Mapping of murine IgE epitopes involved in IgE‐Fcε 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_ER) 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_εR 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_ε3 and C_ε4 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_ε3 domain, a mutant with a truncated C_ε4 domain, a mutant with a 45 amino acid deletion in the carboxy end of C_ε3, and a chimeric human C_ε in which the human C_ε3 was replaced by the homologous mouse C_ε3 domain. These mutants have permitted the localization, to the C_ε3 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_εR binding site, and 95.3 recognizes a site on IgE which is related, but not identical to the Fc_εR binding site. The antigenic determinant recognized by the 51.3 mAb, which is inefficient at blocking the IgE‐Fc_εR interaction, has been mapped to the C_ε4 domain. When tested for binding to the Fc_εR on RBL‐2H3 cells, the point mutant bound to the Fc_εR with twofold reduced affinity, while the C_ε3 deletion mutant and the mutant truncated in C_ε4 lost all receptor binding activity. These data suggest that the Fc_εR binding site can be assigned to the third C region domain, and that the fourth domain, while not directly involved in Fc_εR binding, may play a role in the formation of the H₂L₂ tetrameric IgE molecule, and in stabilizing the conformation of IgE required for Fc_εR binding.