J Biol Chem, Vol. 274, Issue 53, 37673-37678, December 31, 1999

Purification, Characterization, and Amino Acid Sequence of an Embryonic Lectin in Perivitelline Fluid of the Horseshoe Crab^*

Taku Nagai, Shun-ichiro Kawabata^§¶, Fumio Shishikura, and Hiroaki Sugita^**

From the  Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, the ^§ Department of Biology, Kyushu University, Fukuoka 812-8581, the  Department of Biology, Nihon University School of Medicine, Tokyo 173-8610, and the ^** Institute of Biological Sciences, the University of Tsukuba, Ibaraki 305-8572, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hemagglutinating activity in perivitelline fluid of the horseshoe crab embryo dramatically increases during the third and fourth molt of the embryo. A 27-kDa lectin, which we named tachylectin-P (TL-P), was newly identified in perivitelline fluid of the horseshoe crab Tachypleus tridentatus. TL-P preferentially agglutinated human A-type erythrocytes, and the activity was inhibited by N-acetyl group-containing monosaccharides. The amino acid sequence analysis indicated that TL-P is almost structurally the same as a hemocyte-derived lectin with no hemagglutinating activity, tachylectin-1 (TL-1), and that 218 out of 221 amino acid residues in total were conserved between the two lectins. Despite the high sequence similarity, biological and biochemical characteristics of TL-P differed from those of TL-1: (i) unlike TL-1, TL-P agglutinates several animal-derived erythrocytes; (ii) unlike TL-1, TL-P has no significant affinity for bacterial lipopolysaccharides or antibacterial activity; (iii) Based on apparent molecular masses determined by gel filtration, TL-P forms a dimer in solution, while TL-1 is present as a monomer; (iv) and TL-P interacts with endogenous proteins of 13 and 14 kDa present in the perivitelline fluid; however, neither has any affinity for TL-1. We propose that TL-P may have an important role in completing embryonic development by interacting with endogenous glycoproteins or N-acetylhexosamines.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

During early developmental stages of the horseshoe crab embryo, the inner egg membrane is newly formed, and a space between the inner egg membrane and embryo, perivitelline space, is filled with perivitelline fluid (1). The perivitelline fluid contains proteins such as hemagglutinins and hemocyanin, which may have an important role during embryogenesis (2, 3). The embryo in the perivitelline fluid goes through four molts before hatching. Shishikura and Sekiguchi reported that the hemagglutinating activity in perivitelline fluid of the Indo-Pacific horseshoe crab Tachypleus gigas dramatically increases after the third embryonic molting (3). Four kinds of lectins, named tachylectins (TLs)¹ (TL-1 to TL-4), from hemocytes of the Japanese horseshoe crab Tachypleus tridentatus have been identified, and their unique structures and critical roles in host defense systems have been demonstrated (4-8). However, much less is known of perivitelline-derived lectins of horseshoe crabs. A multimeric lectin of 450 kDa composed of a 40-kDa subunit was purified from the perivitelline fluid of T. gigas, using affinity chromatography of bovine submaxillary gland mucin-agarose (3). We report here the isolation, characterization, and amino acid sequence of a lectin of 27 kDa from the perivitelline fluid of T. tridentatus, which we named tachylectin-P (TL-P). The amino acid sequence of TL-P is 99% identical to that of the hemocyte-derived TL-1 (previously named L6) (4), but TL-P and TL-1 significantly differ in biological and biochemical characteristics.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Lipopolysaccharides (LPS) from Escherichia coli O111:B4, Salmonella minnesota (Smooth), and S. minnesota R595 (Re mutant) were from List Biological Laboratories, Inc. (Campbell, CA). CNBr-activated Sepharose 4B, protein A-Sepharose, and reference proteins for molecular weight estimation were from Amersham Pharmacia Biotech. Lysyl endopeptidase and endoproteinase Asp-N were, respectively, from Wako Pure Chemical Industries, Ltd. (Tokyo) and from Roche Molecular Biochemicals (Mannheim, Germany). Sugars and glycoproteins were from Sigma and Wako Pure Chemical Industries, Ltd., Tokyo. Horse and sheep erythrocytes were from Nippon Bio-Laboratories (Tokyo). A hemocyte-derived TL-1 was prepared, as described (4).

Preparation of Perivitelline Fluid-- Adults of T. tridentatus were collected in Kitsuki, Japan and maintained at the Shimoda Marine Research Center of the University of Tsukuba. The eggs of T. tridentatus were inseminated artificially (1). The developmental stage of the embryo was determined according to the normal plate of T. tridentatus, described by Sekiguchi (9). Perivitelline fluid was collected from the eggs after the third or fourth molt of the embryo by making a tear in the inner egg membrane, and then the fluid was stored at 20 °C until use.

Preparation of Bovine Submaxillary Gland Mucin (BSM)-Sepharose 4B-- BSM (20 mg) was coupled with 2 g of CNBr-activated Sepharose, according to the instruction manual.

SDS-PAGE and Immunoblotting-- SDS-PAGE was performed in 12 or 15% slab gels, according to Laemmli (10). The gels were stained with Coomassie Brilliant Blue R-250 or silver staining. For immunoblotting, electrophoresed proteins were transferred to a nitrocellulose membrane by electroblotting, and the membrane was treated with a rabbit polyclonal antibody against TL-1 and visualized as described (11).

Hemagglutination Activity and Antimicrobial Activity-- Hemagglutinating activity (12) and antimicrobial activity (12, 13) were determined as described.

Peptide Mapping and Enzymatic Digestion-- TL-P and TL-1 were reduced, S-alkylated with iodoacetamide, and digested with lysyl endopeptidase (E/S = 1:100 (w/w)) in 0.1 M NH₄HCO₃, containing 2 M urea at 37 °C for 12 h (14). The S-alkylated TL-P was also digested with endoproteinase Asp-N (E/S = 1:40 (w/w)) in 50 mM Tris-HCl, pH 7.5, containing 2 M urea for 12 h at 37 °C. The resulting peptides were separated by reverse-phase HPLC, using Cosmosil 5C18-MS (2.0 × 150 mm; Nacalai Tesque, Kyoto) with a linear gradient of 0-80% acetonitrile in 0.052% trifluoroacetic acid at a flow rate of 0.2 ml/min. The effluent was monitored at 210 nm.

Amino Acid and Sequence Analyses-- Amino acid analysis was performed on a PICO-TAG system (Waters, Millipore Corp., Milford, MA), and sequence analysis was performed using an Applied Biosystems 473A or 477A sequencer.

Reverse Transcription-PCR Analysis-- The degenerate nucleotide sequences of primers used for PCR were based on peptide sequences of TL-P, QWHQIP (residues 2-7), and KQCDAT (residues 199-204). Sense and antisense nucleotides were synthesized with an EcoRI site at the 5' end. Total RNA was extracted from T. tridentatus embryo according to Chomczynski and Sacchi (15), and poly(A)⁺ RNA was purified using Oligotex dt30 (Takara Shuzo Co., Kyoto). First-strand cDNA synthesis from poly(A)⁺ RNA was performed using First SuperScript II RNase H Reverse Transcriptase and random primers (Life Technologies, Inc.). The cDNA template (corresponding to about 0.1 µg of poly(A)⁺ RNA) and 100 pmol of each oligonucleotide 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. The PCR product was subcloned into plasmid Bluescript II SK (Stratagene) for sequence analysis.

Gel Filtration-- Gel filtration was done, using an FPLC system (Amersham Pharmacia Biotech) on a Bio-silect SEC 250-5 column (300 × 7.8 mm, Bio-Rad). Proteins were eluted with 50 mM Tris-HCl, pH 7.5, containing 1 M NaCl and 15% acetonitrile at the flow rate of 0.25 ml/min and monitored at A₂₈₀. The reference proteins were IgG (146,000), bovine serum albumin (67,000), ovalbumin (45,000), carbonic anhydrase (29,000), and myoglobin (17,500).

Enzyme-linked Immunosorbent Assay of TL-P-- A polyclonal antibody against TL-1 prepared previously (11) was biotinylated with EZ-Link NHS-LC-Biotin (Pierce), according to a protocol provided by the manufacturer. Microtiter plates were coated with the nonbiotinylated antibody by incubating overnight at 4 °C. After washing with 20 mM sodium phosphate, pH 7.0, the plates were blocked with 2.5% casein, and 2-fold serial dilutions of samples were added. The bound antigen was assayed, using the biotinylated antibody and streptavidin-biotinylated horseradish peroxidase complex (Amersham Pharmacia Biotech), as described (6).

Immunoprecipitation-- An aliquot of samples, 400 µl, was mixed with 5 µg of TL-P or TL-1 and incubated for 10 h at 4 °C. The anti-TL-1 antibody (5 µg) was added and incubated further for 10 h. Protein A-Sepharose was then mixed and incubated for 3 h at 4 °C. After washing with 50 mM Tris-HCl, pH 8.0, containing 0.5 M NaCl, the pellet was suspended in the Laemmli sampling buffer and subjected to SDS-PAGE under reducing conditions.

Determination of Protein Concentrations-- Protein concentrations were determined by the method of Bradford (16), using bovine serum albumin as a standard.

Component Sugar Analysis-- Sugar contents were analyzed by the method of Takemoto et al. (17). The principle was based on HPLC analysis of sugar derivatives released after pyridylamination of the acid hydrolysates of protein samples.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purification of TL-P-- The pooled perivitelline fluid just after the fourth molt of the embryo was dialyzed against 50 mM Tris-HCl, pH 7.5, containing 0.5 M NaCl and 10 mM CaCl₂ and centrifuged at 3000 rpm for 1 h to remove a flocculent substance. The resulting supernatant was applied to a BSM-Sepharose 4B column (1 × 5 cm) equilibrated with the same buffer. After extensive washing with the buffer, hemagglutinating activity was eluted with 0.4 M GlcNAc present in the same buffer. A typical elution profile is shown in Fig. 1A. The pooled fraction indicated by a bar gave a single band of M_r = 27,000 on SDS-PAGE, under reducing conditions (Fig. 1B). About 0.4 mg of the hemagglutinin was purified from 50 ml of the perivitelline fluid, corresponding to about 700 eggs. We named this newly isolated lectin TL-P.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Purification of TL-P. Experimental details are presented under "Results." A, BSM-Sepharose 4B column chromatography. Fraction tubes indicated by a bar were pooled. B, SDS-PAGE of the crude perivitelline fluid (lane 1) and purified TL-P (lane 2) under reducing conditions.

Hemagglutinating Activity of TL-P-- TL-P agglutinated human A-type erythrocytes at the minimum concentration of 0.85 µg/ml. The activity is equivalent to activities of tachylectins earlier identified from hemocytes, TL-2 (5) and TL-3 (6). TL-P also agglutinated human B-type and animal-derived erythrocytes including horse and sheep, but a 30-fold higher concentration than that of human A-type erythrocytes was required for agglutination (25 µg/ml). Ca²⁺ was not required for the hemagglutinating activity of TL-P, but the activity was enhanced 2-fold by the addition of Ca²⁺ at 10 mM. Therefore, human A-type erythrocytes were used in the presence of 10 mM Ca²⁺ for subsequent hemagglutination assays. The activity was completely inhibited by 0.1 M EDTA, and the inactivation could not be recovered after dialysis against the assay buffer containing 10 mM Ca²⁺, which suggested the presence of metal ion(s) in TL-P.

Effects of Carbohydrates on Hemagglutinating Activity-- Effects of various carbohydrates and several glycoproteins on the hemagglutinating activity of TL-P are summarized in Table I. For mono- and oligosaccharides, D-GalNAc and D-ManNAc inhibited the activity at the minimum inhibitory concentration (MIC) of 12.5 mM. D-GlcNAc and NeuNAc were also inhibited at MIC ranging from 25 to 50 mM, but monosaccharides without the N-acetyl group had no effect. On the other hand, an N-acetyl derivative of amino acid, N-acetyl-glycine, was without effect at concentrations up to 200 mM. These results suggest that the N-acetyl group is important but not sufficient for recognition. BSM or orosomucoid was a potent inhibitor with MIC of 6.25 µg/ml, or 15.6 nM assuming M_r = 4.0 × 10⁵ for BSM (18). In contrast, bacterial LPS from E. coli O111 and S. minnesota R595 had no inhibitory effect.

Amino Acid Sequence of TL-P-- The NH₂-terminal sequence analysis of TL-P facilitated the identification of 20 amino acid residues, and this sequence was identical to that of a hemocyte-derived tachylectin, TL-1 (4). Furthermore, a polyclonal antibody against TL-1 recognized TL-P, as determined by immunoblotting (see Fig. 5B). TL-1 consisting of 221 amino acid residues is also a 27-kDa protein on SDS-PAGE but exhibits no hemagglutinating activity against human erythrocytes (4). These results indicate that while the two lectins are homologous they are not identical. TL-P and TL-1 were digested with lysyl endopeptidase, and their peptide maps were compared by reverse-phase HPLC (Fig. 2, A and B). A definitive difference was observed between the two maps. A peak with a retention time of 53 min in TL-1 disappeared in TL-P, and two peaks named K8 and K9 emerged. Amino acid sequence analysis indicated that peptides K8 and K9 derived from TL-P correspond to positions 162-185 and 186-197 in TL-1, with amino acid substitutions Leu¹⁶⁸ and Arg¹⁹⁷ of TL-1 to Ile and Lys in TL-P, respectively. The peptides derived from TL-P were all sequenced, and peptide K4, corresponding to positions 70-84 of TL-1, was found to contain another amino acid substitution with Ser⁷⁷ in TL-1 to Thr. Each substituted residue was confirmed by amino acid composition analysis of the peptides (data not shown). Furthermore, the sequence analysis of peptides derived from endoproteinase Asp-N digest overlapped the K-peptides. Details of the sequence analysis for individual peptides are summarized in Fig. 3. Essentially all amino acid residues were sequenced except for three residues corresponding to positions 36, 198, and 199. 

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Peptide mapping of the proteolytic digest of TL-P (A) and TL-1 (B). Peptides derived from TL-P and TL-1 digested with lysyl endopeptidase were purified by reversed-phase HPLC, as described under "Experimental Procedures."

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Amino acid sequence of TL-P and its fragments. The arrows represent amino acid residues determined by Edman degradation. K, lysyl endopeptidase-digested fragments; D, endoproteinase Asp-N-digested fragments. The outlined letters represent different amino acid residues different from those of TL-1.

To complete the sequence of TL-P, reverse transcription-PCR and DNA sequence analysis were done, as described under "Experimental Procedures." The deduced amino acid sequence confirmed the three amino acid substitutions of TL-P determined at the protein level; the unidentified residues were Cys³⁶, Leu¹⁹⁸, and Lys¹⁹⁹, all of which are identical to those of TL-1 (Fig. 4A). Consequently, TL-P consists of 221 amino acid residues, the same number as those of TL-1. Only the three amino acid residues differ between the two lectins, which are located in the sequence repeats, III, V, and VI (Fig. 4B).

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Nucleotide and deduced amino acid sequences of TL-P. A, amino acid residues are numbered on the right. The arrows represent regions of primers used for PCR. The outlined letters represent amino acid residues different from those of TL-1. B, schematic models of tandem repeats of TL-P and TL-1.

Antibacterial and LPS Binding Activities of TL-P-- Although TL-1 exhibits no hemagglutinating activity, it does show antibacterial activity against Gram-negative bacteria, such as E. coli B (4). TL-P, however, had no significant antibacterial activity against E. coli B, even at a concentration of 80 µg/ml (Table II). In contrast, TL-1 inhibited the growth of E. coli B by 95% under the same conditions.

Apparent Molecular Masses of TL-P and TL-1 in Solution-- The apparent molecular masses of TL-P and TL-1 in solution were determined by gel filtration (Fig. 5A). TL-P and TL-1 bound weakly to this silica-based resin; therefore, 1 M NaCl and 15% acetonitrile were added to the elution buffer to prevent nonspecific binding. TL-P showed two major peaks of 54 and 27 kDa, corresponding, respectively, to a dimer and a monomer. On the other hand, TL-1 had a single peak of 27 kDa, corresponding to a monomer. The presence of TL-P or TL-1 in each peak was confirmed by immunoblotting, using the anti-TL-1 antibody (Fig. 5B). Hemagglutinating activity was detected only in peak 1, not in peaks 2 and 3, suggesting that the dimeric formation of TL-P is an essential condition for hemagglutination. TL-P present in the monomer fraction (peak 2) may be denatured irreversibly and not form the dimer under the conditions used.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Gel filtration of TL-P and TL-1. A, apparent molecular masses of TL-P (top) and TL-1 (bottom) were determined by gel filtration on a Bio-silect SEC 250-5 column, as described under "Experimental Procedures." The arrows represent elution positions of reference proteins. B, immunoblotting analysis of the three peaks obtained by gel filtration with anti-TL-1 antibody. Lane 1, peak 1; lane 2, peak 2; lane 3, peak 3.

Quantification of TL-P in Perivitelline Fluid-- Hemagglutinating activities in perivitelline fluid prepared just after the third and the fourth molt of the embryo were compared. The perivitelline fluid prepared after the fourth molt had 8 times higher hemagglutinating activity than that after the third molt. The content of TL-P in each perivitelline fluid was quantitated by enzyme-linked immunosorbent assay, as described under "Experimental Procedures": 2.9 ± 0.26 µg/ml in the perivitelline fluid after the third molt and 22.4 ± 2.5 µg/ml in that after the fourth molt. Consequently, TL-P increased 7.7-fold during the two stages at the antigen level, which is consistent with the increase in hemagglutinating activity. The protein concentration in the perivitelline fluid after the fourth molt was quantitated to be 112 µg/ml, using the method of Bradford (16). Hence, TL-P accounts for 20% of the total proteins present in the perivitelline fluid at this stage.

TL-P-binding Proteins in Perivitelline Fluid-- The flocculated material, which emerged in the perivitelline fluid during dialysis, was collected as precipitates by centrifugation. Shishikura and Sekiguchi (19) partially purified several glycoproteins from the flocculated material in perivitelline fluid of T. gigas; these glycoproteins inhibited the hemagglutinating activity of the perivitelline fluid.

To determine if the perivitelline fluid of T. tridentatus contains such glycoproteins, the precipitates were washed with 0.5 M GlcNAc and recentrifuged. When the supernatant was subjected to immunoblotting using the anti-TL-1 antibody, a 27-kDa immunoreactive band was observed (data not shown), a finding that suggests that part of TL-P in the perivitelline fluid is incorporated into the flocculated material during dialysis. After washing with 0.5 M GlcNAc, the precipitates were solubilized in 5 M urea and separated by gel filtration, as shown in Fig. 6A. The first fraction gave two protein bands of 13 and 14 kDa on SDS-PAGE, and the second fraction contained TL-P, in addition to these proteins (Fig. 6B). The 13- and 14-kDa proteins in the first fraction co-precipitated with the anti-TL-1 antibody only in the presence of TL-P, not in the presence of TL-1 (Fig. 6C). Furthermore, the co-precipitation of the 13- and 14-kDa proteins was inhibited in the presence of GlcNAc, suggesting that TL-P specifically interacts with these two proteins through the carbohydrates (Fig. 7). A component sugar analysis of the fraction containing the 13- and 14-kDa proteins showed that it contains Gal, Man, Fuc, Xyl, GlcNAc, and GalNAc at a molar ratio of 1.0:5.3:3.4:12.1:13.2:11.0.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   TL-P-binding substances in the perivitelline fluid. A, gel filtration. The sample was solubilized in 50 mM Tris-HCl, pH 8.0, containing 5 M urea, and 0.5 M NaCl was applied to the same column in Fig. 5, equilibrated with the same buffer containing 2 M urea and 0.5 M NaCl. B, fraction tubes indicated by bars were pooled, and aliquots were subjected to SDS-PAGE under reducing conditions (silver staining). Lane 1, peak 1; lane 2, peak 2. C, the fraction F1 was dialyzed against 50 mM Tris-HCl, pH 8.0, containing 0.5 M NaCl, and immunoprecipitated with anti-TL-1 antibody in the presence or absence of TL-P and TL-1, as described under "Experimental Procedures." Lane 1, in the presence of TL-P; lane 2, in the presence of TL-1; lane 3, in the absence of TL-P and TL-1. The precipitated samples were subjected to SDS-PAGE (15% gel) under reducing conditions (silver staining).

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Inhibition of the co-precipitation of the 13- and 14-kDa proteins by GlcNAc. Immunoprecipitation and SDS-PAGE were performed, as described in the legend to Fig. 6, in the absence (lane 1) or presence of GlcNAc (50 mM (lane 2) or 100 mM (lane 3)).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hemagglutinating activity in the perivitelline fluid of the Japanese horseshoe crab T. tridentatus dramatically increased between the third and the fourth molt of the embryo, as was found to be the case for the Indo-Pacific horseshoe crab T. gigas (3). We identified an embryonic 27-kDa lectin, TL-P, in the perivitelline fluid of T. tridentatus. The increase of the hemagglutinating activity in the perivitelline fluid correlated with the increase of TL-P at the antigen level. These results indicate that the major hemagglutinin present in perivitelline fluid during this period is TL-P.

We previously identified four kinds of hemocyte-derived tachylectins, all of which seem to be involved in innate immunity of the horseshoe crab. TL-1 exhibits no hemagglutinating activity but does bind to LPS and polysaccharides such as agarose and dextran with broad specificity (4). TL-2 specifically agglutinates human A-type erythrocytes and recognizes several LPS and staphylococcal lipoteichoic acids, probably through a high ligand density on pathogens (5). In contrast, TL-3 (6) and TL-4 (7) specifically bind to S-type LPS from several Gram-negative bacteria through a certain sugar moiety on O-antigens.

Sequence analysis of TL-P indicated that TL-P is almost the same as TL-1, except for substitutions of three amino acid residues. This slight difference between primary sequences of the two tachylectins makes for a great difference in biological and biochemical characteristics: (i) unlike TL-1, TL-P agglutinates preferentially human A-type erythrocytes; (ii) unlike TL-1, TL-P has no significant affinity for bacterial lipopolysaccharides and does not exhibit antibacterial activity; (iii) based on apparent molecular masses determined by gel filtration, TL-P forms a dimer in solution, while TL-1 is present as a monomer; (iv) TL-P interacts with endogenous proteins of 13 and 14 kDa present in the perivitelline fluid, yet neither protein has any affinity for TL-1.

TL-1 contains a zinc ion in the molecule, and removal of this zinc by EDTA leads to a loss of its sugar binding affinity (4). Hemagglutinating activity of TL-P was not Ca²⁺-dependent, but the activity was completely inhibited by EDTA, thereby indicating that TL-P also contains metal ion(s) with an important role for the sugar binding. Tectonins from the myxomycete Physarum polycephalum have been reported to have sequence similarity to TL-1 (33% identity), but the physiological role of tectonins remains unknown (20). A sequence homology search showed no significant similarity between TL-P and other lectins derived from embryos or vitelline membranes, including chicken galectins (21, 22), sea urchin lectins (23, 24), and fish egg lectins (25).

D-GlcNAc, D-GalNAc, and D-ManNAc, each of which is a stereoisomer at C-2 or C-4, inhibited the hemagglutinating activity of TL-P at similar concentrations. N-Acetyllactosamine (Gal1,4GlcNAc) also inhibited the hemagglutination of TL-P. These results suggest that the configuration of C-2 and C-4 and a free 4-OH group of the ligand are not essential for TL-P for sugar recognition, and this is not the case for TL-2 with GlcNAc specificity. The importance of the configuration at C-2 and C-4 and a free 4-OH is evident from the crystal structure of TL-2 containing the ligand (26). TL-P probably recognizes the ligand mainly through the N-acetyl group.

The inner egg membrane of horseshoe crab has an ion-permeable nature for Na⁺, Cl, and Ca²⁺, but it does not allow for permeation of substances of higher molecular weights, such as carbohydrates, proteins, and lipids (27). These data suggest that TL-P functions in the perivitelline fluid without secretion through the membrane. Shishikura and Sekiguchi (19) partially purified several glycoproteins from the perivitelline fluid of T. gigas, which inhibit the hemagglutinating activity of the perivitelline fluid. Here we identified candidates of endogenous ligands for TL-P from the perivitelline fluid of T. tridentatus. These substances of 13 and 14 kDa were solubilized from aggregates formed in the perivitelline fluid during dialysis. TL-P could be eluted from precipitates with 0.5 M D-GlcNAc, suggesting that the endogenous ligands are glycoproteins containing N-acetylhexosamines. The potential ligands could be immunoprecipitated with TL-P but not with TL-1, using the anti-TL-1 antibody. The dimeric form of TL-P may lead to an increase in binding affinity for multivalent ligands.

The perivitelline fluid rapidly increases about 5-fold in volume between the third and the fourth molt of the embryo; therefore, the maintenance of osmotic pressure of the perivitelline fluid is an important factor to complete embryonic development (1, 27). Several carbohydrates and glycoproteins have been reported to be major factors to maintain osmotic pressure in perivitelline fluid (27). TL-P occupies the majority of proteins in the perivitelline fluid after the fourth molting, accounting for 20% of the total proteins. TL-P probably has an important role in regulating the carbohydrate concentrations in the perivitelline fluid by interacting with endogenous glycoproteins or N-acetyl hexosamines.

Structural differences between TL-P and TL-1 probably originated during molecular evolution by gene duplication and the accumulation of point mutations from the common ancestral gene. This is considered to be a good example of isolectins that have obtained different biological properties by mutating only a few amino acids of the ancestral lectin.

	ACKNOWLEDGEMENTS

We thank W. Kamada and H. Kondo (Institute of Biological Sciences, University of Tsukuba) for technical assistance with amino acid sequence analysis. We also thank Drs. S. Iwanaga (Professor Emeritus, Kyushu University) and O. Numata and T. Takeda (Institute of Biological Sciences, University of Tsukuba) for helpful discussion.

	FOOTNOTES

^* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (to S. K.) and Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation (to S. 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 GenBank^TM/EMBL Data Bank with accession number(s) AB028144.

^¶ To whom correspondence should be addressed: Dept. of Biology, Kyushu University, Fukuoka 812-8581, Japan. Tel./Fax: 81-92-642-2633 or 81-92-642-2634; E-mail: skawascb@mbox.nc.kyushu-u.ac.jp.

	ABBREVIATIONS

The abbreviations used are: TL, tachylectin; LPS, lipopolysaccharide(s); BSM, bovine submaxillary gland mucin; MIC, minimum inhibitory concentration; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES