title: An intermediate pH unfolding transition abrogates the ability of IgE to interact with its high affinity receptor FcepsilonRIalpha.
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.
authors.affiliation: Biogen Idec, San Diego, California 92122, USA. stephan.demarest@biogenidec.com
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Demarest
authors.forename: Jennifer
authors.initials: J
authors.lastname: Hopp
authors.forename: Julie
authors.initials: J
authors.lastname: Chung
authors.forename: Karen
authors.initials: K
authors.lastname: Hathaway
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Mertsching
authors.forename: Xianjun
authors.initials: X
authors.lastname: Cao
authors.forename: Judy
authors.initials: J
authors.lastname: George
authors.forename: Konrad
authors.initials: K
authors.lastname: Miatkowski
authors.forename: Michael J
authors.initials: MJ
authors.lastname: LaBarre
authors.forename: Michael
authors.initials: M
authors.lastname: Shields
authors.forename: Marilyn R
authors.initials: MR
authors.lastname: Kehry
mesh_terms: D000818:Animals; D016466:CHO Cells; D002152:Calorimetry, Differential Scanning; D003001:Cloning, Molecular; D006224:Cricetinae; D006863:Hydrogen-Ion Concentration; D007073:Immunoglobulin E / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry; D007249:Inflammation; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017455:Receptors, IgE / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D007074:Immunoglobulin G; D017455:Receptors, IgE; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E