Abstract
The interaction of immunoglobulin E (IgE) antibodies with the high-affinity receptor (Fc epsilon RI) is important in anti-parasitic immunity and plays a central role in allergic responses. It has been shown that the human C epsilon 3 domains comprise the binding sites for Fc epsilon RI alpha 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 C epsilon 2-C epsilon 3 interface (residues 332-337), loop BC (residues 362-365), loop DE (residues 393-396), and loop FG (residues 424-427) in canine C epsilon 3 domain were targeted by site-specific mutagenesis. The functional consequences of the mutations to support (i) IgE-mediated, antigen-induced release of beta-hexosaminidase from RBL cells transfected with canine or human Fc epsilon RI alpha and (ii) the affinity of the mutants for the soluble extracellular domain of the alpha-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 Fc epsilon RI alpha binding site, are discussed. (C) 2013 Elsevier Ltd. All rights reserved.