Assembly of B cell coreceptors and activation of FcR by C-reactive protein

B 细胞辅助受体的组装和 C 反应蛋白对 FcR 的激活

• 批准号: 8555842
• 负责人: PETER D. SUN
• 金额: $ 35.17万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8555842
• 关键词: Amino Acids; Anti-Inflammatory Agents; Anti-inflammatory; Antigen-Antibody Complex; Antigens; Autoimmune Diseases; B-Lymphocytes; Bacteria; Binding; Binding Sites; C-reactive protein; Cells; Characteristics; Competitive Binding; Complement; Complement 1q; Complement Activation; Complex; Crystallization; Cytoplasmic Tail; Data; Disulfides; Escherichia coli; Extracellular Domain; Extracellular Structure; Face; Family; Fc Receptor; Glutathione; Goals; Human; Humoral Immunities; ITAM; IgA receptor; Immunoglobulin A; Immunoglobulin G; Immunoglobulins; Inclusion Bodies; Infection; Inflammation; Inflammatory Response; Laboratories; Ligands; Link; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Methods; Molecular; Mus; Natural Immunity; Pathway interactions; Phagocytosis; Phosphorylation; Photons; Plasma Proteins; Production; Property; Proteins; Protomer; R-factor; Receptor Activation; Receptor Aggregation; Receptors, Antigen, B-Cell; Recombinants; Resolution; Role; Signal Transduction; Site; Solutions; Source; Structure; Surface; Surface Immunoglobulins; System; TNF gene; Tryptophan; Tyrosine; Work; antigen binding; base; beamline; beta pleated sheet; combat; crosslink; cytokine; design; disulfide bond; emergency service responder; macrophage; molecular recognition; neutrophil; novel; novel therapeutics; receptor; receptor binding; research study; resistance factors; src-Family Kinases

The B cell antigen receptor (BCR) is a multiprotein structure that consists of a non-covalently associated antigen binding subunit and a signaling subunit. Membrane-bound immunoglobulin (Ig) is an antigen binding subunit that has a very short cytoplasmic tail of only about three amino acids. Signal is transmitted through disulfide-linked heterodimer consisting of Iga and Igb proteins (CD79a and CD79b). Each contain a single immunoreceptor tyrosine-based activation motif (ITAM) within their cytoplasmic tail that initiates signal transduction following BCR aggregation. Aggregation of the BCR results in the phosphorylation of the ITAM tyrosine residues on Iga and Igb primarily by src-family kinases. In efforts to elucidate molecular recognition between Iga and Igb and between the heterodimer and the BCR through structural studies, we have expressed both human and murine extracellular domains of Iga and Igb in a recombinant E. coli system as inclusion bodies. Attempts were made to refold Iga and Igb either individually or in concert. Both Iga and Igb can be refolded individually. Refolding of Iga yielded a mixture of monomeric and homodimeric species as determined by mass spectrometry and SDS-PAGE analysis. The crystallization trials of Iga, however, was not successful. In contrast, the crystallization experiments of both the refolded human and murine Igb yielded crystals that diffracted to 3.8 and 1.7 angstrom resolution, respectively. Subsequently, we have determined the structure of extracellular portion of murine Igb at 1.7 E resolution. The diffraction data were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory. The structure was solved by the molecular replacement method with the final R-factors are R=18.7 and Rfree=19.7%. As predicted, the structure shows a V-type immunoglobulin fold made of two antiparallel beta sheets with a characteristic A-strand switch and conserved disulfide bond between the B and F strands flanked by tryptophan from C strand. There are several distinct features in the murine Igb structure. Among them, a new disulfide bond was observed connecting the N-terminus and the beginning of G strand. In addition, the typical C, C antiparallel strands characteristic for a V-type Ig fold are not present in murine Igb. Instead of C/C strands, the structure shows a much shorter strand bridging two antiparallel beta sheets. There is one free cystein located in FG loop which is blocked by glutathione in the structure. Presumably, this cystein takes part in the formation of a disulfide bond in Iga/Igb heterodimer. Further structural and binding studies are currently underway. Recent evidence suggests that pentraxins interact with Fc receptors raising the possiblity of cross interaction between the complement and the Fc receptor mediated pathways. We are investigating the interaction between SAP/CRP and Fc receptors and determine the structure of their complex. We have recently determined the crystal structure of human SAP in complex with FcRIIa. The 1:1 receptor-SAP recognition is predominantly mediated through the interactions of the ridge helix from two separate SAP protomers with the D1 and D2 domains of the Fc receptor. The complex structure between human SAP and FcRIIa reveals a diagonally bound receptor on each SAP pentamer. Mutational analysis suggests a conserved receptor recognition among pentraxins. The shared binding site for SAP and IgG results in their competition to FcR binding and the inhibition of immune complex-mediated phagocytosis by soluble pentraxins. These results establish the role of innate pentraxins in the FcgR pathway, and have novel therapeutic implications for autoimmune diseases.Unexpectedly, the SAP binding site on FcRIIa overlaps partially with the IgG binding site on the receptor. The solution-based binding experiments confirmed that SAP and CRP competed against IgG for the binding to FcRs. Solutuion binding experiments showed that pentraxins recognize various FcRs and activate FcR-mediated phagocytosis and cytokine secretion. Moreover, soluble SAP and CRP inhibited significantly the immune complex-mediated phagocytosis, indicating a regulatory function for this family of plasma proteins. The result highlights the importance of pentraxins in interfacing between the innate and humoral immunities and suggests new therapeutic applications for pentraxins in autoimmune diseases. We recently identified the major IgA receptor, FcRI as a ligand for pentraxins. We conclude through competitive binding and mutational analysis that CRP binds to a distinct site on FcRI from that of IgA, and that the recognition involves the effector face of CRP in a region overlapping with its C1q and FcR binding site. Furthermore, CRP crosslinking of FcRI resulted in ERK phosphorylation, degranulation and cytokine production in FcRI transfected RBL cells. In neutrophils, CRP binding induced FcRI surface expression and TNF- secretion, and CRP-opsonized bacteria triggered phagocytosis. The impact of this work is two folds. First, the discovery that pentraxins activate FcRI reveals a novel function for pentraxins in inflammation. It implicates a potential pentraxin-mediated synergistic activation of various Fc receptors in neutrophil and macrophage-mediated inflammatory responses. This is particular so since neutrophils and macrophages are the first responders of infection and inflammation. Second, our finding also highlights the innate aspect of antibody receptors that are mediators of humoral immunity. More recently, the mechanism of IVIg has been intensely studied and still remain controversial. We initiated a study to investigate the potential mechansim associated with IVIg-mediated anti-inflammatory property.

B细胞抗原受体（BCR）是一种多蛋白结构，由非共价相关的抗原结合亚基和信号亚基组成。膜结合的免疫球蛋白（IG）是一种抗原结合亚基，其细胞质尾巴非常短，仅约三个氨基酸。信号通过由IGA和IGB蛋白（CD79A和CD79B）组成的二硫键链接异二聚体传输。每种都包含单个免疫受体酪氨酸的活化基序（ITAM）在其细胞质尾部，该基元在BCR聚集后启动信号转导。 BCR的聚集导致ITAM酪氨酸残基在IGA和IGB上的磷酸化，主要是由SRC家庭激酶磷酸化。 为了通过结构研究阐明IgA和IGB之间以及异二聚体和BCR之间的分子识别，我们在重组大肠杆菌系统中以包容性体内表示IGA和IGB的人类和鼠细胞外域。试图单独或共同重新分配IGA和IGB。 IgA和IGB均可单独重折叠。 通过质谱和SDS-PAGE分析确定，IgA的重折叠产生了单体和同型二聚体物种的混合物。 但是，IGA的结晶试验并不成功。 相比之下，重折叠的人和鼠IGB的结晶实验分别产生了衍射为3.8和1.7埃斯特罗姆分辨率的晶体。随后，我们以1.7 E分辨率确定了鼠IGB细胞外部分的结构。衍射数据是在Argonne National Laboratory的高级光子源的东南区域合作访问团队（SER-CAT）22-ID梁线上收集的。通过最终R因子的分子替代方法求解结构，R = 18.7，RFREE = 19.7％。如预期的那样，该结构显示了由两个反平行β板制成的V型免疫球蛋白折叠，具有特征性的A链开关，并在B和F链之间保守的二硫键，侧面是来自C Strand的色氨酸的侧面。鼠IGB结构中有几个不同的特征。其中，观察到了连接N末端和G链的开始的新二硫键。 另外，在鼠IGB中不存在典型的C，C抗平行链特征。该结构不是C/C链，而是显示了一个短得多的链，桥接了两个反平行β板。 FG环中有一个自由的半胱氨酸，在结构中被谷胱甘肽阻塞。据推测，这个半胱氨酸参与了IgA/IGB异二聚体中二硫键的形成。目前正在进行进一步的结构和结合研究。 最近的证据表明，五肽与FC受体相互作用，从而增加了补体和FC受体介导的途径之间交叉相互作用的可能性。 我们正在研究SAP/CRP和FC受体之间的相互作用，并确定其复合物的结构。我们最近确定了与FCRIIA复合物中人类SAP的晶体结构。 1：1受体识别的识别主要是通过从两个单独的SAP原始物与FC受体的D1和D2结构域的脊螺旋相互作用来介导的。人类SAP和FCRIIA之间的复杂结构揭示了每个SAP Pentamer上的对角线受体。 突变分析表明五肽之间的受体识别是保守的。用于SAP和IgG的共享结合位点导致其与FCR结合的竞争，并抑制可溶性五肽的免疫复合物介导的吞噬作用。这些结果确定了先天五肽在FCGR途径中的作用，并对自身免疫性疾病具有新的治疗意义。毫无指示，FCRIIA上的SAP结合位点与受体上的IgG结合位点部分重叠。基于溶液的结合实验证实，SAP和CRP与IgG竞争与FCR的结合。溶质结合实验表明，五肽素识别各种FCR，并激活FCR介导的吞噬作用和细胞因子分泌。 此外，可溶性SAP和CRP显着抑制了免疫复合物介导的吞噬作用，表明该血浆蛋白家族的调节功能。 结果凸显了五卫蛋白在与先天和体液免疫之间接触中的重要性，并提出了五型五肽在自身免疫性疾病中的新治疗应用。 我们最近将主要的IgA受体FCRI确定为五肽的配体。我们通过竞争性结合和突变分析得出结论，CRP与IgA的FCRI上的不同位点结合，并且该识别涉及与其C1Q和FCR结合位点重叠的区域中CRP的效应子面。 此外，FCRI的CRP交联导致FCRI转染的RBL细胞的ERK磷酸化，脱粒和细胞因子产生。 在中性粒细胞中，CRP结合诱导的FCRI表面表达和TNF分泌，以及CRP揭示的细菌引发了吞噬作用。这项工作的影响是两个折。 首先，发现五肽激活FCRI的发现揭示了pentraxins在炎症中的新功能。 这意味着潜在的pentraxin介导的在中性粒细胞和巨噬细胞介导的炎症反应中各种FC受体的协同激活。这是特别的，因为中性粒细胞和巨噬细胞是感染和炎症的第一反应者。 其次，我们的发现还突出了抗体受体的先天性，这些抗体受体是体液免疫的介体。 最近，对IVIG的机制进行了深入研究，并且仍然存在争议。 我们开始了一项研究，以研究与IVIG介导的抗炎特性相关的潜在Mechansim。