The chicken leukocyte receptor complex encodes a family of different affinity FcY receptors

Chicken Ig-like receptors (CHIR) form a large family in the leukocyte receptor complex on microchromosome 31 with inhibitory, activating, and bifunctional receptors. Recently, we characterized CHIR-AB1 as a high-affinity, primordial FcY receptor. Given that the CHIR family represents a multigene family, it is plausible that more than a single receptor binds to IgY. Therefore, after comparing CHIR-AB1-like sequences in databases, we cloned CHIR-AB1 homologues from two individual chickens representing the lines M11 and R11 with primers binding to highly conserved regions. In both lines this approach yielded 18 different CHIR-AB amino acid versions, with one sequence out of each line that was identical with the previously characterized B19 CHIR-AB1 Ig domain and two additional R11-M11 identical sequence pairs. All M11-derived CHIR-AB homologues were then expressed as soluble human Ig fusion proteins. Following standardization of the fusion protein concentration with an ELISA, the IgY, IgM, and IgA binding activities were determined by ELISA. Six fusion proteins recognized IgY, whereas none bound to IgM and IgA. The affinities of selected fusion proteins were determined using surface plasmon resonance yielding an equilibrium binding constant between 25 nM for high binders and 260 nM for low binders. Sequence comparisons and subsequent mutational analysis of selected residues identified five amino acids that are potentially involved in IgY binding. These results imply that multiple FcY receptors of variable affinity are encoded by the CHIR locus and that different chicken lines may express both unique as well as highly conserved FcY receptors.