¹Department of Life Sciences, University of Tokushima

Tokushima, JAPAN

sea-hide@ias.tokushima-u.ac.jp

　

²Graduate School of Integrated Science, Yokohama City University

Yokohama-Kanagawa, JAPAN

　

　

The toxopneustid sea urchin, Toxopneustes pileolus and Tripneustes gratilla have well-developed globiferous pedicellariae with bioactive substances. Two D-galactose-binding lectins (SUL-I and SUL-II) and a heparin-binding lectin (TGL-I) were purified from T. pileolus and T. gratilla using gel filtration chromatography, affinity chromatography, and reverse-phase HLPC. SUL-I and SUL-II from the large flower-like globiferous pedicellariae of T. pileolus are D-galactose-binding proteins with molecular masses 32 kDa and 23 kDa, respectively (Nakagawa et al., 1999a). Furthermore, Contractin A (Nakagawa et al., 1991), a mannose-containing glycoprotein (18 kDa) from the ordinary globiferous pedicellariae of T. pileolus is also a novel lectin that causes smooth muscle contraction and relaxation. On the other hand, TGL-I from the small globiferous pedicellariae of T. gratilla is a Ca²⁺-independent heparin-binding protein with a molecular mass of 23 kDa (Nakagawa et al., 1999a). SUL-I and Contractin A induced mitogenic stimulation on murine splenocytes but SUL-II and TGL-I did not. SUL-I had weak cytotoxic activity on murine splenocytes, and promoted chemotaxis of guinea-pig macrophages. SUL-I did not show a sequence homology to the N-terminal 21 amino acid sequence of SUL-II. However, SUL-I is related to fish egg lectins (Tateno et al., 1998; Hosono et al., 1999). On the other hand. SUL-II showed a sequence homology to Contractin A and UT841 from T. plileolus (Zhang et al., 2001), which may be a phospholipase A₂-like substance. The present results suggest that the toxopneustid sea urchins might be a resource for invertebrate lectins with an interesting mechanism of action.

　

　

Most animal lectins can be classified into two groups: C-type lectins, which are dependent on Ca²⁺ for their carbohydrate binding activity; and galectins, soluble molecules that share characteristic amino acid sequences and specificity for β-galactoside (Drickamer, 1988; Barondes et al., 1994; Kasai and Hirabayashi, 1996). Although in recent years, some β-galactoside binding lectins have also been isolated from marine invertebrates (Yokosawa et al., 1986; Ozeki et al., 1991, 1997; Mikheyskaya et al., 1995), it is not clear that they appear to be a family of galectins. The toxopneustid sea urchins, Toxopneustes pileolus and Tripneustes gratilla have well-developed globiferous pedicellariae with bioactive substances. Some of these bioactive substances caused deleterious effects such as severe pain, syncope respiratory distress, and loss of consciousness (Fujiwara, 1935; Mendes et al., 1963; Alender et al., 1965; Kimura et al., 1975; Mebs, 1984). We have recently purified a D-galactose-binding lectin (SUL-I) from the large flower-like pedicellariae of sea urchin, T. pileolus. SUL-I with a molecular mass of 32 kDa showed chemotactic properties for guinea-pig neutrophils (Nakagawa et al., 1996). Although the physiological roles of SUL-I from the large globiferous pedicellariae of T. pileolus are unknown, it is suggested that the primary role of pedicellarial lectin may be defense and offense against a foreign body. More recently, we have also isolated a coelomic lectin from the coelomic fluid of T. pileolus (unpublished data). It is possible that animal lectins including those within the galectin family may function in a variety of biological processes. Direct evidences for particular functions have recently begun to accumulate for not only vertebrate lectins but also invertebrate lectins. Here we present the results on biological activities of pedicellarial lectins from the toxopneustid sea urchins, T. pileolus and T. gratilla.

　

　

Toxopneustes pileolus (47 specimens) and Tripneustes gratilla (48 specimens) were collected along the coast of Shikoku Island and Okinawa Island Japan, from 1992 through 1996 (Fig.1). Rabbit blood sample was obtained from Nippon Bio-test Lab. (Tokyo, Japan). Sephadex G-200 gel and heparin-Sepharose CL-6B were obtained from Amersham Biosciences Corp. (New Jersey, U.S.A.). Immobilized D-galactose gel was from Pierce (Illinois, U.S.A.). All the other chemicals were reagent grades.

　

Thirty large flower-like pedicellariae per T. pileolus sea urchin specimen (8-10 cm in diameter) were removed with fine forceps. They were extracted with 20 ml of 0.15 M NaCl at 4℃ for twenty-four hours. The 20 ml aliquot of each was centrifuged at 12,000 g for twenty minutes and the supernatant was used as the crude lectin extract (Nakagawa et al., 1996). Briefly, for the first step of purification, the crude extract was applied to a Sephadex G-200 column (2.6 x 80 cm) equilibrated with 0.15 M NaCl solution and was eluted with the same solution at a flow rate of 15 ml/hour. Fractions of 10 ml each were collected and analyzed for absorption at 280 nm and agglutinating activity. For the second step of purification, the gel chromatographic fractions (the second protein peak and third protein peak) were dissolved in 150 mm phosphate buffer and placed on an immobilized D-galactose column (1 x 2 cm). The sample was washed with same buffer and was eluted with 100 mM D-galactose in the same buffer. The 2 ml elution fractions were collected and analyzed for absorption at 280 nm and agglutinating activity. Each of the second peaks was pooled. Final purification was achieved by HPLC using a reverse-phase C₈ column. Two solvents, 0.1% trifluoroacetic acid (TFA) and acetonitrile in 0.08% TFA were used. The fraction was monitored at 230 nm. The main peaks were pooled and analyzed for agglutinating activity and SDS-PAGE, and then used as the purified sea urchin lectins (SUL-I and SUL-II). In the case of T. gratilla, the pedicellarial extract was fractionated as reported previously (Nakagawa et al., 1999a). The venom proteins from the pedicellariae were extracted with 20 ml distilled water at 4℃ for twenty-four hours. Twenty ml aliquots of the extract were centrifuged at 12,000 g for twenty minutes and the supernatant was used as the crude lectin extract. The extract was applied to a Sephadex G-200 column equilibrated with 0.15 M NaCl solution containing 10 mM lactose at flow rate of six ml/hour. Final purification was achieved by heparin-Sepharose CL-6B affinity chromatography. The gel chromatographic fraction (the first protein peak) was dissolved in 6.4 mM phosphate buffer saline (PBS), pH 7.2, and placed on a heparin-Sepharose CL-6B column (1 x 2 cm). The sample was washed with PBS and was eluted with 1.0 M NaCl in PBS. The 2 ml elution fractions were collected and analyzed for absorption at 280 nm and agglutinating activity. The second protein peak was collected and used a purified heparin-binding lectin (Tripneustes gratilla lectin-I, TGL-I). Contractin A from the ordinary globiferous pedicellariae of T. pileolus was purified as reported previously (Nakagawa et al., 1991).

　

　

The agglutinating activity was assayed by using rabbit erythrocytes in microtiter plates. Twenty-five μl of 2% (V/V) suspension of erythrocytes in PBS was added to 50 μl of serial two-fold dilutions of the lectin fractions and purified sea urchin lectins. The plates were incubated at room temperature for one hour. The results were expressed by the minimum concentration of the test solution (μg/ml) required for positive agglutination.

　

The mitogenic activity by murine splenocytes was determined by the cell culture assay using the tetrazolium salt MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide). Fresh splenocytes were taken from female ddY mice (30-40 g) and suspended in a RPMI-1640 medium supplemented with penicillin and streptomycin (100 μg/ml and 100 U/ml). The splenocytes with or without concanavalin A (Con A) and lectin fractions were plated in flat-bottomed microtiter plates incubated at 37℃ in a humidified atmosphere containing 5% CO₂ for sixty-eight hours. Ten μl of MTT (5 mg/ml) was then introduced in each well and the formazan in the cells was extracted with 10% sodium dodecyl sulfate (SDS). The optical density of each well was measured spectrophotometrically with microplate reader (Bio-Rad, model 450) at 570 nm.

　

Macrophages were induced by intraperitoneal injection of 1% glycogen solution into male guinea pigs (350-400 g), and collected by centrifugation in PBS (Ohura et al., 1990). The washed cells were resuspended in Dulbecco's modified Eagle's medium (DMEM) and adjusted to a macrophage density of 1 x 10⁶ cell/ml. The macrophage cell suspensions were plated in the 96-well plate at 100 μl per well and allowed to stand for one hour. Non-adherent cells were washed out by two washings with Eagle's balanced salt solution (EBSS). One-hundred μl EBSS containing fluorescein conjugated Escherichia coli bioparticles adjusted to 1 x 10⁷ cell/ml were added to the adherent macrophages. After incubation for one hour at 37℃ in a 5% CO_2- humidified atmosphere, extracellular fluorescence was quenched by 100-μl trypan blue (0.25g/ml of 13 mM citrate buffer), and the dye was removed. The fluorescence intensity was measured at 485 nm excitation and 530 nm emission using by a Cyto Fluor (PerSeptive Biosystems).

　

The amino acid sequence was determined directly on a Shimadzu PPSQ-10 system protein sequencer.