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Three-finger toxins (3FTxs) have traditionally been obtained via venom fractionation of whole venoms from snakes. This method often yields functional toxins, but it can be difficult to obtain pure isoforms, as it is challenging to separate the many different toxins with similar physicochemical properties that generally exist in many venoms. This issue can be circumvented via the use of recombinant expression. However, achieving the correct disulfide bond formation in recombinant toxins is challenging and requires extensive optimization of expression and purification methods to enhance stability and functionality. In this study, we investigated the expression of α-cobratoxin, a well-characterized 3FTx from the monocled cobra (Naja kaouthia), in three different expression systems, namely Escherichia coli BL21 (DE3) cells with the csCyDisCo plasmid, Escherichia coli SHuffle cells, and Komagataella phaffii (formerly known as Pichia pastoris). While none of the tested systems yielded α-cobratoxin identical to the variant isolated from whole venom, the His 6 -tagged α-cobratoxin expressed in K. phaffii exhibited a comparable secondary structure according to circular dichroism spectra and similar binding properties to the α7 subunit of the nicotinic acetylcholine receptor. The findings presented here illustrate the advantages and limitations of the different expression systems and can help guide researchers who wish to express 3FTxs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Naja atra cobrotoxin and cardiotoxin 3 (CTX3) exhibit neurotoxicity and cytotoxicity, respectively. In the present study, we aimed to investigate whether the carboxyl groups of cobrotoxin play a role in structural constraints, thereby preventing cobrotoxin from exhibiting cytotoxic activity. Six of the seven carboxyl groups in cobrotoxin were conjugated with semicarbazide. Measurement of circular dichroism spectra and Trp fluorescence quenching showed that the gross conformation of semicarbazide-modified cobrotoxin (SEM-cobrotoxin) and cobrotoxin differed. In sharp contrast to cobrotoxin, SEM-cobrotoxin demonstrated membrane-damaging activity and cytotoxicity, which are feature more characteristic of CTX3. Furthermore, both SEM-cobrotoxin and CTX3 induced cell death through AMPK activation. Analyses of the interaction between polydiacetylene/lipid vesicles and fluorescence-labeled lipids revealed that SEM-cobrotoxin and cobrotoxin adopted different membrane-bound states. The structural characteristics of SEM-cobrotoxin were similar to those of CTX3, including trifluoroethanol (TFE)-induced structural transformation and membrane binding-induced conformational change. Conversely, cobrotoxin was insensitive to the TFE-induced effect. Collectively, the data of this study indicate that blocking negatively charged residues confers cobrotoxin with membrane-damaging activity and cytotoxicity. The findings also suggest that the structural constraints imposed by carboxyl groups control the functional properties of snake venom α-neurotoxins during the divergent evolution of snake venom neurotoxins and cardiotoxins., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Venomous snakebites cause >100 000 deaths every year, in many cases via potent depression of human neuromuscular signaling by snake α-neurotoxins. Emergency therapy still relies on antibody-based antivenom, hampered by poor access, frequent adverse reactions, and cumbersome production/purification. Combining high-throughput discovery and subsequent structure-function characterization, we present simple peptides that bind α-cobratoxin (α-Cbtx) and prevent its inhibition of nicotinic acetylcholine receptors (nAChRs) as a lead for the development of alternative antivenoms. Candidate peptides were identified by phage display and deep sequencing, and hits were characterized by electrophysiological recordings, leading to an 8-mer peptide that prevented α-Cbtx inhibition of nAChRs. We also solved the peptide:α-Cbtx cocrystal structure, revealing that the peptide, although of unique primary sequence, binds to α-Cbtx by mimicking structural features of the nAChR binding pocket. This demonstrates the potential of small peptides to neutralize lethal snake toxins in vitro, establishing a potential route to simple, synthetic, low-cost antivenoms.

Snake venom α-neurotoxins, invaluable pharmacological tools, bind with high affinity to distinct subtypes of nicotinic acetylcholine receptor. The combinatorial high-affinity peptide (HAP), homologous to the C-loop of α1 and α7 nAChR subunits, binds biotinylated α-bungarotoxin (αBgt) with nanomolar affinity and might be a protection against snake-bites. Since there are no data on HAP interaction with other toxins, we checked its binding of α-cobratoxin (αCtx), similar to αBgt in action on nAChRs. Using radioiodinated αBgt, we confirmed a high affinity of HAP for αBgt, the complex formation is supported by mass spectrometry and gel chromatography, but only weak binding was registered with αCtx. A combination of protein intrinsic fluorescence measurements with the principal component analysis of the spectra allowed us to measure the HAP-αBgt binding constant directly (29 nM). These methods also confirmed weak HAP interaction with αCtx (>10000 nM). We attempted to enhance it by modification of HAP structure relying on the known structures of α-neurotoxins with various targets and applying molecular dynamics. A series of HAP analogues have been synthesized, HAP[L9E] analogue being considerably more potent than HAP in αCtx binding (7000 nM). The proposed combination of experimental and computational approaches appears promising for analysis of various peptide-protein interactions.

Naja spp. venom is a natural source of active compounds with therapeutic application potential. Phospholipase A 2 (PLA 2 ) is abundant in the venom of Naja spp. and can perform neurotoxicity, cytotoxicity, cardiotoxicity, and hematological disorders. The PLA 2 s from Naja spp. venoms are Asp 49 isoenzymes with the exception of PLA 2 Cys 49 from Naja sagittifera. When looking at the functional aspects, the neurotoxicity occurs by PLA 2 called β-toxins that have affinity for phosphatidylcholine in nerve endings and synaptosomes membranes, and by α-toxins that block the nicotinic acetylcholine receptors in the neuromuscular junctions. In addition, these neurotoxins may inhibit K + and Ca ++ channels or even interfere with the Na + /K + /ATPase enzyme. The disturbance in the membrane fluidity also results in inhibition of the release of acetylcholine. The PLA 2 can act as anticoagulants or procoagulant. The cytotoxicity exerted by PLA 2 s result from changes in the cardiomyocyte membranes, triggering cardiac failure and hemolysis. The antibacterial activity, however, is the result of alterations that decrease the stability of the lipid bilayer. Thus, the understanding of the structural and functional aspects of PLA 2 s can contribute to studies on the toxic and therapeutic mechanisms involved in the envenomation by Naja spp. and in the treatment of pathologies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A crucial mechanism to the formation of native, fully functional, 3D structures from local secondary structures is unraveled in this study. Through the introduction of various amino acid substitutions at four canonical β-turns in a three-fingered protein, Toxin α from Naja nigricollis, we found that the release of internal entropy to the external environment through the globally synchronized movements of local substructures plays a crucial role. Throughout the folding process, the folding species were saturated with internal entropy so that intermediates accumulated at the equilibrium state. Their relief from the equilibrium state was accomplished by the formation of a critical disulfide bridge, which could guide the synchronized movement of one of the peripheral secondary structure. This secondary structure collided with a core central structure, which flanked another peripheral secondary structure. This collision displaced the internal thermal fluctuations from the first peripheral structure to the second peripheral structure, where the displaced thermal fluctuations were ultimately released as entropy. Two protein folding processes that acted in succession were identified as the means to establish the flow of thermal fluctuations. The first process was the time-consuming assembly process, where stochastic combinations of colliding, native-like, secondary structures provided candidate structures for the folded protein. The second process was the activation process to establish the global mutual relationships of the native protein in the selected candidate. This activation process was initiated and propagated by a positive feedback process between efficient entropy release and well-packed local structures, which moved in synchronization. The molecular mechanism suggested by this experiment was assessed with a well-defined 3D structure of erabutoxin b because one of the turns that played a critical role in folding was shared with erabutoxin b., Competing Interests: The authors have declared that no competing interests exist.

Cobra neurotoxin (CNT), a peptide isolated from snake venom of Naja naja atra , shows central analgesic effects in our previous research. In order to help CNT pass through blood-brain barrier (BBB) and improve its central analgesic effects, a new kind of CNT nanocapsules were prepared by double emulsification with soybean lecithin and cholesterol as the shell, and pheophorbide as the photosensitizer added to make it photoresponsive. The analgesic effects were evaluated by hot plate test and acetic acid-induced writhing in mice. The CNT nanocapsules had an average particle size of 229.55 nm, zeta potential of -53.00 mV, encapsulation efficiency of 84.81% and drug loading of 2.98%, when the pheophorbide content was 1% of lecithin weight. Pheophorbide was mainly distributed in outer layer of the CNT nanocapsules and increased the release of the CNT nanocapsules after 650 nm illumination. The central analgesic effects were improved after intraperitoneal injection of CNT at 25 and 50 µg·kg -1 under 650 nm irradiation for 30 min in the nasal cavity. Activation of pheophorbide by red light generated reactive oxygen species which opened the nanocapsules and BBB and helped the CNT enter the brain. This research provides a new drug delivery for treatment of central pain., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

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 A 2 (PLA 2 , 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 PLA 2 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 PLA 2 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 A 2 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., (Copyright © 2017. Published by Elsevier B.V.)

Ethnopharmacological Relevance: The Naja atra (Chinese cobra), primarily distributing in the low or medium altitude areas of southern China and Taiwan, was considered as a medicine in traditional Chinese medicine and used to treat pain, inflammation and arthritis., Aim of the Study: To study the anti-inflammatory and anti-arthritic activities of cobrotoxin (CTX), an active component of the venom from Naja atra., Materials and Methods: Adjuvant-induced arthritis (AA) rats were used as the animal model of rheumatoid arthritis. The anti-arthritic effects of CTX were evaluated through the arthritis score, paw edema and histopathology changes of joints. The anti-inflammation effects were assayed by the level of IL-6, TNF-α, IL-1β and the number of inflammatory cells in peripheral blood, as well as the proliferation of fibroblast-like synoviocytes (FLS). The immune level was valued by the proliferation of T cells and the level of CD4 and CD8., Results: CTX alleviated the disease development of AA rats according to the ameliorating arthritis score, paw edema and histopathology character. At the meanwhile, CTX decreased the levels of IL-6, TNF-α, IL-1β and the numbers of inflammatory cells in peripheral blood. CTX also suppressed the abnormal increasing of CD4+ T cells/ CD8+ T cells ratio, and could significantly inhibit T cell proliferation. Consistent with its effects on inhibiting granuloma's formation, CTX inhibited the proliferation of the cultured FLSs. Further studies on inflammatory signaling in FLSs revealed that CTX could inhibit the NF-κB signaling pathway., Conclusions: CTX has beneficial effects on rheumatoid arthritis by its immune regulation effects and anti-inflammation effects. The inhibition of NF-κB pathway partly contributes to the anti-inflammatory properties of CTX., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Snake venom three finger toxins (3FTxs) are a non-enzymatic family of venom proteins abundantly found in elapids. We have purified a 7579.5 ± 0.591 Da 3FTx named as Nk-3FTx from the venom of Naja kaouthia of North East India origin. The primary structure was determined by a combination of N-terminal sequencing and electrospray ionization liquid chromatography-mass spectrometry/mass spectrometry. Biochemical and biological characterization reveal that it is nontoxic to human cell lines and exhibit mild anticoagulant activity when tested on citrated human plasma. Nk-3FTx was found to affect the compound action potential (CAP) and nerve conduction velocity of isolated toad sciatic nerve. This is the first report of a non-conventional 3FTx from Naja kaouthia venom that reduces CAP for its neurotoxic effect. Further studies can be carried out to understand the mechanism of action and to explore its potential therapeutic application., (© 2015 Wiley Periodicals, Inc.)

Ionotropic receptors of γ-aminobutyric acid (GABAAR) regulate neuronal inhibition and are targeted by benzodiazepines and general anesthetics. We show that a fluorescent derivative of α-cobratoxin (α-Ctx), belonging to the family of three-finger toxins from snake venoms, specifically stained the α1β3γ2 receptor; and at 10 μm α-Ctx completely blocked GABA-induced currents in this receptor expressed in Xenopus oocytes (IC50 = 236 nm) and less potently inhibited α1β2γ2 ≈ α2β2γ2 > α5β2γ2 > α2β3γ2 and α1β3δ GABAARs. The α1β3γ2 receptor was also inhibited by some other three-finger toxins, long α-neurotoxin Ls III and nonconventional toxin WTX. α-Conotoxin ImI displayed inhibitory activity as well. Electrophysiology experiments showed mixed competitive and noncompetitive α-Ctx action. Fluorescent α-Ctx, however, could be displaced by muscimol indicating that most of the α-Ctx-binding sites overlap with the orthosteric sites at the β/α subunit interface. Modeling and molecular dynamic studies indicated that α-Ctx or α-bungarotoxin seem to interact with GABAAR in a way similar to their interaction with the acetylcholine-binding protein or the ligand-binding domain of nicotinic receptors. This was supported by mutagenesis studies and experiments with α-conotoxin ImI and a chimeric Naja oxiana α-neurotoxin indicating that the major role in α-Ctx binding to GABAAR is played by the tip of its central loop II accommodating under loop C of the receptors., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Previous studies showed that venoms of the monocled cobra, Naja kaouthia from Thailand and Malaysia are substantially different in their median lethal doses. The intraspecific venom variations of N. kaouthia, however, have not been fully elucidated. Here we investigated the venom proteomes of N. kaouthia from Malaysia (NK-M), Thailand (NK-T) and Vietnam (NK-V) through reverse-phase HPLC, SDS-PAGE and tandem mass spectrometry. The venom proteins comprise 13 toxin families, with three-finger toxins being the most abundant (63-77%) and the most varied (11-18 isoforms) among the three populations. NK-T has the highest content of neurotoxins (50%, predominantly long neurotoxins), followed by NK-V (29%, predominantly weak neurotoxins and some short neurotoxins), while NK-M has the least (18%, some weak neurotoxins but less short and long neurotoxins). On the other hand, cytotoxins constitute the main bulk of toxins in NK-M and NK-V venoms (up to 45% each), but less in NK-T venom (27%). The three venoms show different lethal potencies that generally reflect the proteomic findings. Despite the proteomic variations, the use of Thai monovalent and Neuro polyvalent antivenoms for N. kaouthia envenomation in the three regions is appropriate as the different venoms were neutralized by the antivenoms albeit at different degrees of effectiveness., Biological Significance: Biogeographical variations were observed in the venom proteome of monocled cobra (Naja kaouthia) from Malaysia, Thailand and Vietnam. The Thai N. kaouthia venom is particularly rich in long neurotoxins, while the Malaysian and Vietnamese specimens were predominated with cytotoxins. The differentially expressed toxin profile accounts for the discrepancy in the lethal dose of the venom from different populations. Commercially available Thai antivenoms (monovalent and polyvalent) were able to neutralize the three venoms at different effective doses, hence supporting their uses in the three regions. While dose adjustment according to geographical region seems possible, changes to standard recommended dosage should only be made if further study validates that the monocled cobras within a population do not exhibit remarkable inter-individual venom variation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Resveratrol polymer has better effects than monomer in some aspects as reported, but most of synthetic methods acquire severe conditions and no analgesic effects are investigated. A novel method is found to synthesize resveratrol polymer by excitation of photosensitizer pheophorbide at red light of 630 nm. The polymer was analyzed by fluorescence spectra and HPLC, further isolated by preparative liquid chromatography and identified as a resveratrol dimer by MS and NMR. Analgesic effects were measured by acetic acid writhing and hot-plate test in mice. The resveratrol dimer has the stronger analgesic effects than monomer, and drug combination of the dimer and cobra neurotoxin enhances and prolongs analgesic effects, suggesting the synergistic action. Simulation of molecular interaction reveals that the dimer spontaneously binds to cobra neurotoxin and makes a complex substance. The dimer can interact with cyclooxygenase-2, μ receptor and nicotine receptor, the synergistic analgesic effects of the complex are attributed to its multiple targets role. The combination of resveratrol dimer and cobra neurotoxin may make up for their deficiencies in analgesic effects, and has prospects in clinical use., (© 2014 The American Society of Photobiology.)

Cobra neurotoxin (NT) has central analgesic effects, but it is difficult to pass through brain blood barrier (BBB). A novel method of red light induction is designed to help NT across BBB, which is based on photosensitizer activation by red light to generate reactive oxygen species (ROS) to open BBB. The effects were evaluated on cell models and animals in vivo with illumination by semiconductor laser at 670nm on photosensitizer pheophorbide isolated from silkworm excrement. Brain microvascular endothelial cells and astrocytes were co-cultured to build up BBB cell model. The radioactivity of (125)I-NT was measured in cells and tissues for NT permeation. Three ways of cranial irradiation, nasal cavity and intravascular irradiation were tested with combined injection of (125)I-NT 20μg/kg and pheophorbide 100μg/kg to rats, and organs of rats were separated and determined the radioactivity. Paw pressure test in rats, hot plate and writhing test in mice were applied to appraise the analgesic effects. NT across BBB cell model increased with time of illumination, and reached stable level after 60min. So did ROS in cells. NT mainly distributed in liver and kidney of rats, significantly increased in brain after illumination, and improved analgesic effects. Excitation of pheophorbide at red light produces ROS to open BBB, help NT enter brain, and enhance its central action. This research provides a new method for drug across BBB to improve its central role., (Copyright © 2014 Elsevier B.V. All rights reserved.)

To identify high-affinity interactions between long-chain α-neurotoxins and nicotinic receptors, we determined the crystal structure of the complex between α-btx (α-bungarotoxin) and a pentameric ligand-binding domain constructed from the human α7 AChR (acetylcholine receptor) and AChBP (acetylcholine-binding protein). The complex buries ~2000 Ų (1 Å=0.1 nm) of surface area, within which Arg³⁶ and Phe³² from finger II of α-btx form a π-cation stack that aligns edge-to-face with the conserved Tyr¹⁸⁴ from loop-C of α7, while Asp³⁰ of α-btx forms a hydrogen bond with the hydroxy group of Tyr¹⁸⁴. These inter-residue interactions diverge from those in a 4.2 Å structure of α-ctx (α-cobratoxin) bound to AChBP, but are similar to those in a 1.94 Å structure of α-btx bound to the monomeric α1 extracellular domain, although compared with the monomer-bound complex, the α-btx backbone exhibits a large shift relative to the protein surface. Mutational analyses show that replacing Tyr¹⁸⁴ with a threonine residue abolishes high-affinity α-btx binding, whereas replacing with a phenylalanine residue maintains high affinity. Comparison of the α-btx complex with that coupled to the agonist epibatidine reveals structural rearrangements within the binding pocket and throughout each subunit. The overall findings highlight structural principles by which α-neurotoxins interact with nicotinic receptors.

Cobra venom (Naja kaouthia) contains a toxin called α-cobratoxin (α-Cbtx). This toxin is a natural protein containing 71 amino acids (MW 7821 Da) with a reported analgesic potency greater than morphine. In 2007, in USA, this substance was found in the barns of a thoroughbred trainer and since then till date, the lack of a detection of this molecule has remained a recurring problem for the horseracing industry worldwide. To solve this problem, the first method for the detection of α-cobratoxin in equine plasma has now been developed. Plasma sample (3 mL) was treated with ammonium sulfate at the isoelectric point of α-Cbtx, and the pellet was dissolved in a phosphate buffer and mixed with methanol for precipitation. The supernatant was then concentrated prior to its extraction on WCX SPE cartridges. The eluate was concentrated with two consecutive filtration steps before the trypsin digestion. The samples were analyzed using a LC-MS/MS Q Exactive instrument at 70,000 resolution on the product ions of the doubly charged precursor of the target peptide ((24)TWCDAFCSIR(33)). The method was validated (n = 18) at 5 μg/L (640 pmol/L) according to the Association of Official Racing Chemists (AORC) requirements. The lower limit of detection was 1 μg/L (130 pmol/L). The present method has made it possible for us to confirm the presence of α-Cbtx in a horse plasma sample 24 h post the administration of α-Cbtx. Thus, the present method provides the first sensitive, specific, and reliable analytical method to confirm the presence of α-Cbtx in equine plasma.

Background: Neurotoxin-Nna (NT), an analgesic peptide separated from the venom of Naja naja atra, has reported to have an exceptional specificity to block transmission of the nerve impulse by binding to the α- subunit of the nicotinic acetylcholine receptor in the membrane. However, little information is available on the anti-inflammatory effects of NT. Therefore, the anti-inflammatory activity of Neurotoxin-Nna was investigated in this study., Methods: The anti-inflammatory effects of NT were evaluated by measuring its influence on several crucial factors in inflammatory pathways, including total antioxidant activity, antinociceptive effects in vivo, nuclear factor kappa B (NF-κB), polymorphonuclear cells (PMN), inducible nitric oxide synthase (iNOS), adhesion molecule (ICAM-1) and tactile hyperalgesia., Results: NT treatment decreased the levels of tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β). NT treatment decreased the total antioxidant status (TAOS) and reduced CFA-induced tactile hyperalgesia in a dose-dependent manner. NT significantly inhibited regulation of NF-kappaB activation and the production of IL-1β, TNF-α, iNOS and CAM-1. Moreover, NT suppressed infiltration of PMN., Conclusions: Our results showed that NT reduced CFA-induced tactile hyperalgesia through inhibition inflammatory pathways in experimental inflammatory rats.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

In Naja kaouthia cobra venom, we have earlier discovered a covalent dimeric form of α-cobratoxin (αCT-αCT) with two intermolecular disulfides, but we could not determine their positions. Here, we report the αCT-αCT crystal structure at 1.94 Å where intermolecular disulfides are identified between Cys(3) in one protomer and Cys(20) of the second, and vice versa. All remaining intramolecular disulfides, including the additional bridge between Cys(26) and Cys(30) in the central loops II, have the same positions as in monomeric α-cobratoxin. The three-finger fold is essentially preserved in each protomer, but the arrangement of the αCT-αCT dimer differs from those of noncovalent crystallographic dimers of three-finger toxins (TFT) or from the κ-bungarotoxin solution structure. Selective reduction of Cys(26)-Cys(30) in one protomer does not affect the activity against the α7 nicotinic acetylcholine receptor (nAChR), whereas its reduction in both protomers almost prevents α7 nAChR recognition. On the contrary, reduction of one or both Cys(26)-Cys(30) disulfides in αCT-αCT considerably potentiates inhibition of the α3β2 nAChR by the toxin. The heteromeric dimer of α-cobratoxin and cytotoxin has an activity similar to that of αCT-αCT against the α7 nAChR and is more active against α3β2 nAChRs. Our results demonstrate that at least one Cys(26)-Cys(30) disulfide in covalent TFT dimers, similar to the monomeric TFTs, is essential for their recognition by α7 nAChR, although it is less important for interaction of covalent TFT dimers with the α3β2 nAChR.

Based on the crystal structure of the extracellular domain (ECD) of the mouse nicotinic acetylcholine receptor (nAChR) alpha1 subunit bound to α-bungarotoxin (α-Btx) we have generated in silico models of the human nAChR α1 bound to α-Btx and α-cobratoxin (α-Cbtx), both in the presence and in the absence of the N-linked carbohydrate chain. To gain further insight into the structural role of glycosylation molecular dynamics (MD) simulations were carried out in explicit solvent so as to compare the conformational dynamics of the binding interface between nAChR α1 and the two toxins. An interesting observation during the course of the MD simulations is the strengthening of the receptor-toxin interaction in the presence of the carbohydrate chain, mediated through a shift in the position of the sugars towards the bound toxin. Critical protein-sugar interactions implicate residues Ser187 and Trp184 of nAChR and Thr6, Ser9, and Thr15 of α-Btx, as well as Thr6 and Pro7 of α-Cbtx. Analysis of the predicted residue-specific intermolecular interactions is intended to inspire biophysical studies on the functional role of glycosylation in the gating mechanism., (© 2010 Wiley-Liss, Inc.)

Snake venoms are a mixture of pharmacologically active proteins and polypeptides that have led to the development of molecular probes and therapeutic agents. Here, we describe the structural and functional characterization of a novel neurotoxin, haditoxin, from the venom of Ophiophagus hannah (King cobra). Haditoxin exhibited novel pharmacology with antagonism toward muscle (alphabetagammadelta) and neuronal (alpha(7), alpha(3)beta(2), and alpha(4)beta(2)) nicotinic acetylcholine receptors (nAChRs) with highest affinity for alpha(7)-nAChRs. The high resolution (1.5 A) crystal structure revealed haditoxin to be a homodimer, like kappa-neurotoxins, which target neuronal alpha(3)beta(2)- and alpha(4)beta(2)-nAChRs. Interestingly however, the monomeric subunits of haditoxin were composed of a three-finger protein fold typical of curaremimetic short-chain alpha-neurotoxins. Biochemical studies confirmed that it existed as a non-covalent dimer species in solution. Its structural similarity to short-chain alpha-neurotoxins and kappa-neurotoxins notwithstanding, haditoxin exhibited unique blockade of alpha(7)-nAChRs (IC(50) 180 nm), which is recognized by neither short-chain alpha-neurotoxins nor kappa-neurotoxins. This is the first report of a dimeric short-chain alpha-neurotoxin interacting with neuronal alpha(7)-nAChRs as well as the first homodimeric three-finger toxin to interact with muscle nAChRs.

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.

Rediocides A and G, the principle components of Trigonostemon reidioides (Kurz) Craib, which is known as Lotthanong in Thai, were investigated for a detoxification mechanism against Naja kaouthia venom by in silico, in vitro, and in vivo methods. Molecular dockings of alpha-cobratoxin with rediocides A and G were performed, and the binding energies were found to be -14.17 and -14.14 kcal/mol, respectively. Rediocides bind to alpha-cobratoxin at the same location as alpha-cobratoxin binds to the nicotinic acetylcholine receptor (nAChR), i.e., at the Asp27, Phe29, Arg33, Gly34, Lys35, and Val37 residues. alpha-Cobratoxin cannot bind to nAChR, because some of its binding sites are occupied with rediocides. From in vitro SDS-PAGE, it was found that rediocides can diminish the bands of alpha-cobratoxin. In the presence of acetylcholine-binding protein (AChBP), it was apparent that rediocides can bind both alpha-cobratoxin and AChBP. From an in vivo test, it was found that injection of rediocides at 0.5 mg/kg immediately after an alpha-cobratoxin dose of three times LD(50) cannot prolong the survival time of mice. However, rediocide can prolong the survival time, if it is injected 30 min before the injection of alpha-cobratoxin. The in vitro SDS-PAGE and the in vivo results support the in silico detoxification mechanism of rediocides against cobra venom at a molecular level.

The contact area of neurotoxin II from Naja naja oxiana when interacting with the membrane-bound nicotinic acetylcholine receptor from Torpedo californica was determined by solid-state, magic-angle spinning NMR spectroscopy. For this purpose, the carbon signals for more than 90% of the residues of the bound neurotoxin were assigned. Differences between the solution and solid-state chemical shifts of the free and bound form of the toxin are confined to distinct surface regions. Loop II of the short toxin was identified as the main interaction site. In addition, loop III of neurotoxin II shows several strong responses defining an additional interaction site. A comparison with the structures of alpha-cobratoxin bound to the acetylcholine-binding protein from snail species Lymnaea stagnalis and Aplysia californica, and of alpha-bungarotoxin bound to an extracellular domain of an alpha-subunit of the receptor reveals different contact areas for long and short alpha-neurotoxins.

A protein with M 7485 Da containing five disulfide bonds was isolated from the venom of cobra Naja oxiana using various types of liquid chromatography. The complete amino acid sequence of the protein was determined by protein chemistry methods, which permitted us to assign it to the group of weak toxins. This is the first weak toxin isolated from the venom of N. oxiana. In a similar way, two new toxins with M 7628 and 7559 Da, which fall into the range of weak toxin masses, were isolated from the venom of the cobra N. kaouthia. The characterization of these proteins using Edman degradation and MALDI mass spectrometry has shown that one of these proteins is a novel weak toxin and the other is the known weak toxin WTX with an oxidized methionine residue in position 9. Such a modification was detected in weak toxins for the first time. A study of the biological activity of the toxin from N. oxiana showed that, like other weak toxins, it can be bound by muscle nicotinic cholinoreceptors and alpha7 nicotinic cholinoreceptors.

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.

Snake venoms have demonstrated antinociceptive activity, and certain isolated neurotoxins have demonstrated significant analgesia in animal models. Here we report a novel analgesic toxin which was isolated from Naja naja atra and was given the name 'najanalgesin'. The LD(50) of the crude venom and najanalgesin were 0.89mg/kg and 2.69mg/kg, respectively. We used the writhing test and hot plate test to evaluate the antinociceptive properties of the crude venom and najanalgesin after intraperitoneal (ip) administration. The analgesic mechanism of najanalgesin was also studied. The response latency time was significantly prolonged in the hot plate test after ip administration of the crude venom of Naja naja atra (0.111-0.445mg/kg) in a dose-dependent manner. Najanalgesin (1mg/kg) elicited almost the same antinociceptive effect as that of the crude venom of Naja naja atra at the dose of 0.445mg/kg and remained for 6h after intraperitoneal injection, shown by hot plate test. The percentage of increase in the latency time for the venom and the najanalgesin 3h after drug administration was 96.2% and 112%, respectively. The number of writhes decreased to almost 1/3, 1/6, and 1/12 of the NS (physiological saline) group after intraperitoneal administration of najanalgesin at 0.25, 0.5, and 1.0mg/kg, respectively. Pretreatment with atropine (1mg/kg) or naloxone (3mg/kg) blocked the antinociception of najanalgesin in the hot plate test. Based on the sequence information, najanalgesin is found to be highly homologous with the conventional CTXs (cardiotoxins). To our knowledge, no study had previously reported that a toxin which was homologous with CTXs possessed the antinociceptive activity. Thus, this is the first report that the antinociceptive effect induced by najanalgesin is mediated by cholinergic and opioidergic mechanisms.

Nicotinic AChRs (nAChRs) represent a paradigm for ligand-gated ion channels. Despite intensive studies over many years, our understanding of the mechanisms of activation and inhibition for nAChRs is still incomplete. Here, we present molecular dynamics (MD) simulations of the alpha7 nAChR ligand-binding domain, both in apo form and in alpha-Cobratoxin-bound form, starting from the respective homology models built on crystal structures of the acetylcholine-binding protein. The toxin-bound form was relatively stable, and its structure was validated by calculating mutational effects on the toxin-binding affinity. However, in the apo form, one subunit spontaneously moved away from the conformation of the other four subunits. This motion resembles what has been proposed for leading to channel opening. At the top, the C loop and the adjacent beta7-beta8 loop swing downward and inward, whereas at the bottom, the F loop and the C terminus of beta10 swing in the opposite direction. These swings appear to tilt the whole subunit clockwise. The resulting changes in solvent accessibility show strong correlation with experimental results by the substituted cysteine accessibility method upon addition of acetylcholine. Our MD simulation results suggest a mechanistic model in which the apo form, although predominantly sampling the "closed" state, can make excursions into the "open" state. The open state has high affinity for agonists, leading to channel activation, whereas the closed state upon distortion has high affinity for antagonists, leading to inhibition.

Walterinnesia aegyptia is a monotypic elapid snake inhabiting in Africa and Mideast. Although its envenoming is known to cause rapid deaths and paralysis, structural data of its venom proteins are rather limited. Using gel filtration and reverse-phase HPLC, phospholipases A(2) (PLAs), three-fingered toxins (3FTxs), and Kunitz-type protease inhibitors (KIns) were purified from the venom of a single specimen of this species caught in northern Egypt. In addition, specific primers were designed and PCR was carried out to amplify the cDNAs encoding members of the three venom families, respectively, using total cDNA prepared from its venom glands. Complete amino acid sequences of two acidic PLAs, three short chain 3FTxs, and four KIns of this venom species were thus deduced after their cDNAs were cloned and sequenced. They are all novel sequences and match the mass data of purified proteins. For members of each toxin family, protein sequences were aligned and subjected to molecular phylogenetic analyses. The results indicated that the PLAs and a Kunitz inhibitor of W. aegyptia are most similar to those of king cobra venom, and its 3FTxs belongs to either Type I alpha-neurotoxins or weak toxins of orphan-II subtype. It is remarkable that both king cobra and W. aegyptia cause rapid deaths of the victims, and a close evolutionary relationship between them is speculated.

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of alpha-cobratoxin (a long-chain alpha-neurotoxin) and heterodimers formed by alpha-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26-30 in polypeptide loop II of alpha-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the alpha-cobratoxin capacity to compete with alpha-bungarotoxin for binding to Torpedo and alpha7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that alpha-cobratoxin dimer not only interacts with alpha7 nAChR but, in contrast to alpha-cobratoxin monomer, also blocks alpha3beta2 nAChR. In the latter activity it resembles kappa-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric alpha3beta2 nAChRs.

We investigate the interactions between the long chain alpha-cobratoxin (Cbtx) and the nicotinic acetylcholine receptor using a rigid body docking procedure. The method, (i) reproduces the binding of Cbtx to Lymnea acetylcholine-binding protein (AChBP); (ii) shows that most of the structures of AChBP obtained in the presence of antagonists are compatible with Cbtx binding; and (iii) reveals a complex between Cbtx and muscle nAChR that corresponds to the basal "resting" state conformation. The structures are made available for further understanding of the allosteric transitions of the nAChR as well as for drug design.

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.

Neuronal nicotinic acetylcholine receptors (nAChRs) containing alpha7 subunit are well represented in the brain and some non-neuronal tissues, and their malfunctioning is associated with diverse pathologies. Therefore, detection and quantification of alpha7 nAChR are important tasks. The affinity-purified antibodies were prepared against the 1-23 and 179-190 fragments of the human and rat alpha7 nAChR extracellular domain. The specificity and selectivity of these alpha7 (1-23) and alpha7 (179-190) antibodies was tested by ELISA in model systems: the E. coli-expressed alpha7 subunit extracellular domain and the pituitary cell line GH(4)C(1) stably expressing human alpha7 nAChR. On the rat brain slices two antibodies and biotinylated alpha-cobratoxin specifically stained the hippocampus region known to be rich in alpha7 nAChR. Western blot analysis revealed that in the human thalamus membranes and in rat brain membranes, antibodies alpha7 (1-23) stained a single band of 62 kDa, while the alpha7 (179-190) antibodies stained a doublet of 53-54 kDa. The results obtained show that utilization of model systems and a combination of several antibodies with appropriately labeled toxins may provide better ways for detection of alpha7 nAChR.

Weak toxins are the "three-fingered" snake venoms toxins grouped together by having an additional disulfide in the N-terminal loop I. In general, weak toxins have low toxicity, and biological targets have been identified for some of them only, recently by detecting the effects on the nicotinic acetylcholine receptors (nAChR). Here the methods of docking and molecular dynamics simulations are used for comparative modeling of the complexes between four weak toxins of known spatial structure (WTX, candoxin, bucandin, gamma-bungarotoxin) and nAChRs. WTX and candoxin are those toxins whose blocking of the neuronal alpha7- and muscle-type nAChR has been earlier shown in binding assays and electrophysiological experiments, while for the other two toxins no such activity has been reported. Only candoxin and WTX are found here to give stable solutions for the toxin-nAChR complexes. These toxins appear to approach the binding site similarly to short alpha-neurotoxins, but their final position resembles that of alpha-cobratoxin, a long alpha-neurotoxin, in the complex with the acetylcholine-binding protein. The final spatial structures of candoxin and WTX complexes with the alpha7 neuronal or muscle-type nAChR are very similar and do not provide immediate answer why candoxin has a much higher affinity than WTX, but both of them share a virtually irreversible mode of binding to one or both these nAChR subtypes. Possible explanation comes from docking and MD simulations which predict fast kinetics of candoxin association with nAChR, no gross changes in the toxin conformation (with smaller toxin flexibility on alpha7 nAChR), while slow WTX binding to nAChR is associated with slow irreversible rearrangement both of the tip of the toxin loop II and of the binding pocket residues locking finally the toxin molecule. Computer modeling showed that the additional disulfide in the loop I is not directly involved in receptor binding of WTX and candoxin, but it stabilizes the structure of loop I which plays an important role in toxin delivery to the binding site. In summary, computer modeling visualized possible modes of binding for those weak toxins which interact with the nAChR, provided no solutions for those weak toxins whose targets are not the nAChRs, and demonstrated that the additional disulfide in loop I cannot be a sound criteria for joining all weak toxins into one group; the conclusion about the diversity of weak toxins made from computer modeling is in accord with the earlier phylogenetic analysis.

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.

Selective reduction on the Cys28-Cys32 disulfide of Ophiophagus hannah neurotoxins, Oh-4 and Oh-5, revealed that isomerization of this disulfide linkage caused the two toxins to have distinct conformation and different retention time on a reversed-phase column. The Cys28-Cys32 disulfide of Oh-4 and Oh-5 was prone to form mixed disulfides with glutathione following pseudo-first-order kinetics. In addition to glutathionylated proteins, Oh-4 could be promoted to convert into Oh-5 by thiol compounds. Isomerization of Oh-5 into Oh-4 was not observed in the presence of thiol compounds. Dethiolation of glutathionylated proteins produced Oh-4 and Oh-5. Oxidation of the partially reduced toxin with reduced Cys28 and Cys32 was exclusively converted into Oh-5 regardless of the absence or presence of GSH/GSSG. Acrylamide quenching studies revealed difference in degree of exposure of the single Trp27 between Oh-4 and Oh-5. Synthesized peptides with substitution of Trp27 or Phe31 with Gly abolished entirely the formation of disulfide-linked dimeric product noted with the peptide of wild-type sequence. These results suggest that disulfide formation and isomerization of Cys28-Cys32 could be regulated by thiolation, and that the bulky aromatic residues Trp27 and Phe31 facilitate favorably the occurrence of disulfide isomerization of Cys28-Cys32.

The refolding kinetics of Cobrotoxin (CBTX), a small all beta-sheet protein is investigated using a variety of biophysical techniques including quenched-flow hydrogen-deuterium (H/D) exchange in conjunction with two-dimensional NMR spectroscopy. Urea-induced equilibrium unfolding of CBTX follows a two-state mechanism with no distinct intermediates. The protein is observed to fold very rapidly within 250 ms. Both the refolding and the unfolding limbs of the chevron plot of CBTX show a prominent curvature suggesting the accumulation of kinetic intermediates. Quenched-flow H/D exchange data suggest the presence of a broad continuum of kinetic intermediates between the unfolded and native states of the protein. Comparison of the native state hydrogen exchange data and the results of the quenched-flow H/D exchange experiments, reveals that the residues constituting the folding core of CBTX are not a subset of the slow exchange core. To our knowledge, this is the first report wherein the refolding of a small all beta-sheet protein is shown to be a multi-step process involving the accumulation of kinetic intermediates.

In this study, alpha-cobrotoxin was incorporated into the microspheres composed of poly(lactide-co-glycolide) (PLGA) and poly[1,3-bis(p-carboxy-phenoxy) propane-co-p-(carboxyethylformamido) benzoic anhydride] (P(CPP:CEFB)) and intranasally delivered to model rats in order to improve its analgesic activity. The microspheres with high entrapment efficiency (>80%) and average diameter of about 25 microm could be prepared by a modified water-in-oil-in-oil (w/o/o) emulsion solvent evaporation method. Scanning electron micrograph (SEM) study indicated that P(CPP:CEFB) content played a considerable role on the morphology and degradation of the microspheres. The presence of P(CPP:CEFB) in the microspheres increased their residence time at the surface of the nasal rat mucosa. The toxicity of the composite microspheres to nasal mucosa was proved to be mild and reversible. A tail flick assay was used to evaluate the antinociceptive activity of the microspheres after nasal administration. Compared with the free alpha-cobrotoxin and PLGA microspheres, PLGA/P(CPP:CEFB) microspheres showed an apparent increase in the strength and duration of the antinociceptive effect at the same dose of alpha-cobrotoxin (80 microg/kg body weight).

Cobrotoxin is known to bind with cysteine residues of biological molecules such as nicotine acetylcholine receptor. Cobrotoxin may modify IKKs and p50 through protein-protein interaction since cysteine residues are present in the kinase domains of IKKalpha and IKKbeta and in the p50 of NF-kappaB. Our surface plasmon resonance analysis showed that cobrotoxin directly binds to p50 (K(d) = 1.54 x 10(-)(5) M), IKKalpha (K(d) = 3.94 x 10(-)(9) M) and IKKbeta (K(d) = 3.4 x 10(-)(8) M) with high binding affinity. Moreover, these protein-protein interactions suppressed the lipopolysaccharide (LPS, 1 microg/mL)- and the sodium nitroprusside (SNP, 200 microM)-induced DNA binding activity of NF-kappaB and NF-kappaB-dependent luciferase activity in astrocytes and Raw 264.7 macrophages. These inhibitory effects were correlated with the inhibition of IkappaB release and p50 translocation. Inhibition of NF-kappaB by cobrotoxin resulted in reductions in the LPS-induced expressions of COX-2, iNOS, cPLA(2), IL-4, and TNF-alpha in astrocytes and in COX-2 expression induced by SNP, LPS, and TNF-alpha in astrocytes. Moreover, these inhibitory effects of cobrotoxin were reversed by adding reducing agents, dithiothreitol and glutathione. In addition, cobrotoxin did not have any inhibitory effect on NF-kappaB activity in cells carrying mutant p50 (C62S), IKKalpha (C178A), and IKKbeta (C179A), with the exception of IKKbeta (K44A) mutant plasmid. Confocal microscopic analysis showed that cobrotoxin is uptaken into the nucleus of cells. These results demonstrate that cobrotoxin directly binds to the sulfhydryl groups of p50 and IKKs, and that this results in reduced IkappaB release and the translocation of p50, thereby inhibiting the activation of NF-kappaB.

The central nervous system of Drosophila melanogaster contains an alpha-bungarotoxin-binding protein with the properties expected of a nicotinic acetylcholine receptor. This protein was purified 5800-fold from membranes prepared from Drosophila heads. The protein was solubilized with 1% Triton X-100 and 0.5 M sodium chloride and then purified using an alpha-cobratoxin column followed by a lentil lectin affinity column. The purified protein had a specific activity of 3.9 micromol of 125I-alpha-bungarotoxin binding sites/g of protein. The subunit composition of the purified receptor was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This subunit profile was identical with that revealed by in situ labeling of the membrane-bound protein using the photolyzable methyl-4-azidobenzoimidate derivative of 125I-alpha-bungarotoxin. The purified receptor reveals two different protein bands with molecular masses of 42 and 57 kDa. From sedimentation analysis of the purified protein complex in H2O and D2O and gel filtration, a mass of 270 kDa was calculated. The receptor has a s(20,w) of 9.4 and a Stoke's radius of 7.4 nm. The frictional coefficient was calculated to be 1.7 indicating a highly asymmetric protein complex compatible with a transmembrane protein forming an ion channel. The sequence of a peptide obtained after tryptic digestion of the 42-kDa protein allowed the specific identification of the Drosophila D alpha5 subunit by sequence comparison. A peptide-specific antibody raised against the D alpha5 subunit provides further evidence that this subunit is a component of an alpha-bungarotoxin binding nicotinic acetylcholine receptor from the central nervous system of Drosophila.

The crystal structure of the snake long alpha-neurotoxin, alpha-cobratoxin, bound to the pentameric acetylcholine-binding protein (AChBP) from Lymnaea stagnalis, was solved from good quality density maps despite a 4.2 A overall resolution. The structure unambiguously reveals the positions and orientations of all five three-fingered toxin molecules inserted at the AChBP subunit interfaces and the conformational changes associated with toxin binding. AChBP loops C and F that border the ligand-binding pocket move markedly from their original positions to wrap around the tips of the toxin first and second fingers and part of its C-terminus, while rearrangements also occur in the toxin fingers. At the interface of the complex, major interactions involve aromatic and aliphatic side chains within the AChBP binding pocket and, at the buried tip of the toxin second finger, conserved Phe and Arg residues that partially mimic a bound agonist molecule. Hence this structure, in revealing a distinctive and unpredicted conformation of the toxin-bound AChBP molecule, provides a lead template resembling a resting state conformation of the nicotinic receptor and for understanding selectivity of curaremimetic alpha-neurotoxins for the various receptor species.

We investigated whether protein stability controls antigen presentation using a four disulfide-containing snake toxin and three derivatives carrying one or two mutations (L1A, L1A/H4Y, and H4Y). These mutations were anticipated to increase (H4Y) or decrease (L1A) the antigen non-covalent stabilizing interactions, H4Y being naturally and frequently observed in neurotoxins. The chemically synthesized derivatives shared similar three-dimensional structure, biological activity, and T epitope pattern. However, they displayed differential thermal unfolding capacities, ranging from 65 to 98 degrees C. Using these differentially stable derivatives, we demonstrated that antigen stability controls antigen proteolysis, antigen processing in antigen-presenting cells, T cell stimulation, and kinetics of expression of T cell determinants. Therefore, non-covalent interactions that control the unfolding capacity of an antigen are key parameters in the efficacy of antigen presentation. By affecting the stabilizing interaction network of proteins, some natural mutations may modulate the subsequent T-cell stimulation and might help microorganisms to escape the immune response.

Cobrotoxin (CBTX) is a highly toxic short neurotoxin, isolated from the Taiwan cobra (Naja naja atra) venom. In the present study for the first time we report the cloning and expression of CBTX in high yields (12mg/L) in Escherichia coli. CBTX fused to the IgG-binding domain of protein A (IgG-CBTX) was expressed in the soluble form. The misfolded CBTX portion (of the overexpressed fusion protein) was refolded under optimal redox conditions. The fusion protein (IgG-CBTX) was observed to undergo auto-catalytic cleavage to yield CBTX with additional 5 amino acids upstream of its N-terminal end. The far UV and near UV circular dichroism spectra of the recombinant CBTX were identical to those of the toxin isolated from the crude venom source. Recombinant CBTX was isotope labeled (15N and 13C) and all the resonances ('H, 13C, and 15N) in the protein have been unambiguously assigned. ' H '5N HSQC spectrum of recombinant CBTX revealed that the protein is in a biologically active conformation. 1H-15Nchemical shift perturbation data showed that recombinant CBTX binds to a peptide derived from the alpha7 subunit of the Torpedo acetylcholine receptor (AchR) with high affinity. The AchR peptide is found to bind to residues located at the tip of Loop-2 in CBTX. The results of the present study provide an avenue to understand the structural basis for the high toxicity exhibited by CBTX. In addition, complete resonance assignments in CBTX (reported in this study) are expected to trigger intensive research towards the design of new pharmacological agents against certain neural disorders.

The sequence of a long-chain neurotoxin (71 amino acid residues, 10 half-cystines) from the venom of the African banded water cobra (Boulengerina annulata) was determined by Edman degradation. It exhibits high sequence similarity with long-chain neurotoxins from the venoms of four species of African cobras (genus Naja), which are collectively more similar to the Boulengerina toxin than to those of Asian Naja species. These results are discussed in the light of phylogenetic hypotheses on the relationships of African cobras.

Solid-state magic-angle spinning nuclear magnetic resonance (NMR) has sufficient resolving power for full assignment of resonances and structure determination of immobilised biological samples as was recently shown for a small microcrystalline protein. In this work, we show that highly resolved spectra may be obtained from a system composed of a receptor-toxin complex. The NMR sample used for our studies consists of a membrane preparation of the nicotinic acetylcholine receptor from the electric organ of Torpedo californica which was incubated with uniformly 13C-,15N-labelled neurotoxin II. Despite the large size of the ligand-receptor complex ( > 290 kDa) and the high lipid content of the sample, we were able to detect and identify residues from the ligand. The comparison with solution NMR data of the free toxin indicates that its overall structure is very similar when bound to the receptor, but significant changes were observed for one isoleucine.

Neurotoxin II from the venom of cobra Naja oxiana is a short type alpha-neurotoxin, which competitively inhibits nicotinic acetylcholine receptor. The toxin gene was expressed as a construct fused with the thioredoxin gene and the linker encoding the enteropeptidase recognition site and a Met residue between the genes. The fusion protein was mainly cleaved by cyanogen bromide, since enteropeptidase was less effective. The yield of neurotoxin II was 6 mg/l of the bacterial culture. The resulting recombinant protein was identified with native neurotoxin II by its N-terminal analysis, mass spectrometry, and NMR spectroscopy. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.

A protein corresponding to the extracellular 1-209 domain of the alpha-subunit of the nicotine acetylcholine receptor from the electric organ of Torpedo californica was prepared using the corresponding cDNA domain by culturing Escherichia coli cells on a synthetic medium supplemented with 5-fluoro-L-tryptophan. The presence of a (His)6 fragment preceding the 1-209 sequence allowed purification of the protein isolated from inclusion bodies by affinity chromatography on Ni-NTA Agarose. The incorporation of 5-fluorotryptophan residues was found by 19F NMR to be approximately 50%. The spectrum of the protein reduced under denaturing conditions and subsequently reoxidized in a dilute solution under denaturing conditions in the presence of 0.05% SDS was sufficiently resolved, which allowed partial assignment of 19F resonances using the Trp60Phe mutant protein. The ability of the prepared domains to specifically bind snake alpha-neurotoxins was demonstrated with the use of radioiodinated alpha-bungarotoxin and trifluoroacetylated alpha-cobratoxin.

A sensitive nonradioactive method for detection of substances interacting with the neuronal nicotinic acetylcholine alpha 7-type receptor (AChR) was proposed. The method uses biotinylated alpha-cobratoxin (Bt-CTX) and is based on the ability of the N-terminal ligand-binding extracellular domain (LBED) of AChR to interact with alpha-cobratoxin (CTX) as does the whole receptor. LBED was produced by heterologic expression of a gene fragment of the alpha 7 subunit of AChR from the rat brain in Escherichia coli cells sorbed on wells of a 96-well plate and incubated with Bt-CTX. The specifically bound Bt-CTX was determined by staining with streptavidin-peroxidase complex. The ability of other compounds to interact with alpha 7-AChR was checked according to the degree with which they inhibit Bt-CTX binding to LBED. Nicotine, carbamylcholine, d-tubocurarin, anabaseine, conotoxin ImI, and neurotoxin II were used as model compounds. The sensitivity of this method was comparable with that of the radioligand method (up to 10 pmol).