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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., (Copyright © 2020 Fouët, Gout, Wicker-Planquart, Bally, De Nardis, Dedieu, Chouquet, Gaboriaud, Thielens, Kleman and Rossi.)

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., (Copyright © 2019 Bally, Dalonneau, Chouquet, Gröbner, Amberger, Kapferer-Seebacher, Stoiber, Zschocke, Thielens, Rossi and Gaboriaud.)

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., (Copyright © 2019 Gröbner, Kapferer-Seebacher, Amberger, Redolfi, Dalonneau, Björck, Milnes, Bally, Rossi, Thielens, Stoiber, Gaboriaud and Zschocke.)

C1r and C1s are the proteases responsible for the activation and proteolytic activity of the C1 complex of the classical complement pathway, respectively. They are assembled into a Ca(2+)-dependent C1s-C1r-C1r-C1s tetramer which in turn associates with the recognition protein C1q. The C1 complex circulates in serum as a zymogen and is activated upon binding of C1q to appropriate targets, such as antigen-antibody complexes. This property is used for the purification of C1r and C1s from human serum after binding of C1 to insoluble immune complexes. Disruption of the bound C1 complex by EDTA releases C1r and C1s which are further separated by ion-exchange chromatography; both proteins can be reassembled in the presence of calcium ions and the reconstituted tetramer isolated by gel filtration. In this chapter, we describe the purification of the activated and proenzyme forms of C1r and C1s and of the proenzyme C1s-C1r-C1r-C1s tetramer as well as methods for their biochemical and functional characterization. The production of recombinant C1s and of the proenzyme tetramer in a baculovirus-insect cell system, and their purification by affinity chromatography is also presented.

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.

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.

The C1 complex of complement is assembled from a recognition protein C1q and C1s-C1r-C1r-C1s, a Ca(2+)-dependent tetramer of two modular proteases C1r and C1s. Resolution of the x-ray structure of the N-terminal CUB(1)-epidermal growth factor (EGF) C1s segment has led to a model of the C1q/C1s-C1r-C1r-C1s interaction where the C1q collagen stem binds at the C1r/C1s interface through ionic bonds involving acidic residues contributed by the C1r EGF module (Gregory, L. A., Thielens, N. M., Arlaud, G. J., Fontecilla-Camps, J. C., and Gaboriaud, C. (2003) J. Biol. Chem. 278, 32157-32164). To identify the C1q-binding sites of C1s-C1r-C1r-C1s, a series of C1r and C1s mutants was expressed, and the C1q binding ability of the resulting tetramer variants was assessed by surface plasmon resonance. Mutations targeting the Glu(137)-Glu-Asp(139) stretch in the C1r EGF module had no effect on C1 assembly, ruling out our previous interaction model. Additional mutations targeting residues expected to participate in the Ca(2+)-binding sites of the C1r and C1s CUB modules provided evidence for high affinity C1q-binding sites contributed by the C1r CUB(1) and CUB(2) modules and lower affinity sites contributed by C1s CUB(1). All of the sites implicate acidic residues also contributing Ca(2+) ligands. C1s-C1r-C1r-C1s thus contributes six C1q-binding sites, one per C1q stem. Based on the location of these sites and available structural information, we propose a refined model of C1 assembly where the CUB(1)-EGF-CUB(2) interaction domains of C1r and C1s are entirely clustered inside C1q and interact through six binding sites with reactive lysines of the C1q stems. This mechanism is similar to that demonstrated for mannan-binding lectin (MBL)-MBL-associated serine protease and ficolin-MBL-associated serine protease complexes.

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.