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The Journal of Immunology, 2006, 176: 4861-4868.
Copyright © 2006 by The American Association of Immunologists

Cloning and Characterization of Mannose-Binding Lectin from Lamprey (Agnathans)1,2

Momoe Takahashi*, Daisuke Iwaki*, Akiko Matsushita*, Munehiro Nakata{dagger}, Misao Matsushita{dagger}, Yuichi Endo* and Teizo Fujita3,*

* Department of Immunology, Fukushima Medical University, Fukushima, Japan, and {dagger} Institute of Glycotechnology and Department of Applied Biochemistry, Tokai University, Hiratsuka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The recognition of pathogens is mediated by a set of pattern recognition molecules that recognize conserved pathogen-associated molecular patterns shared by broad classes of microorganisms. Mannose-binding lectin (MBL) is one of the pattern recognition molecules and activates complement in association with MBL-associated serine protease (MASP) via the lectin pathway. Recently, an MBL-like lectin was isolated from the plasma of a urochordate, the solitary ascidian. This ascidian lectin has a carbohydrate recognition domain, but the collagen-like domain was replaced by another sequence. To elucidate the origin of MBLs, the aim of this study is to determine the structure and function of the MBL homolog in lamprey, the most primitive vertebrate. Using an N-acetylglucosamine (GlcNAc)-agarose column, MBL-like lectin (p25) was isolated from lamprey serum and cDNA cloning was conducted. From the deduced amino acid sequence this lectin has a collagenous region and a typical carbohydrate recognition domain. This lectin also binds mannose, glucose, and GlcNAc, but not galactose, indicating that it is structurally and functionally similar to the mammalian MBLs. Furthermore, it associated with lamprey MASPs, and the MBL-MASP activated lamprey C3 in fluid-phase and on the surface of pathogens. In conjunction with the phylogenetic analysis, it seems likely that the lamprey MBL is an ortholog of the mammalian MBL. Because acquired immunity seems to have been established only from jawed vertebrates onward, the lectin complement pathway in lamprey, as one of the major contributors to innate immunity, plays a pivotal role in defending the body against microorganisms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunity to infection is mediated by two general systems, acquired (or adaptive) and innate (or natural). Innate immunity was formerly thought to be a nonspecific immune response characterized by phagocytosis. However, innate immunity has considerable specificity and is capable of discriminating between pathogens and self (1). The complement system that consists of three activation pathways is engaged in both acquired and innate immunity (2, 3). The classical pathway is activated by Ab-Ag complexes and is a major effector of the acquired immune system that arose in the jawed vertebrate lineage. The other two, the lectin and alternative pathways function in innate immune defense, which is considered to be successful in defending invertebrates against microbial infections (4, 5). The lectin pathway involves carbohydrate recognition by mannose-binding lectin (MBL)4 and ficolins, which are typical pattern recognition molecules, and the subsequent activation of associated unique enzymes, MBL-associated serine proteases (MASPs). 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.

MBL is a C-type lectin that plays a crucial role in the first line of host defense (6). 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 (7). MBL belongs to the collectin family of proteins (8, 9). Human MBL has an apparent molecular mass of ~300–650 kDa and consists of 9–18 monomeric subunits of ~32 kDa each. Each subunit contains an N-terminal region rich in cysteine, a collagen-like domain consisting of 19 tandem repeats of Gly-X-Y triplet sequences (where X and Y represent any amino acid), and a C-terminal carbohydrate recognition domain (CRD). Through its CRD, MBL binds carbohydrates with 3- and 4-hydroxyl groups in the pyranose ring in the presence of Ca2+ (10, 11). Prominent ligands for MBL are thus mannose and N-acetylglucosamine (GlcNAc), whereas carbohydrates that do not fit this steric requirement 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 (9).

Accumulating evidence indicates that adaptive immunity was established at an early stage in the evolution of the jawed vertebrates. The complement system has a more ancient origin, and all major invertebrate deuterostome groups so far studied, sea urchin, ascidians and amphioxus, as well as jawless vertebrates such as lamprey and hagfish have this system (4, 12, 13). Because these animals are believed to have diverged before the emergence of jawed vertebrates, their complement systems are expected to be simpler than those of higher vertebrates. Recent biochemical identification of several components of the lectin pathway from solitary ascidian, Halocynthia roretzi, revealed that the primitive complement system consisting of lectin-MASP complex, C3 and C3 receptor, functions in an opsonic manner (14, 15, 16, 17, 18). An MBL-like lectin and ficolins were identified as the recognition molecules of the lectin pathway in ascidians. The purified MBL-like lectin binds specifically to glucose and was designated glucose-binding lectin (GBL). Sequence analysis of GBL reveals that the C-terminal half contains a CRD that is homologous to C-type lectins, but the collagen-like domain was replaced by another sequence (17). These results raise the possibility that GBL evolved early as a prototype of MBL. To test this hypothesis, we focused on lamprey (agnathans), one of the most primitive vertebrates lacking an adaptive immune system. In this study, we describe an ortholog of mammalian MBL with a collagenous region and a typical CRD in lamprey. This lamprey MBL is associated with serine proteases of the MASP family. When lamprey MBL recognizes yeast as the pathogens, the MBL-MASPs activate C3, a key component of the complement system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Purification of lamprey MBL and C3

Lampreys, Lampetra japonica, were obtained from local dealers in Fukushima, Japan. Serum from lampreys was applied to GlcNAc-agarose (Sigma-Aldrich) equilibrated with Tris buffer (50 mM Tris (pH 7.8), 200 mM NaCl, and 20 mM CaCl2). After the column had been washed with starting buffer, elution was conducted with 0.3 M mannose-containing buffer. The eluted fractions were dialyzed against 25 mM Tris-HCl (pH 7.8), containing 50 mM NaCl and 5 mM CaCl2, and then chromatographed on a Mono Q column (Amersham Biosciences), before being eluted with a linear NaCl gradient to 0.5 M. The preparation was analyzed by SDS-PAGE using the Laemmli system and proteins were stained with Coomassie brilliant blue R-250. Collagenase digestion was conducted by incubating 10 µg of the prepared proteins with collagenase (50 U; Sigma-Aldrich) from Clostridium histolyticum in 50 mM Tris, 150 mM NaCl and 10 mM CaCl2 (pH 7.5) at 37°C for 3 h. Lamprey C3 was purified according to published methods (19) with a modification. Briefly, lamprey serum was precipitated by polyethylene glycol and subjected to ion-exchange chromatography, followed by gel filtration. To examine C3 activation, 8 µg of lamprey C3 was incubated with various amounts of MBL-MASPs in 50 mM Tris, 150 mM NaCl, and 10 mM CaCl2 (pH 7.5) at 37°C for 30 min and the reaction mixture was then subjected to SDS-PAGE.

Preparation of Abs

Monospecific antiserum to lamprey p25 (MBL) or lamprey C3 was raised by immunizing rabbits with purified proteins in CFA. IgG of anti-C3 Ab was isolated using protein A-Sepharose. To remove the natural Abs to yeast for FACS analysis, both Abs were absorbed with an excess amount of yeast. The Abs to MASPs were also prepared by immunizing rabbits with synthetic peptides of MASP-A H chain, and recombinant L chain peptides of MASP-B and MASP-1.

Amino acid sequence analysis

The N-terminal amino acid of lamprey p25 was blocked. Therefore, its internal amino acid sequence was determined. After lamprey p25 was digested with collagenase as described, digested products were subjected to SDS-PAGE under reducing conditions and then electroblotted onto polyvinylidene difluoride membranes (Millipore). The protein bands were stained, excised, and then analyzed using a protein sequencer (model 476A; Applied Biosystems). Lamprey p25 was also digested with Staphylococcus aureus V8 protease (Sigma-Aldrich) according to the method in the Cleveland study (20). Digested peptides were electroblotted and analyzed as described.

Cloning of p25 cDNA

The liver and various tissues were removed immediately before use. RNA was isolated from these tissues using the acid guanidine thiocyanate method, and the poly(A)+ fraction was purified by passage through an oligo(dT) cellulose column (Clontech Laboratories). cDNAs were prepared from liver RNA using Superscript II Reverse Transcriptase (Invitrogen Life Technologies). Four degenerated primers were synthesized based on the amino acid sequence that had been determined for the products of lamprey MBL digested with collagenase and on the conserved sequences of the MBL family: AKGEKGE (5'-YYMMDGGRSMRAARGGRGA-3'), WNDVPCS (5'-KDRCARKNRAYRTCRTTCCA-3'), KGDKGDA (5'-AARGGNGAYAARGGNGAYGC-3'), and NWNDGEPN (5'-TTKGGYTCYYHHKYNTTCCARTT-3'). A part of the p25 cDNA was amplified by a nested RT-PCR using lamprey liver cDNA as a template and two primer sets (5'-YYMMDGGRSMRAARGGRGA-3' and 5'-KDRCARKNRAYRTCRTTCCA-3') for the first; (5'-AARGGNGAYAARGGNGAYGC-3' and 5'-TTKGGYTCYYHHKYNTTCCARTT-3') for the second. PCR products of the expected size were cloned into pGEM-T easy vector (Promega) and sequenced by the dideoxy method using an autosequencer (model 4000; LI-COR). The subcloned 321-bp DNA was 32P-labeled and used as a probe for screening a ZAP cDNA library. A total of 2 x 106 plaques of a liver ZAP II cDNA library were screened. Positive clones were subcloned in pBluescript II (SK+) by in vivo excision (Stratagene) and sequenced by the method previously described.

Northern blot hybridization and RT-PCR

A membrane filter blotted with 0.4 µg of poly(A)+ RNA from various tissues of lamprey was hybridized with a 32P-labeled specific cDNA fragment of the lamprey MBL (nucleotides 49–666) at 42°C overnight in 50% formamide, 5x Denhardt’s solution, 5x SSPE (1x SSPE is 9 mM sodium phosphate, 150 mM NaCl, and 1 mM EDTA (pH 7.4)), 0.5% SDS, and 200 µg/ml salmon sperm DNA. After a final washing at 55°C for 40 min in 0.1x SSC (1.5 mM sodium citrate, and 15 mM NaCl (pH 7.0)) containing 0.1% SDS, the filter was exposed to an autoradiogram imaging screen. The image was then read using a bioimaging analyzing system (BAS 2500; Fujifilm). Furthermore, to confirm the results of Northern blot analysis, RT-PCR was performed using poly(A)+ RNA from various tissues as a template, and a primer set was designed to amplify the collagenous portion of the cDNA (230 bp). The PCR product was visualized on an agarose gel by staining with ethidium bromide.

Construction of the phylogenetic tree

The 15 members of the MBL family and 52 members of the collectin family were aligned at the amino acid sequence level using Clustal W software (21). A pairwise distance matrix was obtained by calculating the proportions of different amino acids. The matrix was then used to construct trees by the neighbor-joining method (22). Bootstrap analysis was used to assess the reliability of branching patterns.

Binding of lamprey MBL to carbohydrates

The following oligosaccharides were conjugated to DPPE (dipalmitoylphosphatidylethanolamine) as previously described (17): mannopentaose (Man5) with a structure of Man{alpha}1–6(Man{alpha}1–3)Man{alpha}1–6(Man{alpha}1–3)Man; penta-N-acetyl-chitopentaose (GN5) structured GlcNAcbeta1–4GlcNAcbeta1–4GlcNAcbeta1–4GlcNAcbeta1–4GlcNAc); and lactose (Lac) as Galbeta1–4Glc. The neoglycolipid-including liposomes were prepared by dispersion of a lipid film of consisting of the neoglycolipid and DPPC (dipalmitoylphosphatidylcholine; 1/10, mol/mol) into PBS, followed by passing through a polycarbonate filter (50-nm pore size) with a LiposoFast extruder (Avestin). The liposomes were then adsorbed (~20,000 RU) onto a HPA sensor chip with a BIAcore 3000 instrument. Liposome without neoglycolipids was also adsorbed as a reference. Binding of lamprey MBL to carbohydrate moiety on the surface of the sensor chip was analyzed. All sensorgrams were recorded at a flow rate of 20 µl/min at 25°C in running buffer (10 mM Tris-HCl (pH 7.3), containing 150 mM NaCl and 10 mM CaCl2).

To determine MBL binding to other carbohydrates, lamprey serum (200 µl) was incubated with 40 µl of mannan-agarose, mannose-agarose, GlcNAc-agarose, glucose-agarose, or N-acetylgalactosamine-agarose beads (Sigma-Aldrich) on ice for 1 h in 50 mM Tris, 150 mM NaCl, and 10 mM CaCl2 (pH 7.5). After washing four times with the same buffer, the beads were treated four times with 25 µl of the corresponding carbohydrates (0.3 M) (total 100 µl), and the eluted materials were analyzed by immunoblotting.

Immunoblotting

After SDS-PAGE (10% gel), proteins were transferred from the gel to a polyvinylidene difluoride membrane, and the blot was probed with rabbit Abs against lamprey MBL, MASP-A, MASP-B, and MASP-1. Peroxidase-conjugated anti-rabbit IgG was used as a secondary Ab and the blot was developed with tetramethylbenzidine solution (Wako Chemical).

Flow cytometry

The binding of lamprey MBL and C3 to yeast was analyzed by flow cytometry. Yeast (AH109) was obtained from Clontech Laboratories and the yeast cells (2 x 107) in GVB (veronal buffered saline supplemented with 0.1% gelatin, 2 mM CaCl2 and 0.5 mM MgCl2) were incubated with lamprey serum, GlcNAc-agarose-treated serum, or/and partially purified MBL-MASPs complex as described below, and then washed three times with GVB to detect MBL binding, and with GVB supplemented with 10 mM EDTA and 0.1% gelatin (EDTA-GVB) to detect C3 binding, respectively. The washed cells were then reacted on ice for 30 min with 20 µl of anti-C3 Ab IgG (0.4 mg/ml) or 15 µl of anti-MBL antiserum and stained on ice for 30 min with 20 µl of 500 µg/ml FITC-conjugated swine anti-rabbit IgG (DakoCytomation Japan). The yeast was washed three times with PBS between each reaction. Reactivities were evaluated on a FACScan flow cytometer (BD Biosciences) and compared with controls consisting of bacteria treated with primary Abs and FITC-conjugated swine anti-rabbit IgG.

Preparation of GlcNAc-agarose-treated serum and partially purified MBL-MASPs complex

Lamprey serum (400 µl) was incubated with 80 µl of GlcNAc-agarose beads on ice for 1 h in 50 mM Tris, 150 mM NaCl, and 10 mM CaCl2, (pH 7.5) in a total of 800 µl. After washing four times with the same buffer, the beads were treated four times with 50 ml of the described buffer containing 0.3 M mannose (total 200 µl), and the eluted materials consisting of crude MBL-MASPs complex were massively dialyzed against PBS.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Purification of lamprey MBL-like lectins, p25

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 described in Materials and Methods, because it is reported that human MBL can be eluted with mannose, and L-ficolin, one of human serum ficolins, can be subsequently eluted with GlcNAc (23). Both of these lectins are found to complex with human MASPs (24, 25, 26). As previously reported (27), the lamprey lectin eluted with GlcNAc from GlcNAc-agarose was identified as the lamprey homolog of mammalian C1q, the first component of the classical complement pathway. Eluates obtained with mannose were further purified by Mono Q chromatography. As shown in Fig. 1A, the 25-kDa band (p25) was stained by SDS-PAGE under reducing conditions. Under nonreducing conditions, a ~300-kDa band was observed, suggesting that p25 was composed of subunits linked to form homopolymers via disulfide bonds. Furthermore, to detect the protein having a collagen-like sequence, we performed the collagenase digestion of the fraction from Mono Q, and found that the band of 25 kDa was reduced to ~15 kDa (Fig. 1B), suggesting that the 25-kDa lectin (p25) has a collagen-like sequence. For cDNA cloning of p25, we determined the N-terminal amino acid sequences of the peptides derived from collagenase and V8 protease digestion of the lamprey p25 because its N-terminal amino acid was blocked.


Figure 1
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  		FIGURE 1. Analysis of lamprey lectin, p25. A, SDS-PAGE of p25. Purified p25 (lanes 1) or human MBL (lanes 2) was subjected to SDS-PAGE under reducing (+2-ME; 12% gel) and nonreducing (–2-ME; 5% gel) conditions. Proteins were stained with Coomassie brilliant blue R-250. Reducing molecular marker sizes are indicated to the left. B, Collagenase digestion of p25. Lamprey p25 was incubated in the absence (lane 1) or presence (lane 2) of collagenase, and subjected to SDS-PAGE followed by protein staining.

 
cDNA cloning of p25

Based on the amino acid sequences of the peptides derived from collagenase digestion of the lamprey p25 and on the conserved sequences of the MBL family, we designed degenerated primers and performed nested PCR. A single band of 321 bp, which was amplified, was cloned and then sequenced by the dideoxy method. The deduced amino acid sequence revealed that it contained the amino acid sequence of the collagenase-digested fragment of p25. A liver cDNA library was screened using this PCR product as a probe and a 2.3-kb long cDNA clone with an open reading frame of 840 bp, which is predicted to encode a 279-aa protein, including a putative leader peptide (52 aa), was isolated (Fig. 2). The predicted molecular mass of the mature protein was 23,217 Da, and there is no N-linked glycosylation site. Sequence analysis revealed that it is a homolog of mammalian MBL, as it contains a collagen-like sequence in the N-terminal half and a CRD in the C-terminal half (Fig. 2). Lamprey MBL shared ~30% identity at the amino acid level with human MBL.


Figure 2
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  		FIGURE 2. Alignment of the entire amino acid sequence of lamprey MBL with mammalian MBL. The entire amino acid sequences of lamprey, human, rat, and mouse MBLs were aligned using ClustalW software with reference to the invariant residues of C-type lectins (31 ). Gaps inserted during alignment are indicated by dashes. The double-thick line shows the collagen-like sequences. Asterisks and dots below the sequences indicate the residues are conserved and similar among these six sequences, respectively. The underlined sequences were directly determined by amino acid sequencer and arrows indicate the sites of the designed primers.

 
Northern blotting

Northern blot analysis was performed using several samples of liver, heart, gill, intestine, blood, and brain. As shown in Fig. 3A, the major transcript of lamprey MBL expressed in the liver is ~2.6-kb long and several faint bands were observed. To clarify the nature of these faint signals, we performed RT-PCR using the same tissue samples as a template. A single band 230-bp long, which corresponds to the collagenous portion, was observed in liver, and no band was detected in the other tissues (Fig. 3B).


Figure 3
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  		FIGURE 3. Northern blotting analysis of lamprey MBL. A, a membrane filter containing poly(A)+ RNA (0.4 µg) from liver, heart, gill, intestine, blood, and brain was hybridized with a 32P-labeled cDNA fragment (nucleotides 49–666). B, RT-PCR revealed a 230 bp band only in liver.

 
Phylogenetic tree of MBL and collectins

The phylogenetic tree was constructed based on the amino acid sequences of 15 members of the MBL family including lamprey MBL. As shown in Fig. 4A, lamprey MBL branches first at the root together with the ancestors for mammalian, chicken, and bony fish MBLs, suggesting an ancient origin. Interestingly, four members of bony fish MBLs form a tight cluster, although binding specificity is different among these MBLs. The mammalian MBLs form a cluster that is divided into two subgroups, MBL-A and MBL-C. To further examine the evolutionary origin of MBL, we constructed another tree including the other members of the collectin family. As shown in Fig. 4B, this tree shows clearly distinct groups for MBL, surfactant proteins SP-D, SP-A, and collectins CL-L1, CL-K1, and CL-P1, although the ascidian collectins (Grails) (28) and ascidian GBL form a subgroup separated from the MBL family. In this tree, lamprey MBL forms again a cluster together with the other MBL members, although the bootstrap percentage for its branching is not so high. Based on these results, it is likely that the lamprey MBL is an ortholog of mammalian MBL.


Figure 4
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  		FIGURE 4. Phylogenetic tree of MBL and collectin. The tree was constructed based on the alignments of the sequences of 15 members of the MBL family (A) and 52 members of the collectin family (B). Numbers on branches are bootstrap percentages supporting a given partitioning.

 
Binding specificity of lamprey MBL for carbohydrates

To determine the specificity of lamprey MBL binding to various carbohydrates, we used surface plasmon resonance. As shown in Fig. 5A, MBL bound to oligosaccharides composed of mannose (Man5) and GlcNAc (GN5), but not lactose (Lac), suggesting that MBL does not bind to terminal galactose. To confirm these results and to determine whether lamprey MBL binds to glucose or N-acetylgalactosamine, we performed additional experiments. The lamprey serum was applied to several carbohydrate-conjugated agarose beads, and eluted materials were analyzed by immunoblotting. The lamprey MBL binds to glucose, in addition to mannose and GlcNAc, but not to N-acetylgalactosamine (Fig. 5B).


Figure 5
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  		FIGURE 5. Binding specificity of lamprey MBL for carbohydrates. A, Binding of lamprey MBL to each neoglycolipid was evaluated in terms of resonance units (RU) obtained with surface resonance analysis as described in Materials and Methods. B, In addition, serum was treated with mannan-agarose (lane 1), mannose-agarose (lane 2), GlcNAc-agarose (lane 3), glucose-agarose (lane 4), and N-acetylgalactose-agarose (lane 5). Eluted materials with the corresponding carbohydrates were analyzed by immunoblotting.

 
Complex formation of MBL with MASPs, and activation of C3 by MBL-MASPs

Next, we determined whether the purified MBL fraction contained the lamprey serine proteases MASPs, which had been reported as homologs of mammalian MASPs (29). To identify MASPs in the MBL preparation, Western blotting was performed using Abs against MASP-A, MASP-B, and MASP-1. In the case of MASP-A, the H chain band (65 kDa) was observed, and in the case of MASP-B and MASP-1, the L chain bands (34 and 35 kDa) were observed (Fig. 6A). To confirm that the complex formed, the MBL preparation was subjected to molecular sieve chromatography using Sepharose 6 (Fig. 6B). In the presence of Ca2+, the main peak appeared in the fraction around 700 kDa (10.67 ml), whereas in the presence of EDTA the main peak (10.67 ml) disappeared, and the two peaks were separately recovered in the peaks of 12.85 and 14.47 ml. By SDS-PAGE and Western blotting analyses, the main peak contained both lamprey MBL (p25) and MASPs, and in the presence of EDTA the peaks of 12.85 and 14.47 ml contained MBL and MASPs, respectively (data not shown). These results indicate that lamprey MBL associates with MASPs in the presence of Ca2+.


Figure 6
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  		FIGURE 6. Western blotting and gel filtration of the lamprey MBL preparation. A, Purified lamprey MBL preparation was probed with anti-MASP-A (lane 1), anti-MASP-B (lane 2), anti-MASP-1 (lane 3), and irrelevant Ab (lane 4). B, The MBL preparation was applied to Sepharose 6 in the presence of either 5 mM CaCl2 or 10 mM EDTA and the both eluted profiles are shown with references. The flow rate is 0.5 ml/min and one fraction is 0.5 ml.

 
In the experiments shown in Fig. 7, we examined whether the MBL-MASP complexes activated lamprey C3. The C3 {alpha}-chain was cleaved by MBL-MASPs in a dose-dependent manner, yielding an {alpha}'-chain. As previously reported (27), purified MASP-A cleaved the C3. Although the precise function of each MASP is not known, it is possible that the binding of lamprey MBL-MASPs to carbohydrates on pathogens results in C3 activation. This possibility is examined in the following experiments.


Figure 7
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  		FIGURE 7. Cleavage of lamprey C3 by MBL-MASPs. Lamprey C3 (8 µg) (lane 1) was incubated at 37°C for 30 min with various amounts of MBL-MASPs: 0.03 µl (lane 2), 0.06 µl (lane 3), 0.12 µl (lane 4), 0.25 µl (lane 5), and 0.5 µl (lane 6) in a total amount of 10 µl. Lamprey C3 was then subjected to SDS-PAGE under reducing conditions followed by protein staining. Lamprey C3 consisted of {alpha}-, beta-, and {gamma}-chains, and the {alpha}-chain was cleaved into an {alpha}'-chain.

 
Next, we asked whether lamprey MBL bound to yeast using lamprey serum by flow cytometry. As shown in Fig. 8A, the MBL bound to yeast and its binding was inhibited by mannose. Previously, C3 in lamprey serum was reported to binds to zymozan, a cell wall of yeast, and to act as an opsonin (19), although the recognition molecule involved has not been identified. Because MBL bound to yeast as shown, to clarify the lamprey complement system, C3 binding to yeast was analyzed by flow cytometry. As shown in Fig. 8B, a considerable amount of C3 bound to yeast in the reconstitution experiments, indicating that MBL recognizes mannan of yeast and the associated MASPs cleavage C3 into C3b that is deposited on their surfaces and acts as an opsonin. From these results, it is concluded that MBL is one of the lectins that act as a recognition molecule in the lectin pathway of the lamprey complement system.


Figure 8
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  		FIGURE 8. The binding of lamprey MBL and C3 to yeast. A, Yeast cells (2 x 107) were incubated on ice for 1 h with 5 µl of lamprey serum (plot 1), 10 µl of GlcNAc-agarose-treated serum (plot 2), or 20 µl of partially purified MBL-MASPs complex (plot 3) in a total volume of 80 µl in GVB (thick line histogram). For the inhibition experiments (dashed line histogram), equal volume of 0.3 M mannose was added to each serum or MBL-MASPs complex before incubation with the yeast. The degree of MBL binding to the yeast was detected with anti-MBL as described in Materials and Methods. The controls (shaded histogram) consist of bacteria treated with anti-MBL Ab and FITC-conjugated swine anti-rabbit IgG. B, To detect C3 binding to yeast, 10 µl of lamprey serum (plot 1), 20 µl of GlcNAc-agarose-treated serum (plot 2), or reconstituted serum consisting of 20 µl of GlcNAc-agarose-treated serum and 10 µl of partially purified MBL-MASPs complex (plot 3) in a total volume of 80 µl in GVB (thick line histogram). The degree of C3 binding to the yeast was detected with anti-C3. The controls (shaded histogram) consist of bacteria treated with anti-C3 Ab and FITC-conjugated swine anti-rabbit IgG.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we isolated a novel lectin present in the serum of a lamprey, Lampetra japonicus, by affinity chromatography using GlcNAc-agarose followed by chromatography on Mono Q, and cloned its cDNA from cDNA libraries of the lamprey liver. The deduced amino acid sequence of the lamprey lectin has a major feature of the mammalian MBL family: it contained a collagen-like domain and a CRD. In comparison with the mammalian MBL family, its N-terminal half has 15 Gly-X-Y triplets without gaps, whereas the mammalian MBLs have 18–20 Gly-X-Y triplets that are interrupted to form a bend in the triple helix (8). Its CRD is a C-type lectin, containing 16 of the 18 highly conserved amino acid residues including four cysteine residues that are involved in disulfide bonds within the domain (30, 31). Five residues (Glu184, Asn186, Glu191, Asn205, and Asp206), which have been reported to bind directly to mannose, GlcNAc, and glucose in the presence of Ca2+ (10, 11), are completely conserved among these proteins (Fig. 2).

As mentioned, sequence analysis of CRDs revealed that Glu185 and Asn187 (EPN) sequences are highly conserved among mammalian and bird MBLs. Galactose-binding CRDs have Gln185 and Asp187 (QPD) sequences at these critical positions (10), and site-directed mutagenesis has shown that mannose-specificity can be changed to galactose-specificity by replacing Glu185 and Asn187 (EPN) with Gln185 and Asp187 (QPD) (11). In bony fish (carp, zebrafish and goldfish) several lectins have been characterized and their deduced primary structure indicates selectivity for galactose (QPD). Recently, another carp MBL with specificity for mannose (EPN) was purified (32). Previously, we purified and cloned an MBL-like lectin (GBL) from a urochordate, the solitary ascidian Halocynthia roretzi (17). Sequence analysis of GBL reveals that the C-terminal half of the ascidian lectin contains a CRD that is homologous to a C-type lectin (EPN), but a collagen-like domain was replaced by another sequence that has an {alpha}-helix structure similar to the configuration of Gly-X-Y repeats. In the present study, we purified the lamprey lectin that has a collagenous region and a typical (EPN) CRD. When lamprey MBL was compared with ascidian GBL in carbohydrate binding specificity, ascidian GBL only binds to glucose, but lamprey MBL binds to mannose, GlcNAc, and glucose-like mammalians MBLs. Therefore, it is possible that the ascidian GBL evolved early as a prototype of MBL, and during evolution GBL may have acquired the broad binding specificity for carbohydrates and the collagen structure characteristic of MBL. Thus, considering the phylogenetic analysis, it seems likely that the lamprey lectin is ancestor of the vertebrate MBL.

Another important finding is that lamprey MBL associates with three types of MASP (MASP-1, MASP-A, and MASP-B). These MASPs have been identified as cDNA sequences, but recently, MASP-A was purified at the protein level (27), whereas MASP-2, C1r, and C1s have not been identified (29). MASPs are classified into three types (MASP-1, MASP-2 and MASP-3) based on the codon encoding the serine residue at the active center of the serine protease domain and the gene organization (4). Lamprey MASP-A and MASP-B were classified into the MASP-3 group in a phylogenetic tree (29), although their gene structure did not support their orthology as MASP-3. In human and mouse, MASP-2 is involved in the activation of C4 and C2, similar to what is observed with C1s, whereas we showed that MASP-1 directly cleaves C3 (33). The functions of MASP-3 have not yet been clarified. Among lamprey MASPs complexed with MBL, we demonstrated that MASP-1 is an ortholog of mammalian MASP-1. Like human MASP-1, lamprey MASP-1 may cleave C3 directly. Also, we demonstrated that MASP-B associated with lamprey MBL. As reported previously, lamprey MASP-A that associated with lamprey C1q, activated C3 (27). Although their precise functions are not known, MASP-1 and MASP-B have been identified in this study at the protein level. Thus, MBL forms complexes with three MASPs, which activate C3, showing the presence of the primordial lectin pathway in lamprey.

Previously, we proposed that the primitive complement system consists of a lectin, an associated protease and C3, the key components of the complement system. It appears in an ascidian, our closest invertebrate relative (4, 5, 13), although the origin of the complement system can be traced back to echinoderms and further back to arthropods because C3 and C2/factor B-like sequence have been identified in sea urchin (12, 34) and horseshoe crab (35). In the ascidian complement system, ficolins and MBL-like lectin (GBL) function as the recognition molecules. In lamprey, by contrast, C1q, the recognition molecule of the classical pathway in mammals, has been identified, but it has lectin-like activity and is associated with MASPs that activate C3 (27). In the present study, we cloned and characterized lamprey MBL that is also associated with MASPs. Interestingly, lamprey C1q bound only to GlcNAc, whereas MBL bound to mannose, GlcNAc, and glucose, showing that lamprey MBL has overlapping and distinct specificities for carbohydrates. In addition, we show that the lamprey complement system consists of at least the lectin-MASP complex and C3, similar to the ascidian system. However, the recent identification of soluble regulatory proteins of the complement system such as lamprey C4-bp (36) and factor H (A. Matsushita and T. Fujita, our unpublished observation) leads to the prediction that the lamprey complement system may be more sophisticated than the ascidian system. Furthermore, C2/factor B-like sequences have been cloned in horseshoe crab, sea urchin, ascidians, and lamprey, though their functions are yet to be 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 and this is currently under investigation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant-in-aid for Scientific Research on Priority Area from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by Core Research for Evolutional Science and Technology, Japan Science and Technology Agency. Back

2 The nucleotide sequence reported in this paper has been deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases under the accession number AB195797. Back

3 Address correspondence and reprint requests to Dr. Teizo Fujita, Department of Immunology, Fukushima Medical University, 1 Hikariga-oka, Fukushima 960-1295, Japan. E-mail address: tfujita{at}fmu.ac.jp Back

4 Abbreviations used in this paper: MBL, mannose-binding lectin; MASP, MBL-associated serine protease; GBL, glucose-binding lectin; CRD, carbohydrate recognition domain; GlcNAc, N-acetylglucosamine. Back

Received for publication June 17, 2005. Accepted for publication January 26, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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M. Nakao, T. Kajiya, Y. Sato, T. Somamoto, Y. Kato-Unoki, M. Matsushita, M. Nakata, T. Fujita, and T. Yano
Lectin Pathway of Bony Fish Complement: Identification of Two Homologs of the Mannose-Binding Lectin Associated with MASP2 in the Common Carp (Cyprinus carpio)
J. Immunol., October 15, 2006; 177(8): 5471 - 5479.
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