The membrane-proximal part of FcepsilonRIalpha contributes to human IgE and antibody binding--implications for a general structural motif in Fc receptors.
The high affinity receptor for human IgE (FcepsilonRI) on tissue mast cells and blood basophils is responsible for immediate hypersensitivity reactions. Binding of human IgE (hIgE) to FcepsilonRI has been shown to be mediated via three independent regions in the extracellular part of the alpha-subunit of FcepsilonRI (ecFcepsilonRIalpha). By site-directed mutagenesis we investigated the contribution of amino acids within the ecFcepsilonRIalpha FG loop (residues Lys154-Leu165) to binding to hIgE and two monoclonal anti-FcepsilonRIalpha antibodies (15/1, 5H5/F8). The mutated receptors were expressed and secreted from eukaryotic cells as amino-terminal fusion to HSA. We show that the proposed loop region contributes partly to hIgE binding and that the epitope of mAb 15/1, which inhibits hIgE/FcepsilonRIalpha interaction, maps to this region whereby a single W156A mutation results in complete loss of mAb 15/1 binding. In contrast, hIgE binding is not affected by the W156A mutation indicating that different amino acid residues within the loop are recognized by the mAbs 15/1 and hIgE. MAb 5H5/F8 does not recognize a receptor mutant truncated to Ile170. By screening a random dodecapeptide library displayed on bacterial flagella the epitope for mAb 5H5/F8 was mapped to P173REKY177 whereas one of the 15/1 binding clones displayed a peptide with an amino acid sequence homologous to Leu158-lle167. Based on the epitopes identified for the inhibitory mAb 15/1 and the non-inhibitory mAb 5H5F8 and on binding data obtained with polyclonal antisera raised against two ecFcepsilonRIalpha peptides, we propose a structural element in the membrane proximal part of ecFcepsilonRIalpha which forms a 3D structure which might facilitate specific and efficient attachment of hIgE.
Nechansky
A
A
Novartis Forschungsinstitut GmbH, Vienna, Austria.
Aschauer
H
H
Kricek
F
F
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001483:Base Sequence; D019556:COS Cells; D002478:Cells, Cultured; D018360:Crystallography, X-Ray; D017931:DNA Primers; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism*; D007313:Insecta; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017455:Receptors, IgE / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D017455:Receptors, IgE; D007073:Immunoglobulin E