Tina Hummelshøj

The Impact of FCN2 Polymorphisms and Haplotypes on the Ficolin‐2 Serum Levels

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.

Synergy between Ficolin-2 and Pentraxin 3 Boosts Innate Immune Recognition and Complement Deposition

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.

A Novel Mannose-binding Lectin/Ficolin-associated Protein Is Highly Expressed in Heart and Skeletal Muscle Tissues and Inhibits Complement Activation

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.

MBL2, FCN1 , FCN2 and FCN3-The genes behind the initiation of the lectin pathway of complement

Mannose-binding lectin (MBL) and the ficolins (Ficolin-1, Ficolin-2 and Ficolin-3) are soluble collagen-like proteins that are involved in innate immune defence. They bind sugar structures or acetylated compounds present on microorganisms and on dying host cells and they initiate activation of the lectin complement pathway in varying degrees. Common variant alleles situated both in promoter and structural regions of the human MBL gene (MBL2) influence the stability and the serum concentration of the protein. Although not as thoroughly investigated as the MBL2 gene polymorphisms the ficolin genes (FCNs) also exhibit genetic variations affecting both the serum concentration, stability and binding capacity of the corresponding proteins. Epidemiological studies have suggested that the genetically determined variations in MBL serum concentrations influence the susceptibility to and the course of different types of diseases, while the importance of the ficolins in general and the genetic variation in the FCNs genes in particular is still largely unresolved. This overview will summarize the current molecular knowledge of the human MBL2, FCN1, FCN2 and FCN3 genes.

Immunodeficiency Associated with FCN3 Mutation and Ficolin-3 Deficiency

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.

Characterization of a polymorphism in the coding sequence of FCN3 resulting in a Ficolin-3 (Hakata antigen) deficiency state

Ficolin-3 (Hakata antigen or H-ficolin) is a soluble pattern recognition molecule in the lectin complement pathway. We speculated whether common genetic variations in the FCN3 gene contribute to deficiency of Ficolin-3. The FCN3 gene was sequenced in 237 healthy Danish Caucasians. The relevance of polymorphisms was assessed with antibodies against Ficolin-3 in a novel ELISA system and by production of recombinant Ficolin-3 variants. Ficolin-3 serum profiles were analyzed by SDS-PAGE and western blotting. Ficolin-3 serum concentration varied 10-fold (median, 24microg/ml; range, 3-54microg/ml). Out of several polymorphisms one FCN3+1637delC causing a reading frame shift and a distortion of the C-terminal end of the molecule with an allele frequency of 0.011 was particularly interesting. In individuals heterozygous for the FCN3+1637delC deletion lowered Ficolin-3 concentration was observed (P=0.025). SDS-PAGE and western blotting of serum revealed a weak band corresponding to the truncated molecule in addition to the normal Ficolin-3 pattern. Characterization of recombinant Ficolin-3 derived from FCN3+1637delC showed that in the homozygous situation this allelic variant would lead to Ficolin-3 deficiency. In conclusion an FCN3+1637delC deletion variant disrupting the possibility for pattern recognition was detected. Characterization of recombinant variant Ficolin-3 shows that homozygosity for the FCN3+1637delC deletion may lead to Ficolin-3 deficiency and may thus be the basis for a novel complement deficiency state.

The genetics of ficolins.

Ficolins constitute a family of proteins whose biological role has been an enigma for many years. Over the past few years it has become evident that ficolins are part of the innate immune system and function as recognition molecules in the complement system. The 3 human ficolins, ficolin-1 (M-ficolin), ficolin-2 (L-ficolin) and ficolin-3 (H-ficolin or Hakata antigen) are encoded by the FCN1, FCN2 and FCN3 genes, respectively. Phylogenetic studies suggest that ficolins are of ancient origin. Ficolin-3 seems to be the most ancient molecule, from a phylogenetic perspective. Searches in databases and phylogenetic tree analysis demonstrate that the ficolin precursor has gone through an expansion involving independent duplication events in the different branches of the evolutionary tree. Of particular interest is the prediction that ficolin-1 appears to be present as an ortholog molecule. All human FCN genes are polymorphic. The FCN2 gene encoding ficolin-2, contains polymorphisms that affect ligand binding, while differences in the serum levels are associated with promoter polymorphisms. Recently, a frame-shift variation in the FCN3 gene was described, leading to ficolin-3 deficiency and defective complement activation. This FCN3 variation was also shown to be associated with immunodeficiency. This survey summarizes the current phylogenetic and inter-individual molecular understanding of the FCN genes.

The innate pattern recognition molecule Ficolin-1 is secreted by monocytes/macrophages and is circulating in human plasma

Ficolin-1 (M-Ficolin) is a pattern recognition molecule of the complement system that is expressed by myeloid cells and type II alveolar epithelial cells. Ficolin-1 has been shown to localize in the secretory granules of these cells and attached to cell surfaces, but whether Ficolin-1 exists a soluble molecule in the extracellular environment or in plasma is unknown. In this study we explored the possibility that Ficolin-1 may be secreted from monocytes, macrophages or immature dendritic cells and may exist in human plasma. Expression of Ficolin-1 was analyzed using real-time quantitative PCR and SDS-PAGE/western blot. Secretion of Ficolin-1 was investigated in cells and plasma from healthy donors through affinity purification using N-acetyl-d-glucosamine-agarose beads and ELISA. Ficolin-1 was found differentially expressed and synthesised by monocytes, macrophages and immature dendritic cells. Notably monocytes and macrophages, but not immature dendritic cells are able to secrete Ficolin-1 into the extracellular environment. Moreover, Ficolin-1 was detected in human plasma from healthy donors with a median concentration of 60.5 ng/ml ranging from 45.7 to 100.4 ng/ml. We show that Ficolin-1 is secreted into the extracellular environment from human monocytes/macrophages, but not immature dendritic cells. Importantly, these results demonstrate that Ficolin-1 exists in human plasma and serum under normal conditions, hereby revising the general assumption that Ficolin-1 is solely a cellular associated protein.

Functional SNPs in the human ficolin (FCN) genes reveal distinct geographical patterns

The ficolin protein family comprises three different molecules encoded by the FCN1, FCN2, and FCN3 genes, respectively, that play roles in innate immunity. The FCN genes in Caucasians are polymorphic and genetic variations may have functional consequences both in relation to function and concentration. The ethnic diversity of the FCN genes is unknown. The promoter and coding regions of the FCNs genes were sequenced in individuals from five different ethnic groups: Caucasians (Denmark, n=60), Japanese (Japan, n=50), South-East Africans (Mozambique, n=50), West-Africans (Ghana, n=50), and Indians (Argentina, n=50). We identified the most common FCN gene polymorphisms in five ethnic groups. Large ethnic differences were observed and the African populations contained several SNPs that were not observed in the other groups. Several variations, that will have major impact on the function of the ficolin proteins, were found. Three novel amino acid variations in Ficolin-1*Gly303Ser, Ficolin-2*Arg103Cys, and Ficolin-2*Thr137Met SNP were predicted by computational analyses to have a major functional physicochemical effect on their respective proteins. Additionally, a Gly43Asp in Ficolin-1 affects the Gly-Xaa-Yaa repeats and a Trp279STOP introduces a stop codon, thereby destroying the fibrinogen-like domain of Ficolin-1. In contrast to FCN1 and FCN2, the number of SNPs in FCN3 was very low. In conclusion, large ethnic differences in the FCN genes that will affect the concentration, structure, and function of the ficolin molecules were detected and which probably will be of pathophysiological relevance in different disease settings.

Functional Analysis of Ficolin-3 Mediated Complement Activation

The recognition molecules of the lectin complement pathway are mannose-binding lectin and Ficolin -1, -2 and -3. Recently deficiency of Ficolin-3 was found to be associated with life threatening infections. Thus, we aimed to develop a functional method based on the ELISA platform for evaluating Ficolin-3 mediated complement activation that could be applicable for research and clinical use. Bovine serum albumin (BSA) was acetylated (acBSA) and chosen as a solid phase ligand for Ficolins in microtiter wells. Binding of Ficolins on acBSA was evaluated, as was functional complement activation assessed by C4, C3 and terminal complement complex (TCC) deposition. Serum Ficolin-3 bound to acBSA in a calcium dependent manner, while only minimal binding of Ficolin-2 and no binding of Ficolin-1 were observed. No binding to normal BSA was seen for any of the Ficolins. Serum C4, C3 and TCC deposition on acBSA were dependent only on Ficolin-3 in appropriate serum dilutions. Deposition of down stream complement components correlated highly significantly with the serum concentration of Ficolin-3 but not with Ficolin-2 in healthy donors. To make the assay robust for clinical use a chemical compound was applied to the samples that inhibited interference from the classical pathway due to the presence of anti-BSA antibodies in some sera. We describe a novel functional method for measuring complement activation mediated by Ficolin-3 in human serum up to the formation of TCC. The assay provides the possibility to diagnose functional and genetic defects of Ficolin-3 and down stream components in the lectin complement pathway.