An intermediate pH unfolding transition abrogates the ability of IgE to interact with its high affinity receptor FcεRIα

The interaction between IgE-Fc (Fc epsilon) and its high affinity receptor Fc epsilon RI on the surface of mast cells and basophils is a key event in allergen-induced allergic inflammation. Recently, several therapeutic strategies have been developed based on this interaction, and some include Fc epsilon-containing moieties. Unlike well characterized IgG therapeutics, the stability and folding properties of IgE are not well understood. Here, we present comparative biophysical analyses of the pH stability and thermostability of Fc epsilon and IgG1-Fc (Fc gamma). Fc gamma was found to be significantly less stable than Fc gamma under all pH and NaCl conditions tested. Additionally, the C epsilon 3C epsilon 4 domains of Fc epsilon were shown to become intrinsically unfolded at pH values below 5.0. The interaction between Fc epsilon and an Fc gamma-Fc epsilon RI alpha fusion protein was studied between pH 4.5 and 7.4 using circular dichroism and a combination of differential scanning calorimetry and isothermal titration calorimetry. Under neutral pH conditions, the apparent affinity of Fc epsilon for the dimeric fusion protein was extremely high compared with published values for the monomeric receptor (K-D < 10(-12) M). Titration to pH 6.0 did not significantly change the binding affinity, and titration to pH 5.5 only modestly attenuated affinity. At pH values below 5.0, the receptor binding domains of Fc epsilon unfolded, and interaction of Fc epsilon with the Fc gamma-Fc epsilon RI alpha fusion protein was abrogated. The unusual pH sensitivity of Fc epsilon may play a role in antigen-dependent regulation of receptor-bound, non-circulating IgE.