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

文献类型： 外文期刊

第一作者： Li, Qingmei

作者： Li, Qingmei;Wang, Ruining;Yang, Jifei;Guo, Junqing;Zhang, Gaiping;Li, Ge;Wang, Xun;Zhang, Gaiping;Wang, Ruining;Zhang, Gaiping

作者机构：

关键词： Fc-binding epitope; BoFc gamma RI; Bovine IgG1; Synthetic peptides; Binding inhibition

期刊名称：JOURNAL OF IMMUNOLOGICAL METHODS （ 影响因子：1.6； 五年影响因子：1.9 ）

ISSN： 0022-1759

年卷期： 2025 年 536 卷

页码：

收录情况： SCI

摘要： Background: Bovine IgG1 Fc receptor (boFc gamma RI) is a homologue to human Fc gamma RI (CD64) that has three extracellular Ig-like domains and can bind bovine IgG1 with high affinity. Identification of the linear epitope for Fcbinding on boFc gamma RI provides new insights for the IgG-Fc gamma interaction and Fc gamma R-targeting drugs development. Methods: The boFc gamma RI molecules were expressed on cell surface of the boFc gamma RI -transfected COS-7 cells. The extracellular domain of boFc gamma RI was expressed in Escherichia coli (E. coli) BL21, and the soluble boFc gamma RI was purified by Ni-chelation chromatography followed by refolding. To identify the Fc-binding epitope on the boFc gamma RI, peptides derived from the membrane-distal extracellular domain (EC2) of boFc gamma RI 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 boFc gamma RI 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 boFc gamma RII with IC50 of 20.27 mu mol/L, and inhibited the rosette formation of bovine IgG1-sensitized RBCs on the boFc gamma RI transfected cells with IC50 of 86.75 mu mol/L. Conclusions: The linear epitope for Fc-binding as well as its crucial residues of EC2 domain on boFc gamma RI was identified using synthetic peptides. The Fc-binding peptide showed well capability of regulating boFc gamma RI-IgG1 interaction on cell surface.

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