An intermediate pH unfolding transition abrogates the ability of IgE to interact with its high affinity receptor FcepsilonRIalpha.
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.
Demarest
Stephen J
SJ
Biogen Idec, San Diego, California 92122, USA. stephan.demarest@biogenidec.com
Hopp
Jennifer
J
Chung
Julie
J
Hathaway
Karen
K
Mertsching
Elisabeth
E
Cao
Xianjun
X
George
Judy
J
Miatkowski
Konrad
K
LaBarre
Michael J
MJ
Shields
Michael
M
Kehry
Marilyn R
MR
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
D007074:Immunoglobulin G; D017455:Receptors, IgE; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E