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In 1963, Lepow and colleagues resolved C1, the first component of the classical pathway, into three components, which they named C1q, C1r, and C1s. All three of these components were demonstrated to be involved in causing hemolysis in vitro. For over 30 years after that seminal discovery, the primary function attributed to C1q was as part of the C1 complex that initiated the classical pathway of the complement cascade. Then, a series of papers reported that isolated C1q could bind to apoptotic cells and facilitate their clearance by macrophages. Since then, rheumatologists have recognized that C1q is an important pattern recognition receptor (PRR) that diverts autoantigen containing extracellular vesicles from immune recognition. This critical function of C1q as a regulator of immune recognition has been largely overlooked in transplantation. Now that extracellular vesicles released from transplants have been identified as a major agent of immune recognition, it is logical to consider the potential impact of C1q on modulating the delivery of allogeneic extracellular vesicles to antigen presenting cells. This concept has clinical implications in the possible use of C1q or a derivative as a biological therapeutic to down-modulate immune responses to transplants.

Autoantibodies against the complement component C1q (anti-C1q) are among the main biomarkers in lupus nephritis (LN) known to contribute to renal injury. C1q, the recognition sub component of the complement classical pathway, forms a heterotetrameric complex with C1r and C1s, and can also associate a central complement regulator and C1 Inhibitor (C1-Inh). However, the frequency and the pathogenic relevance of anti-C1r, anti-C1s and anti-C1-Inh autoantibodies remain poorly studied in LN. In this paper, we screened for anti-C1q, anti-C1r, anti-C1s and anti-C1-Inh autoantibodies and evaluated their association with disease activity and severity in 74 LN patients followed up for 5 years with a total of 266 plasma samples collected. The presence of anti-C1q, anti-C1r, anti-C1s and anti-C1-Inh was assessed by ELISA. IgG was purified by Protein G from antigen-positive plasma and their binding to purified C1q, C1r and C1s was examined by surface plasmon resonance (SPR). The abilities of anti-C1q, anti-C1r and anti-C1s binding IgG on C1 complex formation were analyzed by ELISA. The screening of LN patients’ plasma revealed 14.9% anti-C1q positivity; only 4.2%, 6.9% and 0% were found to be positive for anti-C1r, anti-C1s and anti-C1-Inh, respectively. Significant correlations were found between anti-C1q and anti-dsDNA, and anti-nuclear antibodies, C3 and C4, respectively. High levels of anti-C1q antibodies were significantly associated with renal histologic lesions and correlated with histological activity index. Patients with the most severe disease (A class according to BILAG Renal score) had higher levels of anti-C1q antibodies. Anti-C1r and anti-C1s antibodies did not correlate with the clinical characteristics of the LN patients, did not interfere with the C1 complex formation, and were not measurable via SPR. In conclusion, the presence of anti-C1q, but not anti-C1s or anti-C1r, autoantibodies contribute to the autoimmune pathology and the severity of LN. Bulgarian National Science Fund, Competition for financial support for bilateral projects—2016—Bulgaria-France, Bulgarian Ministry of Education and Science under Grant DNTS/France 01/11, 9 May 2017).

The molecules of the complement system connect the effectors of innate and adaptive immunity and play critical roles in maintaining homeostasis. Among them, the C1 complex, composed of C1q, C1r, and C1s (C1qr2s2), is the initiator of the classical complement activation pathway. While deficiency of C1s is associated with early-onset systemic lupus erythematosus and increased susceptibility to bacteria infections, the gain-of- function variants of C1r and C1s may lead to periodontal Ehlers Danlos syndrome. As C1s is activated under various pathological conditions and associated with inflammation, autoimmunity, and cancer development, it is becoming an informative biomarker for the diagnosis and treatment of a variety of diseases. Thus, more sensitive and convenient methods for assessing the level as well as activity of C1s in clinic samples are highly desirable. Meanwhile, a number of small molecules, peptides, and monoclonal antibodies targeting C1s have been developed. Some of them are being evaluated in clinical trials and one of the antibodies has been approved by US FDA for the treatment of cold agglutinin disease, an autoimmune hemolytic anemia. In this review, we will summarize the biological properties of C1s, its association with development and diagnosis of diseases, and recent progress in developing drugs targeting C1s. These progress illustrate that the C1s molecule is an effective biomarker and promising drug target.

Periodontal Ehlers-Danlos syndrome (pEDS) is a rare condition caused by pathogenic variants in the C1R and C1S genes, encoding subunits C1r and C1s of the first component of the classical complement pathway. It is characterized by early-onset periodontitis with premature tooth loss, pretibial hyperpigmentation and skin fragility. Rare arterial complications have been reported, but venous insufficiency is rarely described. Here we report thirteen novel patients carrying heterozygous pathogenic variants in C1R and C1S including three novel C1S variants (c.962G > C, c.961 T > G and c.961 T > A). In addition to the pEDS phenotype, three patients and one relative displayed widespread venous insufficiency leading to persistent varicose leg ulcers. One patient suffered an intracranial aneurysm with familial vascular complications including thoracic and abdominal aortic aneurysm and dissection and intracranial aneurysm rupture. This work confirms that vascular complications can occur, although they are not frequent, which leads us to propose to carry out a first complete non-invasive vascular evaluation at the time of the diagnosis in pEDS patients. However, larger case series are needed to improve our understanding of the link between complement pathway activation and connective tissue alterations observed in these patients, and to better assess the frequency, type and consequences of the vascular complications.

OBJECTIVE: Inflammation and innate immune responses may contribute to development and progression of Osteoarthritis (OA). Chondrocytes are the sole cell type of the articular cartilage and produce extracellular-matrix molecules. How inflammatory mediators reach chondrocytes is incompletely understood. Previous studies have shown that chondrocytes express mRNA encoding complement proteins such as C1q, suggesting local protein production, which has not been demonstrated conclusively. The aim of this study is to explore C1q production at the protein level by chondrocytes. DESIGN: We analysed protein expression of C1q in freshly isolated and cultured human articular chondrocytes using Western blot, ELISA and flow cytometry. We examined changes in mRNA expression of collagen, MMP-1 and various complement genes upon stimulation with pro-inflammatory cytokines or C1q. mRNA expression of C1 genes was determined in articular mouse chondrocytes. RESULTS: Primary human articular chondrocytes express genes encoding C1q, C1QA, C1QB, C1QC, and secrete C1q to the extracellular medium. Stimulation of chondrocytes with pro-inflammatory cytokines upregulated C1QA, C1QB, C1QC mRNA expression, although this was not confirmed at the protein level. Extracellular C1q bound to the chondrocyte surface dose dependently. In a pilot study, binding of C1q to chondrocytes resulted in changes in the expression of collagens with a decrease in collagen type 2 and an increase in type 10. Mouse articular chondrocytes also expressed C1QA, C1QB, C1QC, C1R and C1S at the mRNA level. CONCLUSIONS: C1q protein can be expressed and secreted by human articular chondrocytes and is able to bind to chondrocytes influencing the relative collagen expression. ispartof: OSTEOARTHRITIS AND CARTILAGE vol:28 issue:5 pages:675-684 ispartof: location:England status: published

Glioblastoma multiforme (GBM) is a prevalent intracranial brain tumor associated with a high rate of recurrence and treatment difficulty. The prediction of novel molecular biomarkers through bioinformatics analysis may provide new clues into early detection and eventual treatment of GBM. Here, we used data from the GTEx and TCGA databases to identify 1923 differentially expressed genes (DEGs). GO and KEGG analyses indicated that DEGs were significantly enriched in immune response and coronavirus disease-COVID-19 pathways. Survival analyses revealed a significant correlation between high expression of C1R, CCL2, and TNFRSF1A in the coronavirus disease-COVID-19 pathway and the poor survival in GBM patients. Cell experiments indicated that the mRNA expression levels of C1R, CCL2, and TNFRSF1A in GBM cells were very high. Immune infiltration analysis revealed a significant difference in the proportion of immune cells in tumor and normal tissue, and the expression levels of C1R, CCL2, and TNFRSF1A were associated with immune cell infiltration of GBM. Additionally, the protein-protein interaction networks of C1R, CCL2, and TNFRSF1A involved a total of 65 nodes and 615 edges. These results suggest that C1R, CCL2, and TNFRSF1A may be used as molecular biomarkers of prognosis and immune infiltration in GBM patients in the future.

Complement evasion is a hallmark of extracellular microbial pathogens such as Borrelia burgdorferi, the causative agent of Lyme disease. Lyme disease spirochetes express nearly a dozen outer surface lipoproteins that bind complement components and interfere with their native activities. Among these, BBK32 is unique in its selective inhibition of the classical pathway. BBK32 blocks activation of this pathway by selectively binding and inhibiting the C1r serine protease of the first component of complement, C1. To understand the structural basis for BBK32-mediated C1r inhibition, we performed crystallography and size-exclusion chromatography–coupled small angle X-ray scattering experiments, which revealed a molecular model of BBK32-C in complex with activated human C1r. Structure-guided site-directed mutagenesis was combined with surface plasmon resonance binding experiments and assays of complement function to validate the predicted molecular interface. Analysis of the structures shows that BBK32 inhibits activated forms of C1r by occluding substrate interaction subsites (i.e., S1 and S1’) and reveals a surprising role for C1r B loop–interacting residues for full inhibitory activity of BBK32. The studies reported in this article provide for the first time (to our knowledge) a structural basis for classical pathway–specific inhibition by a human pathogen.

Significance Antibody-dependent complement activation plays a major role in various pathophysiological processes in our body, including infection, inflammation, autoimmunity, and transplant rejection. In order to activate complement, antibodies should bind to target cells and recruit complement component C1. C1 is a large, multimolecular complex that consists of the antibody recognition protein C1q and a heterotetramer of proteases (C1r2s2). Although it is believed that interactions between C1 and IgGs are solely mediated by C1q, we here show that C1r2s2 proteases affect the capacity of C1q to form an avid complex with surface-bound IgG molecules. Furthermore, we demonstrate that C1q–IgG stability is influenced by IgG oligomerization and that promoting IgG oligomerization improves phagocytosis of the pathogenic bacterium Staphylococcus aureus., Complement is an important effector mechanism for antibody-mediated clearance of infections and tumor cells. Upon binding to target cells, the antibody’s constant (Fc) domain recruits complement component C1 to initiate a proteolytic cascade that generates lytic pores and stimulates phagocytosis. The C1 complex (C1qr2s2) consists of the large recognition protein C1q and a heterotetramer of proteases C1r and C1s (C1r2s2). While interactions between C1 and IgG-Fc are believed to be mediated by the globular heads of C1q, we here find that C1r2s2 proteases affect the capacity of C1q to form an avid complex with surface-bound IgG molecules (on various 2,4-dinitrophenol [DNP]-coated surfaces and pathogenic Staphylococcus aureus). The extent to which C1r2s2 contributes to C1q–IgG stability strongly differs between human IgG subclasses. Using antibody engineering of monoclonal IgG, we reveal that hexamer-enhancing mutations improve C1q–IgG stability, both in the absence and presence of C1r2s2. In addition, hexamer-enhanced IgGs targeting S. aureus mediate improved complement-dependent phagocytosis by human neutrophils. Altogether, these molecular insights into complement binding to surface-bound IgGs could be important for optimal design of antibody therapies.

The complement system is involved in promoting secondary injury after traumatic brain injury (TBI), but the roles of the classical and lectin pathways leading to complement activation need to be clarified. To this end, we aimed to determine the ability of the brain to activate the synthesis of classical and lectin pathway initiators in response to TBI and to examine their expression in primary microglial cell cultures. We have modeled TBI in mice by controlled cortical impact (CCI), a clinically relevant experimental model. Using Real-time quantitative polymerase chain reaction (RT-qPCR) we analyzed the expression of initiators of classical the complement component 1q, 1r and 1s (C1q, C1r, and C1s) and lectin (mannose binding lectin A, mannose binding lectin C, collectin 11, ficolin A, and ficolin B) complement pathways and other cellular markers in four brain areas (cortex, striatum, thalamus and hippocampus) of mice exposed to CCI from 24 h and up to 5 weeks. In all murine ipsilateral brain structures assessed, we detected long-lasting, time- and area-dependent significant increases in the mRNA levels of all classical (C1q, C1s, C1r) and some lectin (collectin 11, ficolin A, ficolin B) initiator molecules after TBI. In parallel, we observed significantly enhanced expression of cellular markers for neutrophils (Cd177), T cells (Cd8), astrocytes (glial fibrillary acidic protein&mdash, GFAP), microglia/macrophages (allograft inflammatory factor 1&mdash, IBA-1), and microglia (transmembrane protein 119&mdash, TMEM119), moreover, we detected astrocytes (GFAP) and microglia/macrophages (IBA-1) protein level strong upregulation in all analyzed brain areas. Further, the results obtained in primary microglial cell cultures suggested that these cells may be largely responsible for the biosynthesis of classical pathway initiators. However, microglia are unlikely to be responsible for the production of the lectin pathway initiators. Immunofluorescence analysis confirmed that at the site of brain injury, the C1q is localized in microglia/macrophages and neurons but not in astroglial cells. In sum, the brain strongly reacts to TBI by activating the local synthesis of classical and lectin complement pathway activators. Thus, the brain responds to TBI with a strong, widespread and persistent upregulation of complement components, the targeting of which may provide protection in TBI.

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, with increasing incidence worldwide. Previous studies have shown the role of the complement system in cSCC progression. In this study, we have investigated the mechanistic role of serine proteinase C1r, a component of the classical pathway of the complement system, in cSCC. Knockout of C1r in cSCC cells using CRISPR/Cas9 resulted in a significant decrease in their proliferation, migration, and invasion through collagen type I compared with that of wild-type cSCC cells. Knockout of C1r suppressed the growth and vascularization of cSCC xenograft tumors and promoted apoptosis of tumor cells in vivo. mRNA-sequencing analysis after C1r knockdown revealed significantly regulated Gene Ontology terms cell-matrix adhesion, extracellular matrix component, basement membrane, and metalloendopeptidase activity and Kyoto Encyclopedia of Genes and Genomes pathway extracellular matrix-receptor interaction. Among the significantly regulated genes were invasion-associated matrix metalloproteinases (MMPs) MMP1, MMP13, MMP10, and MMP12. Knockout of C1r resulted in decreased production of MMP-1, MMP-13, MMP-10, and MMP-12 by cSCC cells in culture. Knockout of C1r inhibited the expression of MMP-13 by tumor cells, suppressed invasion, and reduced the amount of degraded collagen in vivo in xenografts. These results provide evidence for the role of C1r in promoting the invasion of cSCC cells by increasing MMP production.

LRP1 is a large endocytic modular receptor that plays a crucial role in the scavenging of apoptotic material through binding to pattern-recognition molecules. It is a membrane anchored receptor of the LDL receptor family with 4 extracellular clusters of ligand binding modules called cysteine rich complement-type repeats that are involved in the interaction of LRP1 with its numerous ligands. Complement C1q was shown to interact with LRP1 and to be implicated in the phagocytosis of apoptotic cells. The present work aimed at exploring how these two large molecules interact at the molecular level using a dissection strategy. For that purpose, recombinant LRP1 clusters II, III and IV were produced in mammalian HEK293F cells and their binding properties were investigated. Clusters II and IV were found to interact specifically and efficiently with C1q with K Ds in the nanomolar range. The use of truncated C1q fragments and recombinant mutated C1q allowed to localize more precisely the binding site for LRP1 on the collagen-like regions of C1q (CLRs), nearby the site that is implicated in the interaction with the cognate protease tetramer C1r2s2. This site could be a common anchorage for other ligands of C1q CLRs such as sulfated proteoglycans and Complement receptor type 1. The use of a cellular model, consisting in CHO LRP1-null cells transfected with full-length LRP1 or a cluster IV minireceptor (mini IV) confirmed that mini IV interacts with C1q at the cell membrane as well as full-length LRP1. Further cellular interaction studies finally highlighted that mini IV can endorse the full-length LRP1 binding efficiency for apoptotic cells and that C1q has no impact on this interaction.

We have previously reported that complement activation precedes the development of kidney fibrosis; however, little is known about the cellular mechanisms involved in this transition. We hypothesized that increased expression of C1 complex protease C1r, the initiator of complement activation, contributes to tubulointerstitial fibrosis and tested this idea in mice with global deletion of C1r. Although expression of C1r in untreated wild-type (WT) mice was higher in the liver compared with kidney tissue, administration of folic acid (FA) led to upregulation of C1r mRNA and protein levels only in kidney tissue. Immunohistochemistry and in situ hybridization experiments localized increased expression of C1r and C1s proteases to renal tubular epithelial cells. C1r-null mice had reduced acute tubular injury and inflammation measured 2 days after FA administration compared with WT mice. C1r deletion reduced expression of C1s, C3 fragment formation, and organ fibrosis measured 14 days after FA administration. Differential gene expression performed in kidney tissue demonstrated that C1r-null mice had reduced expression of genes associated with the acute phase response, complement, proliferation of connective tissue cells (e.g., platelet-derived growth factor receptor-β), and reduced expression of genes associated with inflammation compared with FA-treated WT mice. In vitro experiments in renal epithelial cells demonstrated that C1s expression is dependent on increased C1r expression and that interferon-γ induces the expression of these two proteases. We conclude that increased expression of C1 complex proteases is associated with increased tissue inflammation and complement C3 formation and represents an important pathogenic mechanism leading to FA-mediated tubulointerstitial fibrosis.

Ehlers-Danlos syndromes (EDS) are clinically and genetically heterogeneous disorders characterized by soft connective tissue alteration like joint hypermobility and skin hyper-extensibility. We previously identified heterozygous missense mutations in the C1R and C1S genes, coding for the complement C1 proteases, in patients affected by periodontal EDS, a specific EDS subtype hallmarked by early severe periodontitis leading to premature loss of teeth and connective tissue alterations. Up to now, there is no clear molecular link relating the nominal role of the C1r and C1s proteases, which is to activate the classical complement pathway, to these heterogeneous symptoms of periodontal EDS syndrome. We aim therefore to elucidate the functional effect of these mutations, at the molecular and enzymatic levels. To explore the molecular consequences, a set of cell transfection experiments, recombinant protein purification, mass spectroscopy and N-terminal analyses have been performed. Focusing on the results obtained on two different C1S variants, namely p.Val316del and p.Cys294Arg, we show that HEK293-F cells stably transfected with the corresponding C1s variant plasmids, unexpectedly, do not secrete the full-length mutated C1s, but only a truncated Fg40 fragment of 40 kDa, produced at very low levels. Detailed analyses of the Fg40 fragments purified for the two C1s variants show that they are identical, which was also unexpected. This suggests that local misfolding of the CCP1 module containing the patient mutation exposes a novel cleavage site, between Lys353 and Cys354, which is not normally accessible. The mutation-induced Fg40 fragment contains the intact C-terminal serine protease domain but not the N-terminal domain mediating C1s interaction with the other C1 subunits, C1r, and C1q. Thus, Fg40 enzymatic activity escapes the normal physiological control of C1s activity within C1, potentially providing a loss-of-control. Comparative enzymatic analyses show that Fg40 retains the native esterolytic activity of C1s, as well as its cleavage efficiency toward the ancillary alarmin HMGB1 substrate, for example, whereas the nominal complement C4 activation cleavage is impaired. These new results open the way to further molecular explorations possibly involving subsidiary C1s targets.

C1r and C1s are serine proteases responsible for activating the classical complement pathway to initiate the complement cascade, which plays a crucial role in eliminating invading pathogenic microbes. In this study, cDNA sequences of C1r and C1s were identified from black rockfish and designated as SsC1r and SsC1s, respectively. In both sequences, two CUB domains, an EGF-like domain, two CCP domains, and a trypsin-like serine protease domain were identified. Multiple sequence alignments with known vertebrate homologs demonstrated that both sequences were highly conserved and, especially, the catalytic and substrate binding residues were completely conserved. In the constructed phylogenetic tree, C1r and C1s formed two separate clusters, which further branched into groups of related organisms. SsC1r and SsC1s joined with their respective teleostean clusters. Transcriptional analysis showed that the highest mRNA expression level was in the liver under normal physiological conditions. Significantly upregulated expression of both mRNAs in spleen and liver after pathologic stress, by intraperitoneal injection with different stimuli, suggested their vital role in immunity. The serine protease domains of SsC1r and SsC1s were cloned and the recombinant proteins were expressed and purified. A protease assay, conducted to confirm their functionality, indicated that both recombinant proteins had proteolytic activity. Taken together, these results indicate that SsC1r and SsC1s have significant properties to aid in the immunity of black rockfish by activating the complement system by proteolytic cleavage.

C1s deficiency is strongly associated with the development of human systemic lupus erythematosus (SLE); however, the mechanisms by which C1s deficiency contributes to the development of SLE have not yet been elucidated in detail. Using ICR-derived-glomerulonephritis (ICGN) mouse strain that develops SLE and very weakly expresses C1s in the liver, we investigated the protective roles of C1s against SLE. A genetic sequence analysis revealed complete deletion of the C1s1 gene, a mouse homolog of the human C1s gene, with partial deletion of the C1ra and C1rb genes in the ICGN strain. This deletion led to the absence of C1r/C1s and a low level of C1q in the circulation. In order to investigate whether the C1r/C1s deficiency induces SLE, we produced a congenic mouse strain by introducing the deletion region of ICGN into the C57BL/6 strain. Congenic mice exhibited no C1r/C1s and a low level of C1q in the circulation, but did not have any autoimmune defects. These results suggest that C1r/C1s deficiency is not sufficient to drive murine SLE and also that other predisposing genes exist in ICGN mice.

The complement components C1r and C1s play a vital role in immunity with the activation of C1 complex in the classical complement pathway against pathogen infection. In this study, Nile tilapia (Oreochromis niloticus) C1r and C1s orthologs (OnC1r and OnC1s) were identified and characterized. The cDNA of OnC1r and OnC1s ORFs consisted of 1902 bp and 2100 bp of nucleotide sequence encoding polypeptides of 633 and 699 amino acids, respectively. The deduced OnC1r and OnC1s proteins both possessed CUB, EGF, CCP and SP domains, which were significantly homology to teleost. Spatial mRNA expression analysis revealed that the OnC1r and OnC1s were highly expressed in liver. After the in vivo challenges of Streptococcus agalactiae (S. agalactiae) and lipopolysaccharide (LPS), the mRNA expressions of OnC1r and OnC1s were significantly up-regulated in liver and spleen, which were consistent with immunohistochemical detection at the protein level. The up-regulation of OnC1r and OnC1s expressions were also demonstrated in head kidney monocytes/macrophages in vitro stimulated with LPS, S. agalactiae, and recombinant OnIFN-γ. Taken together, the results of this study indicated that OnC1r and OnC1s were likely to get involved in the immune response of Nile tilapia against bacterial infection.

Aim: The purpose of this experimental study was to investigate the potential diagnostic value of the platelet activation marker SCUBE1 [signal peptide, CUB (complement C1r/C1s, Uegf, and Bmp1), and EGF (epidermal growth factor)-like domain-containing protein 1] and the adipocytokine chemerin in a prepubertal rat model of testicular torsion (TT). Materials and Methods: Twenty-eight male rats were used for this study. They were randomly assigned into one of the four groups, each containing seven rats. No additional procedure other than a sham operation was performed on the control group (group IV). The other subjects comprised the torsion ischemia groups (groups I, II, and III). Blood specimens were collected after 30 min (group I), 2 h (group II), or 4 h (group III) using the intracardiac method. For group IV, which was the sham operation group, blood specimens were collected after 4 h, and testis tissue specimens were extracted by orchiectomy for histopathological examination. Results: No statistically significant change was determined in SCUBE1 levels of rats exposed to torsion. Also, no significant difference was observed between SCUBE1 levels of rats exposed to torsion and those of the control group. Statistically significant change was determined in chemerin levels during observation in rats exposed to torsion. This change was statistically significant between groups I and III. There was no statistically significant difference between chemerin levels of rats exposed to torsion (groups I, II, and III) and those of the control group (group IV). Conclusion: We observed no statistically significant differences when plasma SCUBE1 and chemerin levels of rats subjected to TT were compared with a control group in this study., Haseki Eğitim ve Araştırma Hastanesi

Marsupials are born immunologically premature, relying on cells and molecules in maternal milk for immune protection. Both immunoglobulin and complement proteins have been identified in marsupial milk, but the expression of specific complement proteins remains largely unexplored. We report partial cDNA sequences for two complement-activating proteins, C3, C1r, CFP and MASP2, in liver tissues from red-tailed phascogale (Phascogale calura). Conservation of functionally relevant motifs were identified in the translated cDNA sequences from phascogale C3, CFP and MASP2 and their eutherian homologues. Gene expression of representative molecules from each of the major complement pathways was also investigated in whole body tissues from 1 to 18 day old animals and liver tissues from 31-day to 14-month old animals. Average complement expression in whole bodies and liver tissues of C1r, CFP, MASP2 and C3 increased significantly in juveniles compared to pouch young, presumably due to the maturation of the young's own complement system. Comparing expression in liver tissues only, we found that the average CFP expression were higher in pouch young compared to juveniles, while results were still statistically similar to the average expression of all tissues for C1r, MASP2 and C3. The average complement expression then significantly decreased as the animals aged into adulthood.

Significance C1 is a large complex that triggers the destruction of invading pathogens via lysis or by stimulation of innate and adaptive immune processes. It is composed of C1q, a protein with a bouquet-like architecture, together with a tetramer assembled from two copies each of the serine proteases C1r and C1s, which activate when C1q binds to a pathogen surface. Here we describe detailed structures that show how C1r and C1s interact via an extensive interface encompassing the N-terminal regions of both proteases. Our findings reveal how the protease tetramer is organized and suggest a mechanism for the assembly and activation of C1., The multiprotein complex C1 initiates the classical pathway of complement activation on binding to antibody–antigen complexes, pathogen surfaces, apoptotic cells, and polyanionic structures. It is formed from the recognition subcomponent C1q and a tetramer of proteases C1r2C1s2 as a Ca2+-dependent complex. Here we have determined the structure of a complex between the CUB1-EGF-CUB2 fragments of C1r and C1s to reveal the C1r–C1s interaction that forms the core of C1. Both fragments are L-shaped and interlock to form a compact antiparallel heterodimer with a Ca2+ from each subcomponent at the interface. Contacts, involving all three domains of each protease, are more extensive than those of C1r or C1s homodimers, explaining why heterocomplexes form preferentially. The available structural and biophysical data support a model of C1r2C1s2 in which two C1r-C1s dimers are linked via the catalytic domains of C1r. They are incompatible with a recent model in which the N-terminal domains of C1r and C1s form a fixed tetramer. On binding to C1q, the proteases become more compact, with the C1r-C1s dimers at the center and the six collagenous stems of C1q arranged around the perimeter. Activation is likely driven by separation of the C1r-C1s dimer pairs when C1q binds to a surface. Considerable flexibility in C1s likely facilitates C1 complex formation, activation of C1s by C1r, and binding and activation of downstream substrates C4 and C4b-bound C2 to initiate the reaction cascade.

Here, we report brain white matter alterations in individuals clinically and genetically diagnosed with periodontal Ehlers–Danlos syndrome, a rare disease characterized by premature loss of teeth and connective tissue abnormalities. Eight individuals of two families clinically diagnosed with periodontal Ehlers–Danlos syndrome were included in the present study and underwent general physical, dental, and neurological examination. Whole exome sequencing was performed, and all patients included in the study underwent MRI of the brain. Whole exome sequencing revealed heterozygous C1R mutations c.926G>T (p.Cys309Phe, Family A) and c.149_150TC>AT (p.Val50Asp, Family B). All adult individuals (n = 7; age range 31 to 68 years) investigated by MRI had brain white matter abnormalities. The MRI of one investigated child aged 8 years was normal. The MRI pattern was suggestive of an underlying small vessel disease that is progressive with age. As observed in other leukoencephalopathies related to microangiopathies, the extent of the white matter changes was disproportionate to the neurologic features. Medical history revealed recurrent headaches or depression in some cases. Neurological examination was unremarkable in all individuals but one had mild cognitive decline and ataxia and experienced a seizure. The observation that periodontal Ehlers–Danlos syndrome caused by missense mutations in C1R is consistently associated with a leukoencephalopathy opens a new pathogenic link between the classical complement pathway, connective tissue, brain small vessels, and brain white matter abnormalities.

The carboxy-terminal domain of the BBK32 protein from Borrelia burgdorferi sensu stricto, termed BBK32-C, binds and inhibits the initiating serine protease of the human classical complement pathway, C1r. In this study we investigated the function of BBK32 orthologues of the Lyme-associated Borrelia burgdorferi sensu lato complex, designated BAD16 from B. afzelii strain PGau and BGD19 from B. garinii strain IP90. Our data show that B. afzelii BAD16-C exhibits BBK32-C-like activities in all assays tested, including high-affinity binding to purified C1r protease and C1 complex, and potent inhibition of the classical complement pathway. Recombinant B. garinii BGD19-C also bound C1 and C1r with high-affinity yet exhibited significantly reduced in vitro complement inhibitory activities relative to BBK32-C or BAD16-C. Interestingly, natively produced BGD19 weakly recognized C1r relative to BBK32 and BAD16 and, unlike these proteins, BGD19 did not confer significant protection from serum killing. Site-directed mutagenesis was performed to convert BBK32-C to resemble BGD19-C at three residue positions that are identical between BBK32 and BAD16 but different in BGD19. The resulting chimeric protein was designated BXK32-C and this BBK32-C variant mimicked the properties observed for BGD19-C. To query the disparate complement inhibitory activities of BBK32 orthologues, the crystal structure of BBK32-C was solved to 1.7Å limiting resolution. BBK32-C adopts an anti-parallel four-helix bundle fold with a fifth alpha-helix protruding from the helical core. The structure revealed that the three residues targeted in the BXK32-C chimera are surface-exposed, further supporting their potential relevance in C1r binding and inhibition. Additional binding assays showed that BBK32-C only recognized C1r fragments containing the serine protease domain. The structure-function studies reported here improve our understanding of how BBK32 recognizes and inhibits C1r and provide new insight into complement evasion mechanisms of Lyme-associated spirochetes of the B. burgdorferi sensu lato complex., Author summary Lyme disease, caused by Borrelia burgdorferi sensu lato complex, is the most common tick-borne infection in the Northern hemisphere. As a pathogen that is transmitted within blood and body fluids, Borrelia species must combat the complement system, which is a soluble host defense pathway that marks invaders for elimination. Previously, we demonstrated that the B. burgdorferi protein BBK32 blocks the activation of the classical pathway of complement by binding to and inhibiting the initiating protease, C1r. Here we show that the BBK32 orthologue from B. garinii, designated BGD19, is unable to neutralize complement-mediated serum killing. In contrast, BBK32 and the B. afzelii orthologue BAD16 potently inhibits serum killing. Comparison of the amino acid sequences of these proteins identified three residues unique to BGD19 that contribute to its reduced inhibitory activity. The three-dimensional structure of BBK32-C, reported here, showed that these residue positions are surface exposed in BBK32, further supporting their role in C1r inhibition. These results render important insight into how Lyme disease-associated spirochetes recognize and block an important human innate immune pathway and provide mechanistic insight into evolutionarily optimized C1r inhibitors.

We report an extremely rare case of combined classical and periodontal Ehlers&minus, Danlos syndrome (EDS) with early severe periodontitis and a generalized lack of attached gingiva. A German family with classical EDS was investigated by physical and dental evaluation and exome and Sanger sequencing. Due to the specific periodontal phenotype in the affected child, an additional diagnosis of periodontal EDS was suspected. Physical and genetic examination of two affected and three unaffected family members revealed a family diagnosis of classical EDS with a heterozygous mutation in COL5A1 (c.1502del, p.Pro501Leufs*57). Additional to the major clinical criteria for classical EDS&mdash, generalized joint hypermobility, hyperelastic skin, and atrophic scarring &mdash, the child aged four years presented with generalized alveolar bone loss up to 80%, early loss of two lower incisors, severe gingival recession, and generalized lack of attached gingiva. Due to these clinical findings, an additional diagnosis of periodontal EDS was suspected. Further genetic analysis revealed the novel missense mutation c.658T&gt, G (p.Cys220Gly) in C1R in a heterozygous state. Early severe periodontitis in association with generalized lack of attached gingiva is pathognomonic for periodontal EDS and led to the right clinical and genetic diagnosis in the present case.

Background: Pulmonary embolism (PE) is a common and potentially life-threatening disorder. Patients with PE often have nonspecific symptoms, and the diagnosis is often delayed. Aim: The aim of our study was to investigate the role of signal peptide-complement C1r/C1s, Uegf, and Bmp1-epidermal growth factor domain-containing protein 1 (SCUBE1) used in the diagnosis of PE. Methods: The study was designed prospectively. A total of 57 patients who were admitted to emergency service with clinically suspected PE were included in the study. The patients diagnosed with PE were defined as PE group (n = 32), and the patients with undetectable embolism on computerized tomographic pulmonary angiography were defined as non-PE group (n = 25). Twenty-five age- and sex-matched healthy cases were chosen for the study. Routine biochemical analysis, complete blood count, D-dimer, SCUBE1, and arterial blood gas analysis were performed early after admission. Results: Mean SCUBE1 levels were higher in the PE group (0.90 ng/mL) than in the non-PE (0.38 ng/mL) and control groups (0.47 ng/mL) (P < 0.01). A cutoff point of 0.49 ng/mL for SCUBE1 indicated 100% sensitivity and 64% specificity in patients with PE. Mean D-dimer levels were not different between PE and non-PE groups (P = 0.591). A multivariable logistic regression analysis (with dichotomous PE groups as the response variable; age, gender, chest pain, syncope, diabetes mellitus, chronic obstructive pulmonary disease, hypertension, D-dimer, neutrophil-lymphocytes ratio, and SCUBE1 variables as predictors) showed that the significant and independent predictors of PE diagnosis were SCUBE1 and chest pain. Conclusion: This study suggests that serum SCUBE1 measurement might be used as a diagnostic biomarker in PE.

Joint hypermobility syndrome (JHS) (also termed Ehlers-Danlos syndrome, hypermobility type) is a heritable connective tissue disorder which is characterized by generalized joint hypermobility, chronic pain, dizziness, fatigue, and minor skin changes. However, it has yet to be determined in patients with JHS whether specific genetic factors are involved in the risk of developing the disorder. Therefore, interventions have been limited to symptomatic treatments, and biomarkers for diagnosis and therapy have not yet been identified. In the present study, to identify potential serum biomarkers for JHS, we examined proteins with differential levels in sera from patients with JHS and in sera from control individuals using isobaric tags for relative and absolute quantitation (iTRAQ) labeling in combination with nano LC-MALDI-TOF/TOF-MS/MS followed by ProteinPilot analysis. In the sera of patients with JHS, a total of 106 proteins with differential levels were identified, and they were further narrowed down to 6 proteins (p

Almitairi et al. (1) present structural information on the interaction between the proteases C1r and C1s, both consisting of six domains, called CUB1-EGF-CUB2-CCP1-CCP2-SP. The authors also propose a model for the C1 complex where the C1r2s2 tetramer is bound to C1q. Using our published and deposited small-angle X-ray scattering (SAXS) data (2) for the C1 complex, Almitairi et al. (1) conduct rigid body modeling and obtain models with a fit to the data (χ2) in the range 2–4, of which only one model with χ2 = 2.9 is presented. In this model, the two C1r molecules interact in the center of the C1 complex through … [↵][1]1To whom correspondence should be addressed. Email: gra{at}mbg.au.dk. [1]: #xref-corresp-1-1

In their letter, Mortensen et al. (1) query our model of zymogen C1. It was assembled from overlapping crystal structures with constraints imposed by known interactions (2). The starting point was the protease subcomponent, C1r2C1s2, which comprises two antiparallel C1r-C1s dimers (mediated via CUB1-EGF-CUB2 contacts) linked through a central interaction between the CCP1-CCP2-SP domains of C1r. During C1 assembly, C1r2C1s2 folds-up, so the CUB1-EGF-CUB2 domains bind to the collagenous stems of C1q. We propose that C1r–C1r interactions are maintained in zymogen C1, preventing one C1r polypeptide from activating its partner. Activation is driven by separation of C1r arms when C1q binds to a surface. Our model is compatible with solution, structural, … [↵][1]1To whom correspondence should be addressed Email: rw73{at}le.ac.uk. [1]: #xref-corresp-1-1

The lack of early diagnostic maneuvers and effective pharmacotherapy for osteoarthritis (OA) is predominantly attributed to current limited understanding of its pathogenesis. In the present study, the alteration of synovial fluid (SF) proteome in OA were analyzed using SWATH-mass spectrometry (SWATH-MS) to further elucidate the pathogenesis of OA. SF samples were collected from 10 OA and 10 rheumatoid arthritis (RA) patients undergoing arthroplasty and 10 patients undergoing arthroscopy for traumatic arthritis (meniscus injury without cartilage lesion). According to the Kellgren-Lawrence (KL) radiographic grading criteria, all the OA and RA patients were classified as KL grade 4. SWATH-MS was applied to identify differentially expressed proteins specifically regulated in OA. Differentially expressed proteins identified by SWATH-MS were subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation. Proteins of interest were quantified by enzyme-linked immunosorbent assay (ELISA) following identification. With the use of SWATH-MS, 131 proteins were identified to be differentially expressed in OA, of which 93 corresponded to upregulation and 38 to downregulation. Complement C1r was the most significantly upregulated protein in OA. Twenty-eight out of the 131 proteins were specifically regulated in OA, of which 17 were upregulated and 11 were downregulated. Dickkopf-related protein 2 (DKK2) was one of the proteins specifically upregulated in OA. Furthermore, KEGG pathway annotation indicated that differentially expressed proteins in OA were enriched in complement and coagulation cascades. ELISA indicated that OA severity was positively correlated with the levels of complement C1r (r=0.549; P

The complement components C1r and C1s play a crucial role in innate immunity via activation of the classical complement cascade system. As initiators of the pathogen-induced signaling cascade, C1r and C1s modulate innate immunity. In order to understand the immune responses of teleost C1r and C1s, Oplegnathus fasciatus C1r and C1s genes (OfC1r and OfC1s) were identified and characterized. The genomic sequence of OfC1r was enclosed with thirteen exons that represented a putative peptide with 704 amino acids (aa), whereas eleven exons of OfC1s represented a 691 aa polypeptide. In addition, genomic analysis revealed that both OfC1r and OfC1s were located on a single chromosome. These putative polypeptides were composed of two CUB domains, an EGF domain, two CCP domains, and a catalytically active serine protease domain. Phylogenetic analysis of C1r and C1s showed that OfC1r and OfC1s were evolutionary close to the orthologs of Pundamilia nyererei (identity = 73.4%) and Oryzias latipes (identity = 58.0%), respectively. Based on the results of quantitative real-time qPCR analysis, OfC1r and OfC1s transcripts were detected in all the eleven different tissues, with higher levels of OfC1r in blood and OfC1s in liver. The putative roles of OfC1r and OfC1s in response to pathogenic bacteria (Edwardsiella tarda and Streptococcus iniae) and virus (rock bream iridovirus, RBIV) were investigated in liver and head kidney tissues. The transcription of OfC1r and OfC1s was found to be significantly upregulated in response to pathogenic bacterial and viral infections. Overall findings of the present study demonstrate the potential immune responses of OfC1r and OfC1s against invading microbial pathogens and the activation of classical signaling cascade in rock bream.

In infection, complement C1q recognizes pathogen-congregated antibodies and elicits complement activation. Among endogenous ligands, C1q binds to DNA and apoptotic cells, but whether C1q binds to nuclear DNA in apoptotic cells remains to be investigated. With UV irradiation-induced apoptosis, C1q initially bound to peripheral cellular regions in early apoptotic cells. By 6 h, binding concentrated in the nuclei to the nucleolus but not the chromatins. When nucleoli were isolated from non-apoptotic cells, C1q also bound to these structures. In vivo, C1q exists as the C1 complex (C1qC1r2C1s2), and C1q binding to ligands activates the C1r/C1s proteases. Incubation of nucleoli with C1 caused degradation of the nucleolar proteins nucleolin and nucleophosmin 1. This was inhibited by the C1 inhibitor. The nucleoli are abundant with autoantigens. C1q binding and C1r/C1s degradation of nucleolar antigens during cell apoptosis potentially reduces autoimmunity. These findings help us to understand why genetic C1q and C1r/C1s deficiencies cause systemic lupus erythematosus.

In PNAS, Mortensen et al. (1) propose a model of the C1 complex of complement derived from small-angle X-ray scattering (SAXS) and electron microscopy (EM) analyses that contradicts previously published models and suggests an intermolecular activation process. This proposal is largely derived from a conjectural structure of C1r2C1s2, the catalytic unit of C1, in which the serine protease (SP) domains of both C1r and C1s are located at the periphery of the tetramer. Such an arrangement is clearly not consistent with the multiple biochemical, biophysical, and structural studies carried out so far (reviewed in ref. 2). Thus, the X-ray structures (2) showing that C1r dimerizes through interactions between the … [↵][1]1To whom correspondence should be addressed. Email: christine.gaboriaud{at}ibs.fr. [1]: #xref-corresp-1-1

To identify a genetic cause of early-onset systemic lupus erythematosus (SLE) in a large consanguineous family from Turkey and to study the mechanisms of the disease.We performed whole-exome sequencing and single-nucleotide polymorphism array genotyping in family members with and without SLE. Protein and gene expression, cytokine profile, neutrophil extracellular trap (NET) formation, and presence of low-density granulocytes were evaluated in patient primary cells and serum samples.We identified a novel, homozygous, loss-of-function mutation (p.Pro445Leufs*11) in the C1R gene. Using the Sanger method of DNA sequencing in 14 family members, we confirmed the presence of the mutation in 4 patients with SLE and in an asymptomatic 9-year-old girl. Complement levels were low in sera from patients with truncated C1r protein. Two siblings with SLE who were available for detailed evaluation exhibited strong type I interferon (IFN) inflammatory signatures despite their disease being clinically inactive at the time of sampling. The type I IFN transcriptional signature in the patients' blood correlated with disease expressivity, whereas the neutrophil signature in peripheral blood mononuclear cells was likely associated with disease severity. The female patient with SLE with the most severe phenotype presented with a stronger neutrophil signature, defined by enhanced NET formation and the presence of low-density granulocytes. Analysis of exome data for modifying alleles suggested enrichment of common SLE-associated variants in the more severely affected patients. Lupus-associated HLA alleles or HLA haplotypes were not shared among the 4 affected subjects.Our findings revealed a novel high-penetrance mutation in C1R as the cause of monogenic SLE. Disease expressivity in this family appears to be influenced by additional common and rare genetic variants.

Anti-nuclear autoantibodies, which frequently target the nucleoli, are pathogenic hallmarks of systemic lupus erythematosus (SLE). Although the causes of these Abs remain broad and ill-defined, a genetic deficiency in C1 complex (C1qC1r2C1s2) or C4 is able to induce these Abs. Considering a recent finding that, in dead cells, nucleoli were targeted by C1q and two nucleolar autoantigens were degraded by C1r/C1s proteases, we considered that C1 could help protect against antinuclear autoimmunity by broadly degrading nucleolar proteins or autoantigens. Nucleoli were isolated to homogeneity and structurally defined. After C1 treatment, cleaved nucleolar proteins were identified by proteomic two-dimensional fluorescence difference gel electrophoresis and mass spectrometry, and further verified by Western blotting using specific Abs. The extent of nucleolar autoantigen degradation upon C1 treatment was estimated using SLE patient autoantibodies. The isolated nucleoli were broadly reactive with SLE patient autoantibodies. These nucleoli lacked significant autoproteolysis, but many nucleolar proteins and autoantigens were degraded by C1 proteases; >20 nucleolar proteins were identified as C1 cleavable. These were further validated by Western blotting using specific Abs. The broad autoantigenicity of the nucleoli may attribute to their poor autoproteolysis, causing autologous immune stimulation upon necrotic exposure. However, C1q targets at these nucleoli to cause C1 protease activation and the cleavage of many nucleolar proteins or autoantigens. This may represent one important surveillance mechanism against antinuclear autoimmunity because C1 genetic deficiency causes anti-nuclear autoantibodies and SLE disease.

Protease-activated receptor-1 (PAR1) is the prototypic member of a family of four G-protein-coupled receptors that signal in response to extracellular proteases. In the peripheral nervous system, the expression and/or the role of PARs are still poorly investigated. High PAR1 mRNA expression was found in the rat dorsal root ganglia and the signal intensity of PAR1 mRNA increased in response to sciatic nerve transection. In the sciatic nerve, functional PAR1 receptor was reported at the level of non-compacted Schwann cell myelin microvilli of the nodes of Ranvier. Schwann cells are the principal population of glial cells of the peripheral nervous system which myelinate axons playing an important role during axonal regeneration and remyelination. The present study was undertaken in order to determine if the activation of PAR1 affects the neurotrophic properties of Schwann cells. Our results suggest that the stimulation of PAR1 could potentiate the Schwann cell ability to favour nerve regeneration. In fact, the conditioned medium obtained from Schwann cell cultures challenged with a specific PAR1 activating peptide (PAR1 AP) displays increased neuroprotective and neurotrophic properties with respect to the culture medium from untreated Schwann cells. The proteomic analysis of secreted proteins in untreated and PAR1 AP-treated Schwann cells allowed the identification of factors differentially expressed in the two samples. Some of them (such as macrophage migration inhibitory factor, matrix metalloproteinase-2, decorin, syndecan 4, complement C1r subcomponent, angiogenic factor with G patch and FHA domains 1) appear to be transcriptionally regulated after PAR1 AP treatment as shown by RT-PCR.

Objective Ischemia-reperfusion (I/R) injury is a major clinical problem linked to vascular surgery. Currently, no drugs to prevent or to treat I/R injury are approved for clinical use. C1 inhibitor (C1 INH) is known to reduce activation of the plasma cascade systems that are involved in the pathophysiologic process of I/R injury. The aim of this study was therefore to investigate the effect of C1 INH on complement deposition and endothelial cell activation in a rat model of hind limb I/R injury. Methods Male Wistar rats (wild type, bred at the central animal facility, University of Bern), weighing 250 to 320 g, were used. The rats underwent 2-hour ischemia and 24-hour reperfusion by unilateral clamping of the femoral artery and additional use of a tourniquet. Five groups were divided according to intravenous treatment 5 minutes before ischemia: 50 IU/kg C1 INH (n = 5); 100 IU/kg C1 INH (n = 7); vehicle control (n = 5); nontreated control (n = 7); and normal, healthy control without intervention (n = 4). At the end, muscle edema, tissue viability, and histologic features were assessed. Deposition of immunoglobulin M, C1r, C4d, and fibrin and expression of plasminogen activator inhibitor 1, heparan sulfate (HS), E-selectin, and vascular cell adhesion molecule 1 were evaluated by fluorescence staining. In addition, high-mobility group box 1 protein was measured in plasma. Results Edema formation was reduced by C1 INH at two dosages, mirrored by improved histologic injury scores and preserved muscle viability. Deposition of immunoglobulin M, C4d, and fibrin was significantly decreased by 100 IU/kg C1 INH compared with nontreated controls. Pretreatment with 100 IU/kg C1 INH also significantly reduced HS shedding and expression of plasminogen activator inhibitor 1 as well as plasma levels of high-mobility group box 1 protein. Conclusions Pretreatment with both 50 and 100 IU/kg C1 INH attenuated reperfusion injury of rat hind limbs. Pretreatment with 100 IU/kg also preserved the endothelial HS layer as well as the natural, profibrinolytic phenotype of the endothelium. Prevention of endothelial cell activation by C1 INH may therefore be a promising strategy to prevent I/R injury in the clinical setting of peripheral vascular diseases and elective surgery on extremities.

Members of a family of collagen-binding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) from Gram-positive bacteria are established virulence factors in several infectious diseases models. Here, we report that these adhesins also can bind C1q and act as inhibitors of the classical complement pathway. Molecular analyses of Cna from Staphylococcus aureus suggested that this prototype MSCRAMM bound to the collagenous domain of C1q and interfered with the interactions of C1r with C1q. As a result, C1r2C1s2 was displaced from C1q, and the C1 complex was deactivated. This novel function of the Cna-like MSCRAMMs represents a potential immune evasion strategy that could be used by numerous Gram-positive pathogens.