Preclinical exploration of combined glucagon inhibition and liver-preferential insulin for treatment of diabetes using in vitro assays and rat and mouse models.

AIMS/HYPOTHESIS: Normalisation of blood glucose in individuals with diabetes is recommended to reduce development of diabetic complications. However, risk of severe hypoglycaemia with intensive insulin therapy is a major obstacle that prevents many individuals with diabetes from obtaining the recommended reduction in HbA1c. Inhibition of glucagon receptor signalling and liver-preferential insulin action have been shown individually to have beneficial effects in preclinical models and individuals with diabetes (i.e. improved glycaemic control), but also have effects that are potential safety risks (i.e. alpha cell hyperplasia in response to glucagon receptor antagonists and increased levels of liver triacylglycerols and plasma alanine aminotransferase activity in response to glucagon receptor antagonists and liver-preferential insulin). We hypothesised that a combination of glucagon inhibition and liver-preferential insulin action in a dual-acting molecule would widen the therapeutic window. By correcting two pathogenic mechanisms (dysregulated glucagon signalling and non-physiological distribution of conventional insulin administered s.c.), we hypothesised that lower doses of each component would be required to obtain sufficient reduction of hyperglycaemia, and that the undesirable effects that have previously been observed for monotreatment with glucagon antagonists and liver-preferential insulin could be avoided. METHODS: A dual-acting glucagon receptor inhibitor and liver-preferential insulin molecule was designed and tested in rodent models (normal rats, rats with streptozotocin-induced hyperglycaemia, db/db mice and mice with diet-induced obesity and streptozotocin-induced hyperglycaemia), allowing detailed characterisation of the pharmacokinetic and pharmacodynamic properties of the dual-acting molecule and relevant control compounds, as well as exploration of how the dual-acting molecule influenced glucagon-induced recovery and spontaneous recovery from acute hypoglycaemia. RESULTS: This molecule normalised blood glucose in diabetic models, and was markedly less prone to induce hypoglycaemia than conventional insulin treatment (approximately 4.6-fold less potent under hypoglycaemic conditions than under normoglycaemic conditions). However, compared to treatment with conventional long-acting insulin, this dual-acting molecule also increased triacylglycerol levels in the liver (approximately 60%), plasma alanine aminotransferase levels (approximately twofold) and alpha cell mass (approximately twofold). CONCLUSIONS/INTERPRETATION: While the dual-acting glucagon receptor inhibitor and liver-preferential insulin molecule showed markedly improved regulation of blood glucose, effects that are potential safety concerns persisted in the pharmacologically relevant dose range.

Crystal structure and functional characterization of the complement regulator mannose-binding lectin (MBL)/ficolin-associated protein-1 (MAP-1).

The human lectin complement pathway activation molecules comprise mannose-binding lectin (MBL) and ficolin-1, -2, and -3 in complex with associated serine proteases MASP-1, -2, and -3 and the non-enzymatic small MBL associated protein or sMAP. Recently, a novel plasma protein named MBL/ficolin-associated protein-1 (MAP-1) was identified in humans. This protein is the result of a differential splicing of the MASP1 gene and includes the major part of the heavy chain but lacks the serine protease domain. We investigated the direct interactions of MAP-1 and MASP-3 with ficolin-3 and MBL using surface plasmon resonance and found affinities around 5 nm and 2.5 nm, respectively. We studied structural aspects of MAP-1 and could show by multi-angle laser light scattering that MAP-1 forms a calcium-dependent homodimer in solution. We were able to determine the crystal structure of MAP-1, which also contains a head-to-tail dimer ∼146 Šlong. This structure of MAP-1 also enables modeling and assembly of the MASP-1 molecule in its entirety. Finally we found that MAP-1 competes with all three MASPs for ligand binding and is able to mediate a strong dose-dependent inhibitory effect on the lectin pathway activation, as measured by levels of C3 and C9.

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.

Structural Investigations of Full-Length Insulin Receptor Dynamics and Signalling.

Insulin regulates glucose homeostasis via binding and activation of the insulin receptor dimer at two distinct pairs of binding sites 1 and 2. Here, we present cryo-EM studies of full-length human insulin receptor (hIR) in an active state obtained at non-saturating, physiologically relevant insulin conditions. Insulin binds asymmetrically to the receptor under these conditions, occupying up to three of the four possible binding sites. Deletion analysis of the receptor together with site specific peptides and insulin analogs used in binding studies show that both sites 1 and 2 are required for high insulin affinity. We identify a homotypic interaction of the fibronectin type III domain (FnIII-3) of IR resulting in tight interaction of membrane proximal domains of the active, asymmetric receptor dimer. Our results show how insulin binding at two distinct types of sites disrupts the autoinhibited apo-IR dimer and stabilizes the active dimer. We propose an insulin binding and activation mechanism, which is sequential, exhibits negative cooperativity, and is based on asymmetry at physiological insulin concentrations with one to three insulin molecules activating IR.

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.

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.

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.

Comparative study of the human ficolins reveals unique features of Ficolin-3 (Hakata antigen).

The ficolins and mannose-binding lectin (MBL) are collagen-like defence proteins that serve as recognition molecules in lectin complement pathway. Differential features that may indicate diverse functions of these proteins are poorly understood. In this study we compared important biological features of the ficolins and MBL. We investigated the tissue distribution of the FCN1-3 and the MBL2 genes encoding the ficolins and MBL by quantitative PCR. Recombinant proteins were produced and structural and biological characteristics were investigated and compared. Our main findings were that FCN3 mRNA was highly expressed in the liver and lung compared with the other genes revealing the lung as the tissue with the highest FCN3 expression pattern. Ficolin-3 revealed higher complement activating capacity compared with Ficolin-2, MBL and Ficolin-1 and was highly resistant to bacterial collagenase treatment, which is different from the other ficolins and MBL. We discovered several unique properties of Ficolin-3 showing that FCN3 is the most highly expressed gene in liver and lung among the lectin complement pathway initiators. Moreover, Ficolin-3 has a high complement activating potential and is the only collagenase proteolytic resistant molecule among the lectin complement pathway initiators.

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 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.

Ficolins and Mannose-Binding Lectin in Danish patients with sarcoidosis.

Mannose-Binding Lectin (MBL) is a prognostic marker in pulmonary diseases. Ficolins, sharing many structural and functional similarities with MBL, may also be involved in the pathogenesis of pulmonary diseases. The objectives of the study were to establish whether plasma concentrations of Ficolin-2, -3, and MBL in Danish patients with sarcoidosis and control persons differed and whether they were of prognostic significance. We retrospectively included 46 consecutive patients (26 male, 20 female) and 51 age- and sex-matched healthy control persons (28 male, 23 female). Information about the patients was obtained from their medical records. We measured plasma concentrations of Ficolin-2, -3, and MBL using ELISA. There was a significant difference in the patients' mean Ficolin-3 plasma level (14.9 microg/ml; +/-2SD: 6.7-23.1) compared with the control persons' (21.6 microg/ml; +/-2SD: 12.7-30.5). The difference was 6.7 microg/ml (95% CI: 5.0-8.4 microg/ml; p<0.001). In the patients, Ficolin-3 correlated inversely with the CD4(+)/CD8(+)-ratio (Spearman's Rho=-0.37; p=0.021; n=39). There were no significant differences in plasma concentrations of Ficolin-2 or MBL between the two groups. Ficolin-3 concentrations were lower in plasma from patients with sarcoidosis. This suggests a possible involvement of Ficolin-3 in the complex pathophysiology of sarcoidosis. However, we could not show the applicability of Ficolin plasma level measurement as a marker of disease activity or of prognostic significance in sarcoidosis.

Ficolin-2 recognizes DNA and participates in the clearance of dying host cells.

Ficolin-2 is a serum opsonin, which has been shown to be a pattern recognition molecule in the lectin complement activation pathway. Because innate immune mechanisms are involved in maintaining tissue homeostasis we hypothesized that Ficolin-2 also participate in the clearance of dying host cells. We found that Ficolin-2 binds to late apoptotic cells, as well as to apoptotic bodies and necrotic cells, but not to early apoptotic cells. We demonstrated that Ficolin-2 binds DNA in a calcium dependent manner and that DNA inhibits the binding to late apoptotic and necrotic cells, suggesting that DNA on permeable dying cells is a plausible ligand. Reconstituting serum deficient of Ficolin-2, C1q and mannose-binding lectin with Ficolin-2 augmented deposition of complement C4 on necrotic cells. Opsonization leads to an enhanced attachment/uptake of necrotic cells by macrophages. In conclusion dying host cells expose ligands with the capacity of binding Ficolin-2, which in turn leads to increased attachment and engulfment. Binding of Ficolin-2 to DNA points at nucleic acid exposed by permeable late apoptotic and necrotic cells as one of the ligands for Ficolin-2. Ficolin-2 may therefore be a scavenger molecule participating in the removal of host cells and maintenance of tissue homeostasis.

Molecular organization of human Ficolin-2.

Human Ficolin-2 (L-Ficolin) is an oligomeric serum protein consisting of a collagen-like stalk and fibrinogen-like recognition domains. The protein binds to arrays of sugars present on different microorganisms, enhances phagocytosis and promotes activation of the lectin complement pathway. So far the detailed oligomeric structure and composition of human Ficolin-2 has not been determined. Recombinant human Ficolin-2 was expressed in Chinese hamster ovary cells and its structure and biological functions were investigated by gel filtration, sucrose density gradient ultracentrifugation, mass spectrometry and surface plasmon resonance spectroscopy. It was revealed that Ficolin-2 has a high molecular weight due to extensive disulfide bridge formation. It was able to bind to different ligands, interact with mannose-binding lectin associated serine proteases and activate the complement system. Mass values of 807 and 403 kDa were determined corresponding to a 24-mer and a 12-mer of 34.4 kDa polypeptides. However, the 24-mer was unstable and the 12-mer is likely the major functional form of the protein. Our results are consistent with the view that Ficolin-2 is built up by a mixture of stable homodimers and homotrimers. Based on our findings we propose a model in which disulfide bridges located in the N-terminal region of the polypeptides explain the oligomerization pattern of human Ficolin-2.

A functional polymorphism in the Eta-1 promoter is associated with allele specific binding to the transcription factor Sp1 and elevated gene expression.

Early T lymphocyte activator 1 (Eta-1), also known as Osteopontin, is a cytokine produced by macrophages and T lymphocytes. It is involved in the regulation of IL-12 and IL-10 expression in macrophages and stimulates the polarization of T cells to the Th1 subset. Three promoter polymorphisms of the human Eta-1 gene, -443T/C, -156delG/G, -66T/G, were investigated for possible influence on gene expression. Electrophoretic mobility shift assays (EMSA) with nuclear extract from the human myeloid leukaemia premonocyte cell line, THP-1, revealed sequence specific binding of the transcription factor Sp1 to the -66T allele but not the -66G allele, and haplotype -443C/-156G/-66T showed a marked increase in promoter activity of a luciferase reporter gene. Thus, a substitution of the T-base with G at position -66 in the Eta-1 promoter modulates the promoter activity of the Eta-1 gene, which might influence the Th1 versus Th2 balance. These observations are discussed in relation to a recently reported related observation on the same gene, and it is argued that discrepancies between reporter gene assays in the two studies may be due to the use of different cell lines and may reflect requirements for different transcription factors in cells involved in immune responses compared with other cells.

The interaction pattern of murine serum ficolin-A with microorganisms.

The ficolins are soluble pattern recognition molecules in the lectin pathway of complement, but the spectrum and mode of interaction with pathogens are largely unknown. In this study, we investigated the binding properties of the murine serum ficolin-A towards a panel of different clinical relevant microorganisms (N = 45) and compared the binding profile with human serum ficolin-2 and ficolin-3. Ficolin-A was able to bind Gram-positive bacteria strains including E. faecalis, L. monocytogenes and some S. aureus strains, but not to the investigated S. agalactiae (Group B streptococcus) strains. Regarding Gram-negative bacteria ficolin-A was able to bind to some E. coli and P. aeruginosa strains, but not to the investigated Salmonella strains. Of particular interest ficolin-A bound strongly to the pathogenic E. coli, O157:H7 and O149 strains, but it did not bind to the non-pathogenic E. coli, ATCC 25922 strain. Additionally, ficolin-A was able to bind purified LPS from these pathogenic strains. Furthermore, ficolin-A bound to a clinical isolate of the fungus A. fumigatus. In general ficolin-2 showed similar selective binding spectrum towards pathogenic microorganisms as observed for ficolin-A indicating specific pathophysiological roles of these molecules in host defence. In contrast, ficolin-3 did not bind to any of the investigated microorganisms and the anti-microbial role of ficolin-3 still remains elusive.

Serum concentration and interaction properties of MBL/ficolin associated protein-1.

Recently, a novel protein named MBL/ficolin associated protein-1 (MAP-1) derived from the MASP1 gene through differential splicing was identified. In the present study, we established biochemical characteristics, determined the serum level and assessed the interactions between the lectin complement pathway (LCP) recognition molecules and MAP-1. We expressed recombinant MAP-1 in CHO DG44 cells, developed a quantitative ELISA assay based on a MAP-1 specific monoclonal capture antibody and measured the serum levels in 100 Danish blood donors. In addition we assessed the association properties between MAP-1 and Ficolin-2, -3 and MBL in serum using ELISA and density gradient ultra centrifugation. When recombinant MAP-1 was subjected to N-glycosidase F treatment the molecular mass decreased from ∼45 kDa to ∼40 kDa equivalent with the calculated molecular mass from the deduced amino acid sequence without the signal peptide. We found that serum MAP-1 was very stable when subjected to repeated freeze and thaw cycles. The mean serum concentration of MAP-1 was found to be 240 ng/ml (range: 115-466 ng/ml). MAP-1 was predominantly found in complex with Ficolin-3 and to a lesser degree with Ficolin-2 and MBL and by use of density gradient ultra centrifugation we could show that the major part of serum MAP-1 circulates in complex with the LCP molecules. In conclusion, these results show that MAP-1 is a highly stable glycosylated human serum protein found in complex with Ficolin-3, Ficolin-2 and MBL.

Allelic lineages of the ficolin genes (FCNs) are passed from ancestral to descendant primates.

The ficolins recognize carbohydrates and acetylated compounds on microorganisms and dying host cells and are able to activate the lectin pathway of the complement system. In humans, three ficolin genes have been identified: FCN1, FCN2 and FCN3, which encode ficolin-1, ficolin-2 and ficolin-3, respectively. Rodents have only two ficolins designated ficolin-A and ficolin-B that are closely related to human ficolin-1, while the rodent FCN3 orthologue is a pseudogene. Ficolin-2 and ficolin-3 have so far only been observed in humans. Thus, we performed a systematic investigation of the FCN genes in non-human primates. The exons and intron-exon boundaries of the FCN1-3 genes were sequenced in the following primate species: chimpanzee, gorilla, orangutan, rhesus macaque, cynomolgus macaque, baboon and common marmoset. We found that the exon organisation of the FCN genes was very similar between all the non-human primates and the human FCN genes. Several variations in the FCN genes were found in more than one primate specie suggesting that they were carried from one species to another including humans. The amino acid diversity of the ficolins among human and non-human primate species was estimated by calculating the Shannon entropy revealing that all three proteins are generally highly conserved. Ficolin-1 and ficolin-2 showed the highest diversity, whereas ficolin-3 was more conserved. Ficolin-2 and ficolin-3 were present in non-human primate sera with the same characteristic oligomeric structures as seen in human serum. Taken together all the FCN genes show the same characteristics in lower and higher primates. The existence of trans-species polymorphisms suggests that different FCN allelic lineages may be passed from ancestral to descendant species.

Tethering of Ficolin-1 to cell surfaces through recognition of sialic acid by the fibrinogen-like domain.

Three Ficolins have been identified in humans: Ficolin-1 (M-Ficolin), Ficolin-2 (L-Ficolin), and Ficolin-3 (H-Ficolin). Ficolin-1 is the least-described of the Ficolins and is expressed by monocytes, granulocytes, and in the lungs. Ficolin-1 is found circulating at low concentrations in serum but is regarded primarily as a secretory molecule that exerts its function locally in inflamed tissues. Ficolin-1 has been reported on the surface of monocytes and granulocytes and was suggested originally to function as a phagocytic receptor. However, the molecule does not contain any obvious transmembrane domain, and no binding partners have been identified. To gain further insight in the physiological role of Ficolin-1, we sought to identify the molecular mechanism responsible for the membrane association of Ficolin-1 to monocytes and granulocytes. We demonstrate that expression of Ficolin-1 on the cell surface is restricted to monocytes and granulocytes. Ficolin-1 is tethered to the cell surface of these cells through its fibrinogen-like domain, and the ligand involved in the binding of Ficolin-1 is shown to be sialic acid. Moreover, rFicolin-1 bound activated but not resting T lymphocytes. Together, these results demonstrate a novel self-recognition mechanism of leukocytes mediated by the fibrinogen-like domain of Ficolin-1.

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.

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.