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Ficolins: Novel pattern recognition molecules of the innate immune response
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Immunobiology 213 (2008) 297–306 www.elsevier.de/imbio
Ficolins: Novel pattern recognition molecules of the innate immune response Valeria L. Runzaa,, Wilhelm Schwaebleb, Daniela N. Ma¨nnela a
Institute of Immunology, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93042 Regensburg, Germany Department of Infection, Immunity and Inflammation, Maurice Shock Medical Sciences Building, University of Leicester, Leicester, UK
b
Received 31 July 2007; accepted 17 October 2007
Abstract Ficolins are members of the collectin family of proteins which are able to recognize pathogen-associated molecular pattern (PAMP) on microbial surfaces. Upon binding to their specific PAMP, ficolins may trigger activation of the immune system by either binding to cellular receptors for collectins or by initiating activation of complement via the lectin pathway. For the latter, the human ficolins (i.e. L-, H- and M-ficolin) and murine ficolin-A were shown to associate with the lectin pathway-specific serine protease MBL-associated serine protease-2 (MASP-2) and catalyse its activation which in turn activates C4 and C4b-bound C2 to generate the C3 convertase C4b2a. There is mounting evidence underlining the lectin nature of ficolins with a wide range of carbohydrate moieties recognized on microbial surfaces. However, not all members of the ficolin family appear to act as lectin pathway recognition components. For example, murine ficolin-B does not associate with MASP-2 and appears to be absent in plasma and other humoral fluids. Its stringent cellular localization points to other functions within the immune response, possibly acting as an intracellular scavenger to target and facilitate clearance of PAMP-bearing debris. When comparing ficolin orthologues from different species, it appears evident that human, murine, and porcine ficolins differ in many aspects, a specific point that we aim to address in this review. r 2007 Elsevier GmbH. All rights reserved. Keywords: Complement system; Ficolin; Innate immunity; Lectin pathway
Introduction Abbreviations: CRD, carbohydrate recognition domain; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; LTA, lipoteichoic acid; LPS, lipopolysaccharide; MASP, MBL-associated serine protease; MBL, mannan-binding lectin; PAMP, pathogen-associated molecular pattern; PCR, polymerase chain reaction; UT, untranslated region. Corresponding author. Tel.: +49 941 944 5465; fax: +49 941 944 5462. E-mail address: [email protected] (V.L. Runza). 0171-2985/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2007.10.009
Innate immunity uses a variety of induced effector mechanisms to fight infections and control them until the causative pathogens are eventually recognized by the adaptive immune system. The complement system represents one of the major humoral systems of the innate host defense and is composed of a cascade of activation events that occur on the surface of pathogens or infected cells and generate active products with various effector functions including opsonization and
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pathogen lysis, chemotaxis, and proinflammatory activation of the cellular immune system. There are three distinct pathways by which the complement system can be activated: the classical, the lectin, and the alternative pathway, all of which converge to generate the same set of activation products. The lectin pathway is initiated when mannan-binding lectin (MBL) or ficolins bind to carbohydrate moieties on bacterial surfaces. This binding promotes the activation of the MBL-associated serine proteases (MASPs) which lead to subsequent cleavage of the C4 and C4b-bound C2. Activation of the lectin pathway leads to the generation of the C3 converting enzyme complex C4b2a which – upon accumulation of the C3 cleavage product C3b – can develop C5 convertase activity. With the cleavage of C5, all enzymatically mediated activation steps are completed, while C5b – the major cleavage fragment of C5 – initiates the assembly of the terminal activation steps of C6–C9, leading to the formation of the membrane attack complex (MAC) through a cascade of intermolecular rearrangements. Ficolins were first documented as transforming growth factor-b1 (TGF-b1)-binding proteins on pig uterus membranes by Ichijo et al. (1991). Their primary structure revealed that they are mainly composed of fibrinogen- and collagen-like domains and, this unique feature gave them their name ficolins (Ichijo et al., 1993). Since the first description of porcine ficolins, other homologous proteins with very similar structural features have been identified at the cDNA and/or protein level in human (Endo et al., 1996; Lu et al., 1996; Sugimoto et al., 1998), rodents (Fujimori et al., 1998; Ohashi and Erickson, 1998), Xenopus (Kakinuma et al., 2003), and invertebrates (Kenjo et al., 2001), showing different locations of biosynthesis suggestive of different local functions. Moreover, it has been shown that ficolins present in human, mouse, and pig are lectins with a common binding specificity for N-acetylglucosamine (GlcNAc) (Matsushita et al., 1996). Since the time of their first descriptions, human ficolins have been intensively investigated and numerous reports have been published on their molecular structure, their expression, their involvement as carbohydrate recognition subcomponents of the lectin pathway, and their gene polymorphisms. The biological functions of murine ficolin, however, are less well understood than those of their human orthologues and gene-targeted ficolin-deficient mouse strains may help to define their roles in the antimicrobial immune defense.
Sites of ficolin expression The published data on ficolins show that these lectins are present in two forms: a serum type (predominantly
synthesized in the liver) and a cell-associated type of ficolin (predominantly synthesized in phagocytic cells). Pigs have two distinct but closely related ficolin genes, named a and b (Ichijo et al., 1993). Ficolin-a is expressed in liver, bone marrow, spleen, lung (Ohashi and Erickson, 1998) and very weakly in the uterus (Ichijo et al., 1993), whereas ficolin-b is expressed in bone marrow and neutrophils (Brooks et al., 2003b). Ficolina and -b share 81–84% identity at the amino acid level. Ficolin-a is the major plasma ficolin and consists of N-glycosylated subunits of 35 kDa (Ohashi and Erickson, 1998) while ficolin-b has an apparent molecular weight of 39 kDa and was found to be synthesized, stored, and secreted by porcine neutrophils but not by peripheral blood monocytes or platelets (Brooks et al., 2003b). Ficolin-b is present in both cytoplasmatic and membrane fractions of neutrophil preparations but its subcellular distribution has not been shown. Amongst human ficolins, L- and H- (Hakata antigen) are the plasma ficolins. The hepatocytes are the primary source of synthesis for L-ficolin and its concentration in sera from 181 blood donors was found to range from 1.1 to 12.8 (median 3.7) mg/ml (Kilpatrick et al., 1999). H-ficolin is also synthesized by hepatocytes as well as by bile duct epithelial cells, and in the lung by ciliated bronchial and type II alveolar epithelial cells (Akaiwa et al., 1999). H-ficolin is found in circulation at a median concentration of 18.4 mg/ml (Krarup et al., 2005). On the other hand, the non-serum-type M-ficolin is expressed in peripheral blood leukocytes. Even though its primary structure lacks an apparent transmembrane domain, M-ficolin was found on the surface of PBMs (Teh et al., 2000). Recently, M-ficolin protein was localized in secretory granules in the cytoplasm of neutrophils, monocytes, and type II alveolar epithelial cells in the lung (Liu et al., 2005b). These observations led to the hypothesis that M-ficolin might act as an acute phase protein that is temporarily stored in the secretory granules of the leukocytes to be released locally and to execute its functions in host defense upon the right stimuli, similar to ficolin-b in pigs. Mice, as well as rats, have two ficolin forms, termed ficolin-A and -B. The ficolin-A gene (Fcna) was first isolated by Fujimori and co-workers in 1998 from a mouse liver library (Fujimori et al., 1998). The protein encoded by this gene located on chromosome 2 is 60%, 59.3%, 59.1%, and 59% identical to those of porcine ficolin-a, -b, human M-ficolin, and L-ficolin, respectively (Ohashi and Erickson, 1998). Ficolin-A is the plasma protein with a molecular mass of 37 kDa, highly expressed in liver and spleen (Fujimori et al., 1998). In a recent report, Liu and co-workers showed that ficolin-A mRNA is expressed during ontogenesis as early as on embryonic day (E) 12.5, displaying an increase in abundance during development, peaking around
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birth, and slightly declining in the adult stages (Liu et al., 2005a). In addition, in situ hybridization studies indicated that ficolin-A mRNA was mainly localized in the linings of the hepatic sinusoids in the liver, and in the red pulp of the spleen. These observations, together with further immunohistochemical analysis revealing a distribution pattern of ficolin-A comparable to the Kupffer cells in liver, suggests that ficolin-A mRNA is expressed by cells of the monocytemacrophage lineage (Liu et al., 2005a). Ficolin-B was first characterized by Ohashi and Erickson in 1998 as a mouse ficolin different from the murine plasma ficolin (ficolin-A), with strong mRNA expression in bone marrow and weak expression in spleen, and a 67% and 73,4% identity on the protein level to the human L- and M-ficolin, respectively. (Ohashi and Erickson, 1998). More recently, ficolin-B mRNA levels were described as progressively increasing from E13.5 to E18.5 with a subsequent rapid postnatal decline to undetectable levels in liver prior to the age of 4 weeks. However ficolin-B mRNA was detected in spleen at all time points examined after birth, indicating a complementary expression of ficolin-A and -B in spleen (Liu et al., 2005a). Regarding the specific cell types expressing ficolin-B, distinct cell lineages of sorted bone marrow-derived cells showed different expression patterns by RT-polymerase chain reaction (PCR) with high levels in myeloid cells (Gr-1+ and Mac-1+) and no expression in the Ter119+ erythroid, the T-cell (CD3e+), or the B-cell (B220+) lineages (Liu et al., 2005a). We recently published the first observation on ficolin-B expression at the protein level. Ficolin-B was found to be expressed by peritoneal macrophages of C57Bl/6 mice by immunocytochemistry, and surprisingly a positive staining was detected only after permeabilization of the cells indicating an intracellular expression and not a cell-association of this lectin (Fig. 1) (Runza et al., 2006). However, the possibility that macrophages secrete ficolin-B upon stimulation as a local mediator was not tested yet and, therefore, cannot be discarded. Furthermore, screening of different immune cells revealed ficolin-B mRNA presence in bone-derived immature dendritic cells but not in mast cells (data not shown).
Molecular structure of ficolins Like collectins, ficolins are built of structural subunits each composed of three identical polypeptide chains. In each chain, a short N-terminal region with one or two cysteine residues is followed by a collagenlike domain characterized by 11 to 19 Gly-X-Y triplets (where X and Y denote any amino acid), a short link region, and a subsequent fibrinogen-like domain. Through their collagen-like domain three polypeptide
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Fig. 1. Peritoneal macrophage-expressed ficolin-B is localized inside the cell. Peritoneal macrophages were either permeabilized (lower panel) or not permeabilized (upper panel), and subjected to ficolin-B immunostaining (Runza et al., 2006).
chains assemble into a trimer to form a subunit. Although ficolins do not have a coiled-coil structure acting as the neck region like MBL (Holmskov et al., 2003; Garlatti et al., 2007), they form active oligomers when normally four subunits join together via disulfide bridges at the N-terminal regions. The polypeptide chains then radiate out in a sertiform structure, giving ficolins the typical ‘‘bouquet’’-like appearance (Fig. 2). Higher or smaller oligomers seem to be less common for ficolins than for MBL (Holmskov et al., 2003). The carbohydrate-binding activity of ficolins is assigned to the fibrinogen-like domain which, like the carbohydrate recognition domain (CRD) of MBL, seems to be Ca++-dependent, although there has been some controversy about this point (Matsushita et al., 1996; Ohashi and Erickson, 1997; Le et al., 1997). The fibrinogen-like domain consists of 200–250 residues and is characterized by the presence of 24 invariant, mostly hydrophobic, amino acids including four cysteines and 40 highly conserved residues. According to the crystal structure resolved for the fibrinogen CRD of tachylectin 5A (a nonself-recognizing lectin from the hemolymph plasma of Tachypleus tridentatus), the contact between the protein and its carbohydrate ligand is mediated by four aromatic side chains that form a funnel in which the methyl group of GlcNAc fits in the center (Kairies et al., 2001). However, the structure of a trimer
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Fig. 2. Structural organization of ficolins. A 12-mer is depicted but lower and higher oligomers have similar organisation.
of the recombinant L-ficolin fibrinogen-like domain was resolved by X-ray crystallography, and revealed that the ligand binding site of L-ficolin is situated at a position different from that seen in the tachylectin, at the opposite side of the globular domain (Krarup et al., 2004). This unexpected finding calls for caution when trying to deduce functionalities from orthologous structures. From the genomic point of view, the exon–intron organization is similar among ficolins from different species: 8–10 exons where the first one encodes the 50 UT, leader peptide and N-terminal amino acids; the second and the third exons encode the collagen-like domain; the fourth one the short link region, and the exons five to eight the fibrinogen-like domain. The last exon also encodes the 30 UT, and in the case of the 9- or 10-exon containing genes (i.e. FCN1, Fcna and Fcnb) the additional exons encode extra segments of Gly-X-Y repeats in the collagen-like domain (Endo et al., 1996, 2004).
Ficolin functions Carbohydrate recognition There is ample evidence that ficolins (both serum- and non-serum types) from different species share a common binding affinity for GlcNAc, by recognition of the nonreducing terminal carbohydrate residue carrying the N-acetyl group. Already in 1998, it was shown that L-ficolin is able to bind to GlcNAc and N-acetylgalactosamine (GalNAc) but not to their precursor sugars (Le et al., 1998). At that time it was also reported that L-ficolin binds to glutathione and CNBr-activated sepharose but not to underivatized sepharose, suggesting that L-ficolin might bind the common structure in these compounds which is an amide group (-CO-NH). Later, Krarup and co-workers showed that L-ficolin binds to Streptococcus pneumoniae 11F and that this interaction could be inhibited by N-acetylated compounds, either sugars (GlcNAc, GalNAc, N-acetlymannosamine)
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or other molecules like N-acetylcysteine, N-acetylglycine, and acetylcholine (Krarup et al., 2004), suggesting a rather general acetyl-binding capacity. However, the presence of an N-acetyl group might affect the binding of ficolins by simply neutralizing the charge on the sugar amine which, without the acetyl substitution, would appear protonated (positively charged). Therefore, it still remains to be understood to what extent the binding of ficolins to acetyl groups is relevant under physiological conditions in vivo. In addition, L-ficolin has been involved in the recognition and phagocytosis of type III group B streptococci (Aoyagi et al., 2005). The capsule of these bacteria possesses an elongated polysaccharide containing non-terminal GlcNAc residues and b-1-3 links between glucose and galactose rings (Kadirvelraj et al., 2006) which, in accordance with a recent work by Garlatti and co-workers, suggests that L-ficolin is able to bind such elongated carbohydrates with acetylated and neutral residues like 1,3-b-Dglucan, present on microbial and apoptotic surfaces (Garlatti et al., 2007). Also interesting is the observation that L-ficolin, but not MBL or H-ficolin, specifically binds to lipoteichoic acid (LTA) which is the common exposed antigen present on Gram-positive bacterial cells (Lynch et al., 2004). This finding indicates that the repertoire of microbial organisms recognized by L-ficolin can not only overlap but also extend that recognized by MBL and, therefore, broadens the spectrum for the innate response towards invading pathogens. Human M-ficolin was found to bind to several neoglycoproteins bearing GlcNAc and GalNAc, and more interestingly sialyl-N-acetyllactosamine (SiaLacNAc) (Liu et al., 2005b). Sialic acid is normally involved in the cellular recognition mediated by molecules that regulate distinct host events such as cellular growth and differentiation. The meaning of this specific binding is, therefore, not clear but suggests that M-ficolin might play different and/or additional roles in local immunity. Likewise, mouse ficolin-B seems to recognize the terminal N-acetylneuraminic acid residue present in molecules like SiaLacNAc and fetuin (Endo et al., 2005), suggesting that the cell-associated ficolins (human M-ficolin, mouse ficolin-B) might have other unique functions, in addition to bacterial recognition, and probably related to cellular host events. Upon binding to their carbohydrate ligands all three human ficolins and murine ficolin-A were shown to activate the complement system in C4b deposition assays where ficolin/MASP-2 complexes are preincubated with the immobilized ligand and then recombinant C4 is added previous to the detection of the C4b split product. On the other hand, even when both mouse ficolins-A and -B show specificity to GlcNAc and GalNAc, interestingly, only ficolin-A can form an active
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complex with MASP-2 able to cleave C4 (Endo et al., 2005) which is in accordance to our own observations (unpublished data). A very recent study on the characterization of MASP-binding site seems to deal with this discrepancy (Girija et al., 2007). There it is shown that the MASP-binding site in the collagen-like domain of MBLs and ficolins is conserved. A lysine residue (Lys56) may play a critical role by forming the key contacts with the MASP, and adjacent residues – mainly methionine or alanine – might help to stabilize the interaction (Girija et al., 2007). Mouse ficolin-B contains the required sequences for binding to MASP (i.e. Lys56). However, an adjacent glutamic acid residue is likely to disrupt MASP binding and activation, explaining the lack of complement activation by mouse ficolin-B. In this regard, it would be interesting to investigate if its porcine (ficolin-b) and rat orthologues are able to bind MASP since their sequences contain the conserved adjacent alanine residues.
Interaction with microorganisms and complement activation L-ficolin binds to the Gram-negative strain Salmonella typhimurium TV119 (rough chemotype strain with exposed GlcNAc) and enhances phagocytosis by polymorphonuclear neutrophils and monocytes. In addition to this opsonic activity, it can also activate complement through a Ca++-dependent association with MASPs (Matsushita et al., 2000). In addition, it has been demonstrated that L-ficolin can bind to Escherichia coli and be eluted with a mixture of monosaccharides (Lu and Le, 1998). Interestingly, L-ficolin/MASP complexes from sera were shown to specifically bind to LTA from Gram-positive bacteria such as Staphylococcus aureus and initiate the C4 turnover (Lynch et al., 2004). This was also shown to be true for other clinically relevant bacteria such as Streptococcus pyogenes and Streptococcus agalactiae (Lynch et al., 2004). In another work on bacterial recognition by ficolins, it was reported that L-ficolin binds to some capsulated S. aureus and S. pneumoniae serotypes but not to non-capsulated strains. These results differed from MBL and H-ficolin binding properties (Krarup et al., 2005) indicating that the binding of each lectin is directed towards a specific and different pathogen-associated molecular pattern (PAMP). The biological significance of H-ficolin as a lectin has been investigated by studying its binding potential to different strains and serotype forms of bacteria including S. pneumoniae, E. coli, S. aureus and Aerococcus viridans. Only A. viridans was found to be recognized and the binding specificity was assigned to a particular polysaccharide, namely PSA (polysaccharide
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A. viridans), present on this microorganism (Matsushita et al., 2002). Previously, it had been shown that the Hakata antigen possessed the potential to agglutinate human erythrocytes coated with lipopolysaccharide (LPS) derived from S. typhimurium and S. minnesota, and that this effect was inhibited by mono- and oligosaccharides of GalNAc, GlcNAc, and D-fucose (Sugimoto et al., 1998). H-ficolin isolated from serum is associated with MASP-1, MASP-2, MASP-3, and MAp19, and the H-ficolin/MASP complex is able to activate complement by cleavage of C4 upon binding to PSA (Matsushita et al., 2002) The cell-associated human M-ficolin coprecipitates with MASP-1 and -2, and the complexes are able to cleave C4 on GlcNAc-coated microplates (Liu et al., 2005b). Regarding its microbial recognition capacity M-ficolin was found to bind to S. aureus and to interact with a smooth-type strain of S. typhimurium (LT2), that possesses additional O-polysaccharides, but not with the rough-type strain TV119. Interestingly, exactly the opposite is true for L-ficolin (Matsushita et al., 1996), indicating that the spectrum of bacterial recognition sites might be different among ficolins. Up to date there are no reports indicating specific microbial recognition epitopes by mouse ficolins. Our preliminary work in this field indicates that ficolin-B binds to a carbohydrate moiety on the murein wall of S. aureus and to some strains of S. pneumoniae (Runza et al., in preparation). Among porcine ficolins, ficolin-a was shown to bind Actinobacillus pleuropneumoniae serotype 5B (APP5), which is the pathogen causing economically significant pneumonic and septicemic diseases in young pigs, in a GlcNAc-dependent manner (Brooks et al., 2003a). In addition, Nahid and co-workers more recently described that native ficolin-a, purified from porcine serum, is able to bind to LPS from Gram-negative bacteria of both the rough- and smooth-types, such as E. coli, S. typhimurium, S. enteriditis, Salmonella abortus equi, Shigella flexeneri, Pseudomonas aeruginosa, and Serratia marcescens. Furthermore, it was also shown that ficolin-a reacts with LTA from Gram-positive bacteria, such as Streptococcus sanguis, S. pyogenes, Bacillus subtilis, and S. aureus (Nahid and Sugii, 2006). On the other hand, it seems that ficolin-b might function locally as a secreted collagenous defense lectin at sites of inflammation where neutrophils are activated. The secreted contents of PMA-activated neutrophils are bactericidal and ficolinb may participate with other antibacterial neutrophil products in tissue antisepsis (Brooks et al., 2003b). However, the microbial targets for ficolin-b are not known and, in general, it has not been shown yet whether porcine ficolins can activate the complement system. Unlike MBL, which is known to bind to the gp120 protein of HIV-1 (Haurum et al., 1993) and to
Leishmania major promastigotes (Green et al., 1994), up to date it is not known whether ficolins are able to recognize viruses, fungi, or parasites, and it therefore still remains as an interesting field to be explored. Table 1 summarizes the main characteristics of human, murine, and porcine ficolins for better comprehension and comparison of both their overlapping and non-overlapping functions in the host defense.
Interaction with host cells In 2001 it was shown that the complement component C1q as well as MBL bind to apoptotic cells, promoting macropinocytosis and removal of dead cell bodies (Ogden et al., 2001). Since then, speculations on the ability of ficolins to recognize apoptotic and necrotic cells have been investigated and a number of reports suggest that both plasma L- and H-ficolins do play a role in the clearance of dying host cells. However, there is controversy about the mechanism triggered after ficolin recognition. Kuraya et al. (2005) described that binding of both human ficolins to apoptotic cells leads to complement activation. On the other hand, Jensen et al. (2007) reported more recently that L-ficolin binds not only to late apoptotic cells but also to apoptotic bodies and necrotic cells (and not to early apoptotic cells) but without complement deposition. They described exposed DNA on permeable late apoptotic and necrotic cells as one of the putative ligands for L-ficolin which opsonizes the cell and leads to an enhanced uptake by macrophages.
Evolution and polymorphisms Cloning and software-based sequence analysis of invertebrate ascidian Halocynthia roretzi and lower vertebrate Xenopus laevis ficolins and their comparison with their mammalian orthologues has allowed Fujita and co-workers to build up phylogenetic trees that depict the evolution of the related genes (Kenjo et al., 2001; Kakinuma et al., 2003). In addition, the fact that mice possess two and humans three forms of ficolins, led Endo et al. (2004) to perform evolutionary studies on the structures and organization of these ficolin genes. Evidence had suggested that L-ficolin was closely related to ficolin-A (serum-type), and M-ficolin to ficolin-B (non-serum type). However, the phylogenetic tree based on amino acid sequences indicated that L-ficolin diverged from the B/M-ficolin lineage, suggesting that mouse ficolin-A is not the orthologue of human L-ficolin, although both are mainly expressed in liver and have similar carbohydrate specificities. These results imply that comparable selective pressures acted independently on both the murine and primate lineages to
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Table 1.
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Expression, sugar specificity and target pathogens of collectins and ficolins Tissues of origin
Tissues of presentation
Sugar specificity and pathogen interaction
Complement activation
Liver
Serum
Yes
Serum, bronchus, alveolus, bile
M-ficolin
Liver (hepatocytes and bile epithetlium), type II alveolar cells Monocytes
GlcNAc/ManNAc44GalNAc/CysNAc/ GlyNAc, acetylcholine, elastin, corticosteroids, 1,3-b-D-glucan, LTA from S. aureus, S. pyogenes, S. agalactiae, B. subtillis S. typhimurium (Ra), E. coli, S. pneumoniae GlcNAc, GalNAc, fucose, glucose, PSA A. viridans
Monocyte surface
GlcNAc-BSA, GalNAc-BSA, SiaLacNAcBSA S. aureus
Yes
Mouse Ficolin-A Ficolin-B
Liver and spleen BM and spleen
Serum Peritoneal macropaghes
GlcNAc, GalNAc GlcNAc, GalNAc, SiaLacNAc, fetuin
Yes No
Liver, BM, spleen, lung, uterus
Serum
GlcNAc, LPS from E. coli, S. typhimurium, S. enteriditis, S. abortus equi, Shigella flexeneri, P. aeruginosa, Serratia mascesecens LTA from S. sanguis, S.pyogenes, B. subtillis, S. aureus APP5 n.d.
n.d.
Human L-ficolin
H-ficolin
Pig Ficolin-a
Ficolin-b
BM, neutrophils
Yes
n.d.
BM, bone marrow; GlcNAc, N-acetyl-D-glucosamine; GalNAc, N-acetyl-D-galactosamine; ManNAc, N-acetyl-D-mannosamine; PSA, polysaccharide A. viridans; HIV, human immunodeficiency virus; IAV, influenza A virus; RSV, respiratory syncytial virus; HSV-1, herpes simplex virus type 1; APP5, Actinobacillus pleuropneumoniae 5B; n.d., not determined.
produce these hepatic serum-type ficolins from a nonserum-type lineage (Endo et al., 2004). Furthermore, the tree suggested that H-ficolin had an ancient origin back to an evolutionary stage before the divergence of the Xenopus lineage, although no H-ficolin had been identified in mice. By computer analysis of the mouse genome database, Endo and others characterized the genomic region homologous to the human H-ficolin gene and identified an orthologue of the mouse H-ficolin gene (67% nucleotide sequence identity to the human sequence) present as a pseudogene on chromosome 4, which renders this gene dysfunctional and silent (Endo et al., 2004). In contrast to the human gene, the first exon of the mouse ficolin-H orthologue has stop codons in all three reading frames, which were generated by base changes and micro-deletions, and no start codon could be identified. A similar pseudogene is found in the rat (Endo et al., 2004). Polymorphisms reported on human ficolins showed that, like MBL, single nucleotide polymorphisms (SNPs) in the promoter region of the L-ficolin gene lead to a significant variation in the plasma concentration of this
lectin. On the other hand, two SNPs in the fibrinogen-like domain coding region (exon 8), which result in amino acid substitution, affect notably the binding capacity to GlcNAc (Hummelshoj et al., 2005; Herpers et al., 2005). In addition, it was also reported that children with recurrent infections have low L-ficolin concentrations (Atkinson et al., 2004). The M-ficolin gene was also characterized and 12 SNPs were detected both in the promoter and in the structural regions albeit no amino acid exchanges were found. Regarding the Hakata antigen, a deletion mutation in one of the tested individuals was detected, creating an early stop codon in exon 5. However, the relevance of this mutation remains unknown (Hummelshoj et al., 2005). A very recent study on mouse MBLs and ficolins shows that Mbl1, Mbl2, Fcna and FcnB genes are highly polymorphic like their human functional equivalents MBL and FCN2 (Phaneuf et al., 2007). In this report several SNPs, which might alter the affinity or specificity of carbohydrate binding, were identified in the fibrinogenlike domains of ficolin-A and -B in 10 inbred strains of mice (Phaneuf et al., 2007).
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Discussion As a novel group of pathogen binding proteins, ficolins are the focus of many recent investigations due to their apparent versatile functions within the immune system. Like MBL, they have been shown to activate the complement cascade, through their association with MASPs, upon binding of their lectin domain to microbial surfaces. It has also been proven that L- and H-ficolins play a role in host homeostasis by promoting the clearance of apoptotic cells suggesting that, again like MBL, ficolins are able to recognize non-self (i.e. pathogens) as well as altered-self (i.e. apoptotic and necrotic cells). Research on these lectins from different species over the last years has shown that there are two types of ficolins: a serum ficolin of hepatic origin and a nonserum cell-associated ficolin. Serum-type ficolins (e.g. L- and H-ficolins in human, ficolin-A in mouse) are able to promote pathogen clearance by activating the lectin pathway of the complement system, whereas cell-associated ficolins (e.g. M-ficolin in human, ficolinB in mouse) might play a distinct role. For example, it has been reported that ficolin-B does not associate to MASPs and, therefore, cannot activate the complement cascade. This observation is in accordance to our own findings that ficolin-B is expressed intracellularly in peritoneal macrophages where MASP-2 is absent and complement activation impossible. Whether upon stimulation ficolin-B is secreted to local areas (as it was shown for ficolin-b in pig) or acts as an intracellular pathogen recognition molecule remains to be elucidated. Preliminary observations from our ficolin-B promoter studies indicate that unmethylated CpG dinucleotides up-regulate the reporter gene expression (data not shown), suggesting a Toll-like receptor 9 (TLR-9)dependent response on ficolin-B biosynthesis (AhmadNejad et al., 2002; Latz et al., 2004). Our recombinant ficolin-B displays binding affinity for a variety of bacterial surface antigens which implies that it may act either intracellularly, resembling the Nod2 protein mechanism (Girardin et al., 2003), or as a secreted protein with opsonizing functions. In any case, these several hypotheses are in need of further investigation which will lead to a better understanding of these multifunctional immune components.
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Immunobiology 213 (2008) 297–306 www.elsevier.de/imbio
Ficolins: Novel pattern recognition molecules of the innate immune response Valeria L. Runzaa,, Wilhelm Schwaebleb, Daniela N. Ma¨nnela a
Institute of Immunology, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93042 Regensburg, Germany Department of Infection, Immunity and Inflammation, Maurice Shock Medical Sciences Building, University of Leicester, Leicester, UK
b
Received 31 July 2007; accepted 17 October 2007
Abstract Ficolins are members of the collectin family of proteins which are able to recognize pathogen-associated molecular pattern (PAMP) on microbial surfaces. Upon binding to their specific PAMP, ficolins may trigger activation of the immune system by either binding to cellular receptors for collectins or by initiating activation of complement via the lectin pathway. For the latter, the human ficolins (i.e. L-, H- and M-ficolin) and murine ficolin-A were shown to associate with the lectin pathway-specific serine protease MBL-associated serine protease-2 (MASP-2) and catalyse its activation which in turn activates C4 and C4b-bound C2 to generate the C3 convertase C4b2a. There is mounting evidence underlining the lectin nature of ficolins with a wide range of carbohydrate moieties recognized on microbial surfaces. However, not all members of the ficolin family appear to act as lectin pathway recognition components. For example, murine ficolin-B does not associate with MASP-2 and appears to be absent in plasma and other humoral fluids. Its stringent cellular localization points to other functions within the immune response, possibly acting as an intracellular scavenger to target and facilitate clearance of PAMP-bearing debris. When comparing ficolin orthologues from different species, it appears evident that human, murine, and porcine ficolins differ in many aspects, a specific point that we aim to address in this review. r 2007 Elsevier GmbH. All rights reserved. Keywords: Complement system; Ficolin; Innate immunity; Lectin pathway
Introduction Abbreviations: CRD, carbohydrate recognition domain; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; LTA, lipoteichoic acid; LPS, lipopolysaccharide; MASP, MBL-associated serine protease; MBL, mannan-binding lectin; PAMP, pathogen-associated molecular pattern; PCR, polymerase chain reaction; UT, untranslated region. Corresponding author. Tel.: +49 941 944 5465; fax: +49 941 944 5462. E-mail address: [email protected] (V.L. Runza). 0171-2985/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2007.10.009
Innate immunity uses a variety of induced effector mechanisms to fight infections and control them until the causative pathogens are eventually recognized by the adaptive immune system. The complement system represents one of the major humoral systems of the innate host defense and is composed of a cascade of activation events that occur on the surface of pathogens or infected cells and generate active products with various effector functions including opsonization and
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pathogen lysis, chemotaxis, and proinflammatory activation of the cellular immune system. There are three distinct pathways by which the complement system can be activated: the classical, the lectin, and the alternative pathway, all of which converge to generate the same set of activation products. The lectin pathway is initiated when mannan-binding lectin (MBL) or ficolins bind to carbohydrate moieties on bacterial surfaces. This binding promotes the activation of the MBL-associated serine proteases (MASPs) which lead to subsequent cleavage of the C4 and C4b-bound C2. Activation of the lectin pathway leads to the generation of the C3 converting enzyme complex C4b2a which – upon accumulation of the C3 cleavage product C3b – can develop C5 convertase activity. With the cleavage of C5, all enzymatically mediated activation steps are completed, while C5b – the major cleavage fragment of C5 – initiates the assembly of the terminal activation steps of C6–C9, leading to the formation of the membrane attack complex (MAC) through a cascade of intermolecular rearrangements. Ficolins were first documented as transforming growth factor-b1 (TGF-b1)-binding proteins on pig uterus membranes by Ichijo et al. (1991). Their primary structure revealed that they are mainly composed of fibrinogen- and collagen-like domains and, this unique feature gave them their name ficolins (Ichijo et al., 1993). Since the first description of porcine ficolins, other homologous proteins with very similar structural features have been identified at the cDNA and/or protein level in human (Endo et al., 1996; Lu et al., 1996; Sugimoto et al., 1998), rodents (Fujimori et al., 1998; Ohashi and Erickson, 1998), Xenopus (Kakinuma et al., 2003), and invertebrates (Kenjo et al., 2001), showing different locations of biosynthesis suggestive of different local functions. Moreover, it has been shown that ficolins present in human, mouse, and pig are lectins with a common binding specificity for N-acetylglucosamine (GlcNAc) (Matsushita et al., 1996). Since the time of their first descriptions, human ficolins have been intensively investigated and numerous reports have been published on their molecular structure, their expression, their involvement as carbohydrate recognition subcomponents of the lectin pathway, and their gene polymorphisms. The biological functions of murine ficolin, however, are less well understood than those of their human orthologues and gene-targeted ficolin-deficient mouse strains may help to define their roles in the antimicrobial immune defense.
Sites of ficolin expression The published data on ficolins show that these lectins are present in two forms: a serum type (predominantly
synthesized in the liver) and a cell-associated type of ficolin (predominantly synthesized in phagocytic cells). Pigs have two distinct but closely related ficolin genes, named a and b (Ichijo et al., 1993). Ficolin-a is expressed in liver, bone marrow, spleen, lung (Ohashi and Erickson, 1998) and very weakly in the uterus (Ichijo et al., 1993), whereas ficolin-b is expressed in bone marrow and neutrophils (Brooks et al., 2003b). Ficolina and -b share 81–84% identity at the amino acid level. Ficolin-a is the major plasma ficolin and consists of N-glycosylated subunits of 35 kDa (Ohashi and Erickson, 1998) while ficolin-b has an apparent molecular weight of 39 kDa and was found to be synthesized, stored, and secreted by porcine neutrophils but not by peripheral blood monocytes or platelets (Brooks et al., 2003b). Ficolin-b is present in both cytoplasmatic and membrane fractions of neutrophil preparations but its subcellular distribution has not been shown. Amongst human ficolins, L- and H- (Hakata antigen) are the plasma ficolins. The hepatocytes are the primary source of synthesis for L-ficolin and its concentration in sera from 181 blood donors was found to range from 1.1 to 12.8 (median 3.7) mg/ml (Kilpatrick et al., 1999). H-ficolin is also synthesized by hepatocytes as well as by bile duct epithelial cells, and in the lung by ciliated bronchial and type II alveolar epithelial cells (Akaiwa et al., 1999). H-ficolin is found in circulation at a median concentration of 18.4 mg/ml (Krarup et al., 2005). On the other hand, the non-serum-type M-ficolin is expressed in peripheral blood leukocytes. Even though its primary structure lacks an apparent transmembrane domain, M-ficolin was found on the surface of PBMs (Teh et al., 2000). Recently, M-ficolin protein was localized in secretory granules in the cytoplasm of neutrophils, monocytes, and type II alveolar epithelial cells in the lung (Liu et al., 2005b). These observations led to the hypothesis that M-ficolin might act as an acute phase protein that is temporarily stored in the secretory granules of the leukocytes to be released locally and to execute its functions in host defense upon the right stimuli, similar to ficolin-b in pigs. Mice, as well as rats, have two ficolin forms, termed ficolin-A and -B. The ficolin-A gene (Fcna) was first isolated by Fujimori and co-workers in 1998 from a mouse liver library (Fujimori et al., 1998). The protein encoded by this gene located on chromosome 2 is 60%, 59.3%, 59.1%, and 59% identical to those of porcine ficolin-a, -b, human M-ficolin, and L-ficolin, respectively (Ohashi and Erickson, 1998). Ficolin-A is the plasma protein with a molecular mass of 37 kDa, highly expressed in liver and spleen (Fujimori et al., 1998). In a recent report, Liu and co-workers showed that ficolin-A mRNA is expressed during ontogenesis as early as on embryonic day (E) 12.5, displaying an increase in abundance during development, peaking around
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birth, and slightly declining in the adult stages (Liu et al., 2005a). In addition, in situ hybridization studies indicated that ficolin-A mRNA was mainly localized in the linings of the hepatic sinusoids in the liver, and in the red pulp of the spleen. These observations, together with further immunohistochemical analysis revealing a distribution pattern of ficolin-A comparable to the Kupffer cells in liver, suggests that ficolin-A mRNA is expressed by cells of the monocytemacrophage lineage (Liu et al., 2005a). Ficolin-B was first characterized by Ohashi and Erickson in 1998 as a mouse ficolin different from the murine plasma ficolin (ficolin-A), with strong mRNA expression in bone marrow and weak expression in spleen, and a 67% and 73,4% identity on the protein level to the human L- and M-ficolin, respectively. (Ohashi and Erickson, 1998). More recently, ficolin-B mRNA levels were described as progressively increasing from E13.5 to E18.5 with a subsequent rapid postnatal decline to undetectable levels in liver prior to the age of 4 weeks. However ficolin-B mRNA was detected in spleen at all time points examined after birth, indicating a complementary expression of ficolin-A and -B in spleen (Liu et al., 2005a). Regarding the specific cell types expressing ficolin-B, distinct cell lineages of sorted bone marrow-derived cells showed different expression patterns by RT-polymerase chain reaction (PCR) with high levels in myeloid cells (Gr-1+ and Mac-1+) and no expression in the Ter119+ erythroid, the T-cell (CD3e+), or the B-cell (B220+) lineages (Liu et al., 2005a). We recently published the first observation on ficolin-B expression at the protein level. Ficolin-B was found to be expressed by peritoneal macrophages of C57Bl/6 mice by immunocytochemistry, and surprisingly a positive staining was detected only after permeabilization of the cells indicating an intracellular expression and not a cell-association of this lectin (Fig. 1) (Runza et al., 2006). However, the possibility that macrophages secrete ficolin-B upon stimulation as a local mediator was not tested yet and, therefore, cannot be discarded. Furthermore, screening of different immune cells revealed ficolin-B mRNA presence in bone-derived immature dendritic cells but not in mast cells (data not shown).
Molecular structure of ficolins Like collectins, ficolins are built of structural subunits each composed of three identical polypeptide chains. In each chain, a short N-terminal region with one or two cysteine residues is followed by a collagenlike domain characterized by 11 to 19 Gly-X-Y triplets (where X and Y denote any amino acid), a short link region, and a subsequent fibrinogen-like domain. Through their collagen-like domain three polypeptide
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Fig. 1. Peritoneal macrophage-expressed ficolin-B is localized inside the cell. Peritoneal macrophages were either permeabilized (lower panel) or not permeabilized (upper panel), and subjected to ficolin-B immunostaining (Runza et al., 2006).
chains assemble into a trimer to form a subunit. Although ficolins do not have a coiled-coil structure acting as the neck region like MBL (Holmskov et al., 2003; Garlatti et al., 2007), they form active oligomers when normally four subunits join together via disulfide bridges at the N-terminal regions. The polypeptide chains then radiate out in a sertiform structure, giving ficolins the typical ‘‘bouquet’’-like appearance (Fig. 2). Higher or smaller oligomers seem to be less common for ficolins than for MBL (Holmskov et al., 2003). The carbohydrate-binding activity of ficolins is assigned to the fibrinogen-like domain which, like the carbohydrate recognition domain (CRD) of MBL, seems to be Ca++-dependent, although there has been some controversy about this point (Matsushita et al., 1996; Ohashi and Erickson, 1997; Le et al., 1997). The fibrinogen-like domain consists of 200–250 residues and is characterized by the presence of 24 invariant, mostly hydrophobic, amino acids including four cysteines and 40 highly conserved residues. According to the crystal structure resolved for the fibrinogen CRD of tachylectin 5A (a nonself-recognizing lectin from the hemolymph plasma of Tachypleus tridentatus), the contact between the protein and its carbohydrate ligand is mediated by four aromatic side chains that form a funnel in which the methyl group of GlcNAc fits in the center (Kairies et al., 2001). However, the structure of a trimer
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Fig. 2. Structural organization of ficolins. A 12-mer is depicted but lower and higher oligomers have similar organisation.
of the recombinant L-ficolin fibrinogen-like domain was resolved by X-ray crystallography, and revealed that the ligand binding site of L-ficolin is situated at a position different from that seen in the tachylectin, at the opposite side of the globular domain (Krarup et al., 2004). This unexpected finding calls for caution when trying to deduce functionalities from orthologous structures. From the genomic point of view, the exon–intron organization is similar among ficolins from different species: 8–10 exons where the first one encodes the 50 UT, leader peptide and N-terminal amino acids; the second and the third exons encode the collagen-like domain; the fourth one the short link region, and the exons five to eight the fibrinogen-like domain. The last exon also encodes the 30 UT, and in the case of the 9- or 10-exon containing genes (i.e. FCN1, Fcna and Fcnb) the additional exons encode extra segments of Gly-X-Y repeats in the collagen-like domain (Endo et al., 1996, 2004).
Ficolin functions Carbohydrate recognition There is ample evidence that ficolins (both serum- and non-serum types) from different species share a common binding affinity for GlcNAc, by recognition of the nonreducing terminal carbohydrate residue carrying the N-acetyl group. Already in 1998, it was shown that L-ficolin is able to bind to GlcNAc and N-acetylgalactosamine (GalNAc) but not to their precursor sugars (Le et al., 1998). At that time it was also reported that L-ficolin binds to glutathione and CNBr-activated sepharose but not to underivatized sepharose, suggesting that L-ficolin might bind the common structure in these compounds which is an amide group (-CO-NH). Later, Krarup and co-workers showed that L-ficolin binds to Streptococcus pneumoniae 11F and that this interaction could be inhibited by N-acetylated compounds, either sugars (GlcNAc, GalNAc, N-acetlymannosamine)
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or other molecules like N-acetylcysteine, N-acetylglycine, and acetylcholine (Krarup et al., 2004), suggesting a rather general acetyl-binding capacity. However, the presence of an N-acetyl group might affect the binding of ficolins by simply neutralizing the charge on the sugar amine which, without the acetyl substitution, would appear protonated (positively charged). Therefore, it still remains to be understood to what extent the binding of ficolins to acetyl groups is relevant under physiological conditions in vivo. In addition, L-ficolin has been involved in the recognition and phagocytosis of type III group B streptococci (Aoyagi et al., 2005). The capsule of these bacteria possesses an elongated polysaccharide containing non-terminal GlcNAc residues and b-1-3 links between glucose and galactose rings (Kadirvelraj et al., 2006) which, in accordance with a recent work by Garlatti and co-workers, suggests that L-ficolin is able to bind such elongated carbohydrates with acetylated and neutral residues like 1,3-b-Dglucan, present on microbial and apoptotic surfaces (Garlatti et al., 2007). Also interesting is the observation that L-ficolin, but not MBL or H-ficolin, specifically binds to lipoteichoic acid (LTA) which is the common exposed antigen present on Gram-positive bacterial cells (Lynch et al., 2004). This finding indicates that the repertoire of microbial organisms recognized by L-ficolin can not only overlap but also extend that recognized by MBL and, therefore, broadens the spectrum for the innate response towards invading pathogens. Human M-ficolin was found to bind to several neoglycoproteins bearing GlcNAc and GalNAc, and more interestingly sialyl-N-acetyllactosamine (SiaLacNAc) (Liu et al., 2005b). Sialic acid is normally involved in the cellular recognition mediated by molecules that regulate distinct host events such as cellular growth and differentiation. The meaning of this specific binding is, therefore, not clear but suggests that M-ficolin might play different and/or additional roles in local immunity. Likewise, mouse ficolin-B seems to recognize the terminal N-acetylneuraminic acid residue present in molecules like SiaLacNAc and fetuin (Endo et al., 2005), suggesting that the cell-associated ficolins (human M-ficolin, mouse ficolin-B) might have other unique functions, in addition to bacterial recognition, and probably related to cellular host events. Upon binding to their carbohydrate ligands all three human ficolins and murine ficolin-A were shown to activate the complement system in C4b deposition assays where ficolin/MASP-2 complexes are preincubated with the immobilized ligand and then recombinant C4 is added previous to the detection of the C4b split product. On the other hand, even when both mouse ficolins-A and -B show specificity to GlcNAc and GalNAc, interestingly, only ficolin-A can form an active
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complex with MASP-2 able to cleave C4 (Endo et al., 2005) which is in accordance to our own observations (unpublished data). A very recent study on the characterization of MASP-binding site seems to deal with this discrepancy (Girija et al., 2007). There it is shown that the MASP-binding site in the collagen-like domain of MBLs and ficolins is conserved. A lysine residue (Lys56) may play a critical role by forming the key contacts with the MASP, and adjacent residues – mainly methionine or alanine – might help to stabilize the interaction (Girija et al., 2007). Mouse ficolin-B contains the required sequences for binding to MASP (i.e. Lys56). However, an adjacent glutamic acid residue is likely to disrupt MASP binding and activation, explaining the lack of complement activation by mouse ficolin-B. In this regard, it would be interesting to investigate if its porcine (ficolin-b) and rat orthologues are able to bind MASP since their sequences contain the conserved adjacent alanine residues.
Interaction with microorganisms and complement activation L-ficolin binds to the Gram-negative strain Salmonella typhimurium TV119 (rough chemotype strain with exposed GlcNAc) and enhances phagocytosis by polymorphonuclear neutrophils and monocytes. In addition to this opsonic activity, it can also activate complement through a Ca++-dependent association with MASPs (Matsushita et al., 2000). In addition, it has been demonstrated that L-ficolin can bind to Escherichia coli and be eluted with a mixture of monosaccharides (Lu and Le, 1998). Interestingly, L-ficolin/MASP complexes from sera were shown to specifically bind to LTA from Gram-positive bacteria such as Staphylococcus aureus and initiate the C4 turnover (Lynch et al., 2004). This was also shown to be true for other clinically relevant bacteria such as Streptococcus pyogenes and Streptococcus agalactiae (Lynch et al., 2004). In another work on bacterial recognition by ficolins, it was reported that L-ficolin binds to some capsulated S. aureus and S. pneumoniae serotypes but not to non-capsulated strains. These results differed from MBL and H-ficolin binding properties (Krarup et al., 2005) indicating that the binding of each lectin is directed towards a specific and different pathogen-associated molecular pattern (PAMP). The biological significance of H-ficolin as a lectin has been investigated by studying its binding potential to different strains and serotype forms of bacteria including S. pneumoniae, E. coli, S. aureus and Aerococcus viridans. Only A. viridans was found to be recognized and the binding specificity was assigned to a particular polysaccharide, namely PSA (polysaccharide
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A. viridans), present on this microorganism (Matsushita et al., 2002). Previously, it had been shown that the Hakata antigen possessed the potential to agglutinate human erythrocytes coated with lipopolysaccharide (LPS) derived from S. typhimurium and S. minnesota, and that this effect was inhibited by mono- and oligosaccharides of GalNAc, GlcNAc, and D-fucose (Sugimoto et al., 1998). H-ficolin isolated from serum is associated with MASP-1, MASP-2, MASP-3, and MAp19, and the H-ficolin/MASP complex is able to activate complement by cleavage of C4 upon binding to PSA (Matsushita et al., 2002) The cell-associated human M-ficolin coprecipitates with MASP-1 and -2, and the complexes are able to cleave C4 on GlcNAc-coated microplates (Liu et al., 2005b). Regarding its microbial recognition capacity M-ficolin was found to bind to S. aureus and to interact with a smooth-type strain of S. typhimurium (LT2), that possesses additional O-polysaccharides, but not with the rough-type strain TV119. Interestingly, exactly the opposite is true for L-ficolin (Matsushita et al., 1996), indicating that the spectrum of bacterial recognition sites might be different among ficolins. Up to date there are no reports indicating specific microbial recognition epitopes by mouse ficolins. Our preliminary work in this field indicates that ficolin-B binds to a carbohydrate moiety on the murein wall of S. aureus and to some strains of S. pneumoniae (Runza et al., in preparation). Among porcine ficolins, ficolin-a was shown to bind Actinobacillus pleuropneumoniae serotype 5B (APP5), which is the pathogen causing economically significant pneumonic and septicemic diseases in young pigs, in a GlcNAc-dependent manner (Brooks et al., 2003a). In addition, Nahid and co-workers more recently described that native ficolin-a, purified from porcine serum, is able to bind to LPS from Gram-negative bacteria of both the rough- and smooth-types, such as E. coli, S. typhimurium, S. enteriditis, Salmonella abortus equi, Shigella flexeneri, Pseudomonas aeruginosa, and Serratia marcescens. Furthermore, it was also shown that ficolin-a reacts with LTA from Gram-positive bacteria, such as Streptococcus sanguis, S. pyogenes, Bacillus subtilis, and S. aureus (Nahid and Sugii, 2006). On the other hand, it seems that ficolin-b might function locally as a secreted collagenous defense lectin at sites of inflammation where neutrophils are activated. The secreted contents of PMA-activated neutrophils are bactericidal and ficolinb may participate with other antibacterial neutrophil products in tissue antisepsis (Brooks et al., 2003b). However, the microbial targets for ficolin-b are not known and, in general, it has not been shown yet whether porcine ficolins can activate the complement system. Unlike MBL, which is known to bind to the gp120 protein of HIV-1 (Haurum et al., 1993) and to
Leishmania major promastigotes (Green et al., 1994), up to date it is not known whether ficolins are able to recognize viruses, fungi, or parasites, and it therefore still remains as an interesting field to be explored. Table 1 summarizes the main characteristics of human, murine, and porcine ficolins for better comprehension and comparison of both their overlapping and non-overlapping functions in the host defense.
Interaction with host cells In 2001 it was shown that the complement component C1q as well as MBL bind to apoptotic cells, promoting macropinocytosis and removal of dead cell bodies (Ogden et al., 2001). Since then, speculations on the ability of ficolins to recognize apoptotic and necrotic cells have been investigated and a number of reports suggest that both plasma L- and H-ficolins do play a role in the clearance of dying host cells. However, there is controversy about the mechanism triggered after ficolin recognition. Kuraya et al. (2005) described that binding of both human ficolins to apoptotic cells leads to complement activation. On the other hand, Jensen et al. (2007) reported more recently that L-ficolin binds not only to late apoptotic cells but also to apoptotic bodies and necrotic cells (and not to early apoptotic cells) but without complement deposition. They described exposed DNA on permeable late apoptotic and necrotic cells as one of the putative ligands for L-ficolin which opsonizes the cell and leads to an enhanced uptake by macrophages.
Evolution and polymorphisms Cloning and software-based sequence analysis of invertebrate ascidian Halocynthia roretzi and lower vertebrate Xenopus laevis ficolins and their comparison with their mammalian orthologues has allowed Fujita and co-workers to build up phylogenetic trees that depict the evolution of the related genes (Kenjo et al., 2001; Kakinuma et al., 2003). In addition, the fact that mice possess two and humans three forms of ficolins, led Endo et al. (2004) to perform evolutionary studies on the structures and organization of these ficolin genes. Evidence had suggested that L-ficolin was closely related to ficolin-A (serum-type), and M-ficolin to ficolin-B (non-serum type). However, the phylogenetic tree based on amino acid sequences indicated that L-ficolin diverged from the B/M-ficolin lineage, suggesting that mouse ficolin-A is not the orthologue of human L-ficolin, although both are mainly expressed in liver and have similar carbohydrate specificities. These results imply that comparable selective pressures acted independently on both the murine and primate lineages to
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Table 1.
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Expression, sugar specificity and target pathogens of collectins and ficolins Tissues of origin
Tissues of presentation
Sugar specificity and pathogen interaction
Complement activation
Liver
Serum
Yes
Serum, bronchus, alveolus, bile
M-ficolin
Liver (hepatocytes and bile epithetlium), type II alveolar cells Monocytes
GlcNAc/ManNAc44GalNAc/CysNAc/ GlyNAc, acetylcholine, elastin, corticosteroids, 1,3-b-D-glucan, LTA from S. aureus, S. pyogenes, S. agalactiae, B. subtillis S. typhimurium (Ra), E. coli, S. pneumoniae GlcNAc, GalNAc, fucose, glucose, PSA A. viridans
Monocyte surface
GlcNAc-BSA, GalNAc-BSA, SiaLacNAcBSA S. aureus
Yes
Mouse Ficolin-A Ficolin-B
Liver and spleen BM and spleen
Serum Peritoneal macropaghes
GlcNAc, GalNAc GlcNAc, GalNAc, SiaLacNAc, fetuin
Yes No
Liver, BM, spleen, lung, uterus
Serum
GlcNAc, LPS from E. coli, S. typhimurium, S. enteriditis, S. abortus equi, Shigella flexeneri, P. aeruginosa, Serratia mascesecens LTA from S. sanguis, S.pyogenes, B. subtillis, S. aureus APP5 n.d.
n.d.
Human L-ficolin
H-ficolin
Pig Ficolin-a
Ficolin-b
BM, neutrophils
Yes
n.d.
BM, bone marrow; GlcNAc, N-acetyl-D-glucosamine; GalNAc, N-acetyl-D-galactosamine; ManNAc, N-acetyl-D-mannosamine; PSA, polysaccharide A. viridans; HIV, human immunodeficiency virus; IAV, influenza A virus; RSV, respiratory syncytial virus; HSV-1, herpes simplex virus type 1; APP5, Actinobacillus pleuropneumoniae 5B; n.d., not determined.
produce these hepatic serum-type ficolins from a nonserum-type lineage (Endo et al., 2004). Furthermore, the tree suggested that H-ficolin had an ancient origin back to an evolutionary stage before the divergence of the Xenopus lineage, although no H-ficolin had been identified in mice. By computer analysis of the mouse genome database, Endo and others characterized the genomic region homologous to the human H-ficolin gene and identified an orthologue of the mouse H-ficolin gene (67% nucleotide sequence identity to the human sequence) present as a pseudogene on chromosome 4, which renders this gene dysfunctional and silent (Endo et al., 2004). In contrast to the human gene, the first exon of the mouse ficolin-H orthologue has stop codons in all three reading frames, which were generated by base changes and micro-deletions, and no start codon could be identified. A similar pseudogene is found in the rat (Endo et al., 2004). Polymorphisms reported on human ficolins showed that, like MBL, single nucleotide polymorphisms (SNPs) in the promoter region of the L-ficolin gene lead to a significant variation in the plasma concentration of this
lectin. On the other hand, two SNPs in the fibrinogen-like domain coding region (exon 8), which result in amino acid substitution, affect notably the binding capacity to GlcNAc (Hummelshoj et al., 2005; Herpers et al., 2005). In addition, it was also reported that children with recurrent infections have low L-ficolin concentrations (Atkinson et al., 2004). The M-ficolin gene was also characterized and 12 SNPs were detected both in the promoter and in the structural regions albeit no amino acid exchanges were found. Regarding the Hakata antigen, a deletion mutation in one of the tested individuals was detected, creating an early stop codon in exon 5. However, the relevance of this mutation remains unknown (Hummelshoj et al., 2005). A very recent study on mouse MBLs and ficolins shows that Mbl1, Mbl2, Fcna and FcnB genes are highly polymorphic like their human functional equivalents MBL and FCN2 (Phaneuf et al., 2007). In this report several SNPs, which might alter the affinity or specificity of carbohydrate binding, were identified in the fibrinogenlike domains of ficolin-A and -B in 10 inbred strains of mice (Phaneuf et al., 2007).
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Discussion As a novel group of pathogen binding proteins, ficolins are the focus of many recent investigations due to their apparent versatile functions within the immune system. Like MBL, they have been shown to activate the complement cascade, through their association with MASPs, upon binding of their lectin domain to microbial surfaces. It has also been proven that L- and H-ficolins play a role in host homeostasis by promoting the clearance of apoptotic cells suggesting that, again like MBL, ficolins are able to recognize non-self (i.e. pathogens) as well as altered-self (i.e. apoptotic and necrotic cells). Research on these lectins from different species over the last years has shown that there are two types of ficolins: a serum ficolin of hepatic origin and a nonserum cell-associated ficolin. Serum-type ficolins (e.g. L- and H-ficolins in human, ficolin-A in mouse) are able to promote pathogen clearance by activating the lectin pathway of the complement system, whereas cell-associated ficolins (e.g. M-ficolin in human, ficolinB in mouse) might play a distinct role. For example, it has been reported that ficolin-B does not associate to MASPs and, therefore, cannot activate the complement cascade. This observation is in accordance to our own findings that ficolin-B is expressed intracellularly in peritoneal macrophages where MASP-2 is absent and complement activation impossible. Whether upon stimulation ficolin-B is secreted to local areas (as it was shown for ficolin-b in pig) or acts as an intracellular pathogen recognition molecule remains to be elucidated. Preliminary observations from our ficolin-B promoter studies indicate that unmethylated CpG dinucleotides up-regulate the reporter gene expression (data not shown), suggesting a Toll-like receptor 9 (TLR-9)dependent response on ficolin-B biosynthesis (AhmadNejad et al., 2002; Latz et al., 2004). Our recombinant ficolin-B displays binding affinity for a variety of bacterial surface antigens which implies that it may act either intracellularly, resembling the Nod2 protein mechanism (Girardin et al., 2003), or as a secreted protein with opsonizing functions. In any case, these several hypotheses are in need of further investigation which will lead to a better understanding of these multifunctional immune components.
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