From the Department of Biochemistry, Fukushima Medical University,
School of Medicine, 1-Hikariga-oka, Fukushima 960-1295, Japan and the
Laboratory of Biomedical Chemistry, Department of Applied
Chemistry, Tokai University, Hiratsuka, Kanagawa 259-1292, Japan
Received for publication, December 27, 2000, and in revised form, March 19, 2001
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ABSTRACT |
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Ficolins are animal lectins with collagen-like
and fibrinogen-like domains. They are involved in the first line of
host defense against pathogens. Human ficolin/P35 as well as
mannose-binding lectin (MBL) activates the complement lectin
pathway in association with MBL-associated serine proteases. To
elucidate the origin and evolution of ficolins, we separated ~40 kDa
(p40) and ~50 kDa (p50) N-acetylglucosamine-binding
lectins from hemolymph plasma of the solitary ascidian. Binding assays
revealed that p40 recognizes N-acetyl groups in association
with a pyranose ring and that p50 recognizes
N-acetylglucosamine alone. Based on the amino acid sequences of the proteins, we isolated two clones each of p40 and p50
from the ascidian hepatopancreas cDNA and determined the entire
coding sequences of these clones. Because all of the clones contained
both collagen-like and fibrinogen-like domains, we concluded that these
were homologs of the mammalian ficolin family and designated ascidian
ficolins (AsFCNs). The fibrinogen-like domain of the AsFCNs shows
45.4-52.4% amino acid sequence identity with the mammalian ficolin
family. A phylogenetic tree of the fibrinogen-like sequences
shows that all the fibrinogen-like domains may have evolved from a
common ancestor that branched off an authentic fibrinogen. These
results suggest that AsFCNs play an important role with respect to
ascidian hemolymph lectin activity and the correlation of different
functions with binding specificity.
Lectins play an important role in innate immunity in animals.
Among them, collectins and ficolins have structural and functional similarities. Collectins are characterized as lectins with a
collagen-like domain and a carbohydrate-recognition domain and include
mannose-binding lectin (MBL)1
(1), SP-A, SP-D (2), conglutinin, and CL-43 (3). These binding
specificities for carbohydrates vary, and calcium ions are required for
binding. MBL in serum has an opsonic activity (4) and the capacity to
activate complement (5). MBL is associated with serine proteases,
termed MBL-associated serine proteases (MASPs), and upon binding of MBL
through its carbohydrate-recognition domain to carbohydrates such as
mannose or N-acetylglucosamine (GlcNAc) of pathogens, MASPs
cleave complement components (6-9). Complement activation by MBL in
association with MASPs is called the lectin pathway (7, 10).
Ficolins are a group of proteins that consist of a collagen-like domain
and a fibrinogen-like domain. They were originally identified as
transforming growth factor- The molecular details of invertebrate innate immune systems and their
possible connection to vertebrate innate immunity have not been fully
elucidated. Ascidians occupy a pivotal intermediary position between
invertebrates and vertebrates. Therefore, studies on the host defense
mechanisms of ascidian could provide us with important information
about the evolution of a primitive innate immune system of vertebrates.
The recent identification of cDNA encoding the third component of
complement (AsC3) (22), two types of MASP (AsMASPa and AsMASPb) (23),
and glucose-binding lectin (GBL) (24) suggests that the lectin
complement pathway is present in ascidian, Halocynthia
roretzi. This implies that lectins homologous to vertebrate MBL or
ficolins might exist in ascidian. Here we describe the isolation and
cloning of GlcNAc-binding lectins with a collagen-like domain and a
fibrinogen-like domain, designated as ascidian ficolins (AsFCNs), from
hemolymph plasma of the solitary ascidian, H. roretzi.
Materials and Reagents--
The solitary ascidian, H. roretzi, was obtained from local dealers in Fukushima, Japan.
Sepharose 4B was purchased from Amersham Pharmacia Biotech (Uppsala,
Sweden). Acidified Sepharose 4B was prepared by treating Sepharose 4B
with 0.1 M HCl at 50 °C for 3 h. GlcNAc-agarose,
fetuin, asialofetuin, bovine serum albumin (BSA) conjugated with
lactose (Lac-BSA), and cellobiose (Cel-BSA) were purchased from Sigma
(St Louis, MO); BSA conjugated with N-acetylgalactosamine
(GalNAc-BSA), N-acetyllactose (LacNAc-BSA), and
NeuAc Purification of Ascidian Ficolin-like Lectin--
Hemolymph was
collected from the solitary ascidian by cutting the tunic matrix
without injuring internal organs, followed by centrifugation. To remove
galactose-binding proteins and nonspecific binding proteins, the
hemolymph plasma was directly applied to an acidified Sepharose 4B
column equilibrated with starting buffer consisting of 25 mM Tris-HCl, pH 7.8, containing 1 M NaCl and 5 mM CaCl2. The fractions that passed through
were applied to a GlcNAc-agarose column. After washing the column with
starting buffer, two major proteins designated p40 and p50 were eluted with starting buffer containing 150 mM GlcNAc. The eluted
fractions were dialyzed against 25 mM Tris-HCl, pH 7.8 containing, 50 mM NaCl, 5 mM CaCl2,
and then chromatographed on a Mono Q column, followed by elution with a
linear NaCl gradient to 0.5 M. The preparation was analyzed
by SDS-polyacrylamide gel electrophoresis (PAGE) using the Laemmli system.
Binding Assay of p40 and p50 with Glycoproteins and
Neoglycoproteins--
The ascidian ficolin-like proteins (p40 and p50)
were labeled with Na125I and IODO-GEN from Pierce,
Rockford, IL. The glycoproteins ovalbumin, bovine pancreatic RNase B,
human fetuin, and human asialofetuin and the neoglycoproteins
lactose-conjugated BSA (Lac-BSA), cellobiose-conjugated BSA (Cel-BSA),
N-acetylglucosamine-conjugated BSA (GlcNAc-BSA), mannose-conjugated BSA (Man-BSA), glucose-conjugated BSA (Glc-BSA), galactose-conjugated BSA (Gal-BSA),
N-acetylgalactosamine-conjugated BSA (GalNAc-BSA),
N-acetyllactosamine-conjugated BSA (LacNAc-BSA), NeuAc
Glycoproteins and neoglycoproteins were dissolved in phosphate-buffered
saline at the concentrations indicated in the figures. 100 µl of each
solution was dot-blotted onto a PVDF membrane (Millipore, Bedford, MA)
using an Immunodot microfiltration apparatus (ATTO, Tokyo, Japan).
The dotted membrane was blocked for 2 h at room temperature with
25 mM Tris-HCl buffer, pH 7.8, containing 1 M NaCl and 3% (w/v) BSA. The membrane was rinsed with 25 mM
Tris-HCl buffer, pH 7.8, containing 1 M NaCl and 5 mM CaCl2 (Buffer A) and then incubated for
2 h at 4 °C with 125I-labeled p40 (400,000 cpm/ml)
or 125I-labeled p50 (350,000 cpm/ml) in Buffer A containing
0.1% (w/v) BSA. After the incubation, the membrane was washed with
Buffer A and air-dried. Binding of the radioactive ascidian lectins was measured using a Bio-imaging analyzer BAS-1800II (Fujifilm, Tokyo, Japan) and is indicated as the intensity of the photostimulated luminescence (PSL).
For inhibition assays, 100 µl of each GlcNAc-BSA solution
(0.00032-5.0 µg/100 µl) was dot-blotted onto a PVDF membrane. The membrane was blocked and rinsed as described above.
125I-labeled p40 and p50 were preincubated for 1 h at
4 °C without or with 1, 10, or 100 mM GlcNAc, 10 mM sodium acetate, or 10 mM N-acetylglycine in Buffer A containing 0.1% (w/v) BSA. The
mixture was then overlaid on the membrane and binding of the
radioactive lectin to the GlcNAc-BSA was determined as described above.
Binding Assay of p40 with Neoglycolipids--
Neoglycolipids
were prepared by reductive amination from dipalmitoyl
phosphatidylethanolamine (DPPE) and the following carbohydrates as
described previously (30): GlcNAc, di-N-acetylchitobiose (GlcNAc2), tri-N-acetylchitotriose (GlcNAc3),
tetra-N-acetylchitotetraose (GlcNAc4),
penta-N-acetylchitopentaose (GlcNAc5),
hexa-N-acetylchitohexaose (GlcNAc6), and
GlcNAc Amino Acid Sequence Analysis--
For amino acid sequencing, p40
or p50 was subjected to SDS-PAGE under reducing conditions and then
electroblotted onto PVDF membranes. The bands were visualized by
Coomassie Blue staining, excised, and then analyzed using an ABI
protein sequencer model 476A. To obtain the internal amino acid
sequences, p40 or p50 was digested with Staphylococcus
aureus V8 protease according to Cleveland's method (31). Digested
peptides were electroblotted, followed by the same method as described above.
Cloning of p40 and p50 cDNA--
Ascidian hepatopancreases
were removed immediately before use. RNA was isolated from the
hepatopancreases and hemocytes using the acid guanidine thiocyanate
method, and the poly(A)+ fraction was purified by passage
through an oligo (dT)-cellulose column (CLONTECH,
Palo Alto, CA). cDNAs were prepared using SUPERSCRIPTTM
II Reverse Transcriptase (Life Technologies, Inc., Rockville, MD).
Four degenerated primers were synthesized based on the
NH2-terminal amino acid sequences of p40 and its V8
protease fragments: TGLRNQ (5'-ACNGGNYTNMGNAAYCA-3'), RNQLQE
(5'-MGNAAYCARYTNCARGA-3'), FQRRMD (5'-TCCATNCKNCKYTGRAA-3'), and WTVFQR
(5'-CKYTGRAANACNGTCCA-3'). A nested PCR was performed to amplify a
portion of the cDNA using ascidian hepatopancreas cDNA as a
template and two primer sets (5'-ACNGGNYTNMGNAAYCA-3' and
5'-TCCATNCKNCKYTGRAA-3' for the first and 5'-MGNAAYCARYTNCARGA-3' and
5'-CKYTGRAANACNGTCCA-3'for the second). PCR products of the expected
size were cloned into pGEM-T easy vector (Promega, Wisconsin, WI) and
sequenced. 5'- and 3'-RACE (rapid amplification of cDNA ends) were
then conducted to complete the cDNA sequence using a kit (Marathon,
CLONTECH). Two sets of gene-specific primers (GSPs)
were synthesized: 5'-AGTTTCTGTTTTCTGATCCACTCTG-3' and
5'-TGTTTTCTGATCCACTCTGTTGTCC-3' for 5'RACE and
5'-CGAAGTAAGAATGGACAACAGAGTG-3' and 5'-AAGAATGGACAACAGAGTGGATCAG-3' for
3'-RACE. The RACE products were cloned and sequenced. A complete
stretch of p40 cDNA was constructed by overlapping the sequences of
these PCR products, and it was designated AsFCN1. We also identified
another cDNA sequence, termed AsFCN2.
Four degenerated primers were synthesized for PCR to amplify the
p50 cDNA. These were based on the amino acid sequences VMQKVM (5'-GTNATGCARAARGTNATG-3'), AEGVTG (5'-GCNGARGGNGTNACNGG-3'), WTVFQR
(5'-CKYTGRAANACNGTCCA-3'), and VYCDLT (5'-GTNARRTCRCARTANAC-3'). A
nested PCR was performed using ascidian hepatopancreas cDNA as a
template and two primer sets (5'-GTNATGCARAARGTNATG-3' and 5'-CKYTGRAANACNGTCCA-3'for the first and 5'-GCNGARGGNGTNACNGG-3' and
5'-GTNARRTCRCARTANAC-3' for the second). PCR products of the expected
size were cloned and sequenced as described. To complete the p50
cDNA sequence, 5'- and 3'-RACE were conducted using GSPs (5'-CTGCTATACCCCTGCATCCATCAGG-3' and 5'-ACCCCTGCATCCATCAGGGACTTCA-3' for 5'-RACE and 5'-CGAACAGAGGTTGGAAATGGAAATC-3' and
5'-GTTGGAAATGGAAATCGAGAAGAAA-3' for 3'RACE). Its clone was designated
AsFCN3. Using different sets of GSPs (5'-TCCTTTCCAGGATATAGCCCCGTAC-3'
and 5'-GGATATAGCCCCGTACTTGCTCACT-3' for 5'-RACE, and
5'-CGCAGAAACCCAGCAA- CAAATCGTC3-' and
5'-ACCCAGCAACAAATCGTCGAAGGAA-3' for 3'-RACE), another cDNA
sequence, designated AsFCN4, was identified.
Nucleotide Sequence Analysis--
DNA sequences were determined
by the dideoxy chain termination method (32) using a DNA sequencer
(Model 4000; LI-COR, Lincoln, NE). The labeling reaction was performed
using a Thermo Sequenase Cycle Sequencing kit (USB Corporation,
Cleveland, OH). Sequencing primers were synthesized by Nisshinbo
(Tokyo, Japan).
Northern Blot Hybridization--
A membrane filter blotted with
2 µg of poly(A)+ fraction from hepatopancreases and
hemocytes was hybridized with 32P-labeled specific cDNA
fragments of the ascidian ficolins (AsFCN1, nucleotides 14-336;
AsFCN3, nucleotides 69-384; AsFCN4, nucleotides 7-326) at 42 °C
overnight in 50% formamide, 5× Denhardt's solution, 5× SSPE (1×
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 55 °C for 40 min in 0.1× SSC (1.5 mM sodium citrate, 15 mM NaCl, pH 7.0)
containing 0.1% SDS, the filter was exposed to an autoradiogram
imaging screen, and the image was then read using a bioimaging
analyzing system (BAS 1000, Fujifilm).
Construction of the Phylogenetic Tree--
The fibrinogen-like
sequences of four ascidian ficolins, of eight members of the mammalian
ficolin family, including human ficolin/P35 (12), ficolin M (13),
ficolin H (15), porcine ficolin- Purification of Ascidian Ficolin-like Lectins, p40 and
p50--
The hemolymph plasma was subjected to affinity chromatography
on a GlcNAc-agarose column, and ascidian ficolin-like lectins were
eluted with GlcNAc like human ficolin/P35. Two major proteins with
molecular masses of ~40 and ~50 kDa by SDS-PAGE under reducing conditions were found in the eluate from GlcNAc-agarose column (Fig.
1A) and were tentatively
designated p40 and p50, respectively. Further purification was achieved
by ion-exchange chromatography on a Mono Q column. As shown in Fig.
1B, each fraction of purified p40 and p50 showed a single
band by SDS-PAGE under reducing conditions. Under non-reducing
conditions, p40 and p50 are resolved mainly into 180 and 240 kDa
components and 150, 300, and 450 components, respectively.
Two-dimensional electrophoresis indicated that these proteins could be
reduced to ~40 kDa and ~50 kDa, respectively (data not shown).
These results suggest that p40 and p50 are composed of subunits linked
to form homopolymers via disulfide bonds, as is the case with human
ficolin/P35.
Binding Specificity of p40 for Glycoproteins, Neoglycoproteins, and
Neoglycolipids--
To investigate the lectin activity of p40, its
binding specificity for various glycoproteins and neoglycoproteins was
evaluated. In a binding assay with neoglycoprotein, p40 bound
GlcNAc-BSA, GalNAc-BSA, and Sia-LacNAc-BSA but not to the other
neoglycoproteins (Fig. 2A). In
particular, p40 recognized GlcNAc with the most sensitivity
(GlcNAc > GalNAc > Sia-LacNAc). Furthermore, it was notable
that p40 did not bind to LacNAc-BSA with carbohydrate structure of
Gal
We next performed similar binding assays using neoglycolipids
prepared by reductive amination from dipalmitoyl
phosphatidylethanolamine (DPPE) and the following carbohydrates as
described previously (30): GlcNAc, GlcNAc2, GlcNAc3, GlcNAc4,
GlcNAc5, GlcNAc6, and GlcNAc Binding Specificity of p50 for Glycoproteins and
Neoglycoproteins--
As shown in Fig.
3A, p50 bound to GlcNAc-BSA in
a dose-dependent manner and bound weakly to GalNAc-BSA, but
did not bind to the others (Man-BSA, Glc-BSA, Gal-BSA, Lac-BSA,
Cel-BSA, LacNAc-BSA, and Sia-LacNAc-BSA). It also did not bind to any
of the glycoproteins tested (data not shown). An inhibition assay with
GlcNAc showed that more than 10 mM GlcNAc completely
inhibited p50 binding to GlcNAc-BSA (Fig. 3B). Thus, it is
likely that p50 exclusively recognizes GlcNAc at non-reducing
terminal.
Amino Acid Sequence Analyses of p40 and p50--
The
NH2-terminal amino acid sequences of p40 (27 amino acids)
and p50 (24 amino acids) were determined. These sequences showed no
significant similarity to other known proteins. In p40, the amino acid
sequence, HNEDLXTGLPNQLQEHXSLPESGVIIE, was
obtained and an additional weak arginine (R) and threonine (T) appeared at positions 1 and 22 (shown in
bold) of the mature protein, respectively. After
fragmentation of p40 and p50 with S. aureus V8 protease,
partial internal amino acid sequences were also determined. The
NH2-terminal sequence (VYCDLTSDGGGWTVFQRRMDGSVDF) of one of
the peptides from p40 was the same as that of a peptide from p50. This
sequence has significant similarity to a portion of the fibrinogen-like
sequences in mammalian ficolin, tenascin and fibrinogen.
cDNA Cloning of p40 and p50--
Using degenerated primers
prepared based on the NH2-terminal amino acid sequences of
p40 and p50, and those of the fragmented peptides, we performed nested
PCR. The cDNA sequence completed by 5'- and 3'-RACE showed that p40
cDNA contains a 972-base pair open-reading frame (ORF) encoding 324 amino acids preceded by a leader peptide of 17 amino acids, which we
designated AsFCN1 (Fig. 4). The predicted
molecular size of mature AsFCN1 was calculated to be 34,799 Da. In the
cDNA cloning of AsFCN1, another cDNA clone was identified with
hepatopancreas cDNA. This cDNA clone, termed AsFCN2, encodes
324 amino acids, which had 92.6% identity with AsFCN1 and had the same
domain structure (Fig. 4). The p50 cDNA contains a 1,068-base pair
ORF encoding 356 amino acids preceded by a leader peptide of 21 amino
acids. The predicted molecular size of mature p50, designated AsFCN3,
was calculated to be 38,229 Da. The overall amino acid sequence
identity between AsFCN1 and AsFCN3 was 69.3%. In the cDNA cloning
of AsFCN3, we also identified another clone, termed AsFCN4. AsFCN4
cDNA contains a 1023-base pair ORF encoding 341 amino acids, which
has 76.2%, 69.2, and 70.9% identities to AsFCN1, 2, and 3, respectively.
Because AsFCN1 and AsFCN3 contained three and four potential
N-linked glycosylation sites, respectively, the molecular
sizes observed on SDS-PAGE seem to result from glycosylation. Both
AsFCN1 and AsFCN3 have a short collagenous sequence, five
Gly-X-Y triplet repeats (where X and
Y represent any amino acid) and a COOH-terminal fibrinogen-like domain, suggesting ficolin-like lectins. As shown in
Fig. 4, alignment of the amino acid sequences of these four AsFCNs
indicated that they are closely similar to each other, especially in
their fibrinogen-like domains. The fibrinogen-like domain sequences of
the AsFCNs showed from 45.4 to 52.4% identity with those of human ficolins.
Northern Blot Hybridization of AsFCNs--
Because the
fibrinogen-like domains of the AsFCNs are highly homologous, the 5'-UT
sequences of AsFCN1, 3, and 4 were used as probes in Northern blots. As
shown in Fig. 5, the major transcripts of
AsFCNs1, 3, and 4 expressed in the hepatopancreas are about 1.3, 1.4, and 1.3 kilobases long, respectively. From a comparison with the signal
intensities, it appears that AsFCN3 and 4 are the major products in
ascidian hepatopancreas. The faint signal observed in the AsFCN1 blot
could result from both AsFCN1 and 2 transcripts because the sequences
of AsFCN1 and AsFCN2 cDNAs are very similar. No detectable signals
of AsFCNs were observed in Northern blots with the poly(A)+
fraction from ascidian hemocytes (data not shown).
Phylogenetic Tree of Ficolins, Fibrinogen-like Domain-bearing
Proteins and Fibrinogens--
The phylogenetic relationships between
AsFCNs and other fibrinogen-like proteins were analyzed by
neighbor-joining trees using the regional amino acid sequences of their
fibrinogen-like domains. As shown in Fig.
6, all of the sequences in
fibrinogen-like proteins examined such as AsFCNs, mammalian ficolins,
tenascins, angiopoietins, and horseshoe crab tachylectins roughly
formed a single large branch. In this tree, four AsFCNs formed a tight
cluster. The branch of the AsFCNs did not originate directly from that
of the mammalian ficolins, which also formed independently a tight
branch, making it difficult to define their origin. Members of the
tenascin family also formed a unique branch between the branches of the AsFCNs and the mammalian ficolins. The phylogenetic relationship of
these three groups is not convincing, because the relevant bootstrap
values are not high enough to support the branching definitely. The
fibrinogen-like sequences of AsFCNs were slightly more similar to those
of mammalian ficolins (45-52% identity) than to those of tenascins
(45-48% identity).
In this study, we isolated two novel ascidian lectins present in
hemolymph from a solitary ascidian, H. roretzi by affinity chromatography using GlcNAc-agarose followed by chromatography on Mono
Q, and cloned four lectins from cDNA libraries of the ascidian
hepatopancreas. The deduced amino acid sequences of these ascidian
lectins predict that they have major features of the mammalian ficolin
family. Because these lectins contained three structural domains
consisting of an NH2-terminal domain containing cysteine
residues, a collagen-like domain, and a fibrinogen-like domain, we
designated these lectins as ascidian ficolin, AsFCN 1, 2, 3, and 4. Most remarkably, these AsFCNs are the first invertebrate lectins to be
identified that have characteristic collagen-like and fibrinogen-like
domains. In comparison with the mammalian ficolin family, AsFCNs have
shorter collagen-like domains (5 Gly-X-Y repeats,
whereas those of the mammalian ficolin family contain 11-19
Gly-X-Y repeats). The fibrinogen-like domains of
AsFCNs are 45.4 to 52.4% identical with those of the mammalian ficolin family, which also specifically recognize GlcNAc.
A phylogenetic tree based on the amino acid sequences of the
fibrinogen-like domains indicated that the fibrinogen-like proteins formed a cluster independent of the primary fibrinogen sequences (Fig.
6), suggesting that all the fibrinogen-like domains may have evolved
from a common ancestor that branched off an authentic fibrinogen. In
this tree, four AsFCNs form a group that is independent of the
mammalian ficolin family. Another phylogenetic tree, which was
constructed based on the entire amino acid sequences of AsFCNs and the
mammalian ficolin family, indicated that the AsFCNs again form a tight
cluster (data not shown). These results suggest that the gene
duplication that produced these four genes was a recent event that most
probably occurred in the tunicate lineage. The tree did not clearly
define the origin of AsFCNs from a common lineage with mammalian
ficolins and suggests that the fibrinogen-like domains of AsFCNs may be
related to those of the tenascin family. However, the slightly higher
identity of the fibrinogen-like sequences of AsFCNs with those of
mammalian ficolins compared to other fibrinogen-like domains
(i.e. 45-48% for tenascins, 36-44% for angiopoietins, and 34-42% for fibrinogens) may support a common ancestor for both
ascidian and mammalian ficolins. An advanced phylogenetic tree
containing the sequences of lower vertebrate ficolins that have not yet
been identified may reveal the definite origin of AsFCNs.
We identified two ascidian ficolin proteins, p40 and p50. Based on the
amino acid sequence of p40, we cloned AsFCN1 and AsFCN2. The alignment
of the amino acid sequences of AsFCN1 and AsFCN2 showed that the
NH2-terminal half was the same except for two amino acids,
histidine (H) and serine (S) of AcFCN1, and
arginine (R) and threonine (T) of AsFCN2,
respectively (Fig. 4). The latter two amino acids were also recognized
as an additional signal in the amino acid sequence analysis of p40.
Therefore, it is possible that p40 contains both AsFCN1 and AsFCN2.
With Northern blot analysis, we showed major expression of AsFCN1, 3 and 4 transcripts in ascidian hepatopancreas (Fig. 5). In this
experiment, we could not design a specific probe for AsFCN2 because of
its high similarity with AsFCN1. The message obtained with the AsFCN1
probe contained that of AsFCN2. Fig. 5 shows lower expression of
AsFCN1/2 in hepatopancreas, which corresponds to the quantity of p40 in
ascidian hemolymph (Fig. 1A). On the other hand, the p50
transcript seems identical to that of the AsFCN3 transcript since a
different size of AsFCN4 transcript is observed in Northern blot
analysis (Fig. 5). In this respect we have not yet identified the
AsFCN4 protein.
Binding studies with neoglycoproteins, glycoproteins, and
neoglycolipids indicate that p40 (AsFCN1/2) is able to bind to an N-acetyl group in association with a pyranose ring. The
horseshoe crab lectins, tachylectin 5A and 5B, which have similar
fibrinogen-like structures but lack collagen-like domains, also
recognize the N-acetyl group (34). In this respect p40
(AsFCN1/2) is similar to the horseshoe crab lectins, but differs in its
requirement for a pyranose ring. In addition, collectin-like lectin has
been identified in a different species of ascidian as a GalNAc-binding lectin with a collagen-like domain, although the complete structure has
not been elucidated (35). It is possible that this lectin and AsFCN1/2
diverged from the same ancestor and have similar physiological
functions through their lectin activities. On the other hand, the
binding specificity of p50 (AsFCN3) involves recognition of GlcNAc
alone (Fig. 3). Thus, each fibrinogen-like domain of AsFCNs has a
lectin activity associated with a different sugar specificity. Although
the AsFCNs have much shorter collagen-like sequences than the mammalian
ficolins, the present study elucidated the structural and functional
features common to the ascidian and mammalian ficolins, including
domain structure, multimeric property, and lectin activity.
In the primitive complement system of ascidian, the activation pathway
consisting of at least of C3 (21), factor B (36), and MASPs (22) has
been demonstrated and corresponds to the mammalian alternative and
lectin pathways. Although an ascidian MBL homolog has not been fully
identified, we have identified a similar lectin, termed glucose-binding
lectin (GBL), which lacks a collagen-like domain in its
NH2-terminal region and which specifically recognizes
glucose residues (23). GBL is associated with ascidian MASPs that
activate ascidian C3 (21). Recently, we showed that human ficolin/P35,
associated with MASPs, activates the lectin complement pathway (20).
Therefore, it is possible that AsFCNs function as the recognition
molecule of the ascidian lectin pathway. Our preliminary experiments
show that p40 (AsFCN1/2) is associated with AsMASPs and activates
ascidian C3. Data are accumulating that show that the engagement of
ligands by MBL or ficolin results in activation of the MASPs, which in
turn activate C3 directly (6). MASPs have been identified in lamprey
(37) and ascidian (22) and C3 in ascidian (21) and sea urchin (38).
This leads to the prediction that MBL or ficolin MASPs and C3 may be
the minimum ancestral components of the complement system. Thus, the identification of AsFCNs described in this report may contribute to the
clarification of the primitive complement system.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-binding proteins on porcine uterus
membrane (11). Ficolins have been identified in mammals including human
(12-15), rodents (16, 17), pig (11), and hedgehog (18) and have
tissue-specific distributions. Serum ficolins from human, mouse, and
pig are lectins with a common binding specificity for GlcNAc (12,
15-17). It is likely that the fibrinogen-like domain of serum ficolins
is responsible for carbohydrate binding (19). In human serum, two types
of ficolin, named ficolin/P35 (ficolin L) (12, 14) and ficolin H
(Hakata antigen) (15), have been identified and both of them have
lectin activity. Another ficolin, termed ficolin M (14), or P35-related protein (13), whose mRNA is found in leukocytes and lung is not
considered to be a serum protein. Recently it has been reported that
ficolin M might act as a phagocytic receptor or adaptor on circulating
monocytes for microorganism recognition (12). Ficolin/P35 acts as an
opsonin and enhances phagocytosis of Salmonella typhimurium by neutrophil (20). It also activates the lectin complement pathway in
association with MASPs (21). These findings suggest that ficolins are
involved in host defense through innate immunity in the vertebrate.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-3N-acetyllactosamine (Sia-LacNAc-BSA) from Dextra Laboratories (UK); neoglycoproteins of Man-BSA, Glc-BSA,
GlcNAc-BSA, and Gal-BSA from E. Y. Laboratories (San Mateo, CA);
GlcNAc from Wako Pure Chemical Industries (Osaka, Japan), and
Na125I and [
-32P]dCTP from Amersham
Pharmacia Biotech.
2-3N-acetyllactosamine-conjugated BSA
(Sia-LacNAc-BSA) were used for binding assays. In each neoglycoprotein,
~19-42 mol of carbohydrate were conjugated to 1 mol of BSA.
Ovalbumin contains high mannose-type and hybrid-type oligosaccharides
(25, 26); RNase B, high mannose-type oligosaccharides (27); human
fetuin and human asialofetuin, both complex-type oligosaccharides and O-linked oligosaccharides (28, 29).
1-2Man
1-6(GlcNAc
1-2Man
1-3)Man (GlcNAc2Man3). The
neoglycolipids were applied to two TLC plates (Merck, Darmstadt, Germany), chromatographed, and developed using
chloroform/methanol/water (60:35:8, by volume). After drying, one TLC
plate was used for the binding assay with 125I-labeled p40
(400,000 cpm/ml). The other TLC plate was sprayed with primulin reagent
and then subjected to a Lumino-image analyzer LAS-1000 (Fujifilm) for
lipid detection of neoglycolipids.
and -
(11), rat ficolin and
mouse ficolin-A (16) and -B (17), of other proteins with a
fibrinogen-like domain, and of fibrinogens were aligned using ClustalW
software (EMBL Data Library, Heidelberg, Germany). 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 (33). Bootstrap analysis was used to assess the
reliability of branching patterns. For each tree, 1000 bootstrap
replications were performed.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (82K):
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Fig. 1.
SDS-PAGE of p40 and p50. A,
eluate from a GlcNAc-agarose column was subjected to SDS-PAGE under
reducing conditions (10% gel). B, SDS-PAGE of purified p40
and p50 under reducing (left) and non-reducing
(right) conditions (a, p40; b, p50).
Proteins were stained with Coomassie Brilliant Blue R-250. Molecular
size markers are indicated on both sides.
1-4GlcNAc, suggesting that this lectin can recognize non-reducing terminal carbohydrate residues carrying
N-acetyl group such as GlcNAc, GalNAc, and NeuAc. Another
binding assay was performed with natural glycoproteins, i.e.
ovalbumin (OVA) with high mannose-type and hybrid-type
oligosaccharides, bovine RNase B (RN) with only high
mannose-type oligosaccharides, human fetuin and human asialofetuin,
both with complex-type oligosaccharides and O-linked
oligosaccharides. The results showed that p40 bound to fetuin, which
has oligosaccharides with N-acetylneuraminic acid at their
non-reducing termini (Fig. 2B). Furthermore, one of the
simplest chemicals containing an N-acetyl groups,
N-acetylglycine, inhibited p40 binding to GlcNAc-BSA
(Fig. 2C).
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Fig. 2.
Carbohydrate recognition by
p40. A, binding of p40 to neoglycoproteins. Nine
neoglycoproteins, BSA conjugated with N-acetylglucosamine
(GlcNAc-BSA), N-acetylgalactosamine (GalNAc-BSA),
N-acetyllactosamine (LacNAc-BSA), NeuAc 2-3
N-acetyllactosamine (Sia-LacNAc-BSA), mannose (Man-BSA),
glucose (Glc-BSA), galactose (Gal-BSA), lactose (Lac-BSA), and
cellobiose (Cel-BSA) were dot-blotted onto a PVDF membrane in a volume
of 100 µl each at the concentration indicated. The membrane was
incubated with 125I-labeled p40 and then analyzed for p40
binding. The level of binding to each dot is indicated as intensity of
the photostimulated luminescence (PSL). B,
binding of p40 to natural glycoproteins. Four natural glycoproteins,
human fetuin (Fet), human asialofetuin (asFet),
ovalbumin (OVA), and RNase B (RN) were
dot-blotted onto a PVDF membrane in a volume of 100 µl each at the
concentration indicated. The membrane was incubated with
125I-labeled p40 and then analyzed for p40 binding as in
Fig. 2A. C, inhibition of p40 binding. The
neoglycoprotein, GlcNAc-BSA in 100 µl was dot-blotted onto a PVDF
membrane at the concentration indicated. 125I-labeled p40
was pre-incubated without (Control) or with 10 mM sodium acetate or 10 mM
N-acetylglycine and then subjected to the binding
assay. D, binding of 125I-labeled p40 to
neoglycolipids. Seven neoglycolipids prepared from GlcNAc
(GlcNAc-DPPE), di-N-acetylchitobiose (GlcNAc2-DPPE),
tri-N-acetylchitotriose (GlcNAc3-DPPE),
tetra-N-acetylchitotetraose (GlcNAc4-DPPE),
penta-N-acetyl-chitopentaose (GlcNAc5-DPPE),
hexa-N-acetylchitohexaose (GlcNAc6-DPPE), and
GlcNAc
1-2Man
1-6(GlcNAc
1-2Man
1-3)Man (GlcNAc2Man3-DPPE)
were spotted on two TLC plates and then chromatographed. One plate was
overlaid with 125I-labeled p40, incubated, and then
analyzed for p40 binding (lower panel). The other plate was
stained with primulin reagent for lipid detection of neoglycolipids
(upper panel).
1-2Man
1-6(GlcNAc
1-2Man
1-3)Man
(GlcNAc2Man3). These conjugates were chromatographed on a TLC plate,
which was then used for a binding assay with 125I-labeled
p40. As shown in Fig. 2D, radiolabeled p40 bound to all the
neoglycolipids with the exception of GlcNAc-DPPE. The failure of p40 to
bind to GlcNAc-DPPE may be caused by a structural change in the
pyranose ring by reductive amination from DPPE. These results suggest
that carbohydrate recognition by p40 may require not only an
N-acetyl group but also an intact pyranose ring structure.
View larger version (27K):
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Fig. 3.
Carbohydrate recognition by p50.
A, binding of p50 to neoglycoproteins. Nine neoglycoproteins
were dot-blotted onto a PVDF membrane in a volume of 100 µl each at
the concentration indicated, as described in the legend to Fig. 2. The
membrane was incubated with 125I-labeled p50 and then
analyzed for p50 binding. The level of binding to each dot is indicated
as intensity of the photostimulated luminescence (PSL).
B, inhibition of p50 binding by GlcNAc. The neoglycoprotein,
GlcNAc-BSA in a volume of 100 µl was dot-blotted onto a PVDF membrane
at the concentration indicated. 125I-labeled p50 was
preincubated without (Control) or with 1, 10, or 100 mM GlcNAc and then subjected to the binding assay.
View larger version (78K):
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Fig. 4.
Alignment of the entire amino acid sequences
of four AsFCNs with human ficolins (HFCN/P35, HFCN M, and HFCN H).
Gaps inserted to increase identity are shown by dashes. The
leader sequences of AsFCN1 and AsFCN3 are underlined and the
following 27 and 24 amino acid sequences were determined from the
NH2-terminal of p40 and p50, respectively. The
collagen-like sequences are boxed. Asterisks and
dots below the sequences indicate the conserved and similar
residues among these seven sequences, respectively. The bold
amino acids show two different residues between AsFCN1 and AsFCN2 in
the NH2-terminal half. HFCN/P35, human
ficolin/P35 (GenBankTM/EBI accession number D49353);
HFCN M, human ficolin/P35-related protein or ficolin M
(NM_002003); HFCN H, human ficolin H or Hakata antigen
(D88587).
View larger version (77K):
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Fig. 5.
Northern blotting analysis of AsFCNs. A
membrane filter containing 2 µg of poly(A)+ fraction from
hepatopancrease was hybridized with 32P-labeled cDNA
fragments (a, AsFCN1 nucleotides 7-326; b,
AsFCN3 nucleotides 14-336; c, AsFCN4 nucleotides
69-384).
View larger version (41K):
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Fig. 6.
Phylogenetic tree of ficolins,
fibrinogen-like domain-bearing proteins, and fibrinogens. The tree
was constructed based on the alignments of sequences of the
fibrinogen-like domain from 43 proteins. The numbers on branches are
bootstrap percentages supporting a given partitioning. PeFIB2:
Petromyzon marinus fibrinogen -2 (P33573),
CFIBA, chichen fibrinogen
(P14448); HFIBA,
human fibrinogen
(P02671); RFIBA, rat fibrinogen
(P06399); CFIBB, chicken fibrinogen
(Q02020);
HFIBB, human fibrinogen
(P02675); RFIBB, rat
fibrinogen
(P14480); BFIBB, bovine fibrinogen
(P02676); BFIBG, bovine fibrinogen
(P12799);
HFIBG, human fibrinogen
(P02679); RFIBG, rat
fibrinogen
(P02680); XFIBG, Xenopus
fibrinogen
(P17634); PeFIBG, Petromyzon
marinus fibrinogen
(P04115); MFGL2, mouse
fibrinogen-like protein 2 (P12804); HFGL2, human
fibrinogen-like protein 2 (Q14314); HAGP1, human
angiopoietin-1 (Q15389); MAGP1, mouse angiopoietin-1
(O08538); BAGP1, bovine angiopoietin-1 (O18920);
HAGP2, human angiopoietin-2 (O15123); BAGP2,
bovine angiopoietin-2 (O77802); MAGP2, mouse angiopoietin-2
(O35608); PoFCN
, porcine ficolin
(L12344);
PoFCN
, porcine ficolin
(L12345); MFCNB,
mouse ficolin B (AF063217); MFCNA, mouse ficolin A
(AB007813); RFCN, rat ficolin (AB026057); PaFIBA,
Parastichopus parvimensis fibrinogen-like protein A
(P19477); CTNC, chicken tenascin C (P10039);
HTNC, human tenascin C (P24821); PoTNC, porcine
tenascin C (Q29116); ZTN, zebrafish tenascin (Q90484);
HTNX, human tenascin X (P22105); PoTNX, porcine
tenascin X (Q29038); HMFA4, human microfibril-associated
glycoprotein 4 (P55083); TL-5A, horseshoe crab
tachylectin-5A (AB024737); TL-5B, tachylectin-5B
(AB024738).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by grants-in-aid for scientific research from the Japan Society for the Promotion of Science.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequences reported in this paper have been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession numbers AB049619, AB049620, AB049621, and AB049622.
§ To whom correspondence should be addressed. Tel.: 81-24-548-2111, Ext. 2230; Fax: 81-24-548-6760; E-mail: tfujita@fmu.ac.jp.
Published, JBC Papers in Press, March 20, 2001, DOI 10.1074/jbc.M011723200
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ABBREVIATIONS |
---|
The abbreviations used are: MBL, mannose-binding lectin; BSA, bovine serum albumin; AsFCN, ascidian ficolin; MASP, MBL-associated serine protease; PAGE, polyacrylamide gel electrophoresis; PSL, photostimulated luminescence; PVDF, polyvinylidene difluoride; DPPE, dipalmitoyl phosphatidylethanolamine; GlcNAc, N-acetylglucosamine; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; ORF, open-reading frame.
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