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.? ? Complement pathway constitues an important part of host immune system against infection. The classical complement pathway is activated when immunoglobulins or pentraxins (including the acute phase reactive protein CRP and SAP) bind to C1q. Our goal is to understand the molecular mechanism of C1q activation. We are currently purifying C1q from human plasma, constructing recombinant expression system for the Fc regions of the immunoglobulins. We hope to be able to determine the crystal structure of C1q in complex with CRP or immunoglobulins. 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.
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