The IFN/receptor interaction is critical in determining the extent of the subsequent biological response (5
). Differences in spectra of biological activities among members of the IFN-α family indicate that each subtype could exhibit differences in receptor binding and/or intracellular signaling.
Our previous results showed that hybrid IFNs with N-terminal portion derived from IFN-α21b competed poorly with IFN-α2b for cellular binding (12
). CM3 is one of these hybrids, and also has a mutation in helix C (Y86K). Structural changes in helix C may influence interaction with IFNAR1, a component of the type I interferon receptor which is considered important for IFN antiproliferative activity (19
). This could explain the tenfold lower antiproliferative activity of CM3 than that of IFN-α2c in 48 hours antiproliferative assay. It did however not explain the 800-fold lower ability of CM3 (with 6-histidine tag) to compete with IFN-α2c (with 6-histidine tag) for receptor binding site (17
This observation raised the question, whether interaction among two individual IFN-αs and IFNAR2-EC contribute to the observed poor competition. To explore this question, we analyzed the binding of IFN-α2c and hybrid CM3 with IFNAR2-EC using defined anti-IFN mAbs in competitive binding ELISA, native electrophoresis followed by Western blot, ESI-MS, SPR analysis and neutralization assay.
The data from competitive binding ELISA appear to show that two IFN-α subtypes could bind simultaneously to IFNAR2-EC without significantly affecting each other's binding to IFNAR2-EC. Only IFNs with N-terminal 6-histidine-tag were used in all ELISA experiment, due to the fact that mAbs only recognized IFNs with 6-histidine-tag after binding to IFNAR2-EC. This could be explained by influence of the 6-histidine-tag on IFN structure (24
In order to approach the surface binding observed by ELISA in a more quantitative way, further analysis of the interaction of IFN-α2c and CM3 with IFNAR2-EC was done using SPR optical biosensing. To exclude any negative effect a tag might have on interaction, we used IFNs without N-terminal 6-histidine-tag in these experiments. SPR optical biosensing is a technology that provides real time information on the course of the interaction, and is similar to ELISA in that it requires the immobilization of one of the interacting compounds. It does not, however, require the use of antibodies. Because of the surface, two experimental designs were possible: one, where the interferons were covalently attached to the chip surface, while the receptor was in solution, and the other, with the reverse configuration. Strikingly different binding curves were detected in the different configurations, pointing to a more complex interaction mechanism.
In the configurations where IFN-α2c or CM3 were immobilized on the chip surface, their interaction with IFNAR2-EC that was in solution was similar. Neither of the soluble interferons had any influence on the kinetics of dissociation of the pre-bound receptor. In the configuration in which IFNAR2-EC was immobilized, IFN-α2c and CM3 each interacted differently with the receptor and CM3 affected the kinetics of dissociation of the pre-formed IFN-α2c/receptor complex. One could hypothesize that during the formation of the interferon-surface-bound receptor complexes, different interferons might undergo small conformational changes, which may be mutually influenced by a transient interaction. With immobilized interferons, the immobilization constraints might prohibit the necessary conformational flexibility. Alternatively, lateral association or oligomerization of the receptor-interferon complex produced by their close proximity on the sensor chip surface might also play a role. This is emphasized by our observation from sedimentation velocity of the self-association in solution of IFN-α2c, but no detectable self-association of CM3. While the SPR data did not confirm the presence of two individual IFN-αs on IFNAR2-EC, they showed that both IFNs influence each other during this interaction. The differences in results between ELISA and SPR could be due to the fact that these methods use different surfaces for immobilization of proteins. This could potentially lead to different results regarding the conformation and aggregation state of the surface-bound molecules. Further, the molecules used were slightly different (his-tag in ELISA versus no his-tag in SPR).
ELISA experiments and SPR analysis required immobilizing the proteins onto a surface. Thus the surface could contribute to structural changes of the proteins. To eliminate that possibility, protein-protein interactions were studied in solution, and detected by native electrophoresis and immunoblot. These experiments showed competition between IFN-α2c and CM3 (with or without 6-histidine tag) for IFNAR2-EC. Moreover, ESI-MS analysis of bands representing IFN-receptor complexes that were cut from native PAGE gels confirmed the presence of two IFNs with the IFNAR2-EC at the same time in the band A. These results suggested that two IFNs could bind directly to IFNAR2-EC. However, binding of CM3 to the IFNAR2-EC portion of the IFNAR2-EC-IFN-α2c complex is probably weak and transient, since it is not able to release IFN-α2c from IFNAR2-EC interaction effectively. The presence of CM3 could affect the conformation of IFN-α2c, which could be an additional explanation why monoclonal antibody (anti-6-histidine-tag) recognized IFN-α2c in the complex less effectively. Further studies and the use of different experimental approaches will be necessary to gain a detailed understanding of these interactions.
The generally observed lower quantity of CM3 (with or without 6-histidine-tag) receptor complexes could be explained by conditions in native electrophoresis that may cause some structural changes on CM3 molecule, which then could reduce interaction with IFNAR2-EC.
Experiments using IFNAR2-EC mutants in native electrophoresis showed that IFNAR2-EC mutations in positions 105 (I105A) and 47 (I47A) completely prevented interaction between CM3 (with or without 6-histidine tag) and the receptor subunit. These results provide evidence that CM3 (with or without 6-histidine tag) and IFN-α2c (with or without 6-histidine tag) could use different amino acids within the binding domain of IFNAR2-EC. Similar results had been observed for IFN-α2c versus IFN-β, where mutating IFNAR2-EC (W102A) proved to be most destructive for IFN-β binding (22
). Because IFNs mutually influence each other during competition, binding domains are probably in close proximity or overlapping (22
Cell-based neutralization assays with IFNAR2-EC show the same neutralization effect on CM3 and IFN-α2c. Thus the utilization of different amino acids in the binding region of IFNAR2 could help explain the better competitive ability of IFN-α2c compared to that of CM3. Moreover, these results strengthen evidence that the mutation in helix C had no impact on interaction with IFNAR2. The difference between the two IFNs observed in antiproliferative assay could be a consequence of interaction with IFNAR1 (19
In conclusion, the results of this study show the complexity of the interaction among different IFN-α subtypes during the process of binding to IFNAR2-EC. They underscore the importance of the differences in interaction on the amino acid level during this process, which could help understand the significance of the existence of the various IFN-αs subtypes.