AMINO-ACID-RESIDUES THAT INFLUENCE FC-EPSILON-RI-MEDIATED EFFECTOR FUNCTIONS OF HUMAN-IMMUNOGLOBULIN-E

Immunoglobulin E (IgE) mediates its effector functions via the Fc regi on of the molecule. IgE binding to and subsequent aggregation of the h igh-affinity receptor (Fc epsilon RI) by allergen plays a pivotal role in type I hypersensitivity responses. Earlier studies implicated the C epsilon 2 and 3 interface and the A-B loop in C epsilon 3 in the IgE -Fc epsilon RI interaction. These regions and glycosylation sites in C epsilon 3 were now targeted by site-specific mutagenesis. IgE binding to Fc epsilon RI was compared with surface plasmon resonance (SPR) me asurements, which assessed the binding of the soluble extracellular do main of Fc epsilon RI to IgE. Kinetic analysis based on a pseudo-first -order model agrees with previous determinations. A more refined SPR-b ased kinetic analysis suggests a biphasic interaction. A model-free em pirical analysis, comparing the binding strength and kinetics of nativ e and mutant forms of IgE, identified changes in the kinetics of IgE-F c epsilon RI interaction. Conservative substitutions introduced into t he A-B loop have a small effect on binding, suggesting that the overal l conformation of the loop is important for the complementary interact ion, but multiple sites across the C epsilon 3 domain may influence Ig E-Fc epsilon RI interactions. Asn394 is essential for the generation o f a functional IgE molecule in mammalian cells. A role of Pro333 in th e maintenance of a constrained conformation at the interface between C epsilon 2-3 emerged by studying the functional consequences of replac ing this residue by Ala and Gly. These substitutions cause a dramatic decrease in the ability of the Ligand to mediate stimulus secretion co upling, although only small changes in the association and dissociatio n rates are observed. Understanding the molecular basis of this phenom enon may provide important information for the design of inhibitors of mast cell degranulation.