Identification of the linear Fc-binding epitope on the bovine IgG1 Fc receptor of (boFcγRI) using synthetic peptides

Background: Bovine IgG1 Fc receptor (boFcγRI) is a homologue to human FcγRI (CD64) that has three extracellular Ig-like domains and can bind bovine IgG1 with high affinity. Identification of the linear epitope for Fc-binding on boFcγRI provides new insights for the IgG-Fcγ interaction and FcγR-targeting drugs development.

Methods: The boFcγRI molecules were expressed on cell surface of the boFcγRI -transfected COS-7 cells. The extracellular domain of boFcγRI was expressed in Escherichia coli (E. coli) BL21, and the soluble boFcγRI was purified by Ni-chelation chromatography followed by refolding. To identify the Fc-binding epitope on the boFcγRI, peptides derived from the membrane-distal extracellular domain (EC2) of boFcγRI were synthesized and conjugated to a carrier protein of IgG-free bovine serum albumin (BSA). Binding of bovine IgG1 to the different peptides was tested by Dot-blot assay, and the peptide showing maximal IgG-binding was further modified by truncation and mutation. The inhibition effect of the Fc-binding peptide was determined by competitive ELISA and Fc-rosetting inhibition assay, respectively.

Results: The minimal effective peptide TNLSHNGI corresponding to the sequence 142-149 of boFcγRI was found to bind bovine IgG1 specifically in Dot-blot suggesting it represents a linear ligand-binding epitope located in the putative E-F loop of the EC2 domain on the receptor. Mutation analysis of the peptide showed that the residues of Thr¹⁴², Asn¹⁴³, Leu¹⁴⁴, Gly¹⁴⁸ and Ile¹⁴⁹ within the linear epitope are critical for IgG1-binding. The Fc-binding peptide inhibited bovine IgG1 binding to the soluble recombinant protein of boFcγRII with IC₅₀ of 20.27 μmol/L, and inhibited the rosette formation of bovine IgG1-sensitized RBCs on the boFcγRI transfected cells with IC₅₀ of 86.75 μmol/L.

Conclusions: The linear epitope for Fc-binding as well as its crucial residues of EC2 domain on boFcγRI was identified using synthetic peptides. The Fc-binding peptide showed well capability of regulating boFcγRI-IgG1 interaction on cell surface.