Expression of recombinant human complement C1q allows identification of the C1r/C1s-binding sites.
ABSTRACT: 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.
Project description: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.
Project description:The association and activation states of complement subcomponents C1r and C1s biosynthesized by Hep G2 cells were studied. C1r and C1s are secreted in stoichiometric amounts; in the presence of Ca2+ they are associated in a complex that sediments similarly to plasma C1r2-C1s2. Both compounds are synthesized as monomer proteins of apparent Mr 86 000. C1r is secreted as a dimer. Secreted C1r is not autoactivatable but undergoes proteolysis by exogenous C1r; secreted C1s is also proteolysed by exogenous C1r. In the presence of immune-complex-bound C1q, secreted C1r and C1s are able to reconstitute C1, but normal activation requires extrinsic C1r2-C1s2.
Project description:BACKGROUND:The incidence of epidermal keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is increasing worldwide. OBJECTIVES:To study the role of the complement classical pathway components C1q, C1r and C1s in the progression of cSCC. METHODS:The mRNA levels of C1Q subunits and C1R and C1S in cSCC cell lines, normal human epidermal keratinocytes, cSCC tumours in vivo and normal skin were analysed with quantitative real-time polymerase chain reaction. The production of C1r and C1s was determined with Western blotting. The expression of C1r and C1s in tissue samples in vivo was analysed with immunohistochemistry and further investigated in human cSCC xenografts by knocking down C1r and C1s. RESULTS:Significantly elevated C1R and C1S mRNA levels and production of C1r and C1s were detected in cSCC cells, compared with normal human epidermal keratinocytes. The mRNA levels of C1R and C1S were markedly elevated in cSCC tumours in vivo compared with normal skin. Abundant expression of C1r and C1s by tumour cells was detected in invasive sporadic cSCCs and recessive dystrophic epidermolysis bullosa-associated cSCCs, whereas the expression of C1r and C1s was lower in cSCC in situ, actinic keratosis and normal skin. Knockdown of C1r and C1s expression in cSCC cells inhibited activation of extracellular signal-related kinase 1/2 and Akt, promoted apoptosis of cSCC cells and significantly suppressed growth and vascularization of human cSCC xenograft tumours in vivo. CONCLUSIONS:These results provide evidence for the role of tumour-cell-derived C1r and C1s in the progression of cSCC and identify them as biomarkers and putative therapeutic targets in cSCC. What's already known about this topic? The incidences of actinic keratosis, cutaneous squamous cell carcinoma (cSCC) in situ and invasive cSCC are increasing globally. Few specific biomarkers for progression of cSCC have been identified, and no biological markers are in clinical use to predict the aggressiveness of actinic keratosis, cSCC in situ and invasive cSCC. What does this study add? Our results provide novel evidence for the role of complement classical pathway components C1r and C1s in the progression of cSCC. What is the translational message? Our results identify complement classical pathway components C1r and C1s as biomarkers and putative therapeutic targets in cSCC.
Project description: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.
Project description:C1, the complex that triggers the classic pathway of complement, is a 790-kDa assembly resulting from association of a recognition protein C1q with a Ca(2+)-dependent tetramer comprising two copies of the proteases C1r and C1s. Early structural investigations have shown that the extended C1s-C1r-C1r-C1s tetramer folds into a compact conformation in C1. Recent site-directed mutagenesis studies have identified the C1q-binding sites in C1r and C1s and led to a three-dimensional model of the C1 complex (Bally, I., Rossi, V., Lunardi, T., Thielens, N. M., Gaboriaud, C., and Arlaud, G. J. (2009) J. Biol. Chem. 284, 19340-19348). In this study, we have used a mass spectrometry-based strategy involving a label-free semi-quantitative analysis of protein samples to gain new structural insights into C1 assembly. Using a stable chemical modification, we have compared the accessibility of the lysine residues in the isolated tetramer and in C1. The labeling data account for 51 of the 73 lysine residues of C1r and C1s. They strongly support the hypothesis that both C1s CUB(1)-EGF-CUB(2) interaction domains, which are distant in the free tetramer, associate with each other in the C1 complex. This analysis also provides the first experimental evidence that, in the proenzyme form of C1, the C1s serine protease domain is partly positioned inside the C1q cone and yields precise information about its orientation in the complex. These results provide further structural insights into the architecture of the C1 complex, allowing significant improvement of our current C1 model.
Project description:The availability of the human genome sequence allowed us to identify a human complement-related, C1r-like protease gene (c1r-LP) located 2 kb centromeric of the C1r gene (c1r). Compared with c1r, c1r-LP carries a large deletion corresponding to exons 4-8 of c1r. The open reading frame of the C1r-LP cDNA predicts a 50 kDa modular protein displaying 52% amino acid residue identity with the corresponding regions of C1r and 75% identity with a previously described murine C1r-LP. The serine protease domain of C1r-LP, despite an overall similarity with the AGY group of complement serine proteases, has certain structural features characteristic of C2 and factor B, thus raising interesting evolutionary questions. Northern blotting demonstrated the expression of C1r-LP mRNA mainly in the liver and ELISA demonstrated the presence of the protein in human serum at a concentration of 5.5+/-0.9 microg/ml. Immunoprecipitation experiments failed to demonstrate an association of C1r-LP with the C1 complex in serum. Recombinant C1r-LP exhibits esterolytic activity against peptide thioesters with arginine at the P1 position, but its catalytic efficiency (kcat/K(m)) is lower than that of C1r and C1s. The enzymic activity of C1r-LP is inhibited by di-isopropyl fluorophosphate and also by C1 inhibitor, which forms stable complexes with the protease. Most importantly, C1r-LP also expresses proteolytic activity, cleaving pro-C1s into two fragments of sizes identical with those of the two chains of active C1s. Thus C1r-LP may provide a novel means for the formation of the classical pathway C3/C5 convertase.
Project description:Complement component C1, the complex that initiates the classical pathway of complement activation, is a 790-kDa assembly formed from the target-recognition subcomponent C1q and the modular proteases C1r and C1s. The proteases are elongated tetramers that become more compact when they bind to the collagen-like domains of C1q. Here, we describe a series of structures that reveal how the subcomponents associate to form C1. A complex between C1s and a collagen-like peptide containing the C1r/C1s-binding motif of C1q shows that the collagen binds to a shallow groove via a critical lysine side chain that contacts Ca(2+)-coordinating residues. The data explain the Ca(2+)-dependent binding mechanism, which is conserved in C1r and also in mannan-binding lectin-associated serine proteases, the serine proteases of the lectin pathway activation complexes. In an accompanying structure, C1s forms a compact ring-shaped tetramer featuring a unique head-to-tail interaction at its center that replicates the likely arrangement of C1r/C1s polypeptides in the C1 complex. Additional structures reveal how C1s polypeptides are positioned to enable activation by C1r and interaction with the substrate C4 inside the cage-like assembly formed by the collagenous stems of C1q. Together with previously determined structures of C1r fragments, the results reported here provide a structural basis for understanding the early steps of complement activation via the classical pathway.
Project description:Heterozygous missense or in-frame insertion/deletion mutations in complement 1 subunits C1r and C1s cause periodontal Ehlers-Danlos Syndrome (pEDS), a specific EDS subtype characterized by early severe periodontal destruction and connective tissue abnormalities like easy bruising, pretibial haemosiderotic plaques, and joint hypermobility. We report extensive functional studies of 16 C1R variants associated with pEDS by in-vitro overexpression studies in HEK293T cells followed by western blot, size exclusion chromatography and surface plasmon resonance analyses. Patient-derived skin fibroblasts were analyzed by western blot and Enzyme-linked Immunosorbent Assay (ELISA). Overexpression of C1R variants in HEK293T cells revealed that none of the pEDS variants was integrated into the C1 complex but cause extracellular presence of catalytic C1r/C1s activities. Variants showed domain-specific abnormalities of intracellular processing and secretion with preservation of serine protease function in the supernatant. In contrast to C1r wild type, and with the exception of a C1R missense variant disabling a C1q binding site, pEDS variants had different impact on the cell: retention of C1r fragments inside the cell, secretion of aggregates, or a new C1r cleavage site. Overexpression of C1R variants in HEK293T as well as western blot analyses of patient fibroblasts showed decreased levels of secreted C1r. Importantly, all available patient fibroblasts exhibited activated C1s and activation of externally added C4 in the supernatant while control cell lines secreted proenzyme C1s and showed no increase in C4 activation. The central elements in the pathogenesis of pEDS seem to be the intracellular activation of C1r and/or C1s, and extracellular presence of activated C1s that independently of microbial triggers can activate the classical complement cascade.
Project description:The activation of human C1, a Ca(2+)-dependent complex proteinase comprising a non-enzymic protein, C1q, and two serine proteinases, C1r and C1s, is based primarily on the intrinsic property of C1r to autoactivate. The aim of the present study was to investigate the mechanisms involved in the regulation of C1r autoactivation, with particular attention to the role of Ca2+ ions. Spontaneous activation of proenzyme C1r was observed upon incubation in the presence of EDTA, whereas Ca2+ ions reduced markedly the activation process. Several lines of evidence indicated that Ca2+ inhibited the intramolecular activation reaction but had little or no effect on the intermolecular activation reaction. C1q caused partial release of this inhibitory effect of Ca2+. Complete stabilization of C1r in its proenzyme form was obtained upon incorporation within the Ca(2+)-dependent C1s-C1r-C1r-C1s tetramer, and a comparable effect was observed when C1s was replaced by its Ca(2+)-binding alpha-fragment. Both tetramers, C1s-C1r-C1r-C1s and C1s alpha-C1r-C1r-C1s alpha, readily associated with C1q to form 16.0 S and 14.7 S complexes respectively in which C1r fully recovered its activation potential. Both complexes showed indistinguishable activation kinetics, indicating that the gamma B catalytic region of C1s plays no role in the mechanism that triggers C1r activation in C1. The collagen-like fragments of C1q retained the ability to bind to C1s-C1r-C1r-C1s, but, in contrast with intact C1q, failed to induce C1r activation in the resulting complex at temperatures above 25 degrees C. On the basis of these observations it is proposed that activation of the serine-proteinase domain of C1r is controlled by a Ca(2+)-dependent intramolecular mechanism involving the Ca(2+)-binding alpha-region, and that this control is released in C1 by a signal originating in C1q and transmitted through the C1q/C1r interface.
Project description:Interactions between proenzymic or activated complement subcomponents of C1 and C1 Inh (C1 inhibitor) were analysed by sucrose-density-gradient ultracentrifugation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The interaction of C1 Inh with dimeric C1r in the presence of EDTA resulted into two bimolecular complexes accounting for a disruption of C1r. The interaction of C1 Inh with the Ca2+-dependent C1r2-C1s2 complex (8.8 S) led to an 8.5 S inhibited C1r-C1s-C1 Inh complex (1:1:2), indicating a disruption of C1r2 and of C1s2 on C1 Inh binding. The 8.5 S inhibited complex was stable in the presence of EDTA; it was also formed from a mixture of C1r, C1s and C1 Inh in the presence of EDTA or from bimolecular complexes of C1r-C1 Inh and C1s-C1 Inh. C1r II, a modified C1r molecule, deprived of a Ca2+-binding site after autoproteolysis, did not lead to an inhibited tetrameric complex on incubation with C1s and C1 Inh. These findings suggest that, when C1 Inh binds to C1r2-C1s2 complex, the intermonomer links inside C1r2 or C1s2 are weakened, whereas the non-covalent Ca2+-independent interaction between C1r2 and C1s2 is strengthened. The nature of the proteinase-C1 Inh link was investigated. Hydroxylamine (1M) was able to dissociate the complexes partially (pH 7.5) or totally (pH 9.0) when the incubation was performed in denaturing conditions. An ester link between a serine residue at the active site of C1r or C1s and C1 Inh is postulated.