The Interaction of Interferon Alphas with the Interferon Alpha Receptor

用ELISA法检测到两种IFN-β能与IFNAR 2-EC结合。 为了检测两种IFN种类（CM 3 -6-组氨酸-标签和IFN-2c-6-组氨酸-标签）与受体亚基IFNAR 2-EC的结合，开发了竞争性夹心IFNAR 2-EC ELISA。我们发现，在用CM 3 -6-组氨酸标签饱和IFNAR 2-EC后，受体亚基仍然能够结合IFN-2c-6-组氨酸标签，其中CM 3 -6-组氨酸标签的置换较低。使用表面等离子体共振生物传感器，我们确定IFN-α杂合物CM 3稳定IFN-2c/IFNAR 2-EC复合物。 对IFN-2c和CM 3在溶液中的寡聚状态的分析表明，IFN- 2c的沉降模式与CM 3的沉降模式形成鲜明对比。 为了更深入地了解可能的结合机制，以及SPR中观察到的受体结合差异，通过沉降速度分析超离心研究了两种IFN分子的寡聚状态。使用分析性超离心沉降速度（AUC/SV）监测IFN通过离心场的迁移速率。结果表明，CM 3的沉降曲线在溶液中不与IFN- 2c相互作用（或仅非常弱）。 对溶液中IFN-受体复合物的分析证实了两种IFN（IFN-2c-6-组氨酸标签，CM 3）和IFNAR 2-EC的存在。天然电泳实验表明，IFN在很大程度上是竞争性的，但也允许检测与两种IFN的三重复合物的群体。 IFNAR 2-EC突变体表明CM 3和IFN-2c可以在IFNAR 2-EC的结合结构域内使用不同的氨基酸。 抗增殖试验中IFN-2c和CM 3之间的差异不太可能是与IFNAR 2相互作用的结果。 用可溶性IFNAR 2-EC中和IFN对Daudi细胞的抗增殖活性显示IFNAR 2-EC中和两种IFN至相同程度，表明IFN-2c和CM 3以相似的亲和力结合IFNAR 2-EC。这一观察结果得出结论，IFN-2c和CM 3在抗增殖试验中的差异不是由于与IFNAR 2相互作用的问题。 N-末端6-组氨酸标签似乎影响IFN- 2c的蛋白质结构。 从本研究的结果，我们可以得出结论，N-末端的6-组氨酸标签可能会影响蛋白质的结构，并导致局部错误折叠和空间位阻的IFN-2c分子。虽然我们没有预测到显著的结构变化，但发生的变化足以影响抗体各自表位的识别，并降低特异性抗病毒和抗增殖活性

Two IFN-αs can bind to IFNAR2-EC as detected by ELISA. To examine the binding of two IFN species (CM3-6-histidine-tag and IFN-α2c-6-histidine-tag) to the receptor subunit IFNAR2-EC, a competitive sandwich IFNAR2-EC ELISA was developed. We found that after saturation of IFNAR2-EC with CM3-6-histidine-tag, the receptor subunit was still able to bind IFN-α2c-6-histidine-tag with low displacement of the CM3-6-Histidine tag. Using a surface plasmon resonance biosensor, we determined that the IFN-alpha hybrid CM3 stabilized IFN-α2c/IFNAR2-EC complex. Analysis of IFN-α2c and CM3 oligomeric state in solution showed that the sedimentation profile of IFN-α2c was in sharp contrast to that of CM3. In order to gain more insight into the possible binding mechanisms, and the differences in receptor binding observed in SPR, the oligomeric state of both IFN molecules was studied by sedimentation velocity analytical ultracentrifugation. The rate of migration of the IFNs through a centrifugal field was monitored using analytical ultracentrifugation sedimentation velocity (AUC/SV). The results showed that the sedimentation profile of CM3 does not (or only very weakly) interact with IFN-α2c in solution. Analysis of IFN-receptor complexes in solution confirmed the presence of two IFNs (IFN-α2c-6-histidine-tag, CM3) and IFNAR2-EC. Native electrophoresis experiments showed the IFNs to be largely competitive, but also allowed detection of a population of triple complexes with both IFNs. IFNAR2-EC mutants suggest that CM3 and IFN-α2c could use different amino acids within the binding domain of IFNAR2-EC. Differences between IFN-α2c and CM3 in the antiproliferative assay are unlikely to be a consequence of interaction with IFNAR2. Neutralization of antiproliferative activities of IFNs on Daudi cells with soluble IFNAR2-EC show that IFNAR2-EC neutralized both IFNs to the same degree, suggesting that IFN-α2c and CM3 are binding to IFNAR2-EC with similar affinity. This observation led to the conclusion that differences between IFN-α2c and CM3 in the antiproliferative assay are not due to problems in interaction with IFNAR2. The N-terminal 6-histidine tag appears to affect the protein structure of IFN-α2c. From the results of this study we could conclude that the N-terminal 6-histidine tag may affect protein structure and cause local misfolding and steric hindrance on the IFN-α2c molecule. While we did not predict dramatic structural changes, the changes that occurred were sufficient to influence the recognition of the antibodies respective epitopes and to reduce specific antiviral and antiproliferative activities