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PLoS Neglected Tropical Diseases Nov 2020Bungarus multicinctus is the most venomous snake distributed in China and neighboring countries of Myanmar, Laos, north Vietnam and Thailand. The high mortality rate of...
Immunoreactivity and neutralization study of Chinese Bungarus multicinctus antivenin and lab-prepared anti-bungarotoxin antisera towards purified bungarotoxins and snake venoms.
Bungarus multicinctus is the most venomous snake distributed in China and neighboring countries of Myanmar, Laos, north Vietnam and Thailand. The high mortality rate of B. multicinctus envenomation is attributed to the lethal components of α-, β-, γ- and κ- bungarotoxins contained in the venom. Although anti-B. multicinctus sera were produced in Shanghai, Taiwan and Vietnam, the most widely clinic used product was term as B. multicinctus antivenin and manufactured by Shanghai Serum Bio-technology Co. Ltd. In the present investigation, high purity α-, β- and γ-bungarotoxins were separately isolated from B. multicinctus crude venom. Rabbit anti- α-, β- and γ-bungarotoxin antisera were prepared by common methods, respectively. LD50 values of α-, β- and γ-bungarotoxins were systematically determined via three administration pathways (intraperitoneal, intramuscular and intravenous injections) in Kunming mice. LD50 values of β-bungarotoxin were closely related with injection routines but those of both α- and γ-bungarotoxins were not dependent on the injection routines. Commercial B. multicinctus antivenin showed strong immunoreaction with high molecular weight fractions of the B. multicinctus but weakly recognized low molecular weight fractions like α- and γ-bungarotoxins. Although B. multicinctus antivenin showed immunoreaction with high molecular weight fractions of Bungarus fasciatus, Naja atra, Ophiophagus hannah venoms but the antivenin only demonstrated animal protection efficacy against O. hannah venom. These results indicated that the high molecular weight fractions of the O. hannah played an important role in venom lethality but those of B. fasciatus and N. atra did not have such a role.
Topics: Animals; Antivenins; Bungarotoxins; Bungarus; China; Elapid Venoms; Immune Sera; Lethal Dose 50; Male; Mice; Neutralization Tests; Ophiophagus hannah; Rabbits
PubMed: 33253321
DOI: 10.1371/journal.pntd.0008873 -
Journal of Neurochemistry Sep 2021The first toxin to give rise to the three-finger protein (TFP) family was α-bungarotoxin (α-Bgt) from Bungarus multicinctus krait venom. α-Bgt was crucial for... (Review)
Review
The first toxin to give rise to the three-finger protein (TFP) family was α-bungarotoxin (α-Bgt) from Bungarus multicinctus krait venom. α-Bgt was crucial for research on nicotinic acetylcholine receptors (nAChRs), and in this Review article we focus on present data for snake venom TFPs and those of the Ly6/uPAR family from mammalians (membrane-bound Lynx1 and secreted SLURP-1) interacting with nAChRs. Recently isolated from Bungarus candidus venom, αδ-bungarotoxins differ from α-Bgt: they bind more reversibly and distinguish two binding sites in Torpedo californica nAChR. Naja kaouthia α-cobratoxin, classical blocker of nAChRs, was shown to inhibit certain GABA-A receptor subtypes, whereas α-cobratoxin dimer with 2 intermolecular disulfides has a novel type of 3D structure. Non-conventional toxin WTX has additional 5th disulfide not in the central loop, as α-Bgt, but in the N-terminal loop, like all Ly6/uPAR proteins, and inhibits α7 and Torpedo nAChRs. A water-soluble form of Lynx1, ws-Lynx1, was expressed in E. coli, its H-NMR structure and binding to several nAChRs determined. For SLURP-1, similar information was obtained with its recombinant analogue rSLURP-1. A common feature of ws-Lynx1, rSLURP-1, and WTX is their activity against nAChRs and muscarinic acetylcholine receptors. Synthetic SLURP-1, identical to the natural protein, demonstrated some differences from rSLURP-1 in distinguishing nAChR subtypes. The loop II fragment of the Lynx1 was synthesized having the same µM affinity for the Torpedo nAChR as ws-Lynx1. This review illustrates the productivity of parallel research of nAChR interactions with the two TFP groups.
Topics: Animals; Binding Sites; Bungarotoxins; Humans; Protein Binding; Protein Structure, Secondary; Receptors, Nicotinic; Snakes; Species Specificity
PubMed: 32648941
DOI: 10.1111/jnc.15123 -
Neuron Jul 2020The high-resolution structure of the nicotinic acetylcholine receptor from Torpedo electric tissue in association with the snake toxin α-bungarotoxin (Rahman et al.,...
The high-resolution structure of the nicotinic acetylcholine receptor from Torpedo electric tissue in association with the snake toxin α-bungarotoxin (Rahman et al., 2020) is presented 50 years after its identification as the first neurotransmitter receptor and ligand-gated ion channel.
Topics: Animals; Anniversaries and Special Events; Bungarotoxins; Receptors, Nicotinic; Snake Venoms; Torpedo
PubMed: 32645305
DOI: 10.1016/j.neuron.2020.05.026 -
Journal of Biochemistry May 1982The two most basic beta-bungarotoxins (beta 3- and beta 4-toxins) and another, less neurotoxic beta-bungarotoxin (beta 5-toxin) were purified from Bungarus multicinctus...
The two most basic beta-bungarotoxins (beta 3- and beta 4-toxins) and another, less neurotoxic beta-bungarotoxin (beta 5-toxin) were purified from Bungarus multicinctus venom, by a combination of CM-Sephadex C-25 column chromatography and Sephadex G-75 gel filtration. The three toxins consisted of two dissimilar polypeptides (A chain, 120 amino acid residues; B chain, 60 residues). The LD50 values of the beta 3- and beta 4-toxins were 0.066 micrograms and 0.072 micrograms/g of mouse, respectively, and their phospholipase A activities were 43.2 and 36.5 units/mg of toxin, respectively. beta 5-Toxin was weaker in neurotoxicity (LD50, 0.13 micrograms/g of mouse) than the others, and its phospholipase activity was 47.6 units/mg of toxin. Each toxin was separated into RCM-A and RCM-B chains after reduction and S-carboxymethylation. The RCM-polypeptides were maleylated and digested with TPCK-trypsin. The tryptic peptides were sequenced with manual Edman degradation or the dansyl-Edman method. The final alignment of the tryptic peptides from the respective RCM-polypeptides was deduced on the basis of the amino acid sequences of the A and B chains of beta 1-bungarotoxin (beta 1-toxin). The amino acid sequences of the A chains of the beta 3- and beta 4-toxins were identical but differed from those of the A chains of the beta 1- and beta 2-toxins by 4 amino acid substitutions in the COOH-terminal portions (residues 109-120) and substitution at position 87. The amino acid sequences of the B chains of the beta 3- and beta 4-toxins differed from each other, but they were identical with those of the B chains of the beta 1- and beta 2-toxins, respectively. The amino acid sequence of the A chain of beta 5-toxin differed from that of the A chain of beta 1-toxin by consecutive substitutions in residues 55-60 and substitutions at positions 23, 87, and 89. The amino acid sequence of the B chain of beta 5-toxin was identical with those of the B chains of beta 1- and beta 3-toxin. From our results on the effects of the amino acid displacements found in the A chains on the neurotoxicity, it was concluded that the COOH-terminal portion in the A chains was not essential to their neurotoxicity, whereas the region of residues 55-60 in the A chains appeared to participate in the constitution of the neurotoxically active site of the beta-toxins.
Topics: Amino Acid Sequence; Animals; Bungarotoxins; Chemical Phenomena; Chemistry; Chickens; Elapid Venoms; Mice; Peptides
PubMed: 7096305
DOI: 10.1093/oxfordjournals.jbchem.a133844 -
International Journal of Molecular... Sep 2023This review covers briefly the work carried out at our institute (IBCh), in many cases in collaboration with other Russian and foreign laboratories, for the last 50... (Review)
Review
This review covers briefly the work carried out at our institute (IBCh), in many cases in collaboration with other Russian and foreign laboratories, for the last 50 years. It discusses the discoveries and studies of various animal toxins, including protein and peptide neurotoxins acting on the nicotinic acetylcholine receptors (nAChRs) and on other ion channels. Among the achievements are the determination of the primary structures of the α-bungarotoxin-like three-finger toxins (TFTs), covalently bound dimeric TFTs, glycosylated cytotoxin, inhibitory cystine knot toxins (ICK), modular ICKs, and such giant molecules as latrotoxins and peptide neurotoxins from the snake, as well as from other animal venoms. For a number of toxins, spatial structures were determined, mostly by H-NMR spectroscopy. Using this method in combination with molecular modeling, the molecular mechanisms of the interactions of several toxins with lipid membranes were established. In more detail are presented the results of recent years, among which are the discovery of α-bungarotoxin analogs distinguishing the two binding sites in the muscle-type nAChR, long-chain α-neurotoxins interacting with α9α10 nAChRs and with GABA-A receptors, and the strong antiviral effects of dimeric phospholipases A2. A summary of the toxins obtained from arthropod venoms includes only highly cited works describing the molecules' success story, which is associated with IBCh. In marine animals, versatile toxins in terms of structure and molecular targets were discovered, and careful work on α-conotoxins differing in specificity for individual nAChR subtypes gave information about their binding sites.
Topics: Animals; Bungarotoxins; Toxins, Biological; Neurotoxins; Animal Experimentation; Cytotoxins
PubMed: 37762187
DOI: 10.3390/ijms241813884 -
Toxins Jan 2022α-bungarotoxin is a large, 74 amino acid toxin containing five disulphide bridges, initially identified in the venom of snake. Like most large toxins, chemical...
α-bungarotoxin is a large, 74 amino acid toxin containing five disulphide bridges, initially identified in the venom of snake. Like most large toxins, chemical synthesis of α-bungarotoxin is challenging, explaining why all previous reports use purified or recombinant α-bungarotoxin. However, only chemical synthesis allows easy insertion of non-natural amino acids or new chemical functionalities. Herein, we describe a procedure for the chemical synthesis of a fluorescent-tagged α-bungarotoxin. The full-length peptide was designed to include an alkyne function at the amino-terminus through the addition of a pentynoic acid linker. Chemical synthesis of α-bungarotoxin requires hydrazide-based coupling of three peptide fragments in successive steps. After completion of the oxidative folding, an azide-modified Cy5 fluorophore was coupled by click chemistry onto the toxin. Next, we determined the efficacy of the fluorescent-tagged α-bungarotoxin to block acetylcholine (ACh)-mediated currents in response to muscle nicotinic receptor activation in TE671 cells. Using automated patch-clamp recordings, we demonstrate that fluorescent synthetic α-bungarotoxin has the expected nanomolar affinity for the nicotinic receptor. The blocking effect of fluorescent α-bungarotoxin could be displaced by incubation with a 20-mer peptide mimicking the α-bungarotoxin binding site. In addition, TE671 cells could be labelled with fluorescent toxin, as witnessed by confocal microscopy, and this labelling was partially displaced by the 20-mer competitive peptide. We thus demonstrate that synthetic fluorescent-tagged α-bungarotoxin preserves excellent properties for binding onto muscle nicotinic receptors.
Topics: Acetylcholine; Bungarotoxins; Cell Line; Cholinesterase Inhibitors; Click Chemistry; Fluorescent Dyes; High-Throughput Screening Assays; Humans; Models, Molecular; Protein Conformation
PubMed: 35202107
DOI: 10.3390/toxins14020079 -
Neuropharmacology Dec 2019The ten types of nicotinic acetylcholine receptor α-subunits show substantial sequence homology, yet some types confer high affinity for α-bungarotoxin, whereas others...
The ten types of nicotinic acetylcholine receptor α-subunits show substantial sequence homology, yet some types confer high affinity for α-bungarotoxin, whereas others confer negligible affinity. Combining sequence alignments with structural data reveals three residues unique to α-toxin-refractory α-subunits that coalesce within the 3D structure of the α4β2 receptor and are predicted to fit between loops I and II of α-bungarotoxin. Mutating any one of these residues, Lys189, Ile196 or Lys153, to the α-toxin-permissive counterpart fails to confer α-bungarotoxin binding. However, mutating both Lys189 and Ile196 affords α-bungarotoxin binding with an apparent dissociation constant of 104 nM, while combining mutation of Lys153 reduces the dissociation constant to 22 nM. Analogous residue substitutions also confer high affinity α-bungarotoxin binding upon α-toxin-refractory α2 and α3 subunits. α4β2 receptors engineered to bind α-bungarotoxin exhibit slow rates of α-toxin association and dissociation, and competition by cholinergic ligands typical of muscle nicotinic receptors. Receptors engineered to bind α-bungarotoxin co-sediment with muscle nicotinic receptors on sucrose gradients, and mirror single channel signatures of their α-toxin-refractory counterparts. Thus the inability of α-bungarotoxin to bind to neuronal nicotinic receptors arises from three unique and interdependent residues that coalesce within the receptor's 3D structure.
Topics: Binding Sites; Bungarotoxins; HEK293 Cells; Humans; Ligands; Molecular Conformation; Mutation; Neurons; Receptors, Nicotinic
PubMed: 31163179
DOI: 10.1016/j.neuropharm.2019.05.037 -
Cell Reports Apr 2017Neuronal nicotinic acetylcholine receptors (nAChRs) participate in diverse aspects of brain function and mediate behavioral and addictive properties of nicotine....
Neuronal nicotinic acetylcholine receptors (nAChRs) participate in diverse aspects of brain function and mediate behavioral and addictive properties of nicotine. Neuronal nAChRs derive from combinations of α and β subunits, whose assembly is tightly regulated. NACHO was recently identified as a chaperone for α7-type nAChRs. Here, we find NACHO mediates assembly of all major classes of presynaptic and postsynaptic nAChR tested. NACHO acts at early intracellular stages of nAChR subunit assembly and then synergizes with RIC-3 for receptor surface expression. NACHO knockout mice show profound deficits in binding sites for α-bungarotoxin, epibatidine, and conotoxin MII, illustrating essential roles for NACHO in proper assembly of α7-, α4β2-, and α6-containing nAChRs, respectively. By contrast, GABA receptors are unaffected consistent with NACHO specifically modulating nAChRs. NACHO knockout mice show abnormalities in locomotor and cognitive behaviors compatible with nAChR deficiency and underscore the importance of this chaperone for physiology and disease associated with nAChRs.
Topics: Animals; Binding Sites; Brain; Bridged Bicyclo Compounds, Heterocyclic; Bungarotoxins; Cell Line; Cognitive Dysfunction; Conotoxins; Humans; Iodine Radioisotopes; Membrane Proteins; Mice; Mice, Knockout; Molecular Chaperones; Neurons; Nicotine; Protein Binding; Protein Subunits; Pyridines; Radiopharmaceuticals; Receptors, Nicotinic
PubMed: 28445721
DOI: 10.1016/j.celrep.2017.04.008 -
British Journal of Pharmacology Jan 19731. alpha-Bungarotoxin isolated from the venom of Bungarus multicinctus was acetylated with [(3)H] acetic anhydride and N-[(3)H] acetyl imidazole. Tri-N-acetyl and...
1. alpha-Bungarotoxin isolated from the venom of Bungarus multicinctus was acetylated with [(3)H] acetic anhydride and N-[(3)H] acetyl imidazole. Tri-N-acetyl and hexa-N-acetyl derivatives were obtained from the former, and N,O-di, N,N,O-tri and N,N,N,O-tetraacetyl derivatives from the latter reaction, respectively.2. There were parallel decreases in both neuromuscular blocking action in the phrenic nerve-diaphragm preparation of rats and depression of acetylcholine response of the rectus abdominis muscle of frogs with increased acetylation. Also, a parallel but greater decrease of toxicity in mice was found.3. N,O-Di and N,N,O-triacetyl toxins were localized mostly in the motor endplate region of the rat diaphragm, whereas a slight nonspecific binding along the whole muscle fibre in addition to the peak in the endplate region was observed with N,N,N,O-tetraacetyl and tri-N-acetyl toxins. In contrast, there was a marked nonspecific binding with hexa-N-acetyl toxin and no peak was observed at the endplate zone.4. The specific binding was saturable and irreversible. The number of toxin-receptive sites in one endplate was 1.9-2.2 x 10(7) for all of the labelled toxins irrespective of their potency.5. (+)-Tubocurarine protected effectively against the binding as well as the irreversible neuromuscular blocking effect of the toxins.6. Denervation of the rat diaphragm caused an increase of toxin-receptive sites beginning from the endplate zone at 1-2 days and then along the whole muscle fibre, reaching the maximum at about 18 days. The total receptive sites increased by about 30-fold.7. The significance of the findings is discussed and it is concluded that N,O-di and N,N,O-tri-[(3)H] acetyl alpha-bungarotoxins are specific and irreversible labelling agents for the cholinergic receptors of skeletal muscle.
Topics: Acetylation; Acetylcholine; Animals; Anura; Bungarotoxins; Denervation; Electric Stimulation; Electrophoresis, Starch Gel; In Vitro Techniques; Lethal Dose 50; Mice; Muscle Contraction; Neuromuscular Junction; Parasympatholytics; Protein Binding; Rats; Receptors, Cholinergic; Receptors, Drug; Snakes; Time Factors; Tritium; Tubocurarine; Venoms
PubMed: 4717015
DOI: 10.1111/j.1476-5381.1973.tb08169.x -
BioMed Research International 2022Bungarus multicinctus is one of the top ten venomous snakes in China. Its venom is mainly neurotoxin-based. Novel antivenom drugs need to be further researched and...
BACKGROUND
Bungarus multicinctus is one of the top ten venomous snakes in China. Its venom is mainly neurotoxin-based. Novel antivenom drugs need to be further researched and developed.
OBJECTIVE
This study aimed to explore the molecular mechanism of Cynanchum paniculatum in treating Bungarus multicinctus bites based on network pharmacology. . The potential active ingredients of Cynanchum paniculatum were screened and their SDF structures were obtained using the PubChem database and imported into the SwissTargetPrediction database, and targets were obtained for the antitoxin effects of Cynanchum paniculatum in the treatment of Bungarus multicinctus bites. The Cynanchum paniculatum-active compound-potential target network and protein-protein interaction network were constructed by using Cytoscape software, and then biological function analysis and KEGG pathway enrichment analysis were performed using the DAVID.
RESULTS
Seven potential active components (cynapanoside C, cynatratoside B, tomentolide A, sitosterol, sarcostin, tomentogenin, and paeonol) and 286 drug targets were obtained, including 30 key targets for the treatment of bungarotoxin toxicity. The active components mainly acted on PIK3CA, MAPK1, MAP2K1, JAK2, FYN, ACHE, CHRNA7, CHRNA4, and CHRNB2, and they antagonized the inhibitory effect of bungarotoxin on the nervous system through cholinergic synapses and the neurotrophin signaling pathway.
CONCLUSIONS
Cynanchum paniculatum exerts a therapeutic effect on Bungarus multicinctus bites through multiple active components, multiple targets, and multiple pathways. The findings provide a theoretical basis for the extraction of active components of Cynanchum paniculatum and for related antivenom experiments.
Topics: Animals; Antivenins; Bungarotoxins; Bungarus; Cynanchum; Neurotoxins
PubMed: 35837378
DOI: 10.1155/2022/3887072