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

Editorial: Updates on the role of surfactant proteins A and D in innate immune responses

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Neutron scattering study of the (γ-B) catalytic domains of complement proteases Cl̄r and Cl̄s

AbstractThe catalytic domains of Cl̄r and Cl̄s, comprising the C-tenninal region of the A chain (γ), disulphide-linked to the B chain, were obtained by limited proteolysis of the native proteases with chymotrypsin and plasmin, respectively, and studied by small angle neutron scattering. For CIs (γ-B), a molar mass of 45 000 ± 5000 gmol, and a relatively large radius of gyration (Rg) of 28 ± 1 Å were determined, excluding a single globular domain. The corresponding values for Cl̄r (γ-B), (90,000 gmol, Rg, = 34 ± 1 Å) are consistent with a dimer involving the loose packing of two (γ-B) subunits. Various models of the dimer are discussed in the light of neutron scattering and other data

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

M-ficolin and leukosialin (CD43): new partners in neutrophil adhesion

M-ficolin specificity for sialylated ligands prompted us to investigate its interactions with the main membrane sialoprotein of human neutrophils, CD43. rM-ficolin bound CD43 and prevented the access of anti-CD43 mAb. Moreover, rM-ficolin reacted exclusively with CD43 on Western blots of neutrophil lysate. We confirmed that M-ficolin is secreted by fMLP-activated neutrophils, and this endogenous M-ficolin also binds to CD43 and competes with anti-CD43 mAb. Anti-CD43 antibody cross-linking or fMLP resulted in M-ficolin and CD43 colocalization on polarized neutrophils. The binding of rM-ficolin to resting neutrophils induced cell polarization, adhesion, and homotypic aggregation as anti-CD43 mAb. The M-ficolin Y271F mutant, unable to bind sialic acid, neither reacted with neutrophils nor modulated their functions. Finally, rM-ficolin activated the lectin complement pathway on neutrophils. These results emphasize a new function of M-ficolin, different from ficolin pathogen recognition, i.e., a participation to neutrophil adhesion potentially important in early inflammation, as nanomolar agonist concentrations are sufficient to mobilize M-ficolin to the neutrophil surface. This multivalent lectin could then endow the antiadhesive CD43, essentially designed to prevent leukocyte aggregation in the blood flow, with new adhesive properties and explain, at least in part, dual-adhesive/antiadhesive roles of CD43 in neutrophil recruitment. J. Leukoc. Biol. 91: 469-474; 2012.Coordenacao de Aperfeicoamentoe Pessoal de Nivel Superior/Comite Francais dEvaluation de la Cooperation Universitaire et Scientifique avec le Bresil (CAPES/COFECUB)Coordenacao de Aperfeicoamentoe Pessoal de Nivel Superior/Comite Francais d'Evaluation de la Cooperation Universitaire et Scientifique avec le Bresil (CAPES/COFECUB) [597/08]BaxterBaxterAmgenAmge

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

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

Caractérisation fonctionnelle structurale de la protease MASP-3 associée à la MBL et aux ficolines

La voie lectine du complément, élément majeur de la défense innée, est déclenchée par la fixation sur des pathogènes de complexes associant une protéine de reconnaissance oligomérique, MBL ou ficolines, et la protéase MASP-2. MASP-1 et -3, ainsi qu'un fragment tronqué de MASP-2 (MAp19) sont également associés aux protéines de reconnaissance mais leurs fonctions sont inconnues. Le travail porté principalement sur la caractérisation de l'assemblage MBUMASP-3. Il a été montré que les 2 formes oligomériques majeures de lai MBL circulent dans le sérum sous forme trimérique et tétramérique et qu'elles fixent les MASPs de manière comparable avec une stoechiométrie MBL:MASP de 1 :2. Par ailleurs, la structure de la région d'interaction CUB1-EGF-CUB2 de MASP-3 a été résolue à 2,35 A et montre un homodimère disposé de manière tête-bêche dans lequel tous les modules sont localisés dans le même plan. Chaque module EGF possède un site de liaison du Ca2+ stabilisant l'interface du dimère et un 2nd ion Ca2+ est fixé à la partie distale de chaque module CUB1. De plus, un 3ème ion Ca2+, fixé dans chaque module CUB2, stabilise la protéine. La caractérisation de mutants ponctuels a mis en évidence des résidus impliqués dans l'interaction MBUMASP-3: K55 de la MBL et y56, F103, H218 et y225 de MASP-3, ces derniers étant également impliqués dans l'interaction avec les ficolines. Ces résidus définissent 2 sites d'interaction dans les modules CUB1 et CUB2 de chaque monomère stabilisés par un ion Ca2+. Ces sites sont homologues et localisés dans le même plan, ce qui suggère qu'un dimère de MASP-3 interagit avec des sites homologues sur 4 triples hélices de collagène de la MBL ou des ficolines.oligomeric recognition protein, MBL or ficolins, and the MASP-2 protease. MASP-1 and -3 and a truncated form of MASP-2 (Map19) are also associated to the recognition protein but their function is unknown. The aim of this work was to characterize the MBUMASP-3 complex assembly. We provide evidence that the two major forms of MBL in human serum are trimers and tetramers and bind the MASP and MAp19 in similar ways with a 1:2 stoichiometry. ln addition, the x-ray structure of the CUB1-EGF-CUB2 interaction domain of MASP was solved and refined to a resolution of 2.35 À. The structure reveals a head-to-tail homodimer in which ail six modules are located within the same plane. A Ca2+ ion bound to each EGF module stabilizes the dimer interface and a second Ca2+ ion is bound to the distal part of each CUB1 module. Moreover, a third Ca2+ ion is bound to each CUB2 module and stabilizes the protein. Using site-directed mutagenesis, we have identified several residues involved in the interaction between MBL and MASP-3: K55 of MBL and y56, F103, H218 an y225 of MASP-3. These residues are also involved in the interaction with the ficolins. These mutations define two binding sites in the CUB and CUB2 modules of each monomer, both stabilized by Ca2+ ions. The fact that t'Jese sites are homo logo us and located within the same plane strongly suggests that a MASP-3 dimer interacts with homologous interaction sites on 4 collagen triple helices of MBL or ficolins.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF