Human Ficolin Research Articles

FCN3 inhibits the progression of hepatocellular carcinoma by suppressing SBDS-mediated blockade of the p53 pathway.

Hepatocellular carcinoma (HCC) is the third-leading cause of cancer deaths globally. Although considerable progress has been made in the treatment, clinical outcomes of HCC patients are still poor. Therefore, it is necessary to find novel prognostic factors upon which prevention and treatment strategies can be formulated. Ficolin-3 (FCN3) protein is a member of the human ficolin family. It activates complement through pathways associated with mannose-binding lectin-associated serine proteases. Herein, we identified that FCN3 was downregulated in HCC tissues and decreased FCN3 expression was closely related to poor prognosis. Overexpression of FCN3 induced apoptosis and inhibited cell proliferation via the p53 signaling pathway. Mechanistically, FCN3 modulated the nuclear translocation of eukaryotic initiation factor 6 (EIF6) by binding ribosome maturation factor (SBDS), which induced ribosomal stress and activation of the p53 pathway. In addition, Y-Box Binding Protein 1 (YBX1) involved in the transcription and translation level regulation of FCN3 to SBDS. Besides, a negative feedback loop in the downstream of FCN3 involving p53, YBX1 and SBDS was identified.

Sialic acid-specific lectin participates in an immune response and ovarian development of the banana shrimp Fenneropenaeus merguiensis.

A sialic acid-specific lectin was purified from the hemolymph of Fenneropenaeus merguiensis by repetitive affinity fetuin-agarose column chromatography. The purified F. merguiensis lectin (called FmL) consisted of two distinct 30.9 and 32kDa subunits with identical N-terminal amino acid sequences of ten residues. FmL was also composed of sugar moieties; glucosamine, glucose, mannose and N-acetyl neuraminic acid but not N-glycolyl neuraminic acid. It was postulated to be a glycoprotein as it was positively stained by glycoprotein staining kit and detected by some bionylated plant lectins. Deglycosylation by either peptide N-glycosidase F or trifluoromethanesulfonic acid turned both types of FmL subunits to 28kDa peptides. The internal peptide sequence of FmL was similar to a fibrinogen-related domain of human ficolin and the horseshoe crab lectin. Determination of the lectin concentrations in the hemolymph was performed by ELISA while its hemaglutinating activity (HA) was tested by hemagglutination. Both specific lectin concentrations and HA increased as shrimp developed ovarian maturation stages 2 to 4. Their constitutive levels were found in pre-vitellogenic females and higher than those of males. Both specific lectin concentrations and HA of FmL were inducible to the highest levels at 12h after F. merguiensis was challenged by pathogenic Vibrio harveyi. The FmL-induced agglutination of V. harveyi was specifically abolished by sialic acid, fetuin and bacterial cell wall components. These findings might indicate the implication in an immune response of FmL to protect the shrimp themselves or their spawning eggs towards pathogenic bacteria in surrounding environment.

Mannose-Binding Lectin and Susceptibility to Schistosomiasis

Background. Human ficolin 2 (encoded by FCN2) and mannose-binding lectin (encoded by MBL2) bind to specific pathogen-associated molecular patterns, activate the complement lectin cascade in a similar manner, and are associated with several infectious diseases. Our recently published study established certain FCN2 promoter variants and ficolin-2 serum levels as protective factors against schistosomiasis. Methods. We used the Nigerian cohort from our recently published study, which included 163 Schistosoma haematobium–infected individuals and 183 matched healthy subjects, and investigated whether MBL deficiency and MBL2 polymorphisms are associated with schistosomiasis. Results. MBL serum levels were significantly higher in controls and were associated with protection (P < .0001). The −550H minor allele was significantly associated with protection (P = .03), and the heterozygous genotypes −550HL were observed to confer protection (P = .03). The MBL2*HYPA haplotype was significantly associated with protection (P = .03), with significantly higher serum MBL levels in controls (P = .00073). The heterozygous 6-bp deletion in the promoter was observed to be a susceptibility factor in schistosomiasis (P = .03). Conclusions. In agreement with findings from our recently published study, the findings reported here support the observation that MBL is also associated with protection in schistosomiasis.

Soluble Host-Defense Lectins

Lectins are proteins that bind to carbohydrates and are distributed widely in animals, plants, bacteria, and so forth. Animal lectins have a role in many biological functions including development and immunity. There are two kinds of immune system in vertebrates: innate immunity and adaptive (or acquired) immunity. Innate immunity plays an important role at an early stage of infection, when phagocytic cells (neutrophils and macrophages), NK cells, and complement are major components. Adaptive immunity takes over from the innate immune system as time passes. In adaptive immunity, antibodies, T cells, and B cells are major players. One of the striking differences between innate immunity and adaptive immunity is in recognition specificity for pathogens. Antibodies and T cells are able to recognize pathogens or their derived peptides with high specificity. On the other hand, the recognition specificity of innate immunity is broad. Invertebrates have innate immunity but not adaptive immunity. Proteins known as pattern-recognition proteins recognize components peculiar to microbes on their surfaces. These components are called pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide, peptidoglycan, lipoteichoic acid, and β-glucan. Many soluble animal lectins involved as pattern-recognition proteins have been identified, and their structures and functions have been characterized. These lectins are termed “soluble host-defense lectins” that include collectins, ficolins, galectins, and pentraxins. Soluble host-defense lectins comprise several groups of proteins defined by their structural features. For instance, collectins are proteins that consist of a collagen-like domain and a carbohydrate-recognition domain and include mannose-binding lectin (MBL), SP-A, SP-D, and CL-K1. Ficolins consist of a collagen-like domain combined with a fibrinogen-like domain and include human L-ficolin and mouse ficolin A. Soluble host-defense lectins bind to PAMPs of pathogens as a recognition molecule and elicit immune effector mechanisms including enhancement of phagocytosis and activation of the complement system. Enhancement of phagocytosis involves specific receptors for soluble host-defense lectins present on the membranes of phagocytes. The complement system is an effector system in immunity. It consists of many proteins involved in a cascade of proteolysis and protein complex assembly that leads to the elimination of pathogens in several ways including enhancement of phagocytosis and direct killing. The system is activated via three pathways (the classical, alternative, and lectin pathways). The lectin pathway is an important component of innate immunity and is activated by the binding of serum ficolins or MBL to carbohydrates on the pathogen surface. These soluble host-defense lectins form complexes with three types of MBL-associated serine proteases (MASPs) and their truncated proteins. It was shown that CL-K1 is also complexed with MASPs. In the lectin pathway, the binding of the ficolin-MASP complex or the MBL-MASP complex (i.e., a complex composed of ficolins or MBL, MASPs, and their truncated proteins) to carbohydrates on the microbe surface initiates the activation of complement, leading to the elimination of microbes. This special issue, including 4 original articles and 6 review articles, focuses on “soluble host-defense lectins” in terms of their structure, function, and physiological and pathological relevance. Many of the papers deal with collectins: the functions of novel collectins including CL-K1 (K. Ohtani et al.), the role of MBL in inflammation (M. Larvie et al.), pathological relevance of MBL in IgA nephrology (I. Ohsawa et al.), anti-influenza virus activity of collectins (W. C. Ng et al.), and diverse functions of pulmonary collectins (S. Ariki et al.). Ficolins are also discussed in two papers regarding the clinical significance of L-ficolin (D. C. Kilpatrick and J. D. Chalmers) and the ability of mouse ficolin B to activate the lectin complement pathway (Y. Endo et al.). M. Cedzynski et al. report a role for MBL and ficolins in the protection against infection in neonates. A paper concerning the role of DC-SIGN, a lectin on dendritic cells, in bacterial interaction (E. Miszczyk et al.) and a paper describing the association between the genetic polymorphisms of CLEC5A, a lectin on myeloid cells, and Kawasaki disease (Y.-L. Yang et al.) are also included in this issue. Misao Matsushita David C. Kilpatrick Bok Luel Lee Nobutaka Wakamiya

Fibrinogen-related proteins in ixodid ticks

BackgroundFibrinogen-related proteins with lectin activity are believed to be part of the tick innate immune system. Several fibrinogen-related proteins have been described and characterised mainly on the basis of their cDNA sequences while direct biochemical evidence is missing. One of them, the haemolymph lectin Dorin M from the tick Ornithodoros moubata was isolated and characterised in more depth.ResultsSeveral fibrinogen-related proteins were detected in the haemolymph of ixodid ticks Dermacentor marginatus, Rhipicephalus appendiculatus, R. pulchellus, and R. sanguineus. These proteins were recognised by sera directed against the tick lectin Dorin M and the haemagglutination activity of the ticks R. appendiculatus and D. marginatus. Cross-reactivity of the identified proteins with antibodies against the fibrinogen domain of the human ficolin was also shown. The carbohydrate-binding ability of tick haemolymph was confirmed by haemagglutination activity assays, and this activity was shown to be inhibited by neuraminic acid and sialylated glycoproteins as well as by N-acetylated hexosamines. The fibrinogen-related proteins were shown to be glycosylated and they were localised in salivary glands, midguts, and haemocytes of D. marginatus. Hemelipoglycoprotein was also recognised by sera directed against the fibrinogen-related proteins in all three Rhipicephalus species as well as in D. marginatus. However, this protein does not contain the fibrinogen domain and thus, the binding possibly results from the structure similarity between hemelipoglycoprotein and the fibrinogen domain.ConclusionsThe presence of fibrinogen-related proteins was shown in the haemolymph of four tick species in high abundance. Reactivity of antibodies directed against ficolin or fibrinogen-related proteins with proteins which do not contain the fibrinogen domain points out the importance of sequence analysis of the identified proteins in further studies. Previously observed expression of fibrinogen-related proteins in haemocytes together with the results of this study suggest involvement of fibrinogen-related proteins in tick immunity processes. Thus, they have potential as targets for anti-tick vaccines and as antimicrobial proteins in pharmacology. Research on fibrinogen-related proteins could reveal further details of tick innate immunity processes.

Peptide Isomerization Induced by pH Change Regulates the S1 Binding Site in Ficolins

Infection-inflammation mediated interactions between human ficolin and the pathogen GlcNAc is associated with local acidosis, leading to antimicrobial action. Therefore, revealing the precise molecular conformation induced by pH-shift is crucial in understanding the immune response. Here, we performed constant-pH molecular dynamics simulations on the L-ficolin fibrinogen-like domain over pH 4.5–9. An unusual cis-Asn244-Cys245 peptide bond prevailed over the pH range in the S1 binding site. Analysis of the hydrogen-bond network at S1 suggested Asn244 to be indispensible for maintaining the cis form of Asn244-Cys245, and the absence of the hydroxyl group on Phe262 accounts for the lack of GlcNAc binding.

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.

Single nucleotide polymorphisms of Ficolin 2 gene in Behçet's disease

Genetic susceptibility to Behçet's disease (BD) is well documented for HLA-B51 positivity. However, BD is not a simple hereditary disease and it is exaggerated by exogenous stimuli such as microorganisms' infections. Ficolin 2 is a lectin that binds to the surface of microbial cells and kills microbial cells through the activation of complement system. Novel single nucleotide polymorphisms (SNPs) of human Ficolin 2 gene (FCN2 gene) have been recently identified in Caucasian people. The aim of the study was to elucidate the contribution of FCN2 gene in the pathogenesis of BD. The frequencies of genotypes and alleles of FCN2 gene SNPs in the promoter regions (-987, -602, -557, -64, -4) and exon 8 (+6359, +6424) were examined in 83 patients with BD and 64 healthy controls by genotyping with a DNA sequencing method. There were no significant differences in genotype and allele frequencies of FCN2 gene SNPs between BD patients and healthy controls. No significant differences in genotype and allele frequencies of FCN2 gene SNPs were detected among different clinical subgroups in BD patients. Significant differences in allele frequencies of FCN gene SNPs at both -557 and -64 sites in the promoter regions were found between HLA-B51 positive groups and HLA-B51 negative groups of BD patients. The significant differences in allele frequencies of FCN2 gene SNPs in the promoter lesions (-557 and -64 sites) among HLA-B51 positive BD patients may reveal the possibility that ficolin may contribute to the innate immunity of BD among HLA-B51 haplotypes in BD patients.

Human M-Ficolin Is a Secretory Protein That Activates the Lectin Complement Pathway

Three types of ficolins have been identified in humans: L-ficolin, M-ficolin, and H-ficolin. Similar to mannose-binding lectin, L-ficolin and H-ficolin are the recognition molecules in the lectin complement pathway. Another human ficolin, M-ficolin, is a nonserum ficolin that is expressed in leukocytes and lung; however, little is known about its physiologic roles. In this study, we report the characterization of M-ficolin in terms of its protein localization and lectin activity. M-ficolin was localized in secretory granules in the cytoplasm of neutrophils, monocytes, and type II alveolar epithelial cells in lung. M-ficolin precipitated with mannose-binding lectin-associated serine proteases (MASP)-1 and MASP-2 in a co-immunoprecipitation assay, indicating that M-ficolin forms complexes with MASP-1 and MASP-2. M-ficolin-MASP complexes activated complement on N-acetylglucosamine (GlcNAc)-coated microplates in a C4 deposition assay. M-ficolin bound to several neoglycoproteins bearing GlcNAc, N-acetylgalactosamine, and sialyl-N-acetyllactosamine, suggesting that M-ficolin can recognize the common carbohydrate residues found in microbes. Indeed, M-ficolin bound to Staphylococcus aureus through GlcNAc. These results indicate that M-ficolin, like its family members, functions as a recognition molecule of the lectin complement pathway and plays an important role in innate immunity.

Ficolin A and ficolin B are expressed in distinct ontogenic patterns and cell types in the mouse

Ficolins are a group of proteins characterized by the presence of collagen-like and fibrinogen-like domains. Two of three human ficolins, L-ficolin and H-ficolin, are serum lectins that form complexes with mannose-binding lectin-associated serine proteases (MASPs) and play important roles in the lectin complement pathway. The other human ficolin, M-ficolin, is a non-serum-type ficolin that is expressed in monocytes. Little is known about the physiological roles of ficolins. In this study, we delineated the ontogeny and cell types that express ficolins in mice. RT-PCR analysis showed that the expression pattern of ficolin A expression was closely similar to that of Masps, suggesting that these molecules may function in coordination as components of the lectin complement pathway. The cell types that express ficolin A mRNA in both adult liver and spleen were identified as macrophages by in situ hybridization. Ficolin B exhibited a distinct ontogeny pattern that switched from embryonic liver to postnatal bone marrow and spleen. The cells that express ficolin B mRNA were identified as belonging to the myeloid cell lineage by magnetic sorting and by subsequent RT-PCR in bone marrow cells. Thus, the different spatial-temporal expression patterns of ficolins A and B suggest that these molecules play distinct roles in the prenatal and postnatal stages.

Cutting edge: complement-activating complex of ficolin and mannose-binding lectin-associated serine protease.

Both ficolins and mannose-binding lectin (MBL) are lectins characterized by the presence of collagen-like and carbohydrate-binding domains in a subunit, although their carbohydrate-binding moieties are quite different. A fibrinogen-like domain is in ficolins, and a carbohydrate recognition domain is in MBL. On binding to pathogens, human MBL activates the complement system via the lectin pathway in association with two types of MBL-associated serine proteases (MASP), MASP-1 and MASP-2 and its truncated form, small MBL-associated protein (sMAP, also called MAp19). We report here that ficolin/P35, a human serum ficolin, was found to copurify with MASPs and sMAP. MASPs that were complexed with ficolin/P35 exhibited proteolytic activities against complement components C4, C2, and C3. The ficolin/P35-MASPs-sMAP complex that was bound to Salmonella typhimurium activated complement. These findings indicate that ficolin/P35 is a second collagenous lectin capable of activating the lectin pathway and thus plays a role in innate immunity.

Oligomeric Structure and Tissue Distribution of Ficolins from Mouse, Pig and Human

Mouse plasma ficolin was purified by GlcNAc affinity and anion-exchange chromatography. Gel-filtration chromatography and gradient sedimentation indicated that mouse plasma ficolin is a 12-mer of ∼35 kDa subunits, and electron microscopy showed the same parachute-like structure previously characterized for the pig ficolin 12-mer. Whereas the predominant form in pig plasma is a 24-mer, mouse and human plasma ficolin showed only the 12-mer form. We conclude that mouse plasma ficolin corresponds to the recently described ficolin A. We have identified a second mouse ficolin gene, ficolin B, which means that pig and mouse each have two ficolin genes, and human has three. One ficolin gene in all species is expressed in liver and is the primary source of plasma ficolin. Expression of this gene in other tissues, and expression of the second ficolin gene, appears to vary in different species.

Purification and binding properties of a human ficolin-like protein

Ficolin was initially identified from porcine uterus as a TGF- β1 binding protein and is considered to have an overall structure similar to that of the complement protein C1q and the collectins. Recent studies have shown that human ficolin is synthesized mainly by monocytes in peripheral blood and that it could potentially bind to sugar structures on microorganisms. The aim of the present investigations was to isolate ficolin from human plasma by affinity chromatography on immobilized sugars. A human serum protein was identified in the GlcNAc eluate from GlcNAc-Sepharose which migrated as a polypeptide of approx. 40 kDa on SDS-PAGE under reducing conditions and was, after further purification by FPLC on a mono-Q column, shown to have an identical N-terminal sequence, over the first 14 residues, to P35, a plasma protein having similar sequence and domain organisation to ficolin. This protein, named the ficolin-like protein, was shown to be sensitive to collagenase and similar to P35 in that it was also disulphide-linked into an oligomer of approx. 320 kDa. However, unlike P35, its binding to GlcNAc was independent of Ca 2+. Gel-filtration studies showed that this ficolin-like protein also had a molecular weight of approx. 320 kDa under non-dissociating conditions. During the course of this study this ficolin-like protein was found to simply bind to CNBr-activated Sepharose which had been inactivated with Tris, and from which it could be eluted with GlcNAc. This ficolin-like protein was also shown to bind to GlcNAc, but not to mannose and maltose. The functional significance of the unusual binding property of this ficolin-like protein is not clear, but it has facilitated the development of a simple method for its purification.

Cloning and Characterization of the Human Lectin P35 Gene and Its Related Gene

We previously cloned a novel human lectin, designated P35, with both collagen-like and fibrinogen-like domains. P35 recognizes GlcNAc residues and is opsonic toward microorganisms. The overall structure of P35 closely resembles those of two pig ficolins that are putative TGF-β1-binding proteins. In this study, we analyzed the exon–intron structure and chromosomal location of the P35 gene as well as its structural relationship to splicing variants. In addition, we isolated another distinct genomic clone corresponding to the upstream region of a P35-related gene that has an exon organization closely resembling that of the P35 gene. The sequences of exons in the P35-related gene were identical to the cDNA sequence reported for “human ficolin.” Northern blotting revealed that the P35 gene is expressed mainly in liver, whereas the P35-related gene is expressed in lung and peripheral blood leukocytes, demonstrating tissue-specific expression of these two genes. Both genes were assigned to a closely related region of chromosome 9 at 9q34.

Human ficolin: cDNA cloning, demonstration of peripheral blood leucocytes as the major site of synthesis and assignment of the gene to chromosome 9.

Pig ficolins and a number of other proteins contain sequences that are homologous to the C-terminal halves of fibrinogen beta- and gamma-chains. To clone the cDNA for human ficolin, two degenerate oligonucleotide primers were synthesized, based on two stretches of protein sequence that were highly conserved among those proteins, and used for PCR with cDNA from a human uterus lambda gt11 library as a template. A PCR product with a predicted size of 300 bp was obtained and this was used to screen a uterus cDNA library. Of the positive clones isolated, two (U1 and U2), containing inserts of 1.7 and 1.1 kb respectively, were found to encode human ficolin. The cDNA-derived amino acid sequence of human ficolin has approx. 75% identity with, and a similar domain organization to, the two pig ficolin sequences, which are characterized by the presence of a leader peptide, a short N-terminal segment followed by a collagen-like region and then by a C-terminal fibrinogen-like domain. The 1.1 kb insert of clone U2 was used in Northern-blot analysis, and a very strong signal for a 1.4 kb mRNA species was detected in mRNA from human peripheral blood leucocytes. This showed that, despite the initial characterization of pig ficolin as a putative receptor on uterine cells for transforming growth factor beta 1, blood leucocytes are probably the major site of human ficolin synthesis. Much weaker signals of the same size were also detected in spleen, lung and thymus and may be due to the presence of tissue macrophages or trapped blood in these tissues. An mRNA species of approx. 1.3 kb in human liver also weakly hybridized to the U2 probe, indicating the presence of a sequence that was distinct from, but related to, ficolin. The gene for human ficolin has been mapped to chromosome 9.