Recombinant Pig Interleukin-4 receptor subunit alpha (IL4R), partial

What is the basic structure of recombinant pig IL4R?

Recombinant pig Interleukin-4 receptor subunit alpha (IL4R) is a fragment protein typically comprising amino acids 33-240 of the extracellular domain. The protein has a molecular mass of approximately 26.2 kDa and belongs to the type I cytokine receptor family, type 4 subfamily . The recombinant protein is commonly expressed with an N-terminal 6xHis-tag to facilitate purification and detection in experimental protocols . The protein is generally produced with greater than 85% purity as determined by SDS-PAGE, making it suitable for a range of biochemical and immunological applications .

The amino acid sequence of the extracellular domain contains several functionally significant regions that mediate ligand binding and signal transduction. The protein structure includes multiple cysteine residues that form disulfide bonds critical for maintaining the tertiary structure necessary for proper ligand recognition .

What are the key functional roles of IL4R in immunological signaling?

IL4R serves as a receptor for both interleukin-4 (IL-4) and interleukin-13 (IL-13), coupling to the JAK1/2/3-STAT6 pathway upon ligand binding . The IL-4 response mediated through this receptor is involved in promoting T helper type 2 (Th2) cell differentiation, a critical process in adaptive immunity . Additionally, IL-4/IL-13 responses through this receptor regulate immunoglobulin E (IgE) production and control chemokine and mucus production at sites of allergic inflammation .

In certain cell types, IL4R can signal through alternative pathways involving the activation of insulin receptor substrates (IRS1/IRS2), providing a mechanism for cross-talk between immunological and metabolic signaling networks . This multi-pathway signaling capability makes IL4R an important molecule in understanding the complex interplay between different physiological systems.

How does the binding mechanism of IL4R compare between species?

While species-specific data on pig IL4R binding is limited in the provided search results, insights from human IL-4 receptor binding mechanisms provide valuable comparative information. Human IL-4 binds to its cellular receptor with a Kd in the subnanomolar range, characteristic of many 4-helix-bundle proteins interacting with members of the hematopoietin receptor superfamily .

High-resolution mutational and kinetic analysis of human IL-4 has revealed that high-affinity binding originates from a continuous patch of mostly polar or charged amino acid side chains located on helices A and C . The binding epitope comprises: (i) a set of side chains determining the dissociation rate (koff) and (ii) a partially overlapping set determining the association rate constant (kon) of the IL-4/IL4-BP complex .

In the human system, the koff epitope is assembled from two juxtaposed main determinants (Glu-9 and Arg-88) surrounded by five side chains (Ile-5, Thr-13, Arg-53, Asn-89, and Trp-91) of lower importance . Researchers working with pig IL4R should consider these homologous regions when designing binding studies, while accounting for potential species-specific differences.

What are the optimal reconstitution and storage conditions for recombinant pig IL4R?

For optimal experimental outcomes, reconstitution of lyophilized recombinant pig IL4R should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL . Prior to opening, vials should be briefly centrifuged to bring contents to the bottom, ensuring no product loss during the reconstitution process .

For long-term storage stability, it is recommended to add glycerol to a final concentration of 5-50% after reconstitution and to aliquot the solution to minimize freeze-thaw cycles . These aliquots should be stored at -20°C for optimal protein stability and activity maintenance . If the product is delivered in liquid form, it typically comes in a Tris/PBS-based buffer with 5%-50% glycerol . For lyophilized powder, the buffer before lyophilization is typically a Tris/PBS-based buffer with 6% Trehalose at pH 8.0 .

Researchers should validate protein stability through activity assays after storage periods to ensure experimental reproducibility. Documentation of storage conditions, reconstitution dates, and freeze-thaw cycles is advisable for rigorous experimental protocols.

What experimental approaches can be used to verify the biological activity of recombinant pig IL4R?

Several complementary approaches can be employed to verify the biological activity of recombinant pig IL4R:

• Binding assays: Surface plasmon resonance (SPR) or biolayer interferometry can be used to determine binding kinetics (kon and koff) and affinity (Kd) between recombinant pig IL4R and its ligands (IL-4 and IL-13). Drawing from human IL4R studies, typical binding affinities should be in the subnanomolar range .

• Cell-based assays: Competitive radioligand binding assays using cells expressing the full-length receptor can verify that the recombinant fragment maintains native binding properties. This approach allows comparison between purified protein binding and cellular receptor binding .

• Functional signaling assays: Since IL4R couples to the JAK1/2/3-STAT6 pathway, STAT6 phosphorylation assays can be used to confirm functional activity . This can be measured using phospho-specific antibodies in Western blot, ELISA, or flow cytometry applications.

• Proliferation assays: T cell proliferation assays similar to those used for human IL-4 studies can assess biological activity . The EC50 values from these assays can be compared with binding affinity values to understand the relationship between receptor binding and cellular responses.

• Structural verification: Circular dichroism spectroscopy can verify proper protein folding, while thermal shift assays can assess protein stability under various conditions.

How can researchers effectively use recombinant pig IL4R in receptor-ligand interaction studies?

To effectively study receptor-ligand interactions using recombinant pig IL4R, researchers should consider the following methodological approaches:

• Immobilization strategies: For biosensor-based assays, proper orientation of the receptor is crucial. Using the His-tag for directed immobilization can ensure that the ligand-binding domain remains accessible . Control experiments comparing different immobilization approaches should be conducted to identify potential artifacts.

• Kinetic analysis: Complete kinetic characterization should include determination of association (kon) and dissociation (koff) rate constants, not just equilibrium binding constants (Kd) . This provides mechanistic insights into the binding process that equilibrium measurements alone cannot reveal.

• Mutagenesis studies: Based on insights from human IL-4 studies, strategic mutation of key residues in the binding interface can help map the functional epitope . Alanine scanning mutagenesis of surface-exposed residues can identify critical interaction points.

• Comparative ligand binding: Since IL4R binds both IL-4 and IL-13, comparative binding studies can reveal differences in recognition mechanisms and potential allosteric effects . Competition assays between these ligands can provide insights into binding site overlap.

• Cross-species comparison: Parallel studies with human, mouse, and pig IL4R can highlight species-specific differences in binding mechanisms that may be relevant for translational research .

How should researchers interpret discrepancies between binding affinity and biological activity?

When interpreting experimental data related to recombinant pig IL4R, researchers should be aware that binding affinity measurements may not directly correlate with biological activity measures. Human IL-4 studies have shown that variants with significantly reduced receptor binding affinity (Kd) often show less pronounced decreases in biological activity (EC50) . For instance, human IL-4 variants with more than 100-fold reductions in receptor binding sometimes exhibited only 10 to 20-fold decreases in T cell proliferation activity .

Several factors may explain this discrepancy:

• Receptor density effects: High receptor expression on target cells may compensate for reduced binding affinity through avidity effects.

• Threshold signaling: Biological responses may require only partial receptor occupancy to achieve maximal effects, creating a non-linear relationship between binding and response.

• Kinetic considerations: The biological activity may correlate better with specific kinetic parameters (kon or koff) rather than equilibrium binding constants .

• Signal amplification: Intracellular signaling cascades often include amplification steps that can magnify small receptor-binding events into robust cellular responses.

To address these complexities, researchers should perform comprehensive analyses that include both equilibrium binding measurements and kinetic rate constants, alongside multiple biological activity assays at different time points.

What analytical methods are most appropriate for quality assessment of recombinant pig IL4R preparations?

For rigorous quality assessment of recombinant pig IL4R preparations, multiple complementary analytical methods should be employed:

• SDS-PAGE analysis: This fundamental technique verifies protein molecular weight and assesses purity, which should exceed 85% for research applications . Both reducing and non-reducing conditions should be tested to evaluate disulfide bond formation.

• Size-exclusion chromatography (SEC): SEC provides information about protein aggregation states and homogeneity, critical factors that can affect functional assays.

• Mass spectrometry: This technique confirms protein identity and can detect post-translational modifications or truncations that might affect activity.

• Circular dichroism (CD): CD spectroscopy evaluates secondary structure content and proper protein folding.

• Dynamic light scattering (DLS): DLS assesses protein monodispersity and can detect soluble aggregates that might not be apparent in other analyses.

• Endotoxin testing: Since bacterial contaminants can influence immunological assays, endotoxin levels should be quantified, particularly for preparations expressed in E. coli systems .

• Functional assays: Binding assays using surface plasmon resonance or biolayer interferometry provide the most direct assessment of functional quality.

A comprehensive quality assessment report should include quantitative results from multiple methods, with acceptance criteria established for each parameter based on the intended experimental application.

How do experimental conditions affect the functional properties of recombinant pig IL4R?

The functional properties of recombinant pig IL4R can be significantly influenced by experimental conditions, which researchers should carefully control and document:

• Buffer composition: The presence of specific ions can modulate receptor-ligand interactions. Tris/PBS-based buffers with controlled pH (typically pH 8.0) provide optimal stability for most applications .

• Glycerol concentration: Addition of 5-50% glycerol enhances stability but may affect binding kinetics in concentration-dependent manner . Researchers should use consistent glycerol concentrations across comparative experiments.

• Protein concentration: Concentration-dependent effects such as self-association can occur at high protein concentrations, potentially altering apparent binding parameters.

• Temperature: Binding kinetics are temperature-dependent, with lower temperatures generally resulting in higher affinity but slower association rates. Standardizing assay temperature (typically 25°C for binding studies) is essential for reproducible results.

• Surface immobilization: For biosensor experiments, the immobilization chemistry and density can significantly impact binding measurements. Control experiments comparing different immobilization approaches should be included in study designs .

• Reducing conditions: The presence of reducing agents can disrupt critical disulfide bonds in the IL4R extracellular domain, dramatically altering binding properties.

Systematic validation of experimental conditions through control experiments and careful documentation of all parameters will enhance data reliability and facilitate comparison between different studies.

What are the key considerations when using recombinant pig IL4R fragments for structural studies?

When designing structural studies using recombinant pig IL4R fragments, researchers should consider several critical factors:

• Domain selection: The extracellular domain (residues 33-240) represents only a portion of the full-length receptor . Researchers should carefully evaluate whether this fragment contains all structural elements necessary for their specific research questions.

• Expression systems: The choice between yeast and E. coli expression systems can significantly impact post-translational modifications and protein folding . Yeast-expressed proteins often exhibit glycosylation patterns that may be important for certain structural studies, while E. coli systems typically provide higher yields but lack glycosylation.

• Tag interference: The N-terminal 6xHis-tag, while useful for purification, may interfere with certain structural studies . Control experiments with tag-cleaved protein or alternatively tagged constructs should be considered.

• Protein stability: The extracellular domain may have different stability characteristics than the full-length receptor embedded in the membrane. Thermal shift assays and limited proteolysis studies can help identify stable constructs suitable for crystallography or cryo-EM.

• Complex formation: For co-crystallization studies with ligands, the binding kinetics (particularly koff rates) will influence complex stability. Drawing from human IL-4 studies, the half-life of receptor-ligand complexes may be approximately 6.5 minutes, potentially challenging for certain structural techniques .

• Comparative modeling: Researchers may need to employ comparative modeling approaches based on human IL4R structures, accounting for species-specific differences in sequence and potentially in structure.

How can researchers effectively investigate species-specific differences in IL4R function?

Investigating species-specific differences in IL4R function requires systematic comparative approaches:

• Sequence-structure-function analysis: Comprehensive sequence alignment of IL4R across species, combined with structural mapping of divergent regions, can identify potentially functionally important differences. Special attention should be paid to the binding epitope regions identified in human IL4R studies, particularly residues corresponding to the key charged pair (Glu-9 and Arg-88) and surrounding residues (Ile-5, Thr-13, Arg-53, Asn-89, and Trp-91) .

• Cross-species binding studies: Experimental designs that test binding of IL-4 and IL-13 from different species to pig IL4R can reveal evolutionary conservation or divergence of recognition mechanisms. This approach should include determination of both equilibrium and kinetic binding parameters.

• Chimeric receptor approaches: Creating chimeric receptors with domains from different species can help pinpoint regions responsible for species-specific binding properties or signaling characteristics.

• Mutagenesis approaches: Targeted mutagenesis introducing species-specific residues can directly test hypotheses about the functional importance of sequence differences. This should focus particularly on residues known to be critical in the human system, such as those forming the koff and kon epitopes .

• Signaling pathway analysis: Comparative analysis of downstream signaling pathways activated by IL4R in different species can reveal functional conservation or divergence beyond binding properties. This should include examination of both JAK-STAT and IRS1/IRS2 pathways .

• Infection model studies: Comparative studies in infection models, similar to those that examined IL4R expression during Toxoplasma gondii and Ascaris suum infections, can provide insights into species-specific in vivo regulation .

What are the current limitations and challenges in studying the complete signaling complex involving IL4R?

Research on the complete IL4R signaling complex faces several significant challenges:

• Heteromeric receptor complexes: IL4R functions in heteromeric complexes with either common gamma chain (for IL-4 signaling) or IL-13Rα1 (for both IL-4 and IL-13 signaling) . Reconstituting these complete complexes in vitro presents technical challenges due to the membrane-anchored nature of these components.

• Membrane microenvironment effects: The lipid composition and membrane microenvironment can significantly influence receptor dimerization, clustering, and signaling that cannot be fully recapitulated using soluble extracellular domains alone .

• Dynamic conformational changes: The signaling process likely involves dynamic conformational changes that are difficult to capture with static structural techniques. Time-resolved methods with appropriate temporal resolution are needed but technically challenging.

• Intracellular signaling components: The interactions between the receptor complex and intracellular signaling components (JAK kinases, STAT6, IRS1/IRS2) represent crucial aspects of the signaling mechanism that are not addressed by studies limited to extracellular domains .

• Cellular context dependency: The signaling outcomes appear to be highly context-dependent, with different cell types exhibiting distinct responses to the same ligand-receptor interaction . This complexity necessitates cell-type specific investigations rather than generalized models.

• Species-specific variations: Significant differences may exist between pig, human, and mouse IL4R signaling complexes, complicating translation between model systems. The reference reporting molecular cloning of swine IL-4 receptor alpha and IL-13 receptor alpha 1 chains represents important groundwork for addressing this challenge .

To overcome these limitations, integrated approaches combining structural biology, single-molecule techniques, cellular imaging, and systems biology will be necessary to fully elucidate the complex signaling mechanisms involving IL4R.