Prokaryotic Expression, Eukaryotic Expression And Perliminary Identification Of A Linear Epitope In HeFcγRⅠ

Fc receptors (FcRs) are a group of important molecules expressed on the surface of immune accessory and effector cells, which bind the Fc region of immunoglobulins with specific affinities and have a number of important biological functions. FcRs play a crucial role in immune regulation by providing a link between the humoral and cellular immune responses. The antibody-mediated inflammatory response is regulated by activating and inhibitory FcRs. Thus, FcR-targeting provides therapies for allergy and autoimmune diseases. In this study, we identified and characterized the linear epitopes on human IgG FcRs (huFcyRs) involved in the binding of human IgG using synthetic peptides, which is the first report to describe linear epitopes for Fc-binding on FcRs. It would be very useful for understanding the molecular basis of FcyR-IgG interactions, and may provide a basis for the design of drugs with a potential for regulating antibody-based inflammation.HuFcyRI has three extracellular Ig-like domains and can bind monomeric human IgG with high affinity. A cDNA for the complete coding region of huFcyRI was subcloned into the expression vector pcDNA3 to express the receptor on cell surface. The binding specificity of huFcγRI was determined by the Fc rosetting assay in which IgG-RBCs formed obvious rosettes on the huFcγRI-tranfected COS-7 cells. Meanwhile, the cDNA encoding the extracellular domain of huFcγRI was subcloned into the prokaryotic expression vector pGEX-6P-1 and expressed in Escherichia coli (E. coli) BL21. However, the recombinant protein could not be recovered efficiently from inclusion bodies. Peptides derived from the EC2 domain of huFcyRI were synthesized to identify the linear epitope for Fc-binding on the receptor. The minimal effective peptide CLYYRNGKAFKFF (122-133) andCTNISHNGTYH (144-153) corresponding was found to bind human IgG specifically in Dot-blot suggesting it represents a linear ligand-binding epitope located in the putative C-C' and E-F loop of the EC2 domain on the receptor.