Recombinant Dendroaspis polylepis polylepis Muscarinic toxin beta

What is Muscarinic toxin beta (MTβ) and how does it relate to other toxins in black mamba venom?

Muscarinic toxin beta (MTβ) is one of three muscarinic toxins isolated from the venom of the black mamba (Dendroaspis polylepis), alongside MTα and MTγ. These toxins belong to the three-finger toxin superfamily, which includes approximately 180 other snake venom components such as alpha-neurotoxins, cardiotoxins, and fasciculins. Unlike alpha-neurotoxins that target nicotinic acetylcholine receptors, muscarinic toxins specifically bind to muscarinic acetylcholine receptors (mAChRs). The muscarinic toxins from black mamba and green mamba (Dendroaspis angusticeps) venoms share 60-98% sequence identity with each other, indicating their evolutionary relationship while allowing for distinct pharmacological profiles .

MTβ functions as part of the black mamba's neurotoxic arsenal, contributing to the venom's potent effects on the nervous system. The venom's fast-acting nature results from the combined action of various neurotoxins, including dendrotoxins and α-neurotoxins, together with muscarinic toxins. This combination of toxins makes black mamba envenomation particularly dangerous, with high fatality rates in untreated victims .

What is the molecular structure of MTβ and what are its key biochemical characteristics?

MTβ is a peptide toxin composed of 65-66 amino acids with four disulfide bonds that are critical for maintaining its three-dimensional structure. The tertiary structure of MTβ likely follows the characteristic three-finger toxin fold, with three extended loops protruding from a central core stabilized by the disulfide bridges . The sequence of MTβ has been determined, revealing its primary structure and relationship to other muscarinic toxins .

How do the amino acid sequences of MTβ and other muscarinic toxins influence their receptor selectivity?

The sequence variations between different muscarinic toxins directly impact their receptor subtype selectivity. For example, MTα and MT4 (from D. angusticeps) differ in only three amino acids (residues 31-33), which are Leu-Asn-His in MTα and Ile-Val-Pro in MT4. This small difference causes a pronounced shift in subtype selectivity, with MTα having high affinity to all subtypes while MT4 is more selective .

All three loops of muscarinic toxins appear to be involved in receptor binding, as demonstrated in mutagenesis studies with MT7. The outer loops of the receptor, especially the second loop connecting transmembrane helices four and five, are important for toxin binding. These outer loops are usually less conserved between receptor subtypes and may be crucial for the subtype selectivity observed with different muscarinic toxins . Since MTβ shares structural characteristics with other muscarinic toxins but has a unique sequence, its specific amino acid composition likely determines its particular receptor selectivity profile and pharmacological properties.

What is known about the receptor binding profile of MTβ compared to other muscarinic toxins?

While specific binding data for MTβ is limited in the available literature, we can draw some comparisons based on related muscarinic toxins. Muscarinic toxins from both black and green mambas exhibit varying degrees of selectivity for different muscarinic receptor subtypes. For instance, MTα has high affinity to all mAChR subtypes, with Ki values of 23 nM (m1), 44 nM (m2), 3 nM (m3), 5 nM (m4), and 8 nM (m5) . In contrast, MT7 (from D. angusticeps) appears to have absolute selectivity for the M1 subtype with affinity values in the pico- to nanomolar range and no detectable binding to M2–M5 subtypes .

The binding constants of MTβ to human muscarinic receptors subtypes m1-m5 have been determined, though the specific values aren't detailed in the available sources. What is noted is that the muscarinic toxins from black mamba are generally less selective than those from the green mamba (Dendroaspis angusticeps) . This suggests that MTβ might have a broader receptor binding profile compared to some of the highly selective green mamba toxins.

Do muscarinic toxins like MTβ interact with receptors beyond the muscarinic acetylcholine receptor family?

Recent research has revealed that muscarinic toxins may target receptors beyond the muscarinic acetylcholine receptor family. Several muscarinic toxins have been found to interact with adrenoceptors with varying affinities. MTα, for example, appears to be specific for the α2B-adrenoceptor with no detectable muscarinic receptor activity, contrary to earlier reports . MT1 displays higher binding affinity for the human α2B-adrenoceptor (IC50 = 2.3 nM) compared to muscarinic receptors (IC50 ≥ 100 nM) .

MT3 has been identified as having a particularly broad spectrum of targets, showing high-affinity binding (IC50 = 1–10 nM) to M4 mAChR, α1A-, α1D-, and α2A-adrenoceptors, and lower affinity binding (IC50 ≥ 25 nM) to α1B- and α2C-adrenoceptors and M1 mAChR . In contrast, MT7 appears to be highly specific for M1 receptors without detectable binding to adrenoceptors. While specific cross-reactivity data for MTβ isn't available in the current search results, these findings suggest that MTβ might also interact with receptors beyond the mAChR family.

What methodologies are most effective for studying MTβ binding to receptors?

Several methodologies have proven effective for studying muscarinic toxin binding to receptors, which would be applicable to MTβ research:

• Radioligand binding assays: These can be performed using membrane preparations expressing the receptor of interest. For example, Sf9 insect cells infected with baculovirus carrying the receptor gene have been effectively used. The binding assay typically involves incubating membrane preparations with radiolabeled ligands (e.g., [3H]prazosin for α1-adrenoceptors, [3H]rauwolscine for α2-adrenoceptors, or [3H]N-methylscopolamine for mAChRs) in the presence or absence of the toxin .

• Functional assays: Calcium mobilization assays using fura-2-loaded cells can be used to measure functional effects of MTβ on receptor signaling. In these assays, the toxin is allowed to bind to receptors for a specific period (e.g., 2-3 minutes for short-term incubations or ≥60 minutes for longer incubations) prior to agonist addition. The fura-2 fluorescence ratio (340 nm/380 nm) is then monitored to assess changes in intracellular calcium levels .

• Dissociation kinetics: To determine the mechanism of toxin binding, radioligand dissociation experiments can be conducted. These involve pre-incubating receptors with radiolabeled ligands, initiating dissociation with an excess of a competitive ligand, and then measuring bound radioactivity at different time points. The effect of the toxin on dissociation rates can provide insights into its binding mechanism .

What expression systems are most suitable for producing recombinant MTβ?

While the search results don't specifically address expression systems for recombinant MTβ, we can consider approaches used for similar toxins and proteins with multiple disulfide bonds:

• Bacterial expression systems (E. coli): These systems are cost-effective and scalable but may present challenges for properly forming the four disulfide bonds present in MTβ. Using specialized E. coli strains such as Origami or SHuffle, which have an oxidizing cytoplasmic environment, can improve correct disulfide bond formation. Alternatively, expressing the toxin as a fusion protein with thioredoxin or other solubility-enhancing tags might facilitate proper folding.

• Yeast expression systems (P. pastoris, S. cerevisiae): These eukaryotic systems provide better machinery for disulfide bond formation and post-translational modifications. Pichia pastoris, in particular, has been successful for expressing disulfide-rich proteins and might be suitable for MTβ.

• Insect cell expression (Sf9, Sf21, High Five): The baculovirus expression system in insect cells represents a good compromise between proper eukaryotic processing and reasonable costs. Given that Sf9 cells have been successfully used to express receptors for studying muscarinic toxin binding, this system might also be appropriate for producing the toxins themselves .

What purification strategies work best for isolating recombinant MTβ while maintaining biological activity?

Effective purification strategies for recombinant MTβ would likely include:

• Affinity chromatography: Using affinity tags such as His-tag, FLAG-tag, or GST fusion partners can facilitate initial capture of the recombinant toxin. These tags can be incorporated during the cloning process and, if necessary, removed by protease cleavage after purification.

• Ion exchange chromatography: Given that three-finger toxins typically have defined charge distributions, ion exchange chromatography (either cation or anion exchange depending on the toxin's isoelectric point) can be effective for separating the target toxin from impurities.

• Reversed-phase HPLC: This technique has been used to fractionate native black mamba venom and isolate muscarinic toxins, including dendrotoxins . For recombinant toxins, RP-HPLC can serve as a polishing step to achieve high purity.

• Size exclusion chromatography: This can be used as a final step to ensure homogeneity and remove any aggregates that might have formed during the purification process.

Throughout the purification process, it's crucial to maintain conditions that preserve the native disulfide bond arrangement, as this is essential for biological activity. This might include avoiding reducing agents and controlling pH and temperature to prevent disulfide scrambling.

How can researchers validate the structural integrity and biological activity of recombinant MTβ?

Validation of recombinant MTβ should include:

• Mass spectrometry: Electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) can confirm that the recombinant toxin has the expected molecular weight and, when combined with enzymatic digestion, can verify the primary sequence.

• Circular dichroism (CD) spectroscopy: This can assess the secondary structure content and compare it to native toxin to ensure proper folding.

• Binding assays: Radioligand displacement assays using receptor-expressing cell membranes can confirm that the recombinant toxin binds to the expected receptor targets with appropriate affinity .

• Functional assays: Measuring the toxin's ability to modulate receptor-mediated responses, such as calcium mobilization in cell-based assays, can validate functional activity .

• Comparison with native toxin: When possible, direct comparison with native toxin isolated from venom is the gold standard for validation. This should include both structural and functional parameters.

How can MTβ be used as a pharmacological tool for studying receptor function?

MTβ and other muscarinic toxins can serve as valuable pharmacological tools for studying receptor function in several ways:

• Receptor subtype identification: The differential binding affinities of various muscarinic toxins for receptor subtypes make them useful for identifying and characterizing receptor populations in native tissues or cell lines.

• Allosteric modulation studies: Muscarinic toxins typically bind non-competitively to receptors, as observed in radioligand displacement binding studies . This suggests they act as allosteric modulators, making them valuable for studying allosteric binding sites and interactions.

• Receptor dissociation kinetics: Muscarinic toxins can be used to study the dissociation kinetics of radioligands from receptors, providing insights into receptor conformational states and binding mechanisms .

• Cross-receptor family studies: Given that some muscarinic toxins like MTα and MT1 have been found to interact with adrenoceptors, these toxins can be used to explore structural and functional relationships between different G-protein-coupled receptor families .

What considerations are important when designing experiments using MTβ for receptor studies?

When designing experiments with MTβ, researchers should consider:

• Non-competitive binding mechanism: Unlike many small-molecule ligands, muscarinic toxins often bind non-competitively to receptors . This should be accounted for in experimental design and data interpretation, particularly in binding assays.

• Incubation time: The time allowed for toxin binding can significantly impact results. For short-term experiments, 2-3 minutes of pre-incubation may be sufficient, while longer-term effects might require ≥60 minutes of incubation .

• Buffer composition: The composition of assay buffers, particularly the presence of divalent cations like Mg2+ and Ca2+, can affect toxin binding to receptors and should be carefully controlled.

• Expression system for receptors: The choice of expression system for the target receptors can influence toxin binding. Sf9 insect cells have been successfully used for expressing receptors in toxin binding studies , but other systems might be appropriate depending on the specific research questions.

• Potential cross-reactivity: The possibility that MTβ might interact with multiple receptor types should be considered when interpreting results, particularly in native tissues with mixed receptor populations.

What methods can be used to develop antibodies or antidotes against MTβ toxicity?

Recent advances in developing recombinant antivenoms suggest several approaches for generating antibodies or antidotes against MTβ:

• Phage display technology: This approach has been successfully used to develop fully human immunoglobulin G (IgG) monoclonal antibodies against black mamba dendrotoxins . A similar approach could be applied to MTβ, involving:

  • Identifying and isolating the toxin

  • Selecting antibody-displaying phages that bind to the toxin

  • Expressing and purifying the selected antibodies as full IgGs

• Combined toxicovenomics and antibody discovery: This integrated approach involves fractionating venom to isolate specific toxins (like MTβ), followed by targeted antibody discovery against those components .

• Oligoclonal antibody mixtures: Rather than a single monoclonal antibody, a cocktail of antibodies targeting different epitopes on MTβ might provide more effective neutralization, similar to the approach used for dendrotoxin neutralization .

• In vivo validation: The protective capacity of antibodies against MTβ would need to be validated in appropriate models. For dendrotoxin antibodies, rodent models were used to assess neutralization of whole venom neurotoxicity .

How might structural modifications of recombinant MTβ alter its receptor selectivity?

Structure-function relationships in muscarinic toxins provide insights for potential modifications:

• Targeted mutations in loop regions: The three finger loops of muscarinic toxins are involved in receptor binding . Mutations in these regions could alter receptor selectivity, as demonstrated by the different selectivity profiles of MTα and MT4, which differ in only three amino acids (residues 31-33) .

• Disulfide bond engineering: The four disulfide bonds in MTβ are crucial for maintaining its tertiary structure. Selective modification of disulfide patterns might alter loop flexibility and orientation, potentially affecting receptor interactions.

• Chimeric toxins: Creating chimeric toxins that combine regions from different muscarinic toxins with distinct selectivity profiles could yield novel pharmacological tools with customized receptor targeting. For example, combining regions from MTβ with the highly selective MT7 might produce toxins with unique selectivity profiles.

• Post-translational modifications: Introducing specific post-translational modifications (e.g., glycosylation, phosphorylation) at strategic positions might influence receptor interactions and tissue distribution of the modified toxin.

What role could computational modeling play in understanding MTβ-receptor interactions?

Computational approaches offer powerful tools for studying MTβ-receptor interactions:

• Homology modeling: When crystal structures are unavailable, homology models of MTβ based on related toxins with known structures can provide insights into its three-dimensional conformation.

• Molecular docking: Docking simulations of MTβ with various receptor subtypes can predict binding modes and key interaction residues, guiding experimental verification.

• Molecular dynamics simulations: These can reveal dynamic aspects of toxin-receptor interactions, including conformational changes upon binding and the stability of the complex over time.

• Structure-based virtual screening: Computational screening of compound libraries against modeled MTβ-receptor complexes could identify potential small-molecule mimetics or antagonists of the toxin.

What are the prospects for using MTβ as a template for developing therapeutic agents?

The unique properties of MTβ and related toxins present several opportunities for therapeutic development:

• Highly selective receptor ligands: The selectivity of some muscarinic toxins for specific receptor subtypes makes them valuable templates for developing targeted therapeutic agents. For example, the absolute selectivity of MT7 for M1 receptors could inspire the design of selective drugs for conditions involving this receptor subtype.

• Allosteric modulators: The non-competitive binding mode of muscarinic toxins suggests they act as allosteric modulators . This property could be exploited to develop drugs that modulate receptor function without competing with endogenous ligands.

• Peptidomimetics: Small-molecule compounds that mimic the key binding elements of MTβ but have improved drug-like properties could be developed based on detailed understanding of toxin-receptor interactions.

• Biotoxin-derived therapeutic antibodies: The approach used to develop recombinant antibodies against black mamba dendrotoxins could be adapted to create therapeutic antibodies targeting MTβ or related toxins, potentially leading to improved antivenom treatments.