The following reagents and chemicals were purchased or obtained from the sources indicated: Dulbecco’s PBS (DPBS) with and without calcium and magnesium (Mediatech Inc., Manassas, VA, USA); Dulbecco’s modified Eagles medium (DMEM); RPMI 1640, 100× Penicillin/Streptomycin, (GIBCO, Invitrogen, Grand Island, NY, USA); heat inactivated fetal bovine serum (FBS; Hyclone, Logan, UT, USA); human serum albumin (HSA; Immuno-US, Rochester, MI, USA); p-nitrophenyl phosphate (pNPP; Pierce, Rockford, IL, USA); and Immu-Mount (Thermo Fisher, Waltham, MA, USA). Alexa-488- or Alexa-594-Streptavidin, Alexa-488- or Alexa-594-F(ab′)₂, goat anti-mouse or anti-rabbit; FITC conjugated goat anti-mouse IgG F(ab′)₂ or sheep anti-rabbit IgG F(ab′)₂ (Invitrogen, Carlsbad, CA, USA); and alkaline phosphatase (AP) – conjugated rabbit anti-goat IgG (Pierce), and C4d-EIA test kit (Quidel, San Diego, CA, USA).

The strategy for the construction of a plasmid containing the full-length (mature form or wild type, WT) or truncated forms of gC1qR cDNA as well as purification of the glutathione-S-transferase (GST)–gC1qR fusion products has been described in detail in our earlier publication (Ghebrehiwet et al., 1994; Lim et al., 1996). The GST–gC1qR fusion products were then cleaved by thrombin (3.2 μg/ml) and the GST-free gC1qR proteins purified on fast protein liquid chromatography (FPLC, Pharmacia) using a Mono-Q ion exchange column. The single peak containing the gC1qR was pooled, concentrated to 1–2 mg/ml, and stored at −80°C in the presence of 50 nM PPACK (d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone) – a specific thrombin inhibitor (Sigma Aldrich).

The cDNA and amino acid sequences of the region of gC1qR to be mutated, were identified and selected from analysis of the crystal structure as described (Jiang et al., 1999; Ghebrehiwet et al., 2002). To generate deletions, the Stratagene QuikChange double-strand mutagenesis kit was used, using complementary sense and antisense primers that bridge the deleted region (Ghebrehiwet et al., 1994; Lim et al., 1996). Maximization of mutagenesis was achieved by bringing the melting temperature (T_M) on each side of the deleted sequence to 60°C as described (Breslauer et al., 1986). Primers were extended using the PfuTurbo polymerase and the Stratagene kit, and mutant constructs were enriched by digestion of the methylated template with Dpn1, and transformed into E. coli. Final constructs were sequenced in all cases to verify sequence integrity. The fusion products were purified as described above and elsewhere (Ghebrehiwet et al., 1994; Lim et al., 1996).

A single residue mutation was performed on Trp 233, which is the only domain that projects conspicuously from the S face in the crystal structure, fairly close to the second anionic loop as described earlier (Ghebrehiwet et al., 2002). Encoded by the codon TGG, this residue was mutated by a single-base change to GGG (Gly) using double-strand mutagenesis, transformed into E. coli and the protein purified as above.

To estimate the organization and integrity of each recombinant protein, gel filtration of purified gC1qR WT as well as the various deletion mutants was carried out by analytical gel filtration on a column (1.4 cm × 30 cm) packed with Superose beads and equilibrated in 50 mM Tris–HCl buffer, pH 7.4, containing 1 mM EDTA in a manner similar to that described earlier (Ghebrehiwet et al., 1994). After adjusting the flow rate to 0.2 ml/min, the column was calibrated with two proteins having molecular weights close to those for the monomeric and trimeric forms of gC1qR, respectively. The peak elution times of these proteins – carbonic anhydrase (29 kDa) and BSA (65 kDa) – were recorded and used as the standard against which the elution time of each gC1qR protein was compared.

Analyses on SDS-PAGE were performed on a 1.5-mm thick slab gels according to the method of Laemmli (1970) with samples being run unreduced or reduced and alkylated by boiling for 5 min in the presence of 0.1 M dithiothreitol and 0.2 M iodoacetamide. After electrophoresis, the gels were stained with Coomassie Brilliant Blue, destained, and dried. Samples for Western blot analysis were first run on SDS-PAGE as above then electro transferred to polyvinyl difluoride (PVDF) or nitrocellulose membranes, blocked with 5% non-fat milk containing TBST (20 mM Tris–HCl, 150 mM NaCl, and 0.05% Tween 20), and the bound proteins probed with the appropriate and specific antibodies. The bound antibodies in turn were visualized by chemiluminescence horseradish peroxidase-conjugated species-specific antibody followed by reaction with 4-chloro-1-naphthol substrate.

Human brain microvascular endothelial cells (HBr-MIVEC) were cultured in Microvascular Endothelial Cell Growth Medium-2 (EGM-2MV; Lonza, Walkersville, MD, USA) and contained 5% heat inactivated FBS. Cells grown to confluence on 2% gelatin (Type B), were first treated with 0.25% trypsin, 0.01% EDTA to dissociate cells from the gelatin matrix by incubation (30 min, 37°C) in 0.01 M Tris-buffered saline. The cells were then washed and then subcultured in DMEM. Experiments were done with cells between passages 3 and 15.

U937 cells were grown in RPMI 1640 containing 10% heat inactivated (2 h 56°C) fetal calf serum and 1% antibiotic antimycotic stock (100×) mixture consisting of penicillin G (10,000 U/ml), amphotericin B (25 μg/ml) and streptomycin sulfate (10,000 μg/ml), and maintained in a humidified 37°C incubator in an atmosphere of 95% air and 5% CO₂.

The purified proteins used in these studies were obtained from the following commercial sources. Single-chain or two-chain high molecular weight kininogen (HK), as well as FXII was from (Enzyme Res., South Bend, IN, USA) and monoclonal anti-HK (clone 115-21) and anti-FXII (clone 179) were a generous gift from Rebecca Rowehl (Hybridoma Core Facility, Stony Brook University), and were originally generated and characterized by Reddigari and Kaplan (1989). The various monoclonal and polyclonal antibodies to recombinant gC1qR used in these studies have been described in previous publications and represent part of the anti-gC1qR antibody databank in our laboratory (Ghebrehiwet et al., 1996).

The ability of the various gC1qR proteins to bind HK was assessed by solid-phase ELISA using microtiter plates (Nunc, CovaLink, NH, Denmark). Because the interactants are sticky proteins, the ELISA experiments were designed to include a measure that will exclude interaction due to “stickiness.” Briefly duplicate wells were first coated (30 min, room temp or O/N, 4°C) with 100 μl (2 μg/ml) of WT or various gC1qR mutants (ΔgC1qR) or BSA in carbonate buffer, pH 9.6 (15 mM Na₂CO₃ and 35 mM NaHCO₃). The unbound proteins were aspirated; the wells washed 2× with TBST (20 mM Tris–HCl pH 7.5, 150 mM NaCl, and 0.05% Tween-20), and the unreacted sites blocked by incubation (30 min, room temp) with 300 μl of 1% non-fat dry milk or blotto (10 mg/ml casein in TBS, pH 8.0). After washing 2× with TBST, 100 μl each of concentrations of HK ranging from 0 to 5 μg/ml in TBSZ (TBS containing 10–50 μM Zinc) were added and incubated (30 min, room temp). The wells were then washed, 1× with TBSZ containing 0.5 M NaCl, and 2× with TBSTZ (containing 10 μM Zinc). After drying, the bound proteins were detected by sequential addition and incubation (30 min, room temp) with 100 μl of 2 μg/ml mAb anti-HK, followed by alkaline phosphatase conjugated rabbit anti-goat or goat anti-mouse IgG and pNPP (freshly dissolved in 10% diethanolamine pH 9.5, containing 5 mM MgCl₂ and 0.02% NaN₃). In experiments in which biotin-labeled HK was employed, the ELISA was developed using alkaline phosphatase conjugated Neutravidin followed by pNPP. The absorbance of the resulting color development in either case was measured at 405 nm using a V_Max Kinetic plate reader (Molecular Devices, Menlo Park, CA, USA). The experiments were done at least three times in duplicates.

Similar protocol was used to determine the interaction between FXII and gC1qR either with gC1qR bound to the duplicate wells and the bound FXII detected by mAb anti-FXII or in the reverse order in which FXII was bound to the plate and biotinylated gC1qR mutants were bound and detected by alkaline phosphatase conjugated Neutravidin followed by pNPP.

The ability of gC1qR to generate bradykinin (BK) was assayed in the following manner. Duplicate microtiter plate wells were first coated (30 min, 22°C) with 1% PEG to block charged sites, then gC1qR mutants were incubated with 100 μl of a mixture (1 μg/ml each) of highly purified HK, PK in the presence of 50 μM zinc and in the presence or absence of FXII (1 μg/ml each). Samples were incubated (30 min, 22°C) and the protein in the samples was precipitated with chilled ethanol, centrifuged at 10,000 × g for 20 min, and the supernatant containing BK was lyophilized and re-suspended in the assay buffer. The amount of BK in the sample was then determined using a commercial kit from Peninsula Laboratories (Peninsula Labs, San Carlos, CA, USA).

For flow cytometry, the cultured cell line, U937, was used. After verifying cell viability by trypan blue exclusion (≥95%), the cells (10⁶/ml DPBS) were preincubated (30 min, 20°C) with isotype-matched Fc fragments, followed by incubation (60 min, 20°C) with either buffer alone, or gC1qR proteins (10 μg/ml). After incubation, the cells were washed in DPBS, and further incubated with a predetermined dilution of mAb anti-gC1qR followed by another incubation with Alexa-488 conjugated and species matched IgG. The cells were then washed, fixed by suspension in 4% paraformaldehyde and then analyzed by flow cytometry. When biotin-labeled gC1qR proteins were used, then the cells were directly incubated with the proteins – thus obviating the blockade with Fc fragments – and the bound gC1qR was detected using Alexa-488 conjugated streptavidin.

Immunofluorescence studies were performed on either U937 cells or cultured HBr-MIVECs. The cells were grown on cover slips and the attached monolayer of cells reacted first with PBS containing 0.1% BSA and 1% heat inactivated human serum to block unreacted sites, followed by incubation with biotinylated gC1qR proteins and FITC conjugated secondary reagent as described above. After fixing for 10 min with 3.7% (v/v) formalin, the slides were examined by 3D imaging using deconvolution microscopy.

The ability of gC1qR and its deletion mutants to bind to U937 cells was tested using a microplate assay with fixed cells as described elsewhere (Kennet, 1984; Ghebrehiwet et al., 1996). Briefly, intact U937 cells (2 × 10⁵ cells/well) were first attached (30 min, 22°C) onto poly-l-lysine (10 μg/ml in PBS, pH 7.4) coated duplicate ELISA wells. The cells were fixed (30 min, 22°C) by addition of an equal volume of glutaraldehyde (0.5% solution in PBS), and the unreacted sites quenched with glycine–BSA (100 mM Glycine, 0.1% BSA in PBS, pH 7.4). Then, biotinylated gC1qR or deletion mutants ranging 0–5 μg/ml were added and the bound proteins detected by standard ELISA procedure as described.

Data are presented as mean ± SEM. Student’s t-tests were performed using statistical software (Excel; Microsoft, Redmond, WA, USA). A value of p = 0.05 was considered to be a significant difference (n values represent separate experiments performed under the same conditions).

After purification to homogeneity as assessed by SDS-PAGE, the deletion proteins were subjected to analytical gel filtration to assess whether deletion of a particular domain affects the trimer formation. As shown in Table 1, three deletion mutants failed to maintain the trimeric structure of the molecule. These domains correspond to amino acid residues 74–95, 204–218, and 212–223. The N-terminal residues 74–95 are part of the αA coiled-coil region of each monomer, which forms extensive intermolecular contacts in the trimeric structure of the molecule (Jiang et al., 1999; Ghebrehiwet et al., 2002). The domain covered by residues 204–218, on the other hand, forms the bulk of the β6 structure of each monomer with residues 212–223 essentially making the entire connecting loop between the β6 and β7 and partially covers the 20-Å donut hole of the trimer (Jiang et al., 1999; Ghebrehiwet et al., 2002).

As shown (Figures 1A,B), deletion of the previously identified HK site (aa 204–218) did not support HK binding. However, additional domains, whose deletion adversely affected HK binding, were also identified. These are domains covered by residues, 74–95, 144–148, 190–192, and 196–202. Furthermore, deletion of residues, which are critical for the trimeric integrity (Table 1) of the gC1qR molecule have also reduced HK binding, indicating that monomeric gC1qR does not bind HK efficiently. The relative locations of residues 144–162 (Figure 1C) and 196–2020 (Figure 1D) in the 3D structure of the trimeric molecule are provided for visual reference.

Since a single residue separates 196–202 and residues 204–218, we hypothesized that this domain, together with residues 204–218, might be part of the HK binding pocket in gC1qR. To test this hypothesis, we generated a synthetic peptide corresponding to residues190–202 (six residues longer) and tested its ability to support HK binding. The results of this experiment showed that biotinylated HK bound to the peptide in a specific and dose-dependent manner (not shown). Further preliminary studies also showed that the interaction of HK with peptide 190–202 was not inhibited by mAb 74.5.2, which recognizes residues 204–218 (not shown). This suggested that either HK binds at multiple sites or residues 190–202 contribute residue(s) for the putative binding pocket. Using this information, we designed experiments to screen for monoclonal antibodies that could inhibit the interaction between HK and peptide 190–202. As shown in Figure 2, binding of biotinylated HK was completely blocked by mAbs 48 and 83 but not by mAbs 69 or 73.

Previous experiments have shown that the binding of FXII to the EC surface is similar to that of HK in that it has the same requirement for zinc for binding and can compete for the same site with HK at comparable molar ratio (Reddigari et al., 1993). This observation led to the postulate that the two proteins may compete for the same receptor site on ECs. Since both HK and FXII can bind gC1qR, we examined whether they share the same or overlapping site(s) on the molecule. As shown in Figure 3, deletion of residues 204–218, which is the major binding site for HK did not affect the binding of FXII. However, several deletion mutants, including those identified as potential additional sites for HK (Figure 1) such as domains 74–95, 144–148, and 196–202 showed remarkably poor binding suggesting that the binding of HK and FXII may be to an overlapping site(s) rather than an identical site on gC1qR.

We have shown previously that gC1qR is capable of serving as a platform for the assembly of the KKS (Joseph et al., 2001a). Experiments were therefore designed to identify the critical site(s) for the assembly of KKS and the generation with BK. Two domains, whose deletion resulted in significantly reduced BK generation, were identified: residues 154–162, and a single point mutation W233G (not shown). Residues 154–162 are part of the disordered segment that form the connecting loop between β4 and β5 in the monomer, whereas Trp233 projects conspicuously from the trimeric structure and is at the beginning of the highly conserved sequence αB coiled-coil (Table 1).

Although gC1qR is a membrane-associated protein, it can nonetheless be secreted into the pericellular milieu by infected cells or cells undergoing active proliferation. This soluble gC1qR can also bind to cells in a specific and dose-dependent manner (Peterson et al., 1997). When biotinylated gC1qR proteins were incubated with the fixed cells, almost all of the deletion mutants were able to bind to the cells with varying degrees and in a dose-dependent manner with the exception of gC1qRΔ144–148, gC1qRΔ174–180, gC1qRΔ190–202, and gC1qRΔ196–202, which bound poorly, suggesting that these domains might be involved in cell attachment (Figure 4). These results were further confirmed when the binding of the deletion mutants to intact U937 cells in solution was tested by flow cytometry (Figure 5). In addition, we also used deconvolution microscopy to test the binding of the deletion mutants to microvascular endothelial cells. As shown in Figure 6, both gC1qRΔ144–148 and gC1qRΔ174–180, which is on the M-face of the protein, had weak to almost no binding to ECs. Therefore these domains – in particular residues 144–148 and 174–180 – may be suitable baits to identify the cell surface molecule to which they bind.