Recombinant Mouse Interleukin-27 receptor subunit alpha (Il27ra)

What is the molecular structure of recombinant mouse IL27RA?

Recombinant Mouse IL27RA (Interleukin-27 receptor subunit alpha) is a transmembrane protein consisting of 520 amino acids in its native form. The full protein has a molecular weight of approximately 57 kDa and is composed of three domains: an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain contains two fibronectin type III domains and a cytokine receptor homology domain, which is responsible for binding to IL-27. The transmembrane domain anchors the protein to the cell membrane, while the intracellular domain is involved in signal transduction .

Commercial recombinant forms typically include only the extracellular domain. For example, typical expression regions include Leu117~Ala350, with a theoretical molecular weight of 29.68 kDa when expressed with an N-terminal His tag .

What are the common synonyms and alternative names for IL27RA?

When searching literature and databases, it's important to recognize that IL27RA is referenced under multiple names:

• TCCR (T-Cell Cytokine Receptor)

• WSX1

• CRL1 (Cytokine receptor-like 1)

• IL27R

• Zcytor1

• Type I T-cell cytokine receptor

Understanding these alternative designations is crucial when conducting comprehensive literature searches for experimental planning.

How should recombinant mouse IL27RA protein be stored and handled for optimal stability?

For maintaining maximal activity of recombinant mouse IL27RA:

• Store at -20°C in appropriate buffer (typically PBS, pH 7.4, containing 0.01% SKL, 5% Trehalose)

• Avoid repeated freeze/thaw cycles

• Aliquot upon first thaw to minimize freeze-thaw damage

• When working with the protein, keep on ice

• For long-term storage beyond 1 month, maintain at -80°C

Proper attention to storage conditions is essential for experimental reproducibility, as protein degradation can lead to inconsistent results.

What is the primary signaling pathway activated by IL27RA?

The primary function of IL27RA is to act as a receptor for IL-27, a cytokine produced by activated immune cells. Upon binding to IL-27, IL27RA forms a heterodimeric receptor complex with IL-6ST (gp130), which initiates a signaling cascade primarily involving the JAK-STAT pathway. This leads to:

• Activation of JAK1, JAK2, and TYK2

• Phosphorylation of STAT1 and STAT3

• Translocation of activated STATs to the nucleus

• Regulation of gene expression affecting T cell differentiation and function

This signaling pathway plays a critical role in the development and function of various immune cells, including T cells, B cells, and natural killer cells .

How does IL27RA signaling regulate T cell development and differentiation?

IL27RA signaling has complex effects on T cell development and differentiation:

• Inhibition of Th17 development: IL-27 signaling through IL27RA suppresses RORγt expression and inhibits Th17 cell differentiation

• Regulation of Treg development: IL-27 signaling restrains regulatory T cell (Treg) development by suppressing Foxp3 expression. T cells lacking IL27RA more readily upregulate Foxp3 expression both in vitro and in vivo

• Promotion of Tr1 cells: IL-27 promotes the differentiation of IL-10-producing Tr1 cells that express IFN-γ but lack Foxp3

• Th1 modulation: IL-27 initially promotes Th1 differentiation by inducing T-bet expression, but subsequently limits excessive Th1 responses

This multifaceted regulation makes IL27RA a crucial target for understanding T cell-mediated immune responses in various disease contexts .

What are the optimal methods for validating recombinant IL27RA protein activity in vitro?

To validate the biological activity of recombinant IL27RA protein, several complementary approaches should be employed:

• Binding assays:

  • Surface Plasmon Resonance (SPR) to measure binding kinetics with IL-27

  • ELISA-based binding assays using immobilized IL-27

• Cell-based functional assays:

  • Inhibition of IL-27-induced STAT1/STAT3 phosphorylation in responsive cell lines

  • Competitive inhibition of IL-27 signaling in reporter cell lines expressing IL27RA

• Quality control validation:

  • SDS-PAGE to confirm purity (>95% is standard for research applications)

  • Endotoxin testing (<1.0 EU per 1μg using LAL method)

  • Mass spectrometry to confirm identity and integrity

Researchers should select validation methods based on their specific experimental questions and downstream applications.

How can IL27RA knockout or knockdown systems be effectively established for functional studies?

Several approaches can be used to establish IL27RA-deficient experimental systems:

• CRISPR/Cas9 gene editing:

  • Design guide RNAs targeting early exons of the Il27ra gene

  • Confirm knockout by sequencing and western blot/flow cytometry

  • Establish clonal cell lines from single edited cells

• siRNA/shRNA knockdown:

  • Multiple siRNA sequences should be tested to identify optimal targeting

  • Validate knockdown efficiency by qPCR and western blot

  • Consider inducible knockdown systems for temporal control

• Working with Il27ra knockout mice:

  • Available Il27ra-/- mice allow isolation of primary cells lacking IL27RA

  • Bone marrow chimeras can be created to study cell-specific knockout effects

  • Adoptive transfer of Il27ra-/- T cells into Rag-deficient mice provides a system to study T cell-specific effects

Controls should include wild-type cells treated identically and rescue experiments with recombinant IL27RA to confirm specificity.

How does IL27RA deficiency affect experimental colitis models?

The impact of IL27RA deficiency on experimental colitis is model-dependent, reflecting the complex roles of IL-27 signaling in intestinal inflammation:

• T cell transfer model:

  • Absence of IL27RA on transferred T cells results in diminished weight loss

  • Reduced colonic inflammation observed

  • Greater fraction of transferred T cells assume a Foxp3+ phenotype in the absence of IL27RA

  • Effector cell proliferation and IFN-γ production are reduced

• Dextran sulfate sodium (DSS)-induced colitis:

  • Conflicting results depending on DSS concentration:

  • High-dose DSS (5-10%): IL27RA-deficient mice show increased susceptibility

  • Low-dose DSS (0.5%): IL27RA-deficient mice are protected

• Helminth-induced IBD:

  • IL27RA deficiency impairs intestinal Th1 response

  • Results in ineffective worm expulsion

  • Delayed onset of colitis

These differential effects highlight the importance of considering the specific inflammatory context when interpreting IL27RA function.

What methodological considerations are important when using IL27RA-deficient models to study autoimmune conditions?

When designing experiments with IL27RA-deficient models for autoimmune research:

• Background strain considerations:

  • Ensure proper backcrossing to relevant strain (typically C57BL/6)

  • Use littermate controls to minimize genetic drift effects

• Cell-specific effects:

  • Consider conditional knockout models for tissue-specific deletion

  • Use bone marrow chimeras to distinguish hematopoietic vs. non-hematopoietic effects

• Compensation mechanisms:

  • Assess expression of related receptors (e.g., IL-6ST, IL-6R) that might compensate

  • Consider acute disruption (inducible systems) vs. germline deletion

• Timing considerations:

  • Monitor disease progression longitudinally rather than at single timepoints

  • Include early and late assessment points to capture temporal dynamics

• Measuring inflammatory markers:

  • Combine histopathology, flow cytometry for immune cell composition, and cytokine measurements

  • Include analysis of Foxp3+ Treg frequency and function as IL27RA deficiency affects this population

How can computational approaches inform structure-function relationships of IL27RA?

Advanced computational approaches provide valuable insights into IL27RA structure-function relationships:

• Homology modeling and molecular dynamics:

  • Building computational models based on crystal structures of related cytokine receptors

  • Simulating IL-27/IL27RA interactions to identify critical binding residues

  • Predicting effects of mutations on receptor stability and ligand binding

• Systems biology approaches:

  • Network analysis integrating IL27RA signaling with other cytokine pathways

  • Predictive modeling of cellular responses to IL-27 in different contexts

  • In silico prediction of transcription factor binding to IL27RA gene regulatory regions

• Virtual screening for small molecule modulators:

  • Identification of potential binding pockets on IL27RA extracellular domain

  • Virtual screening of compound libraries for potential IL27RA modulators

  • Rational design of peptide inhibitors based on IL-27/IL27RA interface

These computational methods can guide experimental design, helping researchers focus on the most promising hypotheses regarding IL27RA function.

What are the current challenges in reconciling contradictory findings about IL27RA function in different disease models?

Several methodological approaches can help address contradictions in IL27RA research:

• Standardization of experimental models:

  • Careful documentation of mouse genetic backgrounds and housing conditions

  • Consistent protocols for disease induction (e.g., DSS concentration, T cell transfer numbers)

  • Detailed reporting of microbial environment, as gut microbiota can influence results

• Cell type-specific analysis:

  • Conditional deletion of IL27RA in specific cell populations

  • Single-cell transcriptomics to identify differential responses across cell types

  • Cell-specific reconstitution experiments in global knockout backgrounds

• Temporal considerations:

  • Time-course experiments to distinguish early vs. late effects

  • Inducible systems for temporal control of IL27RA expression

• Ligand complexity:

  • Consider that IL27RA might have ligands other than IL-27

  • Possible interactions with IL-35 or other IL-27-related heterodimers

  • Evaluation of potential heterodimers consisting of IL-27p28 and cytokine-like factor

• Context-dependent signaling:

  • Analysis of IL27RA signaling in the context of other cytokine signals

  • Investigation of receptor complex composition in different cell types

  • Consideration of tissue-specific microenvironmental factors

What are the most promising therapeutic applications targeting IL27RA signaling?

Based on current understanding of IL27RA biology, several therapeutic approaches warrant investigation:

• For inflammatory bowel diseases:

  • IL27RA blocking antibodies may benefit certain forms of colitis

  • Cell-specific targeting to preserve beneficial effects while blocking detrimental ones

  • Combination approaches targeting both IL27RA and complementary pathways

• For autoimmune conditions:

  • Selective agonists of IL27RA to promote Tr1 cell development

  • Peptide mimetics that preferentially activate anti-inflammatory signaling pathways

  • Small molecule inhibitors of specific downstream signaling components

• Cancer immunotherapy:

  • Modulation of IL27RA signaling to enhance anti-tumor immunity

  • Combination with checkpoint inhibitors to improve efficacy

  • Engineered IL-27 variants with enhanced binding to IL27RA

• Protein engineering approaches:

  • Soluble IL27RA decoy receptors to neutralize excessive IL-27

  • Bifunctional fusion proteins combining IL27RA extracellular domain with other immunomodulatory domains

  • Engineered cell therapies with modified IL27RA signaling properties

Development of these approaches requires careful consideration of the complex and sometimes contradictory roles of IL27RA in different physiological and pathological contexts.