Deciphering site 3 interactions of interleukin 12 and interleukin 23 with their cognate murine and human receptors

Alessandra Esch; Anna Masiarz; Sofie Mossner; Jens M Moll; Joachim Grötzinger; Jutta Schröder; Jürgen Scheller; Doreen M Floss

doi:10.1074/jbc.RA120.013935

. 2020 Jun 9;295(30):10478–10492. doi: 10.1074/jbc.RA120.013935

Deciphering site 3 interactions of interleukin 12 and interleukin 23 with their cognate murine and human receptors

Alessandra Esch ¹, Anna Masiarz ¹, Sofie Mossner ¹, Jens M Moll ¹, Joachim Grötzinger ², Jutta Schröder ¹, Jürgen Scheller ¹, Doreen M Floss ^1,^*

PMCID: PMC7383385 PMID: 32518162

Abstract

Interleukin (IL)–12 and IL-23 belong to the IL-12 type family and are composite cytokines, consisting of the common β subunit p40 and the specific cytokine α subunit p35 and p19, respectively. IL-12 signals via the IL-12Rβ1·IL-12Rβ2 receptor complex, and IL-23 uses also IL-12Rβ1 but engages IL-23R as second receptor. Importantly, binding of IL-12 and IL-23 to IL-12Rβ1 is mediated by p40, and binding to IL-12Rβ2 and IL-23R is mediated by p35 and p19, respectively. Previously, we have identified a W157A substitution at site 3 of murine IL-23p19 that abrogates binding to murine IL-23R. Here, we demonstrate that the analogous Y185R site 3 substitution in murine and Y189R site 3 substitution in human IL-12p35 abolishes binding to IL-12Rβ2 in a cross-species manner. Although Trp¹⁵⁷ is conserved between murine and human IL-23p19 (Trp¹⁵⁶ in the human ortholog), the site 3 W156A substitution in hIL-23p19 did not affect signaling of cells expressing human IL-12Rβ1 and IL-23R, suggesting that the interface of murine IL-23p19 required for binding to IL-23R is different from that in the human ortholog. Hence, we introduced additional hIL-23p19 substitutions within its binding interface to hIL-23R and found that the combined site 3 substitutions of W156A and L160E, which become buried at the complex interface, disrupt binding of hIL-23p19 to hIL-23R. In summary, we have identified substitutions in IL-12p35 and IL-23p19 that disrupt binding to their cognate receptors IL-12Rβ2 and IL-23R in a murine/human cross-species manner.

Keywords: IL-12, IL-23, IL-12Rbeta1, IL-23R, IL-12Rbeta2, STAT, ERK, signal transduction, innate immunity, protein–protein interaction, interleukin, receptr, mitogen-activated protein kinase (MAPK), STAT3

The pro-inflammatory interleukin (IL)–12 family members IL-12 and IL-23 show structural similarities but have different functions in the immune system (1). IL-12 drives the development of TH1 cells mainly through activation of STAT4. Interferon γ–producing TH1 cells are crucial for antimicrobial and antitumor responses (2).” IL-23 is important for the maintenance of TH17 cells, which have antimicrobial and antifungal effector functions and are characterized by production of the cytokines IL-17A, IL-17F, and IL-21 (3 –5). Both TH1 and TH17 cells are involved in the pathogenesis of autoimmune and chronic inflammatory diseases (6). IL-12 and IL-23 are heterodimeric cytokines consisting of a cytokine and a soluble receptor subunit. The cytokine α chains IL-23p19 and IL-12p35 are structurally related to IL-6 and form complexes with the soluble receptor subunit p40 (β chain). IL-23p19 and IL-12p35 belong to the gp130 class of long-chain cytokines. The p40 subunit is similar in the domain structure of typical class I cytokine receptors such as the nonsignaling IL-6 receptor α (IL-6Rα) (7). The subunits of IL-12p35 or IL-23p19 are connected by a disulfide bridge with p40 (8). Whereas IL-12 signals via the receptor complex consisting of IL-12Rβ1·IL-12Rβ2, IL-23 binds and activates the heterodimer of IL-12Rβ1·IL-23R (9, 10).

IL-12 and IL-23 have family-typical STAT activation patterns. IL-12 mainly activates STAT4 but also STAT1, STAT3, and STAT5, whereas IL-23 predominantly activates STAT3 but also STAT1, STAT4, and STAT5 (7). Canonical and noncanonical binding sites for STAT molecules are located in the intracellular domains of IL-12Rβ2 and IL-23R but not in IL-12Rβ1 (11, 12). Apart from binding to Tyk2, IL-12Rβ1 does not contribute to IL-12/IL-23–induced intracellular signaling and is therefore considered to be a ligand-binding receptor (13). However, p40 homodimers induce macrophage migration via IL-12Rβ1 (14), suggesting a role of IL-12Rβ1 in signal transduction (15).

Cytokine receptor interactions for IL-12 family cytokines have been proposed based on the structure of IL-6, IL-6Rα, and gp130 (16 –18). In this “site 1-2-3” model, site 1 interaction is formed between the α chain and the soluble receptor chain. The p35–p40 formation is mediated by charged interactions around the central Arg¹⁸⁹ (+22-aa signal peptide) from human p35, which projects into a deep pocket of p40 (19). The critical hot-spot amino acid residues of site 1 are Ile¹⁷⁶, Ala¹⁷⁸, and Arg¹⁷⁹ in murine p19 (20) (Fig. 1). Site 2 of IL-23p19 and IL-12p35, respectively, should engage IL-12Rβ1 as shown for the cytokine receptor complex of IL-6, IL-6Rα, and gp130 (16 –18). However, the β chain p40 was identified as major mediator for interaction with IL-12Rβ1 (20, 21). Consequently, p40 acts as antagonist of IL-12 and IL-23 signaling (22 –26). This direct interaction of p40 with IL-12Rβ1 is unique among α receptors of the IL-6/IL-12 family (1). Site 3 interactions are found between the cytokine and the N-terminal Ig domain of a receptor subunit (16). Accordingly, site 3 interactions of IL-23p19 and IL-12p35 occur via IL-23R and IL-12Rβ2, respectively.

Figure 1. — **Analysis of the crystal structure of the human IL-23/IL-23R complex (PDB code 5MZV).** The structure is displayed in ribbon representation. Residues of IL-23R and IL-23p19 within 4 Å radius of IL-23p19 or IL-23R, respectively, are displayed in stick representation. The *insets* show magnifications of site 1 and 3 including critical residues Val¹⁷⁵, Ala¹⁷⁷, and Arg¹⁷⁸ of p19; Tyr²⁶⁵ and Tyr³¹⁸ of p40; and central Trp¹⁵⁶ of IL-23p19 and contacted residues of IL-23R in stick representation, respectively.

IL-23R is composed of an N-terminal Ig-like domain (domain 1; D1), a cytokine-binding module consisting of two fibronectin type III domains (domains 2 and 3; D2 and D3), and a long stalk region, followed by the transmembrane domain and the intracellular domain (10). Previously, we have shown that substitution W157A in murine IL-23p19 completely disturbs binding to murine IL-23R (20) (Fig. 1). Later, the crystal structure of the human IL-23p19·IL-23R complex confirmed binding of IL-23p19 to D1 of IL-23R. Moreover, additional site 3 hot-spot substitutions were identified in murine IL-23p19 (21). IL-12Rβ2 consists also of an N-terminal Ig-like domain (D1) and a cytokine-binding module (D2 and D3), which is followed by three additional fibronectin type III domains, the transmembrane domain, and the intracellular domain (9). So far, a functional site 3 in IL-12p35 targeting the IL-12Rβ2 has not been identified. It is tempting to speculate that tyrosine residue Tyr^189/185 located at site 3 in human and murine IL-12p35 (Fig. 2A) might contribute to the specific interaction between p35 and IL-12Rβ2 (16, 27).

Figure 2. — **IL-23p19W157 and IL-12p35Y185 in mouse are functional hot-spot amino acids.** A, structural superpositioning of crystal structures of human IL-23p19 and IL-12p35 (PDB codes 3D87 and 3HMX) with models of their murine counterparts. B, cellular proliferation of Ba/F3-gp130 cells with cDNAs coding for mIL-23R and mIL-12Rβ1. The cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or with the indicated cytokines (10% conditioned cell culture supernatant). Parental Ba/F3-gp130 cells were used as controls. The results of one representative experiment of five are shown. *Error bars* represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). C, Western blotting (WB) of secreted mp40–p19 and mp40–p19W157A from transfected CHO-K1 cells. D, analysis of STAT3 and ERK1/2 activation. Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells were washed, starved, and stimulated with the indicated cytokines (10% conditioned cell culture supernatant) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 µg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2, and ERK. Western blotting data show results of one representative experiment of four. E, co-IP of FLAG-tagged mp40–p35 and mp40–p35Y185R by Fc-tagged smIL-12Rβ1 or smIL-12Rβ2. *Lanes L*, lysates; *lanes* +, coimmunoprecipitates; *lanes C*, controls (without smIL-12Rβ1-Fc or smIL-12Rβ2-Fc). One of two independent experiments is shown. F, cellular proliferation of Ba/F3-gp130 cells with cDNAs coding for mIL-12Rβ2 and mIL-12Rβ1. The assay was performed as described for B. The results of one representative experiment of four are shown. *Error bars* represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). G, Western blotting of secreted mp40–p35 and mp40–p35Y185R from transfected CHO-K1 cells. H, analysis of STAT3 and ERK1/2 activation. The assay was performed with BaF3-gp130-mIL-12Rβ1-mIL-12Rβ2 cells as described for D. Western blotting data show the results of one representative experiment of two. *EGFP*, enhanced green fluorescent protein; ns, not significant; *RFU*, relative fluorescence units. ***, P ≤ 0.001.

Here, we analyzed the signal transduction of modified IL-12 and IL-23 in the well-established pre–murine B-cell line Ba/F3 (12, 28). In murine and human we identified site 3 substitutions in IL-12p35 and IL-23p19 that abrogate signal transduction via IL-12Rβ1·IL-12Rβ2 and IL-12Rβ1·IL-23R complexes, respectively. Site 3 substitutions of murine Y185R and human Y189R in IL-12p35 completely abrogate binding to IL-12Rβ2 in a cross-species manner. Unexpectedly, we did not observe similar results for murine and human IL-23p19 substitutions. A single amino acid site 3 substitution in human IL-23p19 was insufficient to disturb signaling. However, combined substitutions of W156A and L160E disrupted binding of human IL-23p19 toward human IL-23R.

Results

Y185R in murine IL-12p35 results in abrogated IL-12Rβ2 binding and IL-12 signaling

IL-23p19 binds to the N-terminal Ig-like domain of IL-23R via site 3 (20). p19 residues in contact with IL-23R are located in the AB loop and helices B and D (Fig. 1 and Fig. S1). At the top of helix D, aromatic residues are conserved among cytokines of IL-6/IL-12 family. These residues are buried in the interface through complex formation. Several reports demonstrated a key role of these central aromatic amino acids for site 3 interactions of the respective cytokines (21, 29, 30). In case of human p19, Trp¹⁵⁶ is stacked against Gly¹¹⁶, Tyr¹⁰⁰, Leu¹¹³, and Ile²⁸ of IL-23R within the IL-23R·p19 interface (Fig. 1). In murine p19 Trp¹⁵⁷ and p35 Tyr¹⁸⁵, and in human p35 Tyr¹⁸⁹ residues are located at this position (Fig. 2A). Hence, we generated murine IL-23 and IL-12 variants and characterized the proteins regarding their effects on signal transduction. Proliferation of Ba/F3-gp130 cells stably transduced with the cDNAs coding for murine (m)IL-23R and (m)IL-12Rβ1 depended on the external stimulation with murine IL-23 (mp40–p19) or Hyper–IL-6 (HIL-6) (Fig. 2B). We used FLAG-tagged murine Hyper–IL-23, a fusion protein of murine p40 and murine p19 connected by a flexible peptide linker (31). Hyper–IL-6, a fusion protein of IL-6 and soluble IL-6 receptor (sIL-6R), activates Ba/F3 cells via the gp130 receptor (28).

As previously shown, substitution of amino acid Trp¹⁵⁷ to alanine in murine IL-23p19 completely abolished cytokine-induced proliferation of Ba/F3-gp130-mIL-23R-mIL-12Rβ1 cells (20) (Fig. 2B). All IL-23 variants have been transiently expressed in CHO-K1 cells with comparable efficiency (Fig. 2C). STAT3 and ERK were activated in Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells after stimulation with mp40–p19, but not with the inactive variant mp40–p19W157A (Fig. 2D). Sequence and structural alignments indicated that Trp¹⁵⁷ in mIL-23p19 and Tyr¹⁸⁵ in mIL-12p35 are functional site 3 hot spots for binding to the N-terminal Ig-like domain of mIL-23R and mIL-12Rβ2, respectively (16, 21, 27) (compare Fig. 1A and Fig. S1). To prove this hypothesis, soluble (s)mIL-12Rβ2-Fc fusion proteins were incubated with murine IL-12 (mp40–p35), precipitated by protein A–agarose beads and binding of FLAG-tagged mp40–p35 to smIL-12Rβ2-Fc was detected by Western blotting using anti-FLAG antibodies. Surprisingly, mp40–p35Y185R was not precipitated by smIL-12Rβ2-Fc via protein A–agarose, indicating that a single amino acid substitution in IL-12p35 was sufficient to abrogate cytokine–cytokine receptor interactions (Fig. 2E). Both variants were precipitated by a smIL-12Rβ1-Fc fusion protein.

Proliferation of Ba/F3-g130 cells stably transduced with the cDNAs coding for murine (m)IL-12Rβ2 and (m)IL-12Rβ1 depends on stimulation with HIL-6 or murine Hyper–IL-12, a fusion protein of mp40 and mp35 (mp40–p35) connected by a flexible linker (32) (Fig. 2F). Murine IL-12 variants were transiently expressed in CHO-K1 cells with comparable efficiency (Fig. 2G). As previously shown, mp40–p35 activated STAT3 and ERK in Ba/F3-gp130 cells expressing murine IL-12 receptors (32). In contrast, murine p40-p35Y185R failed to induce cellular proliferation and STAT3/ERK phosphorylation, indicating Tyr¹⁸⁵ as functional site 3 hot-spot amino acid (Fig. 2, F and H).

Murine IL-23p19W157A but not IL-12p35Y185R is still active on the corresponding human receptor chains

Next, we were interested in murine and human cross-species activity of IL-12, IL-23, and their variants. First, we used murine cytokines on Ba/F3 cells expressing human IL-23R and IL-12Rβ1 (Fig. S2). Murine IL-23 (mp40–p19) induced cellular proliferation and STAT3/ERK phosphorylation with the human IL-23R and IL-12Rβ1 receptors (Fig. 3, A and B), as described previously (10). Surprisingly, mp40–p19W157A was still able to induce cellular proliferation and STAT3/ERK phosphorylation via the human receptors (Fig. 3, A and B). However, cellular proliferation of Ba/F3 cells with human IL-23 receptors was significantly reduced (Fig. 3A). Analogously, murine IL-12 (mp40–p35) was able to induce cellular proliferation and STAT3/ERK phosphorylation of Ba/F3 cells expressing human IL-12Rβ2 and IL-12Rβ1 (Fig. S2), but mp40–p35Y185R did not activate human receptors (Fig. 3, C and D). Consequently, we suggest that the site 3 interface of human IL-23R is differently composed than its murine counterpart.

Figure 3. — **Murine IL-23p19W157A and murine IL-12p35Y185R show striking signaling differences on Ba/F3 cells with human receptors.** A, cellular proliferation of Ba/F3 cells with cDNAs coding for hIL-23R and hIL-12Rβ1. Cells were cultured for 3 days in the presence of IL-3 (2% conditioned cell culture supernatant of WEHI-3B cells) or with the indicated cytokines (10% conditioned cell culture supernatant). Parental Ba/F3 cells were used as controls. The results of one representative experiment of five are shown. *Error bars* represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). B, analysis of STAT3 and ERK1/2 activation. Ba/F3-hIL-12Rβ1-hIL-23R cells were washed, starved, and stimulated with the indicated cytokines (10% conditioned cell culture supernatant) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 µg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2, and ERK. Western blotting data show results of one representative experiment of four. C, cellular proliferation of Ba/F3 cells with cDNAs coding for hIL-12Rβ2 and hIL-12Rβ1. The assay was performed as described for A. The results of one representative experiment of three are shown. *Error bars* represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). D, analysis of STAT3 and ERK1/2 activation. The assay was performed with Ba/F3-hIL-12Rβ1-hIL-12Rβ2 cells as described for B. Western blotting data show results of one representative experiment of two. *EGFP*, enhanced green fluorescent protein; ns, not significant; *RFU*, relative fluorescence units. ***, P ≤ 0.001.

Human IL-23W156A does not activate murine receptors

Next, we generated human Hyper–IL-23 (hp40–p19) and introduced the corresponding site 3 substitution W156A. As expected, human p40–p19 induced cell proliferation and STAT3/ERK phosphorylation of Ba/F3-gp130 cells with murine receptors. However, hp40–p19W156A was biologically inactive (Fig. 4, A–C). We could also confirm that human IL-12 is not cross-reactive with the murine receptor chains as shown previously (33) (Fig. 4, D–F). Our results demonstrate that modification of site 3 in human IL-23 abrogated IL-23 signaling in cells expressing murine IL-23 receptors.

Figure 4. — **Human IL-23p19W156A is inactive on murine cells.** A, cellular proliferation of Ba/F3-gp130 cells with cDNAs coding for mIL-23R and mIL-12Rβ1. Cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or with the indicated cytokines (10% conditioned cell culture supernatant). Parental Ba/F3-gp130 cells were used as controls. The results of one representative experiment of four are shown. *Error bars* represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). B, Western blotting of secreted hp40–p19 and hp40–p19W156A from transfected CHO-K1 cells. Cytokine variants were transiently expressed with comparable efficiency. C, analysis of STAT3 and ERK1/2 activation. Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells were washed, starved, and stimulated with the indicated cytokines (10% conditioned cell culture supernatant) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 µg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2 and ERK. Western blotting data show results of one representative experiment of four. D, cellular proliferation of Ba/F3-gp130 cells with cDNAs coding for mIL-12Rβ2 and mIL-12Rβ1. The assay was performed as described for A. The results of one representative experiment of three are shown. *Error bars* represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). E, Western blotting of secreted hp40–p35 and hp40–p35Y189R from transfected CHO-K1 cells. Cytokine variants were transiently expressed with comparable efficiency. F, analysis of STAT3 and ERK1/2 activation. The assay was performed with Ba/F3-gp130-mIL-12Rβ1-mIL-12Rβ2 cells as described for C. HIL-6 was included as positive control. Western blotting data show results of one representative experiment of two. *EGFP*, enhanced green fluorescent protein; ns, not significant; *RFU*, relative fluorescence units. ***, P ≤ 0.001.

Human IL-23p19W156A and IL-23p19W156E are still active on the corresponding human receptor chains

Because human p40–p19W156A did not activate murine receptors, we analyzed the biological activity on human receptors. Full-length human IL-23 receptors (hIL-12Rβ1 and hIL-23R) were incubated either with FLAG-tagged human IL-23 (hp40–p19) or FLAG-tagged hp40–p19W156A and precipitated by anti–FLAG-agarose beads (Fig. 5, A and B). Binding of hIL-12Rβ1 or hIL-23R to FLAG-tagged hp40–p19 variants was detected by Western blotting using receptor specific antibodies. Precipitation of hIL-23R failed when using hp40–p19W156A indicating an importance of aa Trp¹⁵⁶ in cytokine–cytokine receptor interaction (Fig. 5B). To analyze biological activity of hp40–p19W156A, we used Ba/F3 cells expressing human IL-23R and human IL-12Rβ1. Surprisingly, Ba/F3-hIL-12Rβ1-hIL-23R cells stimulated with hp40–p19W156A induced cellular proliferation and STAT3/ERK phosphorylation (Fig. 5, C and D). This finding indicates that hp40–p19W156A still interacts with hIL-23R in a cellular system.

Figure 5. — **Human IL-23p19W156A and human IL-12p35Y189R show striking signaling differences on Ba/F3 cells with human receptors.** A, co-IP of FLAG-tagged hp40–p19 and full-length hIL-12Rβ1 or hIL-23R. One of two independent experiments is shown. B, co-IP of FLAG-tagged hp40–p19W156A and full-length hIL-12Rβ1 or hIL-23R. One of two independent experiments is shown. C, cellular proliferation of Ba/F3 cells with cDNAs coding for hIL-23R and hIL-12Rβ1. The cells were cultured for 3 days in the presence of IL-3 (2% conditioned cell culture supernatant of WEHI-3B cells) or with the indicated cytokines (10% conditioned cell culture supernatant). Parental Ba/F3 cells were used as controls. The results of one representative experiment of four are shown. *Error bars* represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). D, analysis of STAT3 and ERK1/2 activation. Ba/F3-hIL-12Rβ1-hIL-23R cells were washed, starved, and stimulated with the indicated cytokines (10% conditioned cell culture supernatant) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 µg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2, and ERK. Western blotting data show results of one representative experiment of four. E, co-IP of FLAG-tagged hp40–p35 and full-length hIL-12Rβ1 or hIL-12Rβ2. One of two independent experiments is shown. F, co-IP of FLAG-tagged hp40–p35Y189R and full-length hIL-12Rβ1 or hIL-12Rβ2. One of two independent experiments is shown. G, cellular proliferation of Ba/F3 cells with cDNAs coding for hIL-12Rβ2 and hIL-12Rβ1. The assay was performed as described for C. The results of one representative experiment of four are shown. *Error bars* represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). H, analysis of STAT3 and ERK1/2 activation. The assay was performed with Ba/F3-hIL-12Rβ1-hIL-12Rβ2 cells as described for D. Western blotting data show results of one representative experiment of three. *EGFP*, enhanced green fluorescent protein; ns, not significant; *RFU*, relative fluorescence units; IP, immunoprecipitates. ***, P ≤ 0.001.

Further, we analyzed the corresponding amino acid site 3 substitution in human Hyper–IL-12 (hp40–p35Y189R) on human IL-12 receptors. Full-length human IL-12Rβ1 and IL-12Rβ2 were incubated either with FLAG-tagged human IL-12 (hp40–p35) or FLAG-tagged hp40–p35Y189R and precipitated by anti–FLAG-agarose beads. As expected, precipitation of hIL-12Rβ2 by hp40–p35Y189R failed (Fig. 5, E and F). To investigate biological activity, Ba/F3 cells expressing human IL-12Rβ2 and IL-12Rβ1 were stimulated with either hp40–p35 or hp40–p35Y189R. Cellular proliferation was only shown for hp40–p35, demonstrating the importance of aa Tyr¹⁸⁹ for cytokine binding (Fig. 5G). However, despite the lack of cellular proliferation, we observed a slight activation of STAT3 in cells stimulated with hp40–hp35Y189R. ERK phosphorylation could not be detected, indicating that Ba/F3-hIL-12Rβ1-hIL-12Rβ2 cell proliferation mainly depends on ERK activation as previously demonstrated for Ba/F3 cells with murine IL-23 receptors (12) (Fig. 5H).

Based on the crystal structure of human IL-23 in complex with human IL-23R (21), we speculate that replacement of Trp¹⁵⁶ with alanine may not weaken the hydrophobic core of the interface sufficiently to break binding of human IL-23 and IL-23R. To test our hypothesis, we replaced Trp¹⁵⁶ with glutamic acid (Glu), a large negatively charged amino acid. Human p40–p19W156E was inactive on Ba/F3-gp130 cells expressing murine IL-23R and IL-12Rβ1 but still active on Ba/F3 cells expressing human IL-23R and IL-12Rβ1 (Fig. 6, A–E). Additionally, we analyzed activity of murine p40–p19W157E on BaF3 cells with either human or murine IL-23 receptors. Murine p40–p19W157E tremendously decreased cellular proliferation and STAT3/ERK activation of Ba/F3 cells with human receptors compared with mp40–p19W157A (Fig. 6, F and G). In contrast, both murine p40–p19 site 3 variants (W157A and W157E) diminished cellular proliferation and STAT3/ERK activation of Ba/F3 cells with murine receptors (Fig. 6, H–J). Taken together, our results indicate that replacement of aromatic Trp¹⁵⁷ with Glu weakens the hydrophobic interaction at the IL-23/IL-23R interface and prevents binding of murine IL-23 to human IL-23R.

Figure 6. — **Replacement of W156 by glutamic acid did not influence activity of hp40–p19 on Ba/F3 cells with human receptors.** A, cellular proliferation of Ba/F3-gp130 cells with cDNAs coding for mIL-23R and mIL-12Rβ1. The cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or with the indicated cytokines (10% conditioned cell culture supernatant). Parental Ba/F3-gp130 cells were used as controls. The results of one representative experiment of two are shown. *Error bars* represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). B, analysis of STAT3 and ERK1/2 activation. Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells were washed, starved, and stimulated with the indicated cytokines (10% conditioned cell culture supernatant) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 µg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2, and ERK. Western blotting data show results of one representative experiment of two. C, cellular proliferation of Ba/F3 cells with cDNAs coding for hIL-23R and hIL-12Rβ1. The cells were cultured for 3 days in the presence of IL-3 (2% conditioned cell culture supernatant of WEHI-3B cells) or with the indicated cytokines (10% conditioned cell culture supernatant). Parental Ba/F3 cells were used as controls. The results of one representative experiment of two are shown. *Error bars* represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). D, analysis of STAT3 and ERK1/2 activation. The assay was performed with Ba/F3-hIL-12Rβ1-hIL-23R cells as described for B. Western blotting data show results of one representative experiment of two. E, Western blotting of secreted hp40–p19, hp40–p19W156A, and hp40–p19W156E from transfected CHO-K1 cells. Cytokine variants were transiently expressed with comparable efficiency. F, cellular proliferation of Ba/F3 cells with cDNAs coding for hIL-23R and hIL-12Rβ1. The assay was performed as described in C. The results of one representative experiment of two are shown. *Error bars* represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). G, analysis of STAT3 and ERK1/2 activation. The assay was performed with Ba/F3-hIL-12Rβ1-hIL-23R cells as described for B. Western blotting data show results of one representative experiment of two. H, cellular proliferation of Ba/F3-gp130 cells with cDNAs coding for mIL-23R and mIL-12Rβ1. The assay was performed as described for A. The results of one representative experiment of two are shown. *Error bars* represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). I, analysis of STAT3 and ERK1/2 activation. The assay was performed with Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells as described for B. Western blotting data show results of one representative experiment of two. J, Western blotting of secreted mp40–p19, mp40–p19W157A and mp40–p19W157E from transfected CHO-K1 cells. Cytokine variants were transiently expressed with comparable efficiency. *EGFP*, enhanced green fluorescent protein; ns, not significant; *RFU*, relative fluorescence units. ***, P ≤ 0.001.

Amino acid substitutions surrounding human IL-23p19W156 did not affect binding to human IL-23R

Sequence alignment of human and murine p19 revealed two differences close to hot-spot site 3 tryptophan (human: Pro¹⁵⁵ and Leu¹⁵⁹; murine: Gln¹⁵⁶ and Pro¹⁶⁰; Fig. 7A). We generated hp40–p19 variants with murine site 3 motif either with (QQAQRPLLR) or without (QQWQRPLLR) tryptophan substitution and tested their biological activity (Fig. 7B). Human p40–p19 carrying the murine site 3 motif including W156A was inactive on Ba/F3 cells expressing murine IL-23 receptors but active on Ba/F3 cells with human IL-23 receptors (Fig. 7, C–F). Additionally, we expressed murine p40–p19 with human site 3 motif either with (QPAQRLLLR) or without (QPWQRLLLR) tryptophan substitution (Fig. 8, A and B). As expected, mp40–p19 with the human site 3 motif containing W157A was inactive on Ba/F3 cells with murine IL-23 receptors (Fig. 8, C and D). However, we detected proliferation and STAT3/ERK activation of Ba/F3 cells expressing human IL-23 receptors (Fig. 8, E and F). These results demonstrate that substitution of amino acids surrounding site 3 of p19 had no effect on cell signaling, and Trp → Ala variants are still active on cells with human IL-23 receptors.

Figure 7. — **Human IL-23 with murine site 3 substitution is still active on Ba/F3 cells with human receptors.** A, alignment of human and murine IL-23p19 site 3 amino acids. B, Western blotting of secreted hp40–p19 variants from transfected CHO-K1 cells. Cytokine variants were transiently expressed with comparable efficiency. C, cellular proliferation of Ba/F3-gp130 cells with cDNAs coding for mIL-23R and mIL-12Rβ1. The cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or with the indicated cytokines (10% conditioned cell culture supernatant). Parental Ba/F3-gp130 cells were used as controls. The results of one representative experiment of four are shown. *Error bars* represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). D, analysis of STAT3 and ERK1/2 activation. Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells were washed, starved, and stimulated with the indicated cytokines (10% conditioned cell culture supernatant) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2, and ERK. Western blotting data show results of one representative experiment of four. E, cellular proliferation of Ba/F3 cells with cDNAs coding for hIL-23R and hIL-12Rβ1. The cells were cultured for 3 days in the presence of IL-3 (2% conditioned cell culture supernatant of WEHI-3B cells) or with the indicated cytokines (10% conditioned cell culture supernatant). Parental Ba/F3 cells were used as controls. The results of one representative experiment of four are shown. *Error bars* represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). F, analysis of STAT3 and ERK1/2 activation. The assay was performed with Ba/F3-hIL-12Rβ1-hIL-23R cells as described for D. Western blotting data show results of one representative experiment of four. *EGFP*, enhanced green fluorescent protein; ns, not significant; *RFU*, relative fluorescence units. ***, P ≤ 0.001.

Figure 8. — **Murine IL-23 with human site 3 substitution is still active on Ba/F3 cells with human receptors.** A, alignment of murine and human IL-23p19 site 3 amino acids. B, Western blotting of secreted mp40–p19 variants from transfected CHO-K1 cells. Cytokine variants were transiently expressed with comparable efficiency. C, cellular proliferation of Ba/F3-gp130 cells with cDNAs coding for mIL-23R and mIL-12Rβ1. The cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or with the indicated cytokines (10% conditioned cell culture supernatant). Parental Ba/F3-gp130 cells were used as controls. The results of one representative experiment of five are shown. *Error bars* represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). D, analysis of STAT3 and ERK1/2 activation. Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells were washed, starved, and stimulated with the indicated cytokines (10% conditioned cell culture supernatant) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 µg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2, and ERK. Western blotting data show results of one representative experiment of four. E, cellular proliferation of Ba/F3 cells with cDNAs coding for hIL-23R and hIL-12Rβ1. The cells were cultured for 3 days in the presence of IL-3 (2% conditioned cell culture supernatant of WEHI-3B cells) or with the indicated cytokines (10% conditioned cell culture supernatant). Parental Ba/F3 cells were used as controls. The results of one representative experiment of five are shown. *Error bars* represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). F, analysis of STAT3 and ERK1/2 activation. The assay was performed with Ba/F3-hIL-12Rβ1-hIL-23R cells as described for D. Western blotting data show results of one representative experiment of four. *EGFP*, enhanced green fluorescent protein; ns, not significant; *RFU*, relative fluorescence units. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Human IL-23p19W156A/L160E is inactive on human IL-23 receptor chains

Bloch et al. (21) demonstrated that murine IL-23p19L161E, an alternative variant, resulted in loss of receptor binding and in vitro biological activity. Leu¹⁶⁰ is conserved between murine and human IL-23p19 (Fig. 9A and Fig. S1). Human p40–p19L160E was active on Ba/F3-gp130 cells expressing murine IL-23R and IL-12Rβ1. However, substitution of both amino acids, W156A/E and L160E, resulted in inactive cytokines (Fig. 9, B–D). When hp40–p19L160E was applied to Ba/F3 cells expressing human IL-23 receptors, cellular proliferation and STAT3/ERK phosphorylation were induced. Interestingly, hp40–p19 containing W156A/E and L160E was inactive on Ba/F3 cells with human IL-23 receptors (Fig. 9, E and F). To investigate cytokine–cytokine receptor binding full-length human IL-23 receptors were incubated either with FLAG-tagged hp40–p19L160E or hp40–p19W156A/L160E and precipitated by anti–FLAG-agarose beads. Consistent with our activity data, hIL-23R could not be precipitated by hp40–p19W156A/L160E (Fig. 9, G and H). In conclusion, our results demonstrate differences in the binding interface of human and murine IL-23 receptor complexes.

Figure 9. — **W156A and L160E in human IL-23p19 diminished cytokine activity on Ba/F3 cells with human receptors.** A, analysis of the crystal structure of human IL-23/IL-23R complex (PDB code 5MZV). Structure is displayed in ribbon representation. Residues of IL-23R and IL-23p19 within 4 Å radius of IL-23p19 or IL-23R, respectively, are displayed in stick representation. *Inset* shows central Trp¹⁵⁶ and Leu¹⁶⁰. Structural superpositioning is shown of crystal structures of human IL-23p19 (PDB code 3D87) with models of their murine counterparts. B, Western blotting of secreted hp40–p19 variants from transfected CHO-K1 cells. Cytokine variants were transiently expressed with comparable efficiency. C, cellular proliferation of Ba/F3-gp130 cells with cDNAs coding for mIL-23R and mIL-12Rβ1. Cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or with the indicated cytokines (10% conditioned cell culture supernatant). Parental Ba/F3-gp130 cells were used as controls. The results of one representative experiment of two are shown. *Error bars* represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). D, analysis of STAT3 and ERK1/2 activation. Ba/F3-gp130-mIL-12Rβ1-mIL-23R cells were washed, starved, and stimulated with the indicated cytokines (10% conditioned cell culture supernatant) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 µg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2, and ERK. Western blotting data show results of one representative experiment of two. E, cellular proliferation of Ba/F3 cells with cDNAs coding for hIL-23R and hIL-12Rβ1. The cells were cultured for 3 days in the presence of IL-3 (2% conditioned cell culture supernatant of WEHI-3B cells) or with the indicated cytokines (10% conditioned cell culture supernatant). Parental Ba/F3 cells were used as controls. The results of one representative experiment of two are shown. *Error bars* represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3). F, analysis of STAT3 and ERK1/2 activation. The assay was performed with Ba/F3-hIL-12Rβ1-hIL-23R cells as described for D. Western blotting data show results of one representative experiment of two. G, co-IP of FLAG-tagged hp40–p19L160E and full-length hIL-12Rβ1 or hIL-23R. One of two independent experiments is shown. H, co-IP of FLAG-tagged hp40–p19W156A/L160E and full-length hIL-12Rβ1 or hIL-23R. One of two independent experiments is shown. *EGFP*, enhanced green fluorescent protein; ns, not significant; *RFU*, relative fluorescence units; IP, immunoprecipitates. ***, P ≤ 0.001.

Discussion

IL-23 is a member of the IL-12 cytokine family, which is composed of IL-12, IL-23, IL-27, IL-35, and IL-39 (7). These cytokines share structural features and are closely related to the cytokines of the IL-6 cytokine family (IL-6, IL-11, IL-27, OSM, LIF, CNTF, CT-1, and CLCF1) (34). IL-6 type and IL-12 type cytokines are summarized as IL-6/IL-12 cytokine family (35). Molecular interaction studies of IL-12 type cytokines with their cognate receptors are rare (21). The site 1-2-3 paradigm for cytokine receptor interaction is generally accepted for members of the IL-12 cytokine family. Structural models for cytokine–cytokine receptor complexes have been hypothesized for IL-12, IL-23, IL-27, and IL-35 analogous to the IL-6·IL-6R·gp130 complex (16, 18). Here, site 1 is defined as interaction interface between two subunits of heterodimeric IL-12 family cytokines. IL-12 type cytokines interact via site 2 with the cytokine-binding homology region of one receptor subunit and via site 3 with the N-terminal Ig-like domain of the second receptor. However, findings about the murine IL-23 receptor complex do not fit this predicted model (20, 21). Site 2 interaction of IL-23 is independent of p19 and exclusively mediated by domains D1 and D2 of p40 (20). Assembly of the IL-23 signaling complex requires sequential recruitment of IL-23R via p19 and IL-12Rβ1 via p40 (21). Trp¹⁵⁷ in murine IL-23p19 (Trp¹⁵⁶ in hp19) was identified as a functional hot-spot amino acid for high affinity binding to murine IL-23R (20, 21). Recently, hydrogen-deuterium exchange MS was applied to identify the binding epitope of IL-23R·IL-23 (36). Consequently, Tyr¹⁸⁹ in human IL-12p35 may play a similar role in binding of IL-12 to IL-12Rβ2 (16, 21).

In our study, we confirmed the proposed critical hot-spot amino acid residues in murine and human IL-12p35 for binding to IL-12Rβ2 via site 3. Substitution of Tyr¹⁸⁵ and Tyr¹⁸⁹ in murine and human IL-12p35, respectively, completely eliminated binding to IL-12Rβ2 and biological activity. Importantly, modified murine IL-12 abolished activity on BaF3 cells with human receptors. As shown by Schoenhaut et al. (33), cross-reactivity of human IL-12 on murine cells could not be demonstrated. In contrast, murine and human IL-23 functions on cells with receptors of both species (10). However, our data show differences in binding of IL-23p19 to IL-23R via site 3. Biological activity of modified murine and human IL-23 is completely diminished on Ba/F3 cells with murine receptors. However, we observed the opposite effect on Ba/F3 cells with human IL-23 receptors. Substitution W157E in murine IL-23p19 significantly reduced biological activity on cells with human receptors.

Site 3 interaction of human IL-23 with human IL-23R requires Trp¹⁵⁶ and Leu¹⁶⁰. Consequently, substitution of both amino acids resulted in an inactive human IL-23. Single substitution of Leu¹⁶⁰ did not influence biological activity of human IL-23, as previously shown for murine IL-23p19L161E in a cellular context (21). Both amino acids, Leu¹⁶⁰ and Trp¹⁵⁶, of human IL-23p19 contact residues Tyr¹⁰⁰, Leu¹¹³, and Ile²⁸ of human IL-23R (Fig. S3A). Thus, hydrophobic interactions lost through Trp¹⁵⁶ substitution might be partially compensated by Leu¹⁶⁰. To test this hypothesis, we generated a molecular model of mIL-23R. This model was superpositioned onto the structure of the hIL-23R·p19·p40 complex (PDB code 5MZV). Residues Leu¹¹³ and Ile²⁸ are conserved within murine IL-23R. However, differences are seen for Tyr¹⁰⁰. At this position His⁷⁷ is located in the murine receptor (Fig. S3A). Consequently, we in silico generated a Y100H substitution of the IL-23R in the IL-23 signaling complex, to mimic the murine situation. Tyr¹⁰⁰ of hIL-23R interacts with Trp¹⁵⁶ and Leu¹¹³, whereas Tyr¹⁰⁰ is within a 4 Å radius of IL-23p19L160. In contrast, the distance between His¹⁰⁰ in the murinized model of the hIL-23·IL-23R signaling complex and IL-23p19L160 is longer than 4 Å. (Fig. S3B). This indicates that in the murine IL-23·IL-23R complex, His⁷⁷ probably does not interact with Leu¹¹³.

In conclusion, despite the structural similarities of IL-6/IL-12 type cytokines, distinct formation of these receptor complexes exists. Our results indicate differences in site 3–binding interfaces of murine and human IL-23 receptor complexes. These detailed structural insights into cytokine–cytokine receptor binding provide new avenues for the development of novel therapeutic strategies.

Experimental procedures

Cells and reagents

Ba/F3 (ACC-300), COS-7 (ACC-60), HEK293T (ACC-635), and CHO-K1 cells (ACC-110) were purchased from the Leibniz Institute DSMZ–German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig, Germany). The packaging cell line Phoenix-Eco was described previously (37). Murine Ba/F3-gp130 cells transduced with murine IL-12Rβ1 and mIL-23R (Ba/F3-gp130-mIL-12Rβ1-mIL-23R), as well as mIL-12Rβ1 and mIL-12Rβ2 (Ba/F3-gp130-mIL-12Rβ1-mIL-12Rβ2), were described previously (12, 32). All cell lines were grown in DMEM high-glucose culture medium (GIBCO^®, Thermo Fisher Scientific) supplemented with 10% fetal calf serum (GIBCO^®, Thermo Fisher Scientific), 60 mg/liter penicillin, and 100 mg/liter streptomycin (Genaxxon Bioscience GmbH, Ulm, Germany) at 37 °C with 5% CO₂ in a water-saturated atmosphere. Proliferation of Ba/F3 cells was maintained by adding 0.2% conditioned cell culture supernatant of IL-3 secreting WEHI-3B cells (DSMZ ACC-26). Either recombinant protein (10 ng/ml) or 0.2% of conditioned cell culture medium from a stable CHO-K1 clone secreting Hyper–IL-6 (final concentration, 10 ng/ml as determined by ELISA) was used to supplement the growth medium of Ba/F3-gp130 cell lines. Phospho-STAT3 (Tyr⁷⁰⁵) (D3A7), STAT3 (124H6), phospho-p44/42 MAPK (ERK1/2) (Thr²⁰²/Tyr²⁰⁴) (D13.14.4E) and p44/42 MAPK (ERK1/2) antibodies were obtained from Cell Signaling Technology (Frankfurt, Germany). Peroxidase-conjugated secondary mAbs and human Fc antibody were obtained from Pierce (Thermo Fisher Scientific). Biotinylated hIL-23R (BAF1400), hIL-12Rβ1 (BAF839), hIL-12Rβ2 (BAF1959), mp40 (BAF499), and hp40 (BAF219) mAbs and hIL-12Rβ1 phycoerythrin-conjugated mAb (FAB839P) were from R&D Systems (Minneapolis, MN). Purified rat anti-human IL-12Rβ2 and APC-streptavidin were purchased from BD Biosciences (Heidelberg, Germany). Alexa Fluor 647–conjugated Fab goat anti-rat IgG (number 112-607-003) was obtained from Dianova (Hamburg, Germany).

Cloning

Cloning of pcDNA3.1 expression vectors (Invitrogen) containing an N-terminal FLAG tag and a C-terminal His₆ tag for murine Hyper–IL-23 (mp40–p19) and murine Hyper–IL-12 (mp40–p35) was described elsewhere (12, 32). Analogous cloning strategies were used to generate expression vectors for human Hyper–IL-23 (hp40–p19) and human Hyper–IL-12 (hp40–p35). Mutations within murine/human p19 or murine/human p35 cDNAs were generated by PCR followed by DpnI digestion of methylated template DNA.

The extracellular domains of murine IL-12Rβ2 (amino acids 1–639) comprising D1–D6 and murine IL-12Rβ1 (amino acids 1–566) containing D1–D5 were C-terminally fused with human Fc tags (soluble (s)mIL-12Rβ2-Fc and smIL-12Rβ1-Fc). The resulting cDNAs were subcloned into p409 eukaryotic expression vector (38).

cDNAs for hIL-23R and hIL-12Rβ1 were obtained from Esther van de Vosse (Leiden University Medical Center, Leiden, The Netherlands), and the cDNA for hIL-12Rβ2 was purchased as cDNA clone (IRATp970H04123D) from Source BioSciences (Nottingham, UK). cDNAs were cloned into pMK-FUSIO coding for the self-processing 2A-peptide resulting in pMK-hIL-12Rβ1-2A-hIL-23R and pMK-hIL-12Rβ1-2A-hIL-Rβ2 (39, 40). For retroviral transduction of Ba/F3 cells, expression cassettes were transferred into pMOWS-puro coding for puromycin resistance (37).

Transfection, transduction, and selection of cells

Ba/F3 cells were retrovirally transduced with pMOWS expression plasmids coding for hIL-12Rβ1-2A-hIL-23R and hIL-12Rβ1-2A-hIL-12Rβ2 as described in (12). Transduced cells were grown in standard DMEM as described above supplemented with 0.2% conditioned cell culture supernatant of IL-3 secreting WEHI-3B cells. Selection of transduced Ba/F3 cells was performed with puromycin (1.5 μg/ml) (Carl Roth GmbH, Karlsruhe, Germany) for at least 2 weeks. Afterward, IL-3 was washed away, and the generated Ba/F3 cell lines were selected for IL-23– and IL-12–dependent growth.

Cell surface detection of cytokine receptors

To detect cell surface expression of the cytokine receptors, stably transduced Ba/F3 cell lines were washed with FACS buffer (PBS containing 1% BSA) and incubated at 5 × 10⁵ cells/100 μl FACS buffer supplemented with antibodies against hIL-23R, hIL-12Rβ1 (R&D Systems), or hIL-12Rβ2 (BD Biosciences) for 1 h. After a single wash with FACS buffer, the cells were incubated in 100 μl of FACS buffer containing APC-streptavidin (BD Biosciences) or Alexa Fluor 647–conjugated Fab goat anti-rat IgG (Dianova) for 1 h. Finally, the cells were washed once with FACS buffer, suspended in 500 μl of FACS buffer, and analyzed by flow cytometry (BD FACSCanto II flow cytometer, BD Biosciences). The data were evaluated using the FCS Express software (De Novo Software, Los Angeles, CA, USA).

Cell viability assay

To remove cytokines, Ba/F3 cell lines were washed three times with sterile PBS. 5 × 10³ cells were suspended in DMEM supplemented with 10% fetal calf serum, 60 mg/liter penicillin, and 100 mg/liter streptomycin and cultured for 3 days in a final volume of 100 μl either with or without cytokines (applied as conditioned medium) as indicated. The CellTiter-Blue^® cell viability assay (Promega, Karlsruhe, Germany) was used to estimate the number of viable cells by recording the fluorescence (excitation, 560 nm; emission, 590 nm) using the Infinite M200 PRO plate reader (Tecan, Crailsheim, Germany) immediately after adding 20 μl of reagent/well (time point 0) and up to 2 h after incubation under standard cell culture conditions. The fluorescent signal from the CellTiter-Blue^® reagent is proportional to the number of viable cells. All of the values were measured in triplicate per experiment. The fluorescence values were normalized by subtraction of time point 0 values. All experiments were performed at least two times, and one representative experiment was selected. The data are presented as means ± S.D. For multiple comparisons, one-way ANOVA, followed by Bonferroni correction, was used (GraphPad Prism 6.0, GraphPad Software Inc.). Statistical significance was set at the level of P ≤ 0.05 (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

Stimulation assays

For analysis of STAT3 and ERK1/2 activation in Ba/F3 cell lines, the cells were starved for at least 4 h in serum-free medium. This was followed by stimulation with cytokines as indicated. Subsequently, the cells were harvested and lysed in 10 mm Tris-HCl, pH 7.8, 150 mm NaCl, 0.5 mm EDTA, 0.5% Nonidet P-40, 1 mm sodium vanadate, and 10 mm MgCl₂ supplemented with complete protease inhibitor mixture tablets (Roche Diagnostics, Mannheim, Germany). Protein concentration of cell lysates was determined by BCA protein assay (Pierce, Thermo Scientific) according to the manufacturer's instructions. Analysis of STAT3 and ERK1/2 activation was done by immunoblotting using 50 μg of proteins from total cell lysates and detection with phospho-STAT3 or phospho-ERK1/2 mAbs. Detection of STAT3 or ERK1/2 served as loading control.

Coimmunoprecipitation (Co-IP)

For co-IP via protein A–agarose, transiently transfected COS-7 cells were lysed in 20 mm Tris-HCl, pH 7.5, 1 mm EDTA, 1 mm EGTA, 150 mm NaCl, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 1 mm sodium vanadate, and 1 complete protease inhibitor mixture tablet/50 ml buffer (Roche Diagnostics) supplemented with 1% Triton X-100 for 1.5 h at 4 °C. Cytokine-containing lysates were mixed with those containing the soluble Fc-tagged receptors. As negative control, the cytokine variant was incubated without receptors. All samples were incubated overnight at 4 °C under gentle agitation. 50 μl of protein A–agarose (Roche Diagnostics) was added and incubated at 4 °C for 4 h under gentle agitation. The samples were washed five times with above mentioned buffer, and the proteins were eluted by adding 50 μl of 5× Laemmli buffer, followed by incubation for 10 min at 95 °C. The resulting supernatants were subjected to Western blotting analysis. Cytokine-containing lysates were loaded as control.

For co-IP via ANTI-FLAG^® M2 affinity gel (Sigma–Aldrich) transiently transfected HEK293T cells were lysed in 50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 150 mm NaCl, and 1 complete protease inhibitor mixture tablet/50 ml buffer (Roche Diagnostics) supplemented with 1% Triton X-100 for 1 h on ice. Cytokine-containing lysates were mixed with those containing the full-length receptors. For negative control, cytokine variants were incubated without receptors and vice versa. 30 μl of ANTI-FLAG^® M2 affinity gel (Sigma–Aldrich) was added and incubated overnight at 4 °C under gentle agitation. The samples were washed three times with above mentioned buffer without 1% Triton X-100, and proteins were eluted by adding 50 μl of 2.5 × Laemmli buffer, followed by incubation for 10 min at 95 °C. The resulting supernatants were subjected to Western blotting analysis. Input samples were loaded as controls.

Western blotting

Defined amounts of proteins from cell lysates or conditioned cell culture supernatants were loaded per lane, separated by SDS-PAGE under reducing conditions, and transferred to polyvinylidene difluoride membranes. The membranes were blocked in 5% fat-free dried skimmed milk in TBS-T (10 mm Tris-HCl, pH 7.6, 150 mm NaCl, 1% Tween 20) and probed with the indicated primary antibodies in 5% fat-free dried skimmed milk in TBS-T (STAT3, ERK1/2, Fc) or 5% BSA in TBS-T (pSTAT3, pERK1/2, mp40, hp40, or receptor specific abs) at 4 °C overnight. After washing, the membranes were incubated with secondary peroxidase-conjugated antibodies or streptavidin–horseradish peroxidase diluted in 5% fat-free dried skimmed milk or BSA in TBS-T for 1 h at room temperature. The Immobilon^TM Western chemiluminescent horseradish peroxidase substrate (Merck) and the ChemoCam Imager (INTAS Science Imaging Instruments GmbH, Göttingen, Germany) were used for signal detection.

Modeling

Protein models were generated via the Phyre2 web portal (41) for Uniprot (Q9EQ14 and P43431). Molecular graphics; structural superpositioning of models with PDB codes 5MZV, 3D87, and 3HMX; and structure-based sequence alignments were generated using UCSF Chimera version 1.13.1, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health Grant P41-GM103311 (42).

Data availability

All data are contained within the article.

Supplementary Material

Supporting Information

supp_295_30_10478__index.html^{(906B, html)}

This article contains supporting information.

Author contributions—A. E., A. M., S. M., and J. Schröder formal analysis; A. E. and A. M. validation; A. E. investigation; S. M. and D. M. F. supervision; J. M. M., J. G., and J. Scheller resources; J. M. M. and J. G. software; J. M. M. visualization; J. Scheller and D. M. F. conceptualization; J. Scheller funding acquisition; J. Scheller and D. M. F. writing-review and editing; D. M. F. data curation; D. M. F. methodology; D. M. F. writing-original draft; D. M. F. project administration.

Funding and additional information—This work was supported by Deutsche Forschungsgemeinschaft Grant SFB1116 (to J. S.).

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Abbreviations—The abbreviations used are:

IL: interleukin
HIL: Hyper-IL
hIL: human IL
IL-12R: IL-12 receptor
IL-23R: IL-23 receptor
aa: amino acid(s)
Dn: domain n
PDB: Protein Data Bank
DMEM: Dulbecco's modified Eagle's medium
MAPK: mitogen-activated protein kinase
ERK: extracellular signal-regulated kinase
ANOVA: analysis of variance
IP: immunoprecipitation
m: murine
s: soluble
PAA: polyacrylamide.

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34. Murakami M., Kamimura D., and Hirano T. (2019) Pleiotropy and specificity: insights from the interleukin 6 family of cytokines. Immunity 50, 812–831 10.1016/j.immuni.2019.03.027 [DOI] [PubMed] [Google Scholar]
35. Rose-John S., Scheller J., and Schaper F. (2015) Family reunion"-A structured view on the composition of the receptor complexes of interleukin-6–type and interleukin-12–type cytokines. Cytokine Growth Factor Rev. 26, 471–474 10.1016/j.cytogfr.2015.07.011 [DOI] [PubMed] [Google Scholar]
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38. Althoff K., Müllberg J., Aasland D., Voltz N., Kallen K., Grötzinger J., and Rose-John S. (2001) Recognition sequences and structural elements contribute to shedding susceptibility of membrane proteins. Biochem. J. 353, 663–672 10.1042/0264-6021:3530663 [DOI] [PMC free article] [PubMed] [Google Scholar]
39. Suthaus J., Tillmann A., Lorenzen I., Bulanova E., Rose-John S., and Scheller J. (2010) Forced homo- and heterodimerization of all gp130-type receptor complexes leads to constitutive ligand-independent signaling and cytokine-independent growth. Mol. Biol. Cell 21, 2797–2807 10.1091/mbc.e10-03-0240 [DOI] [PMC free article] [PubMed] [Google Scholar]
40. Fang J., Qian J. J., Yi S., Harding T. C., Tu G. H., VanRoey M., and Jooss K. (2005) Stable antibody expression at therapeutic levels using the 2A peptide. Nat. Biotechnol. 23, 584–590 10.1038/nbt1087 [DOI] [PubMed] [Google Scholar]
41. Kelley L. A., Mezulis S., Yates C. M., Wass M. N., and Sternberg M. J. (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 10.1038/nprot.2015.053 [DOI] [PMC free article] [PubMed] [Google Scholar]
42. Pettersen E. F., Goddard T. D., Huang C. C., Couch G. S., Greenblatt D. M., Meng E. C., and Ferrin T. E. (2004) UCSF Chimera: a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 10.1002/jcc.20084 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information

supp_295_30_10478__index.html^{(906B, html)}

supp_RA120.013935_160242_1_supp_541455_qbcc9g.pdf^{(630.8KB, pdf)}

Data Availability Statement

All data are contained within the article.

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[B21] 21. Bloch Y., Bouchareychas L., Merceron R., Składanowska K., Van den Bossche L., Detry S., Govindarajan S., Elewaut D., Haerynck F., Dullaers M., Adamopoulos I. E., and Savvides S. N. (2018) Structural activation of pro-inflammatory human cytokine IL-23 by cognate IL-23 receptor enables recruitment of the shared receptor IL-12Rβ1. Immunity 48, 45–58 10.1016/j.immuni.2017.12.008 [DOI] [PMC free article] [PubMed] [Google Scholar]

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[B25] 25. Shimozato O., Ugai S., Chiyo M., Takenobu H., Nagakawa H., Wada A., Kawamura K., Yamamoto H., and Tagawa M. (2006) The secreted form of the p40 subunit of interleukin (IL)-12 inhibits IL-23 functions and abrogates IL-23-mediated antitumour effects. Immunology 117, 22–28 10.1111/j.1365-2567.2005.02257.x [DOI] [PMC free article] [PubMed] [Google Scholar]

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[B27] 27. Beyer B. M., Ingram R., Ramanathan L., Reichert P., Le H. V., Madison V., and Orth P. (2008) Crystal structures of the pro-inflammatory cytokine interleukin-23 and its complex with a high-affinity neutralizing antibody. J. Mol. Biol. 382, 942–955 10.1016/j.jmb.2008.08.001 [DOI] [PubMed] [Google Scholar]

[B28] 28. Fischer M., Goldschmitt J., Peschel C., Brakenhoff J., Kallen K., Wollmer A., Grötzinger J., and Rose-John S. (1997) A bioactive designer cytokine for human hematopoietic progenitor cell expansion. Nat. Biotechnol. 15, 142–145 10.1038/nbt0297-142 [DOI] [PubMed] [Google Scholar]

[B29] 29. Barton V. A., Hudson K. R., and Heath J. K. (1999) Identification of three distinct receptor binding sites of murine interleukin-11. J. Biol. Chem. 274, 5755–5761 10.1074/jbc.274.9.5755 [DOI] [PubMed] [Google Scholar]

[B30] 30. Ciapponi L., Graziani R., Paonessa G., Lahm A., Ciliberto G., and Savino R. (1995) Definition of a composite binding site for gp130 in human interleukin-6. J. Biol. Chem. 270, 31249–31254 10.1074/jbc.270.52.31249 [DOI] [PubMed] [Google Scholar]

[B31] 31. Oppmann B., Lesley R., Blom B., Timans J., Xu Y., Hunte B., Vega F., Yu N., Wang J., Singh K., Zonin F., Vaisberg E., Churakova T., Liu M., Gorman D., et al. (2000) Novel p19 protein engages IL-12p40 to form a cytokine, IL-23 with biological activities similar as well as distinct from IL-12. Immunity 13, 715–725 10.1016/S1074-7613(00)00070-4 [DOI] [PubMed] [Google Scholar]

[B32] 32. Floss D. M., Klöcker T., Schröder J., Lamertz L., Mrotzek S., Strobl B., Hermanns H., and Scheller J. (2016) Defining the functional binding sites of interleukin 12 receptor β1 and interleukin 23 receptor to Janus kinases. Mol. Biol. Cell 27, 2301–2316 10.1091/mbc.E14-12-1645 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B33] 33. Schoenhaut D. S., Chua A. O., Wolitzky A. G., Quinn P. M., Dwyer C. M., McComas W., Familletti P. C., Gately M. K., and Gubler U. (1992) Cloning and expression of murine IL-12. J. Immunol. 148, 3433–3440 [PubMed] [Google Scholar]

[B34] 34. Murakami M., Kamimura D., and Hirano T. (2019) Pleiotropy and specificity: insights from the interleukin 6 family of cytokines. Immunity 50, 812–831 10.1016/j.immuni.2019.03.027 [DOI] [PubMed] [Google Scholar]

[B35] 35. Rose-John S., Scheller J., and Schaper F. (2015) Family reunion"-A structured view on the composition of the receptor complexes of interleukin-6–type and interleukin-12–type cytokines. Cytokine Growth Factor Rev. 26, 471–474 10.1016/j.cytogfr.2015.07.011 [DOI] [PubMed] [Google Scholar]

[B36] 36. Sayago C., Gonzalez Valcarcel I. C., Qian Y., Lee J., Alsina-Fernandez J., Fite N. C., Carrillo J. J., Zhang F. F., Chalmers M. J., Dodge J. A., Broughton H., and Espada A. (2018) Deciphering binding interactions of IL-23R with HDX-MS: mapping protein and macrocyclic dodecapeptide ligands. ACS Med. Chem. Lett. 9, 912–916 10.1021/acsmedchemlett.8b00255 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B37] 37. Ketteler R., Glaser S., Sandra O., Martens U. M., and Klingmuller U. (2002) Enhanced transgene expression in primitive hematopoietic progenitor cells and embryonic stem cells efficiently transduced by optimized retroviral hybrid vectors. Gene Ther. 9, 477–487 10.1038/sj.gt.3301653 [DOI] [PubMed] [Google Scholar]

[B38] 38. Althoff K., Müllberg J., Aasland D., Voltz N., Kallen K., Grötzinger J., and Rose-John S. (2001) Recognition sequences and structural elements contribute to shedding susceptibility of membrane proteins. Biochem. J. 353, 663–672 10.1042/0264-6021:3530663 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B39] 39. Suthaus J., Tillmann A., Lorenzen I., Bulanova E., Rose-John S., and Scheller J. (2010) Forced homo- and heterodimerization of all gp130-type receptor complexes leads to constitutive ligand-independent signaling and cytokine-independent growth. Mol. Biol. Cell 21, 2797–2807 10.1091/mbc.e10-03-0240 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B40] 40. Fang J., Qian J. J., Yi S., Harding T. C., Tu G. H., VanRoey M., and Jooss K. (2005) Stable antibody expression at therapeutic levels using the 2A peptide. Nat. Biotechnol. 23, 584–590 10.1038/nbt1087 [DOI] [PubMed] [Google Scholar]

[B41] 41. Kelley L. A., Mezulis S., Yates C. M., Wass M. N., and Sternberg M. J. (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 10.1038/nprot.2015.053 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B42] 42. Pettersen E. F., Goddard T. D., Huang C. C., Couch G. S., Greenblatt D. M., Meng E. C., and Ferrin T. E. (2004) UCSF Chimera: a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 10.1002/jcc.20084 [DOI] [PubMed] [Google Scholar]

PERMALINK

Deciphering site 3 interactions of interleukin 12 and interleukin 23 with their cognate murine and human receptors

Alessandra Esch

Anna Masiarz

Sofie Mossner

Jens M Moll

Joachim Grötzinger

Jutta Schröder

Jürgen Scheller

Doreen M Floss

Abstract

Figure 1.

Figure 2.

Results

Y185R in murine IL-12p35 results in abrogated IL-12Rβ2 binding and IL-12 signaling

Murine IL-23p19W157A but not IL-12p35Y185R is still active on the corresponding human receptor chains

Figure 3.

Human IL-23W156A does not activate murine receptors

Figure 4.

Human IL-23p19W156A and IL-23p19W156E are still active on the corresponding human receptor chains

Figure 5.

Figure 6.

Amino acid substitutions surrounding human IL-23p19W156 did not affect binding to human IL-23R

Figure 7.

Figure 8.

Human IL-23p19W156A/L160E is inactive on human IL-23 receptor chains

Figure 9.

Discussion

Experimental procedures

Cells and reagents

Cloning

Transfection, transduction, and selection of cells

Cell surface detection of cytokine receptors

Cell viability assay

Stimulation assays

Coimmunoprecipitation (Co-IP)

Western blotting

Modeling

Data availability

Supplementary Material

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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