Recombinant Polistes lanio Tachykinin-like peptide-II

What is Recombinant Polistes lanio Tachykinin-like peptide-II?

Recombinant Polistes lanio Tachykinin-like peptide-II (PllTkP-II) is a synthetically produced version of a naturally occurring peptide isolated from the venom of Polistes lanio, a paper wasp species found predominantly in southeastern Brazil. This 17-amino acid peptide belongs to the tachykinin family and shares structural homology with C-terminal regions of tachykinin-like peptides found in various venomous species and vertebrates. The peptide plays a significant role in mediating inflammatory responses through interactions with tachykinin receptors .

What is the amino acid sequence and structural characteristics of PllTkP-II?

The complete amino acid sequence of PllTkP-II is "ASEPTALGLPRIFPGLM" as identified through mass spectrometric analysis . This sequence shows significant C-terminal homology with mammalian tachykinins, particularly in the "FPGLM" motif which is critical for receptor binding and biological activity. The peptide has a molecular weight within the range of other bioactive peptides identified in P. lanio venom (MW 1173-3581) .

How does PllTkP-II compare to other tachykinins?

PllTkP-II functions similarly to mammalian tachykinins, particularly Substance P, as evidenced by its inflammatory effects being mediated through NK1 receptors. Mass spectrometric analysis revealed that P. lanio venom contains at least two tachykinin-like peptides: QPPTPPEHRFPGLM and ASEPTALGLPRIFPGLM (PllTkP-II), both sharing C-terminal sequence similarities with tachykinins found in Phoneutria nigriventer spider venom and vertebrates . The functional similarities suggest evolutionary conservation of tachykinin-like peptides across diverse venomous species, despite variations in the N-terminal regions.

What are the primary biological effects of PllTkP-II?

PllTkP-II induces potent inflammatory responses characterized by:

• Dose-dependent increases in microvascular permeability in rodent skin

• Long-lasting paw edema formation

• Plasma protein extravasation

• Activation of sensory C-fibers

• Stimulation of neurogenic inflammatory pathways

These effects closely mimic those of endogenous neuropeptide Substance P, suggesting evolutionary convergence toward similar inflammatory mechanisms.

Through what receptor pathways does PllTkP-II exert its effects?

• Significant inhibition of PllTkP-II-induced edema by the NK1 receptor antagonist SR140333

• Lack of inhibition by the NK2 receptor antagonist SR48968

• Reduced inflammatory response in capsaicin-treated animals (which depletes sensory neuropeptides)

• Partial inhibition by histamine H1 receptor antagonist pyrilamine

The data suggest a multi-step inflammatory cascade where PllTkP-II activates NK1 receptors, which subsequently leads to histamine release from dermal mast cells, amplifying the inflammatory response.

What is the neurogenic inflammatory mechanism of PllTkP-II?

PllTkP-II induces neurogenic inflammation through a well-defined pathway:

• The peptide activates sensory C-fibers

• This activation triggers the release of Substance P

• Substance P binds to NK1 receptors on vascular endothelial cells

• NK1 receptor activation increases microvascular permeability

• Histamine is released from dermal mast cells as a secondary mediator

• The combined effect results in plasma extravasation and edema formation

This represents the first described neurovascular mechanism for P. lanio venom-mediated inflammation, distinguishing it from direct mast cell degranulation pathways seen with other venoms.

What experimental models are most suitable for studying PllTkP-II effects?

Based on published research, the following experimental models have proven effective:

The choice of model should be based on the specific research question, with rodent models being particularly useful for in vivo assessment of inflammatory parameters .

How should recombinant PllTkP-II be reconstituted and stored?

For optimal stability and bioactivity:

• Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (recommended default: 50%)

• Store at -20°C for regular storage

• For extended storage, maintain at -20°C or -80°C

• Avoid repeated freeze-thaw cycles

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

Proper storage conditions are crucial for maintaining peptide integrity and biological activity over time.

What methods are effective for quantifying PllTkP-II-induced inflammation?

Several complementary approaches have been validated:

• Plasma extravasation assessment: Intradermal injection of test agents into shaved dorsal skin followed by measurement of 125I-albumin accumulation after a 30-minute period

• Edema measurement: Quantification of tissue swelling following intradermal or subplantar injection of the peptide

• Pharmacological intervention: Pretreating animals with receptor antagonists (e.g., SR140333 for NK1, SR48968 for NK2, HOE 140 for bradykinin B2, Des-Arg9-[Leu8]-BK for B1, pyrilamine for histamine H1) to identify receptor involvement

• Sensory fiber depletion: Capsaicin pretreatment to deplete neuropeptides from sensory neurons, establishing the neurogenic component of inflammation

How can PllTkP-II be used to study receptor-ligand interactions?

Researchers can employ PllTkP-II as a tool to investigate tachykinin receptor pharmacology through:

• Competitive binding assays with known NK1 receptor ligands

• Structure-activity relationship studies by creating sequence variants

• Cross-species receptor comparisons to examine evolutionary conservation

• Functional assays measuring downstream signaling cascades

• Receptor internalization and trafficking studies

The specific interaction of PllTkP-II with NK1 receptors makes it valuable for understanding receptor subtype selectivity mechanisms .

What is the significance of PllTkP-II in comparative venom research?

PllTkP-II represents an important element in understanding evolutionary convergence in venom composition:

• It demonstrates how phylogenetically distant organisms (wasps, spiders, vertebrates) have evolved similar peptide components

• Provides insight into the selection pressures that drive venom evolution

• Allows comparison of receptor targeting strategies across venomous species

• Highlights the conservation of inflammatory mechanisms across animal phyla

• Offers potential templates for developing novel therapeutic agents

Comparative studies between wasp, spider, and snake venom peptides have revealed remarkable structural and functional similarities despite diverse evolutionary origins.

How can contradictory experimental results with PllTkP-II be resolved?

When encountering inconsistent results:

• Peptide purity assessment: Verify recombinant peptide purity (>85% by SDS-PAGE is standard)

• Storage condition validation: Improper storage can lead to peptide degradation and inconsistent activity

• Species-specific differences: Consider variation in receptor pharmacology between experimental animals

• Methodological standardization: Ensure consistent reconstitution protocols and dosing regimens

• Contextual effects: The presence of other inflammatory mediators can modify PllTkP-II activity

• Receptor expression profiling: Quantify NK1 receptor expression in target tissues before experiments

Comprehensive controls and standardized protocols are essential for resolving apparent contradictions in experimental outcomes.

What control experiments should be included when studying PllTkP-II?

A robust experimental design should include:

• Dose-response studies to establish EC50 values (effective concentration eliciting 50% of maximum response)

• Comparison with dialyzed venom to separate peptide effects from other venom components

• Positive controls using known NK1 agonists (e.g., Substance P)

• Vehicle controls to account for potential solvent effects

• Time-course experiments to characterize the temporal profile of inflammatory responses

• Appropriate receptor antagonist controls at validated concentrations

How should differences between native and recombinant PllTkP-II be addressed?

Researchers should account for potential differences by:

• Comparing biological activity between native venom-derived and recombinant peptides

• Assessing post-translational modifications that may be present in native but not recombinant peptides

• Evaluating synergistic effects with other venom components

• Considering the impact of expression tags on recombinant peptide function

• Conducting parallel experiments with both peptide forms when feasible

Native P. lanio venom contains multiple bioactive components that may work synergistically, while recombinant PllTkP-II allows for isolated study of this specific peptide's effects .

What are the technical challenges in working with recombinant tachykinin peptides?

Key challenges include:

• Expression efficiency: Small peptides often express poorly in recombinant systems

• Maintaining structural integrity: Ensuring proper folding and disulfide bond formation

• Purification complexity: Separating the peptide from host cell proteins

• Activity verification: Confirming biological activity matches native peptide

• Stability issues: Preventing degradation during storage and experimentation

• Batch consistency: Achieving reproducible production between batches

Baculovirus expression systems have proven effective for PllTkP-II production, though researchers should verify each batch's activity before use .