Department of Chemical Engineering and Materials Science, Michigan State University, Engineering Building, 428 South Shaw Lane, Room 2100, East Lansing, Michigan 48824, United States.
Veterinary Medicine Research and Development, Zoetis Global Therapeutic Research, 333 Portage Street, Kalamazoo, Michigan 49007, United States.
Biochemistry. 2020 Jun 16;59(23):2171-2181. doi: 10.1021/acs.biochem.0c00176. Epub 2020 Jun 4.
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（IL-31）是一种参与严重炎症性皮肤疾病的主要蛋白。其信号通路通过两种 I 型细胞因子受体介导，即白细胞介素-31 受体 A（也称为 gp130 样受体）和抑瘤素 M 受体（OSMR）。了解这些相互作用中的分子细节将有助于开发作为潜在治疗方法的抗白细胞介素-31 单克隆抗体（mAb）拮抗剂。先前的研究表明，人白细胞介素-31 与白细胞介素-31 受体 A 结合，然后招募抑瘤素 M 受体形成三元复合物。在该模型中，在没有白细胞介素-31 受体 A 的情况下，抑瘤素 M 受体不能与白细胞介素-31 相互作用。在这项工作中，我们使用表面等离子体共振、酶联免疫吸附试验和酵母表面展示表明，猫白细胞介素-31（fIL-31）独立地与猫抑瘤素 M 受体结合。此外，竞争实验表明 OSMR 与白细胞介素-31 受体 A 共享部分重叠表位。然后，我们使用深度突变扫描来绘制两个受体在 fIL-31 上的结合位点。与人类同源物的先前研究一致，IL-31-RA 的结合位点包含 fIL-31 的 E20 和 K82 位置，而 OSMR 的结合位点包含“PADNFERK”基序（P103-K110）和 G38 位置。然而，我们的结果还揭示了一个新的重叠位点，由两个受体之间的 R69、R72、P73、D76、D81 和 E97 位置组成，我们称之为“共享位点”。抑制 OSMR 和白细胞介素-31 受体 A 的抗猫白细胞介素-31 mAb 的构象表位也映射到该共享位点。综上所述，我们的结果表明 fIL-31 通过部分重叠的表位独立地与白细胞介素-31 受体 A 和 OSMR 结合。这些结果表明需要重新检查高等动物中白细胞介素-31 信号转导的假定经典机制。
