| α-Bungarotoxin | |||||||
|---|---|---|---|---|---|---|---|
 Schematic diagram of the three-dimensionalstructure of α-bungarotoxin.Disulfide bonds shown in gold. FromPDB:1IDI.[1] | |||||||
| Identifiers | |||||||
| Organism | Bungarus multicinctus | ||||||
| Symbol | ? | ||||||
| CAS number | 11032-79-4 | ||||||
| UniProt | P60616 | ||||||
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| Names | |
|---|---|
| Other names Alpha-Bgt, Alpha-BTX | |
| Identifiers | |
3D model (JSmol) |
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| ECHA InfoCard | 100.031.139 |
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| Properties | |
| C340H533N97O105S11 | |
| Molar mass | 8012.24 g·mol−1 |
| Appearance | Clear solution |
| >10 mg/mL | |
| Hazards | |
| Occupational safety and health (OHS/OSH): | |
Main hazards | Toxic |
| GHS labelling: | |
 | |
| Danger | |
Except where otherwise noted, data are given for materials in theirstandard state (at 25 °C [77 °F], 100 kPa). | |
α-Bungarotoxin is one of thebungarotoxins, components of thevenom of theelapidTaiwanese banded krait snake (Bungarus multicinctus). It is a type ofα-neurotoxin, a neurotoxic protein that is known to bind competitively and in a relatively irreversible manner to thenicotinic acetylcholine receptor found at theneuromuscular junction, causingparalysis,respiratory failure, and death in the victim.[2] It has also been shown to play an antagonistic role in the binding of the α7nicotinic acetylcholine receptor in the brain, and as such has numerous applications in neuroscience research.
Bungarotoxins are a group oftoxins that are closely related with the neurotoxic proteins predominantly present in the venom ofkraits. These toxins are directly linked to thethree-finger toxin superfamily. Among them, α-bungarotoxin (α-BTX) stands out, being apeptidetoxin produced by theTaiwanese banded krait, also known as the many-banded krait or the Taiwanese or Chinese krait.[3]
The venom of the many-banded krait, like the majority of the snake venoms, involves a combination ofproteins that together lead to a remarkable range of neurologic consequences. The Elapid snake family is known for their potent α-neurotoxic venom, which has a postsynaptic mechanism of action.[citation needed] These neurotoxins primarily affect thenervous system, blocking the nerve impulse transmission,[4] leading toparalysis and potentially death if untreated.[5][6]
In South and Southeast Asia,envenomation from a many-banded krait bite is a common and life-threatening medical condition when not promptly treated. Nevertheless, krait bites usually take place at night and do not show any localsymptoms, so victims are not aware of the bite. This can delaymedical care, which makes it the major cause of mortality associated with krait venom.[7][8]
The first time that the many-banded krait was described was in 1861 by the scientist Edward Blyth. It was characterized by its distinctive black-and-white banded pattern along its body, with a maximum length of 1.85 m. This very venomous species is found in central and southern China and Southeast Asia. Their venom contains variousneurotoxins, including α-BTX.
According to later research on its mechanism of action, α-bungarotoxin binds irreversibly to the postsynapticnicotinic acetylcholine receptor (nAChR) at theneuromuscular junction. It inhibits the action ofacetylcholine competitively, leading torespiratory failure,paralysis and even death.[6][9][10]
α-bungarotoxin specifically targets thenervous system, interfering with the nerve impulse transmission. The primary target of the toxin is the neuromuscular junction ofskeletal muscles, where the motor nerve terminal and the nicotinic acetylcholine receptor are the major target sites.[11] Their neurotoxic effect is often referred to asresistant neurotoxicity. This is because of the damage caused tonerve terminals that leads to acetylcholine depletion at the neuromuscular junction. The regeneration of thesynapses can take days, which prolongs the paralysis and recovery process for the victim.[12] In addition, the severity of the paralysis ranges from mild to life-threatening depending on the degree of envenomation, its composition and the early therapeutic intervention.[11]
Antivenom therapy is the current standard treatment for snake envenoming. In China, theBungarus multicinctus monovalent antivenom (BMMAV) is produced and, in Taiwan the Neuro bivalent antivenom (NBAV). Both antivenoms are immunoreactive to the neurotoxins found in the venom, including the α-BTX, whichneutralize the venomlethality. BMMAV is specifically designed to neutralize the venom of theBungarus multicinctus, therefore being more efficacious compared to NBAV. On the other hand, NBAV targets the venom from multiple species of snakes that produce neurotoxic effects, including theBungarus multicinctus. The use of BMMAV or NBAV might differ based onavailability, regional protocols and the specific venomous snake that is present in the area.[12]
α-Bungarotoxin consists of an 8kDa, singlepolypeptide chain that contains 74amino acid residues. This polypeptide chain is cross-linked by fivedisulfide bridges, categorizing the α-bungarotoxin as atype II α-neurotoxin within thethree-finger toxin family. These disulfide bridges are formed between the specificcysteine residues and are important for the stability and function of the toxin. Furthermore, α-bungarotoxin contains ten residues ofhalf-cysteine per molecule. The specific arrangements of disulfide bridges formed by these cysteine residues result in the11-ring structure within the toxin molecule. This 11-ring structure is particularly essential for the toxin interactions with the target receptors and modulation of theneurotransmission at theneuromuscular junction.[13][14][15]
Theamino acid sequence of the α-bungarotoxin contains a high frequency ofhomodipeptides, with ten pairs present whereserine andprolinedipeptides occur twice in the sequence. Theactive site of the toxin is located in the region from position 24 to position 45 within the sequence. There are some key amino acids commonly found in this region that includecysteine,arginine,glycine,lysine andvaline. As previously mentioned, cysteine is crucial for the disulfide bridges formation in proteins. Arginine and lysine can participate in interactions with negatively charged molecules or residues, so they may play a role in the binding to specificreceptors or substrates. Glycine may contribute to the flexibility and conformational dynamics of the α-bungarotoxin. Lastly, the valine residue may help maintain thehydrophobic core of the toxin.[14]
Similar to other α-neurotoxins within the three-finger toxin family, α-bungarotoxin exhibits atertiary structure that is characterized by three projecting "finger" loops, aC-terminal tail, and a smallglobular core stabilized by fourdisulfide bonds. Notably, an additional disulfide bond is present in the second loop, facilitating a proper binding through the mobility of the tips of fingers I and II. Furthermore,hydrogen bonds contribute to the formation of anantiparallel β-sheet, maintaining the parallel orientation of the second and third loops. The structural integrity of thethree-finger toxin is preserved by four of the disulfide bridges, while the fifth bridge, located on the tip of the second loop, can be reduced without compromising toxicity.
The α-bungarotoxin polypeptide chain shows significant sequencehomology with other neurotoxins fromcobra andsea snake venoms, particularly with theα-toxin fromNaja nivea. Comparing α-bungarotoxin with these homologous toxins from cobra and sea snake venoms, it was revealed that there is a high degree of conservation in certain residues. For instance, there are 18 constant residues, which include theeight half-cysteines, that are observed in all toxin sequences. Therefore, α-bungarotoxin shares commonstructural motifs with other toxins of the three-fingered family. For example,α-cobra toxin, erabutoxin A,[16] and candoxin[17] contain three adjacent loops coming up from a globular, small andhydrophobic core that is cross-linked by four conserved disulfide bridges. This conservation suggests the presence of essential functional elements that are shared among theseneurotoxins.[14][15]
Lastly, the abundance of the disulfide bonds and the limitedsecondary structure that is observed in the α-bungarotoxin explains its exceptional stability, which makes it resistant todenaturation even under extreme conditions such asboiling and exposure tostrong acids.
Due to its very large and complex structure, synthesizing α-bungarotoxin has represented a great challenge for synthetic chemists. [16] A study conducted byO. Brun et al. proposed a mechanism for thechemical synthesis of thisneurotoxin.[13] It involves a strategy utilizing peptide fragments andnative chemical ligation (NCL). Due to its length, synthesizing a full linear peptide using solid-phase peptide synthesis (SPPS) is not achievable, thus, the synthesis was done by choosing threepeptide fragments that can further undergo the native chemical ligation. This method produces a native peptide bond between two fragments by reacting thioester (C-terminal) with cysteine (N-terminal).[18] The synthesis strategy employed was from the C-terminus towards the N-terminus. Firstly, the shorter peptide fragments are synthesized via automated SPPS. The first two peptides have a Trp-Cys ligation point, while the ligation with the last fragment occurs in a Gly-Cys ligation point. Additionally, in this study, an alkyne functionality was introduced at the N-terminus of the peptide chain. This allows the conjugation of different molecules such as fluorophores viabioorthogonal reactions. Byfluorescently labelling the chemically synthesised peptide it was shown it has the same effect and functionality on the nicotinic receptors as the naturally occurring α-bungarotoxin.[13]
Due to the challenging chemical synthesis of the neurotoxin, most studies were conducted using a purified form. To investigate the effects of the α-bungarotoxin, the toxin has to be isolated from the venom of the elapid snake. The purification of the polypeptide is done viacolumn chromatography. Firstly, the venom is dissolved in ammonium acetate buffer and then loaded on the CM-Sephadex column. The elution of the compound is done in two different steps by using an ammonium acetate buffer at aflow rate of 35 nl/h. The steps involve using two linear gradients of buffers while increasing the pH.[19]
α-Bungarotoxin is a peptide, therefore it undergoes the protein synthesis pathway, involving transcription and translation. The specific genes encoding for the protein are transcribed into mRNA, which is then translated via the ribosomes, leading to the synthesis of the prepropeptide. Lastly, post-translational modification and folding occur. The mature peptide is stored in the venom gland until envenomation when it gets released.
The venom of snakes contains numerous proteins and peptide toxins that exhibit high affinity and specificity for a larger range of receptors.[3] α-Bungarotoxin is a nicotinic receptor antagonist that binds irreversibly to the receptor, inhibiting the action of acetylcholine at the neuromuscular junctions. Nicotinic receptors are one of the two subtypes of cholinergic receptors, that respond to theneurotransmitteracetylcholine.[2]
Nicotinic acetylcholine receptors (nAChRs) areligand-gated ion channels, being part of theionotropic receptors. When a ligand is bound to it, it regulates excitability by controlling the ion flow during action potential during neurotransmission, primarily through the activation of voltage-gated ion channels upondepolarization of the plasma membrane. The depolarization is induced by an influx of cations, mainly that of sodium ions. For the overall modulation of cellular excitability, an influx of sodium ions and an efflux of potassium ions into the intracellular space is necessary.[21]
In the central and peripheralnervous system, α-bungarotoxin acts by inducingparalysis in skeletal muscles by binding to a subtype of nicotinic receptors α7. α-Neurotoxins are known as "curare-mimetic toxins" due to their similar effects to thearrow poison tubocurarine. A difference between α-neurotoxins and curare alkaloids is that they bind irreversibly and reversibly specifically. α-Neurotoxins block the action of acetylcholine (ACh) at the postsynaptic membrane by irreversiblyinhibiting the ion flow.[6]
From the same toxin family of bungarotoxins, κ-BTX was shown to actpostsynaptically on α3 and α4 neuronal nicotinic receptors with little effect on the muscular nAChRs, targeted by α-BTX. In contrast, β- and γ-BTX actpresynaptically by reducing ACh release. Neurotoxins are named based on the receptor type they target.[6]
The nicotinic receptors are made up of five subunits each and contain two binding sites for snake venom neurotoxins. The α7-nAChR is a homopentamer consisting of five identical α7 subunits. The α7 receptor is known to have a higher Ca2+ permeability compared to other nicotinic receptors. Changes in Ca2+ intracellularly can activate important cellular pathways such as theSTAT pathway or theNF-κB signalling.[22]
Consistency with experimental data on the amount oftoxin per receptor is evident in the observation that a lone molecule of the toxin is adequate to inhibit channel opening.[23] Some computational studies of the mechanism of inhibition using normal mode dynamics[24] suggest that a twist-like motion caused by ACh binding may be responsible for pore opening and that this motion is inhibited by toxin binding.[24][25]
The following section describes theADME (absorption, distribution, metabolism and excretion) of α-bungarotoxin. It is important to note that there is limited information available on thepharmacokinetics of thisneurotoxin. More research is needed to be able to fully understand the metabolism of this neurotoxin inside the body.
Absorption: α-bungarotoxin enters the body after envenomation into thebloodstream at the bite site. Through the venom, a mixture of proteins and different molecules enter the body.
Distribution: Once in the bloodstream, α-bungarotoxin circulates throughout the body. Its distribution may be influenced by factors such as blood flow, tissue permeability, and the presence of binding proteins. Additionally, knowing it binds to nAChRs, it can be predicted where the neurotoxin would be present:neuromuscular junctions,autonomic ganglia,peripheral nerves, andadrenal medulla. One of the main locations would be also thecentral nervous system (CNS), including thebrain. Specific regions such as thehippocampus,cortex, andbasal ganglia contain these receptors.[26]
Metabolism: The metabolic pathways of this neurotoxins have not been fully understood yet, however, it is thought to be metabolised in theliver. Researching venom metabolism is challenging due to the multiple components present in it. Toxins that are not bound may undergo elimination through opsonization by the reticuloendothelial system, mainly involving the liver and kidneys, or they may undergo degradation through cellular internalization facilitated bylysosomes.[27]
Excretion: It is common for proteins and peptides to be excreted via thehepatic andrenal pathways. In the liver, the amino acids present undergo transamination. This way theamino acids are converted into ammonia and keto acids. Lastly, these substances are excreted via the kidney.[28]
However, it is important to take into account that α-bungarotoxin binds irreversibly to the receptors, which would result in a very low metabolic and excretion rate, as most of the neurotoxin would be present at the receptor sites.[2]
The α-bungarotoxin is among the most well-characterized snake toxins, with its high affinity and specificity for nicotinic acetylcholine receptors. It is acompetitive antagonist at nAChR, where it irreversibly and competitively blocks the receptor at the acetylcholine binding sites.[2] It binds to the α1 subunit contained in muscle nAChRs, as well as subsets of neuronal nAChRs like α7-α10. In addition, it was shown that α-bungarotoxin binds to, and block, a subset ofGABAA receptors where the β3 subunits connect with each other. With this knowledge in mind, researchers can use α-bungarotoxin as an experimental tool for studying the properties ofcholinergic receptors.[6]
In addition, by knowing the different and specificbinding sites, researchers are able to visualize and track receptor localization and dynamics within cells. This technique has been shown to be easy with the use of a 13-amino acid (WRYYESSLEPYPD)[29]mimotope, which forms a high affinity α-bungarotoxin binding site with the receptors.[30]
It has been extensively used in research to study the localization and distribution of these receptors. Through techniques likefluorophore orenzymeconjugation followed by microscopy orimmunohistochemical staining, respectively, could give insights about the complex organization and function of the nervous system. With the mentioned techniques, researchers can work towardards adrug development, and understand the disease mechanism. They can idenitify potentialdrug targets by selectively regulating the activity of certain receptors. Therefore, observe how receptors behave when in contact with the α-bungarotoxin compared to when there is no toxin, researchers can study the mechanism of the toxin.
α-Bungarotoxin is available for purchase from multiple biotechnological companies, such asSigma-Aldrich or Biotium. Researchers may purchase it from there to perform a variety of researches on the toxin.
Regardingbioavailability, researchers performed a study in the spinal cord duringembryonic development in the embryos of chicks. They found that that binding of α-bungarotoxin was specific and saturable within the concentration range of 1-34 mM. Meaning, as the concentration of α-bungarotoxin increased, the binding site became more and more limited. Reaching the maximum number at 34 mM. Once there was no binding sites available anymore,nicotine behaved in a competitive manner and pushed out the already-bound α-bungarotoxin. Another thing they found was that thedissociation constant (Kd) was 8.0 nM - a concentration of α-bungarotoxin where half of the binding site were occupied. Moreover, maximum binding capacity (Bmax) was found to be 106 +/- 12 fmol/mg - the maximum number of binding sites available per unit of protein. Finally,exogenously administered α-bungarotoxin showed to penetrate the spinal cord tissue and bind to its specific sites after 7 days.[31]
The efficacy of α-bungarotoxin can be assessed by analyzing theirbinding affinity. It affects how the signal transmits at the skeletal neuromuscular junction by binding to the postsynaptic nAChRs at high affinity. The affinity of the toxin for this receptor is measured with a dissociation constant (Kd), ranging from 10-11 to 10-9 M. In addition to binding to skeletal neuromuscular junctions, it can specifically bind to different neuronal subsets, such as α7. This binding affinity is only slightly lower with Kd measured in the range of 10-9 to 10-8 M.[32]
It can also be analyzed through receptor inhibition, specifically inhibiting the action of acetylcholine on nAChRs. One study found that 5 mirograms/ml of the toxin completely blocks theendplate potential and extrajunctional acetylcholine sensitivity of surface fibers, within approximately 35 minutes in normal and chronically denervated muscles. They performed a washout period of 6.5 hours, which resulted in a partial recovery of the endplate potential, with anamplitude of 0.72 +/- 0.033 mV in normal muscles. Indenervated muscles, a partial recovery of acetylcholine sensitivity was observed, with an amplitude of 41.02 +/- 3.95 mV/nC compared to a control amplitude of 1215 +/- 197 mV/nC. This same study also found a small population of acetylhcoline receptors (1% of the total population) to react with α-bungarotoxin reversibly. With the toxin, either 20 μMcarbamylcholine ordecamethonium was used simultaneously in normal muscles. Once the toxin and the drug were washed out, the muscle restored a twitch to control levels within 2 hours.[33]
The susceptibility of different species to the venom of a krait snake, which contains alpha-bungarotoxin, varies based on theirgenetic makeup. α-Bungarotoxin binds best to the acetylcholine alpha-subunit containingaromatic amino acid residues at positions 187 and 189 - e.g. shrews, cats and mice. In species like humans and hedgehogs, which have nonaromatic amino acid residues at the same positions, have a decreased binding affinity of α-bungarotoxin. Finally, snakes and mongooses have specific amino acid substitutions at 187, 189, and 194, alpha-subunits, which makes the binding of the toxin non-existent.[34]
In humans, exposure to α-bungarotoxin can lead to various symptoms, such as headache, dizziness, unconsciousness, visual and speech disturbances, and occasionally seizures. Onset of severe abdominal pain and muscularparalysis within 10 hours and may last for 4 days. Finally,respiratory paralysis can lead to death.[35] Additionally, it can also lead to mild symptoms likedermatitis and allergic reactions, or stronger symptoms likeblood coagulation,disseminated intravascular coagulation, tissue injury, andhemorrhage.[3]
In animals, studies have been done to analyze the effect of the α-bungarotoxin on animals. One study showed this toxin causing paralysis in chickens by blocking neuromuscular transmission at the motor end-plate. This led to muscle weakness and ultimately, paralysis.[36]
In ancient days, these venoms were already widespread across the world. Then, folklore medicine utilized plant-based andbioactive inhibitor compounds to treat bites from venomous animals like snakes and scorpions. This approach proved successful in preventingenvenomation, effectively mitigating the harmful effects of venom on the victims. Today, treatment for krait bites involvesantivenom, which can lead to various undesirable and potentially life-threatening side effects, such as nausea,urticarial,hypotension,cyanosis, and severe allergic reactions.[3]
α-Bungarotoxin belongs to a group of bungarotoxins, which are a type of poisonous proteins found in the venom of kraits - among the six most deadly snakes in Asia. Their bite can lead to respiratory paralysis and death.[3] α-Bungarotoxin irreversibly and competitively binds to muscular and neuronal acetylcholine receptors. The paralysis happens due to the neuromuscular transmission at the postsynaptic site being blocked.
LD50 values, representing lethal dose required to cause death in 50%, were studied in mice using different routes of administration.Subcutaneous administration showed that 0.108 mg/kg was needed to kill 50% of mice.Intravenous administration resulted in a slightly higher LD50 value of 0.113 mg/kg. However, when it was administeredintraperitoneally, the LD50 value was 0.08 mg/kg. These values can aid in risk assessment of the toxin.[37]
