Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission

Abstract

Dendritic cells (DCs) are the most potent antigen-presenting cells capable of priming naïve T-cells. Its C-type lectin receptor, DC-SIGN, regulates a wide range of immune functions. Along with its role in HIV-1 pathogenesis through complement opsonization of the virus, DC-SIGN has recently emerged as an adaptor for complement protein C1q on the surface of immature DCs via a trimeric complex involving gC1qR, a receptor for the globular domain of C1q. Here, we have examined the nature of interaction between C1q and DC-SIGN in terms of domain localization, and implications of C1q-DC-SIGN-gC1qR complex formation on HIV-1 transmission. We first expressed and purified recombinant extracellular domains of DC-SIGN and its homologue DC-SIGNR as tetramers comprising of the entire extra cellular domain including the α-helical neck region and monomers comprising of the carbohydrate recognition domain only. Direct binding studies revealed that both DC-SIGN and DC-SIGNR were able to bind independently to the recombinant globular head modules ghA, ghB, and ghC, with ghB being the preferential binder. C1q appeared to interact with DC-SIGN or DC-SIGNR in a manner similar to IgG. Mutational analysis using single amino acid substitutions within the globular head modules showed that Tyr^B175 and Lys^B136 were critical for the C1q-DC-SIGN/DC-SIGNR interaction. Competitive studies revealed that gC1qR and ghB shared overlapping binding sites on DC-SIGN, implying that HIV-1 transmission by DCs could be modulated due to the interplay of gC1qR-C1q with DC-SIGN. Since C1q, gC1qR, and DC-SIGN can individually bind HIV-1, we examined how C1q and gC1qR modulated HIV-1-DC-SIGN interaction in an infection assay. Here, we report, for the first time, that C1q suppressed DC-SIGN-mediated transfer of HIV-1 to activated pooled peripheral blood mononuclear cells, although the globular head modules did not. The protective effect of C1q was negated by the addition of gC1qR. In fact, gC1qR enhanced DC-SIGN-mediated HIV-1 transfer, suggesting its role in HIV-1 pathogenesis. Our results highlight the consequences of multiple innate immune pattern recognition molecules forming a complex that can modify their functions in a way, which may be advantageous for the pathogen.

Keywords: C1q; DC-SIGN; HIV-1; globular head domain; protein–protein interaction.

Figure 1

SDS-PAGE under reducing conditions (12% v/v) showing purified fractions of soluble DC-SIGN tetramer (A), DC-SIGN monomer (B), DC-SIGNR tetramer (C), and DC-SIGNR monomer (D), following purification by mannose-agarose affinity chromatography.

Figure 2

Interaction of C1q, ghA, ghB, and ghC with DC-SIGN tetramer and monomer. (A) Microtiter wells coated with different concentrations (5, 2.5, 1.25, 0.625 µg/well) of DC-SIGN tetramer or monomer were probed with 2 µg/well of C1q. Bound C1q was detected with anti-C1q polyclonal antibodies (1:1,000 in PBS) and Protein A HRP conjugate (1:1,000 in PBS). BSA was used as a negative control protein. (B) Binding of ghA, ghB, and ghC to DC-SIGN tetramer and (C) DC-SIGN monomer involved coating a range of concentrations of the respective proteins on microtiter wells, which were then incubated with a fixed concentration of ghA, ghB, ghC, and MBP (2.5 µg/well in 5 mM CaCl₂ buffer) at 37°C. Binding was detected using anti-MBP monoclonal antibodies (1:5,000 in PBS) and then rabbit anti-mouse IgG-HRP (1:5,000 in PBS). (D) Far western blot to show DC-SIGN tetramer binding to membrane-bound ghA, ghB, and ghC: 15 µg of ghA, ghB, and ghC (BSA and MBP as negative control proteins) were run on a 12% SDS-PAGE gel and then transferred on to nitrocellulose membrane. The blot was incubated with 50 µg of DC-SIGN in PBS overnight at room temperature. The bound DC-SIGN protein was detected using anti-DC-SIGN polyclonal antibodies and Protein A HRP conjugate. Bands were developed using diaminobenzidine tablets dissolved in water.

Figure 3

Interaction of C1q, ghA, ghB, and ghC with DC-SIGNR tetramer and monomer. (A) ELISA to examine binding of C1q to DC-SIGNR tetramer and SIGN-R monomer: DC-SIGNR tetramer or monomer were coated at different concentrations, followed by addition of 2 µg/well of C1q. Bound C1q was probed with anti-C1q polyclonal antibodies (1:1,000 in PBS) and Protein A HRP (1:1,000 in PBS), and the color was developed using o-phenylenediamine dihydrochloride. (B) Binding of ghA, ghB, and ghC to DC-SIGNR tetramer and (C) SIGN-R monomer: different concentrations of DC-SIGNR tetramer (B) and DC-SIGNR monomer (C) were coated on microtiter wells in carbonate buffer and incubated overnight at 4°C and then incubated with ghA, ghB, ghC, and MBP (2.5 µg/well in 5 mM CaCl₂ buffer). Binding was detected using anti-MBP monoclonal antibody and rabbit anti-mouse IgG-HRP conjugate.

Figure 4

Binding of globular head substitution mutants to DC-SIGN. Different concentrations of DC-SIGN tetramer were coated on microtiter wells in carbonate buffer overnight at 4°C. Wells were then incubated with 2.5 µg/well (in CaCl₂) of recombinant globular head wild type and mutant proteins and probed with anti-MBP monoclonal antibody and rabbit anti-mouse IgG-HRP conjugate. Percent binding was calculated for each mutant using binding of the wild-type globular head module as 100%. (A) Binding of ghA, ghA-R162E, and ghA-R162A to DC-SIGN; (B) binding of ghB mutants ghB-L136G, ghB-T175L, ghB-R114Q, ghB-R114A, ghB-R163A, ghB-R163E, ghB-R129E, ghB-R129A, and ghB-H117D to DC-SIGN; (C) binding of ghC mutants ghC-R156E, ghC-L170E, and ghC-H101A to DC-SIGN.

Figure 5

Binding of globular head substitution mutants to DC-SIGNR. Different concentrations of DC-SIGNR tetramer were coated on microtiter wells in carbonate buffer overnight at 4°C and then incubated with 2.5 µg/well of recombinant globular head wild type and mutant proteins and probed with anti-MBP monoclonal antibody and rabbit anti-mouse IgG-HRP conjugate. Percent binding was calculated for each mutant using binding of the wild-type globular head module as 100%. (A) Binding of ghA, ghA-R162E and ghA-R162A to DC-SIGNR; (B) binding of ghB mutants ghB-L136G, ghB-T175L, ghB-R114Q, ghB-R114A, ghB-R163A, ghB-R163E, ghB-R129E, ghB-R129A, and ghB-H117D to DC-SIGNR; (C) binding of ghC mutants ghC-R156E, ghC-L170E, and ghC-H101A to DC-SIGNR.

Figure 6

Competitive inhibition of DC-SIGN: HIV-1 gp120 interaction by globular head modules and gC1qR. (A) ELISA to assess whether gC1qR and ghB directly compete for the same binding site on DC-SIGN: DC-SIGN was coated at 5 µg/well overnight at 4°C. Wells were blocked with 2% BSA in PBS for 2 h at 37°C. gC1qR (5 µg/well) and different concentrations of ghB (5, 2.5, 1.25, 0.625 µg/well) were added in buffer containing 5 mM CaCl₂. Incubation was carried out at 37°C for 1.5 h and 4°C for 1.5 h. Following repeated washes, bound gC1qR was probed using rabbit anti-gC1qR polyclonal antibodies (1:1,000) and Protein A-HRP (1:1,000). Color was developed using o-phenylenediamine dihydrochloride substrate; (B) competition between DC-SIGN tetramer and C1q globular head modules to bind solid-phase gp120. Microtiter wells were coated with 250 ng of gp120. Various concentrations of ghA, ghB, ghC, and C1q and constant 2.5 µg/mL of DC-SIGN were incubated at 37°C for 1 h and then at 4°C for 1 h. The binding of DC-SIGN to gp120 in the presence of globular heads or C1q was detected using rabbit anti-DC antibody (1:500), probed with Protein A HRP (1:5,000). DC-SIGN alone binding to gp120 was used as 100%.

Figure 7

In vitro binding of globular heads modules to DC-SIGN expressed on HEK cells. DC-SIGN-expressing HEK cells (DC-HEK cells) were incubated with recombinant globular head modules (ghA, ghB, ghC) and MBP as control for 30 min at 37°C. DC-HEK cells were fixed with 4% paraformaldehyde, washed and blocked with 5% FCS, and probed with mouse anti MBP antibody to detect the presence of MBP fused globular head modules and rabbit anti DC-SIGN to detect DC-SIGN expressed on the cells. Goat anti-mouse secondary antibody conjugated with Alexa Fluor 568 was used to detect binding of globular heads whereas DC-SIGN expression was visualized using goat anti-rabbit IgG conjugated with Alexa Fluor 488 antibody. Scale bar 20 µm.

Figure 8

HIV transfer assay mediated by DC-SIGN. Cell surface DC-SIGN expressing HEK (DC-HEK) cells were grown in a 12-well plate to form a confluent layer. Different concentrations of proteins were added to the cells and incubated for 2 h for binding. Unbound proteins were removed and cells were challenged with 2.5 ng/mL p24 of HIV-1 (SF-162 strain) for 1 h. Unbound virus was washed off and cells were co-cultured with PHA-activated PBMCs for 24 h. PBMCs were separated from the DC-HEK monolayer and cultured separately for 7 days to determine viral titer of the supernatants collected on days 4 and 7. (A) C1q, ghA, ghB, ghC, and ghABC; (B) gC1qR in presence of C1q, ghA, ghB, ghC, and ghABC. Data represent mean ± SD. P < 0.05 is considered significant. * and # indicate statistical significance in comparison to untreated controls of days 4 and 7, respectively.

Figure 9

Diagrammatic model explaining the possible implications of the tripartite molecular interplay between DC-SIGN, C1q, and gC1qR. (A) By virtue of its ability to bind to DC-SIGN on the cell surface, C1q is likely to inhibit interaction between DC-SIGN and HIV-1 gp120, resulting in the inhibition of viral transfer. (B) On the DC/Monocyte surface, a trimolecular receptor complex is formed between gC1qR, C1q, and DC-SIGN. Although each of these molecules can bind the HIV-1 virus independently, we postulate that it is the binding of the HIV-1 gp-41 to both gC1qR and C1q that initiates the membrane fusion before the final binding of gp120 to DC-SIGN and/or CD4, eventually allowing the internalization of the virus. It is possible that HIV-1 interaction with DC/monocytes causes recruitment of gC1qR to the cell surface, or its secretion, which in turn, can bind to C1q globular heads, thereby neutralizing the protection offered by C1q.