The Importance of Lys-352 of Human Immunoglobulin E in FcϵRII/CD23 Recognition

Abstract

The interaction of immunoglobulin E (IgE) with its low affinity receptor (Fc⑀RII/CD23) plays a central role in the initiation and regulation of type I hypersensitivity responses. We have previously identified the importance of amino acid residues in the A-B loop of the C⑀3 domain of human IgE and implicated a region close to the glycosylation site at asparagine 371 as contributing to IgE-CD23 interaction. These residues were now targeted by site-directed mutagenesis. The IgE-CD23 interaction was assessed by semiquantitative flow cytometry. Replacement of the entire C⑀3 A-B loop (residues 341-356) with the homologous rat IgE sequence resulted in complete loss of human CD23 recognition, as did replacement of residues 346 -353, indicating that class-specific effector residue(s) are contained within these eight amino acids. Lysine 352 within the A-B loop was identified as contributing directly to human CD23 interaction. Mutation to the rodent homologue glycine or glutamate resulted in a significant reduction in binding compared with native IgE, whereas conservative substitution with arginine effected a small, but statistically significant, enhancement of CD23 binding. Mutation of the C⑀3 glycosylation site at asparagine 371 to threonine or glutamine did not significantly affect CD23 recognition. Our results yield new insights into the structural basis of the hIgE-CD23 interaction and hold promise for the rational design of drugs that can manipulate IgE-mediated regulation of the allergic response. Antibodies of the immunoglobulin E (IgE)1 isotype have a central role in the initiation and regulation of allergic disorders (1). IgE mediates these functions via the interaction of the Fc region of the molecule with its Fc receptors. The crystal structure of the high affinity receptor (Fc⑀RI, K A ϳ10 10 M Ϫ1 ) complexed to IgE-Fc has been determined, identifying critical residues involved in the interaction (2). Fc⑀RII/CD23 has been identified as a low affinity receptor for human (h) IgE (K A ϳ10 7 M Ϫ1 ) (3). Structurally, CD23 is not a member of the Ig superfamily but a type II transmembrane receptor expressed on a variety of cells of the immune system (4). In the absence of a high resolution structure, a composite molecular model has been proposed based on homology of the lectin head with the rat mannose-binding protein (5, 6). The model predicts that five heptadic repeats of hydrophobic Leu/Ile residues form a trimeric ␣-helical stalk linking the N-terminal extracellular domain to the transmembrane and C-terminal cytoplasmic domain of the receptor (7).Previous studies based on the interaction of hIgE-Fc with a soluble 16-kDa form of the lectin homology domain expressed in NS-O cells identified this region as containing the IgE binding site(s) (8). More recent studies indicate the necessity of the stalk region for IgE interaction (9). Using site-specific mutagenesis, the IgE binding region of CD23 has been mapped to two discontinuous segments between residues 165-190 and 224 -256 (10). Although several other ligands fo...

A Fluorescent Biosensor Reveals Conformational Changes in Human Immunoglobulin E Fc

“…The IgEFc biosensor undergoes no measurable change in FRET efficiency upon binding to CD23 (FcεRII). Two molecules of derCD23 (the IgE-binding lectin domain) can bind to IgEFc (20) at locations that are distinct from the FcεRI binding site (as indicated by mutagenesis (61)). Clearly the binding mechanisms also differ, and IgEFc can bind to derCD23 in its naturally bent conformation without an apparent measurable change in the bend angle.…”

Crystal structure of IgE bound to its B-cell receptor CD23 reveals a mechanism of reciprocal allosteric inhibition with high affinity receptor FcεRI

“…All of the salt-bridge and hydrogen-bond interactions involve the Cε3 domain with the exception of Arg440 in the linker region, whereas Cε4 contributes only van der Waals interactions. Earlier studies had implicated the AB loop of Cε3, and residue Lys352 in particular, in CD23 binding (22), but this loop is not directly involved at all, and Lys352 makes no contact with CD23; any effects of mutagenesis in this loop must therefore be indirect. It is the EF loop/helix of the Cε3 domain that is centrally placed at the interface (Fig.…”

“…Cε2 was also implicated in FcεRI binding (5), but crystal structures of the sFcεRIα-Fcε3-4 complex (20) (Fcε3-4 is a subfragment of IgE-Fc consisting of a dimer of the Cε3 and Cε4 domains) and most recently the sFcεRIα-IgE-Fc complex, including the Cε2 domains, show that the Cε2 domains exert their influence only indirectly upon the formation of this 1:1 complex (21). The location of the CD23 binding site has also been mapped to Cε3 by mutagenesis (22), and monomeric sCD23 has been shown to bind to Fcε3-4 and IgE-Fc with 2:1 stoichiometry (4, 7). The fact that sCD23 can compete with FcεRI binding, albeit at high concentrations (23), was thought to be due to overlap of the two receptor binding sites.…”

Approaches to target IgE antibodies in allergic diseases

“…This was confirmed in a study using chimeric IgE molecules in which the human Cε3 domain was replaced by mouse Cε3: these chimeric molecules bound to mouse CD23 and concomitantly lost their ability to bind the human receptor (Nissim, Schwarzbaum et al 1993). Thereafter, the CD23 binding site on IgE was more precisely mapped to the A-B loop of the Cε3 domain (residues 341-356), with a key role for lysine 352 (Sayers, Housden et al 2004). More recently, the crystal structure of the soluble head domain of CD23 bound to a Cε3-4 IgE dimer was resolved by Dhaliwal and collaborators (Dhaliwal, Yuan et al 2012).…”