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.

Project Start
Project End
Budget Start
Budget End
Support Year
14
Fiscal Year
2012
Total Cost
$351,707
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Lu, Jinghua; Chu, Jonathan; Zou, Zhongcheng et al. (2015) Structure of FcγRI in complex with Fc reveals the importance of glycan recognition for high-affinity IgG binding. Proc Natl Acad Sci U S A 112:833-8
Lu, Jinghua; Sun, Peter D (2015) Structural mechanism of high affinity FcγRI recognition of immunoglobulin G. Immunol Rev 268:192-200
Ten Broeke, Toine; van Spriel, Annemiek; Sun, Peter et al. (2015) Meeting report on immunoreceptors 2014. FASEB J 29:740-4
Wang, Ruipeng; Sun, Peter D (2014) Natural killer cell-mediated shedding of ULBP2. PLoS One 9:e91133
Lu, Jinghua; Marjon, Kristopher D; Mold, Carolyn et al. (2014) Pentraxins and IgA share a binding hot-spot on FcαRI. Protein Sci 23:378-86
Lu, Jinghua; Sun, Peter D (2014) A rapid and rational approach to generating isomorphous heavy-atom phasing derivatives. FEBS J 281:4021-8
Lu, Jinghua; Yu, Yadong; Zhu, Iowis et al. (2014) Structural mechanism of serum amyloid A-mediated inflammatory amyloidosis. Proc Natl Acad Sci U S A 111:5189-94
Abdi, Kaveh; Singh, Nevil J; Spooner, Eric et al. (2014) Free IL-12p40 monomer is a polyfunctional adaptor for generating novel IL-12-like heterodimers extracellularly. J Immunol 192:6028-36
Tikhonova, Anastasia N; Van Laethem, François; Hanada, Ken-ichi et al. (2012) αβ T cell receptors that do not undergo major histocompatibility complex-specific thymic selection possess antibody-like recognition specificities. Immunity 36:79-91
Wang, Ruipeng; Jaw, Jessica J; Stutzman, Nicole C et al. (2012) Natural killer cell-produced IFN-γ and TNF-α induce target cell cytolysis through up-regulation of ICAM-1. J Leukoc Biol 91:299-309

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