoxins constitute a homologous family of proteins, subclasses with different structural and biological properties exist. 2. By using circular dichroism spectroscopy of twelve cardiotoxins belonging to two structural classes and one non-classified group, this investigation indicated that cardiotoxins differ in their stabilities towards denaturation in acidic aqueous acetonitrile, as used in some reversed-phase high performance liquid chromatography separations. 3. It was also shown that cardiotoxins of the structural class II are in general less stable towards this denaturation than class I and non-classified cardiotoxins.

Four polypeptide components of green mamba venom selectively block certain potassium channels in rat brain synaptosomes.

Venom from the green mamba (Dendroaspis angusticeps) blocked 86Rb efflux through voltage-gated K channels in rat brain synaptosomes. Crude venom inhibited both rapidly inactivating, 4-aminopyridine-sensitive K channels, and noninactivating, phencyclidine-sensitive, K channels. Fractionation of the venom by size exclusion chromatography and cation exchange high performance liquid chromatography yielded four 7000-dalton polypeptides (designated alpha-, beta-, gamma-, and delta-DaTX) that blocked synaptosome K channels. Two of these toxins, alpha- and delta-DaTX (10-100 nM), preferentially blocked the inactivating voltage-gated K channels. The other two toxins, beta- and gamma-DaTX, preferentially blocked the noninactivating voltage-gated K channels. The amino acid composition of these four toxins indicated that alpha-DaTX is identical to dendrotoxin [Br. J. Pharmacol. 77:153-161 (1982)] and toxin C13S2C3 [Hoppe-Seyler's Z. Physiol. Chem. 361:661-674 (1980)]; the composition and partial sequence analysis indicate that delta-DaTX is identical to toxin C13S1C3 [Hoppe-Seyler's Z. Physiol. Chem. 361:661-674 (1980)]. Beta- and gamma-DaTX have not previously been identified. Partial amino acid sequences of beta- and gamma-DaTX and the published sequences of alpha- and delta-DaTX reveal that the C-terminal segments of all four toxins are homologous. The C-terminal segments are also homologous with a number of nontoxic proteinase inhibitors. This raises the possibility that the N-terminal rather than the C-terminal regions are more likely responsible for the K channel blocking activity. The N-terminal portions of alpha- and delta-DaTX have some sequence homologies, but they have no obvious homologies with either beta- or gamma-DaTX. The finding of structurally similar peptide toxins with preferential activities toward different K channels may lead to the development of useful probes of K channel structure and may provide the means to distinguish among different K channels biochemically as well as physiologically.

Classification of elapid snake neurotoxins and cytotoxins according to chain length: evolutionary implications.

The amino acid sequences of the 139 homologous "short" neurotoxins, "long" neurotoxins and cytotoxins so far characterised from elapid snake venoms were compared on the basis of the amino acid deletion/insertion events that have occurred during evolution. Systematic grouping of the toxins according to similarity suggests that the short neurotoxins resemble the cytotoxins more closely than they do the long neurotoxins. The significance of this finding is discussed in relation to the methodology, the conformations of the toxins (as represented by circular dichroism spectra) and the outcome of the study that would have been obtained. had more traditional methods been used. It appears probable that the cytotoxins evolved relatively recently from neurotoxic ancestors.