We addressed the issue of C1q autoantigenicity by studying the structural features of the autoepitopes recognized by the polyclonal anti-C1q antibodies present in Lupus Nephritis (LN) sera. We used six fractions of anti-C1q as antigens and selected anti-idiotypic scFv antibodies from the phage library “Griffin.1”. The monoclonal scFv A1 was the most potent inhibitor of the recognition of C1q and its fragments ghA, ghB and ghC, comprising the globular domain gC1q, by the lupus autoantibodies. It was sequenced and in silico folded by molecular dynamics into a 3D structure. The generated 3D model of A1 elucidated CDR similarity to the apical region of gC1q, thus mapping indirectly for the first time a globular autoepitope of C1q. The V_H CDR2 of A1 mimicked the ghA sequence GSEAD suggested as a cross-epitope between anti-DNA and anti-C1q antibodies. Other potential inhibitors of the recognition of C1q by the LN autoantibodies among the selected recombinant antibodies were the monoclonal scFv F6, F9 and A12.

In the present study we report a novel interaction of human C1q, a primary activator of the Complement system, with human Galectin-3 (Gal-3). We investigated the potential recognition between C1q and Gal-3 on a solid hydrophobic surface by ELISA, by fluorescence spectroscopy, molecular docking and molecular dynamics (MD). The data showed that C1q and Gal-3 had a pronounced affinity for protein-protein interaction and supramolecular binding, locating the binding sites within the globular domains of C1q (gC1q) and on the backside of the carbohydrate recognition domain (CRD) of Gal-3. Fluorescence spectroscopy gave quantitative assessment of the recognition with K_D value of 0.04 μM. MD analysis showed that when the active AAs of the two proteins interacted, electrostatic attraction, aided by a large number of hydrogen bonds, was dominant for the stabilization of the complex. When the contact of C1q and Gal-3 was not limited to active residues, the complex between them was stabilized mainly by Van der Waals interactions and smaller in number but stronger hydrogen bonds. This is the first report analyzing the interaction of Gal-3 with C1q, which could open the way to new applications of this protein-protein complex.

Systemic lupus erythematosus (SLE) is an autoimmune disease that affects multiple organs or organ systems. SLE and one of its most common complications, lupus nephritis (LN), are characterized by chronic inflammation due to the secretion of pro-inflammatory molecules and a tissue deposition of immune complexes formed by autoantibodies and their target antigens, such as double-stranded DNA, other nuclear antigens, and complement proteins. Increased activity of phospholipase A2 enzymes (PLA₂) like calcium-independent PLA₂ (iPLA₂), secretory PLA₂ (sPLA₂), and cellular PLA₂ (cPLA₂) has a crucial role in the maintenance of the inflammatory process during the course of SLE. This study aimed to evaluate PLA₂ activity and identify the dominant type of PLA₂ enzymes in a cohort of Bulgarian patients with SLE and LN. Additionally, we investigated whether the increased PLA₂ activity was correlated with the presence of autoantibodies specific to the complement proteins C3 and factor H.

C1q, the key component of the classical pathway of the Complement system, is known for its vast functional activity including clearance of apoptotic cells. The binding of C1q to apoptotic blebs occurs via an interaction with the phosphatidylserine externalized on the cell surface. In this study, we characterized the interaction between C1q and phosphatidylserine, with emphasis on the structure of the phosphatidylserine-binding site within the globular domains of C1q and the nature of binding of C1q with phosphatidylserine, using both in vitro and in silico methods. We established that all three globular fragments, forming one C1q globular domain, bound phosphatidylserine with the leading role of the phosphatidylserine-binding site pertaining to the A chain of the globular fragment of C1q. We also determined the closest-contact amino acids of C1q participating in the interaction with phosphatidylserine. An important role is suggested for the glycosylated Asn124 residue in the A chain of the globular fragment.

C1q is a primary activator of Complement system and has a physiological role in maintaining the homeostasis. C1q is now acknowledged as a protein of complex functionality, capable of forming multiple binding sites for ligands of various nature by its C-terminal globular domains (gC1q), composed of the globular fragments of the three types of chains, ghA, ghB and ghC. The conformation of the globular fragments is critical for the formation of different binding sites underlying the functional activity of C1q. We studied the conformational flexibility of C1q and its globular fragments ghA, ghB and ghC in different routinely used buffers by the highly sensitive fluorescent spectroscopy. We compared the conformational state of C1q in PBS, pH 7.2; TBS, pH 7.2; carbonate buffer, pH 9.6; NaHCO3, pH 7.2 and HEPES, pH 7.2. HEPES buffer appeared most favourable solute in terms of conformational stability of C1q. The ghB of all three globular fragments followed most closely the native protein.

Background and Aims: C1q is an autoantigen in different autoimmune disorders, Systemic Lupus Erythematosus (SLE) and Lupus Nephritis (LN) among them. The two functional domains of C1q, the collagen-like region (CLR) and the globular head region (gC1q), are frequently recognized by autoantibodies in SLE and LN when C1q is immobilized. We studied whether autoantibodies to C1q in SLE and LN patients recognized C1q as a soluble autoantigen and whether the act of immobilization was a prerequisite for the recognition of C1q autoepitopes localized on gC1q domains. Methods: The interaction of soluble C1q and its globular fragments ghA, ghB, and ghC with immobilized IgG autoantibodies (and vice versa) from sera of 48 patients with SLE and LN was studied with ELISA. Data were compared using Spearman correlation coefficient. Fluorescence spectroscopy was used to study the interaction between C1q and LN IgG autoantibodies both presented in solution. Results: We found that anti-C1q autoantibodies from SLE and LN patients specifically bound C1q and gC1q fragments, ghA, ghB, and ghC, both as immobilized and soluble antigens. Correlation analysis indicated a negative correlation between the levels of autoantibodies against immobilized and soluble C1q and immobilized and soluble gC1q fragments which indicates different epitopes when these proteins were recognized as autoantigens in soluble and immobilized conformations. Conclusions: Serum C1q in patients with SLE is a target molecule for binding from anti-C1q autoantibodies. The gC1q region undergoes a conformational change in an immobilized and a soluble form, thus exposing different epitope-binding sites.