Identification of amino acid residues involved in the interaction of canine IgE with canine and human FcɛRIα

Abstract The interaction of immunoglobulin E (IgE) antibodies with the high-affinity receptor (FcɛRI) is important in anti-parasitic immunity and plays a central role in allergic responses. It has been shown that the human Cɛ3 domains comprise the binding sites for FcɛRIα and crystal structure determination has shown that amino acids in four sites contribute to the high affinity of the interaction. The role of homologous residues within canine IgE-Fc, i.e. amino acids located at Cɛ2–Cɛ3 interface (residues 332–337), loop BC (residues 362–365), loop DE (residues 393–396), and loop FG (residues 424–427) in canine Cɛ3 domain were targeted by site-specific mutagenesis. The functional consequences of the mutations to support (i) IgE-mediated, antigen-induced release of β-hexosaminidase from RBL cells transfected with canine or human FcɛRIα and (ii) the affinity of the mutants for the soluble extracellular domain of the α-chain expressed in Pichia pastoris were determined by Surface Plasmon Resonance (SPR). Kinetic analysis supports the observed effects of IgE mutations on stimulus secretion coupling. Potential applications of this study, leading to the generation of an IgE variant with a disabled FcɛRIα binding site, are discussed.