Mutational Analyses of the Recombinant Globular Regions of Human C1q A, B, and C Chains Suggest an Essential Role for Arginine and Histidine Residues in the C1q-IgG Interaction

Abstract

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.

Interaction of C1q with IgG1, C-reactive Protein and Pentraxin 3: Mutational Studies Using Recombinant Globular Head Modules of Human C1q A, B, and C Chains

“…It is also not clear how many binding sites exist on gC1q, whether they overlap, and whether conformational changes are required for specific binding. The availability of the recombinant forms of ghA, ghB, and ghC have allowed us to mutate a number of residues that have been considered important for C1q-target interactions by a variety of approaches, including chemical modification and mutational studies (10,11), molecular modeling (7), bioinformatics (4), and in silico theoretical calculations of electric moment vectors (12). Thus, we examined their interactions with three well-known and physiologically relevant targets of C1q: IgG1, CRP, and PTX3 (4,12,16,18,20,21).…”

“…Another three mutants were also engineered that included the substitution of Arg B114 to glutamine and of His B117 to either alanine or aspartate (10). Four other mutants (Tyr B175 Leu, Lys B136 Glu, His C101 Ala, and Lys C170 Glu) were generated to study the role of ghB and ghC in target binding in more detail.…”

“…The generation of mutants Arg A162 Ala/Glu, Arg B114 Gln/Glu, His B117 Ala/Asp, Arg B129 Ala/ Glu, Lys B136 Glu, Arg B163 Ala/Glu, Tyr B175 Leu, His C101 Ala, Arg C156 Ala/Glu, and Lys C170 Glu has already been published (10). Additional new mutants were generated via point mutations within the DNA sequences by site-directed mutagenesis using the overlapping PCR approach (17).…”

“…Chemical modification, mutational analysis, and molecular modeling approaches have been used to dissect the complementary IgG binding sites on the gC1q domain (7,10,11,19). Studies using point mutants of ghA, ghB, and ghC and their interactions with heat-aggregated IgG have highlighted that the ghB module has a dominant role in the gC1q-IgG interaction: Arg B114 being the key residue, and Arg B129 , Arg B163 , and His B117 having subsidiary roles in the C1q-IgG interaction (10).…”

“…Our selection of mutants was based on four major premises. (i) Recently, a number of amino acid residues (Arg A162 , Arg B114 , His B117 , Arg B129 , Arg B163 , and Arg C156 ) on gC1q have been identified as important for IgG binding via site-directed mutagenesis of ghA, ghB, and ghC (10). The importance of these residues have been previously shown by chemical modification studies (11).…”