The alternative pathway of complement is important in innate immunity, attacking not only microbes but all unprotected biological surfaces through powerful amplification. It is unresolved how host and nonhost surfaces are distinguished at the molecular level, but key components are domains 19-20 of the complement regulator factor H (FH), which interact with host (i.e., nonactivator surface glycosaminoglycans or sialic acids) and the C3d part of C3b. Our structure of the FH19-20:C3d complex at 2.3-Å resolution shows that FH19-20 has two distinct binding sites, FH19 and FH20, for C3b. We show simultaneous binding of FH19 to C3b and FH20 to nonactivator surface glycosaminoglycans, and we show that both of these interactions are necessary for full binding of FH to C3b on nonactivator surfaces (i.e., for target discrimination). We also show that C3d could replace glycosaminoglycan binding to FH20, thus providing a feedback control for preventing excess C3b deposition and complement amplification. This explains the molecular basis of atypical hemolytic uremic syndrome, where mutations on the binding interfaces between FH19-20 and C3d or between FH20 and glycosaminoglycans lead to complement attack against host surfaces. structure and function | X-ray crystallography | hemolysis | kidney diseases | human mutations P reviously unencountered microbes invading a human body must be rapidly recognized and eliminated. This is the function of innate immunity, which includes the alternative pathway (AP) of complement. AP components can attack targets with hydroxyl or amine groups (i.e., all biological surfaces). This is a powerful defense mechanism, because there is rapid amplification leading to efficient opsonization or target lysis by the membrane attack complex (MAC). The AP attack is, therefore, also potentially dangerous for the host if one's cells and acellular structures are not protected.The AP activation is based on spontaneous hydrolysis of C3 in plasma leading to production of C3b, which then randomly attaches onto any surface hydroxyl or amine group through a reactive thioester located on the C3d part [i.e., thioester domain (TED)] of C3b. If these surface-attached C3b molecules are not quickly inactivated to iC3b and C3d, C3b deposition is rapidly amplified by a positive enzymatic feedback loop, leading to opsonophagocytosis and formation of the lytic membrane attack complex. On host surfaces, which are naturally nonactivators of the AP, efficient down-regulation of bound C3b occurs in three ways: factor I-mediated cleavage of C3b to inactive iC3b, acceleration of the decay of the preformed C3 convertases, or inhibition of factor B binding to C3b. Factor H (FH) is required for all these. It also down-regulates C3b deposition on noncellular surfaces, such as the heparan sulfate-rich glomerular basement membrane. FH is, thus, essential for restricting AP attack against host surfaces while allowing AP attack against foreign surfaces (i.e., for target discrimination) (1). A long-standing central question in complemen...

Summary The complement system of innate immunity plays an important role in regulating humoral immunity in large part through the complement receptor CR2 which forms a co-receptor on B cells during antigen-induced activation and retains antigens on follicular dendritic cells, which are critical for maintenance of germinal centers. Recent studies, identify a third important pathway in which naïve B cells pick-up complement C3-coated immune complexes within the lymphatics via CR2 and deliver them to follicular dendritic cells in the B cell compartment. These findings, combined with the recent report on the co-crystal structure of CR2 and its ligand C3d, present additional opportunities for dissecting a possible role for this pathway in regulating autoimmune responses.

Activation and covalent attachment of complement component C3 to pathogens is the key step in complement-mediated host defense. Additionally, the antigen-bound C3d fragment interacts with complement receptor 2 (CR2; also known as CD21) on B cells and thereby contributes to the initiation of an acquired humoral response. The x-ray crystal structure of human C3d solved at 2.0 angstroms resolution reveals an alpha-alpha barrel with the residues responsible for thioester formation and covalent attachment at one end and an acidic pocket at the other. The structure supports a model whereby the transition of native C3 to its functionally active state involves the disruption of a complementary domain interface and provides insight into the basis for the interaction between C3d and CR2.

A vital role for complement in adaptive humoral immunity is now beyond dispute. The crucial interaction is that between B cell and follicular dendritic cell-resident complement receptor 2 (CR2, CD21) and its Ag-associated ligands iC3b and C3dg, where the latter have been deposited as a result of classical pathway activation. Despite the obvious importance of this interaction, the location of a CR2 binding site within C3d, a proteolytic limit fragment of C3dg retaining CR2 binding activity, has not been firmly established. The recently determined x-ray structure of human C3d suggested a candidate site that was remote from the site of covalent attachment to Ag and consisted of an acidic residue-lined depression, which accordingly displays a significant electronegative surface potential. These attributes were consistent with the known ionic strength dependence of the CR2-C3d interaction and with the fact that a significant electropositive surface was apparent in a modeled structure of the C3d-binding domains of CR2. Therefore, we have performed an alanine scan of all of the residues within and immediately adjacent to the acidic pocket in C3d. By testing the mutant iC3b molecules for their ability to bind CR2, we have identified two separate clusters of residues on opposite sides of the acidic pocket, specifically E37/E39 and E160/D163/I164/E166, as being important CR2-contacting residues in C3d. Within the second cluster even single mutations cause near total loss of CR2 binding activity. Consistent with the proposed oppositely charged nature of the interface, we have also found that removal of a positive charge immediately adjacent to the acidic pocket (mutant K162A) results in a 2-fold enhancement in CR2 binding activity.

No abstract

Fibrinogen-like protein 2 (FGL2) is a multifunctional protein, which has been implicated in the pathogenesis of allograft and xenograft rejection. Previously, FGL2 was shown to inhibit maturation of BM-derived DC and T-cell proliferation. The mechanism of the immunosuppressive activity of FGL2 remains poorly elucidated. Here, we focus on identification of FGL2-specific receptor(s) and their ability to modulate APC activity and allograft survival. Using flow cytometry and surface plasmon resonance analysis, we show that FGL2 binds specifically to Fc gamma receptor (FccR)IIB and FccRIII receptors, which are expressed on the surface of APC, including B lymphocytes, macrophages and DC. Antibody to FccRIIB and FccRIII, or deficiency of these receptors, abrogated FGL2 binding. IntroductionFibrinogen-like protein 2 (FGL2), also known as fibroleukin, was first cloned from cytotoxic T lymphocytes and was classified as a member of the fibrinogen superfamily due to its homology (36%) with fibrinogen b and g chains [1]. Predicted as a type II transmembrane glycoprotein [2], cell membraneassociated FGL2 was shown to exhibit novel prothrombinase activity when associated with cell membranes/phospholipid vesicles [3,4], which has been implicated in the pathogenesis of à These authors contributed equally to this study.Correspondence: Dr. Gary Levy e-mail: glfgl2@attglobal.net DOI 10.1002/eji.200838338 Eur. J. Immunol. 2008. 38: 3114- [7]. The procoagulant activity was shown to depend on the serine 89 at the linear N terminal domain of FGL2 [4].The C terminal globular portion of FGL2 lacks the prothrombinase activity and contains a classical fibrinogen-related domain (FRED), which has been suggested to possess immunomodulatory activity based on several lines of evidence. First, other fibrinogen superfamily members including fibrinogen [8] Results Recombinant FcFGL2 protein binds to APCRecombinant FcFGL2 was generated and purified as described in the Materials and methods. The molecular size of the recombinant proteins was examined by SDS-PAGE, silver staining and Western blotting. FcFGL2 had a molecular weight of approximately 440 kDa under non-reducing conditions, which was confirmed by gel filtration (data not shown), and 110 kDa under reducing conditions (Fig. 1A). These data suggested that FcFGL2 exists as a tetramer, consistent with previous reports [13,21]. The Fc tag had a molecular size of 64 kDa under non-reducing conditions and 33 kDa under reducing conditions, suggesting that Fc is dimeric. Biotinylated FcFGL2 bound to the RAW264.7 cells (mouse macrophage cell lines), BMDC, the B-cell line A20 (which expresses only one Fcg receptor, FcgRIIB), thioglycolate-elicited peritoneal exudates cells (495% macrophages) from C57BL/6J mice, but FcFGL2 did not bind to the B-cell line A20IIA1.6, which does not express FcgRIIB (Fig. 1B) [22]. EL4 cells (a mouse T-cell line) did not bind FcFGL2 (data not shown). As expected, the Fc tag protein alone failed to bind to any of these cells and thus, Fc provided an appropriate negativ...

Degenerative disc disease (DDD) is associated with spinal pain often leading to long-term disability. However, the non-chondrodystrophic canine intervertebral disc is protected from the development of DDD, ostensibly due to its retention of notochordal cells (NC) in the nucleus pulposus (NP). In this study, we hypothesized that secretome analysis of the NC-rich NP will lead to the identification of key proteins that delay the onset of DDD. Using mass-spectrometry, we identified 303 proteins including components of TGFβ- and Wnt-signaling, anti-angiogeneic factors and proteins that inhibit axonal ingrowth in the bioactive fractions of serum free, notochordal cell derived conditioned medium (NCCM). Ingenuity Pathway Analysis revealed TGFβ1 and CTGF as major hubs in protein interaction networks. In vitro treatment with TGFβ1 and CTGF promoted the synthesis of healthy extra-cellular matrix proteins, increased cell proliferation and reduced cell death in human degenerative disc NP cells. A single intra-discal injection of recombinant TGFβ1 and CTGF proteins in a pre-clinical rat-tail disc injury model restored the NC and stem cell rich NP. In conclusion, we demonstrate the potential of TGFβ1 and CTGF to mitigate the progression of disc degeneration and the potential use of these molecules in a molecular therapy to treat the degenerative disc.

In physiological settings, the complement protein C3 is deposited on all bacteria, including invasive pathogens. However, because experimental host-bacteria systems typically use decomplemented serum to avoid the lytic action of complement, the impact of C3 coating on epithelial cell responses to invasive bacteria remains unexplored. Here, we demonstrate that following invasion, intracellular C3-positive Listeria monocytogenes is targeted by autophagy through a direct C3/ATG16L1 interaction, resulting in autophagy-dependent bacterial growth restriction. In contrast, Shigella flexneri and Salmonella Typhimurium escape autophagy-mediated growth restriction in part through the action of bacterial outer membrane proteases that cleave bound C3. Upon oral infection with Listeria, C3-deficient mice displayed defective clearance at the intestinal mucosa. Together, these results demonstrate an intracellular role of complement in triggering antibacterial autophagy and immunity against intracellular pathogens. Since C3 indiscriminately associates with foreign surfaces, the C3-ATG16L1 interaction may provide a universal mechanism of xenophagy initiation.