INTRODUCTION

Arthropods have developed a unique immune system without the acquired immunoglobulin-dependent immunity found in vertebrates. Therefore, innate immunity, the pre-existing and immediate ability to prevent and limit invading microbes and pathogens, is likely to be a major host defense system in arthropods. The hemolymph of horseshoe crabs contains three abundant proteins, hemocyanin, C-reactive protein, and ₂-macroglobulin, and one type of granular cell, accounting for 99% of the total hemocytes (1, 2). The granular cells are extremely sensitive to bacterial endotoxin, i.e. lipopolysaccharides (LPS),¹ and the cells release granular components in response to LPS stimulation (3-6). This response is thought to be important for host defense in engulfing and killing invading microbes, in addition to preventing the leakage of hemolymph. The hemocytes contain large and small granules that selectively store proteins and defense molecules, including serine protease zymogens, a clottable protein that participates in the coagulation cascade, protease inhibitors, antibacterial peptides, lectins, and others (6).

Many kinds of lectins play crucial roles in innate immunity and host defense not only in vertebrates but also in invertebrates, with involvement in processes such as non-self recognition, inflammation, opsonization, cell-cell or cell-extracellular matrix interactions, fertilization, development, and regeneration (7-11). To better understand the biological role of lectins in host defense of the horseshoe crab, we have studied various lectins found in hemolymph of this animal (6). Recently, we identified new horseshoe crab hemocyte-derived lectins, named tachylectin-1 (L6) (12) and tachylectin-2 (L10) (13). Tachylectin-1 displays LPS-binding potential with antibacterial activity toward Gram-negative bacteria, but it has no hemagglutinin activity for human, sheep, and rabbit erythrocytes. On the other hand, tachylectin-2 has hemagglutinating activity against human A-type erythrocytes with specificity for N-acetylglucosamine. Furthermore, tachylectin-2 has agglutination activity against Staphylococcus saprophyticus KD. Both tachylectin-1 and tachylectin-2 are composed of unique tandem sequence repeats with no significant sequence similarity with other known proteins, including various animal and plant lectins. While continuing these studies on horseshoe crab lectins, we have now identified a new hemocyte lectin, which we named tachylectin-4; it has more potent hemagglutinating activity than tachylectin-2, shows a unique binding specificity to O-antigen of LPS, and has sequence similarity to the NH₂-terminal domain with unknown functions of Xenopus pentraxin 1.

EXPERIMENTAL PROCEDURES

Hemocytes lysate from the Japanese horseshoe crab (Tachypleus tridentatus) was prepared as described (14). LPS isolated from Escherichia coli O111:B4, Salmonella minnesota (smooth), and S. minnesota R595 (Re) were from List Biological Laboratories, Inc., Campbell, CA and those isolated from E. coli J5 (Rc), E. coli F583 (Rd), S. minnesota R7 (Rd₁), Salmonella typhimurium (smooth), S. typhimurium TV119 (Ra), and S. typhimurium SL684 (Rc) were from Sigma. ConA-Sepharose and molecular weight standards were from Pharmacia Biotech Inc. L-Fucose-immobilized agarose was from EY Laboratories, Inc., San Mateo, CA. Lysyl endopeptidase was from Wako Pure Chemical Industries, Ltd., Tokyo, Japan. Restriction endonucleases and DNA-modifying enzymes were from Nippon Gene Co. (Toyama, Japan), Toyobo Co., Ltd. (Osaka, Japan), and Takara Shuzo Co. (Kyoto, Japan). [-³²P] dCTP was from Amersham Japan, Tokyo. Sugars and glycoproteins were from Sigma, Nacalai Tesque, Co., Ltd. (Kyoto, Japan), and Seikagaku Kogyo, Co., Ltd. (Tokyo, Japan). A ZipLox cDNA library was constructed from poly(A⁺) RNA extracted from hemocytes, using a TimeSaver^TM cDNA synthesis kit (Pharmacia Biotech Inc.) and ZipLox^TM, EcoRI Arms (Life Technologies, Inc.) (13).

Hemagglutinating (13) and LPS binding (12) activities were determined, as described.

The samples were reduced and S-alkylated with iodoacetamide in 50 mM Tris-HCl, pH 7.5, containing 8 M urea (15). The S-alkylated protein (5 µg) was incubated with 0.1 unit of N-glycosidase F (Boehringer Mannheim) in 50 mM Tris-HCl, pH 7.5, containing 2 M urea at 37 °C for 18 h and subjected to SDS-PAGE under reducing conditions.

Purified tachylectin-4 was applied to a Superdex 200 HR10/30 column, equilibrated with 20 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl at a flow rate of 0.5 ml/min. Reference proteins for molecular weight determination were thyroglobulin (M_r = 669,000), apoferritin (M_r = 443,000), alcohol dehydrogenase (M_r = 150, 000), bovine serum albumin (M_r = 67,000), and carbonic anhydrase (M_r = 30,000).

SDS-PAGE was performed in 15% slab gels, according to Laemmli (16). The gels were stained with Coomassie Brilliant Blue R-250. The reference proteins were phosphorylase b (M_r = 94,000), bovine serum albumin (M_r = 67,000), ovalbumin (M_r = 43,000), carbonic anhydrase (M_r = 30,000), soybean trypsin inhibitor (M_r = 20,000), and -lactoalbumin (M_r = 14,400).

Tachylectin-4 (100 µg) was reduced and then S-alkylated with iodoacetamide (15). The S-alkylated protein was digested with lysyl endopeptidase (enzyme/substrate = 1/100, w/w) in 0.1 M NH₄HCO₃ containing 2 M urea at 37 °C for 24 h. The peptides were separated by reverse-phase HPLC using a Chemcosorb 5-ODS-H column (2.1 × 150 mm, Chemco Scientific Co., Ltd., Osaka). Peptides were eluted from the column with a linear gradient of 0-80% acetonitrile in 0.06% trifluoroacetic acid at a flow rate of 0.2 ml/min. Absorbance was monitored at 210 nm. Amino acid sequence analyses were performed using an Applied Biosystems 473A or 477A sequencer with the chemicals and programs supplied by the manufacturer (Perkin-Elmer Japan Co., Ltd., Tokyo, Japan).

Samples were hydrolyzed in 6 M HCl containing 1% phenol at 110 °C for 20 h in evacuated tubes. The hydrolysates were analyzed using a Pico-Tag system (Waters Japan Co., Ltd., Tokyo, Japan) (17).

The degenerate nucleotide sequences of the primers used for PCR were based on the peptides derived from lysyl endopeptidase digestion (-Asn-Ala-Tyr-Val-Glu-Thr- and -Ile-Thr-Asp-Asp-Tyr-Val-) of tachylectin-4. 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 (0.4 kilobase), containing the sequence of tachylectin-4, was used as a probe to screen the ZipLox cDNA library. The PCR fragment, labeled with [-³²P]dCTP using a Ready-To-Go^TM DNA-labeling kit (Pharmacia Biotech Inc.) served as a probe. After secondary screening, the plasmids containing the cDNA insert were prepared from the positive plaques, following the manual supplied by the manufacturer.

Amino acid sequence was compared with all entries in the SWISS-PROT protein data base by the FASTA homology search system of the European Bioinformatics Institute.

RESULTS

The lysate prepared from 100 g (wet weight) of hemocytes was first fractionated on a dextran sulfate-Sepharose CL-6B column (4.5 × 20 cm) with increasing concentrations of NaCl from the range of 0.15-2.0 M (14), and a typical elution pattern and hemagglutinating activity against human A-type erythrocytes is shown in Fig. 1A. The activity in the flow-through fraction is derived from tachylectin-2 (13). The 0.15 M NaCl fractions containing a new lectin, named tachylectin-4, indicated by a bar were pooled and applied to a ConA-Sepharose column (3 × 15 cm), equilibrated with 20 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl. After washing with equilibration buffer, proteins were eluted with the same buffer containing 1 M -methyl-D-glucoside (Fig. 1B). The lectin fractions indicated by a bar were pooled and dialyzed against 20 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl. The dialyzed sample was then applied to an L-fucose-immobilized agarose column equilibrated with the same buffer. The column was washed extensively with the equilibration buffer, and then proteins were eluted with the same buffer containing 1 M L-fucose (Fig. 1C). All steps of the purification procedures were performed at 4 °C. The purification scheme is summarized in Table I. The tachylectin-4 was purified about 600-fold with a yield of 56%, as calculated from the pooled fraction of dextran-sulfate-Sepharose CL-6B chromatography free from the hemagglutinating activity of tachylectin-2.

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Table I. Purification of tachylectin-4 from horseshoe crab hemocyte lysate Purification step Total proteina Total activity Specific activity Purification Yield mg titers titers/mg -fold % Hemocyte lysate 21,152 604,160 29 Dextran sulfate-Sepharose CL-6B 1,208 243,200 201 1 100 ConA-Sepharose 160 153,600 962 4.8 63 Fucose-agarose 1.1 135,171 122,328 611 56 a For the determination of protein concentrations, A280 = 10 for 1% protein solution, was assumed.

The purified tachylectin-4 gave a doublet on SDS-PAGE under reducing (M_r = 30,000 and 31,500) and non-reducing (M_r = 23,500 and 26,000) conditions (Fig. 1D). On the other hand, an apparent molecular weight of tachylectin-4 in solution was determined by gel filtration to be 470,000, indicating an oligomeric protein. The two subunits of 31.5 and 30 kDa could be separated by reverse-phase HPLC on a YMC C4 column (4.6 × 150 mm, Yamamura Chemical Laboratories Co., Ltd., Kyoto, Japan) (data not shown), and their NH₂-terminal sequences proved to be identical up to 18 residues as follows: Trp-Arg-Met-Leu-Tyr-Leu-Pro-Val-Ile-Val-Lys-Tyr-Gly-X-Met-Lys-Leu-Asp-. Furthermore, amino acid compositions of 20-h hydrolysates and peptide mapping patterns of the two subunits were almost indistinguishable (data not shown). The purified tachylectin-4 was treated with N-glycosidase F and subjected to SDS-PAGE (Fig. 2). The N-glycosidase F treatment resulted in disappearance of the upper band, and the lower band was only observed in the gel with the same mobility as the untreated lower band (lanes 2 and 3), indicating that the different molecular weights between the subunits is caused by partial modification of the subunit with N-linked sugar(s).

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The extinction coefficient of tachylectin-4 at 280 nm for 1.0% solution in Tris-HCl buffer (pH 7.5) was calculated from data derived from amino acid analysis. The value of 14.0 was used for subsequent determinations of tachylectin-4 concentrations.

Tachylectin-4 agglutinated all types of human erythrocytes and A-type erythrocytes were most sensitive (Table II). In the presence of 10 mM CaCl₂, tachylectin-4 agglutinated A-type erythrocytes at the minimum concentration of 0.014 µg/ml, and 60- and 120-fold higher concentrations were required for O-type and B-type erythrocytes, respectively. No hemagglutination was observed for erythrocytes derived from horse, rabbit, and sheep (Table II). The hemagglutinating activity for A-type erythrocytes was enhanced 4-fold by 10 mM Ca²⁺, but other divalent cations including Zn²⁺, Co²⁺, Cu²⁺, and Mn²⁺ at 0.1 mM and 10 mM had no effects. The agglutinating activity was completely inhibited by 10 mM EDTA and 0.2 mM o-phenanthroline, suggesting the presence of metal ion(s) other than Ca²⁺ in tachylectin-4.

Table II. Hemagglutinating of activity of tachylectin-4 Erythrocytes Minimum agglutination concentration µg/ml Human   A-type 0.014   B-type 1.7   O-type 0.86 Horse Not agglutinateda Rabbit Not agglutinated Sheep Not agglutinated a Not agglutinated at 12.5 µg/ml.

Effects of various carbohydrates on the hemagglutinating activity of tachylectin-4 are shown in Table III. For mono- and disaccharides, L-fucose and N-acetylneuraminic acid at the minimum concentration of 100 mM inhibited the hemagglutination. On the other hand, a bacterial cell wall component, LPS, was a more potent inhibitor. An LPS derived from E. coli O111:B4 was the most potent inhibitor at the minimum concentration of 0.1 µg/ml. Interestingly, LPS isolated from rough mutants of E. coli O111:B4 (smooth) such as E. coli J5 (Rc) and E. coli F583 (Rd) showed no inhibitory effect, suggesting that the O-antigen of the S-type E. coli is a determinant for carbohydrate recognition of tachylectin-4. This was the case for other Gram-negative bacteria. LPS derived from S-types of both S. minnesota and S. typhimurium inhibited the hemagglutinating activity at the minimum concentrations of 15.6 and 7.8 µg/ml, respectively, but LPS isolated from several rough mutants had no effect. To confirm the binding activity to O-antigen, hemagglutinating activity was measured using sheep erythrocytes coated with LPS derived from a S-strain (E. coli O111:B4) and a rough mutant (S. minnesota R595 (Re). Tachylectin-4 significantly agglutinated sheep erythrocytes sensitized with LPS of E. coli O111:B4, but not those sensitized with LPS of S. minnesota R595 (Re). This agglutinating activity was inhibited by free LPS derived from E. coli O111:B4, but not from S. minnesota R595 (Re). Lipoteichoic acid, a cell wall component of Gram-positive bacteria, also inhibited the hemagglutination, but lipoteichoic acid from Bacillus subtilis was not so effective (Table III).

Table III. Inhibition of agglutinating activity of tachylectin-4 Component Minimum inhibitory concentrationa mM µg/ml L-Fucose 100 D-Glucose NIb D-Galactose NI D-Mannose NI L-Rhamnose NI D-Xylose NI D-Glucosamine NI D-Galactosamine NI N-Acetylneuraminic acid 100 N-Acetyl-D-glucosamine NI N-Acetyl-D-galactosamine NI N-Acetyl-D-mannosamine NI Lactose NI N-Acetylallolactosamine NI Methyl -D-mannoside NI Methyl -D-glucoside NI Hyaluronic acid NI Laminarin NI LPS: E. coli O111:B4 (smooth) 0.1 LPS: E. coli J5 (Rc) NI LPS: E. coli F583 (Rd) NI LPS: S. minnesota (smooth) 15.6 LPS: S. minnesota R595 (Re) NI LPS: S. minnesota R7 (Rd1) NI LPS: S. typhimurium (smooth) 7.8 LPS: S. typhimurium TV119 (Ra) NI LPS: S. typhimurium SL684 (Rc) NI Lipoteichoic acid: Staphylococcus aureus 31.3 Lipoteichoic acid: Streptococcus faecalis 31.3 Lipoteichoic acid: Streptococcus mutans 62.5 Lipoteichoic acid: Streptococcus sanguis 62.5 Lipoteichoic acid: Streptococcus pyogenes 125 Lipoteichoic acid: B. subtilis 500 a Minimum concentrations required for inhibition of two hemagglutinating titers of tachylectin-4 against human A-type erythrocytes. b NI, not inhibited at 100 mM or 1000 µg/ml.

The tachylectin-4-specific probe of 0.4 kilobase was identified with oligonucleotides corresponding to peptides derived from tachylectin-4, using PCR and DNA sequence analysis. When the probe was used to screen a hemocyte cDNA library (400,000 recombinant phages), one positive clone with a 1.3-kilobase pair insert was subjected to restriction mapping followed by sequence determination of both strands and by sequential exonuclease digestion. The nucleotide and deduced amino acid sequences are shown in Fig. 3. The cDNA included 1,344 nucleotides with an open reading frame of 699 nucleotides. The open reading frame for the cDNA encoded for a mature protein of 232 amino acid residues with the calculated molecular weight of 26,625, and a signal sequence of 23 residues with a typical hydrophobic core. The candidate for an initiation codon ATG was found at nucleotide position 21. The stop codon at position 786 was followed by a polyadenylation signal, AATAAA, starting at position 1322. Amino acid sequences of the isolated peptides derived from tachylectin-4 corresponded exactly to the protein sequence deduced from the cDNA sequence, clearly indicating that the isolated cDNA clone codes for tachylectin-4. The deduced protein sequence contains one potential N-linked glycosylation site at Asn-108. Tachylectin-4 was sensitive to N-glycosidase F treatment (Fig. 2) and contained glucosamine, based on amino acid analysis (data not shown), indicating the presence of an N-linked sugar chain at this site. The isoelectric point of tachylectin-4 was calculated from amino acid composition to be 6.05 (19), which is rather acidic, compared with findings in tachylectin-1 (9.69) and tachylectin-2 (9.63).

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A search of SWISS-PROT showed the sequence similarity of tachylectin-4 to Xenopus laevis pentraxin 1 (20). Tachylectin-4 has a 24% sequence identity to the NH₂-terminal domain of Xenopus pentraxin 1 and the sequence of Pro-120 to Lys-159 of tachylectin-4 is highly conserved in the corresponding region of Xenopus pentraxin 1 (50% identity), as shown in Fig. 4.

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DISCUSSION

We previously identified two kinds of lectins with no significant sequence similarity with other known proteins. These were named tachylectin-1 with LPS binding activity (12) and tachylectin-2 with hemagglutinating activity against human A-type erythrocytes (13). In the present study, we identified a horseshoe crab hemocyte lectin, tachylectin-4, with hemagglutinating activity against human erythrocytes and with binding specificity to O-antigen of bacterial LPS. Tachylectin-4 has different characteristics from both tachylectin-1 and tachylectin-2. 1) The minimum agglutination concentration of tachylectin-4 required for human A-type erythrocytes (0.014 µg/ml) is about 100-fold lower than that of tachylectin-2 (1.6 µg/ml) (13). 2) The addition of Ca²⁺ at 10 mM enhances the hemagglutinating activity of tachylectin-4 by 4-fold, whereas Ca²⁺ is not required for the activity of tachylectin-2 and EDTA has no effect on the activity of tachylectin-2 (13). However, the hemagglutinating activity of tachylectin-4 was completely inhibited by EDTA or o-phenanthroline, indicating that tachylectin-4 contains metal ion(s) with an important role for the sugar binding. 3) The activity of tachylectin-2 is inhibited by 0.1 mM N-acetylglucosamine (13), which had no effect on the activity of tachylectin-4 at 100 mM. On the other hand, L-fucose (6-deoxy-L-galactose) specifically inhibits the activity of tachylectin-4, with no inhibition for tachylectin-2. L-Rhamnose (6-deoxy-L-mannose), a stereoisomer of L-fucose at C-2 and C-4, has no effect on the activity of tachylectin-4, suggesting an important role for the configuration of C-2 or C-4 in sugar recognition. 4) Tachylectin-2 (27 kDa) exists as a monomer in solution (13), whereas tachylectin-4 is present as a high molecular mass oligomer of 470 kDa. Based on an assumption of 30 kDa for one subunit, tachylectin-4 is composed of 15-16 subunits, under physiological conditions. 5) Tachylectin-1 agglutinates sheep erythrocytes coated with LPS derived from both a wild-type (smooth) and a Re mutant of S. minnesota (12). In contrast, the hemagglutinating activity of tachylectin-4 was inhibited by S-type LPS, not by R-type LPS lacking O-antigen.

The LPS from E. coli O111:B4 was most effective, and the minimum concentration required to inhibit hemagglutination for human A-type erythrocytes was 160-fold and 80-fold lower than those of LPS from S. minnesota (smooth) and S. typhimurium (smooth), respectively. The O-antigen of E. coli O111:B4 is built up by a unique repeating unit of a main chain containing D-galactose, D-glucose, and D-N-acetylgalactosamine, and a monosaccharide side chain of colitose (3,6-dideoxy-L-galactose or 3-deoxy-L-fucose) (21). In these monosaccharides, colitose is the most probable candidate for a specific ligand of tachylectin-4, since it is a unique monosaccharide present in the O-antigen of E. coli O111:B4, its structure is similar to that of L-fucose, and three other monosaccharides had no effect on the hemagglutinating activity of tachylectin-4 (Table III). On the other hand, the O-antigen of S. typhimurium is composed of a main chain containing of D-mannose, L-rhamnose, and D-galactose, and two monosaccharide side chains of D-glucose and abequose (3,6-dideoxy-D-galactose or 3-deoxy-D-fucose) (21). Abequose is also a candidate for another ligand, since the hexoses except abequose have no effect on the activity of tachylectin-4 (Table III). Abequose is the D-isomer of colitose, which may cause a reduced affinity to tachylectin-4.

A sequence homology search showed no significant sequence similarity between tachylectin-4 and other known LPS-binding lectins, such as Limulus endotoxin-binding protein-protease inhibitor (22), mammalian plasma LPS-binding protein (23), and Periplaneta lectins from the American cockroach (11, 24, 25), whereas the search indicates the sequence similarity of tachylectin-4 to X. laevis pentraxin 1 (20) (Fig. 4). Pentraxins are decameric or dodecameric proteins composed of identical protomers, arranged in two pentameric or hexameric rings interacting face-to-face (26, 27). They constitute a family of carbohydrate binding proteins with divalent cation dependence, and some pentraxins, such as human C-reactive protein, belong to acute phase proteins, rapidly increasing their concentrations in response to stress, injury or infection (7, 28-30). However, Xenopus pentraxin 1 gene, identified from a Xenopus cDNA library using pentraxin-specific oligonucleotide probes, unexpectedly encodes a novel fusion protein with an NH₂-terminal domain of a unique sequence and the COOH-terminal domain with sequence similarity to C-reactive proteins. The sequence similarity of tachylectin-4 to the NH₂-terminal domain suggests that Xenopus pentraxin 1 is a chimeric protein with two different lectin-like activities.

The binding activities of tachylectin-4 with LPS and lipoteichoic acids suggest that tachylectin-4 plays a important role in recognition of invading bacteria. There is no evidence that horseshoe crabs have immunoglobulins (31), the recognition molecules produced by gene rearrangements, which help to distinguish self from non-self. Horseshoe crabs, however, do have an innate constitutive immune system that includes recognition by cell surface receptors or diverse lectin-like substances, phagocytosis (32), and killing reactions by antimicrobial molecules. Therefore, horseshoe crabs may oppose invaders through a combinatorial method using preexisting components in hemolymph plasma and hemocytes. Co-localization of these defense molecules in granules and their release into the hemolymph, in response to the stimulation of LPS, suggest that they serve synergistically to accomplish an effective host defense system against invading microbes and foreign substances.