Disulfide Linkage Controls the Affinity and Stoichiometry of IgE Fcϵ3–4 Binding to FcϵRI

Abstract

؊1 , similar to that determined previously for an isolated and partially folded C⑀3 domain, demonstrates that substantial reduction in affinity can be achieved by preventing the engagement of one of the two C⑀3 domains. Recent structural data indicate that conformational change in IgE is required to allow both C⑀3 domains to bind, and thus an allosteric inhibitor that prevents access to the second C⑀3 has the potential to reduce the ability of IgE to sensitize allergic effector cells. Immunoglobulin E (IgE)1 is the antibody class that plays a central role in the allergic response (1). Mast cells and basophils express a high-affinity receptor for IgE, Fc⑀RI, and it is the cross-linking of receptor-bound IgE on these cells by multivalent allergens that triggers the immediate release of preformed inflammatory mediators. The affinity of IgE for Fc⑀RI is uniquely high among Ig-receptor complexes, two to five orders of magnitude higher than that of IgG for its receptors Fc␥ RI-III (2). This is due principally to a very slow off-rate. The half-life of IgE bound to Fc⑀RI is hours (3) compared with only minutes for IgG bound to its receptors (4). Inhibition of IgE binding to Fc⑀RI or at least modulation of its binding kinetics is a potential therapeutic strategy.IgE Fc binds to the receptor with a 1:1 stoichiometry (5) using both Fc heavy chains (6). The crystal structure of the complex between the IgE fragment, Fc⑀3-4 (a homodimer of ⑀-chains consisting of the C⑀3 and C⑀4 domains), and a soluble fragment of the IgE-binding ␣-chain of the receptor, sFc⑀RI␣ (6), revealed the precise details of the involvement of the two C⑀3 domains in the complex. The extensive binding interface consists of two subsites, one contributed by each C⑀3 domain, and thus the maintenance of the dimeric site might be expected to be important for high-affinity binding. The C⑀3 domain also contains an attachment site (Asn-394) for N-linked carbohydrate that is conserved in other antibody classes (e.g. Asn-297 in the C␥2 domain of IgG Fc). Although the crystal structure did not reveal any contact with carbohydrate (6), an indirect effect of glycosylation upon receptor binding via stabilization of the polypeptide conformation cannot be ruled out. Indeed, an isolated C⑀3 domain expressed in Escherichia coli and lacking carbohydrate was found to be only partially folded (7) and NMR analysis of the same fragment indicates that it may have a molten-globule-like structure (8).The aim of this study was to determine the contributions of these two factors, dimerization and glycosylation of the C⑀3 domains, to the kinetics and affinity of Fc⑀RI binding. We have analyzed these effects using four variants of the Fc⑀3-4 fragment: (i) disulfide-linked and glycosylated (Fc⑀3-4); (ii) lacking only the disulfide link (redFc⑀3-4); (iii) lacking only glycosylation (deglyFc⑀3-4); and (iv) lacking both structural features (Fc⑀3-4⌬C). Fc⑀3-4 lacks the C⑀2 domains of the complete IgE Fc, but we have shown previously that not only does this fragment display a 1:1 stoichiom...

Chicken IgY Binds Its Receptor at the CH3/CH4 Interface Similarly as the Human IgA:FcαRI Interaction

“…It has been demonstrated by enzymatic digestion and site-directed mutagenesis that the removal of carbohydrates from IgG results in loss of FcR binding, whereas the effect was less pronounced for IgE (22,23). The glycosylation site found on the Fc3 domain (N407) has been conserved in both the mammalian C␥2 and the C3 domain, respectively.…”

Immune responses in dogs with cutaneous adverse food reactions

“…IL-4 or IL-13 are produced by Th2 cells and induce class switching in B cells, giving rise to IgE production. The secreted IgE binds to specialised high affinity receptors (Fc"RI) on mast cells, basophils, Langerhans cells (LC) and dendritic cells from which it dissociates only very slowly (Hunt et al 2005). When an allergen crosslinks the IgE on the mast cells and basophils, these cells respond by an immediate release of their prestored mediators, which are responsible for the allergic symptoms, that occur rapidly.…”

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

“…C⑀4 of Fc⑀ does not participate directly in the binding but is crucial for maintaining high affinity (16 -18). 2 The C⑀2 domain, which is unique to IgE and virtually replaces the hinge found in IgG, is much less involved in binding but plays an important role in controlling the proper IgE/ Fc⑀RI␣ stoichiometry and binding kinetics (17,19).…”