Localization of the Fc epsilon RI binding site to the third constant domain of IgE.

Abstract

To map precisely the binding site of the high affinity receptor (FcεRI) on IgE we have constructed and expressed recombinant human and mouse IgE genes with anti-NIP specificity. Various mutated and chi-meric molecules thus prepared were studied for their ability to bind to rat mast cells or transfected fibro-blasts expressing human FcεRI α chain. To avoid destruction of the FcεRI binding site due to conforma-tional changes, we took advantage of the fact that human IgE does not bind to the rodent receptor, and by exon swapping between the human and mouse constant regions (Cε), generated chimeric human IgE molecules having either one or both of the murine Cε2 and Cε3. We found that replacement of the human Cε3 with the murine homologue was sufficient to confer on the human IgE the ability to bind to the rat FcεRI. Moreover, deletion of the Cε2 did not impair the receptor binding capacity of both human and murine molecules. We therefore conclude that all of the FcεRI binding site can be assigned to third constant region domain of IgE. Results and Discussion Many studies have been carried out in an attempt to map the site on the IgE molecule which accommodates the FcεRI. Several recent studies, utilizing different experimental approaches, implicated the Cε2, Cε3 and their interface as the sites that take part in the FcεRI binding [1, 2]. Because a large part of the data used was derived from manipulations which might have had deleterious effects on the overall conformation of IgE, and since the integrity of the native spatial structure of IgE is required for FcεRI-IgE binding [3], we decided to preserve the native IgE conformation as intact as possible. Because of the substantial homology in sequence and overall tertiary and quar-ternary structure between the mouse and the human IgE molecules [4], and the fact that human IgE does not bind to the rodent receptor, we reasoned it to be an excellent system to study the contribution of different murine Cε fragments to the FcεRI binding site. Thus, we constructed recombinant mouse and human ε heavy chain genes composed of the corresponding Cε exons and VH of an anti-NIP IgG. Following transfection into the λ chain producing J588L myeloma cells, functional recombinant IgE have been obtained which were identical to native IgE in their ability to bind to FcεRI and induce mast cell degranula-tion upon binding of NIP protein [5]. These basic constructs were used to generate chimeric human/mouse IgE molecules by replacing different human exons with their murine homologues [6]. Figure 1 describes schematically the different chimeric molecules and compares