Interaction of human C1q with IgG and IgM: revisited.

The first step of activation of the classical complement pathway involves the binding of the globular C1q domain (gC1q) to the antigen-bound IgG or IgM. To improve our understanding of the mechanism of interaction of gC1q with IgG and IgM, we compared the immunoglobulin binding properties of single-residue mutants of individual globular modules of A and C chains. We found that Lys(A200) and Lys(C170) are significant for binding with both immunoglobulins. In addition, two C1q-specific scFv antibodies known as potent inhibitors of C1q-IgG and -IgM interactions were used in the epitope mapping analysis. A set of important residues, which participate in the C1q epitopes for scFv, were identified: Lys(C170) for the scFv3(V) epitope and Arg(B108) and Arg(B109) for the scFv10(V) epitope. The ability of scFv3(V) and scFv10(V) to bind preformed C1q-IgG or C1q-IgM complexes differed: scFv3(V) retained its ability to bind C1q, while scFv10(V) lost it. Given the different locations of the epitopes and the varying abilities of both antibodies to bind C1q-IgG and C1q-IgM complexes, we found that residues from the apical surface of C1q [where the scFv3(V) epitope was located] were involved in the initial recognition of IgG and IgM, while Arg(B108) and Arg(B109) are able to interact during the initial recognition as well as during the final binding of immunoglobulins. The reported results provide the first experimental evidence supporting the notion that apical and equatorial surfaces of gC1q have consecutive involvement following the gC1q reorientation during the interaction with specific C1q ligands.

Existence of different but overlapping IgG- and IgM-binding sites on the globular domain of human C1q.

C1q is the first subcomponent of the classical complement pathway that binds antigen-bound IgG or IgM and initiates complement activation via association of serine proteases C1r and C1s. The globular domain of C1q (gC1q), which is the ligand-recognition domain, is a heterotrimeric structure composed of the C-terminal regions of A (ghA), B (ghB), and C (ghC) chains. The expression and functional characterization of ghA, ghB, and ghC modules have revealed that each chain has some structural and functional autonomy. Although a number of studies have tried to identify IgG-binding sites on the gC1q domain, no such attempt has been made to localize IgM-binding site. On the basis of the information available via the gC1q crystal structure, molecular modeling, mutational studies, and bioinformatics, we have generated a series of substitution mutants of ghA, ghB, and ghC and examined their interactions with IgM. The comparative analysis of IgM- and IgG-binding abilities of the mutants suggests that the IgG- and IgM-binding sites within the gC1q domain are different but may overlap. Whereas Arg(B108), Arg (B109), and Tyr(B175) mainly constitute the IgM-binding site, the residues Arg(B114), Arg(B129), Arg(B163), and His(B117) that have been shown to be central to IgG binding are not important for the C1q-IgM interaction. Given the location of Arg(B108), Arg (B109), and Tyr(B175) in the gC1q crystal structure, it is likely that C1q interacts with IgM via the top of the gC1q domain.