Toxicon 99 (2015) 51e57
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Neutralizing effect of hemolymph from the shore crab, Thalamita crenata, on paralytic shellfish toxins Huajuan Lin a, b, Chaohua Zhang a, b, Jianmeng Liao c, Feng Yang d, Saiyi Zhong a, b, Peihong Jiang a, Xiao Chen a, Yuji Nagashima e, * a
College of Food Science and Technology, Guangdong Ocean University, Zhanjiang 524088, China Guangdong Provincial Key Laboratory of Aquatic Product Processing and Safety, Zhanjian, Gaunadong Province 524088, China Zhanjiang Institute of Supervision & Test on Quality & Measure, Zhanjiang 524096, China d Zhanjiang Ocean and Fishery Environment Monitor Station, Zhanjiang 524039, China e Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Minato, Tokyo 108-8477, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 January 2015 Received in revised form 16 March 2015 Accepted 18 March 2015 Available online 19 March 2015
Several species of crabs are resistant to paralytic shellfish toxins (PSTs) and/or pufferfish toxin, tetrodotoxin, regardless of toxification by the toxins. The shore crab Thalamita crenata, which inhabits Leizhou Peninsula, China, is tolerant to PST toxicity, and the hemolymph has neutralizing effects against the lethal activity of PST. In the present study, we investigated the PST neutralizing factors in the hemolymph from T. crenata and successfully separated PST-binding proteins by PST-ligand affinity chromatography. The neutralization factors, obtained in the fraction with a molecular weight over 10 kDa by ultrafiltration, were susceptible to proteases such as alcalase, animal complex proteases, pancreatin, and papain. The PST-binding protein had high dose-dependent neutralization effects on PST toxicity. The PST-binding activity of the protein was stable at 25 C and then decreased with an increase in temperature; heating at 65 C for 60 min eliminated the initial activity by two-thirds. The PST-binding activity was strongly inhibited in the presence of Mg2þ and Ca2þ, but not Naþ and Kþ. The PST-binding capability of the protein differed among PST components in descending order of neosaxitoxin, gonyautoxins 1 and 4, saxitoxin, and gonyautoxins 2 and 3, suggesting a structureeactivity relationship in PST binding. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Shore crab Thalamita crenata Paralytic shellfish toxins (PST) Toxicity neutralization PST-binding protein PST-ligand affinity chromatography
1. Introduction Some species of crabs are often toxified by marine bio-toxins, such as paralytic shellfish toxins (PSTs), tetrodotoxin (TTX), and palytoxin. Upon ingestion, these toxified crabs occasionally cause human intoxication, including fatalities, in various parts of the world, and thus constitute a worldwide health threat (Alcala et al., 1988; Llewellyn et al., 2002; Noguchi et al., 2011). Since Hashimoto et al. (1969) discovered toxic crabs, Zosimus aeneus, Platypodia granulosa, and Atergatis floridus in the family Xanthidae, in the Ryukyu and Amami Islands, Japan, toxic crabs have been reported
* Corresponding author. Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Konan, Minato, Tokyo 1088477, Japan. E-mail addresses: [email protected] (H. Lin), [email protected] (C. Zhang), [email protected] (J. Liao), [email protected] (F. Yang), zsylxc@126. com (S. Zhong), [email protected] (P. Jiang), [email protected] (X. Chen), [email protected] (Y. Nagashima). http://dx.doi.org/10.1016/j.toxicon.2015.03.010 0041-0101/© 2015 Elsevier Ltd. All rights reserved.
in at least 20 species from 5 different families (Majidae, Atelecylidae, Portunidae, Xanthidae, and Grapsidae), mainly in the Pacific region, including Australia, Philippines, and Taiwan (Yasumura et al., 1986; Llewellyn and Endean, 1989; Tsai et al., 1996; Negri and Llewellyn, 1998; Ho et al., 2006), as well as Japan (Koyama et al., 1983b; Noguchi et al., 1983; Yasumoto et al., 1983). Among these toxic crabs, xanthid crabs have extremely high levels of PST and/or TTX. Importantly, toxic crabs administered intrahemocoelically with the toxins exhibited much higher resistance to the lethal effects of the toxins than non-toxic crabs. In specimens of A. floridus and Z. aeneus, the minimum lethal dose (MLD) of TTX is 1000e2000 mouse units (MU)/20g body weight (equivalent to 10e20 mg/kg body weight) and the MLD of gonyautoxins (GTXs) is 5000e10000 MU/20g body weight (equivalent to 50e100 mg/kg body weight) (Koyama et al., 1983a; Daigo et al., 1987). Some species of non-toxic grapsid crabs belonging to the genus Hemigrapsus also exhibit weak resistance to PST and TTX. Canadian shore crabs Hemigrapsus oregonesis and Hemigrapsus nudus are tolerant to PST only when previously exposed to PST (Barber et al., 1988a, 1988b).
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In contrast, the Japanese shore crab Hemigrapsus sanguineus is tolerant to TTX throughout the year (Shiomi et al., 1992; Yamamori et al., 1992). There are two general toxicity tolerance mechanisms of the crabs toward PST and/or TTX. The first is that their neurocytes are unresponsive to the toxins, which act as specific blockers of voltage-gated sodium channels, e.g., xanthid crabs containing high levels of toxin (Daigo et al., 1988). The second mechanism is endogenic special substances with toxin-binding capability in the hemolymph, e.g., non-toxic Hemigrapsus crabs (Baber et al., 1988b; Shiomi et al., 1992; Nagashima et al., 2002). We have been screening for PST toxicity of shore crabs that inhabit the Leizhou Peninsula, China, located north of the South China Sea, to monitor coastal environmental pollution and shellfish toxification by harmful algae. Our previous investigation revealed that shore crabs inhabiting this area e Thalamita crenata, Leptodius exaratus, and Metopograpsus latifrons e are non-toxic species based on the toxicity levels in the tissues, despite the fact that 3 of 25 individuals collected from December 2009 to March 2012 had slight PST toxicity of 4.3e4.4 MU/g (Lin et al., 2012). Furthermore, the 100% lethal dose of PST for the three species exceeded 5 MU/ 20g of crab, which was relatively higher than that for other sensitive crab species (Koyama et al., 1983a; Hwang et al., 1990; Yamamori et al., 1992; Nagashima et al., 1998). This indicates that these three species of crabs have weak resistance to PST. The lethal time of mice administered PST solution with the hemolymph from the crabs was significantly prolonged, suggesting that the crab hemolymph neutralized the PST toxicity in mice (Lin et al., 2012). The functional agents in the hemolymph that neutralize the toxicity, however, remain to be clarified. In the present study, we investigated the neutralizing factors of hemolymph from T. crenata against PST toxicity with respect to the molecular range and susceptibility to various proteases, and successfully separated PST-binding proteins by PST-ligand affinity chromatography. The PST-binding proteins were found to bind PST components (e.g., saxitoxin [STX], neoSTX, GTX1&4, and GTX2&3) with different PST-binding capabilities.
The animal experiments were performed according to the rules and regulations of the animal ethics committee of the Guangdong Laboratory Animal Monitoring Institute. 2.2. Neutralizing activity against PST toxicity of hemolymph samples Neutralizing activity against PST toxicity was estimated using a mouse assay as described previously (Lin et al., 2012). Briefly, a PST stock solution was prepared to a suitable concentration. In a control experiment, the PST solution was mixed with an equal volume of 10 mM phosphate buffer (pH 7.4) containing 8 mM EDTA and 150 mM sucrose, and intraperitoneally injected into a group of three or five mice to kill the mice within 4e6 min. In a test experiment, the PST solution (same as used in the control experiment) was mixed with hemolymph samples at 25 C for 30 min and intraperitoneally injected into mice in the same manner as the control. The time to kill the mice was recorded and expressed as mean ± standard deviation (SD), based on Student's t-test to examine the significant differences between groups. A p value less than 0.05 was considered statistically significant. Hemolymph was treated with proteases (alcalase, animal complex protease, pancreatin, and papain) to examine the effect of proteolysis on the neutralizing activity of the hemolymph sample. It was digested with each protease at a final concentration of 2.5 unit/ mL for 12 h at 35 C, and subjected to the mouse assay to estimate the neutralizing activity against PST toxicity. In addition, inactivated proteases were also used. The proteases were denatured by heating in boiling water for 5 min. Here, one unit of protease activity was defined as the release of 1 mg tyrosine/mg protein for 1 min. The hemolymph was separated into two fractions by ultrafiltration (nominal molecular weight cut-off 10 kDa, Millipore Amicon, Bedford, MA, USA), and subjected to the neutralizing activity test described above. 2.3. PST binding activity of hemolymph samples
2. Materials and methods 2.1. Materials Shore crab T. crenata was collected at the coast of the Leizhou Peninsula, China, from March to November 2011. In each sampling, about 30 specimens obtained (body weight of 23e48 g) were immediately transported to the laboratory of Aquatic Product Advanced Processing of the Guangdong Higher Education Institutes. The hemolymph was withdrawn from the carpus of live crabs as described previously with slight modification (Lin et al., 2012) and pooled to use as a hemolymph sample. PSTs used in this study were partially purified from the toxic xanthid crab Z. aeneus by the method of Lin et al. (2011) and contained mainly STXs. PST standards, STX, neoSTX, GTX1&4, and GTX2&3, were supplied from the National Research Council Institute for Marine Biosciences, Canada. Alcalase (alkali endoprotease) from Bacillus licheniformis, animal complex protease (containing endoprotease, peptidase, and exo-protease), pancreatin (containing protease, lipase, and amylase), and papain (EC3.4.22.2) were obtained from Pangbo Biological Engineering Co., Ltd. (Nanning, China). AFEpoxy-650M resin was purchased from Tosoh Corporation (Tokyo, Japan). Bovine serum albumin (BSA) was obtained from SigmaeAldrich Co. (St. Louis, MO, USA). Male KM strain mice (20 ± 2 g of body weight) were kindly supplied by Shuanglin Biological Medicine Co., Ltd. (Zhanjiang, China). The facilities for the experimental animals are fully accredited with license number SYXK 2013-0010.
PST binding activity was measured by HPLC as follows: In the control experiment, the PST solution was mixed with an equal volume of 10 mM phosphate buffer (pH 7.4) containing 8 mM EDTA and 150 mM sucrose, put into a centrifugal ultrafiltration tube (3 kDa cut-off, Millipore Amicon), and centrifuged at 20,000 g for 10 min at 4 C. PST was obtained in the resulting filtrate. In a test experiment, the PST solution was mixed with the hemolymph sample and allowed to stand for 1 h at 4 C. The mixture was subjected to centrifugal ultrafiltration under the same condition as the control. In this case, the filtrate contained free-form PSTs that were not bound to the hemolymph. The bound PSTs were estimated by subtracting free-form PSTs from PSTs in the control. PST was determined by HPLC with a post-column fluorescent reaction. The HPLC system comprised an LC-20AD (Shimadzu, Kyoto, Japan) with an XB-C18 column (4.6 mm 150 mm, Welch Materials Inc., Shanghai, China). The mobile phase and post column reaction conditions for STXs were as described by Oshima (1995) and those for GTXs by Jiang et al. (2013). Triplicate tests were carried out for each sample, and the data were expressed as mean ± SD. The relative PST-binding activity was expressed as the ratio of each sample to the highest sample. In addition, an ELISA test for PST-binding activity was performed in a preliminary experiment using a SKit (ELISA for paralytic shellfish poison, Shin-Nihon Kentei Kyokai, Tokyo, Japan) under the previously described procedure (Sato et al., 2014).
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2.4. PST-ligand affinity chromatography of hemolymph samples A PST-coupled affinity column was prepared according to the method by Takati et al. (2007) with some modifications. Briefly, 5 g of AF-Epoxy-650M resin was suspended in 5 mL isopropanol and treated with a mixture of ethylene diamine and dimethyl formamide for 30 min at room temperature. After washing with 50 mM phosphate buffer (pH 7.4), the resin was further treated with 5% glutaraldehyde for 30 min at room temperature and then washed with the same buffer. A total of 66 mg STXeq of PST (tested by mouse assay) was added to the activated resin and gently stirred for 4 h at room temperature. After removing the excess free PST by washing with the buffer, the PST-coupled AF-Epoxy-650M was established by adding 0.1 M NaBH4. The 10-fold diluted hemolymph was added to the PST-coupled AF-Epoxy-650M and incubated overnight at 4 C under weak agitation. The resin was poured into a glass column with a 1.5-cm diameter. The column was washed with 50 mM phosphate buffer (pH 7.4) and eluted with 1 M TriseHCl buffer (pH 10) at a flow rate of 0.5 mL/min. Fractions with a volume of 1.5 mL were collected and immediately neutralized by adding 1.5 mL of 100 mM glycine buffer (pH 2.5). The absorbed fractions containing PST-binding proteins were pooled and the solvent displaced with 50 mM phosphate buffer (pH 7.4) by ultrafiltration (3 kDa cut-off) repeatedly. 2.5. Stability of PST-binding activity Effects of incubation time, temperature, and bivalent ions on PST-binding activity were evaluated. To test the effects of incubation time, the PST/protein mixture in 50 mM phosphate buffer (pH 7.4) was incubated at 4 C for various times, ranging from 0 to 240 min. To test the effects of temperature, the PST/protein mixture in the same buffer was allowed to stand for 60 min at a temperature of 4 C, 15 C, 25 C, 37 C, 50 C, or 65 C. To test the effects of the bivalent ions, MgCl2 or CaCl2 at a final concentration of 10 mM in 50 mM phosphate buffer (pH 7.4) were screened under the optimal conditions (4 C, 60 min). We similarly tested NaCl and KCl. After incubation, the PST/protein mixture was ultrafiltered using a centrifugal ultrafiltration tube (3 kDa cut-off). The obtained filtrates were applied to HPLC analysis to estimate the PST-binding activity, as described above. 2.6. Analytical methods
Fig. 1. Neutralizing effect of hemolymph on PST toxicity to mice. The results are shown as the means ± standard deviation (SD) of triplicate measurements. Statistical significance (p < 0.05) is indicated by asterisk (*), compared to the control.
with the control (5.2 ± 0.2 min; p < 0.05), indicating that the PST toxicity was diminished by the hemolymph samples. Moreover, this result suggested that there is no significant seasonal variation in the neutralizing effect of the hemolymph on PST toxicity, at least from spring through fall. To analyze the neutralizing factors in the hemolymph, the hemolymph sample was separated by molecular size using ultrafiltration (10 kDa cut-off). The survival time of mice administered >10 kDa fractions was significantly longer than that of the controls (p < 0.05) and comparable to that following administration of the initial hemolymph sample (Fig. 2). This finding indicated that the neutralizing factors against PST toxicity were contained mainly in the fraction with a high molecular size (>10 kDa). The hemolymph sample was treated with four types of proteases, including alcalase (alkali endoprotease), animal complex protease (containing endoprotease, peptidase, and exo-protease), pancreatin (containing protease, lipase, and amylase), and papain. The neutralizing effect of hemolymph against PST toxicity was remarkably decreased, irrespective of the proteases used in this study (Fig. 3). In contrast, when the hemolymph sample was treated with proteases inactivated by heating, the hemolymph neutralizing effect was essentially the same as that of the nontreated hemolymph (Fig. 3). SDS-PAGE analysis confirmed hydrolysis of the hemolymph sample by the proteases (Fig. 4). Both the hemolymph sample (lane 10) and hemolymph treated with inactivated proteases (lanes 2, 4, 6, and 8) had a band at around 80 kDa,
Protein content was measured according to the Bradford procedure, using BSA as the standard protein (Bradford, 1976). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted according to the method originated by Laemmli (1970) using 10% polyacrylamide (containing 0.1% SDS) as a separation gel. Protein samples from the hemolymph were dissolved in 0.125 M TriseHCl buffer (pH 6.8) containing 5% sucrose, 5% 2mercaptoethanol, and 2% SDS, followed by heating in boiling water for 5 min. Native-PAGE for the PST-binding protein fraction was performed under the same SDS-PAGE conditions, except for without SDS and 2-mercaptoethanol agents. The protein bands were stained with Coomassie brilliant blue R-250. 3. Results 3.1. Neutralizing effect of hemolymph against PST toxicity The neutralizing effects of hemolymph from the shore crabs T. crenata collected at March, June, October, and November 2011, on PST toxicity are shown in Fig. 1. All hemolymph samples significantly prolonged survival of the mice (10.7e20.2 min), compared
Fig. 2. Neutralizing effect of hemolymph fractionated by ultrafiltration on PST toxicity to mice. The results are shown as the means ± standard deviation (SD) of triplicate measurements. Statistical significance (p < 0.05) is indicated by asterisks (*, **), compared to the control and the fraction with a molecular weight lower than 10 kDa, respectively.
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mL) was administered to a group of five mice, two mice survived and the time to death of the other three mice was prolonged approximately 4-fold (25.1 ± 4.5 min) compared with the control (5.5 ± 0.2 min) and a mixture of PST and BSA at a final concentration of 2.0 mg protein/mL (6.4 ± 0.4 min). This result demonstrated that the PST-binding protein fraction considerably decreased the toxicity of PST. The neutralizing activity of the fraction was evaluated to be 0.4 MU/mg protein from death time-mouse unit relations for PST (Association of Official Analytical Chemists, 2005). We then examined the effect of the concentration of the PST-binding protein on the neutralizing activity. The PST-binding protein fraction almost linearly increased the survival time of mice in a concentration-dependent manner, up to 5.0 mg/mL (Fig. 6). 3.3. PST-binding ability of the PST-binding protein
Fig. 3. Proteolytic effect on neutralizing activity of hemolymph for PST toxicity. Hemolymph (abbreviated as H) was treated by four types of proteases, alcalase (E1), animal complex protease (E2, containing endoprotease, peptidase, and exo protease), pancreatin (E3, containing protease, lipase, and amylase) and papain (E4). The results are shown as the means ± standard deviation (SD) of triplicate measurements. Statistical significance (p < 0.05) is indicated by asterisk (*), compared to the control.
Fig. 4. SDS-PAGE of protease-treated hemolymph. Lane1; E1 þ H, 2; inactivated E1 þ H, 3; E2 þ H, 4; inactivated E2 þ H, 5; E3 þ H, 6; inactivated E3 þ H, 7; E4 þ H, 8; inactivated E4 þ H, 9; protein maker, 10; hemolymph.
while the hydrolyzates of the hemolymph sample treated with the proteases (lanes 1, 3, 5, and 7) degraded the band. These results demonstrated that the neutralizing activity of hemolymph against PST toxicity was attributed to proteinaceous substances that are susceptible to all tested proteases, including alcalase, animal complex protease, pancreatin, and papain.
3.2. Preparation of PST-binding protein fraction from hemolymph PST-ligand affinity chromatography of the hemolymph sample was performed to isolate the PST toxicity neutralizing factor in the hemolymph. A sharp protein peak was detected at fraction numbers 88-98 in the absorbed fraction, designated the PSTbinding protein fraction (Fig. 5A). The fraction still formed several bands in native-PAGE (Fig. 5B), although the affinity chromatography yielded 41.6 mg of protein from an aliquot of 5 mL hemolymph sample with 289 mg of protein. Table 1 shows the neutralizing activity of the PST-binding protein fraction against PST toxicity. When PST mixed with the PSTbinding protein fraction (a final concentration of 2.0 mg protein/
In the preliminary experiment using a PST-ELISA kit (SKit, ShinNihon Kentei Kyokai), the PST-binding ability of the hemolymph sample (protein concentration of 57.8 mg/mL) was determined to be 3.13 ± 0.11 nmol PST/mL, which was equivalent to 0.054 ± 0.002 nmol PST/mg protein. In addition, the PST-binding ability of the PST-binding protein fraction was estimated to be 0.092 ± 0.010 nmol PST/mg protein. The optimum conditions for binding of the protein with PST were then examined by HPLC methods. The PST-binding activity increased with incubation time up to 60 min, and reached a steady level after 60 min (Fig. 7). The PST-binding activity was stable at temperatures below 25 C, and then decreased with an increase in temperature (Fig. 8). The PST-binding activity decreased to 36.1 ± 7.2% when the temperature was 65 C. Bivalent ions, such as Mg2þ and Ca2þ, strongly inhibited PST-binding activity (Fig. 9). In contrast, the effects of monovalent ions, such as Naþ and Kþ, on PST-binding activity were minimal. The relative PST-binding activity with Naþ and Kþ was comparable with that of the control (Fig. 9). These findings implied that bivalent ions are involved in the PST-ligand binding of the PST-binding protein. The binding ability of the PST-binding protein to PST components, including STX, neoSTX, GTX1&4, and GTX2&3 is summarized in Table 2. The PST-binding ratios for the toxins differed from each other; the maximum of 72.3 ± 1.3% for neoSTX and the minimum of 32.5 ± 1.3% for GTX2&3. This result indicated that the PST structures have a remarkable impact on the PST-binding activity of the PSTbinding protein. 4. Discussion The present study revealed that the hemolymph from the shore crab T. crenata had a neutralizing effect against PST toxicity. The neutralizing effect was attributable to PST-binding proteins in the hemolymph, because the neutralizing effect of the PST-binding protein based on the mouse assay (0.4 MU/mg protein) was similar to that of the protein based on ELISA (0.092 nmol/mg protein), despite the use of a PST solution comprising a mixture of PSTs. It is likely that the PST-binding proteins are endogenous, because the highly neutralizing effect was observed regardless of season and year, based on both the present and previous studies (Lin et al., 2012). Furthermore, we surveyed both the cell density of PSTproducing dinoflagellates and cell toxicity in the Leizhou Peninsula from January to November, 2012. During the monitoring, only one type of PST-producing dinoflagellate, Alexandrium tamarense, was found, which appeared mainly from February through July. During this period, the highest cell density was GTX1&4 > STX > GTX2&3. The structures of both neoSTX and GTX1&4 have a hydroxyl group at N-1, while STX and GTX2&3 have a hydrogen at N-1 (Fig. 10). Therefore, substitution of a hydroxyl group at the N-1 position may affect the PST-binding capability of the proteins, which is very helpful for promoting PST-ligand binding. Second, substitution of the sulfonyl group at the C-11 position may also affect the PST-binding capability by decreasing PST binding with the proteins. These findings indicate
a structureeactivity relationship among PST components. Also, in terms of a structureeactivity relationship, the PST-binding proteins from T. crenata hemolymph differ from those of saxiphilin. Mahar et al. (1991) reported that the addition of a hydroxyl group at N-1 and a a-hydroxysulfate moiety at C-11 in PSTs profoundly affects the binding affinity in competitive displacement experiments: 550-fold and 160-fold reduction in binding affinity in the molecular pairs of neoSTX/STX and C1/B1 (GTX5), respectively. These results suggest that the two positions, N-1 and C-11, have important roles in binding with saxiphilin. Notably, the hemolymph from T. crenata also has an apparent TTX toxicity neutralizing effect (Lin et al., 2012). Llewellyn et al. (1997) surveyed diverse animal species to investigate the phylogenetic distribution of saxiphilin based on [3H]STX binding activity and found that several species of crabs in the family Xanthidae exhibit saxiphilin-like activity. An STX-binding fraction that did not bind TTX was found in the hemolymph of xanthid crabs, Lophozozymus pictor, Liomera tristis, Chlorodiella nigra, and Actaeodes tomentosus (Llewellyn, 1997). In addition, we screened non-toxic crabs for resistance to PST and TTX, and determined that the family Ocypodidae Scopimera globosa and the family Grapsidae Gaetice depressus were resistant to PST, but not to TTX (Nagashima et al., 1998). On the contrary, the family Grapsidae H. sanguineus was tolerant to TTX, but not to PST (Shiomi et al., 1992; Yamamori et al., 1992), and its hemolymph contained a unique protein that selectively binds TTX (Nagashima et al., 2002). In the present study, we focused on the neutralization effects the hemolymph from T. crenata against PST toxicity and PST-binding ability, because the neutralizing effect of the hemolymph against PST was higher than that against TTX (Lin et al., 2012). It is possible that the PST-binding proteins have binding ability to TTX or the hemolymph has TTX neutralizing mechanisms distinct from those of PST-binding proteins. Additional studies are needed to examine the physical and chemical properties of the PST-binding protein, and to elucidate the primary structure. Acknowledgments This study was supported by 2012 Guangdong Science Province
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and Technology Plan, Province International Cooperation Projects (Project Number: [2012] 98); the Earmarked Fund for Modern Agro-industryTechnology Research System of China (CARS-48), and Project of the Establishment of an Asian Research Center of Excellence in Healthy and Safe Marine Food Resources, in Tokyo University of Marine Science and Technology, Japan. The authors thank Shin-Nihon Kentei Kyokai for provision and technical instructions of SKit. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2015.03.010. Ethical statement All animal experiments were performed according to the Chinese regulations for experimental animal, “Regulations on the management of laboratory animal for Guangdong Province”, “Regulations on experimental animal body disposal & medical waste” and “Opinion on guiding to treat experimental animal”. The facilities for experimental animal are accredited by the Guangdong Laboratory Animal Monitoring Institute, and the experimental animal use license was approved by Guangdong Provincial Department of Science and Technology. References Alcala, A.C., Alcala, L.C., Garth, J.S., Yasumura, D., Yasumoto, T., 1988. Human fatality due to ingestion of the crab Demania reynaudii that contained a palytoxin-like toxin. Toxicon 26, 105e107. Association of Official Analytical Chemists, 2005. AOAC official methods 959.08. Paralytic shellfish poison. Biological method. In: Truckses, M.W. (Ed.), AOAC Official Methods of Analysis, l8th ed. AOAC International, Gaithersburg, MD. USA, pp. 79e80. Barber, K.G., Kitts, D.D., Townsley, P.M., 1988a. Seasonal resistance of the shore crab, Hemigrapsus oregonesis, to saxitoxin injections. Bull. Environ. Contam. Toxicol. 40, 190e197. Barber, K.G., Kitts, D.D., Townsley, P.M., Smith, D.S., 1988b. Appearance and partial purification of a high molecular weight protein in crabs exposed to saxitoxin. Toxicon 26, 1027e1034. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254. Daigo, K., Arakawa, O., Noguchi, T., Uzu, A., Hashimoto, K., 1987. Resistibility of two xanthid crabs Zosimus aeneus and Daira perlata against paralytic shellfish poison and tetrodotoxin. Nippon Suisan Gakkaishi 53, 881e884. Daigo, K., Noguchi, T., Miwa, A., Kawai, N., Hashimoto, K., 1988. Resistance of nerves from certain toxic crabs to paralytic shellfish poisons and tetrodotoxin. Toxicon 26, 485e490. Hashimoto, Y., Konosu, S., Inoue, A., Saisho, T., Miake, S., 1969. Screening of toxic crabs in the Ryukyu and Amami islands. Bull. Jpn. Soc. Sci. Fish. 35, 83e87. Ho, P.H., Sai, Y.H., Hwang, C.C., Hwang, P.A., Hwang, J.H., Hwang, D.F., 2006. Paralytic toxins in four species of coral reef crabs from Kenting National Park in southern Taiwan. Food Control 17, 439e445. Hwang, D.-F., Chueh, C.-H., Jeng, S.-S., 1990. Susceptibility of fish, crustacean and mollusk to tetrodotoxin and paralytic shellfish poison. Nippon Suisan Gakkaishi 56, 337e343. Jiang, P., Zhang, C., Qin, X., Lu, H., Lin, H., Liang, Y., 2013. Analysis of gonyautoxins by a post-column derivatization high performance liquid chromatography. Food Sci. 34, 170e174 (in Chinese, with English abstract). Koyama, K., Noguchi, T., Uzu, A., Hashimoto, K., 1983a. Resistibility of toxic and nontoxic crabs against paralytic shellfish poison and tetrodotoxin. Bull. Jpn. Soc. Sci. Fish. 49, 485e489.
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Koyama, K., Noguchi, T., Uzu, A., Hashimoto, K., 1983b. Individual, local, and sizedependent variations in toxicity of the xanthid crab Zosimus aeneus. Bull. Jpn. Soc. Sci. Fish. 49, 1273e1279. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 4668e4673. i Lenare c, B., Krishnan, G., Borukhovich, R., Ruck, B., Turk, V., Moczydlowski, E., 2000. Saxiphilin, a saxitoxin-binding protein with two thyroglobulin type 1 domains, is an inhibitor of papain-like cysteine proteinases. J. Biol. Chem. 275, 15572e15577. Li, Y., Moczydlowski, E., 1991. Purification and partial sequencing of saxiphilin, a saxitoxin-binding protein from the bullfrog, reveals homology to transferrin. J. Biol. Chem. 266, 15481e15487. Lin, H., Nagashima, Y., Zhang, C., Qin, X., 2011. Isolation and identification of PST in xanthid crab Zosimus aeneus collected from coral reef in Japan. J. Fish. China 35, 1540e1546 (in Chinese, with English abstract). 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Toxicon 40, 1463e1469. cs, G.L., Li, Y., Hall, S., Moczydlowski, E., 1991. Pharmacological and Mahar, J., Luka biochemical properties of saxiphilin, a soluble saxitoxin-binding protein from the bullfrog (Rana catesbeiana). Toxicon 29, 53e71. Montoya, N.G., Fulco, V., Carignan, M.O., Carreto, J.I., 2010. Toxin variability in cultured and natural populations of Alexandrium tamarense from southern South America e Evidences of diversity and environmental regulation. Toxicon 56, 1408e1418. Nagashima, Y., Ohgoe, H., Yamamoto, K., Shimakura, K., Shiomi, K., 1998. Resistance of non-toxic crabs to paralytic shellfish poisoning toxins. In: Reguera, B., ndez, M.L., Wyatt, T. (Eds.), Harmful Algae. Xunta de Galicia and Blanco, J., Ferna Intergovernmenal Oceanographic Commission of UNESCO, Paris, pp. 604e606. Nagashima, Y., Yamamoto, K., Shimakura, K., Shiomi, K., 2002. A tetrodotoxinbinding protein in the hemolymph of shore crab Hemigrapsus sanguineus: purification and properties. Toxicon 40, 753e760. Negri, A.P., Llewelly, L., 1998. Comparative analyses by HPLC and the sodium channel and saxiphilin 3H-saxitoxin receptor assays for paralytic shellfish toxins in crustaceans and molluscs from tropical north west Australia. Toxicon 36, 283e298. Noguchi, T., Koyama, K., Uzu, A., Hashimoto, K., 1983. Local variation of toxicity and toxin composition in a xanthid crab Atergatis floridus. Bull. Jpn. Soc. Sci. Fish. 49, 1883e1886. Noguchi, T., Miyazawa, K., Daigo, K., Arakawa, O., 2011. Paralytic shellfish poisoning (PSP) toxin- and/or tetrodotoxin-containing crabs and food poisoning by them. Toxin Rev. 30, 91e102. Oshima, Y., 1995. Postcolumn derivatization liquid chromatographic method for paralytic shellfish toxins. J. AOAC Int. 78, 528e532. Sato, S., Takata, Y., Kondo, S., Kotoda, K., Hongo, N., Kodama, M., 2014. Quantitative ELISA kit for paralytic shellfish toxins coupled with sample pretreatment. J. AOAC Int. 97, 339e344. Shimada, H., Motylkova, I.V., Mogilnikova, T.A., Mikami, K., Kimura, M., 2011. Toxin profile of Alexandrium tamarense (Dinophyceae) from Hokkaido, northern Japan and southern Sakhalin, eastern Russia. Plankton Benthos Res. 6, 35e40. Shiomi, K., Yamaguchi, S., Kikuchi, T., Yamamori, K., Matsui, T., 1992. Occurrence of tetrodotoxin-binding high molecular weight substances in the body fluid of shore crab (Hemigrapsus sanguineus). Toxicon 30, 1529e1537. Takati, N., Mountassif, D., Taleb, H., Lee, K., Blafhen, M., 2007. Purification and partial characterization of paralytic shellfish poison-binding protein from Acanthocardia tuberculatum. Toxicon 50, 311e321. Tsai, Y.H., Hwang, D.F., Chai, T.J., Jeng, S.S., 1996. Occurrence of paralytic toxin in Taiwanese crab Atergatis germaini. Toxicon 34, 467e474. Yamamori, K., Yamaguchi, S., Maehara, E., Matsui, T., 1992. Tolerance of shore crabs to tetrodotoxin and saxitoxin and antagonistic effect of their body fluid against the toxins. Nippon Suisan Gakkaishi 58, 1157e1162. Yasumoto, T., Oshima, Y., Tajiri, M., Kotaki, Y., 1983. Paralytic shellfish toxins in previously unrecorded species of coral reef crabs. Bull. Jpn. Soc. Sci. Fish. 49, 633e636. Yasumura, D., Oshima, Y., Yasumoto, T., Alcala, A.C., Alcala, L.C., 1986. Tetrodotoxin and paralytic shellfish toxins in Philippines crab. Agric. Biol. Chem. 50, 593e598.
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Neutralizing effect of hemolymph from the shore crab, Thalamita crenata, on paralytic shellfish toxins Huajuan Lin a, b, Chaohua Zhang a, b, Jianmeng Liao c, Feng Yang d, Saiyi Zhong a, b, Peihong Jiang a, Xiao Chen a, Yuji Nagashima e, * a
College of Food Science and Technology, Guangdong Ocean University, Zhanjiang 524088, China Guangdong Provincial Key Laboratory of Aquatic Product Processing and Safety, Zhanjian, Gaunadong Province 524088, China Zhanjiang Institute of Supervision & Test on Quality & Measure, Zhanjiang 524096, China d Zhanjiang Ocean and Fishery Environment Monitor Station, Zhanjiang 524039, China e Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Minato, Tokyo 108-8477, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 January 2015 Received in revised form 16 March 2015 Accepted 18 March 2015 Available online 19 March 2015
Several species of crabs are resistant to paralytic shellfish toxins (PSTs) and/or pufferfish toxin, tetrodotoxin, regardless of toxification by the toxins. The shore crab Thalamita crenata, which inhabits Leizhou Peninsula, China, is tolerant to PST toxicity, and the hemolymph has neutralizing effects against the lethal activity of PST. In the present study, we investigated the PST neutralizing factors in the hemolymph from T. crenata and successfully separated PST-binding proteins by PST-ligand affinity chromatography. The neutralization factors, obtained in the fraction with a molecular weight over 10 kDa by ultrafiltration, were susceptible to proteases such as alcalase, animal complex proteases, pancreatin, and papain. The PST-binding protein had high dose-dependent neutralization effects on PST toxicity. The PST-binding activity of the protein was stable at 25 C and then decreased with an increase in temperature; heating at 65 C for 60 min eliminated the initial activity by two-thirds. The PST-binding activity was strongly inhibited in the presence of Mg2þ and Ca2þ, but not Naþ and Kþ. The PST-binding capability of the protein differed among PST components in descending order of neosaxitoxin, gonyautoxins 1 and 4, saxitoxin, and gonyautoxins 2 and 3, suggesting a structureeactivity relationship in PST binding. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Shore crab Thalamita crenata Paralytic shellfish toxins (PST) Toxicity neutralization PST-binding protein PST-ligand affinity chromatography
1. Introduction Some species of crabs are often toxified by marine bio-toxins, such as paralytic shellfish toxins (PSTs), tetrodotoxin (TTX), and palytoxin. Upon ingestion, these toxified crabs occasionally cause human intoxication, including fatalities, in various parts of the world, and thus constitute a worldwide health threat (Alcala et al., 1988; Llewellyn et al., 2002; Noguchi et al., 2011). Since Hashimoto et al. (1969) discovered toxic crabs, Zosimus aeneus, Platypodia granulosa, and Atergatis floridus in the family Xanthidae, in the Ryukyu and Amami Islands, Japan, toxic crabs have been reported
* Corresponding author. Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Konan, Minato, Tokyo 1088477, Japan. E-mail addresses: [email protected] (H. Lin), [email protected] (C. Zhang), [email protected] (J. Liao), [email protected] (F. Yang), zsylxc@126. com (S. Zhong), [email protected] (P. Jiang), [email protected] (X. Chen), [email protected] (Y. Nagashima). http://dx.doi.org/10.1016/j.toxicon.2015.03.010 0041-0101/© 2015 Elsevier Ltd. All rights reserved.
in at least 20 species from 5 different families (Majidae, Atelecylidae, Portunidae, Xanthidae, and Grapsidae), mainly in the Pacific region, including Australia, Philippines, and Taiwan (Yasumura et al., 1986; Llewellyn and Endean, 1989; Tsai et al., 1996; Negri and Llewellyn, 1998; Ho et al., 2006), as well as Japan (Koyama et al., 1983b; Noguchi et al., 1983; Yasumoto et al., 1983). Among these toxic crabs, xanthid crabs have extremely high levels of PST and/or TTX. Importantly, toxic crabs administered intrahemocoelically with the toxins exhibited much higher resistance to the lethal effects of the toxins than non-toxic crabs. In specimens of A. floridus and Z. aeneus, the minimum lethal dose (MLD) of TTX is 1000e2000 mouse units (MU)/20g body weight (equivalent to 10e20 mg/kg body weight) and the MLD of gonyautoxins (GTXs) is 5000e10000 MU/20g body weight (equivalent to 50e100 mg/kg body weight) (Koyama et al., 1983a; Daigo et al., 1987). Some species of non-toxic grapsid crabs belonging to the genus Hemigrapsus also exhibit weak resistance to PST and TTX. Canadian shore crabs Hemigrapsus oregonesis and Hemigrapsus nudus are tolerant to PST only when previously exposed to PST (Barber et al., 1988a, 1988b).
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In contrast, the Japanese shore crab Hemigrapsus sanguineus is tolerant to TTX throughout the year (Shiomi et al., 1992; Yamamori et al., 1992). There are two general toxicity tolerance mechanisms of the crabs toward PST and/or TTX. The first is that their neurocytes are unresponsive to the toxins, which act as specific blockers of voltage-gated sodium channels, e.g., xanthid crabs containing high levels of toxin (Daigo et al., 1988). The second mechanism is endogenic special substances with toxin-binding capability in the hemolymph, e.g., non-toxic Hemigrapsus crabs (Baber et al., 1988b; Shiomi et al., 1992; Nagashima et al., 2002). We have been screening for PST toxicity of shore crabs that inhabit the Leizhou Peninsula, China, located north of the South China Sea, to monitor coastal environmental pollution and shellfish toxification by harmful algae. Our previous investigation revealed that shore crabs inhabiting this area e Thalamita crenata, Leptodius exaratus, and Metopograpsus latifrons e are non-toxic species based on the toxicity levels in the tissues, despite the fact that 3 of 25 individuals collected from December 2009 to March 2012 had slight PST toxicity of 4.3e4.4 MU/g (Lin et al., 2012). Furthermore, the 100% lethal dose of PST for the three species exceeded 5 MU/ 20g of crab, which was relatively higher than that for other sensitive crab species (Koyama et al., 1983a; Hwang et al., 1990; Yamamori et al., 1992; Nagashima et al., 1998). This indicates that these three species of crabs have weak resistance to PST. The lethal time of mice administered PST solution with the hemolymph from the crabs was significantly prolonged, suggesting that the crab hemolymph neutralized the PST toxicity in mice (Lin et al., 2012). The functional agents in the hemolymph that neutralize the toxicity, however, remain to be clarified. In the present study, we investigated the neutralizing factors of hemolymph from T. crenata against PST toxicity with respect to the molecular range and susceptibility to various proteases, and successfully separated PST-binding proteins by PST-ligand affinity chromatography. The PST-binding proteins were found to bind PST components (e.g., saxitoxin [STX], neoSTX, GTX1&4, and GTX2&3) with different PST-binding capabilities.
The animal experiments were performed according to the rules and regulations of the animal ethics committee of the Guangdong Laboratory Animal Monitoring Institute. 2.2. Neutralizing activity against PST toxicity of hemolymph samples Neutralizing activity against PST toxicity was estimated using a mouse assay as described previously (Lin et al., 2012). Briefly, a PST stock solution was prepared to a suitable concentration. In a control experiment, the PST solution was mixed with an equal volume of 10 mM phosphate buffer (pH 7.4) containing 8 mM EDTA and 150 mM sucrose, and intraperitoneally injected into a group of three or five mice to kill the mice within 4e6 min. In a test experiment, the PST solution (same as used in the control experiment) was mixed with hemolymph samples at 25 C for 30 min and intraperitoneally injected into mice in the same manner as the control. The time to kill the mice was recorded and expressed as mean ± standard deviation (SD), based on Student's t-test to examine the significant differences between groups. A p value less than 0.05 was considered statistically significant. Hemolymph was treated with proteases (alcalase, animal complex protease, pancreatin, and papain) to examine the effect of proteolysis on the neutralizing activity of the hemolymph sample. It was digested with each protease at a final concentration of 2.5 unit/ mL for 12 h at 35 C, and subjected to the mouse assay to estimate the neutralizing activity against PST toxicity. In addition, inactivated proteases were also used. The proteases were denatured by heating in boiling water for 5 min. Here, one unit of protease activity was defined as the release of 1 mg tyrosine/mg protein for 1 min. The hemolymph was separated into two fractions by ultrafiltration (nominal molecular weight cut-off 10 kDa, Millipore Amicon, Bedford, MA, USA), and subjected to the neutralizing activity test described above. 2.3. PST binding activity of hemolymph samples
2. Materials and methods 2.1. Materials Shore crab T. crenata was collected at the coast of the Leizhou Peninsula, China, from March to November 2011. In each sampling, about 30 specimens obtained (body weight of 23e48 g) were immediately transported to the laboratory of Aquatic Product Advanced Processing of the Guangdong Higher Education Institutes. The hemolymph was withdrawn from the carpus of live crabs as described previously with slight modification (Lin et al., 2012) and pooled to use as a hemolymph sample. PSTs used in this study were partially purified from the toxic xanthid crab Z. aeneus by the method of Lin et al. (2011) and contained mainly STXs. PST standards, STX, neoSTX, GTX1&4, and GTX2&3, were supplied from the National Research Council Institute for Marine Biosciences, Canada. Alcalase (alkali endoprotease) from Bacillus licheniformis, animal complex protease (containing endoprotease, peptidase, and exo-protease), pancreatin (containing protease, lipase, and amylase), and papain (EC3.4.22.2) were obtained from Pangbo Biological Engineering Co., Ltd. (Nanning, China). AFEpoxy-650M resin was purchased from Tosoh Corporation (Tokyo, Japan). Bovine serum albumin (BSA) was obtained from SigmaeAldrich Co. (St. Louis, MO, USA). Male KM strain mice (20 ± 2 g of body weight) were kindly supplied by Shuanglin Biological Medicine Co., Ltd. (Zhanjiang, China). The facilities for the experimental animals are fully accredited with license number SYXK 2013-0010.
PST binding activity was measured by HPLC as follows: In the control experiment, the PST solution was mixed with an equal volume of 10 mM phosphate buffer (pH 7.4) containing 8 mM EDTA and 150 mM sucrose, put into a centrifugal ultrafiltration tube (3 kDa cut-off, Millipore Amicon), and centrifuged at 20,000 g for 10 min at 4 C. PST was obtained in the resulting filtrate. In a test experiment, the PST solution was mixed with the hemolymph sample and allowed to stand for 1 h at 4 C. The mixture was subjected to centrifugal ultrafiltration under the same condition as the control. In this case, the filtrate contained free-form PSTs that were not bound to the hemolymph. The bound PSTs were estimated by subtracting free-form PSTs from PSTs in the control. PST was determined by HPLC with a post-column fluorescent reaction. The HPLC system comprised an LC-20AD (Shimadzu, Kyoto, Japan) with an XB-C18 column (4.6 mm 150 mm, Welch Materials Inc., Shanghai, China). The mobile phase and post column reaction conditions for STXs were as described by Oshima (1995) and those for GTXs by Jiang et al. (2013). Triplicate tests were carried out for each sample, and the data were expressed as mean ± SD. The relative PST-binding activity was expressed as the ratio of each sample to the highest sample. In addition, an ELISA test for PST-binding activity was performed in a preliminary experiment using a SKit (ELISA for paralytic shellfish poison, Shin-Nihon Kentei Kyokai, Tokyo, Japan) under the previously described procedure (Sato et al., 2014).
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2.4. PST-ligand affinity chromatography of hemolymph samples A PST-coupled affinity column was prepared according to the method by Takati et al. (2007) with some modifications. Briefly, 5 g of AF-Epoxy-650M resin was suspended in 5 mL isopropanol and treated with a mixture of ethylene diamine and dimethyl formamide for 30 min at room temperature. After washing with 50 mM phosphate buffer (pH 7.4), the resin was further treated with 5% glutaraldehyde for 30 min at room temperature and then washed with the same buffer. A total of 66 mg STXeq of PST (tested by mouse assay) was added to the activated resin and gently stirred for 4 h at room temperature. After removing the excess free PST by washing with the buffer, the PST-coupled AF-Epoxy-650M was established by adding 0.1 M NaBH4. The 10-fold diluted hemolymph was added to the PST-coupled AF-Epoxy-650M and incubated overnight at 4 C under weak agitation. The resin was poured into a glass column with a 1.5-cm diameter. The column was washed with 50 mM phosphate buffer (pH 7.4) and eluted with 1 M TriseHCl buffer (pH 10) at a flow rate of 0.5 mL/min. Fractions with a volume of 1.5 mL were collected and immediately neutralized by adding 1.5 mL of 100 mM glycine buffer (pH 2.5). The absorbed fractions containing PST-binding proteins were pooled and the solvent displaced with 50 mM phosphate buffer (pH 7.4) by ultrafiltration (3 kDa cut-off) repeatedly. 2.5. Stability of PST-binding activity Effects of incubation time, temperature, and bivalent ions on PST-binding activity were evaluated. To test the effects of incubation time, the PST/protein mixture in 50 mM phosphate buffer (pH 7.4) was incubated at 4 C for various times, ranging from 0 to 240 min. To test the effects of temperature, the PST/protein mixture in the same buffer was allowed to stand for 60 min at a temperature of 4 C, 15 C, 25 C, 37 C, 50 C, or 65 C. To test the effects of the bivalent ions, MgCl2 or CaCl2 at a final concentration of 10 mM in 50 mM phosphate buffer (pH 7.4) were screened under the optimal conditions (4 C, 60 min). We similarly tested NaCl and KCl. After incubation, the PST/protein mixture was ultrafiltered using a centrifugal ultrafiltration tube (3 kDa cut-off). The obtained filtrates were applied to HPLC analysis to estimate the PST-binding activity, as described above. 2.6. Analytical methods
Fig. 1. Neutralizing effect of hemolymph on PST toxicity to mice. The results are shown as the means ± standard deviation (SD) of triplicate measurements. Statistical significance (p < 0.05) is indicated by asterisk (*), compared to the control.
with the control (5.2 ± 0.2 min; p < 0.05), indicating that the PST toxicity was diminished by the hemolymph samples. Moreover, this result suggested that there is no significant seasonal variation in the neutralizing effect of the hemolymph on PST toxicity, at least from spring through fall. To analyze the neutralizing factors in the hemolymph, the hemolymph sample was separated by molecular size using ultrafiltration (10 kDa cut-off). The survival time of mice administered >10 kDa fractions was significantly longer than that of the controls (p < 0.05) and comparable to that following administration of the initial hemolymph sample (Fig. 2). This finding indicated that the neutralizing factors against PST toxicity were contained mainly in the fraction with a high molecular size (>10 kDa). The hemolymph sample was treated with four types of proteases, including alcalase (alkali endoprotease), animal complex protease (containing endoprotease, peptidase, and exo-protease), pancreatin (containing protease, lipase, and amylase), and papain. The neutralizing effect of hemolymph against PST toxicity was remarkably decreased, irrespective of the proteases used in this study (Fig. 3). In contrast, when the hemolymph sample was treated with proteases inactivated by heating, the hemolymph neutralizing effect was essentially the same as that of the nontreated hemolymph (Fig. 3). SDS-PAGE analysis confirmed hydrolysis of the hemolymph sample by the proteases (Fig. 4). Both the hemolymph sample (lane 10) and hemolymph treated with inactivated proteases (lanes 2, 4, 6, and 8) had a band at around 80 kDa,
Protein content was measured according to the Bradford procedure, using BSA as the standard protein (Bradford, 1976). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted according to the method originated by Laemmli (1970) using 10% polyacrylamide (containing 0.1% SDS) as a separation gel. Protein samples from the hemolymph were dissolved in 0.125 M TriseHCl buffer (pH 6.8) containing 5% sucrose, 5% 2mercaptoethanol, and 2% SDS, followed by heating in boiling water for 5 min. Native-PAGE for the PST-binding protein fraction was performed under the same SDS-PAGE conditions, except for without SDS and 2-mercaptoethanol agents. The protein bands were stained with Coomassie brilliant blue R-250. 3. Results 3.1. Neutralizing effect of hemolymph against PST toxicity The neutralizing effects of hemolymph from the shore crabs T. crenata collected at March, June, October, and November 2011, on PST toxicity are shown in Fig. 1. All hemolymph samples significantly prolonged survival of the mice (10.7e20.2 min), compared
Fig. 2. Neutralizing effect of hemolymph fractionated by ultrafiltration on PST toxicity to mice. The results are shown as the means ± standard deviation (SD) of triplicate measurements. Statistical significance (p < 0.05) is indicated by asterisks (*, **), compared to the control and the fraction with a molecular weight lower than 10 kDa, respectively.
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mL) was administered to a group of five mice, two mice survived and the time to death of the other three mice was prolonged approximately 4-fold (25.1 ± 4.5 min) compared with the control (5.5 ± 0.2 min) and a mixture of PST and BSA at a final concentration of 2.0 mg protein/mL (6.4 ± 0.4 min). This result demonstrated that the PST-binding protein fraction considerably decreased the toxicity of PST. The neutralizing activity of the fraction was evaluated to be 0.4 MU/mg protein from death time-mouse unit relations for PST (Association of Official Analytical Chemists, 2005). We then examined the effect of the concentration of the PST-binding protein on the neutralizing activity. The PST-binding protein fraction almost linearly increased the survival time of mice in a concentration-dependent manner, up to 5.0 mg/mL (Fig. 6). 3.3. PST-binding ability of the PST-binding protein
Fig. 3. Proteolytic effect on neutralizing activity of hemolymph for PST toxicity. Hemolymph (abbreviated as H) was treated by four types of proteases, alcalase (E1), animal complex protease (E2, containing endoprotease, peptidase, and exo protease), pancreatin (E3, containing protease, lipase, and amylase) and papain (E4). The results are shown as the means ± standard deviation (SD) of triplicate measurements. Statistical significance (p < 0.05) is indicated by asterisk (*), compared to the control.
Fig. 4. SDS-PAGE of protease-treated hemolymph. Lane1; E1 þ H, 2; inactivated E1 þ H, 3; E2 þ H, 4; inactivated E2 þ H, 5; E3 þ H, 6; inactivated E3 þ H, 7; E4 þ H, 8; inactivated E4 þ H, 9; protein maker, 10; hemolymph.
while the hydrolyzates of the hemolymph sample treated with the proteases (lanes 1, 3, 5, and 7) degraded the band. These results demonstrated that the neutralizing activity of hemolymph against PST toxicity was attributed to proteinaceous substances that are susceptible to all tested proteases, including alcalase, animal complex protease, pancreatin, and papain.
3.2. Preparation of PST-binding protein fraction from hemolymph PST-ligand affinity chromatography of the hemolymph sample was performed to isolate the PST toxicity neutralizing factor in the hemolymph. A sharp protein peak was detected at fraction numbers 88-98 in the absorbed fraction, designated the PSTbinding protein fraction (Fig. 5A). The fraction still formed several bands in native-PAGE (Fig. 5B), although the affinity chromatography yielded 41.6 mg of protein from an aliquot of 5 mL hemolymph sample with 289 mg of protein. Table 1 shows the neutralizing activity of the PST-binding protein fraction against PST toxicity. When PST mixed with the PSTbinding protein fraction (a final concentration of 2.0 mg protein/
In the preliminary experiment using a PST-ELISA kit (SKit, ShinNihon Kentei Kyokai), the PST-binding ability of the hemolymph sample (protein concentration of 57.8 mg/mL) was determined to be 3.13 ± 0.11 nmol PST/mL, which was equivalent to 0.054 ± 0.002 nmol PST/mg protein. In addition, the PST-binding ability of the PST-binding protein fraction was estimated to be 0.092 ± 0.010 nmol PST/mg protein. The optimum conditions for binding of the protein with PST were then examined by HPLC methods. The PST-binding activity increased with incubation time up to 60 min, and reached a steady level after 60 min (Fig. 7). The PST-binding activity was stable at temperatures below 25 C, and then decreased with an increase in temperature (Fig. 8). The PST-binding activity decreased to 36.1 ± 7.2% when the temperature was 65 C. Bivalent ions, such as Mg2þ and Ca2þ, strongly inhibited PST-binding activity (Fig. 9). In contrast, the effects of monovalent ions, such as Naþ and Kþ, on PST-binding activity were minimal. The relative PST-binding activity with Naþ and Kþ was comparable with that of the control (Fig. 9). These findings implied that bivalent ions are involved in the PST-ligand binding of the PST-binding protein. The binding ability of the PST-binding protein to PST components, including STX, neoSTX, GTX1&4, and GTX2&3 is summarized in Table 2. The PST-binding ratios for the toxins differed from each other; the maximum of 72.3 ± 1.3% for neoSTX and the minimum of 32.5 ± 1.3% for GTX2&3. This result indicated that the PST structures have a remarkable impact on the PST-binding activity of the PSTbinding protein. 4. Discussion The present study revealed that the hemolymph from the shore crab T. crenata had a neutralizing effect against PST toxicity. The neutralizing effect was attributable to PST-binding proteins in the hemolymph, because the neutralizing effect of the PST-binding protein based on the mouse assay (0.4 MU/mg protein) was similar to that of the protein based on ELISA (0.092 nmol/mg protein), despite the use of a PST solution comprising a mixture of PSTs. It is likely that the PST-binding proteins are endogenous, because the highly neutralizing effect was observed regardless of season and year, based on both the present and previous studies (Lin et al., 2012). Furthermore, we surveyed both the cell density of PSTproducing dinoflagellates and cell toxicity in the Leizhou Peninsula from January to November, 2012. During the monitoring, only one type of PST-producing dinoflagellate, Alexandrium tamarense, was found, which appeared mainly from February through July. During this period, the highest cell density was GTX1&4 > STX > GTX2&3. The structures of both neoSTX and GTX1&4 have a hydroxyl group at N-1, while STX and GTX2&3 have a hydrogen at N-1 (Fig. 10). Therefore, substitution of a hydroxyl group at the N-1 position may affect the PST-binding capability of the proteins, which is very helpful for promoting PST-ligand binding. Second, substitution of the sulfonyl group at the C-11 position may also affect the PST-binding capability by decreasing PST binding with the proteins. These findings indicate
a structureeactivity relationship among PST components. Also, in terms of a structureeactivity relationship, the PST-binding proteins from T. crenata hemolymph differ from those of saxiphilin. Mahar et al. (1991) reported that the addition of a hydroxyl group at N-1 and a a-hydroxysulfate moiety at C-11 in PSTs profoundly affects the binding affinity in competitive displacement experiments: 550-fold and 160-fold reduction in binding affinity in the molecular pairs of neoSTX/STX and C1/B1 (GTX5), respectively. These results suggest that the two positions, N-1 and C-11, have important roles in binding with saxiphilin. Notably, the hemolymph from T. crenata also has an apparent TTX toxicity neutralizing effect (Lin et al., 2012). Llewellyn et al. (1997) surveyed diverse animal species to investigate the phylogenetic distribution of saxiphilin based on [3H]STX binding activity and found that several species of crabs in the family Xanthidae exhibit saxiphilin-like activity. An STX-binding fraction that did not bind TTX was found in the hemolymph of xanthid crabs, Lophozozymus pictor, Liomera tristis, Chlorodiella nigra, and Actaeodes tomentosus (Llewellyn, 1997). In addition, we screened non-toxic crabs for resistance to PST and TTX, and determined that the family Ocypodidae Scopimera globosa and the family Grapsidae Gaetice depressus were resistant to PST, but not to TTX (Nagashima et al., 1998). On the contrary, the family Grapsidae H. sanguineus was tolerant to TTX, but not to PST (Shiomi et al., 1992; Yamamori et al., 1992), and its hemolymph contained a unique protein that selectively binds TTX (Nagashima et al., 2002). In the present study, we focused on the neutralization effects the hemolymph from T. crenata against PST toxicity and PST-binding ability, because the neutralizing effect of the hemolymph against PST was higher than that against TTX (Lin et al., 2012). It is possible that the PST-binding proteins have binding ability to TTX or the hemolymph has TTX neutralizing mechanisms distinct from those of PST-binding proteins. Additional studies are needed to examine the physical and chemical properties of the PST-binding protein, and to elucidate the primary structure. Acknowledgments This study was supported by 2012 Guangdong Science Province
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