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Departments of
*
Biochemistry and
Internal Medicine II, Fukushima Medical University School of Medicine, Fukushima, Japan;
Department of Biochemistry, Graduate School of Pharmaceutical Science, Hokkaido University, Sapporo, Japan; and
Department of Applied Biochemistry and Institute of Glycotechnology, Tokai University, Hiratsuka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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36 kDa (p36). p36 cDNA was cloned
from an ascidian hepatopancreas cDNA library. Sequence analysis
revealed that the carboxy-terminal half of the ascidian lectin contains
a carbohydrate recognition domain (CRD) that is homologous to C-type
lectin, but it lacks a collagen-like domain that is present in
mammalian MBLs. Purified p36 binds specifically to glucose but not to
mannose or N-acetylglucosamine, and it was designated
glucose-binding lectin (GBL). The two ascidian MASPs associated with
GBL activate ascidian C3, which had been reported to act as an opsonin.
The removal of GBL-MASPs complex from ascidian plasma using Ab against
GBL inhibits C3-dependent phagocytosis. These observations strongly
suggest that GBL acts as a recognition molecule and that the primitive
complement system, consisting of the lectin-proteases complex and C3,
played a major role in innate immunity before the evolution of an
adaptive immune system in vertebrates. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mannose-binding lectin
(MBL)4
(5, 6, 7) is a C-type lectin that plays a crucial role in the
activation of the lectin pathway (8, 9). MBL-associated
serine proteases (MASP-1/MASP-2) are the enzymes responsible for
complement activation initiated by MBL (10, 11). Human MBL
has an apparent molecular mass of 300–650 kDa and consists of 9–18
subunits of 32 kDa each. Each subunit contains an amino-terminal
region rich in cysteine, a collagen-like domain consisting of 18–20
tandem repeats of Gly-X-Y triplet sequences (where X and Y represent
any amino acid), and a carboxy-terminal carbohydrate recognition domain
(CRD). Through its CRD, MBL recognizes mannose and
N-acetyl-D-glucosamine (GlcNAc)
residues in common on microorganisms including bacteria, fungi,
parasitic protozoans, and viruses (8).
Ficolins are group of animal lectins that consist of collagen-like and fibrinogen-like domains. We recently reported that in addition to MBL, a member of the ficolin family, human ficolin/P35, is associated with MASP-1 and MASP-2, and is able to activate the lectin pathway (12).
Sea squirts, ascidians that were previously called tunicates (subphylum urochordata; phylum chordata) are among our closest invertebrate relatives. Halocynthia roretzi is a large solitary ascidian, native to the coastal waters of Japan. Previously, two ascidian MASP clones were identified (13) and an ascidian C3 (AsC3), the central component of the vertebrate complement system, was isolated (14). These reports suggested the presence of a lectin complement pathway in ascidians, although the lectins, which serve as the recognition molecules, had not been identified. Recently, we isolated ascidian ficolins, which probably functioned as the recognition molecules in a possible primitive complement system (15). MBL has been characterized from mammals, chickens (16), and carp (17). In this study, using mannan-Sepharose affinity chromatography, we isolated an ascidian lectin similar to mammalian MBL and analyzed the ascidian complement system that consisted of lectin-protease complexes and C3, and functions in an opsonic manner. These results indicate that the complement system has played a pivotal role in innate immunity by recognizing pathogens and enhancing phagocytosis since before the evolution of acquired immunity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Solitary ascidians, H. roretzi, were obtained from a local dealer (Ishimiya) in Fukushima, Japan. Hemolymph was collected from the solitary ascidian by cutting the tunic matrix without injuring internal organs, followed by centrifugation. Ascidian hemocytes were collected as described previously (14).
Purification of ascidian lectin, lectin-proteases complex, and C3
To prepare MBL-like lectin for the initial experiment, 1
liter of the plasma was concentrated 4-fold using a Pevicon Cassette
(Millipore, Bedford, MA). The concentrated material was treated
with polyethylene glycol 4000 at a final concentration of 7%. The
precipitate was dissolved in 20 mM Tris-HCl buffer containing 1 M NaCl
and 50 mM CaCl2, pH 7.8 (starting buffer), and
applied to a yeast mannan-Sepharose 4B column (10). After
washing the column, MBL-like lectin was eluted with the starting buffer
containing 0.3 M mannose. The eluted preparation was analyzed by
SDS-PAGE, dialyzed against 25 mM Tris, 25 mM NaCl, 5 mM
CaCl2, pH 7.8, and then applied to a Mono Q
column (Amersham Pharmacia Biotech, Uppsala, Sweden) that had
been equilibrated with the same buffer. Elution was conducted with a
linear NaCl gradient to 0.6 M. Under reducing conditions, a band of 36
KDa (p36) was observed. This preparation was used for amino acid
sequence analysis and for assaying binding to carbohydrates.
To prepare the lectin-protease complexes, hemolymph of the solitary ascidians was collected in the presence of protease inhibitors, 10 mM 6-amino-n-capronic acid, 10 mM benzamidine, 100 µM (p-aminophenyl)methanesulfonylfluoride (p-APMSF; Wako Pure Chemical Industries, Osaka, Japan), 20 µM p-nitrophenyl-p-guanidino-benzoate (Merck, Darmstadt, Germany), and 100 µM Pefabloc (Pentapharm, Basel, Switzerland). After centrifugation, the plasma was applied to a mannan-Sepharose 4B column equilibrated with starting buffer. After washing the column with starting buffer, p36 with minor bands was eluted with starting buffer containing 300 mM glucose. It was purified further on a Mono Q column, using the buffer described above. AsC3 was purified according to the method described (14).
Preparation of Abs
Polyclonal anti-ascidian MASPa Ab was obtained from rabbits immunized with synthetic peptides corresponding to the last 18 amino acid residues of ascidian MASPa (13) that had been coupled to keyhole limpet hemocyanin. The specific anti-MASPa Ab was affinity-purified using peptide-coupled Sepharose. Monospecific antiserum to the ascidian MBL-like proteins (p36) was raised by immunizing rabbits with purified proteins in CFA. The Ab to AsC3 was prepared as described previously (14). IgGs of both Abs were isolated using protein A-Sepharose. To remove the natural Abs to yeast, both IgGs were absorbed with an excess amount of yeast.
Amino acid sequence analysis
The lectin p36 was subjected to SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) and stained by Coomassie Brilliant Blue. The band was cut out and analyzed with a gas-phase protein sequencer (476A; PerkinElmer, Foster City, CA). To obtain the internal amino acid sequences, p36 was digested with Staphylococcus aureus V8 protease according to Cleveland’s method (18), and the two major digested bands (8 and 19 kDa) were analyzed with the protein sequencer. In the lectin-proteases complex, 66 and 70 kDa bands were also sequenced.
cDNA cloning of p36
Four degenerated primers were synthesized based on the amino-terminal amino acid sequences of p36 and its 8-kDa fragment: EDEQYLAC (5'-GARGAYGARCARTAYYTNGC-3'), WGPNEPND (5'-CKYTCMTTNCCNSWRTCRTT-3'), and NDSGNER (5'-TTNGGYTCRTTNGGNCCCCA-3'). A part of the p36 cDNA was amplified by nested RT-PCR with ascidian hepatopancreas mRNA as a template and two primer sets (for 5'-GARGAYGARCARTAYYTNGC-3' and 5'-CKYTCMTTNCCNSWRTCRTT-3' the first and 5'-GARGAYGARCARTAYYTNGC-3' and 5'-TTNGGYTCRTTNGGNCCCCA-3' for the second). PCR products of the expected size were cloned into pGEM-T Easy Vector (Promega, Madison, WI) and sequenced by the dideoxy method using an autosequencer (Model 4000; LI-COR, Lincoln, NE). The subcloned 506-bp DNA was labeled with 32P and used as a probe for screening a ZAP cDNA library. A total of 2 x 106 plaques of an ascidian hepatopancreas ZAP II cDNA library was screened. Positive clones were subcloned in pBluescript II (KS+) by its unique ZAP system and sequenced by the method described above.
Northern blot hybridization
Total RNA from ascidian hepatopancreas or hemocytes, 10 µg/lane, was electrophoresed in a 1.0% agarose-formaldehyde gel and transferred onto a nylon membrane (Hybond-N+; Amersham, Little Chalfont, U.K.) by capillary blotting overnight. The membrane was hybridized with the 32P-radiolabeled insert cDNA (nucleotides 1–911) of a cloned plasmid at 42°C overnight in 50% formamide, 5 x Denhardt’s solution, 5 x SSPE (1 x SSPE is 9 mM sodium phosphate, 150 mM NaCl, 1 mM EDTA, pH 7.4), 0.5% SDS, and 200 µg/ml salmon sperm DNA. After a final washing at 42°C for 30 min in 0.1 x SSC (1.5 mM sodium citrate, 150 mM NaCl, pH 7.0) containing 0.1% SDS, the filter was exposed in an autoradiogram imaging system, and the image was then read using a bioimaging analyzing system (BAS 1000; Fuji Film, Tokyo, Japan).
Assay of p36 binding to neoglycolipids
The ascidian MBL-like protein (p36) was labeled with
Na125I (New England Nuclear, Boston, MA) using
Iodo-Gen (Pierce, Rockford, IL) according to the manufacturer’s
instructions. To separate the biologically active lectin from the
inactive form, the radiolabeled lectin was applied to mannan-Sepharose
and eluted with mannose, followed by massive dialysis against Tris-HCl
buffer pH 7.5 containing 0.15 M NaCl. The oligosaccharides were
conjugated to dipalmitoylphosphatidylethanolamine as described
previously (19). The following 10 oligosaccharides were
used: maltopentaose (Mal5; Glc
1-4Glc1-4Glc1-4Glc1-4Glc),
isomaltopentaose (IsoMal5;
Glc1-6Glc1-6Glc1-6Glc1-6Glc), cellopentaose (Cello5;
Glc
1-4Glc1-4Glc1-4Glc1-4Glc), laminaripentaose (Lami5;
Glc1-3Glc1-3Glc1-3Glc1-3Glc), mannopentaose (Man5) with a
structure of Man1-6(Man1-3)Man1-6(Man1-3)Man,
penta-N-acetyl-chitopentaose (GN5;
GlcNAc1-4GlcNAc1-4GlcNAc1-4GlcNAc1-4GlcNAc),
oligosaccharide with a structure of GlcNAc1-2 Man1-6(GlcNAc1-2
Man1-3)Man (GN2M3), 3'-sialyllactose (3'SL; sialic
acid2-3Gal1-4Glc), 6'-sialyllactose (6'SL; sialic
acid2-6Gal1-4Glc) and lactose (Lac; Gal1-4Glc). Because all
the neoglycolipids thus obtained contained a common lipid, they were
separated according to their oligosaccharide structures using a TLC
system. After developing to 7 cm from the origin, the TLC plates
were air-dried. The plates were then overlaid with
125I-labeled p36, and its binding to the
neoglycolipids was assessed by autoradiography and orcinol staining as
reported previously (20). Similar assays of binding of p36
to oligosaccharides derived from six mammalian glycoproteins was
performed as reported previously (20).
[3H]Diisopropylfluorophosphate ([3H]DFP) labeling
Twenty microliters of DFP [1,3-3H]
([3H]DFP, 740 kBq; New England Nuclear) was
incubated at 4°C for 17 h with 200 µl (100 µg/ml) of
ascidian lectin- protease complex or human C1s (10) in
Tris-HCl buffer pH 7.5 containing 0.15 M NaCl in the presence of 40
µl of BSA (1.6 mg/ml) as a carrier. The proteins were then
precipitated by the addition of 1 ml of chilled acetone, and kept at
-20°C for 2 h. After centrifugation at 15,000 rpm for 10 min,
the precipitates were dried up and dissolved in SDS-PAGE buffer, and
subjected to SDS-PAGE under reducing or nonreducing conditions,
followed by autoradiography.
Immunoblotting
After SDS-PAGE (10% gel), proteins were transferred from the gel to a polyvinylidene difluoride membrane, and the blot was probed with rabbit Ab against ascidian MASPa. Peroxidase-conjugated anti-rabbit IgG was used as a second Ab, and the blot was developed with a Konica Immunostaining HRP kit (Konica, Tokyo, Japan).
Flow cytometry
The binding of AsC3 to yeast was analyzed by flow cytometry. Yeast cells (W303D; 5 x 106) in 50 µl of normal Herbst’s artificial seawater (450 mM NaCl, 9.4 mM KCl, 48 mM MgSO4, 48 mM CaCl2, 32 mM Na2SO4, and 3.2 mM NaHCO3, pH 7.6) were incubated at 20°C for 2 h with ascidian plasma or lectin-proteases complex and AsC3, and then washed three times with Veronal buffer supplemented with 10 mM EDTA and 0.1% gelatin. The washed cells were then reacted at 4°C for 30 min with 10 µl of anti-AsC3 Ab (1 mg/ml) and stained at 4°C for 30 min with 20 µl of 100 µg/ml FITC-conjugated swine anti-rabbit Igs (DAKO Japan, Kyoto, Japan). The yeast was washed twice with 10 mM EDTA and 0.1% gelatin between each reaction. Reactivities were evaluated by FACScan flow cytometry (BD Biosciences, Mountain View, CA).
Phagocytosis assay
Hemolymph was collected from an ascidian, and fresh plasma was prepared. To prepare glucose-binding lectin (GBL)- or C3-depleted plasma, the fresh plasma was treated with each Ab, and the resulting Ag-Ab complexes were removed with protein A-Sepharose. Rabbit normal IgG was used as a control. Each plasma was incubated with yeast (W303D) and after washing, the coated yeast was reacted with the ascidian hemocytes. Hemocytes that ingested at least one yeast were considered positive. The degree of phagocytosis was expressed as the ratio of the number of positive hemocytes to the number of total hemocytes (21).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To purify the ascidian lectin homologous to mammalian MBL, plasma
from H. roretzi was concentrated and precipitated with
polyethylene glycol 4000. The precipitate was dissolved in buffer,
applied to a yeast mannan-Sepharose column and eluted with mannose.
SDS-PAGE of the eluted proteins revealed a major band of 36 kDa
under reducing conditions (Fig. 1
). The
36-kDa protein (p36) was further purified by Mono Q chromatography.
Under nonreducing conditions, p36 appeared as three bands with apparent
molecular mass of 70, 140, and 210 kDa. These results indicate that
the ascidian lectin is composed of homodimers consisting of 36-kDa
subunits bound by means of intermolecular disulfide bonding as is the
case with mammalian MBL.
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cDNA cloning of p36
Based on the amino-terminal amino acid sequences of p36, and the
8-kDa fragment produced by protease digestion, we designed degenerated
primers and performed nested PCR. A single band of 500 bp, which was
amplified, was cloned and then sequenced by the dideoxy method. The
deduced amino acid sequence revealed that it contained the complete
amino acid sequence of the 19-kDa fragment of p36, which was not used
for PCR. An ascidian hepatopancreas cDNA library was screened using
this PCR product as a probe and a 1-kb-long cDNA clone with an open
reading frame of 672 bp, which is predicted to encode a 224 aa protein
including a 17-residue leader peptide, was isolated (Fig. 2). The predicted molecular mass of the
protein was 23,716 Da, indicating that the mature 36-kDa protein may be
glycosylated, because there are three N-linked glycosylation sites.
Sequence analysis revealed that the carboxy-terminal half of the
ascidian protein contains a CRD. Unlike mammalian MBL, the N-terminal
portion of the lectin lacks a collagenous region consisting of
Gly-X-Y triplet repeats (where X and Y are any amino acid)
(8, 9). The nonlectin part of the molecule has no
significant similarity to any known sequences.
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). A short stretch of the nonlectin region of the
protein shows limited similarity to the portion of the MBL collagen
region where the Gly-X-Y triplets are interrupted to form a bend in the
triple helix (9). The p36 CRD has a sequence identity of
22.4% to the human MBL. The CRD is of the C-type, containing 13 of the
18 highly conserved amino acid residues, including four cysteines that
are involved in disulfide bonds within the domain (22).
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+ (23), are
completely conserved among these proteins, indicating that the ascidian
lectin belongs to the mannose-binding family. Northern blotting
Northern blot analysis was performed using two potential sources
of plasma proteins, ascidian hepatopancreas and hemocytes. As shown in
Fig. 3, the p36 probe detected a single
transcript band from hepatopancreas. The p36 mRNA is 1.1 kb in length,
which corresponds to the full size of the cDNA. A positive signal was
not detected in blood cells.
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We evaluated the binding specificity of the ascidian lectin, p36,
for various neoglycolipids. As shown in Fig. 4, p36 bound to oligosaccharides composed
of glucose, but not to those containing mannose, GlcNAc, or galactose.
We performed additional experiments to confirm these results. Purified
p36 was applied to a mannose- or a GLcNAc-Sepharose column, but it did
not bind (data not shown). There are no previous reports of lectins
specific for glucose residues although several lectins that recognize
-1,3-glucan have been described (24, 25). Therefore, we
designated the ascidian lectin GBL. We also performed similar assays of
GBL binding to oligosaccharides derived from six mammalian
glycoproteins (20). It did not bind to the high mannose,
complex, or hybrid-type oligosaccharides of mammalian glycoproteins
(data not shown), suggesting that GBL recognizes only those glucose
residues in the polysaccharides of microorganisms. By flow cytometric
analysis, GBL was shown to bind to yeast and to its cell wall, zymosan,
in the presence of Ca2+, probably through
-1,3-glucan (data not shown). During the preparation of GBL on the
yeast mannan-Sepharose column, GBL probably bound, not to the mannose
residue of mannan, as does mammalian MBL, but to glucose-containing
polysaccharides, probably -1,3-glucan, which was a contaminant in
the yeast mannan used to prepare the column. Also, the purified GBL was
applied to mannan-Sepharose and eluted with glucose. The same results
were obtained with mannose. The reason why the mannose disrupted the
binding of GBL to the mannan-Sepharose is not clear. It is possible
that the mannose used in elution contains the epimer form.
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Next, we determined whether the lectin fraction contained the
ascidian serine proteases MASPa and MASPb, which had been
reported as homologues of mammalian MASP-1 (13, 26).
Ascidian hemolymph was collected in the presence of a range of protease
inhibitors. After centrifugation, the plasma was applied to a
mannan-Sepharose column, eluted with glucose, and further purified on a
Mono Q column. SDS-PAGE of the eluted proteins revealed a major band of
GBL, and minor bands of 43, 66, and 70 kDa (Fig. 5). To identify the serine proteases in
this fraction, we performed the experiments of
[3H]DFP incorporation. The radiolabeled DFP was
incorporated into 43- and 36-kDa bands under reducing conditions, and
into a 110-kDa band under nonreducing conditions (Fig. 5). Western
blotting revealed that anti-ascidian MASPa Ab recognized 36- and
110-kDa bands under reducing and nonreducing conditions, respectively.
To identify the proteases, the 70- and 66-kDa bands were analyzed with
a protein sequencer. The 70- and 66-kDa bands contained the
amino-terminal amino acid sequences APSVKNLTG and AELLTAHFG, which had
been reported as the amino-terminal sequences of the heavy chains of
ascidian MASPa and MASPb, respectively (13). These results
indicate that MASPa and MASPb consist of the 70- and 66-kDa heavy
chains, and the 36- and 43-kDa light chains, respectively. Therefore,
it is clear that GBL associates with both MASPa and MASPb.
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Because human MASP-1 is reported to activate C3 directly
(27), we examined whether the ascidian MASPs associated
with GBL could cleave AsC3. Purified AsC3 was incubated with the
GBL-MASPa-MASPb complex, and proteolytic activation was monitored by
SDS-PAGE. As shown in Fig. 6, the C3
-chain was cleaved in a dose-dependent manner, yielding an
'-chain. Because the primary structures of the proteases suggested
that only MASPa has trypsin-like specificity (13), AsC3 is
probably cleaved by MASPa rather than by MASPb, although this point
needs to be confirmed at the biochemical level.
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, a considerable amount of C3 bound to
yeast in the reconstitution experiments, indicating that GBL recognizes
carbohydrates of yeast, probably -1,3-glucan, and that the
associated MASPs activate C3. From these results, it appears that GBL
is one of the lectins that acts as a recognition molecule in the lectin
pathway of the ascidian complement system.
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To confirm the above results, we performed phagocytosis assays.
Usually, 20–30% of ascidian leukocytes (hemocytes) ingested at least
one noncoated yeast cell, and in the case of yeast treated with
ascidian plasma, 40–60% of hemocytes ingested one or more yeast
cells. As reported previously, this opsonic activity is derived
from C3 (14). In the experiments shown in Fig. 8, we also found that the opsonic effect
of ascidian plasma was eliminated in GBL-depleted plasma in a
dose-related manner. In the presence of EDTA or EGTA, the opsonic
activity could not be detected, suggesting that
Ca2+ is required for the activation of C3 in this
system. In addition, we showed that the purified GBL-MASPs and C3
enhanced phagocytosis. Therefore, it is clear that GBL acts as the
recognition molecule, and as the result, the activated C3 is fixed on
yeast which is phagocytosed through the recently identified ascidian
complement receptor (28).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An MBL homologue has not been yet identified in invertebrates, although
an N-acetyl-galactosamine-binding lectin with a
collagen-like domain has been identified as a collectin-like protein in
a different species of ascidian, Stylea plicata
(29). However, its complete structure has not been
elucidated, and its binding specificity for carbohydrates is different
from that of MBL. Recently, by an affinity chromatography on
GlcNAc-Sepharose, we isolated ascidian ficolins that have short
collagen-like sequences and fibrinogen-like domains (15).
As shown in Fig. 1, GBL is a major protein that binds to
mannan-Sepharose, and an additional band of 40 kDa was identified as
ascidian galactose-specific lectin (Gal-lectin) (30).
Because MBL is mainly bound to mannose and GlcNAc, and we failed to
isolate an MBL homologue that binds to mannose from ascidian hemolymph,
we consider that GBL is the functional counterpart of MBL.
Interestingly, GBL has an helix structure in its amino-terminal
region, which is similar to the configuration of Gly-X-Y repeats. This
raises the possibility that GBL has evolved early as a prototype of
MBL. During evolution GBL may have acquired the broad binding
specificity for carbohydrates and the collagen structure characteristic
of MBL. In this regard it will be of interest to determine the
structure and function of the lectin associated with MASP in lamprey,
one of the most primitive vertebrates, because we have already cloned
the cDNA of lamprey MASP (26).
GBL has a unique binding specificity for carbohydrates, because it
binds to only glucose residues. Although several lectins that recognize
-1,3-glucan have been reported (24, 25), this is the
first report of a lectin that recognizes only the glucose residue. MBL
is reported to bind carbohydrates with 3- and 4-hydroxyl groups in the
pyranose ring in the presence of Ca2+ through the
five conserved residues (Glu184,
Asn186, Glu191,
Asn205, and Asp206) in the
MBL CRD (23). These residues are completely conserved in
GBL and mammalian MBLs, supporting the hypothesis that GBL is a
prototype of MBL. The structural difference between mannose or GlcNAc
and glucose is at the site of the 2-hydroxyl group of the pyranose
ring. Therefore, it is possible that residues other than the five
conserved ones in GBL may be involved in recognizing the 2-hydroxyl
group of glucose.
We presented evidence here that AsC3, along with GBL and MASPs, constitute a simple complement system corresponding structurally and functionally to the mammalian lectin pathway. Its activation mechanism, which depends on lectin-based recognition, clearly indicates that this ancestral complement system is part of the innate immune system. Although the origin of the complement system is still not clear, it can be traced back at least to echinoderms, because C3 and factor B have been identified in sea urchin (3). Recently, a C3 receptor on ascidian hemocytes was identified as the homologue of mammalian complement receptor type 3 or 4 (CR3 or CR4) (28). Taken together, our observations strongly suggest that lectin-protease complex, C3, and C3R may have developed as the minimal ancestral components of the primordial complement system in the tunicate lineage.
Another important finding is that an opsonic activity derived from C3 was not observed in GBL-depleted plasma, suggesting that the only activation mechanism of C3 by a lectin-based recognition in the system using yeast involves GBL. Because the alternative pathway was found as the activation mechanism of serum C3 by zymosan without the involvement of any recognition molecules (31), this pathway may not have emerged in the tunicate lineage, although the possibility of a simple role of factor B protein (3) as an amplifier of C3 deposition cannot be excluded completely. The sophisticated recognition mechanism of the alternative pathway to recognize a broad spectrum of pathogens developed more recently. Because the classical activation pathway is part of acquired immunity, the complement lectin pathway seems to have played a central role in host defense against infection for a long time before the evolution of the adaptive immune system in the vertebrate lineage.
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Acknowledgments |
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Footnotes |
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2 The nucleotide sequence data of ascidian GBL will appear in the DDBJ, EMBL, and GenBank DNA databases under accession number AB000805.
3 Address correspondence and reprint requests to Dr. Teizo Fujita, Department of Biochemistry, Fukushima Medical University School of Medicine, 1-Hikariga-oka, Fukushima 960-1295, Japan. E-mail address: tfujita{at}fmu.ac.jp
4 Abbreviations used in this paper: MBL, mannose-binding lectin; GBL, glucose-binding lectin; MASP, MBL-associated serine protease; CRD, carbohydrate recognition domain; AsC3, ascidian C3; DFP, diisopropylfluorophosphate; Glc, D-glucose; Man, D-mannose, GlcNAc, N-acetyl-D-glucosamine; Gal, D-galactose.
Received for publication June 4, 2001. Accepted for publication August 23, 2001.
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-1,3-glucan recognition protein in the prophenoloxidase activating system from hemolymph of the silkworm Bombyx mori. J. Biol. Chem. 263:12056.-1,3-glucan binding protein from plasma of the crayfish Pacifastacus leniusculus. J. Biol. Chem. 265:9327. subunits from the solitary ascidian Halocynthia roretzi. J. Immunol. 166:1710.This article has been cited by other articles:
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 A. A Bulgakov, M. G Eliseikina, I. Y. Petrova, E. L Nazarenko, S. N Kovalchuk, V. B Kozhemyako, and V. A Rasskazov Molecular and Biological Characterization of a Mannan-Binding Lectin from the Holothurian Apostichopus Japonicus Glycobiology, December 1, 2007; 17(12): 1284 - 1298. [Abstract] [Full Text] [PDF]
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M. Takahashi, D. Iwaki, A. Matsushita, M. Nakata, M. Matsushita, Y. Endo, and T. Fujita Cloning and characterization of mannose-binding lectin from lamprey (agnathans). J. Immunol., April 15, 2006; 176(8): 4861 - 4868. [Abstract] [Full Text] [PDF]
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 M. Matsushita, A. Matsushita, Y. Endo, M. Nakata, N. Kojima, T. Mizuochi, and T. Fujita Origin of the classical complement pathway: Lamprey orthologue of mammalian C1q acts as a lectin PNAS, July 6, 2004; 101(27): 10127 - 10131. [Abstract] [Full Text] [PDF]
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 R. G. Woods, K. E. Roper, M. Gauthier, L. M. Bebell, K. Sung, B. M. Degnan, and M. F. Lavin Gene expression during early ascidian metamorphosis requires signalling by Hemps, an EGF-like protein Development, June 15, 2004; 131(12): 2921 - 2933. [Abstract] [Full Text] [PDF]
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Y. Endo, M. Nonaka, H. Saiga, Y. Kakinuma, A. Matsushita, M. Takahashi, M. Matsushita, and T. Fujita 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., May 1, 2003; 170(9): 4701 - 4707. [Abstract] [Full Text] [PDF]
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 M. S. Quesenberry, H. Ahmed, M. T. Elola, N. O'Leary, and G. R. Vasta Diverse Lectin Repertoires in Tunicates Mediate Broad Recognition and Effector Innate Immune Responses Integr. Comp. Biol., April 1, 2003; 43(2): 323 - 330. [Abstract] [Full Text] [PDF]
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B. Davidson and B. J. Swalla A molecular analysis of ascidian metamorphosis reveals activation of an innate immune response Development, March 12, 2003; 129(20): 4739 - 4751. [Abstract] [Full Text] [PDF]
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G. Ambrus, P. Gal, M. Kojima, K. Szilagyi, J. Balczer, J. Antal, L. Graf, A. Laich, B. E. Moffatt, W. Schwaeble, et al. Natural Substrates and Inhibitors of Mannan-Binding Lectin-Associated Serine Protease-1 and -2: A Study on Recombinant Catalytic Fragments J. Immunol., February 1, 2003; 170(3): 1374 - 1382. [Abstract] [Full Text] [PDF]
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