The Interaction of Interferon Alphas with the Interferon Alpha Receptor

ELISA检测到两种IFN-&#945；s可以与IFNAR2-EC结合。为了检测两种IFN （CM3-6-histidine-tag和IFN- 2c-6-histidine-tag）与受体亚基IFNAR2-EC的结合，我们开发了一种竞争性三明治IFNAR2-EC ELISA。我们发现，在IFNAR2-EC被CM3-6-Histidine -tag饱和后，受体亚基仍然能够结合IFN-&#945；2c-6-histidine-tag，而CM3-6-Histidine -tag的位移较小。使用表面等离子体共振生物传感器，我们确定IFN- α杂化CM3稳定IFN-&#945；2c/IFNAR2-EC配合物。对IFN-&#945；2c和CM3在溶液中的低聚态分析表明，IFN-&#945；2c的沉降剖面与CM3的沉降剖面形成鲜明对比。为了更深入地了解可能的结合机制，以及在SPR中观察到的受体结合的差异，我们使用沉降速度分析超离心研究了两种IFN分子的低聚态。利用分析型超离心沉降速度（AUC/SV）监测ifn通过离心场的迁移速率。结果表明，CM3的沉降剖面与溶液中的IFN-&#945；2c没有（或仅非常弱）相互作用。

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