A Neurotoxic Snake Venom without Phospholipase A2: Proteomics and Cross-Neutralization of the Venom from Senegalese Cobra, Naja senegalensis (Subgenus: Uraeus).

The Senegalese cobra, Naja senegalensis, is a non-spitting cobra species newly erected from the Naja haje complex. Naja senegalensis causes neurotoxic envenomation in Western Africa but its venom properties remain underexplored. Applying a protein decomplexation proteomic approach, this study unveiled the unique complexity of the venom composition. Three-finger toxins constituted the major component, accounting for 75.91% of total venom proteins. Of these, cardiotoxin/cytotoxin (~53%) and alpha-neurotoxins (~23%) predominated in the venom proteome. Phospholipase A2, however, was not present in the venom, suggesting a unique snake venom phenotype found in this species. The venom, despite the absence of PLA2, is highly lethal with an intravenous LD50 of 0.39 µg/g in mice, consistent with the high abundance of alpha-neurotoxins (predominating long neurotoxins) in the venom. The hetero-specific VINS African Polyvalent Antivenom (VAPAV) was immunoreactive to the venom, implying conserved protein antigenicity in the venoms of N. senegalensis and N. haje. Furthermore, VAPAV was able to cross-neutralize the lethal effect of N. senegalensis venom but the potency was limited (0.59 mg venom completely neutralized per mL antivenom, or ~82 LD50 per ml of antivenom). The efficacy of antivenom should be further improved to optimize the treatment of cobra bite envenomation in Africa.

Extraction of α-neurotoxins from equine plasma by receptor based affinity purification.

Venoms were first identified as potential doping agents by the racing industry in 2007 when three vials of cobra venom were seized during an inspection of a stable at Keeneland Racecourse in the USA. Venoms are a complex mixture of proteins, peptides, and other substances with a wide range of biological effects, including inhibiting the transmission of nervous and muscular impulses. As an example of this, cobratoxin, an α-neurotoxin found in cobra venom, is claimed to be an effective treatment for pain. Recent analysis of seized samples identified venom from two different species of snake. Proteomic analysis identified the first sample as cobra venom, while the second sample, in a vial labeled "Conotoxin", was identified as venom from a many banded krait. Cobratoxin, conotoxins, and bungarotoxins (a component of krait venom) are all α-neurotoxins, suggesting a common application for all three venom proteins as potential pain blocking medications. Using a peptide based on the nicotinic acetylcholine receptor, a one-step affinity purification method was developed for the detection of α-neurotoxins in plasma.

Proteomic insights into short neurotoxin-driven, highly neurotoxic venom of Philippine cobra (Naja philippinensis) and toxicity correlation of cobra envenomation in Asia.

The Philippine cobra, Naja philippinensis, is a WHO Category 1 venomous snake of medical importance responsible for fatal envenomation in the northern Philippines. To elucidate the venom proteome and pathophysiology of envenomation, N. philippinensis venom proteins were decomplexed with reverse-phase high-performance liquid chromatography, and protein fractions were subsequently digested with trypsin, followed by nano-liquid chromatography-tandem mass spectrometry analysis and data mining. Three-finger toxins (3FTX, 66.64% of total venom proteins) and phospholipases A2 (PLA2, 22.88%) constitute the main bulk of venom proteome. Other proteins are present at low abundances (<4% each); these include metalloproteinase, serine protease, cobra venom factor, cysteine-rich secretory protein, vespryn, phosphodiesterase, 5' nucleotidase and nerve growth factor. In the three-finger toxin family, the alpha-neurotoxins comprise solely short neurotoxins (SNTX, 44.55%), supporting that SNTX is the principal toxin responsible for neuromuscular paralysis and lethality reported in clinical envenomation. Cytotoxins (CTX) are the second most abundant 3FTX proteins in the venom (21.31%). The presence of CTX correlates with the venom cytotoxic effect, which is more prominent in murine cells than in human cells. From the practical standpoint, SNTX-driven neuromuscular paralysis is significant in N. philippinensis envenomation. Antivenom production and treatment should be tailored accordingly to ensure effective neutralization of SNTX. BIOLOGICAL SIGNIFICANCE: The venom proteome of Naja philippinensis, the Philippine cobra, is unravelled for the first time. Approximately half the protein bulk of the venom is made up of short neurotoxins (44.55% of the total venom proteins). As the only alpha-neurotoxins present in the venom, short neurotoxins are the causative toxins of the post-synaptic blockade and fast-onset neuromuscular paralysis in N. philippinensis envenomation. A substantial amount of cytotoxins (21.31%) was also detected in N. philippinensis venom, supporting that the venom can be cytotoxic although the effect is much weaker in human cells compared to murine cells. The finding is consistent with the low incidence of local tissue necrosis in N. philippinensis envenomation, although this does not negate the need for monitoring and care of bite wound in the patients.

A single-label fluorescent derivatization method for quantitative determination of neurotoxin in vivo by capillary electrophoresis coupled with laser-induced fluorescence detection.

Neurotoxin (NT), a short-chain α-neurotoxin, is the main neurotoxic protein identified from the venom of Naja naja atra. As an effective drug for the analgesis of advanced cancer patients, NT lasts longer than morphine and does not cause addiction. However, achieving a sensitive and high-resolution measurement of NT is difficult because of the extra-low content of NT in vivo. Therefore, developing a novel method to quantify NT is essential to study its pharmacokinetics in vivo. Although NT contains four primary amine groups that could react with the thiourea in fluorescein isothiocyanate (FITC), we developed a simple and reproducible single-label fluorescent derivatization method for NT which is related to the reaction of N-terminal α-amino of NT alone under optimized derivatization conditions. Furthermore, neurotoxin labelled with fluorescein isothiocyanate (NT-FITC) was prepared by high-performance liquid chromatography (HPLC) with a purity value higher than 99.29% and identified by MALDI-TOF/TOF-MS. Finally, NT-FITC could be detected at 0.8 nmol L(-1) in rat plasma using capillary electrophoresis coupled with laser induced fluorescence detection (CE-LIF). In this paper, the established method robustly and reliably quantified NT labelled with FITC via intravenous and intramuscular administrations in vivo. In addition, this work fully demonstrated the pharmacokinetic characteristics of NT in vivo, which could reduce the risk of drug accumulation, optimize therapies, and provide sufficient evidence for the rational use of NT in clinical and research laboratories.

Comparative analysis of the venom proteome of four important Malaysian snake species

Naja kaouthia, Ophiophagus hannah, Bungarus fasciatus and Calloselasma rhodostoma are four venomous snakes indigenous to Malaysia. In the present study, their proteomic profile by two-dimensional gel electrophoresis (2-DE) have been separated and compared.

Comparative analysis of the venom proteome of four important Malaysian snake species

Naja kaouthia, Ophiophagus hannah, Bungarus fasciatus and Calloselasma rhodostoma are four venomous snakes indigenous to Malaysia. In the present study, their proteomic profile by two-dimensional gel electrophoresis (2-DE) have been separated and compared.

Non-lethal polypeptide components in cobra venom.

Snakes from several genera (mostly from Naja genus) belonging to the Elapidae family are usually named cobras. The effect of cobra bites is mainly neurotoxic. This is explained by the presence of highly potent alpha-neurotoxin in their venoms. The other two highly toxic components of cobra venoms are cytotoxins and phospholipases A(2). These three types of toxins constitute a major part of cobra venom. They have attracted the attention of researchers for many years and have been very well studied and thoroughly described. However cobra venoms contain also many other less abundant components which possess very low toxicity or even are not toxic at all. These components, mostly proteins, belong to different structural and functional types, and the reason for their presence in the venom is not always evident. Some of them are known for many years (e.g., nerve growth factor and cobra venom factor); others (e.g., cysteine rich secretory proteins, CRISPs) were discovered only recently. There are non-lethal proteins with unique biological activities that can be used as biochemical tools, while others may be regarded as potential leads for drug design. This review is the first attempt to systemize the available data on non-lethal components of cobra venom.

New method for characterizing highly disulfide-bridged peptides in complex mixtures: application to toxin identification from crude venoms.

Animal venoms are highly complex mixtures that can contain many disulfide-bridged toxins. This work presents an LC-MALDI approach allowing (1) a rapid classification of toxins according to their number of disulfide bonds and (2) a rapid top-down sequencing of the toxins using a new MALDI matrix enhancing in-source decay (ISD). The crude venom is separated twice by LC: the fractions of the first separation are spotted on the MALDI matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) and the others using 1,5-diaminonaphthalene (1,5-DAN). CHCA spots are more convenient for obtaining a precise mass fingerprint of a large number of peptides; however, the analysis of 1,5-DAN spots allows the number of disulfide bridges to be counted owing to their partial in-plume reduction by this particular matrix. Subsequently, the disulfide bonds of all peptides present in the crude venom were reduced by an excess of tris(carboxyethyl)phosphine before the LC separation and were subjected to the same analysis in CHCA and 1,5-DAN. Toxins were sequenced using a TOF/TOF analysis of metastable fragments from CHCA spots and ISD fragmentation from 1,5-DAN spots. Novel conotoxin sequences were found using this approach. The use of 1,5-DAN for ISD top-down sequencing is also illustrated for higher molecular weight toxins such as snake cardiotoxins and neurotoxins (>6500 Da), where sequence coverage >70% is obtained from the c-ion series.

Screening of plants containing Naja naja siamensis cobra venom inhibitory activity using modified ELISA technique.

Enzyme-linked immunosorbent assay (ELISA) has been modified for screening plants with antagonistic activity to Naja naja siamensis cobra venom. Aqueous extracts from plants were investigated for their inhibitory effects on the binding of anti-cobra venom antibody to antigen, cobra venom, fixed onto 96-well microtiter plates. Ingredients in extracts were allowed to react with immobilized venom before the subsequent addition of antivenom antibody. Venom components affected by exposure to the extracts, unable to interact with their specific antibody, were predicted to be unable to bind to their native destinations or natural receptors. Curcuma cf. zedoaria, an old Thai medicinal plant, showed clear inhibitory activity in the ELISA test. Neurotoxin and protein degradative enzymes, major components in venom, were identified as targets of this extract in Western immunoblotting analysis. Ingredients in the extract showed high affinity to the toxin in competition assay by immunoprecipitation. The extract attenuated toxin activity by extending contraction time of diaphragm muscle after envenomation and had a potency to protect cellular proteins from venom degradative enzymes. Curcuma parviflora, with less activity in ELISA, exhibited acceptable results in two experiments but negative results in two experiments, whereas Curcuma longa, having low activity in the ELISA test, never showed any favorable results. Screening of 36 samples could classify plants into an inhibition range of 0 to 86%. This modified ELISA is recommended as a preliminary screening method for inhibitors with a large number of samples.

Purification and characterization of a neurotoxin from the venom of Ophiophagus hannah (king cobra).

A neurotoxin, Oh9-1, from the venom of Ophiophagus hannah was isolated by a combination of ion-exchange chromatography and reverse phase HPLC. Amino acid sequence analysis revealed that Oh9-1 consists of 57 amino acids and eight cysteine residues. This protein was mainly constituted with beta-sheet as evidenced by CD spectrum. Oh9-1 inhibited carbachol-induced muscle contraction in an irreversible manner and the dose for achieving 50% inhibition was approximately fourfold that of alpha-bungarotoxin. Since the residues in alpha-neurotoxins closely involve in the binding with acetylcholine receptors are not highly conserved in this toxin molecule, Oh9-1 represents a novel type of neurotoxin structurally distinct from alpha-neurotoxins.

Isolation of nerve growth factor (NGF) from human body fluids; saliva, serum and urine: comparison between cobra venom and cobra serum NGF.

Several investigators have isolated nerve growth factor (NGF) from various tissues and organs of different animals. There is no published documentation about NGF from body fluids, such as blood serum, saliva, and urine. Contrary to the unsuccessful attempts to detect or isolate NGF in serum in the past, this investigation reports the isolation of NGF from human serum, saliva, and urine. It further reports the comparison of properties between NGFs derived from cobra venom and cobra serum. NGF from serum, saliva, and urine was isolated by high pressure liquid chromatography (HPLC) and was identified as described by Lipps (1998). The identified fractions of NGF were further purified to study the biological and immunological properties. The biological activities of NGFs from human body fluids and cobra serum on PC12 cells were miniscule in comparison to the cobra venom derived NGF. The molecular weights of NGFs from human serum, saliva, and urine were identical, 36.0 kDa. However, the molecular weights of cobra serum and cobra venom NGFs were different, 55.0 kDa and 13.5 kDa, respectively. NGF level is age dependent and varies under different conditions. Using anti-human NGF, diagnostic tests can be developed for neurological disorders. This investigation also emphasizes the replacement of invasive blood collection for serum by use of saliva and urine for clinical diagnostic use in general.

Characterization of monoclonal antibodies against Naja naja oxiana neurotoxin I.

Seven monoclonal antibodies (mAbs) were developed against neurotoxin I (NT-1), a protein from central Asian cobra (Naja naja oxiana) venom which binds specifically to nicotinic acetylcholine receptor (AchR). All of the mAbs cross-reacted with another long-chain post-synaptic neurotoxin, Bungarus multicinctus alpha-bungarotoxin (alpha-BT), but not Naja naja kaouthia alpha-cobratoxin, in an enzyme-linked immunosorbent assay (e.l.i.s.a.). Short-chain post-synaptic neurotoxins like Naja naja atra cobrotoxin, Laticauda semifasciata erabutoxin b, or N. n. oxiana neurotoxin II did not cross-react with the NT-1 mAbs, but an antigen(s) found in Dendroaspis polylepis, Acanthophis antarcticus and Pseudechis australis venoms was immunoreactive. The e.l.i.s.a. readings for dithiothreitol-reduced NT-1 and NT-1 mAbs ranged from 13 to 27% of those for native toxin but reduced alpha-BT was not immunoreactive. Synthetic NT-1 peptides were used in epitope-mapping studies and two, non-contiguous regions (Cys15-Tyr23 and Lys25-Gly33 or Pro17-Lys25 and Asp29-Lys37) were recognized by the NT-1 mAbs. The NT-1 mAbs individually inhibited 31-71% of alpha-BT binding to AchR in vitro and afforded a slight protective effect in vivo with a toxin: antibody mole ratio of 1:1.5. This report is the first to describe mAbs which recognize and protect against a heterologous, long-chain, post-synaptic neurotoxin from snake venom.

Boc-Cys(Npys)-OH (BCNP): an appropriate reagent for the identification of T cell epitopes in cystine and/or cysteine-containing proteins.

Some T cell epitopes become inactive when their thiols are blocked with various irreversible reagents (Régnier-Vigouroux, 1988; Maillère, 1992; Maillère et al., 1993). Blocking protein and peptide thiols with BCNP (Boc-Cys(Npys)-OH) constitutes a most appropriate strategy when searching for thiol-containing T cell epitopes. Free cysteines can thus be readily transformed into disulphide-like moieties which not only resist undesirable oxidative reactions but which also remain susceptible to reduction by antigen presenting cells, a prerequisite for the activity of thiol-dependent T cell epitopes. We describe the use of this reagent in a study of the intact disulphide-rich protein, toxin alpha from Naja nigricollis, and also two disulphide-containing toxin fragments.

Detection of nicotinic receptor ligands with a light addressable potentiometric sensor.

The nicotinic acetylcholine receptor, purified from Torpedo electric organ, was coupled to a light addressable potentiometric sensor (LAPS) to form a LAPS-receptor biosensor. Receptor-ligand complexes containing biotin and urease were captured on a biotinylated nitrocellulose membrane via a streptavidin bridge and detected with a silicon-based sensor. Competition between biotinylated alpha-bungarotoxin and nonbiotinylated ligands formed the basis of this assay. This biosensor detected both agonists (acetylcholine, carbamylcholine, succinylcholine, suberyldicholine, and nicotine) and competitive antagonists (d-tubocurarine, alpha-bungarotoxin, and alpha-Naja toxin) of the receptor with affinities comparable to those obtained using radioactive ligand binding assays. Consistent with agonist-induced desensitization of the receptor, the LAPS-receptor biosensor reported a time-dependent increase in affinity for the agonist carbamylcholine as expected, but not for the antagonists.

Intercomparison of small biomolecules by gel filtration and small angle scattering.

This paper describes how small angle neutron scattering (SANS) can be used to confirm that gel filtration results are free of dimerization effects. After characterization by analytical gel filtration, concentrated solutions (in heavy water, D2O) of a cobra neurotoxin, a cytotoxin, and a cytotoxin analog are studied by SANS. Small differences in shape are shown to be discernible by means of least-square fits to ellipsoidal models. The parallel axis theorem is then invoked to assess dimerization levels statistically. The results are briefly discussed on the basis of function in relation to structure.

[2-D-NMR study of the conformation characteristics of toxin 3 from Naja naja siamensis venom]. / 2-D-OaMR-issledovanie konformatsionnykh osobennostei toksina 3 iz iada kobry Naja naja siamensis.

The spatial structure of "long" toxin 3 Naja naja siamensis in solution has been studied by methods of two-dimensional (2D) 1H NMR spectroscopy. The individual signal assignments for 67 out of 71 residues and analysis of nuclear Overhauser effects between distinct protons of the molecule allowed the comparison of the toxin 3 conformations at different pH values and temperatures. It was shown that the deprotonated imidazole ring of His22 residue (at pH greater than or equal to 7,5) is surrounded by the side chains of Cys17, Pro18, Val23, Cys24, Cys45, Ala46 and Thr48 residues. On the contrary, the protonated imidazole ring of His22 (at pH less than 4,0) is exposed into solvent. Ionization of His22 is accompanied by a change in the Tyr25 aromatic ring orientation and affects the conformational mobility of the Cys17, His22, Cys45 and Ala47 side chains. The revealed conformational features of toxin 3 in solution are discussed in connection with the differences between "long" and "short" neurotoxins in the kinetics of their binding to acetylcholine receptor.

[1H-NMR study of the Naja naja oxiana neurotoxin II and its spin-labeled derivatives. Conformation of "short" neurotoxins]. / 1H-IaMR-issledovanie neirotoksina II Naja naja oxiana i ego spin-mechenykh proizvodnykh. Konformatsiia "korotkikh" neirotoksinov.

In 1H NMR spectra of neurotoxin II N. n. oxiana the chemical shift pH-dependences in H2O and 2H2O solutions were studied, and also the deuterium exchange rates and chemical shift temperature gradients were measured for the amide protons. The spin probe method was applied to assess the degree of exposure into solvent of the amide and side chain protons. With the purpose of establishing mutual disposition of certain neurotoxin II groupings, nuclear Overhauser effect was studied in the 1H NMR spectra, along with the broadening of proton resonances induced by spin labels selectively attached to epsilon-amino groups of Lys26, Lys27, Lys45 or Lys47. The mobility of these labels was determined from the EPR spectra. The methyl resonances of Val and Leu residues were assigned to a definite position in the amino acid sequence. The following pKa were determined: alpha-NH2 Leu1 (9,2), gamma-COOH Glu2 (3,7), alpha-COOH Asn62 (1,3). The protonation of a carboxyl group(s) in neurotoxin II (alpha-COOH Asn62 seems to be involved) decreases the temperature stability of the neurotoxin II conformation. On the basis of studies on neurotoxin II and some other homologous neurotoxins, the model for the "short" neurotoxin folding in solution was proposed. Comparison of experimental data for the disposition of equivalent groups in homologous neurotoxins and in the X-ray structure of erabutoxin b Laticauda semifasciata revealed that the Val46 side chain in solution might change its orientation by 180 degrees with respect to polypeptide backbone. Binding of spin labeled neurotoxin II derivatives to the acetylcholine receptor was discussed in light of the obtained data.