A Conformational Rearrangement upon Binding of IgE to Its High Affinity Receptor

Abstract

One of the critical steps in the allergic reaction is the binding of immunoglobulin E (IgE) to its high affinity receptor (Fc⑀RI). Fc⑀RI is a tetrameric complex composed of an ␣-chain, a ␤-chain, and a dimeric ␥-chain. The extracellular portion of the ␣-chain (␣-t) is sufficient for the binding of IgE. The Fc portion of IgE contains two copies of the Fc⑀RI binding sites. In contrast, the binding stoichiometry is 1:1. Previously, it was hypothesized that the binding of Fc⑀RI to IgE results in a conformational change in IgE that precludes the binding of a second molecule (Presta, L., Shields, R., O'Connel, L., Lahr, S., Porter, J., Gorman, C., and Jardieu, P. (1994) J. Biol. Chem. 269, 26368 -26373). Here we characterize the secondary structure of IgE and ␣-t and analyze their interaction by circular dichroism spectroscopy. Binding experiments show that when IgE interacts with ␣-t there is a 15-26% decrease of the negative ellipticity at 217 nm. Together, the absence of an ␣-helix element in ␣-t and the small contribution of ␣-t to the spectra of the complex indicate that upon binding, a major conformational rearrangement must occur on IgE. In addition, we analyze the thermal unfolding of ␣-t, IgE, and their complex. Despite the several domains that constitute IgE and ␣-t, these molecules unfold cooperatively with two-state kinetics.The first step in the pathogenesis of allergic disease is the production of an immunoglobulin E (IgE) antibody response to an allergen. The B cells of allergic individuals secrete IgE against various specific antigens (allergens). These IgE molecules then bind to the high affinity IgE receptor (Fc⑀RI) 1 present on mast cells and basophils and, through the interaction with a multivalent antigen, trigger the release of vasoactive mediators.The Fc⑀RI is a tetrameric complex composed of an ␣-chain, a ␤-chain, and a dimer of identical disulfide-linked ␥-chains. The ␤-and ␥-chains are required for the insertion of the ␣-chain into the membrane and for signal transduction. The extracellular portion of the ␣-chain (␣-t) is heavily glycosylated, and it is sufficient for the binding of IgE (1, 2).IgE is composed of two heavy chains (⑀) and two light chains (k or ). The ⑀-chains are composed of five domains and they are linked through two disulfide bridges between the two C⑀2 domains. IgE can be cleaved by papain into Fc and Fab fragments, and the Fc portion is solely responsible for binding to the receptor (3, 4). The circular dichroism (CD) spectrum of IgE was first reported by Dorrington and Bennich (5). However, the spectrum was not deconvoluted and the secondary structure content was not reported. Although the ␣-chain of Fc⑀RI has been purified in many laboratories and from several sources, the CD spectrum and the secondary structure composition have not been previously characterized. In this study, we characterize the secondary structure of a chimeric IgE containing human Fc (ch-IgE), mouse IgE (m-IgE), and human ␣-t.Ample attention has been dedicated to the characterization of the Fc⑀RI ...

An Intermediate pH Unfolding Transition Abrogates the Ability of IgE to Interact with Its High Affinity Receptor FcϵRIα

“…5) upon formation of the complex. This is indicative of stabilization of ␤-structures or limited structural changes resulting from the "locking down" of flexible loops similar to what was observed by Sechi and co-workers (34).…”

Mutagenesis Within Human FcεRIα Differentially Affects Human and Murine IgE Binding

“…Spectra obtained for both yeastand mammalian-derived sFc⑀RI␣ (data not shown) exhibited features similar to those previously described (16,29,41). The highly characteristic positive dichroism peak at ϳ230 nm is seen for all seven species and probably reflects the high content of aromatic residues and the two intrachain disulfide bonds.…”

“…A soluble form of the IgE-binding extracellular domains of Fc⑀RI has been previously expressed in a number of bacterial (22), insect (18,45), and mammalian (13,14,29,41,42) systems. We have now produced WT sFc⑀RI␣ and a range of mutants using P. pastoris.…”

Interaction of Human IgE with Fc Epsilon RI Alpha Exposes Hidden Epitopes on IgE

“…It has been speculated that conformational changes of parts of the IgE molecule accompany both receptor attachment and allergen binding [7, 8, 9, 20]. Since the anti–rhCε3 mAbs described in this report were found to recognize an epitope within Cε3 that is hidden in solution but accessible when IgE is coated to microtiter plate wells, we tested these mAbs also for their capability of recognizing IgE bound to FcεRIα.…”

“…Moreover, this bent structure is also postulated to be responsible for the equimolar complex between IgE and cell bound or soluble FcεRIα although the IgE molecule would provide identical epitopes on the two Cε3 domains for receptor binding [8]. Based on data obtained from circular dichroism spectra [9], a conformational rearrangement affecting Cε3 upon receptor bindings has been proposed as an explanation for the 1:1 stoichiometry of the Fcε–FcεRI complex on the cellular surface. The production of biologically active recombinant Fcε and fragments thereof in eukaryotic as well as in bacterial systems has been reported by several groups [10, 11].…”