A Newly Identified Horseshoe Crab Lectin with Specificity for Blood Group A Antigen Recognizes Specific O-Antigens of Bacterial Lipopolysaccharides*

Kei-ichiro InamoriDagger §, Tetsu SaitoDagger , Daisuke IwakiDagger §, Tsutomu Nagira, Sadaaki Iwanaga, Fumio Arisakaparallel , and Shun-ichiro KawabataDagger **Dagger Dagger

From the Departments of Dagger  Molecular Biology, Graduate School of Medical Science and  Biology, Faculty of Science, Kyushu University, Fukuoka 812-8581, the parallel  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
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Abstract
Introduction
References

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
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Abstract
Introduction
References

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 beta -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 lambda ZipLox cDNA library was constructed from poly(A+) RNA extracted from hemocytes, using a TimeSaverTM cDNA synthesis kit (Amersham Pharmacia Biotech) and lambda 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 [alpha -32P]dCTP using a Ready-To-GoTM DNA labeling kit (Amersham Pharmacia Biotech), and this served as a probe to screen the lambda 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.

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.

                              
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Table I
Inhibition of hemagglutinating activity of tachylectin-3

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.

                              
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Table II
Inhibition of hemagglutinating activities of TLs by LPS and LTA

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.

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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Table III
NH2-terminal sequences of Cys-containing peptide, TL-3-K1, derived from tachylectin-3


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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).

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.

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.


    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 alpha 1,3GalNAc or GalNAc alpha 1,3GalNAc in the repeating units of O-antigens is shared in these O-antigens. In addition to this fact, the GalNAc alpha 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 alpha 1,3 [Fuc alpha 1,2] Gal) to inhibit the hemagglutinating activity of tachylectin-3, because the A-trisaccharide also contains the disaccharide of GalNAc alpha 1,3Gal. Lima bean (Phaseolus lunatus) lectin binds to the A-trisaccharide and the A-tetrasaccharide of GalNAc alpha 1,3 [Fuc alpha 1,2] Gal beta 1,4Glc (32). Furthermore, the lectin more strongly binds to the A-hexasaccharide of GalNAc alpha 1-3 [Fuc alpha 1,2] Gal beta 1,3GlcNAc beta 1,3Gal beta 1,4Glc than the A-trisaccharide. Galectin-3 with affinity for beta -galactosides, such as lactose (Gal beta 1,4Glc) and lactosamine (Gal beta 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.

Dagger Dagger 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; Glcalpha OMe, methyl alpha -D-glucoside; Glcbeta OMe, methyl beta -D-glucoside; TL, tachylectin; PAGE, polyacrylamide gel electrophoresis; MES, 2-morpholinoethanesulfonic acid.
    REFERENCES
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Abstract
Introduction
References

  1. Wright, S. D., Ramos, R. A., Tobias, P. S., Ulevitch, R. J., and Mathison, J. C. (1990) Science 249, 1431-1433[Medline] [Order article via Infotrieve]
  2. Tennent, G. A., and Pepys, M. B. (1994) Biochem. Soc. Trans. 22, 74-79[Medline] [Order article via Infotrieve]
  3. Turner, M. W. (1996) Immunol. Today 17, 532-540[CrossRef][Medline] [Order article via Infotrieve]
  4. Iwanaga, S., Miyata, T., Tokunaga, F., and Muta, T. (1992) Thromb. Res. 68, 1-32[Medline] [Order article via Infotrieve]
  5. Iwanaga, S. (1993) Curr. Opin. Immunol. 5, 74-82[Medline] [Order article via Infotrieve]
  6. Muta, T., and Iwanaga, S. (1996) in Progress in Molecular and Subcellular Biology, Invertebrate Immunology (Rinkevich, B., and Müller, W. E. G., eds), pp. 154-189, Springer-Verlag, Berlin
  7. Muta, T., and Iwanaga, S. (1996) Curr. Opin. Immunol. 8, 41-47[CrossRef][Medline] [Order article via Infotrieve]
  8. Kawabata, S., Muta, T., and Iwanaga, S. (1996) in New Directions in Invertebrate Immunology (Söderhäll, K., Iwanaga, S., and Vasta, G. R., eds), pp. 255-284, SOS Publications, Fair Haven, NJ
  9. Iwanaga, S., Kawabata, S., and Muta, T. (1998) J. Biochem. (J. B. Rev.) 123, 1-15
  10. Söderhäll, K., Cerenius, L., and Johansson, M. W. (1994) Ann. N.Y. Acad. Sci. 712, 155-161[Medline] [Order article via Infotrieve]
  11. Toh, Y., Mizutani, A., Tokunaga, F., Muta, T., and Iwanaga, S. (1991) Cell Tissue Res. 266, 137-147
  12. Shigenaga, T., Takayenoki, Y., Kawasaki, S., Seki, N., Muta, T., Toh, Y., Ito, A., and Iwanaga, S. (1993) J. Biochem. 114, 307-316[Abstract]
  13. Saito, T., Kawabata, S., Hirata, M., and Iwanaga, S. (1995) J. Biol. Chem. 270, 14493-14499[Abstract/Free Full Text]
  14. Okino, N., Kawabata, S., Saito, T., Hirata, M., Takagi, T., and Iwanaga, S. (1995) J. Biol. Chem. 270, 31008-31015[Abstract/Free Full Text]
  15. Saito, T., Hatada, M., Iwanaga, S., and Kawabata, S. (1997) J. Biol. Chem. 272, 30703-30708[Abstract/Free Full Text]
  16. Nakamura, T., Morita, T., and Iwanaga, S. (1985) J. Biochem. (Tokyo) 97, 1561-1574[Abstract]
  17. Kawabata, S., and Iwanaga, S. (1997) in Methods in Molecular Biology (Shafer, W. M., ed), Vol. 78, pp. 51-61, Humana Press, Totowa, NJ
  18. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  19. Muta, T., Hashimoto, R., Miyata, T., Nishimura, H., Toh, Y., and Iwanaga, S. (1990) J. Biol. Chem. 265, 22426-22433[Abstract/Free Full Text]
  20. Armstrong, P. B., and Rickles, F. R. (1982) Exp. Cell Res. 140, 15-24[Medline] [Order article via Infotrieve]
  21. Miura, Y., Kawabata, S., Wakamiya, Y., Nakamura, T., and Iwanaga, S. (1995) J. Biol. Chem. 270, 558-565[Abstract/Free Full Text]
  22. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  23. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[CrossRef][Medline] [Order article via Infotrieve]
  24. von Heijne, G. (1983) Eur. J. Biochem. 133, 17-21[Abstract]
  25. Yamanaka, M., Hsu, D., Crisp, T., Dale, B., Grubman, M., and Yilma, T. (1988) Virology 166, 251-253[Medline] [Order article via Infotrieve]
  26. Langedijk, J. P. M., Daus, F. J., and van Oirschot, J. T. (1997) J. Virol. 71, 6155-6167[Abstract]
  27. Saito, T., Kawabata, S., Shigenaga, T., Cho, J., Nakajima, H., Hirata, M., and Iwanaga, S. (1995) J. Biochem. 117, 1131-1137[Abstract]
  28. Kawabata, S., Saito, T., Saeki, K., Okino, N., Mizutanti, A., Toh, Y., and Iwanaga, S. (1997) Biol. Chem. Hoppe-Seyler 378, 289-292
  29. Goldman, R. C., White, D., Ørskov, F., Ørskov, I., Rick, P. D., Lewis, M. S., Bhattacharjee, A. K., and Leive, L. (1982) J. Bacteriol. 151, 1210-1221[Medline] [Order article via Infotrieve]
  30. Knirel, Y. A., and Kochetkov, N. K. (1994) Biochemistry (Moscow) 59, 1325-1383
  31. Sengupta, P., Bhattacharyya, T., Shashkov, A. S., Kochanowski, H., and Basu, S. (1995) Carbohydr. Res. 277, 283-290[CrossRef][Medline] [Order article via Infotrieve]
  32. Sikder, S. K., and Kabat, E. A. (1986) Carbohydr. Res. 151, 247-260[CrossRef][Medline] [Order article via Infotrieve]
  33. Leffler, H., and Barondes, S. H. (1986) J. Biol. Chem. 261, 10119-10126[Abstract/Free Full Text]
  34. Sparrow, C. P., Leffler, H., and Barondes, S. H. (1987) J. Biol. Chem. 262, 7383-7390[Abstract/Free Full Text]
  35. Rini, J. M. (1995) Annu. Rev. Biophys. Biomol. Struct. 24, 551-577[CrossRef][Medline] [Order article via Infotrieve]
  36. Mandrell, R. E., Apicella, M. A., Lindstedt, R., and Leffler, H. (1994) Methods Enzymol. 236, 231-254[Medline] [Order article via Infotrieve]
  37. Sato, S., and Hughes, R. C. (1994) J. Biol. Chem. 269, 4424-4430[Abstract/Free Full Text]
  38. Mey, A., Leffler, H., Hmama, Z., Normier, G., and Revillard, J.-P. (1996) J. Immunol. 156, 1572-1577[Abstract]
  39. Janeway, C. A., Jr. (1989) Cold Spring Harbar Symp. Quant. Biol. 54, 1-13[Medline] [Order article via Infotrieve]
  40. Janeway, C. A., Jr. (1992) Immunol. Today 13, 11-16[CrossRef][Medline] [Order article via Infotrieve]


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