Fibrinogen-like Research Articles

ANGPTL3 accelerates atherosclerotic progression via direct regulation of M1 macrophage activation in plaque

IntroductionThe N-terminal domain of angiopoietin-like protein 3 (ANGPTL3) inhibits lipoprotein lipase activity. Its C-terminal fibrinogen-like (FBN) domain is a ligand of macrophage integrin αvβ3. ObjectivesANGPTL3 might home to plaque where it directly regulates macrophage function via integrin αvβ3 for atherosclerosis progression. MethodsLdlr-/- mice on a high-fat diet and ApoE-/- mice on a chow diet were received adeno-associated virus (AAV)-mediated Angptl3 gene transfer and followed up for 12 weeks. ApoE-/- mice were injected AAV containing FLAG-tagged Angptl3 cDNA for tracing. Atherosclerotic features were compared between Angptl3-/-ApoE-/- mice and ApoE-/- littermates. THP-1 cells were exposed to 0 or 50 μg/ml ANGPTL3 FBN domain for 24 h to evaluate Toll-like receptor (TLR)4 expression using western blot analysis and circulating cytokine and chemokine profiles by the MILLIPLEX MAP assay. Phospho-proteomic profile was established in ANGPTL3-treated macrophages. Integrin β3 deficient THP-1 cells were obtained by sgRNAs targeting RGD sequence using Lentivirus-Cas9 system. ResultsAngptl3 overexpression increased atherosclerotic progression and CD68+ macrophages in plaque (p < 0.05 for all). By immunostaining, FLAG+ cells were identified in plaque of gene transferred ApoE-/- mice. Fluorescent immunostaining detected co-localisation of Angptl3 and CD68 in plaque macrophages. Phospho-proteomic analysis revealed that Angptl3 induced phosphorylation of proteins that were involved in the IL-17 signalling pathway in THP-1 cells. In vitro, ANGPTL3 treatment increased the production of interleukin (IL)-1β and tumour necrosis factor-α in THP-1 cells (p < 0.05 for both). Exposure of ANGPTL3 to THP-1 cells induced Akt phosphorylation which was weakened in integrin β3 deficient ones. ANGPTL3 elevated TLR4 expression via Akt phosphorylation. In response to lipopolysaccharide, nuclear factor-κB activity was 2.2-fold higher in THP-1 cells pre-treated with ANGPTL3 than in untreated cells (p < 0.05). ConclusionsTargeting ANGPTL3 could yield a dual benefit of lowering lipid levels in the blood and suppressing macrophage activation in plaque.

Zebrafish ANGPT4, member of fibrinogen-related proteins, is an LTA-, LPS- and PGN-binding protein with a bacteriolytic activity

Fibrinogen-related proteins (FREPs) are a family of glycoproteins that contain a fibrinogen-like (FBG) domain. Many members of FREPs have been shown to play an important role in innate immune response in both vertebrates and invertebrates. Here we reported the immune functional characterization of ANGPT4, member of FREPs, in zebrafish Danio rerio. Quantitative real time PCR showed that the expression of zebrafish ANGPT4 gene is up-regulated by the challenge with lipoteichoic acid (LTA) or lipopolysaccharides (LPS), hinting its involvement in innate immune response. The recombinant ANGPT4 (rANGPT4) could bind to both gram-positive bacteria Staphylococcus aureus and Bacillus subtilis and the gram-negative bacteria Escherichia coli and Aeromonas hydrophila as well as the pathogen-associated molecular patterns (PAMPs) on the bacterial surfaces including LTA, LPS and peptidoglycan (PGN), suggesting it capable of identifying pathogens via LTA, LPS and PGN. In addition, rANGPT4 also displayed strong bacteriolytic activities against both gram-positive and -negative bacteria tested via inducing membrane depolarization and intracellular ROS production. Moreover, the bacterial clearance assay in vivo showed that the rANGPT4 could also accelerate the clearance of bacteria in zebrafish embryos/larvae. Finally, we showed that the eukaryotically expressed recombinant ANGPT4 maintained antibacterial activity and binding activity to bacteria and LTA, LPS and PGN. All these suggested that ANGPT4 could not only capable of recognizing pathogens via LTA, LPS and PGN, but also capable of killing the Gram-positive and Gram-negative bacteria, in innate immune response. This work also provides further information to understand the biological roles of FREPs and the innate immunity in vertebrates.

Identification and diversity of fibrinogen-related protein (FREP) gene family in Haliotis discus hannai, H. rufescens, and H. laevigata and their responses to Vibrio parahemolyticus infection

Fibrinogen-related proteins (FREPs) are distributed universally in vertebrates and invertebrates. These proteins contain fibrinogen-like (FBG) domains in their C-terminal region and involve in immune responses and other aspects of physiology in invertebrates. In this study, 54 proteins that contain FBG domains or a fibrinogen_c domain were identified in Haliotis discus hannai. Comparatively, 88 and 63 FREPs were identified from the genomes of H. rufescens and H. laevigata. Most FREPs of abalones had a conserved motif containing a bound calcium ion site and a second conserved motif containing a polymerization pocket site. By sequence analysis, 394 SNPs and 11 Indels were identified in 20 FREP genes of the whole genome of H. discus hannai; 992 SNPs and 42 Indels were found in 64 FREPs of H. rufescens, and 192 SNPs and 12 Indels were found in 21 FREPs of H. laevigata. Among these SNPs, 92 missense mutation sites were identified in 26 FREP genes of H. rufescens, and 12 were identified in 8 FREP genes of H. laevigata. Due to the poor genomic integrity, annotations of the SNPs or Indels in H. discus hannai did not yield missense mutant sites. FREP genes with polymorphisms were ubiquitously expressed in all the tested tissues; however, the expression is lowest in the hemolymph. In response to Vibrio parahemolyticus infection, expression of FREP genes was significantly upregulated at different exposure times in gills, hepatopancreas, and hemolymph in H. discus hannai. Overall, this study documented the FREP genes of abalones and shed light on the role of FREPs in the innate immune system of these aquaculture species for the prevention and control of diseases.

Latent TGF-β Activation Is a Hallmark of the Tenascin Family.

Transforming growth factor-β (TGF-β) isoforms are secreted as inactive complexes formed through non-covalent interactions between bioactive TGF-β entities and their N-terminal pro-domains called latency-associated peptides (LAP). Extracellular activation of latent TGF-β within this complex is a crucial step in the regulation of TGF-β activity for tissue homeostasis and immune cell function. We previously showed that the matrix glycoprotein Tenascin-X (TN-X) interacted with the small latent TGF-β complex and triggered the activation of the latent cytokine into a bioactive TGF-β. This activation most likely occurs through a conformational change within the latent TGF-β complex and requires the C-terminal fibrinogen-like (FBG) domain of the glycoprotein. As the FBG-like domain is highly conserved among the Tenascin family members, we hypothesized that Tenascin-C (TN-C), Tenascin-R (TN-R) and Tenascin-W (TN-W) might share with TN-X the ability to regulate TGF-β bioavailability through their C-terminal domain. Here, we demonstrate that purified recombinant full-length Tenascins associate with the small latent TGF-β complex through their FBG-like domains. This association promotes activation of the latent cytokine and subsequent TGF-β cell responses in mammary epithelial cells, such as cytostasis and epithelial-to-mesenchymal transition (EMT). Considering the pleiotropic role of TGF-β in numerous physiological and pathological contexts, our data indicate a novel common function for the Tenascin family in the regulation of tissue homeostasis under healthy and pathological conditions.

A fibrinogen-related protein mediates the recognition of various bacteria and haemocyte phagocytosis in oyster Crassostrea gigas

The family of fibrinogen-related proteins (FREPs) is a group of proteins with fibrinogen-like (FBG) domains, which play important roles as pattern recognition receptors (PRRs) in the innate immune responses. In the present study, a fibrinogen-like protein was identified from the oyster Crassostrea gigas (defined as CgFREP1). The open reading frame of CgFREP1 was of 966 bp that encoded a predicted polypeptide of 321 amino acids comprising a signal peptide and a fibrinogen-like domain. The mRNA expression of CgFREP1 was detected in all the examined tissues. The recombinant CgFREP1 (rCgFREP1) displayed binding activities to lipopolysaccharide (LPS), mannose (MAN), as well as Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus) and Gram-negative bacteria (Vibrio splendidus and Escherichia coli). The rCgFREP1 displayed the agglutinating activity towards M. luteus, V. splendidus and E. coli in the presence of Ca2+. rCgFREP1 was able to enhance the phagocytic activity of haemocytes towards V. splendidus, and exhibited binding activity to the CUB domain of CgMASPL-1. These results suggest that CgFREP1 not only serves as a PRR to recognize and agglutinate different bacteria but also mediates the haemocytes phagocytosis towards V. splendidus.

The fibrinogen-like domain of FREP1 protein is a broad-spectrum malaria transmission-blocking vaccine antigen

FREP1 in mosquito midguts facilitates Plasmodium falciparum parasite transmission. The fibrinogen-like (FBG) domain of FREP1 is highly conserved (>90% identical) among Anopheles species from different continents, suggesting that anti-FBG antibodies may block malaria transmission to all anopheline mosquitoes. Using standard membrane-feeding assays, anti-FREP1 polyclonal antibodies significantly blocked transmission of Plasmodium berghei and Plasmodium vivax to Anopheles gambiae and Anopheles dirus, respectively. Furthermore, in vivo studies of mice immunized with FBG achieved >75% blocking efficacy of P. berghei to A. gambiae without triggering immunopathology. Anti-FBG serum also reduced >81% of P. falciparum infection to A. gambiae Finally, we showed that FBG interacts with Plasmodium gametocytes and ookinetes, revealing the molecular mechanism of its antibody transmission-blocking activity. Collectively, our data support that FREP1-mediated Plasmodium transmission to mosquitoes is a conserved pathway and that targeting the FBG domain of FREP1 will limit the transmission of multiple Plasmodium species to multiple Anopheles species.

A fibrinogen-related protein identified from hepatopancreas of crayfish is a potential pattern recognition receptor.

Fibrinogen-related protein (FREP) family is a large group of proteins containing fibrinogen-like (FBG) domain and plays multiple physiological roles in animals. However, their immune functions in crayfish are not fully explored. In the present study, a novel fibrinogen-like protein (designated as PcFBN1) was identified and characterized from hepatopancreas of red swamp crayfish Procambarus clarkii. The cDNA sequence of PcFBN1 contains an open reading frame (ORF) of 1353 bp encoding a protein of 450 amino acids. Sequence and structural analysis indicated that PcFBN1 contains an FBG domain in C-terminal and a putative signal peptide of 19 amino acids in N-terminal. Semi-quantitative PCR revealed that the main expression of PcFBN1 was observed in hepatopancreas and hemocyte. Temporal expression analysis exhibited that PcFBN1 expression could be significantly induced by heat-killed Aeromonas hydrophila. Tissue distribution and temporal change of PcFBN1 suggested that PcFBN1 may be involved in immune responses of red swamp crayfish. Recombinant PcFBN1 protein binds and agglutinates both gram-negative bacteria Escherichia coli and gram-positive bacteria Micrococcus lysodeikticus. Moreover, binding and agglutination is Ca(2+) dependent. Further analysis indicated that PcFBN1 recognizes some acetyl group-containing substance LPS and PGN. RNAi experiment revealed that PcFBN1 is required for bacterial clearance and survival from A.hydrophila infection. Reduction of PcFBN1 expression significantly decreased the survival and enhanced the number of A.hydrophila in the hemolymph. These results indicated that PcFBN1 plays an important role in the innate immunity of red swamp crayfish as a potential pattern recognition receptor.

Highly diverse fibrinogen-related proteins in the Pacific oyster Crassostrea gigas

Fibrinogen-related proteins (FREPs) are a family of proteins with high sequence diversity, and they play crucial roles in invertebrate immune response. However, few studies have characterized this diversity at the whole-genome level. In the present study, approximately 190 predicted FREPs with more than 200 fibrinogen-like (FBG) domains were identified in the genome of the Pacific oyster (Crassostrea gigas), suggesting a historical expansion of this protein family. A sequence analysis showed high numbers of polymorphisms in C. gigas FREP (CgFREP) genes, which may contribute to the versatile immune function of FREPs. A phylogenetic analysis of molluscan FREP sequences indicated lineage-specific duplication of these genes in C. gigas. Additionally, several CgFREP mRNAs were highly expressed in the gills, digestive glands, and hemocytes. Taken together, these findings will help elucidate FREP immune function and facilitate studies of the functional validation of this gene family.

Tenascin-X promotes epithelial-to-mesenchymal transition by activating latent TGF-β.

Transforming growth factor β (TGF-β) isoforms are secreted as inactive complexes formed through noncovalent interactions between the bioactive TGF-β entity and its N-terminal latency-associated peptide prodomain. Extracellular activation of the latent TGF-β complex is a crucial step in the regulation of TGF-β function for tissue homeostasis. We show that the fibrinogen-like (FBG) domain of the matrix glycoprotein tenascin-X (TNX) interacts physically with the small latent TGF-β complex in vitro and in vivo, thus regulating the bioavailability of mature TGF-β to cells by activating the latent cytokine into an active molecule. Activation by the FBG domain most likely occurs through a conformational change in the latent complex and involves a novel cell adhesion-dependent mechanism. We identify α11β1 integrin as a cell surface receptor for TNX and show that this integrin is crucial to elicit FBG-mediated activation of latent TGF-β and subsequent epithelial-to-mesenchymal transition in mammary epithelial cells.

A new fibrinogen-related protein from Argopecten irradians (AiFREP-2) with broad recognition spectrum and bacteria agglutination activity

Fibrinogen-related proteins (FREPs) are a kind of pattern recognition receptors (PRRs) containing fibrinogen-like (FBG) domains, and they play curial roles in the innate immune response. In the present study, a new FREP protein was identified from bay scallop Argopecten irradians (designated as AiFREP-2). The full-length cDNA of AiFREP-2 was of 1299 bp with an open reading frame of 762 bp encoding a polypeptide of 253 amino acids, including a signal sequence and an FBG domain. The FBG domain in AiFREP-2 was highly similar to those of ficolins, tenascins and other FREPs. The mRNA expression of AiFREP-2 could be detected in all the examined tissues with the highest level in gill. The mRNA expression of AiFREP-2 in hemocytes was significantly up-regulated post the stimulation of lipopolysaccharide (LPS), peptidoglycan (PGN) and β-glucan (GLU) (P < 0.01). The recombinant AiFREP-2 (rAiFREP-2) could bind not only different PAMP ligands including LPS, PGN and GLU, but also various microbes including Gram-negative bacteria (Vibrio anguillarum), Gram-positive bacteria (Staphylococcus aureus) and fungus (Pichia pastoris and Yarrowia lipolytica). Additionally, rAiFREP-2 exhibited obvious agglutination activity towards Gram-negative bacteria V. anguillarum and Gram positive bacteria S. aureus. The results indicated that AiFREP-2 was involved in the immune response against Gram-negative bacteria, Gram-positive bacteria and fungus as a PRR in bay scallop, and the information was helpful to understand the innate immune defense mechanisms of mollusks.

1H, 13C and 15N backbone resonance assignments of the monomeric human M-ficolin fibrinogen-like domain secreted by Brevibacillus choshinensis

M-ficolin, which forms trimer-based multimers, is a pathogen-recognition protein in the innate immune system, and it binds to ligands through its fibrinogen-like (FBG) domain. As the first step toward the elucidation of the molecular basis for pathogen-recognition by the M-ficolin multimers, we assigned the backbone resonances of the monomeric mutant of the M-ficolin FBG domain, recombinantly expressed by Brevibacillus choshinensis. Like the wild-type trimeric FBG domain, the monomeric FBG domain also requires His251, His284 and His297 for the ligand-binding activity, as judged by mutational analyses using zonal affinity chromatography. The secondary structure predicted by the backbone resonance assignments is similar to that of the trimeric FBG domain in the crystal, indicating that the monomeric FBG domain is folded correctly to perform its function.

Binding site of C-reactive protein on M-ficolin

The binding abilities of human C-reactive protein (CRP) with the C-terminal fibrinogen-like (FBG) domain and the full-length form of human M-ficolin were investigated by pull-down and zonal affinity chromatography analyses. Pull-down assays using an N-acetyl-d-glucosamine (GlcNAc)-agarose column demonstrated that CRP binds to the trimeric FBG domains, and that the GlcNAc-binding ability of the FBG domain is unaffected by CRP binding. Interestingly, the full-length M-ficolin, comprising the N-terminal collagen-like (COL) and C-terminal FBG domains, displayed lower affinity for CRP, and the monomeric FBG domain showed virtually no binding to CRP, as qualitatively judged by zonal affinity chromatography using a GlcNAc column. These results indicated that CRP binding requires the trimeric form of the FBG domain, and that the presence of the COL domain reduces the interaction between CRP and M-ficolin. In addition, pull-down assays using a histidine-tag affinity column demonstrated that neither the full-length M-ficolin nor the trimeric FBG domains, immobilized through their C-terminal histidine tags, showed any affinity for CRP, indicating that the CRP binding site is located near Ala326 at the C-terminus of M-ficolin, spatially close to a neck region (around Pro115) between the FBG and COL domains. From these findings, we concluded that CRP binding is enhanced by conformational bending at the neck region of M-ficolin, to avoid steric hindrance by the COL domain. Such a situation may be generated by oligomeric M-ficolin binding to surfaces with widely distributed ligands, such as pathogens.

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.

Anopheles Fibrinogen-related Proteins Provide Expanded Pattern Recognition Capacity against Bacteria and Malaria Parasites

The fibrinogen-related protein family (FREP, also known as FBN) is an evolutionarily conserved immune gene family found in mammals and invertebrates. It is the largest pattern recognition receptor gene family in Anopheles gambiae, with as many as 59 putative members, while the Drosophila melanogaster genome has only 14 known FREP members. Our sequence and phylogenetic analysis suggest that this remarkable gene expansion in the mosquito is the result of tandem duplication of the fibrinogen domain. We found that the majority of the FREP genes displayed immune-responsive transcription after challenge with bacteria, fungi, or Plasmodium, and these expression patterns correlated strongly with gene phylogeny and chromosomal location. Using RNAi-mediated gene-silencing assays, we further demonstrated that some FREP members are essential factors of the mosquito innate immune system that are required for maintaining immune homeostasis, and members of this family have complementary and synergistic functions. One of the most potent anti-Plasmodium FREP proteins, FBN9, was found to interact with both Gram-negative and Gram-positive bacteria and strongly co-localized with both rodent and human malaria parasites in the mosquito midgut epithelium, suggesting that its defensive activity involves direct interaction with the pathogen. Interestingly, FBN9 formed dimers that bound to the bacterial surfaces with different affinities. Our findings indicate that the A. gambiae FREP gene family plays a central role in the mosquito innate immune system and provides an expanded pattern recognition and anti-microbial defense repertoire.

Molecular and phylogenetic analysis of a novel family of fibrinogen-related proteins from mosquito Aedes albopictus cell line

Since lectins have been proposed to function as non-self recognizing molecules, it is important to access the specificity and diversity of lectin molecules that play an important role as pattern recognition receptors (PRRs). Like vertebrates, several lectins containing fibrinogen-like (FBG) domains have been reported as PRRs from different invertebrate species, however, in case of mosquito, very little information is available. In this study, we report identification and characterization of a novel family of fibrinogen-related proteins which exhibits (43–68%) similarities to that of A. gambiae FREPs, isolated from the immune challenged cell line of mosquito Aedes albopictus. Analysis of the longest (790 bp) cDNA (AF-1) appears to encode a 263 deduced amino acid sequence that contains a C-terminal fibrinogen-like domain. Amino acid sequence similarity and phylogenetic analysis showed a high degree of conservation with other known insect fibrinogen-related proteins, suggesting that mosquito A. albopictus FREPs may play a similar role in an innate immunity as pattern recognition receptor molecules; however, further characterization is needed to understand their transcriptional regulation and behavior during microbial infection.

Fibrinogen-related protein from amphioxus Branchiostoma belcheri is a multivalent pattern recognition receptor with a bacteriolytic activity

Fibrinogen-related proteins (FREPs) containing fibrinogen-like (FBG) domain have been shown to be involved in immune responses in both invertebrates and vertebrates, but the underlying mechanisms remain ill-defined. In this study we isolated a cDNA encoding amphioxus ( Branchiostoma belcheri) FREP homolog, BbFREP. BbFREP encoded a protein of 286 amino acids, which included a C-terminal FBG domain and clustered together with human fibrinogen β and γ chains. Quantitative real time PCR revealed that the expression of BbFREP was significantly up-regulated following challenge with lipopolysaccharides (LPS) or lipoteichoic acid (LTA). The recombinant BbFREP expressed in Pichia pastoris was able to specifically recognize the pathogen-associated molecular patterns (PAMPs) on the bacterial surfaces including LPS, peptidoglycan (PGN) and LTA, and displayed strong bacteriolytic activities against both Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus. BbFREP was also able to bind to both E. coli and S. aureus. In situ hybridization indicated that BbFREP was mainly expressed in the hepatic caecum and hind-gut, agreeing basically with the primary expression of vertebrate FREP genes in the liver. All these suggest that BbFREP can function as a pattern recognition receptor with a bacteriolytic activity via interaction with LPS, LTA and PGN. It also bolsters the notion that the hepatic caecum of amphioxus is equivalent to the vertebrate liver, acting as a major tissue in acute phase response.

Evolution and potential function of fibrinogen-like domains across twelve Drosophila species

BackgroundThe fibrinogen-like (FBG) domain consists of approximately 200 amino acid residues, which has high sequence similarity to the C-terminal halves of fibrinogen β and γ chains. Fibrinogen-related proteins (FREPs) containing one or more FBG domains are found universally in vertebrates and invertebrates. In invertebrates, FREPs are involved in immune responses and other aspects of physiology. To understand the complexity of this gene family in Drosophila, we analyzed FREPs in twelve Drosophila species.ResultsUsing the genome data from 12 Drosophila species, we identified FBG domains in each species. The results show that the gene numbers in each species vary from 14 genes up to 43 genes. Using sequence profile analysis, we found that FBG domains have high sequence similarity and are highly conserved throughout. By comparison of structure and sequence conservation, some of the FBG domains in Drosophila melanogaster are predicted to function in recognition of carbohydrates and their derivatives on the surface of microorganisms in innate immunity.ConclusionSequence and structural analyses show that FREP family across 12 Drosophila species contains conserved FBG domains. Expansion of the FREP families in Drosophila is mainly accounted by a major expansion of FBG domains.

Identification and characterization of the fibrinogen-like domain of fibrinogen-related proteins in the mosquito, Anopheles gambiae, and the fruitfly, Drosophila melanogaster, genomes

BackgroundThe fibrinogen-like (FBG) domain, which consists of approximately 200 amino acid residues, has high sequence similarity to the C-terminal halves of fibrinogen β and γ chains. Fibrinogen-related proteins (FREPs), which contain FBG domains in their C-terminal region, are found universally in vertebrates and invertebrates. In invertebrates, FREPs are involved in immune responses and other aspects of physiology. To understand the complexity of this family in insects, we analyzed FREPs in the mosquito genome and made comparisons to FREPs in the fruitfly genome.ResultsBy using the genome data of the mosquito, Anopheles gambiae, 53 FREPs were identified, whereas only 20 members were found in the Drosophila melanogaster genome. Using sequence profile analysis, we found that FBG domains have high sequence similarity and are highly conserved throughout the FBG domain region. By secondary structure analysis and comparison, the FBG domains of FREPs are predicted to function in recognition of carbohydrates and their derivatives on the surface of microorganisms in innate immunity.ConclusionDetailed sequence and structural analysis discloses that the FREP family contains FBG domains that have high sequence similarity in the A. gambiae genome. Expansion of the FREP family in mosquitoes during evolutionary history is mainly accounted for by a major expansion of the FBG domain architecture. The characterization of the FBG domains in the FREP family is likely to aid in the experimental analysis of the ability of mosquitoes to recognize parasites in innate immunity and physiologies associated with blood feeding.

M-ficolin is expressed on monocytes and is a lectin binding to N-acetyl-D-glucosamine and mediates monocyte adhesion and phagocytosis of Escherichia coli.

Ficolins are a group of multimeric proteins that contain collagen-like and fibrinogen-like (FBG) sequences. Three types of ficolins have been characterized: H-, L- and M-ficolins. Both H- and L-ficolins have demonstrated lectin activities. In the present study, the FBG domain of M-ficolin was expressed and shown to bind to N-acetyl-D-glucosamine. M-ficolin mRNA was expressed in monocytes but not in the more differentiated macrophages and dendritic cells. By flow cytometry, surface biotinylation and immunoprecipitation, we showed that M-ficolin was associated with the surface of promonocytic U937 cells. M-ficolin transiently expressed in COS-7 cells was also clearly detected on the cell surface by immunoprecipitation. By flow cytometry, M-ficolin was detected on peripheral blood monocytes but not on lymphocytes or granulocytes. Immobilized rabbit anti-M-ficolin F(ab')2 mediated U937 cell adhesion, and the antibody also inhibited phagocytosis of Escherichia coli K-12 by U937 cells. Therefore, M-ficolin might act as a phagocytic receptor or adaptor on circulating monocytes for micro-organism recognition and may potentially mediate monocyte adhesion.

Ficolins and the Fibrinogen-like Domain

Ficolins are a group of proteins containing collagen-like and fibrinogen-like (FBG) sequences and they have a similar overall structure to Clq and the collectins. There are two types of ficolin in man: L-ficolin and M-ficolin. L-ficolin is synthesized in the liver and secreted into the plasma. It binds to several apparently unrelated structures including sugar residues and enhances phagocytosis of bound bacteria. M-ficolin is synthesized mainly in monocytes and is detected on the monocyte surface. The polypeptide sequences of f icolins, the collectins and C1q diverge mainly in their C-terminal globular regions which are, respectively, FBG domains, Ca 2+-dependent carbohydrate recognition domains (C-type CRD), and collagen-related sequences. The FBG domain consists of 220-250 residues and is found in a number of proteins besides fibrinogen and ficolins. The crystal structure of the FBG domain has been characterized and the elucidation of its binding properties should provide essential insights into its role in ficolins and other proteins.