Aoki Y, Narazaki M, Kishimoto T, Tosato G
Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Blood. 2001 Nov 15;98(10):3042-9. doi: 10.1182/blood.v98.10.3042.
Receptor usage by viral interleukin-6 (vIL-6), a virokine encoded by Kaposi sarcoma- associated herpesvirus, is an issue of controversy. Recently, the crystal structure of vIL-6 identified vIL-6 sites II and III as directly binding to glycoprotein (gp)130, the common signal transducer for the IL-6 family of cytokines. Site I of vIL-6, however, comprising the outward helical face of vIL-6, where human IL-6 (hIL-6) would interact with the specific alpha-chain IL-6 receptor (IL-6R), is accessible and not occupied by gp130. This study examined whether this unused vIL-6 surface is available for IL-6R binding. By enzyme-linked immunosorbent assay, vIL-6 bound to soluble gp130 (sgp130) but not to soluble IL-6R (sIL-6R). Using plasmon surface resonance, vIL-6 bound to sgp130 with a dissociation constant of 2.5 microM, corresponding to 1000-fold lower affinity than that of hIL-6/sIL-6R complex for gp130. sIL-6R neither bound to vIL-6 nor affected vIL-6 binding to gp130. In bioassays, vIL-6 activity was neutralized by 4 monoclonal antibodies (mAbs) recognizing a domain within vIL-6 site I, mapped to the C-terminal part of the AB-loop and the beginning of helix B. The homologous region in hIL-6 participates in site I binding to IL-6R. In addition, binding of vIL-6 to sgp130 was interfered with specifically by the 4 neutralizing anti-vIL-6 mAbs. Based on the vIL-6 crystal structure, the vIL-6 neutralizing mAbs map outside the binding interface to gp130, suggesting that they either produce allosteric changes or block necessary conformational changes in vIL-6 preceding its binding to gp130. These results document that vIL-6 does not bind IL-6R and suggest that conformational change may be critical to vIL-6 function.
由卡波西肉瘤相关疱疹病毒编码的病毒白细胞介素6（vIL-6）的受体使用情况存在争议。最近，vIL-6的晶体结构确定vIL-6的位点II和III直接与糖蛋白（gp）130结合，gp130是白细胞介素6细胞因子家族的共同信号转导子。然而，vIL-6的位点I，即vIL-6向外的螺旋面，人类白细胞介素6（hIL-6）在此处会与特异性α链白细胞介素6受体（IL-6R）相互作用，该位点是可及的且未被gp130占据。本研究检测了这个未被利用的vIL-6表面是否可用于与IL-6R结合。通过酶联免疫吸附测定，vIL-6与可溶性gp130（sgp130）结合，但不与可溶性IL-6R（sIL-6R）结合。使用表面等离子体共振技术，vIL-6与sgp130结合，解离常数为2.5微摩尔，其亲和力比hIL-6/sIL-6R复合物对gp130的亲和力低1000倍。sIL-6R既不与vIL-6结合，也不影响vIL-6与gp130的结合。在生物测定中，vIL-6的活性被4种单克隆抗体（mAb）中和，这些单克隆抗体识别vIL-6位点I内的一个结构域，该结构域定位在AB环的C末端部分和螺旋B的起始处。hIL-6中的同源区域参与位点I与IL-6R的结合。此外，4种中和性抗vIL-6单克隆抗体特异性地干扰了vIL-6与sgp130的结合。基于vIL-6的晶体结构，vIL-6中和性单克隆抗体映射在与gp130的结合界面之外，这表明它们要么产生变构变化，要么在vIL-6与gp130结合之前阻断其必要的构象变化。这些结果证明vIL-6不与IL-6R结合，并表明构象变化可能对vIL-6的功能至关重要。
