Mapping of the CD23 binding site on immunoglobulin E (IgE) and allosteric control of the IgE-Fc epsilonRI interaction

Abstract

IgE, the antibody that mediates allergic responses, acts as part of a self-regulating protein network. Its unique effector functions are controlled through interactions of its Fc region with two cellular receptors, FcεRI on mast cells and basophils and CD23 on B cells. IgE cross-linked by allergen triggers mast cell activation via FcεRI, whereas IgE-CD23 interactions control IgE expression levels. We have determined the CD23 binding site on IgE, using a combination of NMR chemical shift mapping and site-directed mutagenesis. We show that the CD23 and FcεRI interaction sites are at opposite ends of the Cε3 domain of IgE, but that receptor binding is mutually inhibitory, mediated by an allosteric mechanism. This prevents CD23-mediated cross-linking of IgE bound to FcεRI on mast cells and resulting antigen-independent anaphylaxis. The mutually inhibitory nature of receptor binding provides a degree of autonomy for the individual activities mediated by IgE-FcεRI and IgE-CD23 interactions.

FIGURE 1.

NMR mapping of the CD23 and IgE interaction surfaces. A, increasing amounts of unlabeled derCD23 were added to a 200 μm sample of ¹⁵N-labeled Cϵ3; five spectra of the titration are overlaid (red, zero derCD23; magenta, 50 μm; blue, 100 μm; cyan, 150 μm; green, 200 μm). Insets show magnified views of the indicated regions. B, the NMR-derived derCD23 interaction site on Cϵ3 was mapped onto the structure of IgE-Fc (1F6A (6)) and shown as surface representation. For comparison, the residues of IgE that interact with FcϵRI are indicated in green. C, the IgE interaction surface on CD23 was defined previously (Ref. and shown here as a surface representation). The interacting surfaces of IgE and CD23 are colored according to electrostatic potential and coded such that regions with a potential <−4 k_BT are red, whereas those >4 k_BT are blue (k_B, Boltzmann constant; T, absolute temperature).

FIGURE 2.

Competition binding experiments between derCD23 and sFcϵRIα for IgE-Fc. A and B, the binding of derCD23 was tested against IgE-Fc immobilized on a sensor surface (A) and IgE-Fc captured on an FcϵRIα-immobilized surface (B); the start of the derCD23 injection is indicated with an arrow. DerCD23 bound to immobilized IgE-Fc with a K_D of 2.3 μm; no measureable binding was observed for derCD23 to IgE-Fc complexed to FcϵRIα. RU, resonance units. C and D, the binding of IgE-Fc to immobilized derCD23 (C) was compared with the binding of a complex of IgE-Fc·sFcϵRIα (D) to the same surface; the start of the injection of the complex is indicated with an arrow. IgE-Fc bound to derCD23 with a K_D of 2.4 μm, but the IgE-Fc·sFcϵRIα complex did not bind to derCD23. All SPR binding experiments were performed using identical 2-fold serial dilutions of ligands, from 40 μm to 78 nm. E, binding between terbium-labeled derCD23 and Alexa Fluor 647-labeled IgE-Fc was measured in a solution TR-FRET assay in the presence of increasing concentrations of unlabeled αγ-fusion protein as inhibitor: 0 nm (black), 0.5 nm (red), 2.5 nm (blue), and 5 nm (green). F, binding between terbium-labeled αγ-fusion protein and Alexa Fluor 647-labeled IgE-Fc was measured with increasing concentrations of unlabeled derCD23 as inhibitor: 0 μm (black), 25 μm (red), 50 μm (blue), and 185 μm (green).

FIGURE 3.

Soluble CD23 does not cross-link IgE bound to FcϵRI on mast cells. The ability of soluble CD23 to engage IgE on B cells and mast cells was tested. A, after preincubation of IgE, the addition of anti-IgE antibody resulted in activation of the FcϵRIα⁺ LAD-2 mast cell line, as measured by release of β-hexosaminidase. Neither monomeric derCD23 nor trimeric triCD23 was able to cross-link IgE and activate mast cells in this assay. B, in contrast, triCD23 effectively cross-links membrane IgE on the surface of IgE⁺ human tonsillar B cells, resulting in activation of these cells and increased secretion of IgE. B cell cultures were activated with IL-4 and anti-CD40, and soluble CD23 was added at 1 μm; supernatants were harvested 12 days after activation, tested for IgE levels, and compared with levels for cells treated with IL-4/anti-CD40 alone (* = p < 0.05; ** = p < 0.01). The regulatory activities of soluble CD23 on IgE⁺ B cells are described in detail in Ref. .