Heme Interacts with C1q and Inhibits the Classical Complement Pathway

Lubka T Roumenina,Maria Radanova,Boris P Atanasov,Krastio T Popov,Srinivas V Kaveri,Sébastien Lacroix-Desmazes,Véronique Frémeaux-Bacchi,Jordan D Dimitrov

doi:10.1074/jbc.m110.206136

Abstract

C1q is the recognition subunit of the first component of the classical complement pathway. It participates in clearance of immune complexes and apoptotic cells as well as in defense against pathogens. Inappropriate activation of the complement contributes to cellular and tissue damage in different pathologies, urging the need for the development of therapeutic agents that are able to inhibit the complement system. In this study, we report heme as an inhibitor of C1q. Exposure of C1q to heme significantly reduced the activation of the classical complement pathway, mediated by C-reactive protein (CRP) and IgG. Interaction analyses revealed that heme reduces the binding of C1q to CRP and IgG. Furthermore, we demonstrated that the inhibition of C1q interactions results from a direct binding of heme to C1q. Formation of complex of heme with C1q caused changes in the mechanism of recognition of IgG and CRP. Taken together, our data suggest that heme is a natural negative regulator of the classical complement pathway at the level of C1q. Heme may play a role at sites of excessive tissue damage and hemolysis where large amounts of free heme are released.

Highlights

We identify heme as an endogenous negative regulator of the classical complement pathway activation that acts at the level of C1q and may play a role at sites of excessive tissue damage and hemolysis
C1q was allowed to interact with ligand-bound C-reactive protein (CRP) (PnC-CRP as described in Ref. 36) or IgG-containing immune complexes
We demonstrated that heme is able to inhibit IgG- and CRP-mediated complement activation by direct binding to C1q

Summary

EXPERIMENTAL PROCEDURES

Materials—The oxidized form of heme (ferriprotoporphyrin IX chloride) was obtained from Fluka (Taufkirchen, Germany), hematoporphyrin IX was obtained from Sigma-Aldrich. Assessment of Classical Complement Pathway Activation in Presence of Heme—The level of complement activation in presence of C1q, exposed to increased concentrations of hemin was evaluated by C3 deposition ELISA, after addition of C1q-depleted serum. Pooled human IgG at 0.5 mg/ml was applied on the tetanus toxoid-coated plate to form immune complexes. The samples were diluted with washing buffer to a final C1q concentration of 1 nM (C1q molecular mass, 460 kDa) and applied at 50 ␮l/well to the microtiter plates. After incubation for 1 h at 37 °C, the plates were washed and either developed with anti-C1q-HRP (Abcam), or 50 ␮l of C1q-depleted serum (Quidel) diluted 1/400 with the washing buffer was added. The optimal CRP, IgG, C1q concentration, and C1q-depleted serum dilution were determined prior running the experiment.

Fluorescence Spectroscopy

RESULTS

DISCUSSION