Feline Interleukin-31 Shares Overlapping Epitopes with the Oncostatin M Receptor and IL-31RA

Interleukin-31 (IL-31) is a major protein involved in severe inflammatory skin disorders. Its signaling pathway is mediated through two type I cytokine receptors, IL-31RA (also known as the gp130-like receptor) and the oncostatin M receptor (OSMR). Understanding molecular details in these interactions would be helpful for developing antagonist anti-IL-31 monoclonal antibodies (mAbs) as potential therapies. Previous studies suggest that human IL-31 binds to IL-31RA and then recruits OSMR to form a ternary complex. In this model, OSMR cannot interact with IL-31 in the absence of IL-31RA. In this work, we show that feline IL-31 (fIL-31) binds independently with feline OSMR using surface plasmon resonance, an enzyme-linked immunosorbent assay, and yeast surface display. Moreover, competition experiments suggest that OSMR shares a partially overlapping epitope with IL-31RA. We then used deep mutational scanning to map the binding sites of both receptors on fIL-31. In agreement with previous studies of the human homologue, the binding site for IL31-RA contains fIL-31 positions E20 and K82, while the binding site for OSMR comprises the “PADNFERK” motif (P103–K110) and position G38. However, our results also revealed a new overlapping site, composed of positions R69, R72, P73, D76, D81, and E97, between both receptors that we called the “shared site”. The conformational epitope of an anti-feline IL-31 mAb that inhibits both OSMR and IL-31RA also mapped to this shared site. Combined, our results show that fIL-31 binds IL-31RA and OSMR independently through a partially shared epitope. These results suggest reexamination of the putative canonical mechanisms for IL-31 signaling in higher animals.白细胞介素-31（Interleukin-31，IL-31）是参与重度炎症性皮肤病的关键蛋白。其信号通路由两种I型细胞因子受体（type I cytokine receptor）介导：IL-31RA（亦称gp130样受体）与制瘤素M受体（oncostatin M receptor，OSMR）。阐明此类相互作用的分子细节，有助于开发抗IL-31拮抗剂单克隆抗体（monoclonal antibodies，mAbs）作为潜在治疗手段。既往研究表明，人源IL-31先结合IL-31RA，随后招募OSMR形成三元复合物；在此经典模型中，若无IL-31RA存在，OSMR无法与IL-31直接结合。本研究中，我们通过表面等离子体共振（surface plasmon resonance）、酶联免疫吸附实验（enzyme-linked immunosorbent assay）及酵母表面展示（yeast surface display）实验证实，猫源IL-31（feline IL-31，fIL-31）可独立结合猫源OSMR。此外，竞争实验结果显示，OSMR与IL-31RA共享部分重叠的表位。随后我们利用深度突变扫描（deep mutational scanning），绘制了两种受体在fIL-31上的结合位点图谱。与既往针对人源同源蛋白的研究结果一致，IL-31RA的结合位点包含fIL-31的E20与K82位点；而OSMR的结合位点则涵盖“PADNFERK”基序（P103-K110）及G38位点。但本研究同时发现了一处全新的共享结合位点，由R69、R72、P73、D76、D81及E97位点组成，我们将其命名为“共享位点”。一株可同时抑制OSMR与IL-31RA结合的抗猫源IL-31单克隆抗体，其构象表位也恰好位于该共享位点。综合以上结果，我们证实fIL-31可通过部分重叠的表位分别结合IL-31RA与OSMR。本研究结果提示，有必要重新审视高等动物中IL-31信号通路的公认经典机制。