AbstractC1q modulates the differentiation and function of cells committed to the monocyte-derived dendritic cell (DC) lineage. Because the 2 C1q receptors found on the DC surface—gC1qR and cC1qR—lack a direct conduit into intracellular elements, we postulated that the receptors must form complexes with transmembrane partners. In the present study, we show that DC-SIGN, a C-type lectin expressed on DCs, binds directly to C1q, as assessed by ELISA, flow cytometry, and immunoprecipitation experiments. Surface plasmon resonance analysis revealed that the interaction was specific, and both intact C1q and the globular portion of C1q bound to DC-SIGN. Whereas IgG reduced this binding significantly, the Arg residues (162-163) of the C1q-A chain, which are thought to contribute to the C1q-IgG interaction, were not required for C1q binding to DC-SIGN. Binding was reduced significantly in the absence of Ca2+ and by preincubation of DC-SIGN with mannan, suggesting that C1q binds to DC-SIGN at its principal Ca2+-binding pocket, which has increased affinity for mannose residues. Antigen-capture ELISA and immunofluorescence microscopy revealed that C1q and gC1qR associate with DC-SIGN on blood DC precursors and immature DCs. The results of the present study suggest that C1q/gC1qR may regulate DC differentiation and function through the DC-SIGN–mediated induction of cell-signaling pathways.

The endothelial cell receptor complex for kininogen (HK) comprises gC1qR, cytokeratin 1, and urokinase-type plasminogen activator receptor and is essential for activation of the kinin system that leads to bradykinin (BK) generation. Of these, gC1qR/p33 constitutes a high affinity site for HK – the BK precursor – and is therefore critical for the assembly of the kinin-generating cascade. Previous studies have identified a putative HK site within the C-terminal domain (residues 204–218) of gC1qR recognized by mAb 74.5.2. In these studies, we used information from the crystal structure of gC1qR, to engineer several deletion (Δ) mutants and test their ability to bind and/or support BK generation. While deletion of residues 204–218 (gC1qRΔ204–218), showed significantly reduced binding to HK, BK generation was not affected when tested by a sensitive bradykinin immunoassay. In fact, all of the gC1qR deletion mutants supported BK generation with the exception of gC1qRΔ154–162 and a point mutation in which Trp 233 was substituted with Gly. Binding studies also identified the existence of two additional sites at residues 144–162 and 190–202. Moreover, binding of HK to a synthetic peptide 190–202 was inhibited by mAbs 48 and 83, but not by mAb 74.5.2. Since a single residue separates domains 190–202 and 204–218, they may be part of a highly stable HK binding pocket and therefore a potential target for drug design to prevent vascular permeability and inflammation.

The receptor for the globular heads of C1q, gC1qR/p33, is a widely expressed cellular protein, which binds to diverse ligands including plasma proteins, cellular proteins, and microbial ligands. In addition to C1q, gC1qR also binds high molecular weight kininogen (HK), which also has two other cell surface sites, namely, cytokeratin 1 and urokinase plasminogen activator receptor (uPAR). On endothelial cells (ECs), the three molecules form two closely associated bimolecular complexes of gC1qR/cytokeratin 1 and uPAR/cytokeratin 1. However, by virtue of its high affinity for HK, gC1qR plays a central role in the assembly of the kallikrein-kinin system, leading to the generation of bradykinin (BK). BK in turn is largely responsible for the vascular leakage and associated inflammation seen in angioedema patients. Therefore, blockade of gC1qR by inhibitory peptides or antibodies may not only prevent the generation of BK but also reduce Clq-induced or microbial-ligand-induced inflammatory responses. Employing synthetic peptides and gClqR deletion mutants, we confirmed previously predicted sites for C1q (residues 75-96) and HK (residues 204-218) and identified additional sites for both C1q and HK (residues 190-202), for C1q (residues 144-162), and for HIV-1 gp41 (residues 174-180). With the exception of residues 75-96, which is located in the alphaA coiled-coil N-terminal segment, most of the identified residues form part of the highly charged loops connecting the various beta-strands in the crystal structure. Taken together, the data support the notion that gC1qR could serve as a novel molecular target for the design of antibody-based and/or peptide-based therapy to attenuate acute and/or chronic inflammation associated with vascular leakage and infection.

C1‐INH is a serine protease inhibitor (serpin) whose deficiency results in a life threatening disease─angioedema. Its primary function is to regulate key enzymes of both the complement and kinin generating systems; but it also plays a key role in controlling the integrity of C1. These studies were undertaken to test the hypothesis that gC1qR, which activates both the complement and kinin systems, can accelerate complement activation in C1‐INH deficient serum. To achieve this, we first prepared C1‐INH depleted serum (NHS−C1inh) by affinity depletion on protein A‐immobilized anti‐C1‐INH, followed by reconstitution with 20 μg/ml C1q and 20 mM CaCl2 and MgCl2. Depletion was verified by ELISA, and its activity by a standard hemolytic assay. Incubation (60 min, 37º C) of NHS alone, followed by measurement of residual hemolytic activity showed no loss in function when compared to untreated NHS. However, NHS−C1inh lost 30–50% of its function due to spontaneous autoactivation. Importantly, whereas incubation of NHS with 5μg/ml gC1qR or aggregated IgG resulted in complete activation after 60 min, it took only 15 min for the same level of activation in NHS−C1inh. This was reversed when the NHS−C1inh was reconstituted with C1‐INH. The data suggest that gC1qR, can trigger and/or exacerbate the inflammatory process associated with angioedema by enhancing activation of complement. [Supported by NIH‐NIAID Grant R01 AI‐060866]

Surface expressed or soluble gC1qR has been shown to activate complement and to play a role in various inflammatory processes. By peptide mapping and antibody inhibition studies, we had previously identified a binding site for C1q in a gC1qR domain contained within residues 76-93. However, although mAbs 60.11, which recognizes residues 76-93, can inhibit C1q binding to gC1qR, the inhibition is not complete. Similarly, binding of C1q to a truncated form of gC1qR lacking residues 76-93 is reduced by only 40-50% when compared to the full-length gC1qR. These findings therefore prompted us to look for additional binding sites elsewhere in the molecule. To this end, we generated several recombinant gC1qR proteins by deletion of domains of very high negative charge— and which, based on their location in the crystal structure—were predicted to interact with solution-phase ligands. The capabilities of these mutants to bind to C1q were therefore examined by comparing to the binding of the full-length gC1qR. Analyses of the data reveal the presence of two additional binding sites for C1q—one contained within residues 190-192 and another within residues 144-162. These results not only demonstrate well the value of crystal-based structure-function predictions, but also allow the identification of potential targets for drug design to alleviate complement-mediated inflammation. [Supported by NIH-NIAID Grant R01 AI-060866]