Recombinant Human IL17A & IL17F Heterodimer Protein (Active)

What is the IL-17A/F heterodimer and how does it structurally compare to IL-17A and IL-17F homodimers?

The IL-17A/F heterodimer is a cytokine composed of one IL-17A chain and one IL-17F chain, associated in a head-to-head, parallel fashion. This heterodimeric configuration differs from the homodimeric IL-17A:A and IL-17F:F structures. All three cytokines display a cystine-knot-like fold characterized by two β-hairpins connected by intra-chain disulfide linkages. The heterodimer shows remarkable structural similarity to both parent homodimers, with root-mean square deviations of only 0.9 Å and 0.8 Å for the A- and F-chains, respectively (when comparing 81 equivalent Cα atoms) .

The dimerization creates a small hydrophobic core that is otherwise absent in the individual non-globular protein subunits. While the backbone structure is highly conserved, there are notable differences at the heterodimer interface compared to the respective homodimers, with non-conservative changes being accommodated through solvent exposure of side chains or structural adaptations that exploit new opportunities for inter-chain contacts .

What is the molecular composition of recombinant human IL-17A/F heterodimer?

Recombinant human IL-17A/F heterodimer typically comprises:

• Human IL-17A (Gly24-Ala155) from Accession # Q16552

• Human IL-17F (Arg31-Gln163) from Accession # Q96PD4

In crystallographic studies, the full-length human IL-17A/F heterodimer has been produced with an APP-tag (EFRHDS) at the N-terminus of the F-chain by co-expression in HEK293 cells . Commercial preparations may use CHO cell expression systems for production of the recombinant protein .

How was the existence of the IL-17A/F heterodimer initially demonstrated?

The existence of the IL-17A/F heterodimer was first hypothesized based on the sequence homology between IL-17A and IL-17F and their overlapping expression patterns. Researchers confirmed its existence through multiple complementary approaches:

• Recombinant expression systems demonstrating heterodimer formation

• Enzyme-linked immunosorbent assay (ELISA) detection

• Immunoprecipitation followed by Western blotting

• Mass spectrometry analysis

These techniques confirmed that both recombinant expression systems and activated human CD4+ T cells produce not only IL-17A and IL-17F homodimers but also the IL-17A/F heterodimer . This discovery expanded our understanding of IL-17 cytokine biology and suggested new roles for the heterodimer in T cell-mediated immune responses.

What receptors does the IL-17A/F heterodimer engage with, and how does this differ from the homodimers?

• The heterodimer shows intermediate binding affinity to IL-17RA compared to IL-17A (higher affinity) and IL-17F (lower affinity) .

• The biological potencies of these three IL-17 cytokines in inducing IL-6 and CXCL1 expression correlate with their binding affinities toward the IL-17RA receptor .

• Unlike the homodimers, the heterodimer presents two distinct faces - an "A-face" and an "F-face" - both capable of binding either receptor with similar affinity .

This dual-binding capability enables the formation of two topologically-distinct heterotrimeric complexes with potentially different signaling properties, adding complexity to IL-17 signaling biology .

What is the significance of the "two-faced" nature of the IL-17A/F heterodimer?

The "two-faced" nature of the IL-17A/F heterodimer represents a unique characteristic with important biological implications:

• Despite IL-17RA having much higher affinity for IL-17A than IL-17F, crystallographic studies surprisingly revealed that IL-17RA can bind to either the "A-face" or "F-face" of the heterodimer with similar affinity .

• Site-directed mutagenesis experiments have confirmed that IL-17RC also does not discriminate between the two faces of the cytokine heterodimer .

• This dual-binding capability enables the formation of two topologically-distinct heterotrimeric signaling complexes .

The biological significance lies in the potential for distinct downstream signaling pathways depending on which face engages with which receptor, potentially explaining some of the intermediate functional properties observed with the heterodimer compared to the respective homodimers.

How do receptor binding kinetics differ between species for the IL-17A/F heterodimer?

There are notable differences between human and mouse IL-17R systems in terms of ligand recognition:

These interspecies differences are important considerations when designing experiments and interpreting results from animal models. Surface plasmon resonance studies have shown stepwise association of receptor/ligand binding; after either IL-17RA or IL-17RC binds their respective ligand, affinity for the reciprocal subunit increases . This observation is consistent with studies showing ligand-inducible association of IL-17RA and IL-17RC on the cell surface .

What are the key biological functions of the IL-17A/F heterodimer compared to the homodimers?

The IL-17A/F heterodimer exhibits biological activities that can be considered intermediate between IL-17A and IL-17F homodimers in many respects:

• Pro-inflammatory potential: The heterodimer shows intermediate pro-inflammatory activity compared to IL-17A (most potent) and IL-17F (least potent) .

• Cytokine/chemokine induction: The EC50 for inducing effects like IL-6 production is typically between that of IL-17A and IL-17F, correlating with its intermediate receptor binding affinity .

• Tissue protection: Like both homodimers, the heterodimer contributes to protecting skin and mucosal barriers against extracellular pathogens, particularly fungi and bacteria .

The heterodimer's distinctive receptor binding characteristics may allow for fine-tuning of inflammatory responses differently than either homodimer alone, suggesting non-redundant functions in immune regulation.

Which cell types naturally produce the IL-17A/F heterodimer?

The IL-17A/F heterodimer is produced by several immune cell populations:

• CD4+ T helper 17 (Th17) cells - the primary adaptive immune source

• Various innate immune cells residing in barrier tissues, including:

  • γδ T cells

  • Invariant natural killer T (iNKT) cells

  • Lymphoid-tissue inducer (LTi)-like cells

  • Natural killer (NK) cells

  • Paneth cells

  • Neutrophils

Research has confirmed that activated human CD4+ T cells produce the IL-17A/F heterodimer alongside the homodimers of IL-17A and IL-17F . This concurrent expression pattern suggests coordinated regulation of all three cytokine forms during immune responses.

What roles does the IL-17A/F heterodimer play in disease pathogenesis?

• Autoimmune pathology: Studies using IL-17-deficient mice or neutralizing IL-17A activity demonstrated that IL-17-producing cells mediate inflammatory pathology in autoimmune models .

• Allergic inflammation: The IL-17 cytokine family, including the heterodimer, appears to add complexity to the regulation of allergic inflammation beyond the classical Th2 paradigm .

• Host defense: The heterodimer likely contributes to T cell-mediated immune responses against extracellular pathogens at barrier surfaces .

The distinctive binding properties and intermediate activities of the heterodimer suggest it may play non-redundant roles compared to either homodimer alone.

What experimental approaches can be used to specifically detect and quantify IL-17A/F heterodimer in biological samples?

Detecting and quantifying the IL-17A/F heterodimer in biological samples requires methods that can distinguish it from IL-17A and IL-17F homodimers:

• Sandwich ELISA: Using capture and detection antibodies with specificity for different chains (e.g., anti-IL-17A capture and anti-IL-17F detection) to specifically detect the heterodimer.

• Immunoprecipitation coupled with Western blotting: Sequential immunoprecipitation with antibodies against one chain followed by blotting with antibodies against the other chain.

• Mass spectrometry: For definitive identification of heterodimeric complexes, particularly using techniques like:

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)

• Bead-based multiplex assays: Using differentially labeled antibodies against IL-17A and IL-17F to detect co-localization indicative of heterodimer formation.

These methods have been successfully employed to demonstrate the presence of IL-17A/F heterodimers in activated human CD4+ T cells alongside the respective homodimers .

How should recombinant IL-17A/F heterodimer be handled and stored for optimal stability in research applications?

For optimal stability and activity of recombinant IL-17A/F heterodimer in research applications:

• Reconstitution: Typically reconstitute lyophilized protein at 100 μg/mL in 4 mM HCl .

• Storage conditions:

  • For long-term storage: -20°C to -80°C (preferably -80°C)

  • Avoid repeated freeze-thaw cycles by storing in working aliquots

  • For short-term use (≤1 month): 2-8°C after reconstitution

• Carrier considerations: The protein may be available in carrier-free formulations (recommended when the presence of bovine serum albumin could interfere with experiments) or with carrier protein (which enhances stability and shelf-life) .

• Handling: When working with the reconstituted protein, maintain sterile conditions and avoid extended periods at room temperature.

Following these guidelines will help ensure experimental reproducibility and maximize the functional lifetime of the reagent.

What are the most effective experimental systems for studying IL-17A/F heterodimer functions?

Several experimental systems are particularly useful for studying IL-17A/F heterodimer functions:

• Cell-based bioassays:

  • Fibroblast or epithelial cell lines measuring IL-6 or CXCL1 secretion

  • Endothelial cell activation assays

  • Primary keratinocyte or synoviocyte cultures for tissue-specific responses

• Co-culture systems:

  • T cell-fibroblast co-cultures to investigate inflammatory pathways

  • Epithelial-immune cell interactions at barrier surfaces

• Ex vivo tissue explants:

  • Skin, joint, or intestinal tissue explants to assess tissue-specific responses

• Animal models:

  • Knock-in models expressing only specific forms of IL-17

  • Conditional knockout systems for temporal control

  • Humanized mouse models when studying human-specific effects

• Structural and binding studies:

  • Surface plasmon resonance (SPR) for measuring binding kinetics

  • Crystallographic studies for structural analysis

  • Site-directed mutagenesis to examine specific protein-protein interactions

When designing experiments, researchers should consider the species-specific differences in IL-17 receptor binding profiles and the potential existence of two different signaling complexes due to the dual-faced nature of the heterodimer .

How can site-directed mutagenesis be utilized to study the two-faced binding properties of the IL-17A/F heterodimer?

Site-directed mutagenesis represents a powerful approach for dissecting the unique two-faced binding properties of the IL-17A/F heterodimer:

• Strategic mutation selection:

  • Target residues at the A-face and F-face that are unique to each face

  • Focus on amino acids identified in crystal structures as critical for receptor binding

  • Create mutations that selectively disrupt binding to IL-17RA or IL-17RC on only one face

• Experimental validation approaches:

  • Surface plasmon resonance (SPR) to measure binding affinities of mutants

  • Cell-based reporter assays to assess functional consequences

  • Crystallography of mutant proteins to confirm structural changes

• Key regions to target:

  • Site 1: The "skirt region" that undergoes significant receptor-induced conformational changes

  • Site 2: The pre-formed cavity described as a "pocket in the garment"

  • Site 3: Additional interaction regions identified in structural studies

Researchers have already used this approach to demonstrate that IL-17RA can bind to both faces of the heterodimer with similar affinity, and that IL-17RC also does not discriminate between the two faces, enabling the formation of two topologically-distinct heterotrimeric complexes .

What are the methodological challenges in distinguishing IL-17A/F heterodimer-mediated effects from those of IL-17A or IL-17F homodimers?

Distinguishing the specific biological effects of the IL-17A/F heterodimer from those of IL-17A and IL-17F homodimers presents several methodological challenges:

• Co-expression challenges:

  • Natural systems typically produce all three dimers simultaneously

  • Designing systems that exclusively express the heterodimer without homodimers

• Selective targeting approaches:

  • Developing antibodies specific for the heterodimer but not reactive with either homodimer

  • Creating receptor variants that selectively engage the heterodimer

• Experimental strategies:

  • Comparing dose-response curves across a wide concentration range

  • Using mathematical modeling to deconvolute mixed effects

  • Employing genetic approaches with selective expression of modified cytokine chains

• Readout considerations:

  • Identifying biological responses that might be uniquely sensitive to the heterodimer

  • Temporal analysis to detect differences in signaling kinetics or duration

One promising approach involves creating obligate heterodimers through protein engineering, where modified IL-17A and IL-17F chains can only form functional dimers with each other but not with themselves, allowing for clean experimental systems to study heterodimer-specific effects.

How do the topologically-distinct receptor complexes formed by IL-17A/F heterodimer influence downstream signaling pathways?

The ability of the IL-17A/F heterodimer to form two topologically-distinct receptor complexes (depending on which face binds to which receptor) has significant implications for downstream signaling:

• Signaling pathway activation:

  • Different binding orientations may lead to distinct conformational changes in the receptor intracellular domains

  • This could potentially activate different downstream adapter proteins or signaling intermediates

  • The intensity and duration of signaling may differ between the two complex configurations

• Gene expression consequences:

  • Transcriptomic analyses comparing responses to the two different complexes may reveal distinct gene expression signatures

  • The balance between pro-inflammatory and regulatory responses could differ

• Experimental approaches to investigate this phenomenon:

  • Engineering of locked heterodimers that can only engage receptors in one specific orientation

  • Phosphoproteomic analyses to map early signaling events

  • Single-cell analyses to detect potential heterogeneity in responses

• Biological significance:

  • The dual binding capability may allow for fine-tuning of inflammatory responses

  • Different tissue microenvironments may favor one binding mode over the other

The two possible signaling complexes formed by IL-17A/F heterodimer represent an additional layer of complexity in IL-17 biology that likely contributes to the context-dependent effects of these cytokines in different physiological and pathological settings .