Evelyne Gout

Carbohydrate Recognition Properties of Human Ficolins: GLYCAN ARRAY SCREENING REVEALS THE SIALIC ACID BINDING SPECIFICITY OF M-FICOLIN*

Ficolins are oligomeric innate immune recognition proteins consisting of a collagen-like region and a fibrinogen-like recognition domain that bind to pathogen- and apoptotic cell-associated molecular patterns. To investigate their carbohydrate binding specificities, serum-derived L-ficolin and recombinant H- and M-ficolins were fluorescently labeled, and their carbohydrate binding ability was analyzed by glycan array screening. L-ficolin preferentially recognized disulfated N-acetyllactosamine and tri- and tetrasaccharides containing terminal galactose or N-acetylglucosamine. Binding was sensitive to the position and orientation of the bond between N-acetyllactosamine and the adjacent carbohydrate. No significant binding of H-ficolin to any of the 377 glycans probed could be detected, providing further evidence for its poor lectin activity. M-ficolin bound preferentially to 9-O-acetylated 2-6-linked sialic acid derivatives and to various glycans containing sialic acid engaged in a 2-3 linkage. To further investigate the structural basis of sialic acid recognition by M-ficolin, point mutants were produced in which three residues of the fibrinogen domain were replaced by their counterparts in L-ficolin. Mutations G221F and A256V inhibited binding to the 9-O-acetylated sialic acid derivatives, whereas Y271F abolished interaction with all sialic acid-containing glycans. The crystal structure of the Y271F mutant fibrinogen domain was solved, showing that the mutation does not alter the structure of the ligand binding pocket. These analyses reveal novel ficolin ligands such as sulfated N-acetyllactosamine (L-ficolin) and gangliosides (M-ficolin) and provide precise insights into the sialic acid binding specificity of M-ficolin, emphasizing the essential role of Tyr271 in this respect.

Structural Basis for Innate Immune Sensing by M-Ficolin and its Control by a Ph-Dependent Conformational Switch.

Ficolins are soluble oligomeric proteins with lectin-like activity, assembled from collagen fibers prolonged by fibrinogen-like recognition domains. They act as innate immune sensors by recognizing conserved molecular markers exposed on microbial surfaces and thereby triggering effector mechanisms such as enhanced phagocytosis and inflammation. In humans, L- and H-ficolins have been characterized in plasma, whereas a third species, M-ficolin, is secreted by monocytes and macrophages. To decipher the molecular mechanisms underlying their recognition properties, we previously solved the structures of the recognition domains of L- and H-ficolins, in complex with various model ligands (Garlatti, V., Belloy, N., Martin, L., Lacroix, M., Matsushita, M., Endo, Y., Fujita, T., Fontecilla-Camps, J. C., Arlaud, G. J., Thielens, N. M., and Gaboriaud, C. (2007) EMBO J. 24, 623–633). We now report the ligand-bound crystal structures of the recognition domain of M-ficolin, determined at high resolution (1.75–1.8 Å), which provides the first structural insights into its binding properties. Interaction with acetylated carbohydrates differs from the one previously described for L-ficolin. This study also reveals the structural determinants for binding to sialylated compounds, a property restricted to human M-ficolin and its mouse counterpart, ficolin B. Finally, comparison between the ligand-bound structures obtained at neutral pH and nonbinding conformations observed at pH 5.6 reveals how the ligand binding site is dislocated at acidic pH. This means that the binding function of M-ficolin is subject to a pH-sensitive conformational switch. Considering that the homologous ficolin B is found in the lysosomes of activated macrophages (Runza, V. L., Hehlgans, T., Echtenacher, B., Zahringer, U., Schwaeble, W. J., and Mannel, D. N. (2006) J. Endotoxin Res. 12, 120–126), we propose that this switch could play a physiological role in such acidic compartments.

Structural insights into the recognition properties of human ficolins.

Innate immunity relies upon the ability of a variety of recognition molecules to sense pathogens through conserved molecular signatures that are often carbohydrates. Ficolins are oligomeric proteins assembled from collagen-like stalks and fibrinogen-like domains that have the ability to sense these molecular patterns on both pathogens and apoptotic cell surfaces. Three ficolins, termed L, H and M, have been identified in humans. They differ in their localization and concentration in extracellular fluids, their mode of secretion and their recognition properties. From a structural point of view, ficolins are assembled from basal trimeric subunits comprising a collagen-like triple helix and a globular domain composed of 3 fibrinogen-like domains. The globular domains are responsible for sensing danger signals whereas the collagen-like stalks provide a link with immune effectors. This review mainly focuses on the structure and recognition properties of the 3 human ficolins, as revealed by recent crystallographic analysis of their recognition domains. The ligand binding sites have been identified in the 3 ficolins and their recognition mechanisms have been characterized at the atomic level. In the case of M-ficolin, a structural transition at acidic pH disables the binding pocket, and thus likely participates in the functional cycle of this protein.

Human adenovirus serotype 3 (Ad3) and the AD fiber protein bind to a 130-kDa membrane protein on HeLa cells

The fiber protein of adenovirus mediates the interaction of adenovirus with cell membrane receptors. We have produced the Ad3 fiber protein in the baculovirus expression system. Biochemical, morphological and functional analyses showed that the recombinant fiber was properly folded and functionally competent. The specific binding of Ad3 virus to two HeLa membrane proteins of 130 and 100 kDa was demonstrated with an overlay protein binding assay. In the same assay, Ad3 fiber only recognized the 130-kDa protein. Divalent cations seemed to be important for the interaction of both virus and fiber with these proteins.

Deciphering Complement Receptor Type 1 Interactions with Recognition Proteins of the Lectin Complement Pathway

Complement receptor type 1 (CR1) is a membrane receptor expressed on a wide range of cells. It is involved in immune complex clearance, phagocytosis, and complement regulation. Its ectodomain is composed of 30 complement control protein (CCP) modules, organized into four long homologous repeats (A–D). In addition to its main ligands C3b and C4b, CR1 was reported to interact with C1q and mannan-binding lectin (MBL) likely through its C-terminal region (CCP22–30). To decipher the interaction of human CR1 with the recognition proteins of the lectin complement pathway, a recombinant fragment encompassing CCP22–30 was expressed in eukaryotic cells, and its interaction with human MBL and ficolins was investigated using surface plasmon resonance spectroscopy. MBL and L-ficolin were shown to interact with immobilized soluble CR1 and CR1 CCP22–30 with apparent dissociation constants in the nanomolar range, indicative of high affinity. The binding site for CR1 was located at or near the MBL-associated serine protease (MASP) binding site in the collagen stalks of MBL and L-ficolin, as shown by competition experiments with MASP-3. Accordingly, the mutation of an MBL conserved lysine residue essential for MASP binding (K55) abolished binding to soluble CR1 and CCP22–30. The CR1 binding site for MBL/ficolins was mapped to CCP24–25 of long homologous repeat D using deletion mutants. In conclusion, we show that ficolins are new CR1 ligands and propose that MBL/L-ficolin binding involves major ionic interactions between conserved lysine residues of their collagen stalks and surface exposed acidic residues located in CR1 CCP24 and/or CCP25.

Human L-Ficolin Recognizes Phosphocholine Moieties of Pneumococcal Teichoic Acid

Human L-ficolin is a soluble protein of the innate immune system able to sense pathogens through its fibrinogen (FBG) recognition domains and to trigger activation of the lectin complement pathway through associated serine proteases. L-Ficolin has been previously shown to recognize pneumococcal clinical isolates, but its ligands and especially its molecular specificity remain to be identified. Using solid-phase binding assays, serum and recombinant L-ficolins were shown to interact with serotype 2 pneumococcal strain D39 and its unencapsulated R6 derivative. Incubation of both strains with serum triggered complement activation, as measured by C4b and C3b deposition, which was decreased by using ficolin-depleted serum. Recombinant L-ficolin and its FBG-like recognition domain bound to isolated pneumococcal cell wall extracts, whereas binding to cell walls depleted of teichoic acid (TA) was decreased. Both proteins were also shown to interact with two synthetic TA compounds, each comprising part structures of the complete lipoteichoic acid molecule with two PCho residues. Competition studies and direct interaction measurements by surface plasmon resonance identified PCho as a novel L-ficolin ligand. Structural analysis of complexes of the FBG domain of L-ficolin and PCho revealed that the phosphate moiety interacts with amino acids previously shown to define an acetyl binding site. Consequently, binding of L-ficolin to immobilized acetylated BSA was inhibited by PCho and synthetic TA. Binding of serum L-ficolin to immobilized synthetic TA and PCho-conjugated BSA triggered activation of the lectin complement pathway, thus further supporting the hypothesis of L-ficolin involvement in host antipneumococcal defense.

Enhancement of Ebola virus infection via ficolin-1 interaction with the mucin domain of GP glycoprotein

ABSTRACT Ebola virus infection requires the surface viral glycoprotein to initiate entry into the target cells. The trimeric glycoprotein is a highly glycosylated viral protein which has been shown to interact with host C-type lectin receptors and the soluble complement recognition protein mannose-binding lectin, thereby enhancing viral infection. Similarly to mannose-binding lectin, ficolins are soluble effectors of the innate immune system that recognize particular glycans at the pathogen surface. In this study, we demonstrate that ficolin-1 interacts with the Zaire Ebola virus (EBOV) glycoprotein, and we characterized this interaction by surface plasmon resonance spectroscopy. Ficolin-1 was shown to bind to the viral glycoprotein with a high affinity. This interaction was mediated by the fibrinogen-like recognition domain of ficolin-1 and the mucin-like domain of the viral glycoprotein. Using a ficolin-1 control mutant devoid of sialic acid-binding capacity, we identified sialylated moieties of the mucin domain to be potential ligands on the glycoprotein. In cell culture, using both pseudotyped viruses and EBOV, ficolin-1 was shown to enhance EBOV infection independently of the serum complement. We also observed that ficolin-1 enhanced EBOV infection on human monocyte-derived macrophages, described to be major viral target cells,. Competition experiments suggested that although ficolin-1 and mannose-binding lectin recognized different carbohydrate moieties on the EBOV glycoprotein, the observed enhancement of the infection likely depended on a common cellular receptor/partner. In conclusion, ficolin-1 could provide an alternative receptor-mediated mechanism for enhancing EBOV infection, thereby contributing to viral subversion of the host innate immune system. IMPORTANCE A specific interaction involving ficolin-1 (M-ficolin), a soluble effector of the innate immune response, and the glycoprotein (GP) of EBOV was identified. Ficolin-1 enhanced virus infection instead of tipping the balance toward its elimination. An interaction between the fibrinogen-like recognition domain of ficolin-1 and the mucin-like domain of Ebola virus GP occurred. In this model, the enhancement of infection was shown to be independent of the serum complement. The facilitation of EBOV entry into target host cells by the interaction with ficolin-1 and other host lectins shunts virus elimination, which likely facilitates the survival of the virus in infected host cells and contributes to the virus strategy to subvert the innate immune response.

Association between the Presence of Autoantibodies Targeting Ficolin-3 and Active Nephritis in Patients with Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of multiple autoantibodies. Antibodies against Ficolin-3 were previously identified in the sera of some SLE patients, but their prevalence and significance have not been yet investigated. The aims of this study were to determine the prevalence of anti-ficolin-3 antibodies among SLE patients and to investigate their potential as diagnostic and/or prognostic biomarkers in SLE. In this retrospective study, sera from SLE patients (n = 165) were selected from a preexisting declared biological collection. Samples from healthy controls (n = 48) were matched with SLE sera. Disease activity was determined according to the SLEDAI score. Anti-ficolin-3, anti-dsDNA and anti-C1q antibodies levels were measured in sera by ELISA. First, a highly significant difference was found in the anti-ficolin-3 levels between SLE patients and healthy subjects. Anti-ficolin-3 antibodies were detected as positive in 56 of 165 (34%) SLE patients. The titer of anti-ficolin-3 antibodies was correlated with the SLEDAI score (r = 0.38, p<0.0001). The presence of anti-ficolin-3 antibodies was associated with anti-C1q and anti-dsDNA antibodies. Regarding associations with clinical manifestations, the presence of active lupus nephritis was significantly associated with the presence of anti-ficolin-3 antibodies (p≤0.001). This association with renal involvement was higher with anti-ficolin-3 or anti-C1q antibodies than with other auto-antibodies. Interestingly, the combination of anti-ficolin-3 and anti-C1q antibodies demonstrated higher specificity than any other serological biomarker. These results suggest that anti-ficolin-3 antibodies could be useful for the diagnosis of active nephritis in SLE patients.