- Email: [email protected]
Lectin complement system and pattern recognition
ARTICLE IN PRESS
Immunobiology 211 (2006) 283–293 www.elsevier.de/imbio
REVIEW
Lectin complement system and pattern recognition Yuichi Endo, Momoe Takahashi, Teizo Fujita Department of Immunology, Fukushima Medical University, Fukushima 960-1295, Japan Received 1 December 2005; accepted 10 January 2006
Abstract Living organisms have strong defense mechanisms against invading microorganisms as survival strategies. One of the defense mechanisms is the complement system, composed of more than 30 serum and cell surface components. This system collaborates in recognition and elimination of pathogens as a part of both the innate and acquired immune systems. The two collagenous lectins, mannose-binding lectin (MBL) and ficolins, are pattern recognition proteins acting in innate immunity and, upon recognition of the pathogens, they trigger the activation of the lectin complement pathway through attached serine proteases (MASPs). A similar lectin-based complement system, consisting of the lectin-protease complex and C3, is present in ascidians, our closest invertebrate relatives and in lamprey, the most primitive vertebrate. Furthermore, a lamprey N-acetylglucosamine (GlcNAc)-binding lectin was identified as the orthlogue of mammalian C1q, and lamprey MASP is suggested as the prototype of MASP-2/C1r/C1s, indicating that the classical complement pathway arose as a part of the innate immune system. Thus, the complement system is one of the most highly organized innate immune systems in invertebrates and jawless vertebrates, and this system has survived in vertebrates with its core components little changed for 600–700 million years. r 2006 Elsevier GmbH. All rights reserved. Keywords: Complement; Ficolin; Lectin; MASP; MBL
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MBL is one of the typical pattern recognition proteins . . . . . . . . . . . . . . . Ascidian MBL-like lectin, glucose-binding lectin (GBL) and lamprey MBL . Ficolins are another recognition molecule of the lectin pathway . . . . . . . . . Lamprey orthologue of mammalian C1q acts as a GlcNAc-binding lectin . . Origin of the MASP family is traced back to an invertebrate, ascidian . . . . Two lineages of the MASP family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lamprey MASP-A/B is the prototype of MASP-2 and C1r/C1s . . . . . . . . . The primitive complement system in ascidian and lamprey. . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author. Fax: +81 24 548 6760.
E-mail address: [email protected] (T. Fujita). 0171-2985/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2006.01.003
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Introduction Immunity is comprised of innate and adaptive defense mechanisms. The innate immune system is an evolutionarily ancient form and crucial for the first line of defense (Hoffmann et al., 1999). Accumulating evidence indicates that adaptive immunity was established at an early stage of jawed vertebrate evolution. In invertebrates, therefore, the innate immune system is the only defense mechanism against infection. Innate immunity was formerly thought to be a non-specific immune response characterized by phagocytosis. However, innate immunity has considerable specificity and is capable of discriminating between pathogens and self as proposed in the concept of pattern recognition molecules of host. These molecules recognize conserved pathogen-associated molecular patterns shared by large groups of microorganisms, thereby successfully defending invertebrates and vertebrates against infection (Medzhitov and Janeway, 2000). The complement system mediates a chain reaction of proteolysis and assembly of protein complexes, playing a major role in body defense as a part of both the innate and adaptive immune systems (Walport, 2001a, b). Complement was first described in the 1890s as a heatlabile protein in serum that ‘complemented’ heat-stable antibodies in the killing of bacteria. Now, the complement system consists of three activation pathways, classical, alternative and lectin pathways, which merge at the proteolytic activation step of C3, the central component of the complement system. C3 is equipped with a unique intra-molecular thioester bond which is exposed to the molecular surface upon activation and forms a covalent bond with invading microorganisms (Law et al., 1980). This covalent tagging of foreign molecules by C3 is considered to be one of the most important functions of the complement system; bound C3 activation products enhance the phagocytosis of pathogens through C3 receptors on phagocytes, and contribute to the activation of the late complement components, C5–C9, which form a cytolytic complex (the so-called membrane attack complex). The classical pathway is activated by antibody–antigen complexes and is a major effector of antibody-mediated immunity. The recently discovered lectin pathway (Fujita, 2002; Matsushita and Fujita, 1996) activates complement following the recognition of microbial carbohydrate patterns by either mannose-binding lectin (MBL) or ficolins, typical fluid phase pattern recognition molecules (Holmskov et al., 2003; Matsushita and Fujita, 2001, 2002), and the subsequent activation of associated unique enzymes, MBL-associated serine proteases (MASPs) (Matsushita et al., 1998; Schwaeble et al., 2002). The alternative pathway is initiated by the covalent binding of a small amount of C3 to hydroxyl or amine groups on cell surface molecules of
microorganisms and does not involve specific recognition molecules. This pathway also functions to amplify C3 activation (amplification loop) (Walport, 2001a, b). Most components of the classical pathway have their structural and functional counterparts in the alternative or lectin pathways, suggesting that gene duplications played an important role in establishing these three pathways. These duplications could be an epoch-making event, which enabled generation of the classical pathway, considered to be the most modern pathway contrary to its name, from the alternative and lectin pathways (Nonaka et al., 1998). Accumulating evidence indicates that the modern complement system seems to have been established by the emergence of jawed vertebrates, and that the complement system of bony and cartilaginous fish has basically the same set of components as the mammalian complement system (Fujita, 2002; Nonaka, 2001; Fujita et al., 2004a). The development of the complement system is illustrated in Fig. 1. In addition, one of the outstanding advances in recent complement research is the discovery of the lectin pathway. In the lectin pathway, MBL and ficolin act as the recognition molecules and activate complement in association with MASPs, a C1r/C1s-like serine protease that is able to cleave the complement components C4, C2 and C3 (Fujita et al., 2004b). Recent biochemical identification of several components of the lectin pathway from a solitary ascidian, Halocynthia roretzi, revealed that the primitive complement system is one of the most highly organized innate immune systems in invertebrates. Furthermore, in lamprey, the most primitive vertebrate (jawless vertebrate) we found that C1q, the component of the classical pathway, has a lectin activity and acts as the pattern recognition molecule (Matsushita et al., 2004). In this review, we focus on typical pattern recognition proteins, MBL and ficolin, and associated serine protease, MASP in ascidian and lamprey, and further we would like to mention the architecture of the primitive complement system.
MBL is one of the typical pattern recognition proteins MBL is a C-type lectin that plays a crucial role in the first line of host defense (Drickamer et al., 1986; Ezekowitz et al., 1988; Kawasaki et al., 1978; Turner, 1996). The importance of this molecule is underlined by a number of clinical studies linking MBL deficiency with increased susceptibility to a variety of infectious diseases (Jack et al., 2001; Neth et al., 2000; Summerfield et al., 1995; Super et al., 1989). MBL is an oligomer of structural subunits, each of which is composed of three identical 32-kDa polypeptide chains. One polypeptide chain contains an N-terminal region rich in cysteine, a
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Arthropods Deuterostomes Chordates
Vertebrates
Amphioxus Horseshoe crab
Sea urchin
Ascidian
Lectin Pathway
285
Shark Lamprey
Carp
Xenopus
Mammals
Adaptive Immunity
MBL or MBL-like Ficolin MASP-1 MASP-3 MASP-2 sMAP
Classical Pathway C1r/C1s C1q C3/Factor B
Fig. 1. An evolutionary perspective for the recognition molecules and serine proteases involved in complement activation. The representative species for jawed and jawless vertebrates, deuterostome invertebrates and arthropods are illustrated, with marks for the presence or absence of each component. The components of the lectin pathway such as MBL, ficolin and MASP are all traced back to an invertebrate, ascidian. The components of the classical pathway are seen in cartilaginous fish (shark) and higher vertebrates, although lamprey C1q is identified as a lectin. As illustrated by shark in this figure, acquired immunity was established at an early stage of the evolution of the jawed vertebrates. The origin of both C3 and factor B-like sequences are traced back to an arthropod, horseshoe crab, although their precise function in invertebrates is still unclear. A C3-like sequence was recently isolated from a coral Swiftia exserta (Cnidaria). The open circles denote the absence of component in the corresponding species. Open and closed squares show phylogenetically not typical form of MASP/sMAP and lamprey C1q with different function from mammalian C1q, respectively.
collagen-like domain consisting of tandem repeats of Gly X–Y triplet sequences (where X and Y represent any amino acid), a neck region and a C-terminal region. Through the collagen-like domain, MBL associates with serine proteases, MASPs. Trimerization of the polypeptide chains is achieved through the collagenous triplehelical region, thereby resulting in the formation of one subunit. The N-terminal cysteine-rich region is involved in the covalent interaction between the three polypeptide chains of the subunit and is also responsible for covalent binding of several subunits into the oligomeric structure. In short, three polypeptides fold together to form the structural subunit and 3–6 of these subunits join to form a mature protein, which has an apparent molecular mass of 300–650 kDa. The overall structure of MBL is shown in Fig. 2. Human MBL exists in several oligomeric forms such as trimers, tetramers and pentamers (Dahl et al., 2001). MBL belongs to the collectin family of proteins that consist of a collagen-like domain as mentioned above and a carbohydrate recognition domain (CRD) (Holmskov et al., 1994). Through its CRD, MBL binds carbohydrates with 3- and 4-hydroxyl groups in the pyranose ring in the presence of Ca2+ through the five conserved residues (Glu185, Asn187, Glu193, Asn205
and Asp206, numbering in Rat MBL-A) in the MBL CRD (Drickamer, 1992; Weis et al., 1992). Prominent ligands for MBL are thus D-mannose, N-acetylglucosamine (GlcNAc) and glucose, whereas carbohydrates that do not fit this steric requirement, D-galactose and sialic acid, which usually decorate mammalian glycoproteins, have undetectable affinity for MBL. This steric selectivity of MBL, along with differences in the spatial organization of the ligands, allows for the specific recognition of carbohydrates on pathogenic microorganisms including bacteria, fungi, parasitic protozoans and viruses, and avoids recognition of self (Holmskov et al., 2003). In addition, sequence analysis of CRDs in comparison with monosaccharide specificity revealed that Glu185 and Asn187 (EPN type) are highly conserved in CRDs that bind mannose, GlcNAc and glucose. Galactose-binding CRDs have Gln185 and Asp187 (QPD type) at these critical positions (Weis et al., 1992), and site-directed mutagenesis has shown that mannose-specificity can be changed to galactose-specificity by replacing Glu185 and Asn187 (EPN type) with Gln185 and Asp187 (QPD type) (Drickamer, 1992). In addition to mammalian and chicken MBL, several lectins in bony fish were characterized. The deduced
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MBL Collagen-like domain
Hinge
CRD
Ficolin collagen-like domain
Fibrinogen-like domain
Fig. 2. Domain and oligomeric structure of MBL and ficolin. MBL is an oligomer of structural subunits each composed of three identical 32 kDa polypeptides. Each polypeptide contains an N-terminal cysteine-rich region, a collagen-like domain consisting of tandem repeats of Gly X–Y triplet sequences (where X and Y represent any amino acid), a neck region, and a C-terminal CRD. MBL forms several sizes of oligomers. Three–six trimers are combined to the multimer via the Nterminal disulfide-bondings, and the hexameric form is shown in this figure. Ficolin monomer consists of a short N-terminal region, a middle collagen-like domain with Gly X–Y triplets, and a globular fibrinogen-like domain. The fibrinogen-like domain forms a globular structure similar to a CRD. The multimers are probably assembled by crosslinking via disulfide bridges in the N-terminal region. The terameric form of Lficolin shown here and the hexameric form of H-ficolin are proposed based on electron microscopy in human.
primary structure of these lectins in carp, zebrafish and goldfish indicates selectivity for galactose, having QPDtype CRDs (Vitved et al., 2000). Recently, another carp MBL with specificity for mannose (EPN type) was also purified (M. Nakao et al., manuscript submitted).
Ascidian MBL-like lectin, glucose-binding lectin (GBL) and lamprey MBL Recently, we purified and cloned an MBL-like lectin from a urochordate, the solitary ascidian H. roretzi (Sekine et al., 2001). The purified 36-kDa lectin binds specifically to glucose but not to mannose or GlcNAc and it was designated glucose-binding lectin (GBL). Its cDNA has an open reading frame of 672 bp, which is predicted to encode a 224 amino acid protein including a 17-residue leader peptide. The predicted molecular mass of the protein was 23,716 Da, indicating that the mature
protein may be glycosylated, since there are three Nlinked glycosylation sites. Sequence analysis of GBL reveals that its N-terminal collagen-like domain is replaced by a sequence that has an a-helix structure similar to the configuration of Gly X–Y repeats, while its C-terminal half contains a CRD. The CRD is of the Ctype (EPN type), and contains 13 of the 18 highly conserved amino acid residues (including the four cysteine residues which form the disulfide bonds within the domain (Drickamer, 1988). The five residues involved in the binding of MBL to the 3- and 4hydroxyl groups of the pyranose ring are completely conserved in GBL and MBLs of other species, except for the three bony fish GalBLs (Fig. 3). Since the structural difference between mannose and glucose is at the site of the 2-hydroxyl group of the pyranose ring, it is possible that residues other than the five conserved residues in GBL are responsible for recognizing the 2-hydroxyl group of glucose. The above results raise the possibility that GBL has evolved early as a prototype of MBL. To test this hypothesis, we also purified the lectin associated with MASP in lamprey, one of the most primitive vertebrates. We isolated a 2.3 kb-long cDNA clone with an open reading frame of 840 bp, which is predicted to encode a 279-amino acid protein including a putative leader peptide (52 amino acids). The predicted molecular mass of the mature protein was 23,217 Da, and there is no N-linked glycosylation site. Lamprey MBL shares 30% identity at the amino acid level with human MBL. Sequence analysis revealed that it is homologous to the mammalian MBLs, as it contains a collagen-like sequence in the N-terminal half and a typical EPN-type CRD in the C-terminal half. When lamprey MBL was compared to ascidian GBL in carbohydrate-binding specificity, ascidian GBL only bound to glucose, but lamprey MBL bound to mannose, GlcNAc and glucose, like mammalian MBLs. Therefore, in conjunction with the phylogenetic analysis (Fujita et al., 2004b), it seems likely that the lamprey lectin is an orthologue of the mammalian MBL (manuscript submitted) and that during evolution GBL acquired the broad binding specificity for carbohydrates and the collagen structure characteristic of MBL. Furthermore, ascidian GBL and lamprey MBL are associated with MASPs and involved in activation of the lectin pathway.
Ficolins are another recognition molecule of the lectin pathway Ficolins are a group of proteins containing both a collagen-like and fibrinogen-like domain and found in varying tissues. Recent characterization has shown that
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287
Terminal sugar 5
1 3
Sugars
O
6
4
2
OH
OH
D Ca
Hydrogen bonds
E N
N E
Coordination bonds
CRD
Fig. 3. Carbohydrate pattern recognition by the CRDs of MBL and related molecules. (Left panel) Individual CRD recognizes carbohydrates with 3- and 4-hydroxyl groups in the pyranose ring in the presence of Ca2+ through the five conserved residues (E, N, E, N and D). Such a pattern is present in D-mannose, GlcNAc and glucose, but not in D-galactose and sialic acid, which usually decorate mammalian glycoproteins. (Right panel) The partial sequences of CRDs. In zebrafish, goldfish and carp, the homologues of MBL are reported. Since another carp MBL was identified recently, in this figure we used CaGalBL for the previously reported MBL and CaMBL for newly identified lectin similar to MBL. AsGBL, ascidian GBL; LaMBL, lamprey MBL; HuMBL, human MBL; MuMBL, mouse MBL.
ficolins present in serum are lectins with a common binding specificity for GlcNAc (Matsushita and Fujita, 2002). Investigations of two types of human serum ficolins, L-ficolin and H-ficolin (Hakata antigen), revealed that they are associated with MASPs and sMAP, and activate the lectin pathway (Matsushita et al., 2001, 2002). Recently, we reported that L-ficolin binds to lipoteichoic acid, a cell component found in all Gram-positive bacteria, and activate the lectin pathway (Lynch et al., 2004). Also, we reported the characterization of M-ficolin, a non-serum lectin, which is secreted from leukocytes. M-ficolin, which can associate with MASPs, activates the lectin pathway and specifically binds to Staphylococcus aureus (Liu et al., 2005). These findings indicate that ficolins act as pattern recognition molecules of the lectin pathway and thus may significantly contribute to innate immunity. We cloned four ficolin cDNAs from the solitary ascidian, H. roretzi (Kenjo et al., 2001), termed ascidian ficolin1–4 (AsFCN1–4). The deduced amino acid sequences of these ficolins revealed the conserved collagen- and fibrinogen-like domains. The fibrinogenlike domains of AsFCNs show about 50% identity with mammalian ficolins. Two types of GlcNAc-binding lectins are purified from the ascidian body fluids and cloned from hepatopancreas cDNA (AsFCN1/2 and AsFCN3). In addition to these ficolins, cDNAs encoding another ficolin has been cloned from the hepatopancreas (AsFCN4). It is of particular interest to note that when compared with mammalian ficolins, the four ascidian ficolins contain short collagen-like domains with 5 and 7 Gly X–Y repeats and also they have long segments between the collagen-like domain and the fibrinogen-like domain. Although it is not yet clear whether these ficolins are associated with MASPs, we postulate that they may act as the recognition molecule of the lectin pathway.
In lamprey, we failed to find the homologue of ficolin as GlcNAc-binding lectin. However, instead of ficolin, we isolated C1q that has a lectin activity and is associated with the prototype of MASP/C1r/C1s-like serine protease (Matsushita et al., 2004).
Lamprey orthologue of mammalian C1q acts as a GlcNAc-binding lectin Serum from the Lampetra japonica lamprey was subjected to GlcNAc-agarose column chromatography in the presence of Ca2+. The column was sequentially eluted with mannose and with GlcNAc, as shown in Fig. 4. Human MBL can be eluted with mannose, and Lficolin, one of the serum ficolins, can be subsequently eluted with GlcNAc (Matsushita and Fujita, 2002). Both of these lectins are found to complex with MASPs. MASP-like protease activity was monitored by esterolytic activity against Boc–Leu–Ser–Thr–Arg–methylcoumarylamide. Lamprey lectin eluted with mannose from GlcNAc-agarose was identified as MBL. Eluates obtained with GlcNAc were further purified by Mono Q chromatography. Lamprey GlcNAc-binding lectin was an oligomer consisting of 24 kDa subunits. The deduced amino acid sequence of this lectin cDNA revealed that it consists of a collagen-like domain and antibody recognition domain, gC1q domain found in a variety of proteins including mammalian C1q. A phylogenetic tree of the gC1q domains of proteins shows that this lectin and mammalian C1q form a tight cluster (Matsushita et al., 2004). We termed this lectin lamprey C1q (LC1q). LC1q co-purified with MASP-A, MASP-B and MASP-1, serine proteases of the MASP family (unpublished observations), and MASP-A exhibited proteolytic activity against lamprey C3 (Matsushita et al., 2004). These
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kDa
GlcNAc elution
Mannose elution
100 75 50 37 25 LaMBL LC1q
15
Fig. 4. SDS-PAGE analysis of lamprey lectins. Lamprey serum was applied to GlcNAc-agarose, and eluted with mannose and subsequently with GlcNAc. The eluted materials were analyzed by SDS-PAGE under reducing conditions (12% gel). Proteins were stained with Coomassie Brilliant Blue R-250. Molecular size markers are indicated on the sides. Lamprey MBL (LaMBL) and C1q (LC1q) are also indicated.
results suggest that C1q may have emerged as a lectin and may have functioned as an initial recognition molecule of the complement system in innate immunity prior to the establishment of adaptive immunity such as immunoglobulins in the cartilaginous fish.
Origin of the MASP family is traced back to an invertebrate, ascidian MASP is a key enzyme of the lectin complement pathway. MASP proteins and/or cDNAs have been isolated from representative vertebrate species including human, mouse, chicken, Xenopus, carp, shark and lamprey, and from two species of invertebrates, amphioxus (cephalochordates) and ascidian (urochordates) (Endo et al., 2003, 1998; Ji et al., 1997) (Fig. 1). Ascidian H. roretzi has two MASPs, designated as MASPa and MASPb, which are the most ancient sequences of MASP identified so far. Amphioxus is one of the most highly organized invertebrates and the closest relative of vertebrates, which occupies a critical position between lamprey and ascidian in phylogeny. Amphioxus Branchiostoma belcheri has at least two MASPs, termed amphioxus MASP-1 and MASP-3, which are the orthologues of jawed vertebrate MASP-1 and MASP3, respectively (Endo et al., 2003). Lamprey L. japonica (cyclostome), which is one of the most primitive vertebrates (jawless vertebrate) and lacks acquired immunity, has at least three kinds of MASP, termed lamprey MASP-1, MASP-A and MASP-B (Endo et al., 2003). As mentioned above, we isolated the orthologues of mammalian MBL and C1q from lamprey plasma. Lamprey MBL and C1q associates with all three types of lamprey MASPs, and the lectin–MASP complexes cleave lamprey C3 into C3b, indicating that the lamprey
complement system is similar to both the classical and lectin pathways in mammals. Mammals including human and mouse, and amphibia Xenopus laevis have three types of MASP, MASP-1, MASP-2 and MASP-3, suggesting a closely similar system of the lectin pathway in these species (Endo et al., 2003). MASP-3 generates from the MASP1/3 gene by alternative splicing and therefore shares the common heavy (H)-chain with the MASP-1 counterpart (Dahl et al., 2001). The mammalian MASP2 gene generates two protein products, MASP-2 and its truncated form with no proteolytic activity, termed as sMAP or MAp19, by alternative splicing (Stover et al., 1999; Takahashi et al., 1999). Recently, we demonstrated that sMAP competes with MASP in binding to MBL and ficolin, and therefore suppressed complement activation, suggesting its regulatory role in the lectin complement pathway (Iwaki and Fujita, 2005). Thus, in human and mouse, six molecules of the MASP family are present, MASP-1, MASP-2, MASP-3, sMAP, C1r and C1s, showing the sophisticated systems of the lectin and classical pathways for complement activation. Interestingly, we identified a C1r/C1slike sequence in shark, probably confirming the cartilaginous fish origin of adaptive immunity. A truncated form of MASP was also identified in carp, although it is large in molecular size and its function is unknown. sMAP-like sequences have not been identified so far in shark, lamprey or invertebrates. To date, a MASP-like molecule has not been identified in lower invertebrates such as sea urchin.
Two lineages of the MASP family Based on the primary structures, exon organization of the genes and usage of codon for the active center serine,
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we identified two lineages in the MASP family (Endo et al., 2003, 1997). The TCN-type, including MASP-1 and ascidian MASPa and MASPb, has a histidine-loop disulfide bridge in the protease domain, a TCN codon at the active site serine, and split exons for the protease domain. The AGY-type, including MASP-2, MASP-3, lamprey MASP-A/B and C1r/C1s, is characterized by the absence of the histidine-loop disulfide bridge, an AGY codon at the active site serine and a single exon for the protease domain. The histidine-loop disulfide bridge, a disulfide bond surrounding the active center histidine, Heavy ch ain A
CUB
EGF CUB
is seen in all serine proteases. The only exception is the AGY-type MASP/C1r/C1s. The precise function of this secondary structure is still unknown. All of the amphioxus/vertebrate MASP/C1r/C1s genes have an intron-less exon encoding an AGY-type L-chain, which is located downstream of the H-chainencoding region (Fig. 5). In contrast, two ascidian MASP genes have no such exon in their corresponding regions. This difference suggests that a processed intronless region might have been inserted between the regions encoding the H-chain and TCN-type L-chain of a
Light chain CCP
Protease
C Chordates
S-S
sMAP or Map19 B 48.4 90.6 60.7
46.9
99.8 67.8
42.1
100
100
1
100
23.4
85.9
98.9 98.8 36.5 3 72.7 62.2 2
51.7
100
75.8 35.3 47.9
289
100
asMASPb Inverte asMASPa -brate amMASP-3 amMASP-1 MASP laMASP-1 shMASP-1 MASP-1 xeMASP-1 muMASP-1 huMASP-1 laMASP-B laMASP-A caMASP-3 shMASP-3 chMASP-3 xeMASP-3b 100 MASP-3 xeMASP-3a muMASP-3 100 huMASP-3 caMASP-2 chMASP-2 MASP-2 xeMASP-2 muMASP-2 huM ASP-2 huC1r huC1s caC1rsA C1r/C1s caC1rsB shC1rs
1
asMASPa/b Cephalochordates
TCN type 2 3
MASP-3 MASP-1 Lamprey
AGY type
MASP-A/B Cartilaginous fish (shark)
MASP-2/C1r/C1s Adaptive Immunity
Fig. 5. Structure and phylogeny of the MASP gene family. (A) The domain structure of MASP/C1r/C1s. MASP-1, MASP-2, MASP-3, subcomplements of component 1, C1r and C1s consist of six domains: two C1r/C1s/Uegf/bone morphogenetic protein 1 (CUB) domains, an epidermal growth factor (EGF)-like domain, two complement control protein (CCP) domains or short consensus repeats (SCRs), and a serine protease domain. Histidine (H), aspartic acid (D) and serine (S) residues are essential for formation of the active center of the serine proteases. The pro-enzyme form of an MASP/C1r/C1s is activated by proteolytic cleavage between the second CCP and protease domains, resulting in the two peptides linked by a disulfide bond, heavy (H) and light (L) chains. (B) Phylogenetic tree of the MASP family. Twenty-eight members of this family, which includes our recently identified shark MASP-1 and C1r/s-like sequences (manuscript in preparation), were aligned by Clustal W using L-chain sequences. The tree was constructed by the neighbor-joining method. Numbers on branches are bootstrap percentages. hu, human; mu, mouse; ch, chicken; xe, Xenopus; ca, carp; sh, shark; la, lamprey; am, amphioxus; as, ascidian. The tree suggests that this family has evolved to generate MASP-1, MASP-2, MASP-3, C1r/C1s and primitive invertebrate MASP. The critical branching points are marked by arrow heads with numbers: 1, generation of the MASP1/3 gene; 2, branching of C1r/C1s; 3, branching of MASP-2. (C) A model of the evolution of the MASP/C1r/C1s family. H in box represents the genomic region encoding the H-chain, and L/T and L/A the genomic regions encoding TCN-type and AGY-type L-chains, respectively. Prior to the emergence of cephalochordates, retroposition of partially processed TCN-type MASP gene and base changes from TCN to AGY at the active site serine would generate a prototype of the MASP-1/3 gene. All of the amphioxus/vertebrate MASP/C1r/C1s genes evolved from this ancestor by gene duplications. The arrowheads with numbers show the evolutionary events corresponding to the branching in the tree shown in B.
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prototype gene as a first step of evolution of the MASP family gene. The insertion of the intron-less region might have occurred after the divergence of urochordates (ascidian), but before the divergence of cephalochordates (amphioxus), since amphioxus MASP gene has such a structure. From the structures of two amphioxus MASP, it can be speculated that an ancestral sequence inserted had the structural features of a TCNtype L-chain, and that the change from the TCN-type to the AGY-type occurred in at least two steps, the base change from TCN to AGY and the loss of an intron in the early step and the loss of a histidine-loop disulfide bridge in the late step. In short, the amphioxus MASP gene, evolved from ascidian gene, producing the orthologues of MASP-1 and MASP-3 in the vertebrate lineage. It is likely that all of the vertebrate MASP/C1r/C1s genes evolved from an ancestral amphioxus MASP1/3type gene-by-gene duplication as a second step, since all of the amphioxus/vertebrate MASP/C1r/C1s genes, MASP2 gene, C1r and C1s genes, have an intron-less exon encoding an AGY-type L-chain in common and these genes lack the TCN-type (MASP-1) L-chainencoding region, which is replaced by an unrelated sequence in the human MASP2 gene (Stover et al., 2001). Thus, the absence of the TCN-type L-chainencoding region in some genes, such as the human MASP2, and human C1r and C1s genes, is explained by the loss of a TCN-type-encoding region during evolution. Interestingly, the lamprey MASP-A gene has no TCN-type L-chain-encoding region. These developments of MASP genes are also supported by phylogenetic evidence that, unlike the MASP2 and C1r/C1s genes, the MASP1/3 gene has an ancient origin that can be traced back at least to the amphioxus (cephalochordate) lineage. The complement system seems to have developed step by step into a higher organized system involving retrotransposition (or partial gene duplication) to generate the MASP1/3 gene, gene duplication to generate the MASP2/C1r/C1s genes, probably before the cartilaginous fish lineage, and by alternative processing of MASP-2 mRNA to produce the truncated form, sMAP.
Lamprey MASP-A/B is the prototype of MASP-2 and C1r/C1s A question arises from the phylogenetic trees as to whether either (or both) lamprey MASP-A or MASP-B is an orthologue of MASP-2, MASP-3 or C1r/C1s. Although these two sequences occupy a position corresponding to a putative lamprey MASP-3, we have not succeeded in cloning a mammalian/amphibian-type MASP-3 from lamprey, which should have an H-chain
common to its MASP-1 counterpart, and lamprey MASP-1 H-chain has a different sequence from MASP-A/B. Therefore, the simple answer is that MASP-A/B arose as the prototype of AGY-type MASP such as MASP-2, C1r and C1s. This assumption is strongly supported by the fact that lamprey C1q associates with MASP-A/B and that the MASP-A gene lacks the TCN-type L-chain-encoding region. If this is the case, lamprey MASP-A/B developed to jawed vertebrate MASP-2/C1r/C1s by gene duplication as shown in Fig. 5.
The primitive complement system in ascidian and lamprey Ascidians (tunicates) occupy a pivotal intermediary position between invertebrates and vertebrates. H. roretzi is the best-studied complement system among non-vertebrate animals. The accumulated evidence indicates that complement originated as a lectin-based opsonization system. As described above, the MBL-like lectin, GBL, was purified as a major protein from the ascidian body fluid. It associates with two ascidian MASPs, and GBL–MASPs complex activates ascidian C3 (Sekine et al., 2001). In addition, we have isolated ascidian ficolins as GlcNAc-binding lectins that have characteristic features of mammalian ficolins (Kenjo et al., 2001). Although it is presently unknown whether these ficolins associate with MASPs and activate complement, these observations indicate that ficolins probably, as well as GBL, act as the recognition molecules of the primitive ascidian complement system in a similar manner to the mammalian lectin pathway. C3 was identified as the main opsonic factor in ascidian plasma (Nonaka et al., 1999), and a C3 receptor was also identified on ascidian hemocytes as the homologue of mammalian complement receptor type 3 or 4 (CR3 or CR4) (Miyazawa et al., 2001; Miyazawa and Nonaka, 2004). Interestingly, antibodies that are specific for GBL, C3 (Sekine et al., 2001) and C3 receptor (Miyazawa et al., 2001) completely inhibited the phagocytosis of yeast in ascidians, which indicates that complement-mediated phagocytosis is a central part of the physiological function of this primitive complement system. Furthermore, yeast treated with purified GBL–MASPs complex and C3 enhanced the phagocytosis by hemocytes (Sekine et al., 2001). These observations strongly suggest that GBL–MASP complex, C3, and its receptor may have developed as the minimal ancestral components of the primordial complement system in the ascidian lineage as shown in Fig. 6 (Fujita, 2002). The classical and lytic pathways of the complement system seem to have emerged at the cartilaginous fish
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Lectin
Bacteria
MASP C3
C3b
C3 receptor
Phagocyte
Fig. 6. Functional model of an ancient lectin-based complement system. The lectin–protease (MASP) complex, C3 and C3 receptor are probably the minimal ancestral components of the primordial complement system which functioned in an opsonic manner and appeared in the ascidian lineage. The complement system of lamprey (the most primitive vertebrate) is also lectinbased and similar to the ascidian system, but C1q subcomponent acts as the recognition molecule of the lamprey complement system. The identification of the soluble regulatory proteins, C4bp-like and factor H-like proteins suggests a more developed and sophisticated complement system exists in lamprey.
stage, coincident with the emergence of adaptive immunity (Nonaka and Miyazawa, 2002). The complement system of lamprey, the most primitive vertebrate, was also thought to lack the classical and lytic pathway, suggesting that lamprey has a lectin-based complement system similar to the ascidian system. However, recently we purified two lectins from lamprey serum using GlcNAc-agarose: one was eluted with mannose, and the other with GlcNAc (Fig. 4), and according to cDNA cloning, the former was identified as lamprey MBL and surprisingly, the latter is the homologue of C1q as described above. Both lamprey MBL and C1q were associated with MASP which exhibits a proteolytic activity against lamprey C3. Therefore, the lamprey complement system consists of at least the lectin–MASP complex and C3. However, identification of lamprey C1q clearly indicates that the classical pathway originates in lamprey stage. Also, the recent identification of soluble regulatory proteins of the complement system such as lamprey C4-bp (Kimura et al., 2004) and factor H (unpublished observation) leads to the prediction that the lamprey complement system may be more sophisticated than the ascidian system. Furthermore, factor Blike sequences have been cloned in horseshoe crab (Zhu et al., 2005), sea urchin (Smith et al., 1998), ascidians (Nonaka et al., 1999) and lamprey (Nonaka and Yoshizaki, 2004), though their functions are not yet clarified. As the alternative pathway was thought to be an ancient mechanism, it is of particular interest to solve the entire molecular architecture of complement system in lamprey.
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Conclusion The identification and functional characterization of the lectin-based activation mechanisms of the complement system have provided new insights into the role of complement in innate immunity, which enables molecular patterns that specifically characterize microorganisms to be detected. In addition, the lectin pathway and the classical pathway are closely related with respect to the structures and functions of the components involved. The classical pathway is activated by binding of C1q followed by activation of C1r and C1s, while the lectin pathway is activated by recognition of carbohydrates on pathogens via MBL and ficolins, associated with MASP. The ascidian complement system, consisting of MBL-like lectin, ficolins, two MASPs, C3 and C3 receptor, functions in an opsonic manner, and it constitutes a primordial complement system. Furthermore, we purified lamprey MBL and C1q, associated with lamprey MASPs, which cleaved C3, suggesting that the lamprey complement system corresponds to both the lectin and classical pathways. From an evolutionary point of view, it is clear that the primitive lectin pathway in innate immunity has evolved into the classical pathway to serve as an effector system of adaptive immunity.
Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and for CREST, Japan Science and Technology Agency.
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Immunobiology 211 (2006) 283–293 www.elsevier.de/imbio
REVIEW
Lectin complement system and pattern recognition Yuichi Endo, Momoe Takahashi, Teizo Fujita Department of Immunology, Fukushima Medical University, Fukushima 960-1295, Japan Received 1 December 2005; accepted 10 January 2006
Abstract Living organisms have strong defense mechanisms against invading microorganisms as survival strategies. One of the defense mechanisms is the complement system, composed of more than 30 serum and cell surface components. This system collaborates in recognition and elimination of pathogens as a part of both the innate and acquired immune systems. The two collagenous lectins, mannose-binding lectin (MBL) and ficolins, are pattern recognition proteins acting in innate immunity and, upon recognition of the pathogens, they trigger the activation of the lectin complement pathway through attached serine proteases (MASPs). A similar lectin-based complement system, consisting of the lectin-protease complex and C3, is present in ascidians, our closest invertebrate relatives and in lamprey, the most primitive vertebrate. Furthermore, a lamprey N-acetylglucosamine (GlcNAc)-binding lectin was identified as the orthlogue of mammalian C1q, and lamprey MASP is suggested as the prototype of MASP-2/C1r/C1s, indicating that the classical complement pathway arose as a part of the innate immune system. Thus, the complement system is one of the most highly organized innate immune systems in invertebrates and jawless vertebrates, and this system has survived in vertebrates with its core components little changed for 600–700 million years. r 2006 Elsevier GmbH. All rights reserved. Keywords: Complement; Ficolin; Lectin; MASP; MBL
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MBL is one of the typical pattern recognition proteins . . . . . . . . . . . . . . . Ascidian MBL-like lectin, glucose-binding lectin (GBL) and lamprey MBL . Ficolins are another recognition molecule of the lectin pathway . . . . . . . . . Lamprey orthologue of mammalian C1q acts as a GlcNAc-binding lectin . . Origin of the MASP family is traced back to an invertebrate, ascidian . . . . Two lineages of the MASP family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lamprey MASP-A/B is the prototype of MASP-2 and C1r/C1s . . . . . . . . . The primitive complement system in ascidian and lamprey. . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author. Fax: +81 24 548 6760.
E-mail address: [email protected] (T. Fujita). 0171-2985/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2006.01.003
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Introduction Immunity is comprised of innate and adaptive defense mechanisms. The innate immune system is an evolutionarily ancient form and crucial for the first line of defense (Hoffmann et al., 1999). Accumulating evidence indicates that adaptive immunity was established at an early stage of jawed vertebrate evolution. In invertebrates, therefore, the innate immune system is the only defense mechanism against infection. Innate immunity was formerly thought to be a non-specific immune response characterized by phagocytosis. However, innate immunity has considerable specificity and is capable of discriminating between pathogens and self as proposed in the concept of pattern recognition molecules of host. These molecules recognize conserved pathogen-associated molecular patterns shared by large groups of microorganisms, thereby successfully defending invertebrates and vertebrates against infection (Medzhitov and Janeway, 2000). The complement system mediates a chain reaction of proteolysis and assembly of protein complexes, playing a major role in body defense as a part of both the innate and adaptive immune systems (Walport, 2001a, b). Complement was first described in the 1890s as a heatlabile protein in serum that ‘complemented’ heat-stable antibodies in the killing of bacteria. Now, the complement system consists of three activation pathways, classical, alternative and lectin pathways, which merge at the proteolytic activation step of C3, the central component of the complement system. C3 is equipped with a unique intra-molecular thioester bond which is exposed to the molecular surface upon activation and forms a covalent bond with invading microorganisms (Law et al., 1980). This covalent tagging of foreign molecules by C3 is considered to be one of the most important functions of the complement system; bound C3 activation products enhance the phagocytosis of pathogens through C3 receptors on phagocytes, and contribute to the activation of the late complement components, C5–C9, which form a cytolytic complex (the so-called membrane attack complex). The classical pathway is activated by antibody–antigen complexes and is a major effector of antibody-mediated immunity. The recently discovered lectin pathway (Fujita, 2002; Matsushita and Fujita, 1996) activates complement following the recognition of microbial carbohydrate patterns by either mannose-binding lectin (MBL) or ficolins, typical fluid phase pattern recognition molecules (Holmskov et al., 2003; Matsushita and Fujita, 2001, 2002), and the subsequent activation of associated unique enzymes, MBL-associated serine proteases (MASPs) (Matsushita et al., 1998; Schwaeble et al., 2002). The alternative pathway is initiated by the covalent binding of a small amount of C3 to hydroxyl or amine groups on cell surface molecules of
microorganisms and does not involve specific recognition molecules. This pathway also functions to amplify C3 activation (amplification loop) (Walport, 2001a, b). Most components of the classical pathway have their structural and functional counterparts in the alternative or lectin pathways, suggesting that gene duplications played an important role in establishing these three pathways. These duplications could be an epoch-making event, which enabled generation of the classical pathway, considered to be the most modern pathway contrary to its name, from the alternative and lectin pathways (Nonaka et al., 1998). Accumulating evidence indicates that the modern complement system seems to have been established by the emergence of jawed vertebrates, and that the complement system of bony and cartilaginous fish has basically the same set of components as the mammalian complement system (Fujita, 2002; Nonaka, 2001; Fujita et al., 2004a). The development of the complement system is illustrated in Fig. 1. In addition, one of the outstanding advances in recent complement research is the discovery of the lectin pathway. In the lectin pathway, MBL and ficolin act as the recognition molecules and activate complement in association with MASPs, a C1r/C1s-like serine protease that is able to cleave the complement components C4, C2 and C3 (Fujita et al., 2004b). Recent biochemical identification of several components of the lectin pathway from a solitary ascidian, Halocynthia roretzi, revealed that the primitive complement system is one of the most highly organized innate immune systems in invertebrates. Furthermore, in lamprey, the most primitive vertebrate (jawless vertebrate) we found that C1q, the component of the classical pathway, has a lectin activity and acts as the pattern recognition molecule (Matsushita et al., 2004). In this review, we focus on typical pattern recognition proteins, MBL and ficolin, and associated serine protease, MASP in ascidian and lamprey, and further we would like to mention the architecture of the primitive complement system.
MBL is one of the typical pattern recognition proteins MBL is a C-type lectin that plays a crucial role in the first line of host defense (Drickamer et al., 1986; Ezekowitz et al., 1988; Kawasaki et al., 1978; Turner, 1996). The importance of this molecule is underlined by a number of clinical studies linking MBL deficiency with increased susceptibility to a variety of infectious diseases (Jack et al., 2001; Neth et al., 2000; Summerfield et al., 1995; Super et al., 1989). MBL is an oligomer of structural subunits, each of which is composed of three identical 32-kDa polypeptide chains. One polypeptide chain contains an N-terminal region rich in cysteine, a
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MBL or MBL-like Ficolin MASP-1 MASP-3 MASP-2 sMAP
Classical Pathway C1r/C1s C1q C3/Factor B
Fig. 1. An evolutionary perspective for the recognition molecules and serine proteases involved in complement activation. The representative species for jawed and jawless vertebrates, deuterostome invertebrates and arthropods are illustrated, with marks for the presence or absence of each component. The components of the lectin pathway such as MBL, ficolin and MASP are all traced back to an invertebrate, ascidian. The components of the classical pathway are seen in cartilaginous fish (shark) and higher vertebrates, although lamprey C1q is identified as a lectin. As illustrated by shark in this figure, acquired immunity was established at an early stage of the evolution of the jawed vertebrates. The origin of both C3 and factor B-like sequences are traced back to an arthropod, horseshoe crab, although their precise function in invertebrates is still unclear. A C3-like sequence was recently isolated from a coral Swiftia exserta (Cnidaria). The open circles denote the absence of component in the corresponding species. Open and closed squares show phylogenetically not typical form of MASP/sMAP and lamprey C1q with different function from mammalian C1q, respectively.
collagen-like domain consisting of tandem repeats of Gly X–Y triplet sequences (where X and Y represent any amino acid), a neck region and a C-terminal region. Through the collagen-like domain, MBL associates with serine proteases, MASPs. Trimerization of the polypeptide chains is achieved through the collagenous triplehelical region, thereby resulting in the formation of one subunit. The N-terminal cysteine-rich region is involved in the covalent interaction between the three polypeptide chains of the subunit and is also responsible for covalent binding of several subunits into the oligomeric structure. In short, three polypeptides fold together to form the structural subunit and 3–6 of these subunits join to form a mature protein, which has an apparent molecular mass of 300–650 kDa. The overall structure of MBL is shown in Fig. 2. Human MBL exists in several oligomeric forms such as trimers, tetramers and pentamers (Dahl et al., 2001). MBL belongs to the collectin family of proteins that consist of a collagen-like domain as mentioned above and a carbohydrate recognition domain (CRD) (Holmskov et al., 1994). Through its CRD, MBL binds carbohydrates with 3- and 4-hydroxyl groups in the pyranose ring in the presence of Ca2+ through the five conserved residues (Glu185, Asn187, Glu193, Asn205
and Asp206, numbering in Rat MBL-A) in the MBL CRD (Drickamer, 1992; Weis et al., 1992). Prominent ligands for MBL are thus D-mannose, N-acetylglucosamine (GlcNAc) and glucose, whereas carbohydrates that do not fit this steric requirement, D-galactose and sialic acid, which usually decorate mammalian glycoproteins, have undetectable affinity for MBL. This steric selectivity of MBL, along with differences in the spatial organization of the ligands, allows for the specific recognition of carbohydrates on pathogenic microorganisms including bacteria, fungi, parasitic protozoans and viruses, and avoids recognition of self (Holmskov et al., 2003). In addition, sequence analysis of CRDs in comparison with monosaccharide specificity revealed that Glu185 and Asn187 (EPN type) are highly conserved in CRDs that bind mannose, GlcNAc and glucose. Galactose-binding CRDs have Gln185 and Asp187 (QPD type) at these critical positions (Weis et al., 1992), and site-directed mutagenesis has shown that mannose-specificity can be changed to galactose-specificity by replacing Glu185 and Asn187 (EPN type) with Gln185 and Asp187 (QPD type) (Drickamer, 1992). In addition to mammalian and chicken MBL, several lectins in bony fish were characterized. The deduced
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MBL Collagen-like domain
Hinge
CRD
Ficolin collagen-like domain
Fibrinogen-like domain
Fig. 2. Domain and oligomeric structure of MBL and ficolin. MBL is an oligomer of structural subunits each composed of three identical 32 kDa polypeptides. Each polypeptide contains an N-terminal cysteine-rich region, a collagen-like domain consisting of tandem repeats of Gly X–Y triplet sequences (where X and Y represent any amino acid), a neck region, and a C-terminal CRD. MBL forms several sizes of oligomers. Three–six trimers are combined to the multimer via the Nterminal disulfide-bondings, and the hexameric form is shown in this figure. Ficolin monomer consists of a short N-terminal region, a middle collagen-like domain with Gly X–Y triplets, and a globular fibrinogen-like domain. The fibrinogen-like domain forms a globular structure similar to a CRD. The multimers are probably assembled by crosslinking via disulfide bridges in the N-terminal region. The terameric form of Lficolin shown here and the hexameric form of H-ficolin are proposed based on electron microscopy in human.
primary structure of these lectins in carp, zebrafish and goldfish indicates selectivity for galactose, having QPDtype CRDs (Vitved et al., 2000). Recently, another carp MBL with specificity for mannose (EPN type) was also purified (M. Nakao et al., manuscript submitted).
Ascidian MBL-like lectin, glucose-binding lectin (GBL) and lamprey MBL Recently, we purified and cloned an MBL-like lectin from a urochordate, the solitary ascidian H. roretzi (Sekine et al., 2001). The purified 36-kDa lectin binds specifically to glucose but not to mannose or GlcNAc and it was designated glucose-binding lectin (GBL). Its cDNA has an open reading frame of 672 bp, which is predicted to encode a 224 amino acid protein including a 17-residue leader peptide. The predicted molecular mass of the protein was 23,716 Da, indicating that the mature
protein may be glycosylated, since there are three Nlinked glycosylation sites. Sequence analysis of GBL reveals that its N-terminal collagen-like domain is replaced by a sequence that has an a-helix structure similar to the configuration of Gly X–Y repeats, while its C-terminal half contains a CRD. The CRD is of the Ctype (EPN type), and contains 13 of the 18 highly conserved amino acid residues (including the four cysteine residues which form the disulfide bonds within the domain (Drickamer, 1988). The five residues involved in the binding of MBL to the 3- and 4hydroxyl groups of the pyranose ring are completely conserved in GBL and MBLs of other species, except for the three bony fish GalBLs (Fig. 3). Since the structural difference between mannose and glucose is at the site of the 2-hydroxyl group of the pyranose ring, it is possible that residues other than the five conserved residues in GBL are responsible for recognizing the 2-hydroxyl group of glucose. The above results raise the possibility that GBL has evolved early as a prototype of MBL. To test this hypothesis, we also purified the lectin associated with MASP in lamprey, one of the most primitive vertebrates. We isolated a 2.3 kb-long cDNA clone with an open reading frame of 840 bp, which is predicted to encode a 279-amino acid protein including a putative leader peptide (52 amino acids). The predicted molecular mass of the mature protein was 23,217 Da, and there is no N-linked glycosylation site. Lamprey MBL shares 30% identity at the amino acid level with human MBL. Sequence analysis revealed that it is homologous to the mammalian MBLs, as it contains a collagen-like sequence in the N-terminal half and a typical EPN-type CRD in the C-terminal half. When lamprey MBL was compared to ascidian GBL in carbohydrate-binding specificity, ascidian GBL only bound to glucose, but lamprey MBL bound to mannose, GlcNAc and glucose, like mammalian MBLs. Therefore, in conjunction with the phylogenetic analysis (Fujita et al., 2004b), it seems likely that the lamprey lectin is an orthologue of the mammalian MBL (manuscript submitted) and that during evolution GBL acquired the broad binding specificity for carbohydrates and the collagen structure characteristic of MBL. Furthermore, ascidian GBL and lamprey MBL are associated with MASPs and involved in activation of the lectin pathway.
Ficolins are another recognition molecule of the lectin pathway Ficolins are a group of proteins containing both a collagen-like and fibrinogen-like domain and found in varying tissues. Recent characterization has shown that
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Terminal sugar 5
1 3
Sugars
O
6
4
2
OH
OH
D Ca
Hydrogen bonds
E N
N E
Coordination bonds
CRD
Fig. 3. Carbohydrate pattern recognition by the CRDs of MBL and related molecules. (Left panel) Individual CRD recognizes carbohydrates with 3- and 4-hydroxyl groups in the pyranose ring in the presence of Ca2+ through the five conserved residues (E, N, E, N and D). Such a pattern is present in D-mannose, GlcNAc and glucose, but not in D-galactose and sialic acid, which usually decorate mammalian glycoproteins. (Right panel) The partial sequences of CRDs. In zebrafish, goldfish and carp, the homologues of MBL are reported. Since another carp MBL was identified recently, in this figure we used CaGalBL for the previously reported MBL and CaMBL for newly identified lectin similar to MBL. AsGBL, ascidian GBL; LaMBL, lamprey MBL; HuMBL, human MBL; MuMBL, mouse MBL.
ficolins present in serum are lectins with a common binding specificity for GlcNAc (Matsushita and Fujita, 2002). Investigations of two types of human serum ficolins, L-ficolin and H-ficolin (Hakata antigen), revealed that they are associated with MASPs and sMAP, and activate the lectin pathway (Matsushita et al., 2001, 2002). Recently, we reported that L-ficolin binds to lipoteichoic acid, a cell component found in all Gram-positive bacteria, and activate the lectin pathway (Lynch et al., 2004). Also, we reported the characterization of M-ficolin, a non-serum lectin, which is secreted from leukocytes. M-ficolin, which can associate with MASPs, activates the lectin pathway and specifically binds to Staphylococcus aureus (Liu et al., 2005). These findings indicate that ficolins act as pattern recognition molecules of the lectin pathway and thus may significantly contribute to innate immunity. We cloned four ficolin cDNAs from the solitary ascidian, H. roretzi (Kenjo et al., 2001), termed ascidian ficolin1–4 (AsFCN1–4). The deduced amino acid sequences of these ficolins revealed the conserved collagen- and fibrinogen-like domains. The fibrinogenlike domains of AsFCNs show about 50% identity with mammalian ficolins. Two types of GlcNAc-binding lectins are purified from the ascidian body fluids and cloned from hepatopancreas cDNA (AsFCN1/2 and AsFCN3). In addition to these ficolins, cDNAs encoding another ficolin has been cloned from the hepatopancreas (AsFCN4). It is of particular interest to note that when compared with mammalian ficolins, the four ascidian ficolins contain short collagen-like domains with 5 and 7 Gly X–Y repeats and also they have long segments between the collagen-like domain and the fibrinogen-like domain. Although it is not yet clear whether these ficolins are associated with MASPs, we postulate that they may act as the recognition molecule of the lectin pathway.
In lamprey, we failed to find the homologue of ficolin as GlcNAc-binding lectin. However, instead of ficolin, we isolated C1q that has a lectin activity and is associated with the prototype of MASP/C1r/C1s-like serine protease (Matsushita et al., 2004).
Lamprey orthologue of mammalian C1q acts as a GlcNAc-binding lectin Serum from the Lampetra japonica lamprey was subjected to GlcNAc-agarose column chromatography in the presence of Ca2+. The column was sequentially eluted with mannose and with GlcNAc, as shown in Fig. 4. Human MBL can be eluted with mannose, and Lficolin, one of the serum ficolins, can be subsequently eluted with GlcNAc (Matsushita and Fujita, 2002). Both of these lectins are found to complex with MASPs. MASP-like protease activity was monitored by esterolytic activity against Boc–Leu–Ser–Thr–Arg–methylcoumarylamide. Lamprey lectin eluted with mannose from GlcNAc-agarose was identified as MBL. Eluates obtained with GlcNAc were further purified by Mono Q chromatography. Lamprey GlcNAc-binding lectin was an oligomer consisting of 24 kDa subunits. The deduced amino acid sequence of this lectin cDNA revealed that it consists of a collagen-like domain and antibody recognition domain, gC1q domain found in a variety of proteins including mammalian C1q. A phylogenetic tree of the gC1q domains of proteins shows that this lectin and mammalian C1q form a tight cluster (Matsushita et al., 2004). We termed this lectin lamprey C1q (LC1q). LC1q co-purified with MASP-A, MASP-B and MASP-1, serine proteases of the MASP family (unpublished observations), and MASP-A exhibited proteolytic activity against lamprey C3 (Matsushita et al., 2004). These
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kDa
GlcNAc elution
Mannose elution
100 75 50 37 25 LaMBL LC1q
15
Fig. 4. SDS-PAGE analysis of lamprey lectins. Lamprey serum was applied to GlcNAc-agarose, and eluted with mannose and subsequently with GlcNAc. The eluted materials were analyzed by SDS-PAGE under reducing conditions (12% gel). Proteins were stained with Coomassie Brilliant Blue R-250. Molecular size markers are indicated on the sides. Lamprey MBL (LaMBL) and C1q (LC1q) are also indicated.
results suggest that C1q may have emerged as a lectin and may have functioned as an initial recognition molecule of the complement system in innate immunity prior to the establishment of adaptive immunity such as immunoglobulins in the cartilaginous fish.
Origin of the MASP family is traced back to an invertebrate, ascidian MASP is a key enzyme of the lectin complement pathway. MASP proteins and/or cDNAs have been isolated from representative vertebrate species including human, mouse, chicken, Xenopus, carp, shark and lamprey, and from two species of invertebrates, amphioxus (cephalochordates) and ascidian (urochordates) (Endo et al., 2003, 1998; Ji et al., 1997) (Fig. 1). Ascidian H. roretzi has two MASPs, designated as MASPa and MASPb, which are the most ancient sequences of MASP identified so far. Amphioxus is one of the most highly organized invertebrates and the closest relative of vertebrates, which occupies a critical position between lamprey and ascidian in phylogeny. Amphioxus Branchiostoma belcheri has at least two MASPs, termed amphioxus MASP-1 and MASP-3, which are the orthologues of jawed vertebrate MASP-1 and MASP3, respectively (Endo et al., 2003). Lamprey L. japonica (cyclostome), which is one of the most primitive vertebrates (jawless vertebrate) and lacks acquired immunity, has at least three kinds of MASP, termed lamprey MASP-1, MASP-A and MASP-B (Endo et al., 2003). As mentioned above, we isolated the orthologues of mammalian MBL and C1q from lamprey plasma. Lamprey MBL and C1q associates with all three types of lamprey MASPs, and the lectin–MASP complexes cleave lamprey C3 into C3b, indicating that the lamprey
complement system is similar to both the classical and lectin pathways in mammals. Mammals including human and mouse, and amphibia Xenopus laevis have three types of MASP, MASP-1, MASP-2 and MASP-3, suggesting a closely similar system of the lectin pathway in these species (Endo et al., 2003). MASP-3 generates from the MASP1/3 gene by alternative splicing and therefore shares the common heavy (H)-chain with the MASP-1 counterpart (Dahl et al., 2001). The mammalian MASP2 gene generates two protein products, MASP-2 and its truncated form with no proteolytic activity, termed as sMAP or MAp19, by alternative splicing (Stover et al., 1999; Takahashi et al., 1999). Recently, we demonstrated that sMAP competes with MASP in binding to MBL and ficolin, and therefore suppressed complement activation, suggesting its regulatory role in the lectin complement pathway (Iwaki and Fujita, 2005). Thus, in human and mouse, six molecules of the MASP family are present, MASP-1, MASP-2, MASP-3, sMAP, C1r and C1s, showing the sophisticated systems of the lectin and classical pathways for complement activation. Interestingly, we identified a C1r/C1slike sequence in shark, probably confirming the cartilaginous fish origin of adaptive immunity. A truncated form of MASP was also identified in carp, although it is large in molecular size and its function is unknown. sMAP-like sequences have not been identified so far in shark, lamprey or invertebrates. To date, a MASP-like molecule has not been identified in lower invertebrates such as sea urchin.
Two lineages of the MASP family Based on the primary structures, exon organization of the genes and usage of codon for the active center serine,
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we identified two lineages in the MASP family (Endo et al., 2003, 1997). The TCN-type, including MASP-1 and ascidian MASPa and MASPb, has a histidine-loop disulfide bridge in the protease domain, a TCN codon at the active site serine, and split exons for the protease domain. The AGY-type, including MASP-2, MASP-3, lamprey MASP-A/B and C1r/C1s, is characterized by the absence of the histidine-loop disulfide bridge, an AGY codon at the active site serine and a single exon for the protease domain. The histidine-loop disulfide bridge, a disulfide bond surrounding the active center histidine, Heavy ch ain A
CUB
EGF CUB
is seen in all serine proteases. The only exception is the AGY-type MASP/C1r/C1s. The precise function of this secondary structure is still unknown. All of the amphioxus/vertebrate MASP/C1r/C1s genes have an intron-less exon encoding an AGY-type L-chain, which is located downstream of the H-chainencoding region (Fig. 5). In contrast, two ascidian MASP genes have no such exon in their corresponding regions. This difference suggests that a processed intronless region might have been inserted between the regions encoding the H-chain and TCN-type L-chain of a
Light chain CCP
Protease
C Chordates
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sMAP or Map19 B 48.4 90.6 60.7
46.9
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1
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asMASPb Inverte asMASPa -brate amMASP-3 amMASP-1 MASP laMASP-1 shMASP-1 MASP-1 xeMASP-1 muMASP-1 huMASP-1 laMASP-B laMASP-A caMASP-3 shMASP-3 chMASP-3 xeMASP-3b 100 MASP-3 xeMASP-3a muMASP-3 100 huMASP-3 caMASP-2 chMASP-2 MASP-2 xeMASP-2 muMASP-2 huM ASP-2 huC1r huC1s caC1rsA C1r/C1s caC1rsB shC1rs
1
asMASPa/b Cephalochordates
TCN type 2 3
MASP-3 MASP-1 Lamprey
AGY type
MASP-A/B Cartilaginous fish (shark)
MASP-2/C1r/C1s Adaptive Immunity
Fig. 5. Structure and phylogeny of the MASP gene family. (A) The domain structure of MASP/C1r/C1s. MASP-1, MASP-2, MASP-3, subcomplements of component 1, C1r and C1s consist of six domains: two C1r/C1s/Uegf/bone morphogenetic protein 1 (CUB) domains, an epidermal growth factor (EGF)-like domain, two complement control protein (CCP) domains or short consensus repeats (SCRs), and a serine protease domain. Histidine (H), aspartic acid (D) and serine (S) residues are essential for formation of the active center of the serine proteases. The pro-enzyme form of an MASP/C1r/C1s is activated by proteolytic cleavage between the second CCP and protease domains, resulting in the two peptides linked by a disulfide bond, heavy (H) and light (L) chains. (B) Phylogenetic tree of the MASP family. Twenty-eight members of this family, which includes our recently identified shark MASP-1 and C1r/s-like sequences (manuscript in preparation), were aligned by Clustal W using L-chain sequences. The tree was constructed by the neighbor-joining method. Numbers on branches are bootstrap percentages. hu, human; mu, mouse; ch, chicken; xe, Xenopus; ca, carp; sh, shark; la, lamprey; am, amphioxus; as, ascidian. The tree suggests that this family has evolved to generate MASP-1, MASP-2, MASP-3, C1r/C1s and primitive invertebrate MASP. The critical branching points are marked by arrow heads with numbers: 1, generation of the MASP1/3 gene; 2, branching of C1r/C1s; 3, branching of MASP-2. (C) A model of the evolution of the MASP/C1r/C1s family. H in box represents the genomic region encoding the H-chain, and L/T and L/A the genomic regions encoding TCN-type and AGY-type L-chains, respectively. Prior to the emergence of cephalochordates, retroposition of partially processed TCN-type MASP gene and base changes from TCN to AGY at the active site serine would generate a prototype of the MASP-1/3 gene. All of the amphioxus/vertebrate MASP/C1r/C1s genes evolved from this ancestor by gene duplications. The arrowheads with numbers show the evolutionary events corresponding to the branching in the tree shown in B.
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prototype gene as a first step of evolution of the MASP family gene. The insertion of the intron-less region might have occurred after the divergence of urochordates (ascidian), but before the divergence of cephalochordates (amphioxus), since amphioxus MASP gene has such a structure. From the structures of two amphioxus MASP, it can be speculated that an ancestral sequence inserted had the structural features of a TCNtype L-chain, and that the change from the TCN-type to the AGY-type occurred in at least two steps, the base change from TCN to AGY and the loss of an intron in the early step and the loss of a histidine-loop disulfide bridge in the late step. In short, the amphioxus MASP gene, evolved from ascidian gene, producing the orthologues of MASP-1 and MASP-3 in the vertebrate lineage. It is likely that all of the vertebrate MASP/C1r/C1s genes evolved from an ancestral amphioxus MASP1/3type gene-by-gene duplication as a second step, since all of the amphioxus/vertebrate MASP/C1r/C1s genes, MASP2 gene, C1r and C1s genes, have an intron-less exon encoding an AGY-type L-chain in common and these genes lack the TCN-type (MASP-1) L-chainencoding region, which is replaced by an unrelated sequence in the human MASP2 gene (Stover et al., 2001). Thus, the absence of the TCN-type L-chainencoding region in some genes, such as the human MASP2, and human C1r and C1s genes, is explained by the loss of a TCN-type-encoding region during evolution. Interestingly, the lamprey MASP-A gene has no TCN-type L-chain-encoding region. These developments of MASP genes are also supported by phylogenetic evidence that, unlike the MASP2 and C1r/C1s genes, the MASP1/3 gene has an ancient origin that can be traced back at least to the amphioxus (cephalochordate) lineage. The complement system seems to have developed step by step into a higher organized system involving retrotransposition (or partial gene duplication) to generate the MASP1/3 gene, gene duplication to generate the MASP2/C1r/C1s genes, probably before the cartilaginous fish lineage, and by alternative processing of MASP-2 mRNA to produce the truncated form, sMAP.
Lamprey MASP-A/B is the prototype of MASP-2 and C1r/C1s A question arises from the phylogenetic trees as to whether either (or both) lamprey MASP-A or MASP-B is an orthologue of MASP-2, MASP-3 or C1r/C1s. Although these two sequences occupy a position corresponding to a putative lamprey MASP-3, we have not succeeded in cloning a mammalian/amphibian-type MASP-3 from lamprey, which should have an H-chain
common to its MASP-1 counterpart, and lamprey MASP-1 H-chain has a different sequence from MASP-A/B. Therefore, the simple answer is that MASP-A/B arose as the prototype of AGY-type MASP such as MASP-2, C1r and C1s. This assumption is strongly supported by the fact that lamprey C1q associates with MASP-A/B and that the MASP-A gene lacks the TCN-type L-chain-encoding region. If this is the case, lamprey MASP-A/B developed to jawed vertebrate MASP-2/C1r/C1s by gene duplication as shown in Fig. 5.
The primitive complement system in ascidian and lamprey Ascidians (tunicates) occupy a pivotal intermediary position between invertebrates and vertebrates. H. roretzi is the best-studied complement system among non-vertebrate animals. The accumulated evidence indicates that complement originated as a lectin-based opsonization system. As described above, the MBL-like lectin, GBL, was purified as a major protein from the ascidian body fluid. It associates with two ascidian MASPs, and GBL–MASPs complex activates ascidian C3 (Sekine et al., 2001). In addition, we have isolated ascidian ficolins as GlcNAc-binding lectins that have characteristic features of mammalian ficolins (Kenjo et al., 2001). Although it is presently unknown whether these ficolins associate with MASPs and activate complement, these observations indicate that ficolins probably, as well as GBL, act as the recognition molecules of the primitive ascidian complement system in a similar manner to the mammalian lectin pathway. C3 was identified as the main opsonic factor in ascidian plasma (Nonaka et al., 1999), and a C3 receptor was also identified on ascidian hemocytes as the homologue of mammalian complement receptor type 3 or 4 (CR3 or CR4) (Miyazawa et al., 2001; Miyazawa and Nonaka, 2004). Interestingly, antibodies that are specific for GBL, C3 (Sekine et al., 2001) and C3 receptor (Miyazawa et al., 2001) completely inhibited the phagocytosis of yeast in ascidians, which indicates that complement-mediated phagocytosis is a central part of the physiological function of this primitive complement system. Furthermore, yeast treated with purified GBL–MASPs complex and C3 enhanced the phagocytosis by hemocytes (Sekine et al., 2001). These observations strongly suggest that GBL–MASP complex, C3, and its receptor may have developed as the minimal ancestral components of the primordial complement system in the ascidian lineage as shown in Fig. 6 (Fujita, 2002). The classical and lytic pathways of the complement system seem to have emerged at the cartilaginous fish
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Lectin
Bacteria
MASP C3
C3b
C3 receptor
Phagocyte
Fig. 6. Functional model of an ancient lectin-based complement system. The lectin–protease (MASP) complex, C3 and C3 receptor are probably the minimal ancestral components of the primordial complement system which functioned in an opsonic manner and appeared in the ascidian lineage. The complement system of lamprey (the most primitive vertebrate) is also lectinbased and similar to the ascidian system, but C1q subcomponent acts as the recognition molecule of the lamprey complement system. The identification of the soluble regulatory proteins, C4bp-like and factor H-like proteins suggests a more developed and sophisticated complement system exists in lamprey.
stage, coincident with the emergence of adaptive immunity (Nonaka and Miyazawa, 2002). The complement system of lamprey, the most primitive vertebrate, was also thought to lack the classical and lytic pathway, suggesting that lamprey has a lectin-based complement system similar to the ascidian system. However, recently we purified two lectins from lamprey serum using GlcNAc-agarose: one was eluted with mannose, and the other with GlcNAc (Fig. 4), and according to cDNA cloning, the former was identified as lamprey MBL and surprisingly, the latter is the homologue of C1q as described above. Both lamprey MBL and C1q were associated with MASP which exhibits a proteolytic activity against lamprey C3. Therefore, the lamprey complement system consists of at least the lectin–MASP complex and C3. However, identification of lamprey C1q clearly indicates that the classical pathway originates in lamprey stage. Also, the recent identification of soluble regulatory proteins of the complement system such as lamprey C4-bp (Kimura et al., 2004) and factor H (unpublished observation) leads to the prediction that the lamprey complement system may be more sophisticated than the ascidian system. Furthermore, factor Blike sequences have been cloned in horseshoe crab (Zhu et al., 2005), sea urchin (Smith et al., 1998), ascidians (Nonaka et al., 1999) and lamprey (Nonaka and Yoshizaki, 2004), though their functions are not yet clarified. As the alternative pathway was thought to be an ancient mechanism, it is of particular interest to solve the entire molecular architecture of complement system in lamprey.
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Conclusion The identification and functional characterization of the lectin-based activation mechanisms of the complement system have provided new insights into the role of complement in innate immunity, which enables molecular patterns that specifically characterize microorganisms to be detected. In addition, the lectin pathway and the classical pathway are closely related with respect to the structures and functions of the components involved. The classical pathway is activated by binding of C1q followed by activation of C1r and C1s, while the lectin pathway is activated by recognition of carbohydrates on pathogens via MBL and ficolins, associated with MASP. The ascidian complement system, consisting of MBL-like lectin, ficolins, two MASPs, C3 and C3 receptor, functions in an opsonic manner, and it constitutes a primordial complement system. Furthermore, we purified lamprey MBL and C1q, associated with lamprey MASPs, which cleaved C3, suggesting that the lamprey complement system corresponds to both the lectin and classical pathways. From an evolutionary point of view, it is clear that the primitive lectin pathway in innate immunity has evolved into the classical pathway to serve as an effector system of adaptive immunity.
Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and for CREST, Japan Science and Technology Agency.
References Dahl, M.R., Thiel, S., Matsushita, M., Fujita, T., Willis, A.C., Christensen, T., Vorup-Jensen, T., Jensenius, J.C., 2001. MASP-3 and its association with distinct complexes of the mannan-binding lectin complement activation pathway. Immunity 15, 127–135. Drickamer, K., 1988. Two distinct classes of carbohydraterecognition domains in animal lectins. J. Biol. Chem. 263, 9557–9560. Drickamer, K., 1992. Engineering galactose-binding activity into a C-type mannose-binding protein. Nature 360, 183–186. Drickamer, K., Dordal, M.S., Reynolds, L., 1986. Mannosebinding proteins isolated from rat liver contain carbohydrate-recognition domains linked to collagenous tails. Complete primary structures and homology with pulmonary surfactant apoprotein. J. Biol. Chem. 261, 6878–6887. Endo, Y., Takahashi, M., Nakao, M., Saiga, H., Sekine, H., Matsushita, M., Nonaka, M., Fujita, T., 1998. Two
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