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1.
J Biol Chem ; 300(5): 107236, 2024 May.
Article in English | MEDLINE | ID: mdl-38552741

ABSTRACT

The complement system serves as the first line of defense against invading pathogens by promoting opsonophagocytosis and bacteriolysis. Antibody-dependent activation of complement occurs through the classical pathway and relies on the activity of initiating complement proteases of the C1 complex, C1r and C1s. The causative agent of Lyme disease, Borrelia burgdorferi, expresses two paralogous outer surface lipoproteins of the OspEF-related protein family, ElpB and ElpQ, that act as specific inhibitors of classical pathway activation. We have previously shown that ElpB and ElpQ bind directly to C1r and C1s with high affinity and specifically inhibit C2 and C4 cleavage by C1s. To further understand how these novel protease inhibitors function, we carried out a series of hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments using ElpQ and full-length activated C1s as a model of Elp-protease interaction. Comparison of HDX-MS profiles between unbound ElpQ and the ElpQ/C1s complex revealed a putative C1s-binding site on ElpQ. HDX-MS-guided, site-directed ElpQ mutants were generated and tested for direct binding to C1r and C1s using surface plasmon resonance. Several residues within the C-terminal region of ElpQ were identified as important for protease binding, including a single conserved tyrosine residue that was required for ElpQ- and ElpB-mediated complement inhibition. Collectively, our study identifies key molecular determinants for classical pathway protease recognition by Elp proteins. This investigation improves our understanding of the unique complement inhibitory mechanism employed by Elp proteins which serve as part of a sophisticated complement evasion system present in Lyme disease spirochetes.


Subject(s)
Borrelia burgdorferi , Complement Pathway, Classical , Borrelia burgdorferi/immunology , Borrelia burgdorferi/metabolism , Borrelia burgdorferi/genetics , Complement Pathway, Classical/immunology , Bacterial Outer Membrane Proteins/metabolism , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/immunology , Bacterial Outer Membrane Proteins/chemistry , Humans , Lipoproteins/metabolism , Lipoproteins/genetics , Lipoproteins/chemistry , Lipoproteins/immunology , Complement C1s/metabolism , Complement C1s/genetics , Complement C1s/chemistry , Protein Binding , Lyme Disease/immunology , Lyme Disease/microbiology , Lyme Disease/metabolism , Lyme Disease/genetics , Complement C1r/metabolism , Complement C1r/genetics
2.
Clin Immunol ; 252: 109646, 2023 07.
Article in English | MEDLINE | ID: mdl-37209807

ABSTRACT

BACKGROUND: Kikuchi-Fujimoto disease (KFD) is a self-limited inflammatory disease of unknown pathogenesis. Familial cases have been described and defects in classical complement components C1q and C4 have been identified in some patients. MATERIAL AND METHODS: We describe genetic and immune investigations of a 16 years old Omani male, a product of consanguineous marriage, who presented with typical clinical and histological features of KFD. RESULTS: We identified a novel homozygous single base deletion in C1S (c.330del; p. Phe110LeufsTer23) resulting in a defect in the classical complement pathway. The patient was negative for all serological markers of SLE. In contrast, two female siblings (also homozygous for the C1S mutation), one has autoimmune thyroid disease (Hashimoto thyroiditis) and a positive ANA and the other sibling has serology consistent with SLE. CONCLUSION: We report the first association between C1s deficiency and KFD.


Subject(s)
Histiocytic Necrotizing Lymphadenitis , Adolescent , Humans , Male , Complement C1s/genetics , Histiocytic Necrotizing Lymphadenitis/genetics , Histiocytic Necrotizing Lymphadenitis/complications , Histiocytic Necrotizing Lymphadenitis/pathology , Loss of Function Mutation
3.
Front Immunol ; 14: 1157421, 2023.
Article in English | MEDLINE | ID: mdl-36960056

ABSTRACT

Periodontal Ehlers-Danlos syndrome (pEDS) is an autosomal dominant disorder characterized by early-onset periodontitis leading to premature loss of teeth, lack of attached gingiva and thin and fragile gums leading to gingival recession. Connective tissue abnormalities of pEDS typically include easy bruising, pretibial plaques, distal joint hypermobility, hoarse voice, and less commonly manifestations such as organ or vessel rupture. pEDS is caused by heterozygous missense mutations in C1R and C1S genes of the classical complement C1 complex. Previously we showed that pEDS pathogenic variants trigger intracellular activation of C1r and/or C1s, leading to extracellular presence of activated C1s. However, the molecular link relating activated C1r and C1s proteases to the dysregulated connective tissue homeostasis in pEDS is unknown. Using cell- and molecular-biological assays, we identified activated C1s (aC1s) as an enzyme which degrades collagen I in cell culture and in in vitro assays. Matrix collagen turnover in cell culture was assessed using labelled hybridizing peptides, which revealed fast and comprehensive collagen protein remodeling in patient fibroblasts. Furthermore, collagen I was completely degraded by aC1s when assays were performed at 40°C, indicating that even moderate elevated temperature has a tremendous impact on collagen I integrity. This high turnover is expected to interfere with the formation of a stable ECM and result in tissues with loose compaction a hallmark of the EDS phenotype. Our results indicate that pathogenesis in pEDS is not solely mediated by activation of the complement cascade but by inadequate C1s-mediated degradation of matrix proteins, confirming pEDS as a primary connective tissue disorder.


Subject(s)
Complement C1s , Ehlers-Danlos Syndrome , Humans , Collagen Type I/genetics , Ehlers-Danlos Syndrome/genetics , Ehlers-Danlos Syndrome/pathology , Mutation, Missense , Complement C1s/genetics
4.
Genes (Basel) ; 12(12)2021 12 15.
Article in English | MEDLINE | ID: mdl-34946939

ABSTRACT

The presence of complement activation products at sites of pathology in post-mortem Alzheimer's disease (AD) brains is well known. Recent evidence from genome-wide association studies (GWAS), combined with the demonstration that complement activation is pivotal in synapse loss in AD, strongly implicates complement in disease aetiology. Genetic variations in complement genes are widespread. While most variants individually have only minor effects on complement homeostasis, the combined effects of variants in multiple complement genes, referred to as the "complotype", can have major effects. In some diseases, the complotype highlights specific parts of the complement pathway involved in disease, thereby pointing towards a mechanism; however, this is not the case with AD. Here we review the complement GWAS hits; CR1 encoding complement receptor 1 (CR1), CLU encoding clusterin, and a suggestive association of C1S encoding the enzyme C1s, and discuss difficulties in attributing the AD association in these genes to complement function. A better understanding of complement genetics in AD might facilitate predictive genetic screening tests and enable the development of simple diagnostic tools and guide the future use of anti-complement drugs, of which several are currently in development for central nervous system disorders.


Subject(s)
Alzheimer Disease/genetics , Alzheimer Disease/immunology , Complement Activation/genetics , Brain/pathology , Clusterin/genetics , Complement Activation/immunology , Complement C1s/genetics , Complement System Proteins/genetics , Genetic Predisposition to Disease/genetics , Genome-Wide Association Study/methods , Humans , Polymorphism, Single Nucleotide/genetics , Receptors, Complement 3b/genetics
5.
Sci Rep ; 11(1): 11926, 2021 06 07.
Article in English | MEDLINE | ID: mdl-34099740

ABSTRACT

Endometriosis (EMS) is a disease that shows immune dysfunction and chronic inflammation characteristics, suggesting a role of complement system in its pathophysiology. To find out the hub genes and pathways involved in the pathogenesis of EMs, three raw microarray datasets were recruited from the Gene Expression Omnibus database (GEO). Then, a series of bioinformatics technologies including gene ontology (GO), Hallmark pathway enrichment, protein-protein interaction (PPI) network and gene co-expression correlation analysis were performed to identify hub genes. The hub genes were further verified by the Real-time quantitative polymerase chain reaction (RT-PCR) and Western Blot (WB). We identified 129 differentially expressed genes (DEGs) in EMs, of which 78 were up-regulated and 51 were down-regulated. Through GO functional enrichment analysis, we found that the DEGs are mainly enriched in cell adhesion, extracellular matrix remodeling, chemokine regulation, angiogenesis regulation, epithelial cell proliferation, et al. In Hallmark pathway enrichment analysis, coagulation pathway showed great significance and the terms in which included the central complement factors. Moreover, the genes were dominating in PPI network. Combined co-expression analysis with experimental verification, we found that the up-regulated expression of complement (C1S, C1QA, C1R, and C3) was positively related to tissue factor (TF) in EMs. In this study, we discovered the over expression complement and the positive correlation between complement and TF in EMs, which suggested that interaction of complement and coagulation system may play a role within the pathophysiology of EMS.


Subject(s)
Blood Coagulation Factors/genetics , Complement System Proteins/genetics , Endometriosis/genetics , Gene Expression Profiling/methods , Blood Coagulation Factors/metabolism , Complement C1q/genetics , Complement C1q/metabolism , Complement C1r/genetics , Complement C1r/metabolism , Complement C1s/genetics , Complement C1s/metabolism , Complement C3/genetics , Complement C3/metabolism , Complement System Proteins/metabolism , Endometriosis/metabolism , Female , Gene Ontology , Gene Regulatory Networks , Humans , Protein Interaction Maps/genetics , Signal Transduction/genetics , Thromboplastin/genetics , Thromboplastin/metabolism
6.
Genes (Basel) ; 12(3)2021 03 20.
Article in English | MEDLINE | ID: mdl-33804666

ABSTRACT

Late-onset Alzheimer's disease (LOAD), the most common cause of dementia, and a huge global health challenge, is a neurodegenerative disease of uncertain aetiology. To deliver effective diagnostics and therapeutics, understanding the molecular basis of the disease is essential. Contemporary large genome-wide association studies (GWAS) have identified over seventy novel genetic susceptibility loci for LOAD. Most are implicated in microglial or inflammatory pathways, bringing inflammation to the fore as a candidate pathological pathway. Among the most significant GWAS hits are three complement genes: CLU, encoding the fluid-phase complement inhibitor clusterin; CR1 encoding complement receptor 1 (CR1); and recently, C1S encoding the complement enzyme C1s. Complement activation is a critical driver of inflammation; changes in complement genes may impact risk by altering the inflammatory status in the brain. To assess complement gene association with LOAD risk, we manually created a comprehensive complement gene list and tested these in gene-set analysis with LOAD summary statistics. We confirmed associations of CLU and CR1 genes with LOAD but showed no significant associations for the complement gene-set when excluding CLU and CR1. No significant association with other complement genes, including C1S, was seen in the IGAP dataset; however, these may emerge from larger datasets.


Subject(s)
Alzheimer Disease/genetics , Clusterin/genetics , Complement C1s/genetics , Receptors, Complement 3b/genetics , Age of Onset , Complement Activation , Databases, Genetic , Genetic Predisposition to Disease , Genome-Wide Association Study , Humans
7.
Clin Genet ; 100(2): 206-212, 2021 08.
Article in English | MEDLINE | ID: mdl-33890303

ABSTRACT

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 13 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.


Subject(s)
Ehlers-Danlos Syndrome/etiology , Mutation , Adolescent , Adult , Aged , Aortic Aneurysm, Abdominal/genetics , Child, Preschool , Complement C1r/genetics , Complement C1s/genetics , Ehlers-Danlos Syndrome/genetics , Female , Heterozygote , Humans , Male , Middle Aged , Varicose Ulcer/etiology , Varicose Ulcer/genetics , Young Adult
8.
Aging (Albany NY) ; 13(4): 5120-5135, 2021 02 01.
Article in English | MEDLINE | ID: mdl-33535173

ABSTRACT

Chronic obstructive pulmonary disease (COPD) is a heterogeneous condition associated with high morbidity and mortality. This study aimed to use weighted gene co-expression network analysis (WGCNA) to explore the molecular pathogenesis of the emphysema phenotype of COPD. After obtaining lung mRNA expression profiles from ten patients with the emphysema phenotype of COPD and eight controls, emphysema-associated gene modules were identified with WGCNA. Among 13 distinct modules, the green-yellow and brown modules showed the strongest correlations with emphysema severity and lung function and were thus selected as hub modules. On gene ontology analysis, these two modules were mainly enriched in immune response, B cell receptor (BCR) signaling pathway, extracellular matrix (ECM) organization, and collagen fibril organization. Pathway analysis primarily showed enrichment in BCR signaling pathways, ECM receptor interaction, and NF-κB and TGF-ß signaling pathways for the two hub modules. Several genes, including FCRLA, MS4A1, CD19, FKBP10, C1S and HTRA1, among others, were identified as hub genes. Our results shed light on the potential genetic mechanisms underlying the pathogenesis of the emphysema phenotype of COPD. However, further research will be needed to confirm the involvement of the identified genes and to determine their therapeutic relevance.


Subject(s)
Gene Regulatory Networks , Pulmonary Disease, Chronic Obstructive/genetics , Pulmonary Emphysema/genetics , Aged , Antigens, CD19/genetics , Case-Control Studies , Complement C1s/genetics , Extracellular Matrix/genetics , Female , High-Temperature Requirement A Serine Peptidase 1/genetics , Humans , Male , Middle Aged , NF-kappa B/genetics , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Emphysema/metabolism , RNA, Messenger , Receptors, Antigen, B-Cell/genetics , Receptors, Fc/genetics , Tacrolimus Binding Proteins/genetics , Transcriptome , Transforming Growth Factor beta/genetics
9.
Osteoarthritis Cartilage ; 28(5): 675-684, 2020 05.
Article in English | MEDLINE | ID: mdl-31634584

ABSTRACT

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.


Subject(s)
Chondrocytes/metabolism , Complement C1q/genetics , Complement C1r/genetics , Complement C1s/genetics , Osteoarthritis, Knee/genetics , RNA, Messenger/metabolism , Animals , Cartilage, Articular/cytology , Collagen Type II/genetics , Collagen Type X/genetics , Gene Expression Regulation , Humans , Mice , Osteoarthritis, Knee/metabolism , Pilot Projects
10.
Am J Physiol Renal Physiol ; 317(5): F1293-F1304, 2019 11 01.
Article in English | MEDLINE | ID: mdl-31509012

ABSTRACT

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-ß), 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.


Subject(s)
Complement C1r/metabolism , Kidney Diseases/enzymology , Animals , Cell Line , Complement C1r/genetics , Complement C1s/genetics , Complement C1s/metabolism , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Folic Acid/pharmacology , Gene Expression Regulation, Enzymologic , Humans , Inflammation , Kidney/cytology , Kidney Diseases/genetics , Male , Mice , Mice, Knockout , RNA, Messenger/genetics , RNA, Messenger/metabolism
11.
Biotechnol Bioeng ; 116(9): 2130-2145, 2019 09.
Article in English | MEDLINE | ID: mdl-31087560

ABSTRACT

Proteolysis associated with recombinant protein expression in Chinese Hamster Ovary (CHO) cells has hindered the development of biologics including HIV vaccines. When expressed in CHO cells, the recombinant HIV envelope protein, gp120, undergoes proteolytic clipping by a serine protease at a key epitope recognized by neutralizing antibodies. The problem is particularly acute for envelope proteins from clade B viruses that represent the major genetic subtype circulating in much of the developed world, including the US and Europe. In this paper, we have identified complement Component 1's (C1s), a serine protease from the complement cascade, as the protease responsible for the proteolysis of gp120 in CHO cells. CRISPR/Cas9 knockout of the C1s protease in a CHO cell line was shown to eliminate the proteolytic activity against the recombinantly expressed gp120. In addition, the C1s-/- MGAT1- CHO cell line, with the C1s protease and the MGAT1 glycosyltransferase knocked out, enabled the production of unclipped gp120 from a clade B isolate (BaL-rgp120) and enriched for mannose-5 glycans on gp120 that are required for the binding of multiple broadly neutralizing monoclonal antibodies (bN-mAbs). The availability of this technology will allow for the scale-up and testing of multiple vaccine concepts in regions of the world where clade B viruses are in circulation. Furthermore, the proteolysis issues caused by the C1s protease suggests a broader need for a C1s-deficient CHO cell line to express other recombinant proteins that are susceptible to serine protease activity in CHO cells. Similarly, the workflow described here to identify and knockout C1s in a CHO cell line can be applied to remedy the proteolysis of biologics by other CHO proteases.


Subject(s)
CRISPR-Cas Systems , Complement C1s/genetics , Complement C1s/metabolism , Gene Knockout Techniques , HIV Envelope Protein gp120/biosynthesis , HIV-1 , Proteolysis , Animals , CHO Cells , Cricetulus , HIV Envelope Protein gp120/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics
12.
Nucleic Acid Ther ; 29(2): 82-91, 2019 04.
Article in English | MEDLINE | ID: mdl-30817230

ABSTRACT

Kallikrein is the key contact system mediator responsible for the conversion of high-molecular-weight kininogen into the inflammatory vasodilator peptide bradykinin, a process regulated by C1-esterase inhibitor (C1-INH). In hereditary angioedema (HAE), genetic mutations result in deficient or dysfunctional C1-INH and dysregulation of the contact system leading to recurrent, sometimes fatal, angioedema attacks. IONIS-PKKRx is a second-generation 2'-O-(2-methoxyethyl)-modified chimeric antisense oligonucleotide, designed to bind and selectively reduce prekallikrein (PKK) mRNA in the liver. IONIS-PKKRx demonstrated dose-dependent reduction of human prekallikrein hepatic mRNA and plasma protein in transgenic mice and dose- and time-dependent reductions of plasma PKK in Cynomolgus monkeys. Similar dose-dependent reductions of plasma PKK levels were observed in healthy human volunteers accompanied by decreases in bradykinin generation capacity with an acceptable safety and tolerability profile. These results highlight a novel and specific approach to target PKK for the treatment of HAE and other diseases involving contact system activation and overproduction of bradykinin.


Subject(s)
Angioedemas, Hereditary/therapy , Bradykinin/genetics , Complement C1s/genetics , Prekallikrein/genetics , Angioedemas, Hereditary/blood , Angioedemas, Hereditary/genetics , Animals , Animals, Genetically Modified/blood , Bradykinin/blood , Complement C1 Inhibitor Protein/pharmacology , Complement C1s/antagonists & inhibitors , Dose-Response Relationship, Drug , Healthy Volunteers , Humans , Liver/drug effects , Liver/metabolism , Macaca fascicularis/blood , Mice , Oligodeoxyribonucleotides, Antisense/genetics , Oligodeoxyribonucleotides, Antisense/pharmacology , Prekallikrein/antagonists & inhibitors
13.
Genes Immun ; 20(2): 121-130, 2019 02.
Article in English | MEDLINE | ID: mdl-29550838

ABSTRACT

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.


Subject(s)
Complement C1r/genetics , Complement C1s/genetics , Lupus Erythematosus, Systemic/genetics , Animals , Complement C1r/deficiency , Complement C1s/deficiency , Female , Gene Deletion , Mice , Mice, Inbred ICR
14.
Front Immunol ; 10: 2962, 2019.
Article in English | MEDLINE | ID: mdl-31921203

ABSTRACT

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.


Subject(s)
Complement C1r , Complement C1s , Ehlers-Danlos Syndrome , Mutation, Missense , Periodontal Diseases , Amino Acid Substitution , Complement C1r/genetics , Complement C1r/immunology , Complement C1s/genetics , Complement C1s/immunology , Ehlers-Danlos Syndrome/genetics , Ehlers-Danlos Syndrome/immunology , Ehlers-Danlos Syndrome/pathology , HEK293 Cells , Humans , Periodontal Diseases/genetics , Periodontal Diseases/immunology , Periodontal Diseases/pathology , Protein Folding
15.
Dev Comp Immunol ; 81: 279-290, 2018 04.
Article in English | MEDLINE | ID: mdl-29247723

ABSTRACT

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.


Subject(s)
Complement C1r/genetics , Complement C1s/genetics , Fish Proteins/genetics , Liver/physiology , Perciformes/immunology , Animals , Cloning, Molecular , Complement C1r/metabolism , Complement C1s/metabolism , Conserved Sequence/genetics , Fish Proteins/metabolism , Immunity, Innate , Perciformes/genetics , Phylogeny , Protein Domains/genetics , Proteolysis , Sequence Alignment , Transcriptome , Vertebrates
16.
Fish Shellfish Immunol ; 70: 240-251, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28882800

ABSTRACT

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.


Subject(s)
Cichlids/genetics , Cichlids/immunology , Complement C1r/genetics , Complement C1s/genetics , Fish Diseases/immunology , Gene Expression Regulation/immunology , Immunity, Innate/genetics , Amino Acid Sequence , Animals , Complement C1r/chemistry , Complement C1r/metabolism , Complement C1s/chemistry , Complement C1s/metabolism , Computational Biology , Fish Proteins/chemistry , Fish Proteins/genetics , Fish Proteins/metabolism , Gene Expression Profiling/veterinary , Lipopolysaccharides/pharmacology , Organ Specificity , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment/veterinary , Streptococcal Infections/veterinary , Streptococcus agalactiae/physiology , Up-Regulation
17.
Proc Natl Acad Sci U S A ; 114(5): 986-991, 2017 01 31.
Article in English | MEDLINE | ID: mdl-28104818

ABSTRACT

The complement system is an important antimicrobial and inflammation-generating component of the innate immune system. The classical pathway of complement is activated upon binding of the 774-kDa C1 complex, consisting of the recognition molecule C1q and the tetrameric protease complex C1r2s2, to a variety of activators presenting specific molecular patterns such as IgG- and IgM-containing immune complexes. A canonical model entails a C1r2s2 with its serine protease domains tightly packed together in the center of C1 and an intricate intramolecular reaction mechanism for activation of C1r and C1s, induced upon C1 binding to the activator. Here, we show that the serine protease domains of C1r and C1s are located at the periphery of the C1r2s2 tetramer both when alone or within the nonactivated C1 complex. Our structural studies indicate that the C1 complex adopts a conformation incompatible with intramolecular activation of C1, suggesting instead that intermolecular proteolytic activation between neighboring C1 complexes bound to a complement activating surface occurs. Our results rationalize how a multitude of structurally unrelated molecular patterns can activate C1 and suggests a conserved mechanism for complement activation through the classical and the related lectin pathway.


Subject(s)
Complement C1r/chemistry , Complement C1s/chemistry , Complement Pathway, Classical/physiology , Complement C1r/genetics , Complement C1r/metabolism , Complement C1s/genetics , Complement C1s/metabolism , Enzyme Activation , Genes, Synthetic , HEK293 Cells , Humans , Immunity, Innate , Microscopy, Electron , Models, Molecular , Protein Conformation , Recombinant Proteins/chemistry , Scattering, Small Angle , Structure-Activity Relationship , X-Ray Diffraction
18.
Am J Hum Genet ; 99(5): 1005-1014, 2016 Nov 03.
Article in English | MEDLINE | ID: mdl-27745832

ABSTRACT

Periodontal Ehlers-Danlos syndrome (pEDS) is an autosomal-dominant disorder characterized by early-onset periodontitis leading to premature loss of teeth, joint hypermobility, and mild skin findings. A locus was mapped to an approximately 5.8 Mb region at 12p13.1 but no candidate gene was identified. In an international consortium we recruited 19 independent families comprising 107 individuals with pEDS to identify the locus, characterize the clinical details in those with defined genetic causes, and try to understand the physiological basis of the condition. In 17 of these families, we identified heterozygous missense or in-frame insertion/deletion mutations in C1R (15 families) or C1S (2 families), contiguous genes in the mapped locus that encode subunits C1r and C1s of the first component of the classical complement pathway. These two proteins form a heterotetramer that then combines with six C1q subunits. Pathogenic variants involve the subunit interfaces or inter-domain hinges of C1r and C1s and are associated with intracellular retention and mild endoplasmic reticulum enlargement. Clinical features of affected individuals in these families include rapidly progressing periodontitis with onset in the teens or childhood, a previously unrecognized lack of attached gingiva, pretibial hyperpigmentation, skin and vascular fragility, easy bruising, and variable musculoskeletal symptoms. Our findings open a connection between the inflammatory classical complement pathway and connective tissue homeostasis.


Subject(s)
Complement C1r/genetics , Complement C1s/genetics , Ehlers-Danlos Syndrome/genetics , Gene Deletion , Mutation, Missense , Periodontitis/genetics , Adolescent , Adult , Child , Child, Preschool , Chromosome Mapping , Chromosomes, Human, Pair 12/genetics , Ehlers-Danlos Syndrome/diagnosis , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Exome , Female , Genetic Loci , Humans , Male , Pedigree , Periodontitis/diagnosis , Protein Conformation , Young Adult
19.
BMC Nephrol ; 17(1): 128, 2016 09 09.
Article in English | MEDLINE | ID: mdl-27613243

ABSTRACT

BACKGROUND: This study sought to investigate crucial genes correlated with diabetic nephropathy (DN), and their potential functions, which might contribute to a better understanding of DN pathogenesis. METHODS: The microarray dataset GSE1009 was downloaded from Gene Expression Omnibus, including 3 diabetic glomeruli samples and 3 healthy glomeruli samples. The differentially expressed genes (DEGs) were identified by LIMMA package. Their potential functions were then analyzed by the GO and KEGG pathway enrichment analyses using the DAVID database. Furthermore, miRNAs and transcription factors (TFs) regulating DEGs were predicted by the GeneCoDis tool, and miRNA-DEG-TF regulatory network was visualized by Cytoscape. Additionally, the expression of DEGs was validated using another microarray dataset GSE30528. RESULTS: Totally, 14 up-regulated DEGs and 430 down-regulated ones were identified. Some DEGs (e.g. MTSS1, CALD1 and ACTN4) were markedly relative to cytoskeleton organization. Besides, some other ones were correlated with arrhythmogenic right ventricular cardiomyopathy (e.g. ACTN4, CTNNA1 and ITGB5), as well as complement and coagulation cascades (e.g. C1R and C1S). Furthermore, a series of miRNAs and TFs modulating DEGs were identified. The transcription factor LEF1 regulated the majority of DEGs, such as ITGB5, CALD1 and C1S. Hsa-miR-33a modulated 28 genes, such as C1S. Additionally, 143 DEGs (one upregulated gene and 142 downregulated genes) were also differentially expressed in another dataset GSE30528. CONCLUSIONS: The genes involved in cytoskeleton organization, cardiomyopathy, as well as complement and coagulation cascades may be closely implicated in the progression of DN, via the regulation of miRNAs and TFs.


Subject(s)
Diabetic Nephropathies/genetics , MicroRNAs/genetics , Transcription Factors/genetics , Transcriptome , Actinin/genetics , Calmodulin-Binding Proteins/genetics , Cardiomyopathies/genetics , Complement C1r/genetics , Complement C1s/genetics , Cytoskeleton/genetics , Databases, Genetic , Down-Regulation , Gene Expression Profiling , Humans , Integrin beta Chains/genetics , Lymphoid Enhancer-Binding Factor 1/genetics , Microfilament Proteins/genetics , Neoplasm Proteins/genetics , Oligonucleotide Array Sequence Analysis , Up-Regulation , alpha Catenin/genetics
20.
Fish Shellfish Immunol ; 46(2): 656-68, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26241508

ABSTRACT

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.


Subject(s)
Complement C1r/genetics , Complement C1s/genetics , Fish Diseases/immunology , Fish Proteins/genetics , Immunity, Innate , Perciformes , Amino Acid Sequence , Animals , Complement C1r/chemistry , Complement C1r/metabolism , Complement C1s/chemistry , Complement C1s/metabolism , DNA Virus Infections/immunology , DNA Virus Infections/virology , Edwardsiella tarda/physiology , Enterobacteriaceae Infections/immunology , Enterobacteriaceae Infections/microbiology , Fish Diseases/microbiology , Fish Diseases/virology , Fish Proteins/chemistry , Fish Proteins/metabolism , Head Kidney/virology , Iridoviridae/physiology , Liver/virology , Molecular Sequence Data , Organ Specificity , Phylogeny , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment/veterinary , Streptococcal Infections/immunology , Streptococcal Infections/microbiology , Streptococcus/physiology
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