Abstract
The interaction between IgE-Fc (Fcepsilon) and its high affinity receptor FcepsilonRI 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 Fcepsilon-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 Fcepsilon and IgG1-Fc (Fcgamma). Fcepsilon was found to be significantly less stable than Fcgamma under all pH and NaCl conditions tested. Additionally, the Cepsilon3Cepsilon4 domains of Fcepsilon were shown to become intrinsically unfolded at pH values below 5.0. The interaction between Fcepsilon and an Fcgamma-FcepsilonRIalpha 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 Fcepsilon for the dimeric fusion protein was extremely high compared with published values for the monomeric receptor (KD < 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 Fcepsilon unfolded, and interaction of Fcepsilon with the Fcgamma-FcepsilonRIalpha fusion protein was abrogated. The unusual pH sensitivity of Fcepsilon may play a role in antigen-dependent regulation of receptor-bound, non-circulating IgE.
Highlights
Cross-linking of cell surface IgE1⁄7Fc⑀RI complexes with multivalent antigens or anti-Immunoglobulin E (IgE) antibodies leads to receptor signaling and cellular degranulation, the release of preformed secretory granules containing histamine, serotonin, various lipids, proteases, and other acute inflammatory agents, and the expression and release of inflammatory cytokines [1]
Using circular dichroism (CD)3 and differential scanning calorimetry (DSC), we demonstrate that Fc⑀ is much less thermostable than Fc␥
Because marked decreases in Fc⑀ stability were observed as the pH was decreased, we investigated whether pH-dependent stability changes attenuated the ability of IgE to interact with its high affinity receptor, Fc⑀RI␣
Summary
Subcloning of Fc⑀, Fc␥, Fc␥-C⑀2, Fc␥-C⑀2C⑀3, and Fc␥Fc⑀RI␣-Fc⑀ was subcloned from mRNA extracted from human blood B cells (CD20ϩ and CD20ϩ IgMϪ). The Fc⑀ and Fc␥-C⑀2 proteins were co-dialyzed against 10 mM sodium phosphate, 15 mM NaCl, pH 2.5, diluted to 1 mg/ml with dialysate, and scanned under identical conditions as described for the buffer panel. For measurement of the thermodynamic interaction parameters between Fc⑀ and Fc␥-Fc⑀RI␣, each protein was dialyzed against a panel of buffer solutions at pH 4.5, 5.0, 5.5, 6.0, and 7.4 as described above for the CD experiments. Using the dimeric molecular weight for apo- and holo-Fc⑀ and the monomeric molecular weight for apo- and holo-Fc␥-Fc⑀RI␣, 2.5 M protein solutions were prepared using matched dialysates for dilution and incubated for at least 2 h at 4 °C prior to DSC analysis. The parameters were input into expressions derived by Brandts and Lin [29] for two proteins that demonstrate independent unfolding transitions in isolation but whose unfolding becomes thermodynamically coupled upon formation of a complex
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