Mutagenesis Within Human FcεRIα Differentially Affects Human and Murine IgE Binding

Abstract

Soluble fragments of the α-chain of FcεRI, the high-affinity receptor for IgE, compete with membrane-bound receptors for IgE and may thus provide a means to combat allergic responses. Mutagenesis within FcεRIα is used in this study, in conjunction with the crystal structure of the FcεRIα/IgE complex, to define the relative importance of specific residues within human FcεRIα for IgE binding. We have also compared the effects of these mutants on binding to both human and mouse IgE, with a view to evaluating the mouse as an appropriate model for the analysis of future agents designed to mimic the human FcεRIα and attenuate allergic disease. Three residues within the C-C′ region of the FcεRI α2 domain and two residues within the α2 proximal loops of the α1 domain were selected for mutagenesis and tested in binding assays with human and mouse IgE. All three α2 mutations (K117D, W130A, and Y131A) reduced the affinity of human IgE binding to different extents, but K117D had a far more pronounced effect on mouse IgE binding, and although Y131A had little effect, W130A modestly enhanced binding to mouse IgE. The mutations in α1 (R15A and F17A) diminished binding to both human and mouse IgE, with these effects most likely caused by disruption of the α1/α2 interface. Our results demonstrate that the effects of mutations in human FcεRIα on mouse IgE binding, and hence the inhibitory properties of human receptor-based peptides assayed in rodent models of allergy, may not necessarily reflect their activity in a human IgE-based system.

The Key Role of Protein Flexibility in Modulating IgE Interactions

“…Expression and Purification of sFc⑀RI␣-The production of a soluble fragment of human sFc⑀RI␣ (Val 1 to Lys 176 ) for mammalian expression in the mouse myeloma cell line NSO (29) and subsequent cloning and expression of a shorter construct (Val 1 to Ala 172 ) in yeast Pichia pastoris have been detailed previously (29,30). The pPICZ␣ vector (Invitrogen) containing the shorter sFc⑀RI␣ construct with an N-terminal polyhistidine tag was transformed into P. pastoris strain SMD1168H.…”

“…Purified recombinant sFc⑀RI␣ was concentrated and buffer exchanged into PBS, pH 5.0. The protein concentration was spectrophotometrically determined using an extinction coefficient at 280 nm of 2.56 for a 1 mg/ml solution (30).…”

Platelet-Related Variants Identified by Exomechip Meta-analysis in 157,293 Individuals

“…The affected amino acid is extracellularly positioned near the interface of two Ig-like domains, an area of the protein critical for FC-IgE interaction as shown through its crystal structure, biochemical data, and mutagenesis studies. [34][35][36][37] Other variants in FCER1A, a subunit of the allergy response IgE receptor and basophil differentiation factor, have previously been associated with IgE levels and monocyte counts. 38,39 Increased platelet activation has been postulated to contribute to or be a consequence of allergic and inflammatory responses.…”

Catalytic folding of the Cε3 domain by its high affinity receptor

“…The production of a soluble fragment of human sFcεRIα (Val 1 to Lys 176 ) for mammalian expression in the mouse myeloma cell line NS0 [24] and subsequent cloning and expression of a shorter construct (Val 1 to Ala 172 ) in yeast Pichia pastoris have been detailed previously [24,25]. The pPICZα vector (Invitrogen) containing the sFcεRIα (Val 1 to Ala 172 ) construct with an N‐terminal polyhistidine tag was transformed into P. pastoris strain SMD1168H.…”