Analysis of the Interaction between Globular Head Modules of Human C1q and Its Candidate Receptor gC1qR

Abstract

The heterotrimeric globular head (gC1q) domain of human C1q is made up of the C-terminal ends of the three individual chains, ghA, ghB, and ghC. A candidate receptor for the gC1q domain is a multi-functional pattern recognition protein, gC1qR. Since understanding of gC1qR and gC1q interaction could provide an insight into the pleiotropic functions of gC1qR, this study was undertaken to identify the gC1qR-binding site on the gC1q domain, using the recombinant ghA, ghB, and ghC modules and their substitution mutants. Our results show that ghA, ghB, and ghC modules can interact with gC1qR independently, thus reinforcing the notion of modularity within the gC1q domain of human C1q. Mutational analysis revealed that while Arg162 in the ghA module is central to interaction between gC1qR and C1q, a single amino acid substitution (arginine to glutamate) in residue 114 of the ghB module resulted in enhanced binding. Expression of gC1qR and C1q in adherent monocytes with or without pro-inflammatory stimuli was also analyzed by qPCR; it showed an autocrine/paracrine basis of C1q and gC1qR interaction. Microscopic studies revealed that C1q and gC1qR are colocalized on PBMCs. Cell proliferation assays indicated that ghA, ghB, and ghC modules were able to attenuate phytohemagglutinin-stimulated proliferation of PBMCs. Addition of gC1qR had an additive effect on the anti-proliferative effect of globular head modules. In summary, our results identify residues involved in C1q-gC1qR interaction and explain, to a certain level, their involvement on the immune cell surface, which is relevant for C1q-induced functions including inflammation, infection, and immunity.

Keywords: C1q; cell proliferation; gC1qR; globular head; protein–protein interaction.

Figure 1

(A) Expression and purification of recombinant gC1qR. Twelve percent (v/v) SDS-PAGE under reducing conditions. E. coli BL21 (λDE3) strain, transformed with plasmid T7-gC1qR and induced with IPTG, overexpressed a ~33 kDa protein (induced) as compared to uninduced cells. Following lysis and sonication of the bacterial cells, the overexpressed gC1qR appeared in the soluble fraction sonicate, which was further purified on a Q-Sepharose column. (B) Western blotting to show immunoreactivity of recombinant gC1qR: 10 μg of recombinant gC1qR protein was run on a 12% v/v gel and transferred onto a nitrocellulose membrane, which was probed with anti-gC1qR polyclonal antibody. BSA was used as a negative control protein.

Figure 2

SDS-PAGE (12% w/v) under reducing conditions of purified fusion proteins following affinity chromatography. MBP fusion proteins containing wild type and mutant globular head modules were purified using an amylose resin column (A). Purified ghA, ghB and ghC (B) purified substitution mutants of ghA module; (C) purified mutants of ghB module, R163A, R163E, R114Q, R114A, R129A, R129E, H117D, and T175L; (D) purified ghC, R156Q, R156E, and H101A.

Figure 3

(A) ELISA to show binding of gC1qR to C1q: microtiter wells, coated with different concentrations of human C1q (5, 2.5, 1.25, and 0.625 μg/well), were incubated with 2.5 μg of gC1qR. Bound gC1qR was detected by anti-gC1qR polyclonal antibody followed by Protein A-HRP conjugate. MBP was used as a negative control protein. (B) ELISA to assess interaction of gC1qR with ghA, ghB, and ghC modules. gC1qR (1 μg/well in 100 μl) was coated on microtiter wells and then incubated with various concentrations of wild type of ghA, ghB, and ghC. MBP was used as a negative control. Following washing, bound protein was detected using anti-MBP monoclonal antibody and goat anti-mouse IgG HRP conjugate. (C) Far-western blot analysis to show ghA, ghB, and ghC binding to gC1qR: 15 μg of gC1qR was first run on the SDS-PAGE under reducing conditions, transferred onto a PDVF membrane and then probed with 10 μg each of ghA, ghB, and ghC. Lanes 1 through 3 show interaction of gC1qR with ghA, ghB, and ghC, respectively.

Figure 4

ELISA to assess interaction between gC1qR and gh substitution mutants. Microtiter wells were coated with different quantities (0.25, 0.5, and 1 μg/well) of gC1qR. After blocking and washing, the wells were incubated with 2.5 μg/well of (A) ghA, R162A and R162E; (B) ghB, R114A, R114Q, R163A, R163E, R129A, R129E, T175L, and H117D; and (C) ghC, for 90 min at 37°C and 90 min at 4°C. Bound proteins were detected with anti-MBP monoclonal antibody followed by goat anti-mouse IgG–HRP conjugate. Data are representative of three experiments. (D) Ligand blot to show binding of ghA mutants R162A and R162E to gC1qR: PVDF membrane strips containing gC1qR were reacted with ghA, R162A and R162E, and then probed with anti-MBP monoclonal antibody followed by goat anti-mouse IgG–HRP.

Figure 5

Interaction of ghA, ghB, and ghC with monocyte/macrophages. PMBCs (1 × 10⁶) were seeded on 13 mm coverslips and incubated in complete RPMI 1640 medium for 2 weeks at 37°C in 5% CO₂ incubator. Cells were treated with 10 μg of each globular head module and incubated with serum-free RPMI 1640 medium for 1 h at 37°C. After washing with PBS, cells were fixed with 4% PFA, permeabilzed with Triton X-100, and probed with anti-gC1qR polyclonal antibody and anti-MBP monoclonal antibody to reveal gC1qR and bound globular head modules, respectively. Cells were washed and treated with Alexa Fluor 488 conjugated secondary goat anti-rabbit antibody and Alexa Fluor 647 conjugated secondary donkey anti-mouse antibody and the nucleus was stained with Hoechst 33342. Cells were then examined under Leica fluorescence microscope with 40× magnification. In the merged images gC1qR is green; globular heads are red; and nucleus is blue. Arrows point to bound globular heads with colocalization of globular heads and gC1qR seen in orange in the merged images. Scale bars: 10 μm.

Figure 6

Expression of C1q (A) and gC1qR (B) by adherent human monocytes in vitro. The expression of C1q and gC1qR was measured using real-time qPCR and the data were normalized via 18S rRNA gene expression as a control. Relative expression (RQ) was calculated by using the 2 h time point as the calibrator. The RQ value was calculated using the formula: RQ = 2^−∆∆Ct. Assays were conducted twice in triplicates. Error bars represent ±SEM. A two-side t-test was performed on the data. All samples showed significant expression compared to the calibrator (p ≤ 0.01), except where noted: *0.01 < p < 0.05; ns: not significant (p ≥ 0.05). LPS was added to cultures at a 20 ng/μl concentration.

Figure 7

Anti-proliferative effects of ghA, ghB, and ghC modules. Human PBMCs from healthy individuals were stimulated with PHA (1 μg/ml) for 3 days with or without recombinant gC1qR (1.25, 2.5, 5, 10, and 20 μg/ml), ghA, ghB, or ghC (20 μg/ml) followed by a 16 h pulse in the presence of tritiated methyl thymidine ([³H]TdR). Each experiment was carried out in triplicates. (A) Each of ghA, ghB, ghC, and gC1qR inhibited PHA-stimulated proliferation of PBMCs; (B) ghA, ghB, and ghC coincubated with gC1qR inhibit PHA-stimulated proliferation of PBMCs.