Participation of the N-Terminal Region of Cε3 in the Binding of Human IgE to Its High-Affinity Receptor FcεRI

Henry, Alistair J.; Cook, Justin P. D.; McDonnell, James M.; Mackay, Graham A.; Shi, Jimin; Sutton, Brian J.; Gould, Hannah J.

doi:10.1021/bi971299+

Cited by 69 publications

(71 citation statements)

References 52 publications

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“…3) were all typical of a folded protein consisting principally of ␤-structure and in agreement with data previously published for Fc⑀3-4 fragments (10,14,20). This indicates that neither the refolding of the protein expressed in E. coli nor deglycosylation of the NS-0 material affected the folding of the fragment.…”

Section: Expression and Purification Of Fc⑀3-4 And Fc⑀3-4⌬c-supporting

confidence: 90%

“…4C). As we found in our previous studies of Fc⑀3-4 fragments (15,20), only a biphasic model could satisfactorily fit the observed binding curves for Fc⑀3-4 and deglyFc⑀3-4. Representative curve fits and residuals are shown in Figs.…”

Section: Expression and Purification Of Fc⑀3-4 And Fc⑀3-4⌬c-supporting

confidence: 46%

“…SA, Switzerland). Methods and kinetic analysis have been described previously (3,15,20). In these experiments, coupling density was typically restricted to 500 response units, flow rate of 20 l/min, and exposure time to analyte of 360 s.…”

Section: Methodsmentioning

confidence: 99%

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

Hunt

Beavil

Calvert

et al. 2005

Journal of Biological Chemistry

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؊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...

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Section: Expression and Purification Of Fc⑀3-4 And Fc⑀3-4⌬c-supporting

confidence: 90%

Section: Expression and Purification Of Fc⑀3-4 And Fc⑀3-4⌬c-supporting

confidence: 46%

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

Hunt

Beavil

Calvert

et al. 2005

Journal of Biological Chemistry

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“…Human mast cells and basophils express the high affinity IgE receptor (Fc⑀RI), which has an affinity for IgE that is uniquely high: K a ϳ 10 with IgE, IgE-Fc, and a subfragment of IgE-Fc consisting of only the C⑀3 and C⑀4 domains termed Fc⑀3-4 (21), that the high affinity is due to the slow dissociation rate from Fc⑀RI (22,23) and that the C⑀2 domains are in part responsible (24). With a half-life on tissue mast cells of 2-3 weeks (25), IgE can effectively and persistently sensitize these cells for rapid degranulation upon encounter with antigen (allergen).…”

Section: ؊1mentioning

confidence: 99%

Avian IgY Binds to a Monocyte Receptor with IgG-like Kinetics Despite an IgE-like Structure

Taylor¹,

Gould

Sutton

et al. 2008

Journal of Biological Chemistry

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An ancestor of avian IgY was the evolutionary precursor of mammalian IgG and IgE, and present day chicken IgY performs the function of human IgG despite having the domain structure of human IgE. The kinetics of IgY binding to its receptor on a chicken monocyte cell line, MQ-NCSU, were measured, the first time that the binding of a non-mammalian antibody to a nonmammalian cell has been investigated (k ؉1 ‫؍‬ 1.14 ؎ 0. 46 ). IgE has an extra pair of immunoglobulin domains when compared with IgG. Their presence reduces the dissociation rate of IgE from its receptor 20-fold, thus contributing to the high affinity of IgE. To assess the effect of the equivalent domains on the kinetics of IgY binding, IgY-Fc fragments with and without this domain were cloned and expressed in mammalian cells. In contrast to IgE, their presence in IgY has little effect on the association rate and no effect on dissociation. Whatever the function of this extra domain pair in avian IgY, it has persisted for at least 310 million years and has been co-opted in mammalian IgE to generate a uniquely slow dissociation rate and high affinity.IgY is the principal serum antibody of amphibians, reptiles, and birds and shares a common ancestor with both mammalian IgG and IgE (1, 2). The cDNA sequence of the chicken upsilon () heavy chain (3) reveals that, like more basal amphibian IgY (4, 5), avian IgY contains a domain pair (C2) 4 that has been conserved in mammalian IgE (as C⑀2) but truncated to form the "hinge" region in mammalian IgG (3). Thus, an orthologous domain pair must have existed in the common (IgY-like) ancestor prior to its duplication and subsequent divergence in the mammalian lineage. Avian IgY is the closest extant protein to this ancestor and therefore the most logical choice for study of the evolution of modern IgG and IgE from an ancient IgY-like ancestor.IgG, the principal mammalian serum antibody, aggregates and facilitates opsonization of antigens, activates complement, and provides protection for the fetus following transport across the placenta. IgE does not activate complement nor cross the placenta but can sensitize effector cells (principally mast cells and basophils) and typically mediates anaphylactic reactions (6). It is well known for its involvement in allergic disease but probably also provides defense against parasitic infections (7).Avian IgY appears to combine the functions of mammalian IgG and IgE. As the major serum antibody, it provides defense against infection (8) but also mediates anaphylaxis (9). Chicken mast cells activated by IgY are responsible for local anaphylactic reactions in the gut, which play a role in defense against protozoan infections (10, 11). Antibody-dependent hypersensitivity and fatal systemic anaphylactic shock have also been demonstrated in chickens (9, 12, 13) and shown to be mediated by basophils (14), which are much more numerous than mast cells in birds (in contrast to mammals in which the reverse is true) (15). These phenomena constitute indirect evidence for the presence of IgY...

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“…Extensive discussion of the SPR methodology and data analysis relating to Fc⑀RI␣ and IgE interactions has been previously described (29,38). Briefly, purified sFc⑀RI␣ was coupled to a BIAcore CM5 sensor chip using an aldehyde coupling technique, which uses protein carbohydrate to form stable linkage between the chip and protein.…”

Section: Spr Analysis Of Bindingmentioning

confidence: 99%

Mutagenesis Within Human FcεRIα Differentially Affects Human and Murine IgE Binding

Mackay

Hulett

Cook

et al. 2002

The Journal of Immunology

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Soluble fragments of the α-chain of FcεRI, the high-affinity receptor for IgE, compete with membrane-bound receptors for IgE and may thus provide a means to combat allergic responses. Mutagenesis within FcεRIα is used in this study, in conjunction with the crystal structure of the FcεRIα/IgE complex, to define the relative importance of specific residues within human FcεRIα for IgE binding. We have also compared the effects of these mutants on binding to both human and mouse IgE, with a view to evaluating the mouse as an appropriate model for the analysis of future agents designed to mimic the human FcεRIα and attenuate allergic disease. Three residues within the C-C′ region of the FcεRI α2 domain and two residues within the α2 proximal loops of the α1 domain were selected for mutagenesis and tested in binding assays with human and mouse IgE. All three α2 mutations (K117D, W130A, and Y131A) reduced the affinity of human IgE binding to different extents, but K117D had a far more pronounced effect on mouse IgE binding, and although Y131A had little effect, W130A modestly enhanced binding to mouse IgE. The mutations in α1 (R15A and F17A) diminished binding to both human and mouse IgE, with these effects most likely caused by disruption of the α1/α2 interface. Our results demonstrate that the effects of mutations in human FcεRIα on mouse IgE binding, and hence the inhibitory properties of human receptor-based peptides assayed in rodent models of allergy, may not necessarily reflect their activity in a human IgE-based system.

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Participation of the N-Terminal Region of Cε3 in the Binding of Human IgE to Its High-Affinity Receptor FcεRI

Cited by 69 publications

References 52 publications

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

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

Avian IgY Binds to a Monocyte Receptor with IgG-like Kinetics Despite an IgE-like Structure

Mutagenesis Within Human FcεRIα Differentially Affects Human and Murine IgE Binding

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