Interaction of the Low-Affinity Receptor CD23/FcεRII Lectin Domain with the Fcε3−4 Fragment of Human Immunoglobulin E

Abstract

CD23/Fc epsilonRII, the low-affinity receptor for IgE, is a multifunctional protein of importance in blood cell development and the immune system. We have studied the interaction of CD23 with IgE in solution using hydrodynamic methods applied to recombinant fragments of both ligands: sCD23, corresponding to the soluble lectin domain of CD23, and Fc epsilon3-4, a dimer of the C epsilon3-C epsilon4 sequence of IgE. The hydrodynamic, spectroscopic, and biological properties of these fragments suggest that they have a fully native structure. Sedimentation equilibrium studies on mixtures of sCD23 and Fc epsilon3-4 indicate that IgE has two binding sites for CD23, each characterized by affinities of approximately 10(5) M(-1). Analysis of the sedimentation as a function of temperature allows conclusions to be drawn about the thermodynamics of binding at the two sites. Binding at the first site is characterized by large changes in enthalpy (delta H(degree)To = -2.1 +/- 3.3 kcal mol(-1)) and heat capacity (delta Cp(degree) = -320 +/- 320 cal mol(-1) K(-1)), whereas binding at the second site is characterized by small changes in enthalpy (delta H(degree)To = 0.1 +/- 5.6 kcal mol(-1)) and heat capacity (delta Cp(degree) = -140 +/- 550 cal mol(-1) K(-1)). In native CD23, there are two or three lectin domains, associated through an alpha-helical coiled-coil stalk. The predicted structure of the CD23 oligomers and symmetry considerations rule out the possibility of two lectin domains from one oligomer binding to identical sites in IgE. The notion of two types of interaction in the 2:1 complex between CD23 and IgE is consistent with the thermodynamic data presented.

Crystal structure of IgE bound to its B-cell receptor CD23 reveals a mechanism of reciprocal allosteric inhibition with high affinity receptor FcεRI

“…4A may reflect the slightly lower affinity reported for sFcεRIα binding to Fcε3-4 compared with IgE-Fc (5, 21, 38)]. derCD23 can similarly cause almost complete displacement of sFcεRIα, although much higher concentrations are required, greater than 100 μM, to overcome its lower affinity (K D ∼ 10 −5 -10 −6 M) (4)(5)(6)(7)(8).…”

“…Folding was assessed by 1D-1 H NMR at 500 MHz (large dispersion and strong signals of methyl groups between 1.0 and −1.0 ppm). Human IgE-Fc (N265Q, N371Q) was expressed in NS0 cells and purified by affinity chromatography with sFcεRIα-IgG 4 -Fc fusion protein as previously described (4,45). The genes for recombinant human Fcε3-4 (Cys328-Lys547, with N-terminal ADP) and sFcεRIα-Cys-His (Val1-Lys176, with C-terminal cysteine and His 6 tag) were synthesized by DNA2.0 and cloned as HindIII/EcoRI fragments into proprietary mammalian expression vectors.…”

“…In contrast, CD23 (FcεRII), expressed on B cells, consists of three C-type lectin "head" domains connected to the membrane by a trimeric α-helical coiled-coil "stalk" (3). A single head domain binds to IgE-Fc with lower affinity (K A = 10 5 -10 6 M −1 ) than FcεRI (4)(5)(6)(7)(8), although avidity of the trimer can substantially enhance this interaction (7,(9)(10)(11). Membrane CD23 (mCD23) is cleaved from the cell surface by endogenous proteases such as ADAM10 (12,13) to yield soluble trimeric and monomeric forms (sCD23), which have been implicated in both positive and negative feedback mechanisms for the regulation of IgE synthesis by B cells that have switched to IgE production (1,7,8,(14)(15)(16).…”

A Monomeric Chicken IgY Receptor Binds IgY with 2:1 Stoichiometry

“…Sedimentation Equilibrium-Sedimentation equilibrium experiments were performed on smCHIR-AB1, Fc2-4, and mixtures of the two using a Beckman Optima XL-A analytical ultracentrifuge as described previously (21). The samples were dialyzed into 0.125 M sodium chloride, 50 mM Tris, pH 7.4, 0.05% sodium azide (TBSa), and the data were acquired as an average of 25 absorbance measurements at a wavelength of 280 nm and a radial spacing of 0.001 cm.…”

“…The carbohydrates led to a V at 4°C of 0.692 for smCHIR-AB1 and 0.715 for Fc2-4 calculated using SEDNTERP (23). The equilibrium data for the 1:1, 2:1, and 1:2 mixtures of smCHIR-AB1 and Fc2-4 were simultaneously fitted to a range of models using the experimental buoyant molecular masses, M(1 Ϫ V ), determined for the individual components as described previously (21). By using the buoyant molecular masses, no errors associated with estimating density or partial specific volume are included in the fit.…”