The long pentraxin 3 (PTX3) is a multifunctional soluble pattern recognition molecule that is crucial in innate immune protection against opportunistic fungal pathogens such as Aspergillus fumigatus. The mechanisms that mediate downstream effects of PTX3 are largely unknown. However, PTX3 interacts with C1q from the classical pathway of the complement. The ficolins are recognition molecules of the lectin complement pathway sharing structural and functional characteristics with C1q. Thus, we investigated whether the ficolins (Ficolin-1, -2, and -3) interact with PTX3 and whether the complexes are able to modulate complement activation on A. fumigatus. Ficolin-2 could be affinity-isolated from human plasma on immobilized PTX3. In binding studies, Ficolin-1 and particularly Ficolin-2 interacted with PTX3 in a calcium-independent manner. Ficolin-2, but not Ficolin-1 and Ficolin-3, bound A. fumigatus directly, but this binding was enhanced by PTX3 and vice versa. Ficolin-2-dependent complement deposition on the surface of A. fumigatus was enhanced by PTX3. A polymorphism in the FCN2 gene causing a T236M amino acid change in the fibrinogen-like binding domain of Ficolin-2, which affects the binding to GlcNAc, reduced Ficolin-2 binding to PTX3 and A. fumigatus significantly. These results demonstrate that PTX3 and Ficolin-2 may recruit each other on pathogens. The effect was alleviated by a common amino acid change in the fibrinogen-like domain of Ficolin-2. Thus, components of the humoral innate immune system, which activate different complement pathways, cooperate and amplify microbial recognition and effector functions.

Ficolin‐2 (L‐ficolin), derived from the FCN2 gene, is an innate immunity pattern recognition molecule found in human serum in which inter‐individual variation in serum appears to be under genetic control. To validate and extend this finding, we developed a sandwich ELISA for detection of human Ficolin‐2 in serum samples and identified FCN2 genotypes with a Taq Man‐based minor groove binder assay and by sequencing. Serum samples were applied to gel‐permeation chromatography and fractions were analysed by an ELISA, SDS‐PAGE and subsequently Western blotting. In 214 Danish blood donors, the median Ficolin‐2 serum concentration was determined to 5.4 μg/ml (range: 1.0–12.2 μg/ml). An ELISA, SDS‐PAGE and Western blot analysis of gel‐permeation chromatography fractions showed that Ficolin‐2 comprises a mixture of covalently and non‐covalently linked Ficolin‐2 oligomers independent of the individual genotypes. The variation in serum concentration was associated with three polymorphisms in the promoter and one polymorphism in the structural part of the FCN2 gene. Further analysis indicated that two particular alleles on the same haplotype determined a low Ficolin‐2 concentration. Our results show that inter‐individual variation of Ficolin‐2 concentration is associated with polymorphisms in the promoter and the structural part of the FCN2 gene.

The human lectin complement pathway involves circulating complexes consisting of mannose-binding lectin (MBL) or three ficolins (ficolin-1, -2, and -3) in association with three MBL/ ficolin-associated serine proteases (MASP) (MASP-1, -2, and -3) and a nonenzymatic sMAP. MASP-1 and MASP-3 (MASP1 isoforms 1 and 2, respectively) are splice variants of the MASP1 gene, whereas MASP-2 and sMAP are splice variants of the MASP2 gene. We have identified a novel serum protein of 45 kDa that is associated with MBL and the ficolins. This protein is named MBL/ficolin-associated protein 1 (MAP-1 corresponding to MASP1 isoform 3). The transcript generating MAP-1 (MASP1_v3) contains exons 1-8 and a novel exon encoding an in-frame stop codon. The corresponding protein lacks the serine protease domains but contains most of the common heavy chain of MASP-1 and MASP-3. Additionally MAP-1 contains 17 unique C-terminal amino acids. By use of quantitative PCR and MAP-1-specific immunohistochemistry, we found that MAP-1 is highly expressed in myocardial and skeletal muscle tissues as well as in liver hepatocytes with a different expression profile than that observed for MASP-1 and MASP-3. MAP-1 co-precipitated from human serum with MBL, ficolin-2, and ficolin-3, and recombinant MAP-1 was able to inhibit complement C4 deposition via both the ficolin-3 and MBL pathway. In conclusion we have identified a novel 45-kDa serum protein derived from the MASP1 gene, which is highly expressed in striated muscle tissues. It is found in complex with MBL and ficolins and may function as a potent inhibitor of the complement system in vivo.Activation of the complement system is accomplished via three different initiation pathways: the alternative pathway, the classical pathway, and the lectin pathway (1). In humans, four recognition molecules of the lectin pathway have been described: mannose-binding lectin (MBL), 3 ficolin-1 (also called M-ficolin), ficolin-2 (also called L-ficolin), and ficolin-3 (also called H-ficolin or Hakata antigen) (2). MBL and the ficolins bind structures on different classes of microorganisms and are involved in sequestration and removal of dying host cells (2, 3). Three MBL/ficolin-associated serine proteases have been described so far (MASP-1, MASP-2, and MASP-3), as well as a protein lacking a serine protease domain named sMAP or MAp19 (4). Present consensus places MASP-2 as the main initiator of the lectin complement pathway by cleaving C4 and C2 to form the C4b2a complex leading to further downstream complement activation (5). Although the functions of the other MASPs are poorly understood, MASP-1 appears to play a role as an amplifier of complement activation (6, 7). Additionally, MASP-1 is able to cleave fibrinogen to fibrin, whereas MASP-2 is able to generate active thrombin by cleavage of prothrombin (8,9). No conclusive biological function has yet been attributed to MASP-3 and sMAP. MASP-1 and MASP-2 were originally named after their association with MBL (5, 10). Subsequently, MASP-3 and sMAP (also named Map19) we...

The ficolin 1, 2 and 3 (derived from the FCN1, 2 and 3 genes, respectively) are homologous soluble pattern recognition molecules of importance for innate immunity, comprising collagen-like and fibrinogen-like domains, binding to sugar groups on different types of microorganisms. Serum concentration of Ficolin-2 varies considerably in healthy individuals. Thus, we speculated whether this could be due to variations in the FCN2 gene. We sequenced the promoter region and the exons and intron-exon boundaries of FCN2 in Danish Caucasians. For comparison, FCN1 and FCN3 were also investigated. Ficolin-2 concentrations were measured in serum and the functional relevance of amino acid substituting polymorphisms in FCN2 was investigated by binding to and recovery from N-acetylglucosamine (GlcNAc). Both FCN1 and FCN2 contained polymorphisms in the promoters and structural parts of the genes, but only polymorphisms in FCN2 resulted in amino acid exchanges. FCN2 promoter polymorphisms were associated with marked changes in the Ficolin-2 serum concentration, whereas two polymorphisms clustered in the exon encoding the fibrinogen-like domain were associated with increased and decreased GlcNAc binding, respectively. In FCN3, only a single frame-shift deletion in exon 5 was detected. These results show that the FCN genes are polymorphic and that particularly FCN2 harbors functional polymorphic sites that regulate both the expression as well as the function of Ficolin-2, which may have pathophysiological implications for innate immunity.

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Ficolin-3, encoded by the FCN3 gene and expressed in the lung and liver, is a recognition molecule in the lectin pathway of the complement system. Heterozygosity for an FCN3 frameshift mutation (rs28357092), leading to a distortion of the C-terminal end of the molecule, occurs in people without disease (allele frequency among whites, 0.01). We describe a patient with recurrent infections who was homozygous for this mutation, who had undetectable serum levels of ficolin-3, and who had a deficiency in ficolin-3-dependent complement activation.

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