Identification Of A Linear Epitope For Fc-binding In Mouse FcγRIII

IgG Fc receptors (FcγR) are a group of very important molecules expressed on the surface of immune accessory and effector cells, FcγRs bind the Fc region of IgG with specific affinities. FcγRs play a very important role in immune regulation by providing a link between the cellular and humoral immune responses. Mouse IgG Fc receptor III (moFcγRIII) is a homologue to human FcγRIII (CD16) which binds mouse IgG via the membrane-distal extracellular doman 2 (EC2). The aim of this study is to abtain a linear epitope in moFcγRIII.To obtain an activated protein product equivalent to the extracellular domain of the moFcγRIII, the cDNA encoding moFcγRIII was subcloned into the prokaryotic expression vector pET-28a for production of the recombinant protein. Expression of moFcγRⅢwas induced 3 h by IPTG, the target protein was expressed in the form of inclusion bodies. After ultrasonic washing, the recombinant protein was then recovered by pH8.0 buffer using a rapid dilution method from inclusion bodies. Detection by ELISA showed that IgG-binding activity recombinant moFcγRⅢprotein was obtained. cDNA for the complete coding region of moFcγRIII and moγ-chain were subcloned into the mammalian expression vector pcDNA3, respectively. COS-7 cells were then cotransfected with the Pvu I lined recombinant plasmids with lipofectamine. After G418-selection and cloning, PCR and resetting showed that the co-transfected COS-7 cells with stable surface expression of moFcγRIII were obtained and these showed about 95% rosetting with mouse IgG-sensitized chicken red blood cells (IgG-RBCs), Flow cytometry showed that about 93.5% positive cells with FITC-moIgG, which provide an ideal platform for moFcγRIII functional study.To identify the linear epitope for Fc-binding on moFcγRIII, peptides derived from the EC2 domain of moFcγRIII corresponding to the homologous region of huFcγRIII were synthesized and coupled to the carrier protein of BSA. Binding of mouse IgG to the different peptides was tested by Dot-blot assay. The effective peptide SFFHNEKSVRYH corresponding to the sequence 119-130 of moFcγRIII possesses binding capability for mouse IgG indicating the receptor bears a linear ligand-binding epitope, which is located in the putative C-C'loop of the EC2 domain. Competitive ELISA showed that the peptide containing the IgG-binding epitope efficiently inhibited the binding of mouse IgG to soluble moFcγRIII, the IC50 is up to 20μmol/L; Rosetting inhibition test showed that the peptide can effectively inhibit the rosette formation of IgG-RBCs with co-transfected COS-7 cells expressing moFcγRIII, the IC50 is up to 80μmol/L. The Fc-binding epitope peptide showed the capability of modulating the interaction of IgG and moFcγRIII on cell surface, and thus could be candidate for the development of FcR-targeting drugs. In this study, we identified the linear epitopes in moFcγRIII involved using synthetic peptides, which is the first report to describe linear epitopes for Fc-binding on moFcγRIII. It would be very useful for understanding the molecular basis of FcγR-IgG interactions, and may provide a basis for the design of drugs with a potential for regulating antibody-based inflammation.