The first step in the activation of the classical complement pathway by immune complexes involves the binding of the globular domain (gC1q) of C1q to the Fc regions of aggregated IgG or IgM. Each gC1q domain is a heterotrimer of the C-terminal halves of one A (ghA), one B (ghB), and one C (ghC) chain. Our recent studies have suggested a modular organization of gC1q, consistent with the view that ghA, ghB, and ghC are functionally autonomous modules and have distinct and differential ligand-binding properties. Although C1q binding sites on IgG have been previously identified, the complementary interacting sites on the gC1q domain have not been precisely defined. The availability of the recombinant constructs expressing ghA, ghB, and ghC has allowed us, for the first time, to engineer single-residue substitution mutations and identify residues on the gC1q domain, which are involved in the interaction between C1q and IgG. Because C1q is a charge pattern recognition molecule, we have sequentially targeted arginine and histidine residues in each chain. Consistent with previous chemical modification studies and the recent crystal structure of gC1q, our results support a central role for arginine and histidine residues, especially Arg114 and Arg129 of the ghB module, in the C1q-IgG interaction.

The interaction of C1q, the first subcomponent of the human complement, with its immunoglobulin ligands from immune complexes is the crucial step in the activation of the classical complement pathway. Thus the mechanism of these interaction and the factors, which influence them, are from high interest. In the present study the effect of immobilization of the interacting proteins on a solid support in ELISA was investigated. The obtained results lead us to the conclusion, that the immobilization process may have a significant influence on the binding activity of tested proteins, especially when hydrophobic interactions are involved.

A new and improved ELISA method for immune complex detection [scFv9(G)-C1qELISA] was developed. The assay is based on the C1q-solid phase binding test. C1q was immobilized on microtiter ELISA plates, coated with the single-chain antibody scFv9(G), specific to the collagen-like region of C1q. The obtained results indicated a significantly reduced nonspecific background, when C1q was immobilized via the scFv9(G)-capture molecule. The comparative analysis between the two wildly used methods for IC detection (C1q-SP-RIA and QUIDEL CIC-C1q ELISA) and the new improved Fv9(G)-C1q ELISA shows a good agreement. The method is simple, reproducible and may be a good alternative for the routine C1q-SP-RIA.

C1q, the recognition subunit of the classical complement pathway, is a multifunctional protein involved in a great number of immunological processes. The molecular basis of the functional diversity of C1q is its ability to recognize a broad range of ligands via the gC1q region. One of the most important ligands of C1q is the human C-reactive protein (CRP), a major acute phase protein, which plays a crucial role in keeping the tolerance to self-structures. The aim of the present study is to evaluate the contribution of the C1q globular head fragments rghA, rghB and rghC to the recognition of CRP. The obtained results indicate that all three C1q-chains are involved in the CRP-binding, and that rghB is the most important one. The pH-dependence of the interactions between rghA/B/C and CRP demonstrates that different amino acid residues from the three chains take part in the formation of the CRP-binding site. The analysis of the CRP-binding activities of biotinylated and nonbiotinylated rghB indicates that Tyr residues from the ghB are the most likely to play a crucial role in the interaction with CRP and that probably only one Lys residue from the rghB is involved as well.