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.

Venomics of Naja sputatrix, the Javan spitting cobra: A short neurotoxin-driven venom needing improved antivenom neutralization.

The venom proteome of Naja sputatrix (Javan spitting cobra) was elucidated through reverse-phase HPLC, nano-ESI-LCMS/MS and data mining. A total of 97 distinct protein forms belonging to 14 families were identified. The most abundant proteins are the three-finger toxins (3FTXs, 64.22%) and phospholipase A2 (PLA2, 31.24%), followed by nerve growth factors (1.82%), snake venom metalloproteinase (1.33%) and several proteins of lower abundance (<1%) including a variety of venom enzymes. At subproteome, the 3FTx is dominated by cytotoxins (48.08%), while short neurotoxins (7.89%) predominate over the long neurotoxins (0.48%) among other neurotoxins of lesser toxicity (muscarinic toxin-like proteins, 5.51% and weak neurotoxins, 2.26%). The major SNTX, CTX and PLA2 toxins were isolated with intravenous median lethal doses determined as 0.13, 1.06 and 0.50µg/g in mice, respectively. SABU, the Indonesia manufactured homologous tri-specific antivenom could neutralize the CTX and PLA2 fraction with moderate potency (potency=0.14-0.16mg toxin per ml antivenom). The SNTX, however, was very poorly neutralized with a potency level of 0.034mg/ml, indicating SNTX as the main limiting factor in antivenom neutralization. The finding helps elucidate the inferior efficacy of SABU reported in neutralizing N. sputatrix venom, and supports the call for antivenom improvement. BIOLOGICAL SIGNIFICANCE: The Javan spitting cobra, Naja sputatrix is by itself a unique species and should not be confused as the equatorial and the Indochinese spitting cobras. The distinction among the spitting cobras was however unclear prior to the revision of cobra systematics in the mid-90's, and results of some earlier studies are now questionable as to which species was implicated back then. The current study successfully profiled the venom proteome of authenticated N. sputatrix, and showed that the venom is made up of approximately 64% three-finger toxins (including neurotoxins and cytotoxins) and 31% phospholipases A2 by total venom proteins. The findings verified that the paralyzing components in the venom i.e. neurotoxins are predominantly the short-chain subtype (SNTX) far exceeding the long-chain subtype (LNTX) which is more abundant in the venoms of monocled cobra and Indian common cobra. The neurotoxicity of N. sputatrix venom is hence almost exclusively SNTX-driven, and effective neutralization of the SNTX is the key to early reversal of paralysis. Unfortunately, as shown through a toxin-specific assay, the immunological neutralization of the SNTX using the Indonesian antivenom (SABU) was extremely weak, implying that SABU has limited therapeutic efficacy in treating N. sputatrix envenomation clinically. From the practical standpoint, actions need to be taken at all levels from laboratory to production and policy making to ensure that the shortcoming is overcome.

Identification of Immunoreactive Peptides of Toxins to Simultaneously Assess the Neutralization Potency of Antivenoms against Neurotoxicity and Cytotoxicity of Naja atra Venom.

Assessing the neutralization capability of nonlethal but medically relevant toxins in venom has been a challenging task. Nowadays, neutralization efficacy is evaluated based simply on the survival rates of animals injected with antivenom together with a predefined dose of venom, which can determine potency against neurotoxicity but not validate the capability to neutralize cytotoxin-induced complications. In this study, a high correlation with in-vivo and in-vitro neutralization assays was established using the immunoreactive peptides identified from short-chain neurotoxin and cytotoxin A3. These peptides contain conserved residues associated with toxin activities and a competition assay indicated that these peptides could specifically block the antibody binding to toxin and affect the neutralization potency of antivenom. Moreover, the titers of peptide-specific antibody in antivenoms or mouse antisera were determined by enzyme-linked immunosorbent assay (ELISA) simultaneously, and the results indicated that Taiwanese bivalent antivenom (BAV) and Vietnamese snake antivenom-Naja (SAV-Naja) exhibited superior neutralization potency against the lethal effect of short-chain neurotoxin (sNTX) and cytotoxicity of cardiotoxin/cytotoxin (CTX), respectively. Thus, the reported peptide ELISA shows not only its potential for antivenom prequalification use, but also its capability of justifying the cross-neutralization potency of antivenoms against Naja atra venom toxicity.

Geographical venom variations of the Southeast Asian monocled cobra (Naja kaouthia): venom-induced neuromuscular depression and antivenom neutralization.

The Southeast Asian monocled cobras (Naja kaouthia) exhibit geographical variations in their venom proteomes, especially on the composition of neurotoxins. This study compared the neuromuscular depressant activity of the venoms of N. kaouthia from Malaysia (NK-M), Thailand (NK-T) and Vietnam (NK-V), and the neutralization of neurotoxicity by a monospecific antivenom. On chick biventer cervicis nerve-muscle preparation, all venoms abolished the indirect twitches, with NK-T venom being the most potent (shortest t90, time to 90% twitch inhibition), followed by NK-V and NK-M. Acetylcholine and carbachol failed to reverse the blockade, indicating irreversible/pseudo-irreversible post-synaptic neuromuscular blockade. KCl restored the twitches variably (NK-M preparation being the least responsive), consistent with different degree of muscle damage. The findings support that NK-T venom has the most abundant curarimimetic alpha-neurotoxins, while NK-M venom contains more tissue-damaging cytotoxins. Pre-incubation of tissue with N. kaouthia monovalent antivenom (NKMAV) prevented venom-induced twitch depression, with the NK-T preparation needing the largest antivenom dose. NKMAV added after the onset of neuromuscular depression could only halt the inhibitory progression but failed to restore full contraction. The findings highlight the urgency of early antivenom administration to sequester as much circulating neurotoxins as possible, thereby hastening toxin elimination from the circulation. In envenomed mice, NKMAV administered upon the first neurological sign neutralized the neurotoxic effect, with the slowest full recovery noticed in the NK-T group. This is consistent with the high abundance of neurotoxins in the NK-T venom, implying that a larger amount or repeated dosing of NKMAV may be required in NK-T envenomation.

Snake venomics of monocled cobra (Naja kaouthia) and investigation of human IgG response against venom toxins.

The venom proteome of the monocled cobra, Naja kaouthia, from Thailand, was characterized by RP-HPLC, SDS-PAGE, and MALDI-TOF-TOF analyses, yielding 38 different proteins that were either identified or assigned to families. Estimation of relative protein abundances revealed that venom is dominated by three-finger toxins (77.5%; including 24.3% cytotoxins and 53.2% neurotoxins) and phospholipases A2 (13.5%). It also contains lower proportions of components belonging to nerve growth factor, ohanin/vespryn, cysteine-rich secretory protein, C-type lectin/lectin-like, nucleotidase, phosphodiesterase, metalloproteinase, l-amino acid oxidase, cobra venom factor, and cytidyltransferase protein families. Small amounts of three nucleosides were also evidenced: adenosine, guanosine, and inosine. The most relevant lethal components, categorized by means of a 'toxicity score', were α-neurotoxins, followed by cytotoxins/cardiotoxins. IgGs isolated from a person who had repeatedly self-immunized with a variety of snake venoms were immunoprofiled by ELISA against all venom fractions. Stronger responses against larger toxins, but lower against the most critical α-neurotoxins were obtained. As expected, no neutralization potential against N. kaouthia venom was therefore detected. Combined, our results display a high level of venom complexity, unveil the most relevant toxins to be neutralized, and provide prospects of discovering human IgGs with toxin neutralizing abilities through use of phage display screening.

Improved method for the isolation, characterization and examination of neuromuscular and toxic properties of selected polypeptide fractions from the crude venom of the Taiwan cobra Naja naja atra.

An improved chromatographic method was developed to isolate and purify polypeptides and proteins from the crude venom of the Taiwan cobra Naja naja atra. The procedure devised is simple, easy to reproduce, and enables large scale isolation of almost all polypeptides and proteins in this cobra venom. Six pure polypeptide fractions of the venom were isolated and characterized using gel filtration on Sephadex G50 (medium), ion exchange chromatography on SP-Sephadex C25, desalting on Sephadex G25 (fine) and preparative HPLC on a RPC 18 column. The neuromuscular activity of these fractions was tested on the chick biventer cervicis nerve-muscle preparation and their toxicity (LD(50)) was determined after i.v. administration in mice. Their antinociceptive activity was tested in the mouse abdominal test by i.v. application. Two of these polypeptide samples had major physiological effects: one acted as a cardiotoxin causing reversible myocardial contractures with no effect on muscle twitches elicited by nerve stimulation (NS); another was a neurotoxin that blocked muscle contractions in response to NS and exogenously added acetylcholine. The cardiotoxic fraction was identified as CTX I, a well-known cardiotoxin present in this venom, and the neurotoxin was identified as neurotoxin-α with an LD50 in mice of 0.075 mg/kg.

Chromatography, mass spectrometry, and molecular modeling studies on ammodytoxins.

The ammodytoxins (Atxs) are neurotoxic phospholipases which occur in Vipera ammodytes ammodytes (Vaa) snake venom. There are three Atx isoforms, A, B, and C, which differ in only five amino acid positions at the C-terminus but differ substantially in their toxicity. The objective of this study was to establish an analytical method for unambiguous identification of all three isoforms and to use the method to assess a procedure for purification of the most toxic phospholipase, AtxA, from the venom. Isolation procedure for AtxA consisted of isolation of Atx-cross-reactive material (proteins recognized by anti-Atx antibodies), by use of an affinity column, then cation exchange on CIM (Convective Interaction Media) disks. The purification procedure was monitored by means of reversed-phase chromatography (RPC) and mass spectrometry (MS). Although previous cation exchange of the pure isoforms enabled separate elution of AtxA from B and C, separation of AtxA from Atxs mixture was not accomplished. RPC was not able to separate the Atx isoforms, whereas an MS based approach proved to be more powerful. Peptides resulting from tryptic digestion of Atxs which enable differentiation between the three isoforms were successfully detected and their sequences were confirmed by post-source decay (PSD) fragmentation. Separation of Atx isoforms by ion-exchange chromatography is most presumably prevented by Atxs heterodimer formation. The tendency of Atxs to form homodimers and heterodimers of similar stability was confirmed by molecular modeling.

Inhibition of the nicotinic acetylcholine receptors by cobra venom α-neurotoxins: is there a perspective in lung cancer treatment?

Nicotine exerts its oncogenic effects through the binding to nicotinic acetylcholine receptors (nAChRs) and the activation of downstream pathways that block apoptosis and promote neo-angiogenesis. The nAChRs of the α7 subtype are present on a wide variety of cancer cells and their inhibition by cobra venom neurotoxins has been proposed in several articles and reviews as a potential innovative lung cancer therapy. However, since part of the published results was recently retracted, we believe that the antitumoral activity of cobra venom neurotoxins needs to be independently re-evaluated.We determined the activity of α-neurotoxins from Naja atra (short-chain neurotoxin, α-cobrotoxin) and Naja kaouthia (long-chain neurotoxin, α-cobratoxin) in vitro by cytotoxicity measurements in 5 lung cancer cell lines, by colony formation assay with α7nAChRs expressing and non-expressing cell lines and in vivo by assessing tumor growth in an orthotopic Non-Obese Diabetic/Severe Combined Immunodeficient (NOD/SCID) mouse model system utilizing different treatment schedules and dosages.No statistically significant reduction in tumor growth was observed in the treatment arms in comparison to the control for both toxins. Paradoxically α-cobrotoxin from Naja atra showed the tendency to enhance tumor growth although, even in this case, the statistical significance was not reached.In conclusion our results show that, in contrast with other reports, the nAChR inhibitors α-cobratoxin from N. kaouthia and α-cobrotoxin from N. atra neither suppressed tumor growth nor prolonged the survival of the treated animals.

Virtual screening against alpha-cobratoxin.

alpha-Cobratoxin (Cbtx), the neurotoxin isolated from the venom of the Thai cobra Naja kaouthia , causes paralysis by preventing acetylcholine (ACh) binding to nicotinic acetylcholine receptors (nAChRs). In the current study, the region of the Cbtx molecule that is directly involved in binding to nAChRs is used as the target for anticobratoxin drug design. The crystal structure (1YI5) of Cbtx in complex with the acetylcholine binding protein (AChBP), a soluble homolog of the extracellular binding domain of nAChRs, was selected to prepare an alpha-cobratoxin active binding site for docking. The amino acid residues (Ser182-Tyr192) of the AChBP structure, the binding site of Cbtx, were used as the positive control to validate the prepared Cbtx active binding site (root mean square deviation < 1.2 A). Virtual screening of the National Cancer Institute diversity set, a library of 1990 compounds with nonredundant pharmacophore profiles, using AutoDock against the Cbtx active site, revealed 39 potential inhibitor candidates. The adapted in vitro radioligand competition assays using [(3)H]epibatidine and [(125)I]bungarotoxin against the AChBPs from the marine species, Aplysia californica (Ac), and from the freshwater snails, Lymnaea stagnalis (Ls) and Bolinus truncates (Bt), revealed 4 compounds from the list of inhibitor candidates that had micromolar to nanomolar interferences for the toxin binding to AChBPs. Three hits (NSC42258, NSC121865, and NSC134754) can prolong the survival time of the mice if administered 30 min before injection with Cbtx, but only NSC121865 and NSC134754 can prolong the survival time if injected immediately after injection with Cbtx. These inhibitors serve as novel templates/scaffolds for the development of more potent and specific anticobratoxin.

Presence of alpha7 nicotinic acetylcholine receptors on dorsal root ganglion neurons proved using knockout mice and selective alpha-neurotoxins in histochemistry.

In complex tissues where multiple subtypes of nicotinic acetylcholine receptors (nAChRs) are expressed, immunohistochemistry has been the most popular tool for investigation of nAChR subunit distribution. However, recent studies with nAChR subunit knockout mice demonstrated that a large panel of antibodies is unsuitable. Thus, we aimed to develop a histochemical method for selective labeling of alpha7 nAChR with neurotoxins, utilizing alpha7 nAChR-transfected cells, dorsal root ganglia (DRG) and spinal cord from wild-type and knockout mouse. The specificity of Alexa Fluor 488-conjugated alpha-bungarotoxin (Alexa-alphaBgt) was demonstrated in binding to alpha7-transfected cells inhibited by long-chain alpha-cobratoxin (CTX), but not short-chain alpha-neurotoxin II (NTII). In contrast, binding to Torpedo muscle-type nAChRs and to motor end plates in mouse tongue sections was prevented by both CTX and NTII. In tissue sections of DRG, expressing all neuronal nAChR subunits, only CTX precluded Alexa-alphaBgt labeling of neurons, with no staining for alpha7 nAChR knockout tissue. It proved that alpha7 nAChRs are the major alphaBgt-binding sites in mouse DRG. Corresponding results were obtained for terminals in the spinal cord. Thus, we present a protocol utilizing Alexa-alphaBgt and non-labeled CTX/NTII that allows specific histochemical detection of alpha7 nAChR with a spatial resolution at the level of single axon terminals.

INN-toxin, a highly lethal peptide from the venom of Indian cobra (Naja naja) venom-Isolation, characterization and pharmacological actions.

A novel toxic polypeptide, INN-toxin, is purified from the venom of Naja naja using combination of gel-permeation and ion-exchange chromatography. It has a molecular mass of 6951.6Da as determined by MALDI-TOF/MS and the N-terminal sequence of LKXNKLVPLF. It showed both neurotoxic as well as cytotoxic activities. INN-toxin is lethal to mice with a LD(50) of 1.2mg/kg body weight. IgY raised in chicks against basic peptide pool neutralized the toxicity of INN-toxin. INN-toxin did not inhibit cholinesterase activity. It is toxic to Ehrlich ascites tumor (EAT) cells, but it is not toxic to leukocyte culture. The toxin appears to be specific in its mode of action. Interaction of N-bromosuccinamide (NBS) with the peptide resulted in the modification of tryptophan residues and loss of lethal toxicity of INN-toxin.

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.

Behavioural effects in mice and intoxication symptomatology of weak neurotoxin from cobra Naja kaouthia.

Weak neurotoxins belong to the superfamily of three-finger toxins from snake venoms. In general, weak toxins have a low toxicity and, contrary to other three-finger toxins, their molecular targets are not well characterized: in vitro tests indicate that these may be nicotinic acetylcholine receptors. Here, we report the influence of intraperitoneal and intravenous injections of weak neurotoxin from Naja kaouthia venom on mouse behaviour. Dose-dependent suppression of orientation-exploration and locomotion activities as well as relatively weak neurotropic effects of weak neurotoxin were observed. The myorelaxation effect suggests a weak antagonistic activity against muscle-type nicotinic acetylcholine receptors. Neurotoxic effects of weak neurotoxin were related to its influence on peripheral nervous system. The symptomatology of the intoxication was shown to resemble that of muscarinic agonists. Our data suggest that, in addition to interaction with nicotinic acetylcholine receptors observed earlier in vitro, weak neurotoxin interacts in vivo with some other molecular targets. The results of behavioural experiments are in accord with the pharmacological profile of weak neurotoxin effects on haemodynamics in mice and rat indicating the involvement of both nicotinic and muscarinic acetylcholine receptors.

Effect of nicotine on neuromuscular transmission in mouse motor synapses.

Nicotine (10 nM) inhibits rhythmic activity of the neuromuscular synapse in mice. This effect was prevented by alpha-cobratoxin and apamin. Hence, the effects of nicotine are realized via presynaptic neuronal nicotinic cholinoceptors and Ca(2+)-activated potassium channels.

NMR-based binding screen and structural analysis of the complex formed between alpha-cobratoxin and an 18-mer cognate peptide derived from the alpha 1 subunit of the nicotinic acetylcholine receptor from Torpedo californica.

The alpha18-mer peptide, spanning residues 181-198 of the Torpedo nicotinic acetylcholine receptor alpha1 subunit, contains key binding determinants for agonists and competitive antagonists. To investigate whether the alpha18-mer can bind other alpha-neurotoxins besides alpha-bungarotoxin, we designed a two-dimensional (1)H-(15)N heteronuclear single quantum correlation experiment to screen four related neurotoxins for their binding ability to the peptide. Of the four toxins tested (erabutoxin a, erabutoxin b, LSIII, and alpha-cobratoxin), only alpha-cobratoxin binds the alpha18-mer to form a 1:1 complex. The NMR solution structure of the alpha-cobratoxin.alpha18-mer complex was determined with a backbone root mean square deviation of 1.46 A. In the structure, alpha-cobratoxin contacts the alpha18-mer at the tips of loop I and II and through C-terminal cationic residues. The contact zone derived from the intermolecular nuclear Overhauser effects is in agreement with recent biochemical data. Furthermore, the structural models support the involvement of cation-pi interactions in stabilizing the complex. In addition, the binding screen results suggest that C-terminal cationic residues of alpha-bungarotoxin and alpha-cobratoxin contribute significantly to binding of the alpha18-mer. Finally, we present a structural model for nicotinic acetylcholine receptor-alpha-cobratoxin interaction by superimposing the alpha-cobratoxin.alpha18-mer complex onto the crystal structure of the acetylcholine-binding protein (Protein Data Bank code ).

A novel short neurotoxin, cobrotoxin c, from monocellate cobra (Naja kaouthia) venom: isolation and purification, primary and secondary structure determination, and tertiary structure modeling.

A novel short neurotoxin, cobrotoxin c (CBT C) was isolated from the venom of monocellate cobra (Naja kaouthia) using a combination of ion-exchange chromatography and FPLC. Its primary structure was determined by Edman degradation. CBT C is composed of 61 amino acid residues. It differs from cobrotoxin b (CBT B) by only two amino acid substitutions, Thr/Ala11 and Arg/Thr56, which are not located on the functionally important regions by sequence similarity. However, the LD50 is 0.08 mg/g to mice, i.e. approximately five-fold higher than for CBT B. Strikingly, a structure-function relationship analysis suggests the existence of a functionally important domain on the outside of Loop III of CBT C. The functionally important basic residues on the outside of Loop III might have a pairwise interaction with alpha subunit, instead of gamma or delta subunits of the nicotinic acetylcholine receptor (nAChR).

A unique approach for high level expression and production of a recombinant cobra neurotoxin in Escherichia coli.

In this report, we describe a simple approach to produce a large quantity of a recombinant cobra neurotoxin containing four pairs of disulfide bonds. A cDNA encoding the toxin was fused, in frame, to the carboxyl termini of thioredoxin via a linker sequence encoding two amino acids, Asp and Pro. Due to the presence of thioredoxin, a soluble form of the fusion protein was expressed in a compartment, sensitive to osmotic pressure, in Escherichia coli. The fusion protein was released into the solution with low ionic strength under an osmotic shock treatment, and purified in a single step using an ion exchange chromatography column. The purified protein was treated in diluted hydrochloric acid to induce hydrolysis of the protein at the Asp-Pro linker site. Then, the recombinant neurotoxin was purified by gel filtration of the acid-treated sample. When the biological activity of the purified toxin was assayed, it was as potent as the natural toxin. Using this protocol, approximately 12 mg of pure recombinant neurotoxin can be produced from one liter of bacterial culture. More importantly, this protocol can be easily used for the production of the toxin at a larger scale with low cost. The approach outlined in this report will be suitable for the production of other recombinant proteins especially those of the 'three-finger' family.

Production of polyclonal antibodies in mice against cobratoxin, botulinum toxin and ricin without altering their toxicity or use of adjuvant.

Purified venom components, botulinum toxin and ricin have been successfully used as immunogenes, after converting to toxoids and using adjuvant for production of polyclonal antibodies in animals. This communication reports that polyclonal antibodies specific to cobratoxin, botulinum toxin and ricin were generated in Balb/C mice. The toxins were used for immunization without adjuvant and without altering their toxicity or converting them to toxoids. Initially, lethal dose for botulinum toxin, cobratoxin and ricin were determined in mice and found to be 1 microg, 4 microg and 2 microg, respectively. For the production of antibodies mice were injected with half lethal dose of the toxins in natural form four times, two weeks apart. The potency of antitoxins was assayed by enzyme-linked immunosorbent assay. High titer antibodies were generated by botulinum toxin, cobratoxin and ricin after three injections consisting of half mouse lethal dose. Such minute amounts of botulinum toxin, cobratoxin and ricin in their natural form were able to produce high titer antibodies, perhaps because these toxins may fall in the category of super-antigens.

Expression and mutagenesis studies of cobrotoxin from Taiwan cobra.

The cDNA encoding cobrotoxin was constructed from the cellular RNA isolated from the venom glands of Naja naja atra (Taiwan cobra). The cDNA was subcloned into the expression vector pET20b(+) and transformed into BL21(DE3) Escherichia coli strain. Expressed cobrotoxin was isolated from inclusion bodies of E. coli and subjected to refolding into its folded structure. The refolded cobrotoxin was purified by high-performance liquid chromatography and exhibited a neurotoxicity in inhibiting acetylcholine-induced muscle contractions. Recombinant cobrotoxin showed a tendency to isomerize its disulfide bonds as that observed with native cobrotoxin. An appreciable decrease in the rate of isomerization reaction was observed when Glu-38 was replaced with Gln-38 or Lys-47 was replaced with Glu-47 or Gln-47. These results reflect that the element in controlling the disulfide isomerization of cobrotoxin is closely associated with the charged side chains in the cobrotoxin molecule.

Functional architectures of animal toxins: a clue to drug design?

Toxic proteins are produced by a diversity of venomous animals from various phyla. They are often of small size, possess a large density of disulfide bonds and exert multiple functions directed toward a variety of molecular targets, including a diversity of enzymes and ion channels. The aim of this brief and non-exhaustive review is three-fold. First, the structural context associated with the functional diversity of animal toxins is presented. Among various situations, it is shown that toxins with a similar fold can exert different functions and that toxins with unrelated folds can exert similar functions. Second, the functional sites of some animal toxins are presented. Their comparison shed light on how (i) distinct functions can be exerted by similarly folded toxins and (ii) similar functions can be shared by structurally distinct toxins. Third, it is shown that part of the functional site of foreign proteins can be grafted on an animal toxin scaffold, opening new perspectives in the domain of protein engineering.