Analysis of C3 gene variants in patients with idiopathic recurrent spontaneous pregnancy loss

Miscarriage is the most common complication of pregnancy. Approximately 1% of couples trying to conceive will experience recurrent miscarriages, defined as three or more consecutive pregnancy losses and many of these cases remain idiopathic. Complement is implicated both in the physiology and pathology of pregnancy. Therefore, we hypothesized that alterations in the C3 gene could potentially predispose to this disorder. We performed full Sanger sequencing of all exons of C3, in 192 childless women, with at least two miscarriages and without any known risk factors. All exons carrying non-synonymous alterations found in the patients were then sequenced in a control group of 192 women. None of the identified alterations were significantly associated with the disorder. Thirteen identified non-synonymous alterations (R102G, K155Q, L302P, P314L, Y325H, V326A, S327P, V330I, K633R, R735W, R1591G, G1606D, and S1619R) were expressed recombinantly, upon which C3 expression and secretion were determined. The L302P and S327P were not secreted from the cells, likely due to misfolding and intracellular degradation. Y325H, V326A, V3301I, R1591G, and G1606D yielded approximately half C3 concentration in the cell media compared with wild type (WT). We analyzed the hemolytic activity of the secreted C3 variants by reconstituting C3-depleted serum. In this assay, R1591G had impaired hemolytic activity while majority of remaining mutants instead had increased activity. R1591G also yielded more factor B activation in solution compared with WT. R1591G and G1606D showed impaired degradation by factor I, irrespectively if factor H, CD46, or C4b-binding protein were used as cofactors. These two C3 mutants showed impaired binding of the cofactors and/or factor I. Taken together, several alterations in C3 were identified and some of these affected the secretion and/or the function of the protein, which might contribute to the disorder but the degree of association must be evaluated in larger cohorts

Multiple domains of MASP-2, an initiating complement protease, are required for interaction with its substrate C4

The complement system is fundamental to both innate and adaptive immunity and can be initiated via the classical, lectin or alternative pathways. Cleavage of C4 by MASP-2, the initiating protease of the lectin pathway, is a crucial event in the activation of this pathway, preceding the eventual formation of the C3 convertase (C4bC2a) complex on the pathogen surface. Interactions required for the cleavage of C4 by MASP-2 are likely to be facilitated by the initial binding of C4 to an exosite on the protease. We have shown that both proteolytically active and catalytically inactive CCP1-CCP2-serine protease (CCP1-CCP2-SP) forms bind C4 with similar affinity. Interestingly, proteins containing the CCP1-CCP2 domains or the SP domain alone bound C4 with much lower affinity than the CCP1-CCP2-SP protein, suggesting that the CCP domains cooperate positively with the active site to mediate efficient binding and cleavage of C4. In addition, mutation of residue K342 to alanine in the CCP1 domain abolished binding to both C4 and C4b in its CCP1-CCP2 form, suggesting a key electrostatic role for this amino acid. The presented data indicates that all of the domains are required in order to mediate high affinity interaction with C4. (C) 2011 Elsevier Ltd. All rights reserved

Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells-A Potential Involvement in Regulation of Gene Transcription

Beside its classical role as a serum effector system of innate immunity, evidence is accumulating that complement has an intracellular repertoire of components that provides not only immune defense, but also functions to maintain cellular homeostasis. While complement proteins, mainly the central component C3, have been detected in B cells, their exact function and source remain largely unexplored. In this study, we investigated the expression and origin of intracellular C3 in human B cells together with its role in B cell homeostasis. Our data provide evidence that endogenous expression of C3 is very low in human B cells and, in accordance with the recent publication, the main origin of intracellular C3 is the serum. Interestingly, we found that both serum-derived and purified C3 are able to enter the nucleus of viable B cells, suggesting its potential involvement in regulation of gene transcription. ELISA, gel shift assay, confocal microscopy, and chromatin immunoprecipitation proved that C3 and C3a strongly bind to nuclear DNA, and among the interacting genes there are key factors of lymphocyte development and differentiation. The strong interaction of C3 with histone proteins and its potential ability to induce chromatin rearrangement suggest that C3/C3a might regulate DNA transcription via chromatin remodeling. Our data reveal a novel, hitherto undescribed role of C3 in immune cell homeostasis, which further extends the repertoire how complement links innate and adaptive immunity and regulates basic processes of the cells

Alternative translation and retrotranslocation of cytosolic C3 that detects cytoinvasive bacteria

Complement C3 was originally regarded as a serum effector protein, although recent data has emerged suggesting that intracellular C3 can also regulate basic cellular processes. Despite the growing interest in intracellular C3 functions, the mechanism behind its generation has not been demonstrated. In this study we show that C3 can be expressed from an alternative translational start site, resulting in C3 lacking the signal peptide, which is therefore translated in the cytosol. In contrast to the secreted form, alternatively translated cytosolic C3 is not glycosylated, is present mainly in a reduced state, and is turned over by the ubiquitin–proteasome system. C3 can also be retrotranslocated from the endoplasmic reticulum into the cytosol, structurally resembling secreted C3. Finally, we demonstrate that intracellular cytosolic C3 can opsonize invasive Staphylococcus aureus within epithelial cell, slowing vacuolar escape as well as impacting bacterial survival on subsequent exposure to phagocytes. Our work therefore reveals the existence and origin of intracellular, cytosolic C3, and demonstrates functions for cytosolic C3 in intracellular detection of cytoinvasive pathogens

Identification of a Catalytic Exosite for Complement Component C4 on the Serine Protease Domain of C1s

The classical pathway of complement is crucial to the immune system, but it also contributes to inflammatory diseases when dys-regulated. Binding of the C1 complex to ligands activates the pathway by inducing autoactivation of associated C1r, after which C1r activates C1s. C1s cleaves complement component C4 and then C2 to cause full activation of the system. The interaction between C1s and C4 involves active site and exosite-mediated events, but the molecular details are unknown. In this study, we identified four positively charged amino acids on the serine protease domain that appear to form a catalytic exosite that is required for efficient cleavage of C4. These residues are coincidentally involved in coordinating a sulfate ion in the crystal structure of the protease. Together with other evidence, this pointed to the involvement of sulfate ions in the interaction with the C4 substrate, and we showed that the protease interacts with a peptide from C4 containing three sulfotyrosine residues. We present a molecular model for the interaction between C1s and C4 that provides support for the above data and poses questions for future research into this aspect of complement activation. The Journal of Immunology, 2012, 189: 2365-2373

Neisseria gonorrhoeae co-opts C4b-binding protein to enhance complement-independent survival from neutrophils.

Neisseria gonorrhoeae (Gc) is a human-specific pathogen that causes the sexually transmitted infection gonorrhea. Gc survives in neutrophil-rich gonorrheal secretions, and recovered bacteria predominantly express phase-variable, surface-expressed opacity-associated (Opa) proteins (Opa+). However, expression of Opa proteins like OpaD decreases Gc survival when exposed to human neutrophils ex vivo. Here, we made the unexpected observation that incubation with normal human serum, which is found in inflamed mucosal secretions, enhances survival of Opa+ Gc from primary human neutrophils. We directly linked this phenomenon to a novel complement-independent function for C4b-binding protein (C4BP). When bound to the bacteria, C4BP was necessary and sufficient to suppress Gc-induced neutrophil reactive oxygen species production and prevent neutrophil phagocytosis of Opa+ Gc. This research identifies for the first time a complement-independent role for C4BP in enhancing the survival of a pathogenic bacterium from phagocytes, thereby revealing how Gc exploits inflammatory conditions to persist at human mucosal surfaces

The structural basis for complement inhibition by gigastasin, a protease inhibitor from the giant Amazon leech

Complement is crucial to the immune response, but dysregulation of the system causes inflammatory disease. Complement is activated by three pathways: classical, lectin, and alternative. The classical and lectin pathways are initiated by the C1r/C1s (classical) and MASP-1/MASP-2 (lectin) proteases. Given the role of complement in disease, there is a requirement for inhibitors to control the initiating proteases. In this article, we show that a novel inhibitor, gigastasin, from the giant Amazon leech, potently inhibits C1s and MASP-2, whereas it is also a good inhibitor of MASP-1. Gigastasin is a poor inhibitor of C1r. The inhibitor blocks the active sites of C1s and MASP-2, as well as the anion-binding exosites of the enzymes via sulfotyrosine residues. Complement deposition assays revealed that gigastasin is an effective inhibitor of complement activation in vivo, especially for activation via the lectin pathway. These data suggest that the cumulative effects of inhibiting both MASP-2 and MASP-1 have a greater effect on the lectin pathway than the more potent inhibition of only C1s of the classical pathway