Abstract
The C epsilon 2 and C epsilon 4 domains are considered as scaffolds, allowing C epsilon 3 domains to assume an appropriate orientation to interact with Fc epsilon RI (Wurzburg and Jardetzky, 2002; Hunter et al., 2008). Human/canine IgE chimeric antibodies were expressed to assess the nature of the contribution of C epsilon 2 and Cs4 domains to bind to and induce target cell degranulation via Fc epsilon RI alpha. Our results indicate that for (1) C epsilon 3 domains in IgE of canine and human origin are the only necessary region for binding to Fc epsilon RI alpha. (2) The interaction of canine IgE with human sFc epsilon RI alpha is significantly enhanced by contributions from both c epsilon 2 and Cs4 domains of dog origin. (3) The canine/human IgE chimeric antibody construct rapidly dissociates from its the receptor when the canine C epsilon 2 and Cs4 domains are replaced by the homologous human Fc domains which do not confer a conformation on the C epsilon 3 domain to facilitate stable interaction with canine Fc epsilon RI alpha. 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 sFc epsilon RI alpha (k(a) = 5.30 x 102 M-1 s(-1)) and a 330-fold increase in the dissociation from canine sFc epsilon RI alpha (ICD = 6.9 x 10(-6) M-1), compared to the wild type IgE kinetic constants (K-a = 6.30 x 104 M-1 s(-1); K-D = 2.1 x 10(-8) M-1). Although canine IgE does engage human Fc epsilon RI alpha, canine C epsilon 2 and c epsilon 4 do not contribute to the high-affinity of interaction with human Fc epsilon RI alpha. Upon replacement of human C epsilon 2 and C epsilon 4 domain by the canine homologues, human IgE C epsilon 3 only retains a low affinity for the human receptor, which shows that C epsilon 2 and C epsilon 4 domains in human IgE Fc contribute significantly to the interaction with its cognate receptor. (C) 2013 Elsevier Ltd. All rights reserved.