Recombinant Laticauda laticaudata Short neurotoxin OKI-10

What is Short neurotoxin OKI-10 and what is its source organism?

Short neurotoxin OKI-10 is a potent neurotoxin derived from Laticauda laticaudata, commonly known as the Blue-ringed sea krait or Blue-lipped sea krait. It belongs to the short neurotoxin family, which typically contains 60-62 amino acid residues with four disulfide bridges. The toxin binds to muscle nicotinic acetylcholine receptors (nAChR) and prevents acetylcholine from binding, thereby inhibiting neuromuscular transmission . Laticauda laticaudata is an amphibious sea snake found primarily in the Ryukyu-Taiwan region and other parts of the Western Pacific .

What is the molecular structure of Recombinant Laticauda laticaudata Short neurotoxin OKI-10?

The recombinant Short neurotoxin OKI-10 comprises positions 21-82 of the full-length protein with the amino acid sequence: RRCFNQQSSE PQTNKSCPPG ENSCYRKQWR DHRGTIIERG CGCPTVKPGV KLRCCESEDC NN. It has a molecular weight of approximately 9,249 Da . Like other short neurotoxins, it likely possesses a three-finger fold structure stabilized by disulfide bridges, which is characteristic of three-finger toxins (3FTX) found in elapid venoms .

What are the standard protocols for reconstituting and storing Recombinant Short neurotoxin OKI-10?

For optimal results when working with recombinant Short neurotoxin OKI-10:

Reconstitution Protocol:

• The lyophilized protein should be briefly centrifuged to ensure all material is at the bottom of the vial.

• Reconstitute in sterile, ultra-pure water or appropriate buffer depending on experimental requirements.

• Gentle mixing is recommended rather than vortexing to prevent protein denaturation.

Storage Recommendations:

• Store the lyophilized form at -20°C for long-term stability.

• For extended storage, maintain at -80°C to prevent degradation.

• Working aliquots can be stored at 4°C for up to one week.

• Avoid repeated freeze-thaw cycles as this may compromise protein activity .

How can researchers verify the activity of Recombinant Short neurotoxin OKI-10 in their experiments?

Verification of Short neurotoxin OKI-10 activity can be achieved through several complementary approaches:

• Electrophysiological Assays: Patch-clamp techniques to measure inhibition of acetylcholine-induced currents in cells expressing nicotinic acetylcholine receptors.

• Competitive Binding Assays: Using radiolabeled α-bungarotoxin or other known nAChR ligands to measure competitive displacement by OKI-10.

• Functional Assays: Ex vivo nerve-muscle preparations (e.g., mouse phrenic nerve-diaphragm preparation) to assess neuromuscular transmission blockade.

• Cell Viability Assays: In cell lines expressing nAChRs to determine cytotoxicity profiles at varying concentrations.

Activity should be compared against established reference standards where possible, with the understanding that recombinant toxins may exhibit variations in potency compared to native proteins.

What genetic factors influence the expression and potency of Short neurotoxin OKI-10 in different Laticauda laticaudata populations?

Genetic analysis of Laticauda laticaudata populations reveals significant genetic differentiation among island groups in the Ryukyu-Taiwan region. Sequence analyses of mitochondrial cytochrome b gene have identified at least four distinct haplotypes across 136 individuals studied . This genetic differentiation is particularly pronounced among islands separated by deep straits, suggesting geographic isolation plays a crucial role in genetic divergence.

The genetic isolation may influence neurotoxin expression patterns, potentially leading to variations in venom composition and potency across different populations. Population pairwise FST analyses revealed significant genetic differentiations even among islands within the same basin for L. laticaudata, indicating stronger site fidelity or philopatry compared to related species like L. semifasciata . These genetic variations could correspond to differences in neurotoxin potency or structural variations that might affect receptor binding affinities.

How does recombinant expression system choice affect the structure and function of Short neurotoxin OKI-10?

The choice of expression system significantly impacts the quality and activity of recombinant Short neurotoxin OKI-10:

The critical concern for Short neurotoxin OKI-10 expression is ensuring proper disulfide bridge formation, as these structural elements are essential for the three-finger toxin fold and functional binding to acetylcholine receptors . Comparing recombinant toxins expressed in different systems against native toxin standards is advisable to validate structural and functional integrity.

What methodological approaches are effective for studying the interaction between Short neurotoxin OKI-10 and nicotinic acetylcholine receptors?

Several complementary methodologies can elucidate the interaction between Short neurotoxin OKI-10 and nAChRs:

• X-ray Crystallography and Cryo-EM: These structural biology techniques can resolve the three-dimensional structure of OKI-10 bound to its receptor, revealing binding sites and molecular interactions. This requires successful co-crystallization of the toxin-receptor complex or stable complex formation for cryo-EM analysis.

• Surface Plasmon Resonance (SPR): Provides real-time binding kinetics (kon and koff rates) and affinity constants (KD) between OKI-10 and immobilized nAChR or receptor fragments. This approach allows quantitative comparison with other neurotoxins.

• Isothermal Titration Calorimetry (ITC): Measures thermodynamic parameters of binding interactions, providing insights into enthalpy and entropy contributions to binding energy.

• Site-Directed Mutagenesis: Systematic mutation of residues in both OKI-10 and nAChR followed by binding and functional assays can identify critical residues for the interaction.

• Molecular Dynamics Simulations: Computational approaches can model the dynamic interaction between OKI-10 and nAChR, predicting conformational changes and energetics of binding.

By integrating multiple approaches, researchers can develop a comprehensive understanding of the molecular basis for OKI-10's high affinity and specificity for nAChRs.

How does Short neurotoxin OKI-10 compare functionally with neurotoxins from other Laticauda species?

While specific comparative data between OKI-10 and other Laticauda neurotoxins is limited in the provided sources, general patterns of neurotoxin evolution and function in related species can be informative. Erabutoxins from Laticauda semifasciata represent well-characterized short-chain neurotoxins that share functional similarities with OKI-10 .

Based on studies of related species, short-chain alpha-neurotoxins (SNTX) like OKI-10 generally exhibit:

• Similar potency to long-chain neurotoxins (LNTX) in animal models (LD₅₀ ~0.1–0.2 μg/g in rodents, intravenously)

• Weaker binding affinity to human nicotinic acetylcholine receptors compared to LNTX

• Limited antigenicity and potentially lower neutralization by antivenom

The molecular evolution of these neurotoxins appears to follow geographical distribution patterns, with SNTX evolving to replace LNTX in certain Asiatic species moving eastward in their range expansion .

What phylogenetic insights can be gained from comparative analysis of Short neurotoxin OKI-10 with other three-finger toxins?

Phylogenetic analysis of three-finger toxins (3FTXs) including Short neurotoxin OKI-10 can provide valuable insights into evolutionary relationships and functional adaptations:

• Evolutionary Origins: 3FTXs constitute >50% of total venom proteins in many elapid species, suggesting strong evolutionary selection for this toxin family .

• Functional Diversification: The 3FTX family has diversified into multiple subtypes with varied functions (neurotoxic, cytotoxic, cardiotoxic), reflecting adaptive radiation in response to ecological pressures.

• Geographical Patterns: A phenotypic venom dichotomy exists in related species, with SNTX replacing LNTX as species dispersed eastward through the Asia-Pacific region, potentially representing convergent evolution in response to similar ecological pressures .

• Molecular Evolution: Sequence comparison of OKI-10 with other 3FTXs could identify conserved structural elements essential for folding versus variable regions associated with target specificity and potency.

The neurotoxin genes typically contain conserved exon-intron structures, with variations in the mature toxin regions that influence binding specificity and potency. A complete phylogenetic analysis would require sequence data from multiple species to reconstruct evolutionary relationships and selective pressures acting on these toxins.

What are the technical challenges in producing high-purity recombinant Short neurotoxin OKI-10 for structural studies?

Producing high-purity recombinant Short neurotoxin OKI-10 for structural studies presents several technical challenges:

Approaches to overcome these challenges include:

• Utilizing eukaryotic expression systems such as mammalian cells for complex disulfide bond formation

• Incorporating solubility-enhancing fusion tags that can be cleaved post-purification

• Developing optimized refolding protocols specific to Short neurotoxin OKI-10

• Implementing quality control procedures to verify structural integrity and homogeneity

How might Short neurotoxin OKI-10 be utilized in developing novel research tools for neuroscience?

Short neurotoxin OKI-10 offers significant potential as a research tool in neuroscience:

• Receptor Subtype Characterization: The specific binding properties of OKI-10 to nAChR subtypes could be leveraged to develop probes for identifying and quantifying receptor subtypes in tissues.

• Fluorescent Conjugates: Conjugation with fluorescent molecules could create tools for visualizing nAChR distribution and trafficking in live cells and tissues.

• Affinity Chromatography: Immobilized OKI-10 could be used for purification of nAChRs from complex biological samples.

• Structure-Based Drug Design: Detailed understanding of OKI-10's binding mechanism could guide the development of peptide-based therapeutics targeting nAChRs.

• Biotechnological Applications: Engineered variants of OKI-10 could serve as biosensors for detecting compounds that interact with nAChRs, potentially useful in drug discovery or environmental monitoring.

Future research directions might focus on:

• Development of recombinant OKI-10 variants with enhanced specificity for particular nAChR subtypes

• Creation of minimized functional domains that retain binding specificity but with improved production characteristics

• Integration of OKI-10-based tools into high-throughput screening platforms for neuropharmacology

What are effective strategies for analyzing structure-function relationships in Short neurotoxin OKI-10?

Elucidating structure-function relationships in Short neurotoxin OKI-10 requires an integrated approach:

• Alanine Scanning Mutagenesis: Systematic replacement of residues with alanine to identify critical amino acids for binding and toxicity.

• Chimeric Toxin Construction: Creating hybrid toxins by exchanging domains between OKI-10 and related neurotoxins to map functional regions.

• Truncation Analysis: Generating truncated versions of OKI-10 to determine the minimal functional domain.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): This technique can provide information about protein dynamics and solvent accessibility, revealing regions involved in conformational changes upon binding.

• Computational Approaches:

  • Molecular dynamics simulations to study conformational flexibility

  • In silico docking to predict binding orientations with nAChR subtypes

  • Quantum mechanics/molecular mechanics (QM/MM) to analyze detailed binding energetics

A successful structure-function analysis would typically progress through these stages:

• Initial structural characterization (X-ray crystallography, NMR)

• In silico prediction of potential functional residues

• Experimental validation through mutagenesis

• Functional assays to quantify effects on binding and toxicity

• Refinement of structural models based on experimental findings

This iterative approach can reveal the molecular basis for OKI-10's specificity and provide insights for designing variants with altered properties.

What bioinformatic approaches are useful for analyzing Short neurotoxin OKI-10 in the context of venom evolution?

Bioinformatic approaches provide powerful tools for understanding Short neurotoxin OKI-10 in an evolutionary context:

• Sequence Alignment and Phylogenetic Analysis:

  • Multiple sequence alignment of OKI-10 with other 3FTXs

  • Construction of phylogenetic trees to infer evolutionary relationships

  • Calculation of sequence conservation scores to identify functionally important regions

• Selection Pressure Analysis:

  • Calculation of nonsynonymous/synonymous substitution rates (dN/dS) to identify regions under positive selection

  • Codon-based tests for detecting adaptive evolution in specific lineages

  • Identification of episodic selection using branch-site models

• Ancestral Sequence Reconstruction:

  • Inference of ancestral toxin sequences to track evolutionary changes

  • Experimental resurrection of ancestral toxins to study functional evolution

• Protein Structure Prediction and Comparison:

  • Homology modeling of OKI-10 based on related toxins with known structures

  • Structural alignment to identify conserved structural elements versus variable regions

  • Prediction of binding sites and comparison across related toxins

• Genomic Context Analysis:

  • Analysis of gene structure and regulatory regions

  • Identification of potential gene duplication events

  • Investigation of syntenic relationships with other venom genes

These approaches can reveal how Short neurotoxin OKI-10 evolved its specific properties and how it relates to the broader adaptive radiation of three-finger toxins in sea snakes and other elapids.