The role of C epsilon 2, C epsilon 3, and C epsilon 4 domains in human and canine IgE and their contribution to Fc epsilon RI alpha interaction

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 (0130 and 0050). Human/canine IgE chimeric antibodies were expressed to assess the nature of the contribution of C epsilon 2 and C epsilon 4 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 C epsilon 4 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 C epsilon 4 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 (ka = 5.30 102 M-1 s-1) and a 330-fold increase in the dissociation from canine sFc epsilon RI alpha (KD = 6.9 10-6 M-1), compared to the wild type IgE kinetic constants (Ka = 6.30 104 M-1 s-1; KD = 2.1 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.