Identification of Amino Acid Residues in Human IgM Fc Receptor (FcµR) Critical for IgM Binding.
Identification of Amino Acid Residues in Human IgM Fc Receptor (FcµR) Critical for IgM Binding.
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DOI：
10.3389/fimmu.2020.618327
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发表时间：
2020
影响因子：
7.3
通讯作者：
Kubagawa H
中科院分区：
文献类型：
--
作者：
Skopnik CM;Al-Qaisi K;Calvert RA;Enghard P;Radbruch A;Sutton BJ;Kubagawa H
非免疫的“天然”和抗原诱导的“免疫”的免疫球蛋白M对于预防感染和调节自身抗原的免疫反应都很重要。其Fc受体(FcµR)在这些IgM效应器功能中的作用已开始被探索。在本研究中，我们利用人和小鼠FcµR在IgM配体结合上的差异(结构性结合)和小鼠FcµR的瞬时结合(瞬时结合)，在建立稳定表达突变型或野生型(WT)受体的细胞系之前，用小鼠等效物取代了人FcµR的非保守氨基酸残基。以受体特异性单抗和IgM副蛋白为配体，用流式细胞仪检测8种不同突变的FcµR细胞和WT受体细胞的细胞表面表达和IgM结合情况。Asn66、Lys79-Arg83和Asn109三个位点分别可能位于人FcµR Ig样结构域的CDR2、DE环和CDR3中，负责结构性的IgM结合。有趣的是，用相应的小鼠残基Gln和Leu替换推测的CDR1中的Glu41和Met42，无论是单一的还是更显著的组合，都增强了受体的表达和IgM结合。在预测的人FcµR的A？链中，Lys24-Gly27的四个氨基酸延伸似乎是维持其在质膜上正确的受体构象所必需的，因为当这些突变时，受体表达和IgM结合潜力都降低了。计算结构模拟分析的结果与这些突变数据一致，并确定了FcµR与IgM结合的一种可能模式，涉及FcµR的Asn66、Arg83和Asn109等环，主要与人IgM的Cµ4结构域(包括Gln510)相互作用，其次是Cµ3结构域(包括Glu398)。据我们所知，这是第一份描述人FcµR与IgM Fc结合的关键氨基酸残基的实验报告。
Both non-immune “natural” and antigen-induced “immune” IgM are important for protection against infections and for regulation of immune responses to self-antigens. The roles of its Fc receptor (FcµR) in these IgM effector functions have begun to be explored. In the present study, by taking advantage of the difference in IgM-ligand binding of FcµRs of human (constitutive binding) and mouse (transient binding), we replaced non-conserved amino acid residues of human FcµR with mouse equivalents before establishment of cell lines stably expressing mutant or wild-type (WT) receptors. The resultant eight-different mutant FcµR-bearing cells were compared with WT receptor-bearing cells for cell-surface expression and IgM-binding by flow cytometric assessments using receptor-specific mAbs and IgM paraproteins as ligands. Three sites Asn66, Lys79-Arg83, and Asn109, which are likely in the CDR2, DE loop and CDR3 of the human FcµR Ig-like domain, respectively, were responsible for constitutive IgM binding. Intriguingly, substitution of Glu41 and Met42 in the presumed CDR1 with the corresponding mouse residues Gln and Leu, either single or more prominently in combination, enhanced both the receptor expression and IgM binding. A four-aa stretch of Lys24-Gly27 in the predicted A ß-strand of human FcµR appeared to be essential for maintenance of its proper receptor conformation on plasma membranes because of reduction of both receptor expression and IgM-binding potential when these were mutated. Results from a computational structural modeling analysis were consistent with these mutational data and identified a possible mode of binding of FcµR with IgM involving the loops including Asn66, Arg83 and Asn109 of FcµR interacting principally with the Cµ4 domain including Gln510 and to a lesser extent Cµ3 domain including Glu398, of human IgM. To our knowledge, this is the first experimental report describing the identification of amino acid residues of human FcµR critical for binding to IgM Fc.