Cloning and Characterization of
Mannose-Binding Lectin from Lamprey
(Agnathans)
This information is current as
of June 15, 2017.
Momoe Takahashi, Daisuke Iwaki, Akiko Matsushita,
Munehiro Nakata, Misao Matsushita, Yuichi Endo and Teizo
Fujita
J Immunol 2006; 176:4861-4868; ;
doi: 10.4049/jimmunol.176.8.4861
http://www.jimmunol.org/content/176/8/4861
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References
The Journal of Immunology
Cloning and Characterization of Mannose-Binding Lectin from
Lamprey (Agnathans)1,2
Momoe Takahashi,* Daisuke Iwaki,* Akiko Matsushita,* Munehiro Nakata,†
Misao Matsushita,† Yuichi Endo,* and Teizo Fujita3*
I
mmunity 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-asso-
*Department of Immunology, Fukushima Medical University, Fukushima, Japan, and
†
Institute of Glycotechnology and Department of Applied Biochemistry, Tokai University, Hiratsuka, Japan
Received for publication June 17, 2005. Accepted for publication January 26, 2006.
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.
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.
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: [email protected]
4
Abbreviations used in this paper: MBL, mannose-binding lectin; MASP, MBLassociated serine protease; GBL, glucose-binding lectin; CRD, carbohydrate recognition domain; GlcNAc, N-acetylglucosamine.
Copyright © 2006 by The American Association of Immunologists, Inc.
ciated 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
0022-1767/06/$02.00
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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. The Journal of Immunology, 2006, 176: 4861– 4868.
4862
CHARACTERIZATION OF LAMPREY MANNOSE-BINDING LECTIN
ascidian, Halocynthia roretzi, revealed that the primitive complement system consisting of lectin-MASP complex, C3 and C3 receptor, functions in an opsonic manner (14 –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.
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 ␮g 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 ␮g 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
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.
of the MBL family: AKGEKGE (5⬘-YYMMDGGRSMRAARGGRGA3⬘), WNDVPCS (5⬘-KDRCARKNRAYRTCRTTCCA-3⬘), KGDKGDA
(5⬘-AARGGNGAYAARGGNGAYGC-3⬘), and NWNDGEPN (5⬘-TTKG
GYTCYYHHKYNTTCCARTT-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⬘-KDRCAR
KNRAYRTCRTTCCA-3⬘) for the first; (5⬘-AARGGNGAYAARGGN
GAYGC-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 32Plabeled and used as a probe for screening a ZAP cDNA library. A total of
2 ⫻ 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 ␮g 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, 5⫻ Denhardt’s solution, 5⫻ SSPE (1⫻ SSPE is 9 mM sodium
phosphate, 150 mM NaCl, and 1 mM EDTA (pH 7.4)), 0.5% SDS, and 200
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Materials and Methods
The Journal of Immunology
4863
␮g/ml salmon sperm DNA. After a final washing at 55°C for 40 min in
0.1⫻ 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.
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
␮l) was incubated with 40 ␮l 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 ␮l of the corresponding carbohydrates (0.3
M) (total 100 ␮l), 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. Peroxidaseconjugated anti-rabbit IgG was used as a secondary Ab and the blot was
developed with tetramethylbenzidine solution (Wako Chemical).
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␣1– 6(Man␣1–3)Man␣1– 6(Man␣1–
3)Man; penta-N-acetyl-chitopentaose (GN5) structured GlcNAc␤1–
4GlcNAc␤1– 4GlcNAc␤1– 4GlcNAc␤1– 4GlcNAc); and lactose (Lac) as
Gal␤1– 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 ␮l/min at 25°C in running
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 ⫻ 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 MBLMASPs 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 ␮l of anti-C3
Ab IgG (0.4 mg/ml) or 15 ␮l of anti-MBL antiserum and stained on ice for
30 min with 20 ␮l of 500 ␮g/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
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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.
4864
CHARACTERIZATION OF LAMPREY MANNOSE-BINDING LECTIN
protease digestion of the lamprey p25 because its N-terminal
amino acid was blocked.
cDNA cloning of p25
Northern blotting
FIGURE 3. Northern blotting analysis of lamprey MBL. A, a membrane
filter containing poly(A)⫹ RNA (0.4 ␮g) 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.
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 ␮l) was incubated with 80 ␮l 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 ␮l. 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 ␮l), and the eluted materials consisting of crude
MBL-MASPs complex were massively dialyzed against PBS.
Results
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 –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
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).
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.
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 Nacetylgalactosamine, 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).
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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 collagenlike 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.
The Journal of Immunology
4865
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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.
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⫹.
In the experiments shown in Fig. 7, we examined whether the
MBL-MASP complexes activated lamprey C3. The C3 ␣-chain
was cleaved by MBL-MASPs in a dose-dependent manner, yielding an ␣⬘-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.
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
4866
CHARACTERIZATION OF LAMPREY MANNOSE-BINDING LECTIN
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.
Discussion
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 collagenlike 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 sitedirected 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,
FIGURE 6. Western blotting and gel filtration of the lamprey MBL
preparation. A, Purified lamprey MBL preparation was probed with antiMASP-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.
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 ␣-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,
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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 mannanagarose (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.
The Journal of Immunology
4867
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
FIGURE 8. The binding of lamprey MBL and C3 to yeast. A, Yeast
cells (2 ⫻ 107) were incubated on ice for 1 h with 5 ␮l of lamprey serum
(plot 1), 10 ␮l of GlcNAc-agarose-treated serum (plot 2), or 20 ␮l of
partially purified MBL-MASPs complex (plot 3) in a total volume of 80 ␮l
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 ␮l of lamprey serum (plot 1), 20 ␮l
of GlcNAc-agarose-treated serum (plot 2), or reconstituted serum consisting of 20 ␮l of GlcNAc-agarose-treated serum and 10 ␮l of partially purified MBL-MASPs complex (plot 3) in a total volume of 80 ␮l 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.
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
The authors have no financial conflict of interest.
References
1. Hoffmann, J. A., F. C. Kafatos, C. A. Janeway, and R. A. Ezekowitz. 1999.
Phylogenetic perspectives in innate immunity. Science 284: 1313–1318.
2. Walport, M. J. 2001. Complement: first of two parts. N. Engl. J. Med. 344:
1058 –1066.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 7. Cleavage of lamprey C3 by MBL-MASPs. Lamprey C3 (8
␮g) (lane 1) was incubated at 37°C for 30 min with various amounts of
MBL-MASPs: 0.03 ␮l (lane 2), 0.06 ␮l (lane 3), 0.12 ␮l (lane 4), 0.25 ␮l
(lane 5), and 0.5 ␮l (lane 6) in a total amount of 10 ␮l. Lamprey C3 was
then subjected to SDS-PAGE under reducing conditions followed by protein staining. Lamprey C3 consisted of ␣-, ␤-, and ␥-chains, and the
␣-chain was cleaved into an ␣⬘-chain.
4868
CHARACTERIZATION OF LAMPREY MANNOSE-BINDING LECTIN
21. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence
weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673– 4680.
22. Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for
reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406 – 425.
23. Matsushita, M., and T. Fujita. 2002. The role of ficolins in innate immunity.
Immunobiology 205: 490 – 497.
24. Matsushita, M., and T. Fujita. 1992. Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine
protease. J. Exp. Med. 176: 1497–1502.
25. Thiel, S., T. Vorup-Jensen, C. M. Stover, W. Schwaeble, S. B. Laursen,
K. Poulsen, A. C. Willis, P. Eggleton, S. Hansen, U. Holmskov, et al. 1997. A
second serine protease associated with mannan-binding lectin that activates complement. Nature 386: 506 –510.
26. Dahl, M. R., S. Thiel, M. Matsushita, T. Fujita, A. C. Willis, T. Christensen,
T. Vorup-Jensen, and J. C. Jensenius. 2001. MASP-3 and its association with
distinct complexes of the mannan-binding lectin complement activation pathway.
Immunity 15: 127–135.
27. Matsushita, M., A. Matsushita, Y. Endo, M. Nakata, N. Kojima, T. Mizuochi, and
T. Fujita. 2004. Origin of the classical complement pathway: Lamprey orthologue
of mammalian C1q acts as a lectin. Proc. Natl. Acad. Sci. USA 101:
10127–10131.
28. Nonaka, M., and F. Yoshizaki. 2004. Primitive complement system of invertebrates. Immunol. Rev. 198: 203–215.
29. Endo, Y., M. Nonaka, H. Saiga, Y. Kakinuma, A. Matsushita, M. Takahashi,
M. Matsushita, and T. Fujita. 2003. Origin of mannose-binding lectin-associated
serine protease (MASP)-1 and MASP-3 involved in the lectin complement pathway traced back to the invertebrate, amphioxus. J. Immunol. 170: 4701– 4707.
30. Drickamer, K., M. S. Dordal, and L. Reynolds. 1986. Mannose-binding 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.
31. Drickamer, K. 1988. Two distinct classes of carbohydrate-recognition domains in
animal lectins. J. Biol. Chem. 263: 9557–9560.
32. Nakao, M., J. Mutsuro, M. Nakahara, Y. Kato, and T. Yano. 2003. Expansion of
genes encoding complement components in bony fish: biological implications of
the complement diversity. Dev. Comp. Immunol 27: 749 –762.
33. Matsushita, M., and T. Fujita. 1995. Cleavage of the third component of complement (C3) by mannose-binding protein-associated serine protease (MASP)
with subsequent complement activation. Immunobiology 194: 443– 448.
34. Smith, L. C., L. A. Clow, and D. P. Terwilliger. 2001. The ancestral complement
system in sea urchins. Immunol. Rev. 180: 16 –34.
35. Zhu, Y., S. Thangamani, B. Ho, and J. L. Ding. 2005. The ancient origin of the
complement system. EMBO J. 24: 382–394.
36. Kimura, Y., N. Inoue, A. Fukui, H. Oshiumi, M. Matsumoto, M. Nonaka,
S. Kuratani, T. Fujita, and T. Seya. 2004. A short consensus repeat-containing
complement regulatory protein of lamprey that participates in cleavage of lamprey complement 3. J. Immunol. 173: 1118 –1128.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
3. Walport, M. J. 2001. Complement: second of two parts. N. Engl. J. Med. 344:
1140 –1144.
4. Fujita, T. 2002. Evolution of the lectin-complement pathway and its role in innate
immunity. Nat. Rev. Immunol. 2: 346 –353.
5. Fujita, T., M. Matsushita, and Y. Endo. 2004. The lectin-complement pathway:
its role in innate immunity and evolution. Immunol. Rev. 198: 185–202.
6. Ezekowitz, R. A. 2003. Role of the mannose-binding lectin in innate immunity.
J. Infect. Dis. 187: S335–S339.
7. Jack, D. L., N. J. Klein, and M. W. Turner. 2001. Mannose-binding lectin: targeting the microbial world for complement attack and opsonophagocytosis. Immunol. Rev. 180: 86 –99.
8. Holmskov, U., R. Malhotra, R. B. Sim, and J. C. Jensenius. 1994. Collectins:
collagenous C-type lectins of the innate immune defense system. Immunol. Today
15: 67–74.
9. Holmskov, U., S. Thiel, and J. C. Jensenius. 2003. Collections and ficolins: humoral lectins of the innate immune defense. Annu. Rev. Immunol. 21: 547–578.
10. Weis, W. I., K. Drickamer, and W. A. Hendrickson. 1992. Structure of a C-type
mannose-binding protein complexed with an oligosaccharide. Nature 360:
127–134.
11. Drickamer, K. 1992. Engineering galactose-binding activity into a C-type mannose-binding protein. Nature 360: 183–186.
12. Nonaka, M. 2001. Evolution of the complement system. Curr. Opin. Immunol.
13: 69 –73.
13. Fujita, T., Y. Endo, and M. Nonaka. 2004. Primitive complement system–recognition and activation. Mol. Immunol. 41: 103–111.
14. Nonaka, M., K. Azumi, X. Ji, C. Namikawa-Yamada, M. Sasaki, H. Saiga,
A. W. Dodds, H. Sekine, M. K. Homma, M. Matsushita, et al. 1999. Opsonic
complement component C3 in the solitary ascidian, Halocynthia roretzi. J. Immunol. 162: 387–391.
15. Miyazawa, S., K. Azumi, and M. Nonaka. 2001. Cloning and characterization of
integrin ␣ subunits from the solitary ascidian, Halocynthia roretzi. J. Immunol.
166: 1710 –1715.
16. Kenjo, A., M. Takahashi, M. Matsushita, Y. Endo, M. Nakata, T. Mizuochi, and
T. Fujita. 2001. Cloning and characterization of novel ficolins from the solitary
ascidian, Halocynthia roretzi. J. Biol. Chem. 276: 19959 –19965.
17. Sekine, H., A. Kenjo, K. Azumi, G. Ohi, M. Takahashi, R. Kasukawa,
N. Ichikawa, M. Nakata, T. Mizuochi, M. Matsushita, et al. 2001. An ancient
lectin-dependent complement system in an ascidian: novel lectin isolated from
the plasma of the solitary ascidian, Halocynthia roretzi. J. Immunol. 167:
4504 – 4510.
18. Miyazawa, S., and M. Nonaka. 2004. Characterization of novel ascidian ␤ integrins as primitive complement receptor subunits. Immunogenetics 55: 836 – 844.
19. Nonaka, M., T. Fujii, T. Kaidoh, S. Natsuume-Sakai, N. Yamaguchi, and
M. Takahashi. 1984. Purification of a lamprey complement protein homologous
to the third component of the mammalian complement system. J. Immunol. 133:
3242–3249.
20. Cleveland, D. W. 1983. Peptide mapping in one dimension by limited proteolysis
of sodium dodecyl sulfate-solubilized proteins. Methods Enzymol. 96: 222–229.