C1q: A Molecular Bridge to Innate and Adaptive Immunity

Like in most fields in science, the complement system is also in a fast gear of self-evolution, with new discoveries of unanticipated pathways and functions capable of cross-talk with other biologic systems. An emerging area that has been gaining much attention recently is the role of locally synthesized complement proteins, which mediate a plethora of cellular functions thereby enhancing either health or disease. Prominent among the locally synthesized proteins is C1q, the first subcomponent of the complement classical pathway, which has been shown to regulate a wide range of immunological and pathological processes in autocrine or paracrine manners. Although the main source of the local synthesis of C1q has been attributed to the potent antigen presenting cells such as macrophages and dendritic cells, where it plays as a powerful molecular sensor of danger and regulator of self-tolerance, the list of the type of cells that locally synthesize C1q has been expanding over the years and now includes various types of proliferating and non-proliferating cells including malignant cells. The aim of the current “Frontiers Topic” is, therefore, to compile a comprehensive list of review and research articles by experts in the field, so that they can be a major source of information for researchers and clinicians alike.This work is supported by a Singapore National University Health System seed fund (R-182-000229-750), a Singapore Ministry of Education Tier 2 grant (MOE2012-T2-2-122), and a Singapore National Medical Research Council Open-funding Individual Research Grant (NMRC/OFIRG/0013/2016)

Isolation, cDNA cloning, and overexpression of a 33-kD cell surface glycoprotein that binds to the globular 'heads' of C1q

This work describes the functional characterization, cDNA cloning, and expression of a novel cell surface protein. This protein designated gC1q-R, was first isolated from Raji cells and was found to bind to the globular 'heads' of C1q molecules, at physiological ionic strength, and also to inhibit complement-mediated lysis of sheep erythrocytes by human serum. The NH 2-terminal amino acid sequence of the first 24 residues of the C1q- binding protein was determined and this information allowed the synthesis of two degenerate polymerase chain reaction primers for use in the preparation of a probe in the screening of a B cell cDNA library. The cDNA isolated, using this probe, was found to encode a pre-pro protein of 282 residues. The NH 2 terminus of the protein isolated from Raji cells started at residue 74 of the predicted pre-pro sequence. The cDNA sequence shows that the purified protein has three potential N-glycosylation residues and is a highly charged, acidic molecule. Hence, its binding to C1q may be primarily but not exclusively due to ionic interactions. The 'mature' protein, corresponding to amino acid residues 74-282 of the predicted pre-pro sequence, was overexpressed in Escherichia coli and was purified to homogeneity. This recombinant protein was also able to inhibit the complement-mediated lysis of sheep erythrocytes by human serum and was shown to be a tetramer by gel filtration in nondissociating conditions. Northern blot and RT-PCR studies showed that the C1q-binding protein is expressed at high levels in Raji and Daudi cell lines, at moderate levels in U937, Molt-4, and HepG2 cell lines, and at a very low level in the HL60 cell line. However, it is not expressed in the K562 cell line. Comparison of gC1q-R NH 2-terminal sequence with that of the receptor for the collagen-like domain of C1q (cC1q-R) showed no similarity. Furthermore, antibodies to gC1q-R or an 18-amino acid residue- long NH 2-terminal synthetic gC1q-R peptide did not cross-react with antibodies to cC1q-R. Anti-gC1q-R immunoblotted a 33-kD Raji cell membrane protein, whereas anti cC1q-R recognized a molecule of ~60 kD. The NH 2- terminal sequence of gC1g-R appears to be displayed extracellularly since anti-gC1g-R peptide reacted with surface molecules on lymphocytes, polymorphonuclear leukocytes, and platelets, as assessed by flow cytometric and confocal laser scanning microscopic analyses. In addition, all or part of the gC1q binding domain may reside within the 24 amino acid stretch of the NH 2-terminal sequence of gC1q-R since the 18 amino acid residue long- synthetic peptide corresponding to this region inhibited serum C1q hemolytic activity. The data presented in this report suggest that there are at least two types of C1q-R which appear to be expressed on the same type of cells and these receptors individually or in concert may contribute to the diversity of C1q-mediated responses.published_or_final_versio

Soluble gC1qR is an autocrine signal that induces B1R expression on endothelial cells

Bradykinin (BK) is one of the most potent vasodilator agonists known and belongs to the kinin family of proinflammatory peptides. BK induces its activity via two G protein-coupled receptors: BK receptor 1 (B1R) and BK receptor 2. Although BK receptor 2 is constitutively expressed on endothelial cells (ECs), B1R is induced by IL-1β. The C1q receptor, receptor for the globular heads of C1q (gC1qR), which plays a role in BK generation, is expressed on activated ECs and is also secreted as soluble gC1qR (sgC1qR). Because sgC1qR can bind to ECs, we hypothesized that it may also serve as an autocrine/paracrine signal for the induction of B1R expression. In this study, we show that gC1qR binds to ECs via a highly conserved domain consisting of residues 174-180, as assessed by solid-phase binding assay and deconvolution fluorescence microscopy. Incubation of ECs (24 h, 37°C) with sgC1qR resulted in enhancement of B1R expression, whereas incubation with gC1qR lacking aa 174-180 and 154-162 had a diminished effect. Binding of sgC1qR to ECs was through surface-bound fibrinogen and was inhibited by anti-fibrinogen. In summary, our data suggest that, at sites of inflammation, sgC1qR can enhance vascular permeability by upregulation of B1R expression through de novo synthesis, as well as rapid translocation of preformed B1R

The human gC1qR/p32 gene, C1qBP. Genomic organization and promoter analysis

gC1qR is an ubiquitously expressed cell protein that interacts with the globular heads of C1q (gC1q) and many other ligands. In this study, the 7.8-kilobase pair (kb) human gC1qPJp32 (C1qBP) gene was cloned and found to consist of 6 exons and 5 introns. Analysis of a 1.3-kb DNA fragment at the 5′-flanking region of this gene revealed the presence of multiple TATA, CCAAT, and Sp1 binding sites. Luciferase reporter assays performed in different human cell lines demonstrated that the reporter gene was ubiquitously driven by this 1.3-kb fragment. Subsequent 5′ and 3′ deletion of this fragment confined promoter elements to within 400 base pairs (bp) upstream of the translational start site. Because the removal of the 8-bp consensus TATATATA at -399 to -406 and CCAAT at -410 to -414 did not significantly affect the transcription efficiency of the promoter, GC-rich sequences between this TATA box and the translation start site may be very important for the promoter activity of the C1qBP gene. One of seven GC-rich sequences in this region binds specifically to PANC-1 nuclear extracts, and the transcription factor Sp1 was shown to bind to this GC-rich sequence by the supershift assay. Primer extension analysis mapped three major transcription start regions. The farthest transcription start site is 49 bp upstream of the ATG translation initiation codon and is in close proximity of the specific SP1 binding site.postprin

Magnitude and Causes of Maternal Deaths at Health Facilities in Eritrea in 2007.

Objective: To measure the level of maternal mortality in health facilities as well as the magnitude and proportion of obstetric complications in health facilities in Eritrea. Methods: The study was a cross-sectional survey of all hospitals and health centers in Eritrea and a random sample of around a third of health stations. Medical records of all patients who encountered obstetric complications in 2007 were reviewed. Findings: The main causes of obstetric complications among hospital admissions in 2007 were abortion complications (45.6%), obstructed/prolonged labor (18.4%), abnormal fetal presentation (10.3%) and preeclampsia/ eclampsia (7.7%). The number of maternal deaths at facilities was relatively small. Out of the 6,315 patients who were admitted for obstetric complications in 2007, 41 were classified as maternal deaths. The leading causes of maternal deaths included pre-eclampsia/ eclampsia in 22.0 percent of the cases, abortion complications in 19.5 percent of the cases and postpartum sepsis in 17.1 percent of the cases and post-partum hemorrhage in 14.6 percent of cases. The case-fatality rate for obstetric complications was low at 0.75 percent. The majority of maternal deaths (65 percent) occurred in the post-partum period, while 32 percent occurred during the ante-partum period, and 3 percent during intra-partum or during labor or delivery Conclusion: Over all it can be concluded that the Eritrean health system is performing well with the current demand for services. The issue of abortion requires special attention because it is the leading obstetric complication, which accounts for 46 percent of maternal complications and is responsible for one fifth of maternal deaths. Although the case fatality rate of all obstetric complications combined is not high (0.75 percent), the cause specific case fatality rates for the leading causes of maternal mortality was high Keywords: Maternal mortality, obstetric complications, abortion, case fatality rat

Is the a-chain the engine that drives the diversity of C1q functions? Revisiting its unique structure

The immunopathological functions associated with human C1q are still growing in terms of novelty, diversity, and pathologic relevance. It is, therefore, not surprising that C1q is being recognized as an important molecular bridge between innate and adaptive immunity. The secret of this functional diversity, in turn, resides in the elegant but complex structure of the C1q molecule, which is assembled from three distinct gene products: A, B, and C, each of which has evolved from a separate and unique ancestral gene template. The C1q molecule is made up of 6A, 6B, and 6C polypeptide chains, which are held together through strong covalent and non-covalent bonds to form the 18-chain, bouquet-of-flower-like protein that we know today. The assembled C1q protein displays at least two distinct structural and functional regions: the collagen-like region (cC1q) and the globular head region (gC1q), each being capable of driving a diverse range of ligand- or receptor-mediated biological functions. What is most intriguing, however, is the observation that most of the functions appear to be predominantly driven by the A-chain of the molecule, which begs the question: what are the evolutionary modifications or rearrangements that singularly shaped the primordial A-chain gene to become a pluripotent and versatile component of the intact C1q molecule? Here, we revisit and discuss some of the known unique structural and functional features of the A-chain, which may have contributed to its versatility

Decidual endothelial cells express surface-bound C1q as a molecular bridge between endovascular trophoblast and decidual endothelium

This study was prompted by the observation that decidual endothelial cells (DECs), unlike endothelial cells (ECs) of blood vessels in normal skin, kidney glomeruli and brain, express surface-bound C1q in physiologic pregnancy. This finding was unexpected, because deposits of C1q are usually observed in pathologic conditions and are associated with complement activation. In the case of DECs, we failed to detect immunoglobulins and C4 co-localized with C1q on the cell surface. Surprisingly, DECs expressed mRNA for the three chains of C1q and secreted detectable level of this component in serum-free medium. The ability to synthesize C1q is acquired by DECs during pregnancy and is not shared by ECs obtained from endometrium and from other sources. Cell-associated C1q has a molecular weight similar to that of secreted C1q and is released from DECs following treatment with heparinase or incubation at low pH. This suggests that C1q binds to DECs and it is not constitutively expressed on the cell surface. C1q is localized at contact sites between endovascular trophoblast and DECs and acts as an intercellular molecular bridge because adhesion of endovascular trophoblast to DECs was inhibited by antibodies to C1q and to a receptor recognizing its globular portion expressed on trophoblast. © 2008 Elsevier Ltd. All rights reserved

Cell Surface Expression and Function of the Macromolecular C1 Complex on the Surface of Human Monocytes

The synthesis of the subunits of the C1 complex (C1q, C1s, C1r), and its regulator C1 inhibitor (C1-Inh) by human monocytes has been previously established. However, surface expression of these molecules by monocytes has not been shown. Using flow cytometry and antigen-capture enzyme-linked immunosorbent assay, we show here for the first time that, in addition to C1q, peripheral blood monocytes, and the monocyte-derived U937 cells express C1s and C1r, as well as Factor B and C1-Inh on their surface. C1s and C1r immunoprecipitated with C1q, suggesting that at least some of the C1q on these cells is part of the C1 complex. Furthermore, the C1 complex on U937 cells was able to trigger complement activation via the classical pathway. The presence of C1-Inh may ensure that an unwarranted autoactivation of the C1 complex does not take place. Since C1-Inh closely monitors the activation of the C1 complex in a sterile or infectious inflammatory environment, further elucidation of the role of C1 complex is crucial to dissect its function in monocyte, dendritic cell, and T cell activities, and its implications in host defense and tolerance

Serum complement activation on heterologous platelets is associated with arterial thrombosis in patients with systemic lupus erythematosus and antiphospholipid antibodies

Complement plays a major role in inflammation and thrombosis associated with systemic lupus erythematosus (SLE) and the antiphospholipid syndrome (APS). A cross-sectional retrospective analysis was performed to evaluate serum complement fixation on platelets and thrombotic incidence using banked sera and clinical data from patients with SLE (n = 91), SLE with antiphospholipid antibodies (aPL) or APS (n = 78) and primary aPL (n = 57) or APS (n = 96). In-situ complement fixation was measured as C1q and C4d deposition on heterologous platelets using an enzyme-linked immunosorbent assay approach. Platelet activation by patient serum in the fluid phase was assessed via serotonin release assay. Enhanced in-situ complement fixation was associated with the presence of IgG aPL and IgG anti-β2 glycoprotein 1 antibodies (P < 0.05) and increased platelet activation (P < 0.005). Moreover, enhanced complement fixation, especially C4d deposition on heterologous platelets, was positively associated with arterial thrombotic events in patients with SLE and aPL (P = 0.039). Sera from patients with aPL possess an enhanced capacity for in-situ complement fixation on platelets. This capacity may influence arterial thrombosis risk in patients with SLE