Mapping of murine IgE epitopes involved in IgE‐Fc_ε receptor interactions

Abstract The generation of anti‐IgE monoclonal antibodies has permitted the identification of various serological epitopes on the IgE molecule. The relationship of the sites on IgE recognized by such antibodies to the Fc epsilon receptor (Fc E R) interaction site has been determined using cross‐inhibition studies. However, interpretation of this type of experiment is limited by problems of steric hindrance. Thus, to accomplish precise mapping on the IgE molecule of the Fc ε R interaction site and the binding sites of various anti‐IgE mAb, we employed site‐directed mutagenesis of the IgE heavy chain gene. To this end we have constructed and expressed a recombinant murine constant E heavy chain (C ε ) gene bearing a (4‐hydroxy‐3‐nitrophenyl)acetic acid (NP)‐binding V H region. Several site‐specific mutants in the C ε 3 and C ε 4 domains of this recombinant C ε gene were prepared and expressed by transfection into the light chain‐producing J558L myeloma cell line. The resulting IgE antibodies were tested for binding to mast cells and to various anti‐IgE mAb. The mutants produced include a proline to histidine point mutant at amino acid residue 404 in the C ε 3 domain, a mutant with a truncated C ε 4 domain, a mutant with a 45 amino acid deletion in the carboxy end of C ε 3, and a chimeric human C ε in which the human C ε 3 was replaced by the homologous mouse C ε 3 domain. These mutants have permitted the localization, to the C ε 3 domain, of the epitopes recognized by the 84.1C and 95.3 anti‐IgE mAb. The 84.1C mAb recognizes a site on IgE which is identical or very close to the Fc ε R binding site, and 95.3 recognizes a site on IgE which is related, but not identical to the Fc ε R binding site. The antigenic determinant recognized by the 51.3 mAb, which is inefficient at blocking the IgE‐Fc ε R interaction, has been mapped to the C ε 4 domain. When tested for binding to the Fc ε R on RBL‐2H3 cells, the point mutant bound to the Fc ε R with twofold reduced affinity, while the C ε 3 deletion mutant and the mutant truncated in C ε 4 lost all receptor binding activity. These data suggest that the Fc ε R binding site can be assigned to the third C region domain, and that the fourth domain, while not directly involved in Fc ε R binding, may play a role in the formation of the H 2 L 2 tetrameric IgE molecule, and in stabilizing the conformation of IgE required for Fc ε R binding.