Recombinant Lachesana tarabaevi M-zodatoxin-Lt8b (cit 1-2)
Description
Overview of Lachesana tarabaevi Venom Peptides
Latarcins are short, linear antimicrobial and cytolytic peptides isolated from the venom of the spider Lachesana tarabaevi. They exhibit broad-spectrum activity against Gram-positive and Gram-negative bacteria, fungi, and tumor cells, primarily through membrane destabilization via a "carpet-like" mechanism . While "M-zodatoxin-Lt8b" is not explicitly mentioned in the provided sources, latarcins such as Ltc-2a and Ltc-3a have been structurally and functionally characterized:
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Structural Motif: Latarcins adopt an amphipathic α-helical conformation in membrane-mimicking environments .
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Mechanism: They disrupt microbial membranes by forming transient pores or inducing lipid bilayer curvature .
Rational Design of Synthetic Analogs
The development of synthetic analogs (e.g., Lt-MAP1, Lt-MAP2, Lt-MAP3) from latarcin-3a demonstrates key principles that could apply to recombinant variants like "M-zodatoxin-Lt8b":
Key Design Modifications
| Parameter | Lt-MAP1 | Lt-MAP2 | Lt-MAP3 |
|---|---|---|---|
| Charge | +2 | +4 | +5 |
| Hydrophobicity | Moderate | High | High |
| MIC Range | 8–128 μg/ml | 8–128 μg/ml | 4–128 μg/ml |
| Antitumor EC₅₀ | Inactive | 88.7 μM (C1498) | Inactive |
Source:
Functional Outcomes
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Antimicrobial Activity: Lt-MAP3 showed the strongest antibacterial effects against Acinetobacter baumannii and Escherichia coli (MIC = 4–64 μg/ml) .
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Antitumor Activity: Lt-MAP2 reduced viability of myeloid leukemia cells (C1498) to 18% at 50 μM .
Methodologies for Recombinant Peptide Production
While the search results focus on synthetic peptides, recombinant production of latarcins would likely involve:
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Gene Synthesis: Codon-optimized sequences for expression in E. coli or yeast systems.
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Purification: Affinity chromatography (e.g., His-tag systems) followed by HPLC .
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Structural Validation: MALDI-TOF mass spectrometry and NMR for confirmation of disulfide bonds and α-helical content .
Critical Research Gaps and Opportunities
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Selectivity Optimization: Natural latarcins (e.g., Ltc-1, Ltc-2a) show hemolytic activity, whereas analogs like Lt-MAP2 achieve tumor cell selectivity (no hemolysis at 128 μg/ml) .
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Mechanistic Studies: Molecular dynamics simulations could refine understanding of lipid-peptide interactions for improved therapeutic windows .
Comparative Data on Latarcin Activities
Table 1. Biological Activities of Select Latarcins
| Peptide | Antibacterial MIC (μg/ml) | Antitumor EC₅₀ (μM) | Hemolysis (128 μg/ml) |
|---|---|---|---|
| Ltc-3a | 4–128 | 51–74.4 | None |
| Ltc-2a | 2–16 | Not tested | High |
| Lt-MAP2 | 8–128 | 64.8–88.7 | None |
Sources:
Future Directions
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Multifunctional Engineering: Combining antimicrobial and antitumor epitopes into single constructs.
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Delivery Systems: Nanocarriers to enhance stability and biodistribution in vivo.
Product Specs
Target Background
Q&A
Basic Research Questions
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What is the molecular composition of Lachesana tarabaevi venom and how does M-zodatoxin-Lt8b fit into this profile?
Lachesana tarabaevi venom has a unique molecular composition, relying primarily on linear cytolytic polypeptides without disulfide bridges, unlike most other spider venoms that contain disulfide-rich neurotoxic peptides. Approximately 80% of the total venom protein exhibits membrane-active properties . The venom contains 33 membrane-interacting polypeptides (ranging from 18-79 amino acid residues) that comprise five major groups: repetitive polypeptide elements (Rpe), latarcins (Ltc), met-lysines (MLys), cyto-insectotoxins (CIT), and latartoxins (LtTx) . M-zodatoxin-Lt8b belongs to the CIT family, specifically the CIT 1-2 subfamily, which constitutes the primary toxic component accounting for the venom's potency .
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What structural characteristics define CIT family toxins from Lachesana tarabaevi?
CIT family toxins from Lachesana tarabaevi display several distinctive structural features:
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They are larger peptides (61-79 amino acid residues) compared to other venom components
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They possess a modular structure consisting of two shorter Ltc-like peptides joined together
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They adopt amphipathic α-helical structures in membrane-mimicking environments
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They lack disulfide bridges that are common in other spider toxins
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Toxins from the CIT 1 and 2 families specifically demonstrate that the two parts act synergistically when covalently linked
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What is the general mechanism of action for membrane-active peptides from Lachesana tarabaevi?
Peptides from Lachesana tarabaevi, including the CIT family toxins, primarily act through a "carpet-like" model of membrane disruption. In this mechanism, peptides accumulate parallel to the membrane surface until reaching a threshold concentration, at which point they disrupt membrane integrity . Planar lipid bilayer studies indicate these peptides cause scaled membrane destabilization at physiological membrane potential . This mechanism allows them to produce lytic effects on diverse cell types, including Gram-positive and Gram-negative bacteria, erythrocytes, and yeast at micromolar concentrations . Some latarcins, such as Ltc-3a, have additionally demonstrated inhibition of E. coli ATP synthase with amidated C-terminal .
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How are precursor proteins for Lachesana tarabaevi toxins structured and processed?
For every latarcin and related toxin from Lachesana tarabaevi, a precursor protein sequence has been identified. Based on structural features, these precursors are categorized into three distinct groups:
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Simple precursors with a conventional prepropeptide structure
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Binary precursors with a typical modular organization
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Complex precursors which are cleaved into mature chains of two different types
The presence of an inverted processing quadruplet motif (iPQM) has been confirmed to mark cleavage sites in spider toxin precursors that are processed into several mature chains, as evidenced by the isolation of Rpe peptides that are encoded by the same genes as antimicrobial peptides Ltc 4a and 4b .
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What analytical methods are used to confirm the identity of recombinant Lachesana tarabaevi toxins?
The identity and purity of recombinant or synthetic peptides derived from Lachesana tarabaevi are typically confirmed using Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-ToF-MS) . The methodology involves:
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Resuspending peptides in ultra-pure water
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Mixing with a saturated solution of α-cyano-4-hydroxycinnamic acid (10 mg/ml in 50% acetonitrile, 0.1% trifluoroacetic acid) in a 1:3 ratio
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Direct application onto a massive plate
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Acquisition of monoisotopic mass spectra in reflected mode with a range of 700–3,500 m/z
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MS/MS spectra acquisition using the MS/MS LIFT method
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Manual determination of primary sequence using FlexAnalysis 3.3 software
Advanced Research Questions
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What methodological approaches are most effective for evaluating antimicrobial activity of recombinant M-zodatoxin-Lt8b?
The evaluation of antimicrobial activity for recombinant peptides derived from Lachesana tarabaevi involves multiple complementary approaches:
| Test Type | Methodology | Measurement | Interpretation |
|---|---|---|---|
| Minimum Inhibitory Concentration (MIC) | 96-well microplate dilution method with 2–5 × 10^5 CFU/ml bacterial concentration | Spectrophotometric reading at 600 nm after 18h incubation | Lowest concentration with no significant bacterial growth |
| Minimum Bactericidal Concentration (MBC) | Plating 10 μl from MIC wells onto MHA | Visual inspection after 24h incubation | Lowest concentration with no visible bacterial growth |
| Anti-Biofilm Activity | Crystal violet staining of adherent cells after peptide treatment | Absorbance at 595 nm after solubilization with 60% ethanol | Concentration preventing biofilm formation |
These tests should be conducted against both Gram-positive bacteria (e.g., S. aureus) and Gram-negative bacteria (e.g., E. coli, P. aeruginosa) with appropriate controls such as ciprofloxacin. All experiments should be performed in biological and technical triplicates for statistical validity .
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How can researchers optimize recombinant expression systems for CIT family toxins?
While specific expression systems for M-zodatoxin-Lt8b are not directly detailed in the literature, optimization strategies for recombinant expression of membrane-active peptides typically include:
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Selection of appropriate host systems that can tolerate potentially toxic peptides
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Use of fusion partners to reduce toxicity to the expression host
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Employment of periplasmic or extracellular secretion to minimize host cell damage
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Codon optimization for the expression host
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Temperature and induction condition optimization to balance yield and proper folding
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Consideration of the complex precursor structures identified in Lachesana tarabaevi toxins, which may require specialized processing enzymes
Understanding the precursor protein structure is particularly important as CIT toxins have evolved through the joining of two shorter membrane-active peptides into one larger molecule . Expression systems must accurately process these complex structures to maintain the synergistic activity between the domains.
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What structural modifications can enhance the selectivity and potency of M-zodatoxin-Lt8b for specific cellular targets?
Based on research with rationally designed peptide analogs derived from latarcin-3a, several structural modifications can potentially enhance selectivity and potency:
These modifications should be guided by understanding the relationship between physical-chemical parameters (charge, hydrophobicity, polypeptide chain length, and amino acid composition) and interaction with specific target membranes .
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How does the modular structure of CIT family toxins contribute to their functional synergy?
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Complementary membrane targeting, where each domain interacts with different aspects of the membrane
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Increased local concentration effect, as both active components are delivered simultaneously to the target site
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Structural stabilization that enhances resistance to degradation in physiological environments
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Optimized amphipathicity created by the combination of the two domains
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Potential cooperative binding to membrane targets that enhances the membrane-disrupting capabilities
This evolutionary development through the joining of two shorter membrane-active peptides into one larger molecule represents a significant advancement in venom efficacy and offers insights for the rational design of novel antimicrobial compounds .
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What techniques are most effective for studying the membrane interaction dynamics of recombinant M-zodatoxin-Lt8b?
While specific techniques for studying M-zodatoxin-Lt8b are not detailed in the provided literature, several approaches have proven effective for related Lachesana tarabaevi toxins:
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Circular Dichroism (CD) spectroscopy to analyze secondary structure formation in membrane-mimicking environments
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Planar lipid bilayer studies to observe membrane destabilization at physiological membrane potential
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Fluorescent dye leakage assays to quantify membrane permeabilization
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Surface plasmon resonance to measure binding kinetics to model membranes
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Molecular dynamics simulations to visualize peptide-membrane interactions at the atomic level
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Confocal microscopy with fluorescently labeled peptides to track cellular localization
These techniques can provide complementary information about the mechanism of action, helping researchers understand how the modular structure of CIT toxins contributes to their membrane-disrupting capabilities and cytolytic effects.
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How do synthetic peptide analogs derived from Lachesana tarabaevi toxins compare in efficacy to natural peptides?
Research comparing synthetic peptide analogs (Lt-MAP1, Lt-MAP2, and Lt-MAP3) derived from latarcin-3a with the natural peptide revealed distinct activity profiles:
| Peptide | Antibacterial Activity | Antitumor Activity | Hemolytic Activity |
|---|---|---|---|
| Ltc-3a (natural) | Active against various bacteria | Moderate activity | Low at tested concentrations |
| Lt-MAP1 | MIC between 4-128 μg/ml | Moderate activity | None at 128 μg/ml |
| Lt-MAP2 | MIC between 4-128 μg/ml | Best antitumor activity | None at 128 μg/ml |
| Lt-MAP3 | Best antibacterial activity | Moderate activity | None at 128 μg/ml |
The synthetic analogs demonstrated differential activities against various targets, with Lt-MAP3 showing optimal antibacterial properties and Lt-MAP2 exhibiting superior antitumor activity. Importantly, none of the synthetic peptides promoted hemolysis at concentrations up to 128 μg/ml, indicating good selectivity for microbial cells over mammalian erythrocytes .
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What strategies can overcome challenges in maintaining structural integrity during recombinant expression of M-zodatoxin-Lt8b?
Several strategies can address challenges in maintaining structural integrity during recombinant expression of complex peptides like M-zodatoxin-Lt8b:
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Expression as fusion proteins with solubility-enhancing partners (e.g., thioredoxin, SUMO, or MBP)
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Use of specialized E. coli strains optimized for difficult protein expression
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Low-temperature induction to slow folding and prevent aggregation
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Co-expression with molecular chaperones to assist proper folding
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Purification under native conditions to preserve structural elements
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Refolding protocols optimized for amphipathic α-helical peptides if inclusion bodies form
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Consideration of eukaryotic expression systems for complex post-translational modifications
Understanding the natural precursor processing is crucial, as Lachesana tarabaevi toxin precursors are split into three groups (simple, binary, and complex precursors) with different processing requirements . Design of expression constructs should account for these natural processing pathways to maximize the yield of correctly folded, biologically active recombinant toxin.
