The Human C1q Globular Domain: Structure and Recognition of Non-Immune Self Ligands

C1q, the ligand-binding unit of the C1 complex of complement, is a pattern recognition molecule with the unique ability to sense an amazing variety of targets, including a number of altered structures from self, such as apoptotic cells. The three-dimensional structure of its C-terminal globular domain, responsible for its recognition function, has been solved by X-ray crystallography, revealing a tightly packed heterotrimeric assembly with marked differences in the surface patterns of the subunits, and yielding insights into its versatile binding properties. In conjunction with other approaches, this same technique has been used recently to decipher the mechanisms that allow this domain to interact with various non-immune self ligands, including molecules known to provide eat-me signals on apoptotic cells, such as phosphatidylserine and DNA. These investigations provide evidence for a common binding area for these ligands located in subunit C of the C1q globular domain, and suggest that ligand recognition through this area down-regulates C1 activation, hence contributing to the control of the inflammatory reaction. The purpose of this article is to give an overview of these advances which represent a first step toward understanding the recognition mechanisms of C1q and their biological implications

Interaction of C1q With Pentraxin 3 and IgM Revisited: Mutational Studies With Recombinant C1q Variants

Pentraxins and complement defense collagens are soluble recognition proteins that sense pathogens and altered-self elements, and trigger immune responses including complement activation. PTX3 has been shown to interact with the globular recognition domains (gC1q) of the C1q protein of the classical complement pathway, thereby modulating complement activity. The C1q-PTX3 interaction has been characterized previously by site-specific mutagenesis using individual gC1q domains of each of the three C1q chains. The present study is aimed at revisiting this knowledge taking advantage of full-length recombinant C1q. Four mutations targeting exposed amino acid residues in the gC1q domain of each of the C1q chains (LysA200Asp-LysA201Asp, ArgB108Asp-ArgB109Glu, TyrB175Leu, and LysC170Glu) were introduced in recombinant C1q and the interaction properties of the mutants were analyzed using surface plasmon resonance. All C1q mutants retained binding to C1r and C1s proteases and mannose-binding lectin-associated serine proteases, indicating that the mutations did not affect the function of the collagen-like regions of C1q. The effect of these mutations on the interaction of C1q with PTX3 and IgM, and both the PTX3- and IgM-mediated activation of the classical complement pathway were investigated. The LysA200Asp-LysA201Asp and LysC170Glu mutants retained partial interaction with PTX3 and IgM, however they triggered efficient complement activation. In contrast, the ArgB108Asp-ArgB109Glu mutation abolished C1q binding to PTX3 and IgM, and significantly decreased complement activation. The TyrB175Leu mutant exhibited decreased PTX3- and IgM-dependent complement activation. Therefore, we provided evidence that, in the context of the full length C1q protein, a key contribution to the interaction with both PTX3 and IgM is given by the B chain Arg residues that line the side of the gC1q heterotrimer, with a minor participation of a Lys residue located at the apex of gC1q. Furthermore, we generated recombinant forms of the human PTX3 protein bearing either D or A at position 48, a polymorphic site of clinical relevance in a number of infections, and observed that both allelic variants equally recognized C1q

The serine protease domain of MASP-3: enzymatic properties and crystal structure in complex with ecotin.

International audienceMannan-binding lectin (MBL), ficolins and collectin-11 are known to associate with three homologous modular proteases, the MBL-Associated Serine Proteases (MASPs). The crystal structures of the catalytic domains of MASP-1 and MASP-2 have been solved, but the structure of the corresponding domain of MASP-3 remains unknown. A link between mutations in the MASP1/3 gene and the rare autosomal recessive 3MC (Mingarelli, Malpuech, Michels and Carnevale,) syndrome, characterized by various developmental disorders, was discovered recently, revealing an unexpected important role of MASP-3 in early developmental processes. To gain a first insight into the enzymatic and structural properties of MASP-3, a recombinant form of its serine protease (SP) domain was produced and characterized. The amidolytic activity of this domain on fluorescent peptidyl-aminomethylcoumarin substrates was shown to be considerably lower than that of other members of the C1r/C1s/MASP family. The E. coli protease inhibitor ecotin bound to the SP domains of MASP-3 and MASP-2, whereas no significant interaction was detected with MASP-1, C1r and C1s. A tetrameric complex comprising an ecotin dimer and two MASP-3 SP domains was isolated and its crystal structure was solved and refined to 3.2 Å. Analysis of the ecotin/MASP-3 interfaces allows a better understanding of the differential reactivity of the C1r/C1s/MASP protease family members towards ecotin, and comparison of the MASP-3 SP domain structure with those of other trypsin-like proteases yields novel hypotheses accounting for its zymogen-like properties in vitro

X-Ray Structure of the Human Calreticulin Globular Domain Reveals a Peptide-Binding Area and Suggests a Multi-Molecular Mechanism

In the endoplasmic reticulum, calreticulin acts as a chaperone and a Ca2+-signalling protein. At the cell surface, it mediates numerous important biological effects. The crystal structure of the human calreticulin globular domain was solved at 1.55 Å resolution. Interactions of the flexible N-terminal extension with the edge of the lectin site are consistently observed, revealing a hitherto unidentified peptide-binding site. A calreticulin molecular zipper, observed in all crystal lattices, could further extend this site by creating a binding cavity lined by hydrophobic residues. These data thus provide a first structural insight into the lectin-independent binding properties of calreticulin and suggest new working hypotheses, including that of a multi-molecular mechanism

Periodontal Ehlers-Danlos Syndrome Is Caused by Mutations in C1R and C1S, which Encode Subcomponents C1r and C1s of Complement

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

Exploration cristallographique de certains trésors cachés de la défense innée

Après un court aperçu de mon C.V, cette présentation sera l'occasion de faire un bilan des résultats accumulés depuis l'an 2000 et d'évoquer les projets à venir. Vous pourrez ainsi mieux savoir ce que des études cristallographiques nous ont appris sur le fonctionnement de certaines nano-machines qui participent activement à la défense de notre organisme. Ce sujet faisant intervenir des complexes macromoléculaires flexibles de haut poids moléculaire (>> 500 kDa), nous avons utilisé une stratégie de dissection structurale et fonctionnnelle. Cette stratégie nous a permis de découvrir et analyser des structures supportant des propriétés fonctionnelles très variées: très forte spécificité de protéases modulaires, contrôle de l'activation de ces protéases, multispécificité (fixation spécifique sur différentes cibles) de domaines de reconnaissance, sites d'assemblage de ces complexes macromoléculaires .... En assemblant les pièces du puzzle, nous commençons à pouvoir déchiffrer un mécanisme étonnant qui permet de convertir un signal d'étirement mécanique en un signal d'activation d'une cascade enzymatique très contrôlée conduisant à des activités biologiques complexes et variées. Beaucoup d'énigmes restent à élucider, et quelques exemples seront brièvement évoqués

C1r

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Caractérisation de l'interaction des collagènes de défense avec la calréticuline de Trypanosoma cruzi et CR1/CD35

Les collagènes de défense (C1q, MBL, ficolines) sont capables de reconnaître de nombreux motifs à la surface des éléments du non soi ou du soi altéré, via leurs domaines globulaires C-terminaux. Ils peuvent également interagir avec certains récepteurs présents à la surface des cellules humaines ou de pathogènes. Nous nous sommes intéressés dans un premier temps à la calréticuline de Trypanosoma Cruzi (TcCRT), une protéine qui interviendrait dans les mécanismes d'évasion de ce parasite. Dans le but de réaliser des études fonctionnelles et structurales de la TcCRT, nous avons produit différents fragments recombinants. Nous ne sommes cependant pas parvenus à obtenir un échantillon nous permettant d'accomplir nos objectifs, nous conduisant à reporter nos efforts sur l'étude d'un autre récepteur, CR1/CD35. Il a été montré précédemment que CR1/CD35 pouvait interagir avec C1q et la MBL, probablement par ses modules CCP 22-30. Cette interaction pourrait être impliquée dans l'élimination des complexes immuns, la phagocytose ou encore des mécanismes de signalisation cellulaire. A l'aide d'un fragment recombinant comprenant les modules CCP 22-30 de CR1, nous avons confirmé par SPR l'interaction avec C1q et la MBL. Nous avons également montré pour la première fois que CR1 pouvait interagir avec les ficolines L, H et M par ce même domaine. Nos résultats indiquent que cette interaction prendrait majoritairement place dans la région collagène de C1q, de la MBL et de la ficoline L, probablement à proximité du site de fixation des protéases. L'utilisation de fragments tronqués de CR1 CCP 22-30, nous permet de proposer l'hypothèse que les modules CCP 24 et 25 de CR1 seraient le site majoritaire de fixation des collagènes de défense. Ces données ouvrent la voie à des études structurales et fonctionnelles visant à approfondir notre connaissance des interactions CR1 collagène de défense et de leur rôle physiologique.The defence collagens (C1q, MBL, ficolins) are able to recognize various patterns on non-self or altered-self surfaces through their globular domains. They can also interact with receptors at the surface of human cells or pathogens. First, we were interested in the calreticulin from Trypanosoma cruzi, a protein which may be involved in the evasion mechanisms of that parasite. To achieve structural and functional studies, we produced recombinant fragments from TcCRT. Unfortunately, we couldn't obtain any sample suitable for our studies, so we decided to focus on another receptor, CR1/CD35. It has been shown previously by other teams that C1q and MBL bind to CR1/CD35, probably through CCP modules 22 to 30, close to the cell membrane. This interaction could be involved in several biologic mechanisms: elimination of immune complexes, phagocytosis, cell signaling. We produced a recombinant fragment including the CCP modules 22 to 30 of CR1 and confirmed its interaction with C1q and MBL using SPR. We also showed for the first time that L-, H- and M-ficolins bind to CR1 through CCP modules 22 to 30. Our results point out that the CR1 binding site of C1q, MBL and L-ficolin is located in the collagen stalks, most probably at or in close proximity to the serine protease interaction site. By using CR1 CCP 22-30 truncated fragments, we suggest that CCP modules 24 to 25 could be the main binding site for the defence collagens. These data open the way for structural and functional studies aiming at improving our knowledge of the CR1 defense collagen interactions and of their physiological role.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Conception, synthèse et évaluation biologique de nouveaux dérivés de l'acronycine à potentialité antitumorale

L objet de ce travail est l obtention de nouveaux dérivés de l acronycine, alcaloïde naturel. Deux séries ont été développées. La première a abouti à l obtention d un nouveau composé en série acronycine, la triazoloacronycine, ainsi que plusieurs intermédiaires potentiellement intéressants. La deuxième série développée aura permis la synthèse de nouveaux analogues de la psorospermine, furanoxanthone naturelle, en série benzo[b]acronycine. Pour cela, une méthode mettant en jeu des micro-organismes a notamment été mise au point. L'activité cytotoxique des composés préparés a été évaluée in vitro sur des modèles de leucémies murines L1210 et de tumeurs solides KB-3-1. La triazoloacronycine s est révélée être quatre fois plus active que le composé naturel. Les analogues de la psorospermine également développés, possèdent des activités de l ordre du nanomolaire et ont fait l objet d une étude approfondie de leur mécanisme d action in vitro. Ce sont des alkylants de l ADN ciblant le grand sillon.The aim of this work is the preparation of natural occuring alcaloïd acronycine new derivatives. Two series were developped. The first one led to the synthesis of the triazoloacronycine new compound in acronycine series and of some interesting intermediates. The second one led to the synthesis of new psorospermin naturally occuring furanoxanthone. analogs in the benzo[b]acronycine series. For this, a new method involving micro-organism has been developped. The cytotoxic activity of the prepared compounds was evaluated on murine leukemic L1210 and human carcinoma KB-3-1 cells in vitro. Triazoloacronycine compound is four time more active than the naturaly occuring alcaloid. The psorospermin-acronycine analogs have a nanomolar activity and their mechanism of action was deeply studied. Those compounds are DNA alkylating agents targetting the major groovePARIS-BIUP (751062107) / SudocSudocFranceF

Structures of the MASP Proteases and Comparison with Complement C1r and C1s

International audienceSeveral recognition proteins of the defence collagen family associate with proteases to initiate the complement cascade. The associated proteases, which are the subject of this review, mediate the proteolytic activation trigger. MBL-associated serine proteases (MASPs) mainly activate the lectin complement pathway (LP), while C1r and C1s activate the classical complement pathway (CP). This chapter will briefly introduce the current structural knowledge on these effector proteases and question what we know on MASPs structures, their common properties and how they differ from the C1r/s proteases. We will primarily focus on the structural comparison of the N-terminal domains, where the collectin binding sites are located and highlight novel aspects on their interaction with collagens. We also aim to highlight further molecular details associated to functional specificities, and to mention questions remaining open and needing further investigations. This chapter complements other reviews that describe the main general lines of complement activation mechanisms (Merle et al. 2015), the proposed structure-based scheme of the lectin pathway activation (Kjaer et al. 2013) and previous comparisons of LP and CP proteases (Gál et al. 2007; Pike and Wijeyewickrema 2013)