Search

The stability of the complex between IgE and its high-affinity receptor, FcepsilonRI, on mast cells is a critical factor in the allergic response. The long half-life of the complex of IgE bound to this receptor in situ ( approximately 2 weeks, compared with only hours for the comparable IgG complex) contributes to the permanent sensitization of these cells and, hence, to the immediate response to allergens. Here we show that the second constant domain of IgE, Cepsilon2, which takes the place of the flexible hinge in IgG, contributes to this long half-life. When the Cepsilon2 domain is deleted from the IgE Fc fragment, leaving only the Cepsilon3 and Cepsilon4 domains (Cepsilon3-4 fragment), the rate of dissociation from the receptor is increased by greater than 1 order of magnitude. We report the structure of the Cepsilon2 domain by heteronuclear NMR spectroscopy and show by chemical shift perturbation that it interacts with FcepsilonRIalpha. By sedimentation equilibrium we show that the Cepsilon2 domain binds to the Cepsilon3-4 fragment of IgE. These interactions of Cepsilon2 with both FcepsilonRIalpha and Cepsilon3-4 provide a structural explanation for the exceptionally slow dissociation of the IgE-FcepsilonRIalpha complex.

IgE antibodies mediate antiparasitic immune responses and the inflammatory reactions of allergy and asthma. We have solved the crystal structure of the human IgE-Fc Cepsilon3-Cepsilon4 domains to 2.3 A resolution. The structure reveals a large rearrangement of the N-terminal Cepsilon3 domains when compared to related IgG-Fc structures and to the IgE-Fc bound to its high-affinity receptor, FcepsilonRI. The IgE-Fc adopts a more compact, closed configuration that places the two Cepsilon3 domains in close proximity, decreases the size of the interdomain cavity, and obscures part of the FcepsilonRI binding site. IgE-Fc conformational flexibility may be required for interactions with two distinct IgE receptors, and the structure suggests strategies for the design of therapeutic compounds for the treatment of IgE-mediated diseases.

IgE Abs mediate allergic responses by binding to specific high affinity receptors (FcepsilonRI) on mast cells and basophils. Therefore, the IgE/FcepsilonRI interaction is a target for clinical intervention in allergic disease. An anti-IgE mAb, termed BSW17, is nonanaphylactogenic, although recognizing IgE bound to FcepsilonRI, and interferes with binding of IgE to FcepsilonRI. Thus, BSW17 represents a candidate Ab for treatment of IgE-mediated disorders. By panning BSW17 against random peptide libraries displayed on phages, we defined mimotopes that mimic the conformational epitope recognized on human IgE. Two types of mimotopes, one within the Cepsilon3 and one within the Cepsilon4 domain, were identified, indicating that this mAb may recognize either a large conformational epitope or eventually two distinct epitopes on IgE. On the basis of alignments of the two mimotopes with the human IgE sequence, we postulate that binding of BSW17 to the Cepsilon3 region predominantly blocks binding of IgE to FcepsilonRI, leading to neutralization of IgE. Moreover, binding of BSW17 to the Cepsilon4 region may explain how BSW17 recognizes FcepsilonRI-bound IgE, and binding to this region may also interfere with degranulation of IgE sensitized cells (basophils and mast cells). As a practical application of these findings, mimotope peptides coupled to a carrier protein may be used for the development of a peptide-based anti-allergy vaccine by induction of anti-IgE Abs similar to the current approach of using humanized nonanaphylactogenic anti-IgE Abs as a passive vaccine.

The characterization of the site on the IgE molecule which accommodates the high affinity receptor for IgE (Fc epsilon RI) should allow the design of IgE analogues which can be utilized to block allergic responses. Using chimeric human IgE molecules in which different constant region domains were exchanged with their murine homologues, we demonstrate here that the C epsilon 3 in its native configuration is essential for the binding to the alpha subunit of the human Fc epsilon RI. Deletion of the human C epsilon 2 from such chimeric molecules did not impair their ability to interact with the Fc epsilon RI, indicating that C epsilon 2 is not directly involved in the human Fc epsilon RI binding site and that C epsilon 3 alone is necessary and sufficient to account for most of the human Fc epsilon RI-binding capacity.

Identification of the precise region(s) on the IgE molecule that take part in the binding of IgE to its high affinity receptor (Fc epsilon RI) may lead to the design of IgE analogues able to block the allergic response. To localize the Fc epsilon RI-binding domain of mouse IgE, we attempted to confer on human IgE, which normally does not bind to the rodent receptor, the ability to bind to the rat Fc epsilon RI. Employing exon shuffling, we have expressed chimeric epsilon-heavy chain genes composed of a mouse (4-hydroxy-3-nitrophenyl)acetic acid (NP)-binding VH domain, and human C epsilon in which various domains were replaced by their murine counterparts. This has enabled us to test the Fc epsilon RI-binding of each mouse IgE domain while maintaining the overall conformation of the molecule. All of the chimeric IgE molecules which contain the murine C epsilon 3, bound equally to both the rodent and human receptor, as well as to monoclonal antibodies recognizing a site on IgE which is identical or very close to the Fc epsilon RI binding site. Deletion of the second constant region domain did not impair either the binding capacity of the mutated IgE or its ability to mediate mast cell degradation. These results assign the third epsilon domain of IgE as the principal region involved in the interaction with the Fc epsilon RI.