The role of Cɛ2, Cɛ3, and Cɛ4 domains in human and canine IgE and their contribution to FcɛRIα interaction

The Cɛ2 and Cɛ4 domains are considered as scaffolds, allowing Cɛ3 domains to assume an appropriate orientation to interact with FcɛRI ( Wurzburg and Jardetzky, 2002; Hunter et al., 2008 ). Human/canine IgE chimeric antibodies were expressed to assess the nature of the contribution of Cɛ2 and Cɛ4 domains to bind to and induce target cell degranulation via FcɛRIα. Our results indicate that for (1) Cɛ3 domains in IgE of canine and human origin are the only necessary region for binding to FcɛRIα. (2) The interaction of canine IgE with human sFcɛRIα is significantly enhanced by contributions from both Cɛ2 and Cɛ4 domains of dog origin. (3) The canine/human IgE chimeric antibody construct rapidly dissociates from its the receptor when the canine Cɛ2 and Cɛ4 domains are replaced by the homologous human Fc domains which do not confer a conformation on the Cɛ3 domain to facilitate stable interaction with canine FcɛRIα. Kinetic constants for the binding of this chimera to the soluble extracellular domain of the receptor indicate an approximate 120-fold decrease in the affinity for canine sFcɛRIα ( k a = 5.30 × 10 2 M −1 s −1 ) and a 330-fold increase in the dissociation from canine sFcɛRIα ( K D = 6.9 × 10 −6 M −1 ), compared to the wild type IgE kinetic constants ( K a = 6.30 × 10 4 M −1 s −1 ; K D = 2.1 × 10 −8 M −1 ). Although canine IgE does engage human FcɛRIα, canine Cɛ2 and Cɛ4 do not contribute to the high-affinity of interaction with human FcɛRIα. Upon replacement of human Cɛ2 and Cɛ4 domain by the canine homologues, human IgE Cɛ3 only retains a low affinity for the human receptor, which shows that Cɛ2 and Cɛ4 domains in human IgE Fc contribute significantly to the interaction with its cognate receptor.