Fc Alpha/u R Study Of Ligand Binding Sites

Binding of the Fc regions of antibodies to cell surface immunoglobulin Fc receptors results in various immune responses, which plays important roles in immune defense. The human Fc alpha/mu receptor (hFca/μR, CD351) is a receptor that has dual specificity for IgA and IgM. It is a type I transmembrane protein with a short extracellular domain (EC)1, an Ig variable (V) region-like EC2domain and a stalk-like EC3domain in the extracellular region. The EC2domain shares43%homology with domain1(D1) of polymeric immunoglobulin receptor (pIgR), which is also capable of binding both polymeric IgA and IgM. Thus it is predicted that EC2could also served as the IgA and IgM binding domain for Fca/μR. In this work, we aimed to find out the regions that are responsible for ligand binding, and to get a better understanding of the structural requirements of hFca/μR for its IgA and IgM binding.To find out which amino acids in hFca/μR EC2might contribute to IgA and IgM binding, we started by creating a homology model using human pIgR D1as a template. The hFca/μR EC2contains three complementarity-determining region (CDR)-like loops, i.e., APSSVNRHQ for the CDR1-like loop, STNQY for the CDR2-like loop and SENNM for the CDR3-like loop. All three CDR-like loops are exposed, resulting in potential binding sites for IgA and IgM.To define the subregions for ligand binding on hFcα/μR EC2, we generated three chimaeric receptors by splice overlap extension PCR (SOE-PCR). These chimaeric receptors were composed of the hFcα/μR backbone but having the CDR1-, CDR2-and CDR3-like loops of EC2replaced by their counterpart loops from human pIgR D2, which itself does not bind to IgA or IgM. COS-7and Hela cells stably expressing WT or chimaeric hFcα/μR were established by lentiviral transduction. And then flow cytometry and confocal microscopy analysis were used to compare IgA and IgM binding by wild type hFcα/μR or chimaeric receptors. Results showed that substitution of either the CDR1-or the CDR2-like loop abrogated IgA and IgM binding. In comparison, substitution of CDR3-like loop resulted in significant loss of IgM binding but only caused slightly decrease on IgA binding.To identify the key residues involved in ligand binding, point mutations were introduced into each of the CDRs by SOE-PCR. The seven mutated amino acids were V29A, R31A, R31E, NQ54-55AA, E98A, E98R and NN99-100AA, which amino acids were either replaced with opposite charged residues or small noncharged Ala. Then COS-7and Hela cells stably expressing point mutated hFcoc/μR were established by lentiviral transduction. Compared with wild type hFcα/μR, flow cytometry analysis showed that changing the positively charged Arg31in the CDR1-like loop abolished IgA binding, and decreased IgM binding by60%-70%. Ala substitution at Val29resulted in an approximately60%and40%reduction in IgA and IgM binding respectively. NQ54-55AA, in which both Asn54-Gln55were replaced by alanines, resulted in50%-60%reduction in IgA and IgM binding. Surprisingly, we found that IgA binding was almost doubled when negatively charged Glu98within CDR3was replaced with neutral Ala, while IgM binding were decreased. Similar results were shown for confocal macroscopic analysis.In summary, these data provided evidence that all three CDR-like loops in hFcα/μR EC2were involved in the ligand-receptor interaction. The positively charged Arg31in the CDRl-like loop was indispensable for both IgA and IgM binding. Compared to CDR1-and CDR2-like loops of hFca/μR EC2, the contribution of CDR3-like loop to ligand binding was different. It might play a role for structural integrity of Fca/μR.