Innate immunity relies critically upon the ability of a few pattern recognition molecules to sense molecular markers on pathogens, but little is known about these interactions at the atomic level. Human L‐ and H‐ficolins are soluble oligomeric defence proteins with lectin‐like activity, assembled from collagen fibers prolonged by fibrinogen‐like recognition domains. The X‐ray structures of their trimeric recognition domains, alone and in complex with various ligands, have been solved to resolutions up to 1.95 and 1.7 Å, respectively. Both domains have three‐lobed structures with clefts separating the distal parts of the protomers. Ca2+ ions are found at sites homologous to those described for tachylectin 5A (TL5A), an invertebrate lectin. Outer binding sites (S1) homologous to the GlcNAc‐binding pocket of TL5A are present in the ficolins but show different structures and specificities. In L‐ficolin, three additional binding sites (S2–S4) surround the cleft. Together, they define an unpredicted continuous recognition surface able to sense various acetylated and neutral carbohydrate markers in the context of extended polysaccharides such as 1,3‐β‐D‐glucan, as found on microbial or apoptotic surfaces.

(CUB 2 ), plus one and two water molecules, respectively. To identify the residues involved in interaction of MASP-1 and -3 with MBL and L-and H-ficolins, 27 point mutants of human MASP-3 were generated, and their binding properties were analyzed using surface plasmon resonance spectroscopy. These mutations map two homologous binding sites contributed by modules CUB 1 and CUB 2 , located in close vicinity of their Ca 2؉ -binding sites and stabilized by the Ca 2؉ ion. This information allows us to propose a model of the MBL-MASP-1/3 interaction, involving a major electrostatic interaction between two acidic Ca 2؉ ligands of MASP-1/3 and a conserved lysine of MBL. Based on these and other data, a schematic model of a MBL⅐MASP complex is proposed.The lectin pathway of complement is increasingly recognized as an important component of innate immunity against pathogens. This pathway is triggered by oligomeric lectins that recognize patterns of neutral and acetylated carbohydrates on the surface of pathogens and share the ability to associate with and trigger activation of modular proteases termed mannanbinding lectin-associated serine proteases (MASPs) 3 (1, 2). Four such oligomeric lectins have been described: mannanbinding lectin (MBL) and ficolins L, H, and M (3-7). These proteins all assemble as oligomers of homotrimeric subunits, each comprising N-terminal collagen-like triple helices prolonged by recognition domains endowed with lectin-like binding activities. There are three different MASPs (MASP-1, -2, and -3) (4,8,9), and these feature modular structures homologous to those of C1r and C1s, the proteases of the C1 complex of complement, with an N-terminal CUB module (10), an epidermal growth factor (EGF)-like module belonging to the Ca 2ϩ -binding subset (11), a second CUB module, two complement control protein (CCP) modules (12), and a chymotrypsin-like serine protease domain. MASP-1 and MASP-3 are alternative splicing products of the MASP1/3 gene and include different serine protease domains but share identical CUB 1 -EGF-CUB 2 -CCP 1 -CCP 2 segments (9). Likewise, alternative splicing of the MASP2 gene generates MBL-associated protein 19 (MAp19), a truncated protein comprising the N-terminal CUB 1 -EGF segment of MASP-2 prolonged by four residues specific to MAp19 (13,14). From a functional standpoint, the ability of MASP-2 to trigger the lectin pathway of complement is clearly established (8). In contrast, whether MASP-1 is involved in this pathway is still a controversial issue (15,16), and the function of MASP-3 remains elusive.Studies on human (17-20) and rat (21, 22) proteins have established that the MASPs and MAp19 each associate as homodimers through their CUB 1 -EGF segment. In turn, the MASPs and MAp19 each form individual Ca 2ϩ -dependent complexes with MBL and the ficolins. The interaction involves a major site located in the CUB 1 -EGF moiety of each protein but is strengthened by module CUB 2 (18,19,22,23). Resolution of the x-ray structure of human MAp19 has allowed identification of a Ca 2ϩ...

F icolins are members of the defense collagen family that comprises oligomeric proteins with globular recognition domains able to sense danger signals, such as pathogen-or apoptotic cell-associated molecular patterns, and collagen-like stalks providing the link with immune effectors (1, 2). Ficolins are assembled from homotrimeric subunits comprising a collagen-like triple helix and a lectin-like domain composed of three fibrinogenlike (FBG) 3 domains. Two cysteines at the N-terminal end of the polypeptide chains form interchain disulfide bonds that mediate assembly into higher oligomerization structures (3, 4). In humans, L-and H-ficolins have been characterized in serum whereas Mficolin is mainly expressed by the monocytic cell lineage (5-7). In addition to humans, ficolins have been identified in different mammalian species including rodents and pigs (8, 9), which have two related ficolin genes called A and B and ␣ and ␤, respectively, orthologous to the human L-and M-ficolin genes, respectively (10). To date, H-ficolin has only been identified in humans and it has been reported recently that the mouse and rat homologues of the H-ficolin gene are pseudogenes, which accounts for the absence of the corresponding protein in rodents (10). Like mannan-binding lectin (MBL), ficolins are able to activate the lectin complement pathway in response to recognition of neutral carbohydrates and N-acetyl groups on pathogens and damaged cells. This results from the ability of MBL and ficolins to associate with and trigger activation of MBL-associated serine protease (MASP)-2. Activated MASP-2 cleaves the complement proteins C2 and C4, thereby triggering the complement cascade (11-13). Three other MBL/ficolins-associated proteins have been described, the MASP-1 and MASP-3 proteases (14, 15) and a truncated form of MASP-2 called MAp19 (19-kDa MBL-associated protein) or sMAp (16,17). MASP-3 has no known physiological substrates whereas MASP-1 cleaves with a low efficiency a few protein substrates, among which are fibrinogen and coagulation factor XIII (18). It has been proposed recently that MASP-1 might contribute to the activation of the lectin pathway, but this issue is still controversial (19,20). Complement activation results in opsonization of microbes and apoptotic cells with C3-derived fragments, thus promoting their clearance through interaction with C3 receptors on The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the Commissariat à l'Energie Atomique, the Centre National de la Recherche Scientifique, the Université Joseph Fourier (Grenoble, France).

Mannan-binding lectin (MBL)-associated serine proteases (MASP-1, -2, and -3) are homologous modular proteases that each associate with MBL and L- and H-ficolins, which are oligomeric serum lectins involved in innate immunity. To investigate its physicochemical, interaction, and enzymatic properties, human MASP-3 was expressed in insect cells. Ultracentrifugation analysis indicated that rMASP-3 sedimented as a homodimer (s20,w = 6.2 ± 0.1 S) in the presence of Ca2+, and as a monomer (s20,w = 4.6 ± 0.1 S) in EDTA. As shown by surface plasmon resonance spectroscopy, it associated with both MBL (KD = 2.6 nM) and L-ficolin (KD = 7.2 nM). The protease was produced in a single-chain, proenzyme form, but underwent slow activation upon prolonged storage at 4°C, resulting from cleavage at the Arg430-Ile431 activation site. Activation was prevented in the presence of protease inhibitors iodoacetamide and 1,10-phenanthroline but was not abolished upon substitution of Ala for the active site Ser645 of MASP-3, indicating extrinsic proteolysis. In contrast, the corresponding mutations Ser627→Ala in MASP-1 and Ser618→Ala in MASP-2 stabilized the latter in their proenzyme form. Likewise, the MASP-1 and MASP-2 mutants were each activated by their active counterparts, but MASP-3 S645A was not. Activated MASP-3 did not react with C1 inhibitor; had no activity on complement proteins C2, C4, and C3; and only cleaved the N-carboxybenzyloxyglycine-l-arginine thiobenzyl ester substrate to a significant extent. Based on these observations, it is postulated that MASP-3 activation and control involve mechanisms that are different from those of MASP-1 and -2.

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Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys55Ala abolished interaction with the MASPs and MAp19 and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys55Gln and Lys55Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys55Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3. Mutation Arg47Glu inhibited interaction with MAp19 and decreased the ability of MBL to trigger the lectin pathway. Mutant Arg47Lys showed no interaction with the MASPs or MAp19, likely resulting from a defect in oligomerization. In contrast, mutation Arg47Ala had no impact on the interaction with the MASPs and MAp19, nor on the ability of MBL to trigger the lectin pathway. Mutation Pro53Ala only had a slight effect on the interaction with MASP-1 and -3, whereas mutations at residues Leu49 and Leu56 were ineffective. In conclusion, the MASP binding site of MBL involves a sequence stretch centered on residue Lys55, which may form an ionic bond representing the major component of the MBL-MASP interaction. The binding sites for MASP-2/MAp19 and MASP-1/3 have common features but are not strictly identical.

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.

Ficolins and pentraxins are soluble oligomeric pattern-recognition molecules that sense danger signals from pathogens and altered self-cells and might act synergistically in innate immune defense and maintenance of immune tolerance. The interaction of M-ficolin with the long pentraxin pentraxin 3 (PTX3) has been characterized using surface plasmon resonance spectroscopy and electron microscopy. M-ficolin was shown to bind PTX3 with high affinity in the presence of calcium ions. The interaction was abolished in the presence of EDTA and inhibited by N-acetyl-D-glucosamine, indicating involvement of the fibrinogen-like domain of M-ficolin. Removal of sialic acid from the single N-linked carbohydrate of the C-terminal domain of PTX3 abolished the interaction. Likewise, an M-ficolin mutant with impaired sialic acid-binding ability did not interact with PTX3. Interaction was also impaired when using the isolated recognition domain of M-ficolin or the monomeric C-terminal domain of PTX3, indicating requirement for oligomerization of both proteins. Electron microscopy analysis of the M-ficolin–PTX3 complexes revealed that the M-ficolin tetramer bound up to four PTX3 molecules. From a functional point of view, immobilized PTX3 was able to trigger M-ficolin–dependent activation of the lectin complement pathway. These data indicate that interaction of M-ficolin with PTX3 arises from its ability to bind sialylated ligands and thus differs from the binding to the short pentraxin C-reactive protein and from the binding of L-ficolin to PTX3. The M-ficolin–PTX3 interaction described in this study represents a novel case of cross-talk between soluble pattern-recognition molecules, lending further credit to the integrated view of humoral innate immunity that emerged recently.