Genetic Insights into the Impact of Complement in Alzheimer's Disease.

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.

Multi-omics analysis reveals the interaction between the complement system and the coagulation cascade in the development of endometriosis.

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.

Complement C1r serine protease contributes to kidney fibrosis.

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.

C1r and C1s from Nile tilapia (Oreochromis niloticus): Molecular characterization, transcriptional profiling upon bacterial and IFN-γ inductions and potential role in response to bacterial infection.

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.

Crucial genes associated with diabetic nephropathy explored by microarray analysis.

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.

Characterization of rock bream (Oplegnathus fasciatus) complement components C1r and C1s in terms of molecular aspects, genomic modulation, and immune responsive transcriptional profiles following bacterial and viral pathogen exposure.

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.

Expression of recombinant human complement C1q allows identification of the C1r/C1s-binding sites.

Complement C1q is a hexameric molecule assembled from 18 polypeptide chains of three different types encoded by three genes. This versatile recognition protein senses a wide variety of immune and nonimmune ligands, including pathogens and altered self components, and triggers the classical complement pathway through activation of its associated proteases C1r and C1s. We report a method for expression of recombinant full-length human C1q involving stable transfection of HEK 293-F mammalian cells and fusion of an affinity tag to the C-terminal end of the C chain. The resulting recombinant (r) C1q molecule is similar to serum C1q as judged from biochemical and structural analyses and exhibits the characteristic shape of a bunch of flowers. Analysis of its interaction properties by surface plasmon resonance shows that rC1q retains the ability of serum C1q to associate with the C1s-C1r-C1r-C1s tetramer, to recognize physiological C1q ligands such as IgG and pentraxin 3, and to trigger C1r and C1s activation. Functional analysis of rC1q variants carrying mutations of LysA59, LysB61, and/or LysC58, in the collagen-like stems, demonstrates that LysB61 and LysC58 each play a key role in the interaction with C1s-C1r-C1r-C1s, with LysA59 being involved to a lesser degree. We propose that LysB61 and LysC58 both form salt bridges with outer acidic Ca(2+) ligands of the C1r and C1s CUB (complement C1r/C1s, Uegf, bone morphogenetic protein) domains. The expression method reported here opens the way for deciphering the molecular basis of the unusual binding versatility of C1q by mapping the residues involved in the sensing of its targets and the binding of its receptors.

Prohaptoglobin is proteolytically cleaved in the endoplasmic reticulum by the complement C1r-like protein.

Many secretory proteins are synthesized as proforms that become biologically active through a proteolytic cleavage in the trans-Golgi complex or at a later stage in the secretory pathway. Haptoglobin (Hp) is unusual in that it is cleaved in the endoplasmic reticulum before it enters the Golgi. Here, we present evidence that the recently discovered complement C1r-like protein (C1r-LP) mediates this cleavage. C1r-LP has not previously been shown to possess proteolytic activity, despite its homology to trypsin-like Ser proteinases. We demonstrate that coexpression of the proform of Hp (proHp) and C1r-LP in COS-1 cells effected cleavage of proHp in the endoplasmic reticulum. This cleavage depended on proteolytic activity of C1r-LP because mutation of the putative active-site Ser residue abolished the reaction. Furthermore, incubation of affinity-purified C1r-LP and proHp led to the cleavage of the latter protein. ProHp appeared to be cleaved at the expected site because substitution of Gly for Arg-161 blocked the reaction. C1r-LP showed specificity for proHp, in that it did not cleave the proform of complement C1s, a protein similar to Hp particularly around the cleavage site. C1r-LP accounts for at least part of the endogenous proHp-cleavage activity because suppression of the C1r-LP expression by RNA interference reduced the cleavage of proHp by up to 45% in the cells of a human hepatoma cell line (HepG2).

Increased expression and secretion of r-Gsp protein, rat counterpart of complement C1s precursor, during cyclic AMP-induced differentiation in rat C6 glioma cells.

The gene, termed r-gsp, was originally isolated during identification of differentiation-associated molecules in rat C6 glial cells. Its mRNA expression was markedly increased during cAMP-induced glial cell differentiation. The deduced amino acid sequence of r-gsp was homologous to those of complement C1s precursors of hamsters and humans. In the present study, we raised anti-peptide antibody against r-Gsp protein and analyzed its change during cAMP-induced differentiation. The 90-kDa r-Gsp protein increased time-dependently and reached the maximal level ( approximately 7.6-fold increase) at 24 h in response to dibutyryl cyclic AMP (dbcAMP) and theophylline. Moreover, it was secreted into the medium and then was cleaved to form disulfide-linked fragments, one of which was 30 kDa, similar to C1s, suggesting its processing in the extracellular space. In fact, the partially purified r-Gsp from culture medium was cleaved by active human C1r to form a 30-kDa polypeptide. Moreover, secreted r-Gsp protein cleaved human C4alpha to yield C4alpha' and associated with human serum C1-esterase inhibitor, strongly suggesting that r-Gsp protein is rat C1s. However, in C6 cells overexpressing r-Gsp, their morphology and proliferation rate were similar to those in parent C6 cells. These results suggest that r-Gsp protein could not induce glial differentiation alone, and suggest that r-Gsp protein was secreted as a proenzyme and processed in culture medium. Its possible role in glial cell differentiation will be discussed.

Functional characterization of human mannose-binding lectin-associated serine protease (MASP)-1/3 and MASP-2 promoters, and comparison with the C1s promoter.

The 5'-flanking regions of the genes encoding human mannose-binding lectin-associated serine protease (MASP)-1/3 and MASP-2, key enzymes in the lectin complement pathway, were isolated and characterized. The features of their promoters were compared with those of the human gene for C1s, the effector component of the classical pathway. The sequences upstream from the transcription start sites of the three genes contained the elements essential for transcription and liver-specific expression. Transient expression of constructs of these genes fused to the luciferase reporter gene confirmed their liver-specific expression and showed that the MASP promoters were slightly up-regulated by the presence of IL-1beta. The stimulatory effects of IL-1beta on MASP1/3 and MASP2 gene expression were abolished by the simultaneous presence of IL-6. MASP-1/3 promoter activity was also down-regulated by IFN-gamma. In contrast, C1s promoter activity was strongly up-regulated by IL-6, IL-1beta and IFN-gamma. These results indicate that IL-6 and IFN-gamma affect the expression of the MASP genes in a different fashion from that of the C1s gene, implying differential regulatory effects of these cytokines on the biosynthesis of lectin pathway-specific serine proteases and classical pathway-specific serine proteases.

Selective complement C1s deficiency caused by homozygous four-base deletion in the C1s gene.

The complement system plays an important role in defense mechanisms by promoting the adherence of microorganisms to phagocytic cells and lysis of foreign organisms. Deficiencies of the first complement components, C1r/C1s, often cause systemic lupus erythematosus-like syndromes and severe pyogenic infections. Up to now no genetic analysis of the C1r/C1s deficiencies has been carried out. In the present work, we report the first genetic analysis of selective C1s deficiency, the patient having a normal amount of C1r. C1s RNA with a normal size was detected in patient's subcutaneous fibroblasts (YKF) by RNA blot analysis and RT-PCR. The amount of C1s RNA was approximately one-tenth of the RNA from the human chondrosarcoma cell line, HCS2/8. In contrast, the levels of C1r and beta-actin RNA of YKF were similar to that of HCS2/8. Sequence analysis of C1s cDNA revealed a deletion at nucleotides 1087-1090 (TTTG), creating a stop codon (TGA) at position 94 downstream of the mutation site. Direct sequencing of the gene between the primers designed on intron 9 and exon 10 indicated the presence of the deletion on exon 10 of the gene. Quantitative Southern blot hybridization suggested the mutation was homozygous. The 4-bp deletion on exon 10 was also found in the patient's heterozygous mother who had normal hemolytic activity.

Site-directed mutagenesis of hamster complement C1S: characterization with an active form-specific antibody and possible involvement of C1S in tumorigenicity.

We have previously shown that non-transformed mouse A31 cells became tumorigenic when they were transfected with hamster C1s cDNA expression plasmid BCMGSNeoCS. In the present study, mutations were introduced into the cDNA at the activation cleavage site, Arg423(AGG) and the active center Ser617(AGC). These amino-acids were replaced by His423(CAC) and Thr617(ACC), respectively. The mutated cDNAs were inserted into BCMGSNeo and transfected to A31 and its polyoma-virus-transformed SEA7 cells. C1s produced from these transfectants lost their enzyme activity. Transfectants of these mutated C1s cDNA did not form tumors in nude mice, To distinguish between active and inactive C1s in situ, we have developed novel antibodies, one directed to the NH2-terminal neoepitope of the L chain and the other specific for uncleaved inactive C1s. These antibodies were used to characterize C1s produced by transfectants, so as to determine whether or not it was cleaved at the right position.

Substrate specificities of the protease of mouse serum Ra-reactive factor.

Ra-reactive factor (RaRF) is a serum bactericidal factor whose function seems to be to activate C in a manner similar to that of C1, but with activation triggered by binding to bacterial polysaccharides instead of to immune complexes. It is composed of multiple polysaccharide-binding subunits associated with a novel serine protease, and its overall structural organization is similar to that of C1. This similarity extends to the serine protease component, which shares a similar modular construction and about 40% sequence identity with the C1r and C1s subcomponents of C1. In this study, we examined the substrate specificity of mouse RaRF by assaying its ability to cleave C components C3, C4, and C5, and its activity against the murine C4 isotype, sex-limited protein. Our results revealed that RaRF preferentially cleaves the C4 alpha-chain with specific activities 20- to 100-fold greater than either human or murine C1s, and that RaRF also cleaves the C3 alpha-chain, but with a lower efficiency than C4 alpha. We also found that RaRF is much less sensitive than C1s to mutations near the proteolytic site and that the two proteases show different reactivities against synthetic substrates. Hence, although the RaRF protease and C1s have similar structures and play similar roles in C activation, they also display clear differences in substrate range and in the details of their substrate recognition mechanisms. Finally, we found that RaRF does not cleave sex-limited protein even at a level 100-fold higher than necessary for C4 cleavage.

Tumorigenicity of BALB3T3 A31 cells transfected with hamster-complement-C1s cDNA.

Hamster-complement-C1s cDNA was inserted into an expression plasmid BCMGSNeo (BCMGSNeoHACS). BALB/c mouse fibroblast A31 cells, which do not produce C1s, were transfected with BCMGSNeoHACS and the transfectants were selected with G418. Normal C1s production by the transfectants was confirmed by Northern and immunoblot analysis and by an esterase assay. To examine the tumorigenicity of the transfectants, 1 x 10(6) cells were injected s.c. into 6-week-old BALB/c nu/nu mice. Three C1s cDNA transfectants (A3CS9, A3CS12, A3CS13) formed tumors whereas both A31 and A31 transfected with the vector alone (A3BCM1 and A3BCM3) did not. The tumors derived from the transfectants showed invasive growth, and many capillaries were observed in the tumors. A tumor derived from A3CS13 was examined immunohistochemically and found to be reactive with an anti-C1s monoclonal antibody. Tumor cells were cultured in vitro again and C1s secreted into the culture medium was examined by immunoblot analysis. C1s synthesized by the tumor cells derived from A3CS13 maintained its biological functions. Tumor cells derived from A3CS9 and A3CS12 cells, however, produced C1s having abnormal disulfide bonds.

A 100-kDa protein in the C4-activating component of Ra-reactive factor is a new serine protease having module organization similar to C1r and C1s.

Ra-reactive factor (RaRF), a C-dependent bactericidal factor in mice, is composed of one polysaccharide-binding component and one C4/C2-activating component. The former is an oligomer of 28-kDa protein corresponding to the mannose-binding protein of mice. The 100-kDa protein, P100, has been shown to be present in the C4/C2-activating component. This protein generates 29- and 70-kDa polypeptide chains when reduced. In this study, we determined the nucleotide sequence of cDNA coding for P100. cDNAs were prepared by reverse transcription PCR and cassette-ligation-mediated PCR on mRNA from BALB/c mouse liver, using primers synthesized by reference to the sequence determined in a previous study. The results of cDNA sequencing indicate that the precursor protein of P100 containing a 24-residue signal peptide consists of 704 amino acid residues. Taking the results of the previous electrophoretic study into consideration, it is thought that the cleavage of mature P100 protein generates a 29-kDa chain of 251 residues and a 70-kDa chain of 429 residues. Although homology in the amino acid sequence of P100 with that of human C1r and C1s subcomponents of C was less than 40%, a striking similarity in domain organization was found among these proteins, indicating that P100 is a new C4-activating serine protease structurally similar to C1r and C1s. Northern hybridization showed that the liver was the primary site of the expression of the P100 gene.

Identification of the disulfide bonds of human complement C1s.

C1s, one of the three subcomponents of C1, the first component of the complement system, is a complex serine protease. To determine the disulfide-bonding pattern, fragments of C1s were generated by cleavage with pepsin, thermolysin, or subtilisin. Disulfide bonds have been identified by several methods, for example, direct observation of the phenylthiohydantoin derivative of cystine during Edman degradation of isolated peptides and placement in the known cDNA sequence. All of the 26 half-cystines are linked in disulfide bonds occurring at positions 50-68, 120-132, 128-141, 143-156, 160-187, 219-236, 279-326, 306-339, 344-388, 371-406, 410-534, 580-603, and 613-644. All of the disulfide bonds of the earlier described substructures of C1s, the EGF-homologous part, the two SCR units, and the two domains typical for C1s and C1r are localized within these domains.

Structure and function of C1r and C1s: current concepts.

C1r and C1s, the constituent proteins of C1s-C1r-C1r-C1s, the Ca2+ -dependent catalytic unit of C1, are homologous serine proteinases that share a common activation pattern and have similar structural organizations at the monomeric level. In both cases, activation occurs through cleavage of a single Arg-Ile bond, which converts the single-chain proenzymes into active proteinases comprising two chains linked by a single disulphide bridge. Both NH2-terminal A chains are sub-divided into five structural units (I-V) including a single copy of an Epidermal Growth Factor-like segment (II) and two different pairs of internal repeats (I/III and IV/V). Regions I and III have no equivalent in other proteins, whereas regions IV and V are homologous to short consensus repeats found, in particular, in complement proteins C2, B, H, C4b-binding protein and CR1. The COOH-terminal B chains are homologous to the catalytic chains of serine proteinases, but lack the "histidine-loop", a disulphide bridge common to all other known mammalian serine proteinases. Overall sequence comparison of C1r and C1s reveals 40% amino acid identity and conservation of all cysteine residues. In contrast, C1r and C1s widely differ from each other by their glycosylation patterns: both proteins contain Asn-linked carbohydrates, but four glycosylation sites are present on C1r, and only two on C1s.(ABSTRACT TRUNCATED AT 250 WORDS)

Complete cDNA sequence of human complement Cls and close physical linkage of the homologous genes Cls and Clr.

Overlapping molecular clones encoding the complement subcomponent Cls were isolated from a human liver cDNA library. The nucleotide sequence reconstructed from these clones spans about 85% of the length of the liver Cls messenger RNAs, which occur in three distinct size classes around 3 kilobases in length. Comparisons with the sequence of Clr, the other enzymatic subcomponent of Cl, reveal 40% amino acid identity and conservation of all the cysteine residues. Beside the serine protease domain, the following sequence motifs, previously described in Clr, were also found in Cls: (a) two repeats of the type found in the Ba fragment of complement factor B and in several other complement but also noncomplement proteins, (b) a cysteine-rich segment homologous to the repeats of epidermal growth factor precursor, and (c) a duplicated segment found only in Clr and Cls. Differences in each of these structural motifs provide significant clues for the interpretation of the functional divergence of these interacting serine protease zymogens. Hybridizations of Clr and Cls probes to restriction endonuclease fragments of genomic DNA demonstrate close physical linkage of the corresponding genes. The implications of this finding are discussed with respect to the evolution of Clr and Cls after their origin by tandem gene duplication and to the previously observed combined hereditary deficiencies of Clr and Cls.