A Newly Identified Horseshoe Crab Lectin with Specificity for
Blood Group A Antigen Recognizes Specific O-Antigens of Bacterial
Lipopolysaccharides*
Kei-ichiro
Inamori
§,
Tetsu
Saito
,
Daisuke
Iwaki
§,
Tsutomu
Nagira¶,
Sadaaki
Iwanaga¶,
Fumio
Arisaka
, and
Shun-ichiro
Kawabata
¶**
From the Departments of
Molecular Biology, Graduate
School of Medical Science and ¶ Biology, Faculty of Science,
Kyushu University, Fukuoka 812-8581, the
Department of Life
Science, Faculty of Bioscience and Bioengineering, Tokyo Institute
of Technology, Yokohama 226-8501, and the ** Core Research for
Evolutional Science and Technology, Japan Science and Technology
Corporation, Tokyo 101-0062, Japan
 |
ABSTRACT |
A 14-kDa lectin, named
tachylectin-3, was newly identified from hemocytes of the Japanese
horseshoe crab, Tachypleus tridentatus. This lectin
exhibited hemagglutinating activity against human A-type erythrocytes,
but not against the B- and O-types of erythrocytes and animal
erythrocytes, including those of sheep, rabbit, horse, and bovine. The
hemagglutinating activity of tachylectin-3 was equivalent to that of a
previously identified lectin, named tachylectin-2, with affinity for
N-acetyl-D-glucosamine or
N-acetyl-D-galactosamine. However, the activity
of tachylectin-3 was not inhibited by these two
N-acetylhexosamines at 100 mM but was inhibited
by a blood group A-pentasaccharide at a minimum inhibitory
concentration of 0.16 mM. Furthermore, the hemagglutinating
activity was strongly inhibited by bacterial S-type lipopolysaccharides
(LPSs) from Gram-negative bacteria but not by R-type LPSs lacking
O-antigens. One of the most effective S-type LPSs was from
Escherichia coli O111:B4, with a minimum inhibitory
concentration of 6 ng/ml. These data suggest that tachylectin-3
specifically recognizes Gram-negative bacteria through the unique
structural units of O-antigens. Ultracentrifugation analysis revealed
that tachylectin-3 is present in dimer in solution. A cDNA coding
for tachylectin-3 was isolated from a hemocyte cDNA library.
Tachylectin-3 consisted of two repeating sequences, each with a partial
sequence similarity to rinderpest virus neuraminidase. Tachylectin-3
and three previously isolated types of tachylectins were all
predominantly expressed in hemocytes and released from hemocytes in
response to external stimuli. These lectins present at injured sites
suggest that they probably serve synergistically to accomplish an
effective host defense against invading microbes.
 |
INTRODUCTION |
Immunity to infectious agents is mediated by two general systems,
innate and acquired. Acquired immunity, found only in vertebrates, is
one function of B and T lymphocytes, which produce an infinite multitude of specific antigen receptors and antibodies through somatic
gene rearrangement. On the other hand, innate immunity is
phylogenetically older than acquired immunity, and a certain form of
innate immunity is present in all multicellular organisms. Non-self-recognizing proteins involved in innate immunity seem to
recognize mainly carbohydrate moieties on pathogens; for instance, a
receptor CD14 (1) on macrophages for bacterial lipopolysaccharides (LPSs),1 C-reactive protein
(2), and the mannose-binding lectin of the collectin family in
mammalian plasma (3). Furthermore, the innate immunity in invertebrates
is also triggered by polysaccharides, such as LPSs and
-1,3-glucans,
as seen in the hemolymph coagulation system in horseshoe crab (4-9)
and the phenoloxidase-mediated melanization system in crustaceans and
insects (10).
In the Japanese horseshoe crab, Tachypleus tridentatus, one
of the major defense systems is carried by hemolymph that contains granular hemocytes comprising 99% of the total hemocytes (11). These
granular hemocytes are filled with two populations of secretory granules, named large (L) and small (S) granules. These granules selectively store defense molecules, such as clotting serine protease zymogens, a clottable protein coagulogen, protease inhibitors, lectins,
and antimicrobial peptides (4-9). The hemocytes are highly sensitive
to LPSs, and these defense molecules stored in both granules are
secreted by exocytosis after stimulation with LPSs. This response is
important for the host defense related to engulfing and killing
invading microbes, in addition to preventing the leakage of hemolymph.
We reported evidence of several types of hemocyte-derived lectins,
named tachylectins, which may play in a functional role of the innate
immunity of this animal. Tachylectin-1, identical to an
L-granule-derived protein L6 (12), binds to both S- and R-types of LPSs
and inhibits the growth of Gram-negative bacteria (13). Tachylectin-2,
identical to another L-granule-derived protein, L10 (12), exhibits
binding specificity to D-GlcNAc or D-GalNAc and
agglutinates a certain strain of Staphylococcus (14).
Tachylectins-4 has affinity for L-fucose and binds to S-type LPSs from several Gram-negative bacteria through O-specific polysaccharides (O-antigens) (15). We have now identified a new type of
the hemocyte-derived lectin, named tachylectin-3, which differs
structurally and functionally from those of the tachylectins mentioned
above. Tachylectins-3 has specificity for a blood group A antigen and
recognizes specific O-antigens of bacterial lipopolysaccharides.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Hemocyte lysate from T. tridentatus
was prepared as described (16), except that the procedure was carried
out in the presence of 10 mM CaCl2, and NaCl
was added to the lysate to give a final concentration of 0.5 M. LPSs from Escherichia coli O111:B4, E. coli O55:B5, Salmonella minnesota, and S. minnesota R595 were from List Biological Laboratories, Inc.
(Campbell, CA). LPSs from E. coli O127:B8, E. coli O128:B12, E. coli O26:B6, Klebsiella pneumoniae, Shigella flexneri 1A, Pseudomonas
aeruginosa 10, Vibrio cholerae Inaba 569B, E. coli EH100, E. coli J5, E. coli F583, S. minnesota R7, Salmonella typhimurium, S. typhimurium TV119, and S. typhimurium SL684 and LTA
from Staphylococcus aureus, Bacillus subtilis,
Streptococcus faecalis, Streptococcus mutans,
Streptococcus pyogenes, and Streptococcus sanguis
were from Sigma. LPSs from V. cholerae O22 and V. cholerae O139 were kindly provided by Drs. S. Kondo and K. Hisatsune of Josai University. Sepharose CL-6B was from Amersham
Pharmacia Biotech. CM-Toyopearl 650M was from TOSOH Corp. (Tokyo,
Japan). Lysyl endopeptidase was from Wako Pure Chemical Industries,
Ltd. (Tokyo, Japan). A
ZipLox cDNA library was constructed from
poly(A+) RNA extracted from hemocytes, using a TimeSaverTM
cDNA synthesis kit (Amersham Pharmacia Biotech) and
ZipLoxTM EcoRI arms (Life Technologies,
Inc.).
Hemagglutinating Activity and Inhibition
Assay--
Hemagglutinating activity and the inhibition assay were
done as described (14, 17).
Analytical Ultracentrifugation--
Sedimentation equilibrium
run for tachylectin-3 was performed in 50 mM MES, pH 6.0, containing 0.25 M NaCl by Beckman XL-A at 18,000 rpm at
20 °C.
Isolation of Tachylectin-3-derived Peptides--
Tachylectin-3
was reduced, S-alkylated with iodoacetamide, and then digested with
lysyl endopeptidase (E/S = 1/100) at 37 °C for 24 h. The digest
was separated by reverse-phase high performance liquid chromatography
using a Chemcosorb 5-ODS-H column (2.1 × 150 mm, Chemco
Scientific Co., Ltd., Osaka, Japan), and the isolated peptides were
subjected to amino acid and sequence analyses.
Tachylectin-3-specific DNA Probes and Screening of cDNA
Library--
The degenerate nucleotide sequences of primers used for
PCR were based on peptides derived from lysyl endopeptidase digestion (CVTDNT and TWNCIK) of tachylectin-3. Sense and antisense nucleotides were synthesized with an EcoRI site at the 5' end. Reactions
for PCR contained the cDNA template (corresponding to 0.1 µg of
poly(A)+ RNA) and 100 pmol each of the primer were carried
out in a Perkin-Elmer Cetus thermal cycler. The PCR products were
treated with EcoRI and purified with agarose gel
electrophoresis. Fragments of interest were then ligated into plasmid
Bluescript II SK (Stratagene, La Jolla, CA) for sequence analysis, as
described (18). One clone (0.15 kilobase pair), containing the sequence
of tachylectin-3, was labeled with [
-32P]dCTP using a
Ready-To-GoTM DNA labeling kit (Amersham Pharmacia Biotech), and this
served as a probe to screen the
ZipLox cDNA library.
Determination of Disulfide Linkages--
Tachylectin-3 was
dissolved in 0.1 M Tris-HCl, pH 6.8, containing 2 M urea and 0.01% SDS, and digested with lysyl
endopeptidase at 37 °C for 16 h. The digest was separated by
reverse-phase high performance liquid chromatography on a
µBondasphere 5C8 column (2.1 × 150 mm, Nihon Waters Ltd.,
Tokyo). Two peptides containing disulfide bonds were identified by
amino acid analysis after performic acid oxidation. The positions of
disulfide linkages were confirmed directly by amino acid sequence
analysis, as described (19).
Amino Acid and Sequence Analyses--
Amino acid analysis was
performed on a PICO-TAG system (Waters, Millipore, Milford, MA) or a
Hitachi L-8500 amino acid analyzer. Protein concentration for
determining the extinction coefficient of tachylectin-3 was calculated
from the amino acid mass/A280. An internal
standard, norleucine, was added to the protein hydrolysates to allow
correction for losses. Amino acid sequence analysis was performed using
an Applied Biosystems 473A or 477A sequencer.
Exocytosis of Horseshoe Crab Hemocytes--
Exocytosis of
hemocytes with ionophore A23187 (Sigma) was done as described (20,
21).
Biotinylation of Polyclonal Antibodies against
Tachylectins--
An antiserum against tachylectin-3 was raised in
rabbits, as described (13, 14). Polyclonal antibodies against
tachylectin-1 (13), tachylectin-2 (14) and tachylectin-3 were purified
from rabbit antisera with the Ampure PA kit (Amersham Pharmacia
Biotech). The IgG fractions obtained were dialyzed against 0.1 M NaHCO3 and biotinylated with EZ-LinkTM
NHS-LC-Biotin, according to a protocol provided by the manufacturer (Pierce).
Enzyme-linked Immunosorbent Assay (ELISA)--
Microtiter plates
were coated with non-biotinylated antibodies against tachylectins
(0.5-10 µg/ml in 20 mM sodium phosphate, pH 7.0, containing 0.1 M NaCl) by incubating overnight at 4 °C. After washing with the same buffer, the plates were blocked with 2.5%
casein in washing buffer, and 2-fold serial dilutions of samples were
added, incubated at 37 °C for 1 h, and then washed. Biotinylated antibodies were added and incubated at 37 °C for 1 h and washed. Streptavidin-biotinylated horseradish peroxidase complex
(Amersham Pharmacia Biotech) was added and incubated at 37 °C for
1 h. The enzyme activity of horseradish peroxidase was detected
with o-phenylenediamine at 490 nm, using a microplate reader, model 3550 (Bio-Rad). For measurement of coagulogen, a monoclonal antibody (14B1) was used instead of biotinylated IgG, and
was detected by horseradish peroxidase conjugated goat anti-mouse IgG
(Bio-Rad).
SDS-PAGE and Immunoblotting--
SDS-PAGE was performed
according to the method of Laemmli (22). For immunoblotting, gels were
transferred to nitrocellulose membranes overnight at 30 V using an
electroblot apparatus (Bio-Rad). The membranes were then treated with
antiserum and incubated with horseradish peroxidase-conjugated goat
anti-rabbit IgG and visualized using an ECL kit, according to protocol
provided by the manufacturer (Amersham Pharmacia Biotech).
Reverse Transcription-PCR Analysis--
Total RNA was extracted
from various tissues of T. tridentatus according to
Chomczynski and Sacchi (23). First-strand cDNA synthesis from 0.1 µg of poly(A)+ RNA was performed using SuperScriptTM II
Rnase H
reverse transcriptase (Life Technologies, Inc.)
and random primers. One one-hundredth of the first-strand of cDNA
and 200 pmol of each primer were subjected to PCR (30 cycles) with
denaturation at 94 °C for 0.5 min, annealing at 50 °C for 1 min,
and extension at 70 °C for 1 min. PCR products were analyzed on a
1.5% agarose gel and visualized following ethidium bromide staining.
Homology Search--
A computer-assisted homology search of
tachylectin-3 was made using the DNA Data Bank of Japan homology search
system and programs FASTA, Version 3.0, and BLAST, Version 1.4.9.
 |
RESULTS |
Purification of Tachylectin-3--
The lysate prepared from
12 g (wet weight) of hemocytes was applied to a Sepharose CL-6B
column (2.0 × 7.0 cm) equilibrated with 20 mM
Tris-HCl, pH 7.5, containing 0.5 M NaCl and 10 mM CaCl2, and washed extensively with the same
buffer. The typical elution pattern is shown in Fig.
1A. The flow-through fraction
showed hemagglutinating activity against human A-type erythrocytes.
This hemagglutinating activity depended heavily on tachylectin-2
and tachylectin-4, and both lectins had no affinity to Sepharose CL-6B (data not shown). However, a significant level of hemagglutinating activity was retained in the eluate, despite extensive washing, as
indicated in Fig. 1A, open circles. These data suggested the possible presence of a new lectin with weak binding affinity to the
resin. Fractions indicated by a solid bar were pooled and concentrated
with polyethylene glycol 20,000, and dialyzed against 50 mM
MES, pH 6.0, containing 25 mM NaCl. The dialyzed sample was
applied to a CM-Toyopearl column (2.0 × 4.5 cm) equilibrated with
the same buffer. After washing with equilibration buffer, hemagglutinating activity was eluted with a linear gradient of 25-500
mM NaCl, in the same buffer (Fig. 1B). The
pooled fractions indicated by a solid bar (Fig.
1B) gave a single protein band of 14 kDa on SDS-PAGE, under
reducing and nonreducing conditions (Fig. 1C). About 1.0 mg
of the protein could be purified reproducibly from 10 g of
hemocytes. We named this newly isolated lectin tachylectin-3.

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Fig. 1.
Elution profiles for tachylectin-3 from a
Sepharose CL-6B column and a CM-Toyopearl column and SDS-PAGE of
purified tachylectin-3. Experimental details are presented under
"Results." A, Sepharose CL-6B chromatography.
B, CM-Toyopearl chromatography. C, SDS-PAGE of
the retained fraction of Sepharose CL-6B chromatography (lane
1) and purified tachylectin-3 under reducing (lane 2)
and nonreducing (lane 3) conditions.
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The extinction coefficient of tachylectin-3 at 280 nm for 1% solution
in deionized water was calculated from the data on amino acid analysis,
and a value of 20.8 was used for subsequent determinations of protein concentrations.
Hemagglutinating Activity of Tachylectin-3--
Tachylectin-3
agglutinated trypsin-treated human A-type erythrocytes but not
trypsin-treated human B- and O-types of erythrocytes and native
animal-derived erythrocytes, including those of sheep, rabbit, horse,
and bovine. The hemagglutinating activity of tachylectin-3 for
trypsin-treated A-type erythrocytes was 8-fold more potent than that
for the native erythrocytes. Therefore, trypsinized A-type erythrocytes
were used for subsequent hemagglutination assays. The minimum
concentration required for agglutination of the trypsin-treated A-type
erythrocytes was 0.51 µg/ml. Ca2+ (10 mM),
Mg2+ (50 mM), or EDTA (100 mM) had
no apparent effects on the hemagglutinating activity.
The hemagglutinating activity of tachylectin-3 in 50 mM
MES, pH 6.0, containing 0.25 M NaCl, was gradually
decreased by repetitive freezing and thawing, and 10 cycles of freezing
and thawing led to a 90% loss of the initial hemagglutinating
activity. However, tachylectin-3 could be stored in the same buffer at
4 °C for at least 6 months without losing hemagglutinating activity.
Effects of Carbohydrates on Hemagglutination--
Monosaccharides,
including GlcNAc and GalNAc, did not inhibit the hemagglutinating
activity of tachylectin-3, even at 100 mM, as shown in
Table I. However, a blood group
A-pentasaccharide completely inhibited the hemagglutination at the
minimum inhibitory concentration (MIC) of 0.16 mM. On the
other hand, a blood group A-trisaccharide had no inhibitory effect at
2.5 mM, although it did inhibit the hemagglutinating
activity of tachylectin-2 at MIC of 0.63 mM.
LPSs from various Gram-negative bacteria were also examined, as shown
in Table II. S-type LPSs were potent
inhibitors, whereas R-type LPSs were not inhibitory. LPSs derived from
E. coli O111:B4, E. coli O55:B5, E. coli O127:B8, and V. cholerae O22 strongly inhibited
the hemagglutination at MIC of 0.006 µg/ml. However, LPSs isolated
from rough mutants of E. coli O111:B4, such as E. coli J5 (Rc), showed no inhibitory effect, suggesting that the O-antigen is important for carbohydrate recognition of tachylectin-3. On the other hand, S-type LPS from P. aeruginosa 10 had no
inhibitory effect. LTA, a cell wall component of Gram-positive
bacteria, also inhibited the hemagglutinating activity of
tachylectin-3. An LTA from S. aureus was the most potent
inhibitor, but the MIC of 3.1 µg/ml was significantly higher than
that of LPS from E. coli O111:B4 (0.006 µg/ml). These
results indicate that tachylectin-3 recognizes specific O-antigens of
LPSs derived from several Gram-negative bacteria.
Tachylectin-2, one of the hemocyte-derived lectins (14), exhibits
specificity against blood group A antigen, and it has high affinity for
monosaccharides, such as GlcNAc and GalNAc (14). Such being the case,
the inhibitory effects of LPSs and LTAs on the hemagglutinating
activity of tachylectin-2 were also examined and compared with those of
tachylectin-3 (Table II). S-type LPS from S. minnesota (S)
was the most effective inhibitor at an MIC of 12.5 µg/ml, and R-type
LPS from S. typhimurium TV119 (Ra) was also effective at an
MIC of 25 µg/ml, indicating that tachylectin-2 has no specificity for
the O-antigen. The LTA from S. aureus was the best inhibitor
with MIC of 6.3 µg/ml.
Analytical Ultracentrifugation--
Tachylectin-2 is present as a
monomeric protein in solution, as judged from ultracentrifugal analysis
(14); this ultracentrifugal analysis was also carried out for
tachylectin-3. The concentration gradients obtained for tachylectin-3
in the sedimentation equilibrium run could be well simulated, based on
the assumption of the presence of a single species with a molecular
weight of 28,850 ± 270, thus indicating that tachylectin-3 exists
as a dimer in solution.
Isolation of the cDNA Clone and the Nucleotide Sequence of
Tachylectin-3--
The tachylectin-3-specific probe of 0.15 kilobase
was identified with oligonucleotide-primers corresponding to peptides
derived from tachylectin-3, using PCR and DNA sequence analyses.
Screening a hemocyte cDNA library with the probe gave a positive
clone with a 0.6-kilobase insert, and it was subjected to restriction
mapping followed by sequence determination of both strands. The
nucleotide and deduced amino acid sequences are shown in Fig.
2. The cDNA included 618 nucleotides
with an open reading frame of 441 nucleotides. A stop codon, TGA
(nucleotide position 34), was followed by an initiation Met beginning
at position 79. The open reading frame for the cDNA encoded for a
mature protein of 123 amino acid residues and a signal sequence of 24 residues with a typical hydrophobic core (24). The stop codon at
position 519 was followed by two polyadenylation signals, AATAAA,
starting at positions 583 and 605. Amino acid sequences of the isolated
peptides derived from tachylectin-3 agreed with the protein sequence
deduced from the cDNA sequence. Glucosamine and galactosamine were
not detectable in tachylectin-3 by amino acid analysis. The deduced
sequence contained one potential N-linked glycosylation site
at Asn114 with the sequence Asn114-Ile-Ser, but
the residue was identified as phenylthiohydantoin-Asn by peptide
sequencing, indicating the absence of N-linked sugar chains
in tachylectin-3. The calculated molecular weight from the deduced
amino acid sequence was 13,723, which is in good agreement with that of
the purified protein estimated on SDS-PAGE (Mr = 14,000). The isoelectric point of tachylectin-3, calculated from the
amino acid composition, was 9.05, which is close to points for
tachylectin-1 (9.69) and tachylectin-2 (9.63), but not to that of
tachylectin-4 (6.05).

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Fig. 2.
Nucleotide and deduced amino acid sequences
of tachylectin-3. Nucleotides and amino acid residues are numbered
on the right. Single underlines represent
sequences determined by amino acid sequence analysis of isolated
peptides. An asterisk represents the termination
codon.
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Assignment of Disulfide Linkages in
Tachylectin-3--
Tachylectin-3 contains six cysteine residues, as
shown in Fig. 2. Tachylectin-3 was treated with iodoacetamide in the
presence of 8 M urea, under nonreducing conditions. Amino
acid analysis revealed that no cysteine residues were
S-alkylated, indicating the absence of SH-Cys and the
presence of three disulfide bridges in tachylectin-3. The intact
tachylectin-3 was digested with lysyl endopeptidase, and two
disulfide-containing peptides, TL3-K1 and TL3-K2, were isolated, as
described under "Experimental Procedures." By amino acid analysis,
TL3-K2 was found to be composed of two chains,
Thr44-Lys49 and
Arg86-Lys103, indicating the presence of
Cys47-Cys98. On the other hand, TL3-K1
contained four cysteines and was composed of three peptide chains,
Thr1-Lys14,
Thr50-Lys71, and
Pro19-Lys43, linked by two disulfides.
Sequence analysis of TL3-K1 revealed that phenylthiohydantoin-cystine
was recovered at the 10th and 11th cycles of Edman degradation, clearly
indicating that the two disulfide bonds were
Cys2-Cys60 and
Cys28-Cys58 (Table
III and Fig.
3A).

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Fig. 3.
Repetitive sequence and disulfide bond
locations of tachylectin-3 and alignment of the amino acid sequence
with hemagglutinin-neuraminidase of rinderpest virus.
A, repetitive sequence of tachylectin-3. Consensus amino
acid residues in the repeating units are indicated in boldface,
large letters. B, alignment of the amino acid sequences of each
repeat of TL-3 with that of corresponding portion of
hemagglutinin-neuraminidase of rinderpest virus strain Kabete O
(HEMA-RINDK).
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Sequence Similarity to Other Proteins--
A search of the DNA
Data Bank of Japan showed a partial sequence similarity to
hemagglutinin-neuraminidase of rinderpest virus (25, 26), as shown in
Fig. 3B. The sequence identities between the 39 amino acids
in each repetitive sequence (positions 10-48 and 66-104) of
tachylectin-3 and the sequence (positions 432-470) of the
hemagglutinin-neuraminidase were 33 and 23%, respectively. The
sequence similarity suggests that the region 432-470 of neuraminidase plays a important role in sugar binding.
Subcellular Localization and Exocytosis of
Tachylectin-3--
Tachylectins isolated from hemocytes, such as
tachylectin-1 and tachylectin-2, are located in L-granules of hemocytes
(13, 14). Anti-tachylectin-3 antiserum was used to identify
localization of tachylectin-3 in hemocytes. Isolated L- and S-granules
from hemocytes (12) were treated with 2% SDS at 100 °C for 3 min and subjected to SDS-PAGE under reducing conditions for immunoblotting. The antiserum reacted with the 14-kDa protein in the extract of L-granules, as shown in Fig.
4A, but immunoreactive
materials were not detected in the extract of S-granules, indicating
that tachylectin-3 is located in L-granules of hemocytes.

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Fig. 4.
Subcellular localization and release of
tachylectin-3 from hemocytes. A, subcellular
localization of tachylectin-3. One µg each of large and small granule
samples was subjected to SDS-PAGE under reducing conditions. Lane
1, large granules; lane 2, small granules; lane
3, purified tachylectin-3. B, fresh hemocytes were
incubated with 10 mM ionophore A23187, and the exocytosed
fluid was subjected to ELISA using anti-tachylectin-3 antibody.
Closed and open circles represent presence and
absence of the ionophore A23187, respectively. The inset
shows Western blot analysis on the exocytosed fluid, using
anti-tachylectin-3 antiserum.
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To determine whether or not tachylectin-3 could be released from
hemocytes in response to external stimuli, hemocytes were treated with
the calcium ionophore A23187 that induces exocytosis of horseshoe crab
hemocytes (20, 21). Tachylectin-3 was indeed released into the
extracellular fluid, as detected by immunoblotting and ELISA (Fig.
4B). Similarly, the release of other hemocyte-derived lectins, including tachylectin-1 and tachylectin-2, was also confirmed, using specific polyclonal antibodies (data not shown).
The contents of these three tachylectins in the exocytosed fluid were
quantitated by ELISA as follows: 104.1 ± 20.9 µg/ml (tachylectin-1), 16.6 ± 1.7 µg/ml (tachylectin-2), and
11.7 ± 0.4 µg/ml (tachylectin-3). The content of coagulogen,
the most abundant protein in L-granules, was also quantitated to be
271.6 ± 12.0 µg/ml. Therefore, the three tachylectins must be
present in L-granules of hemocytes at the ratio of
tachylectin-1:tachylectin-2:tachylectin-3 = 38:6:4, with the
assumption of coagulogen being 100.
Expression of Tachylectin-3 in Various Tissues--
To investigate
tissue-specific expression of tachylectin-3, reverse transcription PCR
analysis was performed using poly(A)+ RNA derived from
hemocytes, heart, hepatopancreas, stomach, and skeletal muscle. Big
defensin, an antibacterial peptide isolated from hemocytes, is
expressed in all tissues tested (27, 28); therefore, oligonucleotide
primers for big defensin were used as a positive control of the
experiments. As shown in Fig. 5, tachylectin-3 was highly expressed in hemocytes, and significant expression was also observed in heart and muscle, but not in
hepatopancreas and stomach. Expressions of other tachylectins in these
tissues were also examined. In all cases, the primary expression site was in hemocytes, whereas expression patterns in other tissues differed. The expression pattern of tachylectin-1 was similar to that
of tachylectin-3, although a small amount of the PCR product of
tachylectin-1 was detected in the stomach. Tachylectin-2 was expressed
all the tissues tested, whereas the expression of tachylectin-4 was
restricted mainly to hemocytes.

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Fig. 5.
Comparison of tissue-specific expression
patterns of tachylectin-3 and other previously identified
tachylectins. Tissue-specific expressions of tachylectin-3 and
other tachylectins were investigated by reverse transcription-PCR
analysis, as described under "Experimental Procedures." Lane
1, hemocytes; lane 2, heart; lane 3,
hepatopancreas; lane 4, stomach; and lane 5,
skeletal muscle. TL, tachylectin; BD, big
defensin.
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 |
DISCUSSION |
We newly identified a member of horseshoe crab hemocyte-derived
lectins, named tachylectin-3. This lectin agglutinated specifically human A-type erythrocytes, like tachylectin-2 with specificity for
D-GlcNAc or D-GalNAc (14). The hemagglutinating
activity of tachylectin-3 was equivalent to that of tachylectin-2, but the activity of tachylectin-3 was not inhibited by the addition of 100 mM D-GlcNAc or D-GalNAc (Table I).
This activity of tachylectin-3 was completely inhibited by the blood
group A-pentasaccharide at 0.16 mM. Furthermore, the
activity was more strongly inhibited by S-type LPSs isolated from
several strains of Gram-negative bacteria, but it was not inhibited by
R-type LPSs (Table II). One of the most effective LPSs was from
E. coli O111:B4 with MIC of 0.006 µg/ml or 0.38 nM, based on an average molecular weight (Mr = 15,800) of LPS from E. coli
O111 (29). This LPS is also the best ligand for tachylectin-4, but a
much higher concentration was required for recognition by tachylectin-4
(MIC = 0.1 µg/ml) (15). Therefore, tachylectin-3, more strongly
than tachylectin-4, specifically recognizes a certain chemical
structure of O-antigens.
Fig. 6 shows chemical structures of the
A-pentasaccharide and repeating units of the O-antigens specifically
recognized by tachylectin-3. In our previous report, we suggested that
colitose (3-deoxy-L-fucose), a specific constituent of the
O-antigens from several strains of E. coli, is the most
probable ligand for tachylectin-4 (15). Colitose, however, is not a
specific ligand for tachylectin-3, because the inhibitory potency of
LPSs from E. coli O127 and E. coli O128
containing no colitose in their O-antigens is equal to that of the
colitose containing LPSs from E. coli O111 and E. coli O55 (30, 31). Therefore, tachylectin-3 possibly recognizes more specific structural units of O-antigens, similar to that for blood
group A antigen.

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|
Fig. 6.
Chemical structures of the A-pentasaccharide
and the repeating unit of O-antigens. Structures of the
A-pentasaccharide and the repeating unit of O-antigens of LPSs
possessing potent inhibitory activity for the hemagglutinating activity
of tachylectin-3.
|
|
Comparison of the chemical structures of the A-pentasaccharide and the
O-antigens suggests a potential ligand for tachylectin-3 (Fig. 6,
underlined). A disaccharide of Gal
1,3GalNAc or GalNAc
1,3GalNAc in the repeating units of O-antigens is shared in these O-antigens. In addition to this fact, the GalNAc
1,3Gal structure of
the A-pentasaccharide is very similar to those of the repeating units
of O-antigens. However, the potential candidate cannot explain the
inability of the A-trisaccharide (GalNAc
1,3 [Fuc
1,2] Gal) to
inhibit the hemagglutinating activity of tachylectin-3, because the
A-trisaccharide also contains the disaccharide of GalNAc
1,3Gal. Lima bean (Phaseolus lunatus) lectin binds to the
A-trisaccharide and the A-tetrasaccharide of GalNAc
1,3 [Fuc
1,2] Gal
1,4Glc (32). Furthermore, the lectin more strongly
binds to the A-hexasaccharide of GalNAc
1-3 [Fuc
1,2] Gal
1,3GlcNAc
1,3Gal
1,4Glc than the A-trisaccharide. Galectin-3
with affinity for
-galactosides, such as lactose (Gal
1,4Glc) and
lactosamine (Gal
1,4GlcNAc), also has an affinity for the
A-tetrasaccharide, and the affinity is about 30-fold higher than that
of lactose (33, 34). Like these lectins, tachylectin-3 may recognize an
annexed structure, in addition to the disaccharide of the O-antigens.
Tachylectin-3 probably contains interaction sites for the extended
oligosaccharides in addition to the primary binding sites, termed as
subsite multivalency (35). Furthermore, tachylectin-3 forms a dimer in
solution, and this dimeric form may lead to the increase in affinity
for appropriate multivalent ligands, such as O-antigens of LPS, termed as subunit multivalency (35). Further studies are required for identification of specific ligand of tachylectin-3.
Mammalian lectins have been suggested to play an important role in
host-pathogen interactions by specific recognition with cell surface
substances of bacteria (36). For example, galectin-3, expressed in the
cytosol of activated macrophages or epithelial cells, is secreted in
response to inflammatory stimuli through nonclassical pathways (37).
Furthermore, galectin-3 binds to LPS and is likely to mediate
interactions between host and pathogens (38). In the horseshoe crab,
four types of lectins have been identified in the circulating
hemocytes, and all are predominantly expressed in hemocytes and
released from hemocytes in response to the external stimuli (Figs. 4
and 5). Hence, they probably have a major functional role in host
defense, including recognition, opsonization, and killing of invading microbes.
Tachylectin-1 interacts with LPSs derived from both a wild-type and a
rough-mutant of S. minnesota probably through the
2-keto-3-deoxyoctonate disaccharide, an essential constituent of LPS in
most Gram-negative bacteria (13). Tachylectin-1 also binds to
polysaccharides such as agarose and dextran, and it is the most
abundant lectin between tachylectins so far identified. This broad
specificity of tachylectin-1, in addition to the abundant quantity
stored in secretory granules of hemocytes, may be very important for
primary pattern recognition (39, 40) against invading microbes.
Tachylectin-2 recognizes staphylococcal LTA and several kinds of LPSs
(Table II). In contrast, tachylectin-4 specifically binds to the S-type
LPS from E. coli O111 through a certain sugar moiety on the
O-antigen (15). Our newly identified tachylectin-3 exhibits much higher
specificity than tachylectin-4 against O-antigens from several
Gram-negative bacteria. Different types of lectins have been noted in
hemolymph plasma (9). Therefore, the innate immunity system of
horseshoe crab may recognize invading pathogens through a combinatorial method by using lectins with high specificity and lectins with broad
specificity against substances exposed on pathogens. An encounter with
these lectins derived from hemocytes and hemolymph plasma at injured
sites, in response to the stimulation of LPSs, suggest that they may
serve, synergistically, to provide an effective host defense against
invading microbes and foreign substances.
 |
ACKNOWLEDGEMENTS |
We thank Drs. S. Kondo and K. Hisatsune
(Josai University) for providing LPSs of V. cholerae O22 and
V. cholerae O139 and for helpful discussions. We also thank
W. Kamada for expert technical assistance with peptide sequencing and
amino acid analyses and M. Ohara for helpful comments on the manuscript.
 |
FOOTNOTES |
*
This work was supported by a Grant-in-Aid for Scientific
Research from Ministry of Education, Science, Sports and Culture of
Japan (to S.-i. K.) and Core Research for Evolutional Science and
Technology of Japan Science and Technology Corp. (to S.-i. K.).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 sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB017484.
§
Research fellows of the Japan Society for the Promotion of Science.

To whom correspondence should be addressed: Dept. of Biology,
Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan. Tel.
and Fax: 81-92-642-2634 or 2633; E-mail:
skawascb{at}mbox.nc.kyushu-u.ac.jp.
The abbreviations used are:
LPS, lipopolysaccharide; LTA, lipoteichoic acid; ELISA, enzyme-linked
immunosorbent assay; PCR, polymerase chain reaction; MIC, minimum
inhibitory concentration; Glc, D-glucose; Gal, D-galactose; Fuc, L-fucose; GlcNAc, N-acetyl-D-glucosamine; GalNAc, N-acetyl-D-galactosamine; Glc
OMe, methyl
-D-glucoside; Glc
OMe, methyl
-D-glucoside; TL, tachylectin; PAGE, polyacrylamide gel
electrophoresis; MES, 2-morpholinoethanesulfonic acid.
 |
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