α-Bungarotoxin is one of the bungarotoxins, components of the venom of the elapid Taiwanese banded krait snake (Bungarus multicinctus). It is a type of α-neurotoxin, a neurotoxic protein that is known to bind competitively and in a relatively irreversible manner to the nicotinic acetylcholine receptor found at the neuromuscular junction, causing paralysis, respiratory failure, and death in the victim. It has also been shown to play an antagonistic role in the binding of the α7 nicotinic acetylcholine receptor in the brain, and as such has numerous applications in neuroscience research.
History Bungarotoxins are a group of toxins that are closely related with the neurotoxic proteins predominantly present in the venom of kraits. These toxins are directly linked to the three-finger toxin superfamily. Among them, α-bungarotoxin (α-BTX) stands out, being a peptide toxin produced by the Taiwanese banded krait, also known as the many-banded krait or the Taiwanese or Chinese krait.
The venom of the many-banded krait, like the majority of the snake venoms, involves a combination of proteins 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. These neurotoxins primarily affect the nervous system, blocking the nerve impulse transmission, leading to paralysis and potentially death if untreated.
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 local symptoms, so victims are not aware of the bite. This can delay medical care, which makes it the major cause of mortality associated with krait venom.
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 various neurotoxins, including α-BTX.
Mechanism According to later research on its mechanism of action, α-bungarotoxin binds irreversibly to the postsynaptic nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction. It inhibits the action of acetylcholine competitively, leading to respiratory failure, paralysis and even death.
α-bungarotoxin specifically targets the nervous system, interfering with the nerve impulse transmission. The primary target of the toxin is the neuromuscular junction of skeletal muscles, where the motor nerve terminal and the nicotinic acetylcholine receptor are the major target sites. Their neurotoxic effect is often referred to as resistant neurotoxicity. This is because of the damage caused to nerve terminals that leads to acetylcholine depletion at the neuromuscular junction. The regeneration of the synapses can take days, which prolongs the paralysis and recovery process for the victim. 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.
Treatment Antivenom therapy is the current standard treatment for snake envenoming. In China, the Bungarus 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, which neutralize the venom lethality. BMMAV is specifically designed to neutralize the venom of the Bungarus 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 the Bungarus multicinctus. The use of BMMAV or NBAV might differ based on availability, regional protocols and the specific venomous snake that is present in the area.
Structure and available forms α-Bungarotoxin consists of an 8 kDa, single polypeptide chain that contains 74 amino acid residues. This polypeptide chain is cross-linked by five disulfide bridges, categorizing the α-bungarotoxin as a type II α-neurotoxin within the three-finger toxin family. These disulfide bridges are formed between the specific cysteine residues and are important for the stability and function of the toxin. Furthermore, α-bungarotoxin contains ten residues of half-cysteine per molecule. The specific arrangements of disulfide bridges formed by these cysteine residues result in the 11-ring structure within the toxin molecule. This 11-ring structure is particularly essential for the toxin interactions with the target receptors and modulation of the neurotransmission at the neuromuscular junction.
The amino acid sequence of the α-bungarotoxin contains a high frequency of homodipeptides, with ten pairs present where serine and proline dipeptides occur twice in the sequence. The active 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 include cysteine, arginine, glycine, lysine and valine. 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 specific receptors or substrates. Glycine may contribute to the flexibility and conformational dynamics of the α-bungarotoxin. Lastly, the valine residue may help maintain the hydrophobic core of the toxin.
Similar to other α-neurotoxins within the three-finger toxin family, α-bungarotoxin exhibits a tertiary structure that is characterized by three projecting "finger" loops, a C-terminal tail, and a small globular core stabilized by four disulfide 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 an antiparallel β-sheet, maintaining the parallel orientation of the second and third loops. The structural integrity of the three-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 sequence homology with other neurotoxins from cobra and sea snake venoms, particularly with the α-toxin from Naja 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 the eight half-cysteines, that are observed in all toxin sequences. Therefore, α-bungarotoxin shares common structural motifs with other toxins of the three-fingered family. For example, α-cobra toxin, erabutoxin A, and candoxin contain three adjacent loops coming up from a globular, small and hydrophobic core that is cross-linked by four conserved disulfide bridges. This conservation suggests the presence of essential functional elements that are shared among these neurotoxins.
Lastly, the abundance of the disulfide bonds and the limited secondary structure that is observed in the α-bungarotoxin explains its exceptional stability, which makes it resistant to denaturation even under extreme conditions such as boiling and exposure to strong acids.
Synthesis
Chemical synthesis Due to its very large and complex structure, synthesizing α-bungarotoxin has represented a great challenge for synthetic chemists. A study conducted by O. Brun et al. proposed a mechanism for the chemical synthesis of this neurotoxin. It involves a strategy utilizing peptide fragments and native 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 three peptide 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). 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 via bioorthogonal reactions. By fluorescently 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.
Purification 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 via column 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 a flow rate of 35 nl/h. The steps involve using two linear gradients of buffers while increasing the pH.
Biosynthesis 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.
Mechanism of action
The venom of snakes contains numerous proteins and peptide toxins that exhibit high affinity and specificity for a larger range of receptors. 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 the neurotransmitter acetylcholine.
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels, being part of the ionotropic 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 upon depolarization 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.
In the central and peripheral nervous system, α-bungarotoxin acts by inducing paralysis 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 the arrow 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 irreversibly inhibiting the ion flow.