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. 2023 Aug 14;13(9):302. doi: 10.1007/s13205-023-03724-7

Canine interleukin-31 binds directly to OSMRβ with higher binding affinity than to IL-31RA

Yuxin Zheng 1,2,3,#, Jing Zhang 2,3,#, Tianling Guo 2,3, Jin Cao 1,2,3, Lixian Wang 2,3, Jie Zhang 2,3, Xuefei Pang 2,3, Feng Gao 2,3, Hua Sun 1, Haixia Xiao 2,3,✉
PMCID: PMC10425310  PMID: 37588794

Abstract

Interleukin-31 (IL-31) is a pro-inflammatory cytokine involved in skin inflammation and tumor progression. The IL-31 signaling cascade is initiated by its binding to two receptors, IL-31 receptor alpha (IL-31RA) and oncostatin M receptor subunit beta (OSMRβ). The previous study suggested that human IL-31 (hIL-31) directly interacts with IL-31RA and OSMRβ, independently, but the binding ability of hIL-31 to IL-31RA is stronger than to OSMRβ. In different to its human ortholog, feline IL-31 (fIL-31) has a higher binding affinity for feline OSMRβ. However, the binding pattern of canine IL-31 to its receptors remains to be elucidated. In this study, we purified the recombinant canine IL-31 (rcIL-31) protein and revealed its secondary structure to be mainly composed of alpha-helices. Moreover, in vitro studies show that rcIL-31 has the ability to induce the phosphorylation of signal transducer activator of transcription 3 (STAT3) and STAT5 in DH-82 cells. In the following, the binding efficacies of bioactive rcIL-31 for its individual receptor components have been measured using a flow cytometry assay. The result demonstrates that correctly refolded rcIL-31 binds independently with cIL-31RA and cOSMRβ which were expressed on the cell surface. Of note, rcIL-31 has a greater than tenfold higher affinity to OSMRβ than to IL-31RA. Additionally, we demonstrated that D1–D4, especially D4 of cOSMRβ, is crucial for its binding to cIL-31. Furthermore, this study proved that rcIL-31 has a high binding affinity to the soluble cOSMRβ with a KD value of 3.59 × 10–8 M. The results presented in the current study will have a significant implication in the development of drugs or antibodies against diseases induced by cIL-31 signaling.

Keywords: Canine interleukin-31, IL-31 receptor alpha, Oncostatin M receptor beta, Binding affinity, STAT phosphorylation

Introduction

Interleukin-31 (IL-31) is a recently identified pro-inflammatory cytokine produced by CD4+ type 2 helper T cells (TH2), macrophages, dendritic cells, fibroblasts, and keratinocytes (Bilsborough et al. 2006; McCandless et al. 2014). IL-31 belongs to the IL-6 cytokine family, but it does not mediate signaling through a receptor complex containing gp130. Instead, its receptor heterodimer is composed of IL-31 receptor alpha (IL-31RA) and the oncostatin M receptor β (OSMRβ) that is a co-receptor with another family member, oncostatin M (OSM) (Diveu et al. 2003; Ghilardi et al. 2002). IL-31RA is widely expressed in many different cells, including macrophages (Lewis et al. 2017), keratinocytes (Bilsborough et al. 2006; Lee et al. 2018), some dorsal root ganglia neurons (Lewis et al. 2017; Xu et al. 2020), bronchial and alveolar epithelial cells (Jawa et al. 2008) and pulmonary fibroblasts (Ferretti et al. 2017; Yaseen et al. 2020), as well as CD14- and CD56-positive blood cells (Ghilardi et al. 2002). Moreover, IL-31RA is overexpressed in luminal breast cancer cells (Tal Kan et al. 2020) and lesional skin (Olszewska et al. 2022). Whereas, OSMRβ is expressed at relatively high levels in keratinocytes, neural cells, fibroblast and epithelial cells (Lee et al. 2018). To be addressed, OSMRβ is upregulated in many cancer tissues in patients with decreased survival (Geethadevi et al. 2021; McCollum et al. 2022; Yu et al. 2019; Zhu et al. 2020).

The binding of IL-31 to its specific receptors, IL-31RA and OSMRβ, triggers the strong activation of the STAT3 and STAT5 pathways (Dreuw et al. 2004). The activation of downstream transcription factor then further regulates the expression of genes which are involved in various physiological processes such as inflammation (Bilsborough et al. 2006), cell differentiation, and cancer progression (Geethadevi et al. 2021). As such, IL-31 and its two receptors become major therapeutic targets in several diseases, including atopic dermatitis (AD) (Marsella and De Benedetto 2017; Yang et al. 2022), allergic respiratory diseases (Ip et al. 2007), hematological diseases (Zhang et al. 2017), tumor progression (Caffarel and Coleman 2014; Di Maira et al. 2022; Lee et al. 2021) and tumor prognosis (West et al. 2012). To date, several monoclonal antibodies (mAb) targeting IL-31 or its receptors have been developed, and two of them have been approved (Kabashima et al. 2018; Marsella et al. 2020; Nemoto et al. 2016; Richards et al. 2020; Stander et al. 2020). Among those mAbs, nemolizumab, a humanized anti-IL-31RA mAb, is the first approval of mAb for the treatment of itches associated with AD in 2022. Moreover, lokivetmab, a caninized anti-IL-31 mAb, was the first mAb to be approved in animals, and it has been widely used for the treatment of AD in dogs since 2017 (Marsella et al. 2020). In addition to those licensed ones, an investigational fully human mAb in the forefront is vixarelimab (KPL-716), a mAb targeting OSMRβ that met the primary efficacy endpoint in a phase 2 clinical trial for treatment of prurigo nodularis (ClinicalTrials.gov Identifier: NCT03816891). To be noted, vixarelimab is the only mAb in development that targets two signaling pathways induced by IL-31 and OSM (Richards et al. 2020).

The understanding of the interacting pattern and binding affinity of IL-31 to its receptors will certainly facilitate the development of drugs for the treatment of diseases induced by IL-31 pathways. Up to now, the binding abilities of IL-31 to IL-31RA and OSMRβ have been well studied in species of human and feline (Diveu et al. 2004;  Le Saux et al. 2010; Medina-Cucurella et al. 2020; Chen et al. 2023). However, little is known about the interaction between IL-31 and its receptors in the canine ortholog. Therefore, the aim of this work was to carry out a detailed analysis of the binding activity of canine IL-31 (cIL-31) to IL-31RA and OSMRβ. To meet this end, we firstly expressed and purified cIL-31, and characterized its structural composition. Afterwards, the receptor binding ability and the biological activity of cIL-31 have been further investigated and confirmed.

Material and methods

Cell lines

Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin and 10% fetal bovine serum (FBS). Canine macrophage DH-82 cells were grown with MEM containing 15% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37 °C with 5% CO2.

Preparation of recombinant cIL-31 and cOSMRβ proteins

The condon-optimized gene fragment of cIL-31 (amino acids 25–159, GenBank: NP_001159386.1) was inserted into pET21a and then transformed into Escherichia coli (E. coli) BL21 (DE3). Recombinant cIL-31 proteins were expressed in the form of inclusion bodies and then refolded according the method previously described (Xiao et al. 2019). Briefly, aliquots of inclusion bodies were added dropwise into the refolding buffer (100-mM Tris, 2-mM EDTA, 400-mM l-arginine, 5-mM reduced glutathione, 0.5-mM oxidized glutathione, pH 8.0) and kept on stirring for overnight. Subsequently, the refolded cIL-31 protein was concentrated using the Amicon® stirred cell and then exchanged with buffer containing 20-mM Tris, 50-mM NaCl, 2% glycerol and pH 8.0. The refolded proteins were purified by size-exclusion chromatography on a HiLoad 16/600 Superdex™ 75 PG column (Cytiva) and then further recycled using Superdex™ 75 10/300 GL (Cytiva).

The purified recombinant cIL-31 (rcIL-31) proteins were biotinylated using the EZ-LinkTM NHS-PEG4-Biotin Biotinylation Kit (Thermo Scientific) according to the manufacturer’s instructions. Briefly, 1.5 mg/ml of rcIL-31 was mixed with the reconstituted NHS-PEG4-Biotin compound and 10 mM of sulfosuccinimidyl-6-[biotin-amido]-hexanoate. The reaction mixture was incubated at room temperature (RT) for more than 30 min (min), then desalted with a PD MiniTrap™ G-25 column (Cytiva).

Circular dichroism measurements

The CD spectra were collected with a Chirascan CD spectropolarimeter (Applied Photophysics, Surrey, UK). Refolded rcIL-31 proteins were diluted to a final concentration of 0.1 mg/ml in PBS. The CD data were recorded in the far-UV range of 190–260 nm at 25.0 °C for an average of three scans with a bandwidth of 1.0 nm and a sensitivity of 0.5 s time-per-point. The secondary structural elements of rcIL-31 were analyzed by submitting the raw data to the software CDNN using the CDNN algorithm with a reference set.

Flow cytometry (FACS) assay

Codon-optimized gene segments of cIL-31RA (GenBank, XP_013963900.1) and full-length or different truncated mutants of cOSMRβ (GenBank, NP_001310434) were inserted into the pEGFP-N1 vector, respectively. The recombinant plasmid, pEGFP-cIL-31RA or pEGFP-cOSRMβ, was transiently transfected into HEK293T cells using linear polyethylenimine (PEI, Mw 40,000, Polysciences). At 4–6 h post-transfection, cells were washed with PBS and supplemented with DMEM. At 48 h post-transfection, cell cultures were trypsinized into single cells, aliquoted into a 96-well plate, and then incubated with various concentrations of biotinylated rcIL-31 (rcIL-31-Biotin) at RT for 1 h. In the following, cells were washed twice and incubated with PE/Cyanine7-conjugated streptavidin (BioLegend) at RT for 1 h. At last, the cells were washed twice and then supplemented with PBS to be analyzed by flow cytometry (Beckman MoFlo XDP). The binding percentage of rcIL-31 to IL-31RA was calculated based on the ratio of rcIL-31-Biotin and IL-31RA-EGFP double-positive cells to all IL-31RA-EGFP-positive cells. So is it with the binding of rcIL-31 to different cOSMRβ constructs.

Surface plasmon resonance assay

The binding affinity of rcIL-31 to cOSMRβ was measured by SPR assay using Biacore T200 (Cytiva). Biotinylated rcIL-31 was immobilized to the streptavidin (SA) biosensor chip in PBST (0.005% Tween-20 in PBS). The association of immobilized rcIL-31-Biotin with cOSMRβ protein at concentrations of 7.81, 15.63, 31.25, 62.5, 125, and 250 nM was measured for 5 min, followed by a 5-min long dissociation phase. The data were analyzed using a 1:1 binding model with global fitting algorithms, and the KD value was calculated with Biacore T200 evaluation software.

STAT phosphorylation assay

DH-82 cells were used for the STAT phosphorylation assay because it was confirmed that both cIL-31RA and cOSMRβ express on this cell line (Hwang et al. 2021), and STAT phosphorylation could be induced in response to the cIL-31 treatment (Hwang et al. 2021). The confluent DH-82 cells were seeded into a 96-well tissue culture plate and incubated at 37 °C overnight. Confluent cells were treated with 10 ng/ml of canine IFNγ for 24 h and then serum-starved for 2 h. In the following, cells were stimulated with different concentrations of rcIL-31 for 5–10 min. Next, cultures were lysed with 50 μl/well of lysis buffer from AlphaLISA® SureFire Ultra™ HV kits (PerkinElmer) for 10 min on a shaker. Finally, the phosphorylation of STAT3 (Y705) and STAT/5 (Y694/699) in the cell lysates was tested according to the protocol of the AlphaLISA® SureFire Ultra™ HV kits, and the plates were read using a Multi-Mode Microplate Reader (BioTek Synergy Neo2, USA).

Comparison of amino acid sequences

Protein sequences of IL-31, IL-31RA, and OSMRβ from canine, feline and human were retrieved from the GenBank database or UniProt: cIL-31 (NP_001159386.1), cIL-31RA (XP_013963900.1), cOSMRβ (XP_005619431), fIL-31 (XP_011286140), fIL-31RA (A0A337S7E6), fOSMRβ (M3W8Y0), hIL-31 (NP_001014358), hIL-31RA (NP_620586.3) and hOSMRβ (NP_001310434). Homology among different species for each protein was performed by the CLC Sequence Viewer and Lasergene Megalign program (Version 7.1, DNASTAR Inc.).

Results

Determination of the secondary structure for rcIL-31 protein

Recombinant rcIL-31 protein was expressed as inclusion bodies in E. coli and then successfully refolded into monomeric form (Fig. 1a). To characterize the actual secondary structure of refolded rcIL-31, CD spectra in the far-UV and near-UV ranges were measured for this protein. The CD spectrum revealed that the refolded rcIL-31 has the characteristic α-helix signature peaks at 192, 208 and 222 nm (Fig. 1b). According to this prediction, about 96.10% of amino acids of refolded rcIL-31 are folded into α-helices (Fig. 1c). This result is in good agreement with the 3D model structure of cIL-31 predicted by AlphaFold (Fig. 1d), which shows that mature cIL-31 mainly contains 4 α-helices. The 3D structure of the refolded rcIL-31 was similar overall to those cytokines of the IL-6 family members, such as IL-6 (PDB: 7NXZ) (Reif et al. 2021), interleukin-11 (PDB: 4MHL) (Putoczki et al. 2014), as well as oncostatin M (OSM) (PDB: 1EVS) (Deller et al. 2000). The CD result, as well as the 3D structure prediction, suggests that refolded rcIL-31 has either the native structure or the one very close to it.

Fig. 1.

Fig. 1

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Purification of rcIL-31 and the determination of its secondary structural compositions. a Size-exclusion chromatography analysis of refolded rcIL-31 proteins using a Superdex™ 75 10/300 GL. The peak fraction of monomeric rcIL-31 was confirmed on SDS-PAGE gel under both reducing conditions in the presence of dithiothreitol (+ DTT) and non-reducing condition with DTT (− DTT). b Secondary structure patterns were revealed by circular dichroism. The data were representative of three independent experiments using three different batches of rcIL-31 proteins. c The actual elements of rcIL-31 were analyzed using CDNN software. The data were the average of three experiments and had a high degree of confidence (total summary < 105%). d The model of 3D structure for the extracellular domain of cIL-31 (AF-C7G0W1-F1-model_v4) was predicted by AlphaFold server developed by the DeepMind group (Jumper et al. 2021). This model shows that soluble rcIL-31 is mainly composed of four α-helices, αA, αB, αC, and αD, which are colored with red, orange, yellow, and green, respectively

Canine IL-31 binds to membrane-anchored cIL-31RA and cOSMRβ, independently

To assess the cell-surface binding capability of rcIL-31 to its receptors, 293T cells expressed with cIL-31RA-EGFP or cOSMRβ-EGFP, were incubated with rcIL-31-Biotin. The binding signals were picked up using PE/Cyanine7-conjugated streptavidin and then analyzed by FACS. The flow data showed that the percentage of double-positive cells (rcIL-31-Biotin and cIL-31RA-EGFP) to all cIL-31RA-EGFP-positive cells was 73.0% when the cells were incubated with rcIL-31-Biotin at a concentration of 20 μg/ml (Fig. 2a). At the same concentration, rcIL-31-Biotin and cOSMRβ-EGFP double-positive cells have a much higher ratio to all cOSMRβ-EGFP-positive cells, which is 95.40% (Fig. 2b). At the same time, we used hIL-31RA-EGFP as a negative control and found that rcIL-31-Biotin presented no binding to hIL-31RA-EGFP (Fig. 2c). To determine the binding constants of rcIL-31-Biotin to cIL-31RA-EGFP- and cOSMRβ-EGFP-positive cells, the ratio of rcIL-31-Biotin- and EGFP-fused protein double-positive cells to all EGFP-fusion protein-positive cells was converted into percent bound, and two saturation curves were plotted against concentrations of rcIL-31-Biotin. The concentrations corresponding to 50% of the maximal effect (EC50) of rcIL-31-Biotin to cIL-31RA-EGFP- and cOSMRβ-EGFP-positive cells were 8.54 μg/ml and 0.81 μg/ml, respectively (Fig. 2d). The binding affinity of rcIL-31 for cOSMRβ is 10.5 times as high as that for cIL-31RA. Obviously, cIL-31RA and cOSMRβ expressed on the cell surface could independently attach to soluble rcIL-31 in a dose-dependent manner in the FACS assay. Moreover, rcIL-31 has a much stronger binding ability to cOSMRβ than to cIL-31RA.

Fig. 2.

Fig. 2

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Binding efficacy of rcIL-31 to membrane-anchored receptors, cIL-31RA and cOSMRβ. a–c Flow cytometric analysis of the binding ability of rcIL-31 to cIL-31RA, cOSMRβ and hOSMRβ, which were fused with EGFP at their C-terminals. Biotinylated rcIL-31 proteins (rcIL-31-Biotin), at a concentration of 20.0 μg/ml, were incubated with HEK293T cells, which were expressed with cIL-31RA-EGFP, cOSMRβ-EGFP or hIL-31RA-EGFP on the cell membrane. The binding signals were picked up by PE/Cyanine7-labeled streptavidin. The data were representative of three independent experiments with similar results. d Binding curves of rcIL-31 to membrane-expressed cOSMRβ and cIL-31RA were established based on the binding percentages at a series of dilutions of rcIL-31-Biotin. The binding percentage was calculated based on the ratio of rcIL-31-Biotin- and EGFP-fusion protein double-positive cells to all EGFP-fusion protein-positive cells. The concentrations corresponding to 50% of the maximal effect (EC50) were calculated based on these curves built with GraphPad software. The experiment was repeated three times, and an error bar indicates the mean standard deviation (n = 6)

The region responsible for the interaction of cOSMRβ with cIL-31

The extracellular region of cOSMRβ is composed of seven distinct domains (D1, D2, D3, D4, D5, D6, and D7) (Fig. 3a). To determine the region responsible for the binding of cOSMRβ to cIL-31, truncated mutants of cOSMRβ fused with EGFP were constructed (Fig. 3a). The binding efficacies of biotinylated rcIL-31 to those truncated mutants, as well as the full length of cOSMRβ-EGFP-fusion proteins, were quantified using  a FACS assay and presented as the bound percentage of rcIL-31-Biotin- and EGFP-fused protein double-positive cells to all EGFP-fused protein-positive cells. The results showed that truncated mutant D4_D567, rather D3_D567, is able to directly interact with rcIL-31, and the percentage of rcIL-31-Biotin- and EGFP-fused protein double-positive cells to all D4_D567-positive cells is 21.2% (Fig. 3b). Whereas, the construct, D34_D567, including five continuous domains, D3–D7, demonstrated a relative higher binding percentage, 57.7%, to 20 μg/ml of rcIL-31-Biotin. Furthermore, the truncated mutant D1234, which contains four domains, D1–D4, has 99.0% bound to 20 μg/ml of rcIL-31 (Fig. 3b). However, the other truncated mutants, including D567, D3_D567, D12_D567 and D23_D567, had no binding ability to rcIL-31 (Fig. 3b). These results suggest that D4 of cOMSRβ has much more contribution to the direct binding of cIL-31. Meanwhile, it also proved that D4 directly binds to IL-31 and then initiates the sequential interaction of D3 with this cytokine. The first two domains of cOSMRβ, D1 and D2, alone cannot bind to cIL-31, but these two domains, together with D3 and D4, enhance the binding efficacy of cOSMRβ to this cytokine. In brief, the first four domains of extracellular cOSMRβ, especially D4, are necessary for the interaction with cIL-31.

Fig. 3.

Fig. 3

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Determination of the binding domains of cOSMRβ with cIL-31. a Schematic representation of EGFP-fused full-length or different truncates of cOSMRβ constructs used in this study. SP signal peptide, D1 domain 1, TM transmembrane domain, and CT cytoplasmic domain. b Binding ability of different cOSMRβ-EGFP-fusion proteins to rcIL-31 measured by flow cytometry. Biotinylated rcIL-31 at a concentration of 20.0 μg/ml was incubated with HEK293T cells, which were expressed with different cOSMRβ-EGFP-fusion proteins on the cell membrane. The binding signals were picked up by PE/Cyanine7-labeled streptavidin. The binding percentage was calculated based on the ratio of rcIL-31-Biotin- and EGFP-fusion protein double-positive cells to all EGFP-fusion protein-positive cells. The data were the statistical results of three independent experiments, and an error bar indicates the mean standard deviation (n = 6)

Binding kinetics of rcIL-31 to cOSMRβ

The SPR assay was applied to determine the binding kinetics of rcIL-31 to soluble cOSMRβ, which was expressed and purified based on the method described previously (Zheng et al. 2022). The SPR data showed that rcIL-31 has a relative high binding affinity to cOSMRβ with a KD value of 35.9 nM (Fig. 4a). This binding occurred with fast-on (Kon = 1.12 × 105 M−1 s−1) and quick-off (Koff = 4.00 × 10–3 s−1). However, rcIL-31 did not show any cross-reactivity with BSA protein (Fig. 4b). To confirm the right confirmation of rcIL-31, we also purified a caninized anti-cIL-31 monoclonal antibody (mAb), 34D03 (Bammert and Dunham 2017), and tested the binding affinity between rcIL-31 and this mAb using the SPR method. The SPR data suggested that rcIL-31 has a high binding affinity to 34D03 with a KD value of 3.73 × 10–11 M (Fig. 4c), which is similar to the published data, 29.1 pM conducted by Zoetis (Bammert and Dunham 2017). In the current study, the binding of caninized 34D03 to rcIL-31 occurred with fast-on (Kon = 1.86 × 106 M−1 s−1) and slow-off (Koff = 6.94 × 10–5 s−1). Similarly, rcIL-31 does not show any binding to the negative control IgG molecule (Fig. 4d).

Fig. 4.

Fig. 4

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Binding affinity of rcIL-31 to soluble rcOSMRβ and a caninized mAb, 34D03. The binding affinities of rcIL-31 to rcOSMRβ and its specific mAb, 34D03 (a, c) were measured using a surface plasmon resonance (SPR) assay. Bovine serum albumin (BSA) and an mAb that does not bind to rcIL-31 were used as negative controls (b, d). All of these binding curves were built using GraphPad Prism software, and the dissociation constant KD values were determined using Biacore T200 evaluation software. Representative data are shown from three independent experiments with similar results. The values were normalized to the cell samples which were not treated with rcIL-31

Phosphorylation of STAT3/5 induced by rcIL-31

After determining the structural composition of rcIL-31 and its binding ability with the receptors, the biological activity of STAT3/5 activation was evaluated using DH-82 cells, and the pSTAT signaling was assessed using AlphaLISA pSTAT3 and pSTAT5 kits. The AlphaLISA assay showed that there was no significant difference in terms of pSTAT3 induced by four dilutions (5 μg/ml, 2.5 μg/ml, 1.25 μg/ml and 0.6 μg/ml) of rcIL-31. Whereas, the phosphorylation of STAT5 induced by rcIL-31 occurs in a dose-dependent manner between the concentration of 5 μg/ml and the two lower doses, 1.25 μg/ml and 0.6 μg/ml (Fig. 5). Altogether, the results suggested that rcIL-31 could activate both pSTAT3 and pSTAT5 signaling pathways even at the lowest concentration of 0.6 μg/ml (Fig. 5).

Fig. 5.

Fig. 5

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Dose-dependent changes in STAT3/5 phosphorylation by DH-82 cells in response to rcIL-31. DH-82 cells were stimulated with graded doses of rcIL-31 for 10 min, followed by the quantification of pSTAT3/5 using the AlphaLISA assay. The data were averages from three parallel experiments (n = 6). *p < 0.05 and **p < 0.01

Homology analysis of IL-31 and its receptors from three species

Multiple sequence alignments of IL-31 from canine, feline and human revealed that amino acids composed of alpha-helix A and B from canine and feline have higher homology than their human ortholog. Of note, one position on binding site 2, E21, and one on binding site 3, K111 (amino acid numbering based on the mature hIL-31 protein), are strictly conserved among three species, and the other two positions on site 2 are different, with E83 and H87 in hIL-31, and K83 and Q87 in both cIL-31 and fIL-31 (Fig. 6a). Among the binding sites of IL-31 for OSMRβ including the G38 and “PADNFERK” motif (P104–K111), G38, P104, D106, and E109 are conserved for three species, and the rest are variable (Fig. 6a). Similar to IL-31, the cytokine-binding domains (CBD) of IL-31RA and OSMRβ are also different from those of the other three species (Fig. 6b–d). It is obvious that canine and feline share much higher identities in the sequence of IL-31, IL-31RA and OSMRβ, which are 75.7%, 89.9%, and 83.8%, respectively (Fig. 6d). However, these two species have a relative lower similarity with human receptors. The corresponding identities of IL-31, IL-31RA and OSMRβ between canine and human are around 56.3%, 68.1% and 73.1%, individually (Fig. 6d). The higher identities of IL-31 and its receptors shared by canine and feline might be an explanation for the similar binding activities of cIL-31 and fIL-31 to their two receptors.

Fig. 6.

Fig. 6

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Multiple sequence alignments of IL-31, IL-31RA and OSMRβ from different species. a Sequence alignments of mature IL-31 proteins from canine, feline and human were performed with the CLC Sequence Viewer and the Lasergene DNASTAR Megalign program. Four alpha-helices, αA, αB, αC and αD, are marked with squares in the colors of marine, green, yellow, and magenta, respectively. The amino acids, E21, E83, H87, and K111 of hIL-31, which were proved to be decisive positions in the interaction with its receptors, were emphasized with black edged boxes (Le Saux et al. 2010). The binding sites of fIL-31 to fOSMRβ, G38 and the “PADNFERK” (P104-K111) motif are framed with the green dashed line. Whereas, the overlapping binding site of fIL-31 to both fIL-31RA and fOSMRβ is in the frame of the blue dashed line (Medina-Cucurella et al. 2020). b Protein sequence alignment of cytokine-binding domains (CBD) of IL-31RA from three species. c The first four domains, including one Ig-like domain and three CBDs of OSMRβ were aligned by the Clustal W method using the Lasergene Megalign program. d Percentage identity matrix obtained with Clustal W alignments of IL-31, IL-31RA and OSMRβ from canine, feline, and human

Discussion

The IL-31 cytokine and its receptors are important therapeutic targets for pruritus and some cancers (Arita et al. 2008; Di Salvo et al. 2021; Furue et al. 2018; Kabashima and Irie 2021). In human and feline orthologs, the interactions between IL-31 and two receptors have been well studied (Diveu et al. 2004; Le Saux et al. 2010; Medina-Cucurella et al. 2020). However, molecular communication between canine IL-31 and its receptors remains to be fully elucidated. To meet this end, we purified rcIL-31 and investigated the binding affinity of this recombinant ligand to the membrane-anchored receptors, cIL-31RA and cOSMRβ, after confirming the right structural composition. Our study demonstrated that rcIL-31 has the ability to independently recognize its two receptors, and the binding affinity of rcIL-31 to cOSMRβ is higher than to cIL-31RA in FACS analysis (Fig. 2). More importantly, we provided experimental evidence to show that D4 of cOSMRβ directly interacts with cIL-31, and the first three domains, D1–D3, make considerable contributions to the binding of cOSMRβ to this cytokine. In similar to our results with the canine ortholog, feline IL-31 can directly bind to fOSMRβ and fIL-31RA in both ELISA and FACS assays (Medina-Cucurella et al. 2020). The similar binding activity of cIL-31 and fIL-31 might be caused by the high identities of IL-31 and its receptors in canine and feline species (Fig. 6). The high identities provide the possibility that cIL-31 and fIL-31 are capable of cross-reacting with each other’s receptors. As a matter of fact, these two cytokines were able to bind to DH-82 cells and induced pSTAT signaling (Bammert and Dunham 2017). This rationality is further proved by two mouse mAbs developed by Zoetis. One is anti-IL-31 mAb 34D03 (Bammert and Dunham 2017), and the other is anti-OSMRβ 19F07 (Bammert and Gonzales 2022). The mAb, 34D03 obtained from the mouse immunized with cIL-31, is cross-reacting with fIL-31 and has ability to block the signaling transduction triggered by cIL-31 and fIL-31 (Bammert and Dunham 2017). Besides, mAb, 19F07, isolated from cOSMRβ-immunized mouse presents the cross-reactivity with fOSMRβ, so as to inhibit the fIL-31 signaling (Bammert and Gonzales 2022).

Previous studies showed that human IL-31 directly interacts with hIL-31RA-hFc (hIL-31RA fused with human Fc fragment) and fails to independently bind to hOSMRβ-hFc in the immunoprecipitation assay (Diveu et al. 2004; Le Saux et al. 2010). After the initiation of the binary complexes, hIL-31/hIL-31RA, hOSMRβ is recruited, and the ternary complex, hIL-31/hIL-31RA/hOSMRβ, forms to activate downstream signal transduction (Le Saux et al. 2010). However, recently, there was a new discovery that hIL-31 binds both membrane-anchored hOSMRβ and hIL-31RA independently, and the binding affinity to hOSMRβ is far weaker than that to hIL-31RA in the FACS assay (Chen et al. 2023). This recent study is much more convincible, because the interaction of hIL-31 with cell surface-expressed receptors detected by the FACS assay is much more sensitive. Furthermore, the possible conformational distortion or the hidden presence of CBD in the soluble Fc-fused receptors might influence their binding ability to IL-31. The different activities of IL-31 between human and other two species might be the result of low sequence homology (Fig. 6). The results from previous and our studies together demonstrated that human, canine, or feline IL-31 binds IL-31RA and OSMRβ, independently, and the ternary complex forms in later.

Conclusion

This study provides critical information about the characteristics and functionality of rcIL-31 proteins expressed in E. coli. This protein is well-refolded and presents proper biological activities, including binding to its receptors and inducing phosphorylation of STAT3/5. More importantly, the binding results demonstrated that rcIL-31 recognizes IL-31RA and OSMRβ, independently, and the binding affinity to OSMRβ is higher than to IL-31RA. Moreover, it is the first time to provide experimental evidence to show that four domains, D1–D4, especially D4 of cOSMRβ, play an important role in the binding to cIL-31. This behavior of cIL-31 is different with hIL-31, but is the same with fIL-31. The understanding of the interacting pattern and binding affinity of cIL-31 to its receptors will certainly facilitate the development of mAbs and small molecular drugs targeting the diseases induced by cIL-31 signaling pathways.

Acknowledgements

This work is supported by Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-KJGG-014 and TSBICIP-IJCP-001) and the National key research and development program of China (2020YFA0907104).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Conflict of interest

The authors declare that there are no conflicts of interests in this manuscript.

Footnotes

Yuxin Zheng and Jing Zhang contributed equally.

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Associated Data

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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