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Novel proteinaceous toxins from the nematocyst venom of the Okinawan sea anemone Phyllodiscus semoni Kwietniewski
BBRC Biochemical and Biophysical Research Communications 294 (2002) 760–763 www.academicpress.com
Novel proteinaceous toxins from the nematocyst venom of the Okinawan sea anemone Phyllodiscus semoni Kwietniewski Hiroshi Nagai,a,*,1 Naomasa Oshiro,b Kyoko Takuwa-Kuroda,a Setsuko Iwanaga,b Masatoshi Nozaki,b and Terumi Nakajimaa a
Suntory Institute for Bioorganic Research, 1-1-1 Wakayamadai, Shimamoto, Mishima, Osaka 618-8503, Japan Okinawa Prefectural Institute of Health and Environment, 2085 Ozato, Ozato-son, Okinawa 901-1202, Japan
b
Received 16 May 2002
Abstract The Okinawan sea anemone Phyllodiscus semoni is known to cause cases of severe stinging. We isolated P. semoni toxins 60A and 60B (PsTX-60A and PsTX-60B; ca. 60 kDa) as the major toxins from the isolated nematocysts of this species for the first time. PsTX-60A and PsTX-60B showed lethal toxicity to the shrimp Palaemon paucidence when administered via intraperitoneal injection (LD50 values: 800–900 and 800 lg/kg, respectively) and hemolytic activity toward a 0.8% suspension of sheep red blood cells (ED50 values: 600 and 300 ng/ml, respectively). Furthermore, we sequenced the cDNA encoding PsTX-60A. The deduced amino acid sequence of PsTX-60A did not show any similarity to previously reported proteins. The N-terminal amino acid sequence of PsTX60B showed homology with that of PsTX-60A. These toxins represent a novel class of cytolytic proteinaceous toxins. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Sea anemone; Phyllodiscus semoni; Protein; Hemolysis; Toxin; PsTX; Nematocyst
The Okinawan sea anemone Phyllodiscus semoni Kwietniewski, 1897 is called ‘‘unbachi-isoginchaku’’ in Japanese which means ‘‘wasp-sea-anemone,’’ because this species causes cases of severe stinging [1–3]. This species is distributed widely in Okinawa Island, Japan. The characteristic symptoms of stinging by this species are severe dermatitis with swelling, local fever, and ulceration. However, the toxins of P. semoni have not yet been isolated or characterized. Understanding the mode of action of the toxin is essential to develop effective remedies against P. semoni stings. The isolation and characterization of P. semoni toxins is the first step in such a project. Our study focused on the major cytolytic and fatal toxins that exist in the nematocyst, which is the organ responsible for the P. semoni sting.
*
Corresponding author. Fax: +81-3-5463-0398. E-mail address: nagai@tokyo-u-fish.ac.jp (H. Nagai). 1 Present address: Tokyo University of Fisheries, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan.
In cnidarians, the toxins of anthozoans (sea anemones) have been studied extensively by comparing them with those of hydrozoan or scyphozoan species. Several polypeptide and protein toxins from sea anemones have been reported [4,5]. Herein we report the first isolation of P. semoni toxins 60A and 60B (PsTX-60A and PsTX-60B), novel hemolytic, and lethal proteins (60 kDa) from the nematocysts of this dangerous sea anemone and their bioactivities as well as the cDNA and amino acid sequences for PsTX60A.
Materials and methods General experimental procedure. Amino acid sequences were analyzed by a PSQ-1 protein sequencer (Shimadzu, Kyoto, Japan). Nucleotide sequences were determined using a BigDye Terminator Cycle Sequencing Kit (Applera, Norwalk, CT) and the ABI PRISM 310 (Applera). Animal material. The sea anemone P. semoni Kwietniewski (Anthozoa, Aliciidae) specimens were collected along the coast of Itoman, Okinawa, Japan, in August 2000 and maintained in an aquarium prior to use. The samples were identified by Dr. Hiro’omi Uchida of
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 5 4 7 - 8
H. Nagai et al. / Biochemical and Biophysical Research Communications 294 (2002) 760–763 Kushimoto Marine Park. Specimens for confirming identification were deposited at Kushimoto Marine Park, Wakayama, Japan. Hemolytic activity test. A few microliters of toxic fractions or purified toxin solution were incubated in a plastic tube at room temperature with 1 ml of a 0.8% suspension of sheep red blood cells (Nikken-Seibutu-Igaku-Kenkyusho, Japan) in phosphate-buffered saline (PBS) (+). After 4 h the tubes were centrifuged at 3000g for 10 min. The concentration of hemoglobin released into the supernatant was measured on a microplate reader (MPR-4Ai, Tosoh, Japan) at 550 nm. A 100% hemolysis value was obtained by the addition of 0.1 mg/ml saponin. Shrimp toxicity assay. The fractionated samples (each 10 ll) were injected intraperitoneally into the shrimp Palaemon paucidence. Fatal toxicity was observed 12 h after injection. The purified toxin was diluted in PBS(+) and 10 ll was injected intraperitoneally into P. paucidence (five shrimps for each toxin concentration). Preparation of isolated nematocysts of P. semoni. The nematocysts from P. semoni were isolated using a modification of the reported methods [6]. Globular vesicles (ca. 1 mm in diameter), which are organs surrounding the nematocysts, were separated from the skin of living P. semoni using tweezers and dispersed in 1 M sodium citrate solution. To eliminate the mucus and contaminants surrounding the vesicles, the sample suspension was kept at 5 °C in a 50-ml conical centrifuge tube and the supernatant was removed by decanting. The vesicles were washed by this method a few times for 2–3 days. Then, the sample suspension was autolyzed in a 1 M sodium citrate solution at 5 °C. After a week of autolysis, the suspension was well vortexed to liberate nematocysts from the vesicles. Free nematocysts were separated from the vesicles by pouring the suspension through fine-mesh nylon netting. The filtrate was allowed to settle in a 50-ml conical centrifuge tube for 3 h at 5 °C. The supernatant was then removed by decanting and the nematocysts were washed at least three times using 1 M sodium citrate solution. Finally, the isolated nematocysts were obtained and stored at )80 °C. Extraction and isolation of toxins from isolated nematocysts. The isolated nematocysts were centrifuged (12,000g, 5 min). The supernatant was eliminated by pipetting. To obtain the crude extracts, 10 mM phosphate buffer (pH 6.0) was added to the isolated nematocysts. The obtained crude toxin solution was centrifuged (12,000g, 15 min) and the supernatant was filtered through a 0.45-lm membrane. The filtrate was applied to an ion-exchange HPLC, TSK-GEL CM-5PW column (7:5 75 mm, 0.5 ml/min), which had been equilibrated with 10 mM phosphate buffer containing 0.3 M NaCl (pH 6.0) and then the column was washed with 10 mM phosphate buffer (pH 6.0). The adsorbed components were eluted with a gradient solvent system (0–0.7 M NaCl: 0–90 min). Most of the toxicity was observed in the non-adsorbed fraction. Weak toxicity was observed in the fraction that was adsorbed to the column and eluted with the salt gradient solvent system. The column-adsorbed fraction with weak toxicity was further purified (results to be published elsewhere). The non-adsorbed fraction with most of the toxicity was concentrated with the Amicon ultrafiltration system. Then, the sample was applied to a gel-permeation HPLC, Superdex 200 column (15 280 mm, 1 ml/min), which had been equilibrated with 0.5 M NaCl and 10 mM phosphate buffer (pH 7.0). Finally, PsTX-60A and PsTX-60B were isolated. The isolation of the toxins was guided by the hemolytic activity and the shrimp toxicity tests. All HPLC elution was monitored with a UV detector at 210 or 280 nm. The separation of each fraction was monitored by SDS–PAGE according to standard methods [7]. The protein concentration of the sample was measured using a BCA protein-assay reagent (Pierce) following manufacturer’s instructions. Protein sequencing of PsTX-60A and -60B. PsTX-60A and -60B (30 lg each), the isolated proteinaceous toxins from P. semoni nematocysts, were introduced into the PSQ-1 protein sequencer (Shimadzu) to analyze the N-terminal amino acid sequences of these toxins.
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Cloning of PsTX-60A cDNA. Total RNA was isolated from the intact tentacles of P. semoni with TRIzol Reagent (Invitrogen, Carlsbad, CA) following manufacturer’s instructions. Degenerative 30 -rapid amplification of cDNA ends (RACE) was performed by the following method. First-strand cDNA was synthesized from 1 lg total RNA using a 50 /30 -RACE kit (Boehringer–Mannheim, Germany) and the oligo dT-anchor primer. The cDNA was then amplified by the first 30 -RACE reaction using the degenerate primer 60KA-30 -1: 50 -GAY AAR GAR ACI GAR CAY ATH GAR AT-30 (corresponding to peptide sequence: DKETEHIEI) and the PCR anchor primer, followed by reamplification of the primary PCR products using the degenerate primer 60KA-30 -2: 50 -ATH WSI MGI GGI GCI YTI GGI CAR GG-30 (corresponding to peptide sequence: ISRGALGQG) and the PCR anchor primer. The secondary PCR products were subcloned into the pCR2.1 vector and sequenced. Amplification was performed using Ex Taq Polymerase (Takara, Kyoto, Japan) under the following conditions: 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 3 min; and 72 °C for 7 min. The 1464-bp product obtained from the reaction was cloned into the pCR2.1 vector (Invitrogen) and nucleotide sequences were analyzed using the BigDye Terminator Cycle Sequencing Kit (Applera) and the ABI PRISM 310 (Applera) automated sequencer. This 1464-bp cDNA was then treated by 50 -RACE. 50 -RACE was performed by the following method: first-strand cDNA was synthesized from 1 lg total RNA using a 50 /30 -RACE kit (Boehringer– Mannheim, Germany) and a gene-specific primer 60KA-50 -5 (50 -ATT CGT CCA TTG GAA GGT CC-30 ). The first 50 -RACE reaction was completed using the oligo dT-anchor primer and the primer 60KA-50 -4 (50 -CAC CCG TTG CTT CAA ATT GC-30 ). The second 50 -RACE reaction was also completed using the PCR anchor primer and the primer 60KA-50 -1 (50 -CTT AGC AAA TCC TCT CTG TGG-30 ). Amplification for 50 -RACE was performed using Ex Taq Polymerase (Takara) under the following conditions: 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 2 min; and 72 °C for 7 min. The secondary PCR products were subcloned into the pCR2.1 vector and sequenced as described above.
Results and discussion Preparation of isolated nematocysts of P. semoni The preparation of pure venom from a suspension of nematocysts free of cytoplasmic contaminants is important for investigating unambiguously the toxins
Fig. 1. Purification scheme for PsTX-60A and PsTX-60B from P. semoni.
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H. Nagai et al. / Biochemical and Biophysical Research Communications 294 (2002) 760–763
Isolation of PsTX-60A and PsTX-60B from the isolated nematocysts Fig. 2. N-terminal amino acid sequences of PsTX-60A and PsTX-60B. The alignment of the sequences of PsTX-60A (top) and PsTX-60B (bottom) is shown by the single-letter amino acid code. Identical residues are marked by asterisks. Conserved residues are marked by dots.
that are actually involved in stinging, because it has been shown that the toxins obtained from the tentacles are sometimes not detected in the nematocysts, which are the organs responsible for the cnidarian sting [8,9]. We could isolate nematocysts successfully from P. semoni using a modification of the reported method by Hessinger and Lenhoff [6]. The isolation of nematocysts allowed us to obtain the toxins which actually exist in the nematocysts of P. semoni.
Addition of 10 mM phosphate buffer (pH 6.0) to the isolated nematocysts caused complete discharge of the nematocysts so that we could obtain crude venom from them. The toxin was isolated using the methods in Materials and methods (Fig. 1). The molecular weight of each of the isolated toxins, P. semoni toxins 60A and 60B (PsTX-60A and PsTX-60B), as determined by SDS– PAGE, was ca. 60 kDa. The molecular weight of PsTX60A was slightly higher than that of PsTX-60B. Both PsTX-60A and -60B caused 50% hemolysis of an 0.8% suspension of sheep red blood cells at the concentrations of 600 and 300 ng/ml, respectively. Furthermore, PsTX-60A and -60B were fatally toxic to the shrimp P. paucidence at 800–900 and 800 lg/kg (LD50 value, i.p.), respectively. Most of the total lethality and hemolytic activities in the nematocyst could be explained
Fig. 3. Nucleotide and amino acid sequences of PsTX-60A. The complete gene sequence of PsTX-60A and its translation product are illustrated. The deduced amino acid sequence is shown starting from the first ATG codon of the open reading frame. The asterisks indicate in-frame stop codons (TAG and TAA). Nucleotide and amino acid numbers are shown at the right. The box indicates the N-terminal sequence of the mature PsTX-60A. The DDBJ Accession Number of this sequence is AB063315.
H. Nagai et al. / Biochemical and Biophysical Research Communications 294 (2002) 760–763
by these toxins. Thus, these toxins are the major toxins in the nematocysts. Protein sequencing of PsTX-60A and PsTX-60B The isolated PsTX-60A and PsTX-60B were sequenced. Sequences of 26 (PsTX-60A) and 24 (PsTX60B) amino acids were elucidated at the N-termini (Fig. 2). These showed 65.2% homology with each other, but no homology with other known proteins using a BLAST search [10]. This suggests that PsTX-60A and -60B are novel biologically active proteins. Cloning and sequencing of PsTX-60A cDNA On the basis of the N-terminal amino acid sequence, degenerative mixed oligonucleotide primers were designed. 30 -Rapid amplification of cDNA ends (RACE) using these primers was performed on total RNA obtained from P. semoni. The obtained PCR product (ca. 1450 bp), which included a polyadenylation site, was sequenced. This sequence was then used to design primers for 50 -RACE. Analysis of the 50 -RACE data revealed the remaining 50 -terminal sequence. Finally, the sequence of the full-length cDNA (1665 bp) that encodes PsTX-60A was clarified. The 50 -untranslated region contains a stop codon (TAG) in the same reading frame as the first initiation codon at position 52. A 1503-bp region prior to the first termination codon (TAA) encodes a 501-amino acid protein (see Fig. 3). Analysis of deduced amino acid sequence of PsTX-60A The deduced PsTX-60A sequence contains 501 amino acids from the putative initiating methionine to the putative last tyrosine. The peptide sequence for the N-terminus is found in the deduced molecule. This confirmed that the reading frame for PsTX-60A was correct. The N-terminal sequence of mature PsTX-60A starts with aspartic acid at position 36. Thus, the preceding part, which consists of 35 amino acids, should be a signal peptide and a propart. PsTX-60A (466 amino acids) has 15 Cys residues and its calculated isoelectric point is 7.35. Anderluh and Macek [5] recently reviewed the peptide and protein toxins obtained thus far from sea anemones and they categorized these toxins into four groups. Comparison of the deduced amino acid sequence for PsTX-60A with those of other proteins using
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the BLAST algorithm [10] revealed no significant similarity. Thus, PsTX-60A is representative of a novel class of cytolytic proteinaceous toxins and also of a fifth family of sea anemone polypeptide toxin. Because of its similarity with PsTX-60A, PsTX-60B should also be in the same family.
Acknowledgments The authors thank Dr. T. Koyama of Nagoya University and Mr. T. Maenosono of Okinawa Prefectural Institute of Health and Environment for helping with sample collection; Dr. H. Uchida of Kushimoto Marine Park Center for sample identification and biological information; Mr. Y. Araki, Okinawa Prefectural Central Health Center for epidemiological and biological information; Ms. E. Iwakoshi of Suntory Institute for Bioorganic Research (SUNBOR) for assistance in peptide sequencing and also Dr. H. Minakata of SUNBOR; and Dr. M. Nakao of Suntory Limited for their insightful discussions. N.O. thanks Okinawa International Exchange and Human Resources Development Foundation for scholarships. This research was supported by Grants-in-Aid from The Ministry of Education, Science, Sports and Culture of Japan (Nos. 11660210 and 12045267 to H.N.).
References [1] M. Kurokawa, in: Kaiyosei Kiken Seibutsu Taisakujigyo Houkokusho, Okinawa Prefectural Institute for Health and Environment, Okinawa, Japan, 1994, p. 6 (in Japanese). [2] M. Nakamoto, H. Uezato, Stings of box-jellyfish and sea anemones, Rinsho Hifuka 52 (1998) 29–33 (in Japanese). [3] M. Mizuno, K. Nishikawa, Y. Yuzawa, T. Kanie, H. Mori, Y. Araki, N. Hotta, S. Matsuo, Acute renal failure after a sea anemone sting, Am. J. Kidney Dis. 36 (2000) E10. [4] P. Macek, Polypeptide cytolytic toxins from sea anemones (Actiniaria), FEMS Microbiol. Immunol. 105 (1992) 121–130. [5] G. Anderluh, P. Macek, Cytolytic peptide and protein toxins from sea anemones (Anthozoa: Actiniaria), Toxicon 40 (2002) 111–124. [6] D.A. Hessinger, H.M. Lenhoff, Assay and properties of the pure venom from the nematocysts of the acontia of the sea anemone Aiptasia pallida, Arch. Biochem. Biophys. 159 (1973) 629–638. [7] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [8] A.W. Bernheimer, L.S. Avigad, A cholesterol-inhibitable cytolytic protein from the sea anemone Metridium senile, Biochem. Biophys. Acta 541 (1978) 96–106. [9] H. Nagai, K. Takuwa, M. Nakao, M. Ito, M. Miyake, M. Noda, T. Nakajima, Novel proteinaceous toxins from the box jellyfish (sea wasp) Carybdea rastoni, Biochem. Biophys. Res. Commun. 275 (2000) 582–588. [10] S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman, Basic local alignment search tool, J. Mol. Biol. 215 (1990) 403– 410.
Novel proteinaceous toxins from the nematocyst venom of the Okinawan sea anemone Phyllodiscus semoni Kwietniewski Hiroshi Nagai,a,*,1 Naomasa Oshiro,b Kyoko Takuwa-Kuroda,a Setsuko Iwanaga,b Masatoshi Nozaki,b and Terumi Nakajimaa a
Suntory Institute for Bioorganic Research, 1-1-1 Wakayamadai, Shimamoto, Mishima, Osaka 618-8503, Japan Okinawa Prefectural Institute of Health and Environment, 2085 Ozato, Ozato-son, Okinawa 901-1202, Japan
b
Received 16 May 2002
Abstract The Okinawan sea anemone Phyllodiscus semoni is known to cause cases of severe stinging. We isolated P. semoni toxins 60A and 60B (PsTX-60A and PsTX-60B; ca. 60 kDa) as the major toxins from the isolated nematocysts of this species for the first time. PsTX-60A and PsTX-60B showed lethal toxicity to the shrimp Palaemon paucidence when administered via intraperitoneal injection (LD50 values: 800–900 and 800 lg/kg, respectively) and hemolytic activity toward a 0.8% suspension of sheep red blood cells (ED50 values: 600 and 300 ng/ml, respectively). Furthermore, we sequenced the cDNA encoding PsTX-60A. The deduced amino acid sequence of PsTX-60A did not show any similarity to previously reported proteins. The N-terminal amino acid sequence of PsTX60B showed homology with that of PsTX-60A. These toxins represent a novel class of cytolytic proteinaceous toxins. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Sea anemone; Phyllodiscus semoni; Protein; Hemolysis; Toxin; PsTX; Nematocyst
The Okinawan sea anemone Phyllodiscus semoni Kwietniewski, 1897 is called ‘‘unbachi-isoginchaku’’ in Japanese which means ‘‘wasp-sea-anemone,’’ because this species causes cases of severe stinging [1–3]. This species is distributed widely in Okinawa Island, Japan. The characteristic symptoms of stinging by this species are severe dermatitis with swelling, local fever, and ulceration. However, the toxins of P. semoni have not yet been isolated or characterized. Understanding the mode of action of the toxin is essential to develop effective remedies against P. semoni stings. The isolation and characterization of P. semoni toxins is the first step in such a project. Our study focused on the major cytolytic and fatal toxins that exist in the nematocyst, which is the organ responsible for the P. semoni sting.
*
Corresponding author. Fax: +81-3-5463-0398. E-mail address: nagai@tokyo-u-fish.ac.jp (H. Nagai). 1 Present address: Tokyo University of Fisheries, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan.
In cnidarians, the toxins of anthozoans (sea anemones) have been studied extensively by comparing them with those of hydrozoan or scyphozoan species. Several polypeptide and protein toxins from sea anemones have been reported [4,5]. Herein we report the first isolation of P. semoni toxins 60A and 60B (PsTX-60A and PsTX-60B), novel hemolytic, and lethal proteins (60 kDa) from the nematocysts of this dangerous sea anemone and their bioactivities as well as the cDNA and amino acid sequences for PsTX60A.
Materials and methods General experimental procedure. Amino acid sequences were analyzed by a PSQ-1 protein sequencer (Shimadzu, Kyoto, Japan). Nucleotide sequences were determined using a BigDye Terminator Cycle Sequencing Kit (Applera, Norwalk, CT) and the ABI PRISM 310 (Applera). Animal material. The sea anemone P. semoni Kwietniewski (Anthozoa, Aliciidae) specimens were collected along the coast of Itoman, Okinawa, Japan, in August 2000 and maintained in an aquarium prior to use. The samples were identified by Dr. Hiro’omi Uchida of
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 5 4 7 - 8
H. Nagai et al. / Biochemical and Biophysical Research Communications 294 (2002) 760–763 Kushimoto Marine Park. Specimens for confirming identification were deposited at Kushimoto Marine Park, Wakayama, Japan. Hemolytic activity test. A few microliters of toxic fractions or purified toxin solution were incubated in a plastic tube at room temperature with 1 ml of a 0.8% suspension of sheep red blood cells (Nikken-Seibutu-Igaku-Kenkyusho, Japan) in phosphate-buffered saline (PBS) (+). After 4 h the tubes were centrifuged at 3000g for 10 min. The concentration of hemoglobin released into the supernatant was measured on a microplate reader (MPR-4Ai, Tosoh, Japan) at 550 nm. A 100% hemolysis value was obtained by the addition of 0.1 mg/ml saponin. Shrimp toxicity assay. The fractionated samples (each 10 ll) were injected intraperitoneally into the shrimp Palaemon paucidence. Fatal toxicity was observed 12 h after injection. The purified toxin was diluted in PBS(+) and 10 ll was injected intraperitoneally into P. paucidence (five shrimps for each toxin concentration). Preparation of isolated nematocysts of P. semoni. The nematocysts from P. semoni were isolated using a modification of the reported methods [6]. Globular vesicles (ca. 1 mm in diameter), which are organs surrounding the nematocysts, were separated from the skin of living P. semoni using tweezers and dispersed in 1 M sodium citrate solution. To eliminate the mucus and contaminants surrounding the vesicles, the sample suspension was kept at 5 °C in a 50-ml conical centrifuge tube and the supernatant was removed by decanting. The vesicles were washed by this method a few times for 2–3 days. Then, the sample suspension was autolyzed in a 1 M sodium citrate solution at 5 °C. After a week of autolysis, the suspension was well vortexed to liberate nematocysts from the vesicles. Free nematocysts were separated from the vesicles by pouring the suspension through fine-mesh nylon netting. The filtrate was allowed to settle in a 50-ml conical centrifuge tube for 3 h at 5 °C. The supernatant was then removed by decanting and the nematocysts were washed at least three times using 1 M sodium citrate solution. Finally, the isolated nematocysts were obtained and stored at )80 °C. Extraction and isolation of toxins from isolated nematocysts. The isolated nematocysts were centrifuged (12,000g, 5 min). The supernatant was eliminated by pipetting. To obtain the crude extracts, 10 mM phosphate buffer (pH 6.0) was added to the isolated nematocysts. The obtained crude toxin solution was centrifuged (12,000g, 15 min) and the supernatant was filtered through a 0.45-lm membrane. The filtrate was applied to an ion-exchange HPLC, TSK-GEL CM-5PW column (7:5 75 mm, 0.5 ml/min), which had been equilibrated with 10 mM phosphate buffer containing 0.3 M NaCl (pH 6.0) and then the column was washed with 10 mM phosphate buffer (pH 6.0). The adsorbed components were eluted with a gradient solvent system (0–0.7 M NaCl: 0–90 min). Most of the toxicity was observed in the non-adsorbed fraction. Weak toxicity was observed in the fraction that was adsorbed to the column and eluted with the salt gradient solvent system. The column-adsorbed fraction with weak toxicity was further purified (results to be published elsewhere). The non-adsorbed fraction with most of the toxicity was concentrated with the Amicon ultrafiltration system. Then, the sample was applied to a gel-permeation HPLC, Superdex 200 column (15 280 mm, 1 ml/min), which had been equilibrated with 0.5 M NaCl and 10 mM phosphate buffer (pH 7.0). Finally, PsTX-60A and PsTX-60B were isolated. The isolation of the toxins was guided by the hemolytic activity and the shrimp toxicity tests. All HPLC elution was monitored with a UV detector at 210 or 280 nm. The separation of each fraction was monitored by SDS–PAGE according to standard methods [7]. The protein concentration of the sample was measured using a BCA protein-assay reagent (Pierce) following manufacturer’s instructions. Protein sequencing of PsTX-60A and -60B. PsTX-60A and -60B (30 lg each), the isolated proteinaceous toxins from P. semoni nematocysts, were introduced into the PSQ-1 protein sequencer (Shimadzu) to analyze the N-terminal amino acid sequences of these toxins.
761
Cloning of PsTX-60A cDNA. Total RNA was isolated from the intact tentacles of P. semoni with TRIzol Reagent (Invitrogen, Carlsbad, CA) following manufacturer’s instructions. Degenerative 30 -rapid amplification of cDNA ends (RACE) was performed by the following method. First-strand cDNA was synthesized from 1 lg total RNA using a 50 /30 -RACE kit (Boehringer–Mannheim, Germany) and the oligo dT-anchor primer. The cDNA was then amplified by the first 30 -RACE reaction using the degenerate primer 60KA-30 -1: 50 -GAY AAR GAR ACI GAR CAY ATH GAR AT-30 (corresponding to peptide sequence: DKETEHIEI) and the PCR anchor primer, followed by reamplification of the primary PCR products using the degenerate primer 60KA-30 -2: 50 -ATH WSI MGI GGI GCI YTI GGI CAR GG-30 (corresponding to peptide sequence: ISRGALGQG) and the PCR anchor primer. The secondary PCR products were subcloned into the pCR2.1 vector and sequenced. Amplification was performed using Ex Taq Polymerase (Takara, Kyoto, Japan) under the following conditions: 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 3 min; and 72 °C for 7 min. The 1464-bp product obtained from the reaction was cloned into the pCR2.1 vector (Invitrogen) and nucleotide sequences were analyzed using the BigDye Terminator Cycle Sequencing Kit (Applera) and the ABI PRISM 310 (Applera) automated sequencer. This 1464-bp cDNA was then treated by 50 -RACE. 50 -RACE was performed by the following method: first-strand cDNA was synthesized from 1 lg total RNA using a 50 /30 -RACE kit (Boehringer– Mannheim, Germany) and a gene-specific primer 60KA-50 -5 (50 -ATT CGT CCA TTG GAA GGT CC-30 ). The first 50 -RACE reaction was completed using the oligo dT-anchor primer and the primer 60KA-50 -4 (50 -CAC CCG TTG CTT CAA ATT GC-30 ). The second 50 -RACE reaction was also completed using the PCR anchor primer and the primer 60KA-50 -1 (50 -CTT AGC AAA TCC TCT CTG TGG-30 ). Amplification for 50 -RACE was performed using Ex Taq Polymerase (Takara) under the following conditions: 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 2 min; and 72 °C for 7 min. The secondary PCR products were subcloned into the pCR2.1 vector and sequenced as described above.
Results and discussion Preparation of isolated nematocysts of P. semoni The preparation of pure venom from a suspension of nematocysts free of cytoplasmic contaminants is important for investigating unambiguously the toxins
Fig. 1. Purification scheme for PsTX-60A and PsTX-60B from P. semoni.
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H. Nagai et al. / Biochemical and Biophysical Research Communications 294 (2002) 760–763
Isolation of PsTX-60A and PsTX-60B from the isolated nematocysts Fig. 2. N-terminal amino acid sequences of PsTX-60A and PsTX-60B. The alignment of the sequences of PsTX-60A (top) and PsTX-60B (bottom) is shown by the single-letter amino acid code. Identical residues are marked by asterisks. Conserved residues are marked by dots.
that are actually involved in stinging, because it has been shown that the toxins obtained from the tentacles are sometimes not detected in the nematocysts, which are the organs responsible for the cnidarian sting [8,9]. We could isolate nematocysts successfully from P. semoni using a modification of the reported method by Hessinger and Lenhoff [6]. The isolation of nematocysts allowed us to obtain the toxins which actually exist in the nematocysts of P. semoni.
Addition of 10 mM phosphate buffer (pH 6.0) to the isolated nematocysts caused complete discharge of the nematocysts so that we could obtain crude venom from them. The toxin was isolated using the methods in Materials and methods (Fig. 1). The molecular weight of each of the isolated toxins, P. semoni toxins 60A and 60B (PsTX-60A and PsTX-60B), as determined by SDS– PAGE, was ca. 60 kDa. The molecular weight of PsTX60A was slightly higher than that of PsTX-60B. Both PsTX-60A and -60B caused 50% hemolysis of an 0.8% suspension of sheep red blood cells at the concentrations of 600 and 300 ng/ml, respectively. Furthermore, PsTX-60A and -60B were fatally toxic to the shrimp P. paucidence at 800–900 and 800 lg/kg (LD50 value, i.p.), respectively. Most of the total lethality and hemolytic activities in the nematocyst could be explained
Fig. 3. Nucleotide and amino acid sequences of PsTX-60A. The complete gene sequence of PsTX-60A and its translation product are illustrated. The deduced amino acid sequence is shown starting from the first ATG codon of the open reading frame. The asterisks indicate in-frame stop codons (TAG and TAA). Nucleotide and amino acid numbers are shown at the right. The box indicates the N-terminal sequence of the mature PsTX-60A. The DDBJ Accession Number of this sequence is AB063315.
H. Nagai et al. / Biochemical and Biophysical Research Communications 294 (2002) 760–763
by these toxins. Thus, these toxins are the major toxins in the nematocysts. Protein sequencing of PsTX-60A and PsTX-60B The isolated PsTX-60A and PsTX-60B were sequenced. Sequences of 26 (PsTX-60A) and 24 (PsTX60B) amino acids were elucidated at the N-termini (Fig. 2). These showed 65.2% homology with each other, but no homology with other known proteins using a BLAST search [10]. This suggests that PsTX-60A and -60B are novel biologically active proteins. Cloning and sequencing of PsTX-60A cDNA On the basis of the N-terminal amino acid sequence, degenerative mixed oligonucleotide primers were designed. 30 -Rapid amplification of cDNA ends (RACE) using these primers was performed on total RNA obtained from P. semoni. The obtained PCR product (ca. 1450 bp), which included a polyadenylation site, was sequenced. This sequence was then used to design primers for 50 -RACE. Analysis of the 50 -RACE data revealed the remaining 50 -terminal sequence. Finally, the sequence of the full-length cDNA (1665 bp) that encodes PsTX-60A was clarified. The 50 -untranslated region contains a stop codon (TAG) in the same reading frame as the first initiation codon at position 52. A 1503-bp region prior to the first termination codon (TAA) encodes a 501-amino acid protein (see Fig. 3). Analysis of deduced amino acid sequence of PsTX-60A The deduced PsTX-60A sequence contains 501 amino acids from the putative initiating methionine to the putative last tyrosine. The peptide sequence for the N-terminus is found in the deduced molecule. This confirmed that the reading frame for PsTX-60A was correct. The N-terminal sequence of mature PsTX-60A starts with aspartic acid at position 36. Thus, the preceding part, which consists of 35 amino acids, should be a signal peptide and a propart. PsTX-60A (466 amino acids) has 15 Cys residues and its calculated isoelectric point is 7.35. Anderluh and Macek [5] recently reviewed the peptide and protein toxins obtained thus far from sea anemones and they categorized these toxins into four groups. Comparison of the deduced amino acid sequence for PsTX-60A with those of other proteins using
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the BLAST algorithm [10] revealed no significant similarity. Thus, PsTX-60A is representative of a novel class of cytolytic proteinaceous toxins and also of a fifth family of sea anemone polypeptide toxin. Because of its similarity with PsTX-60A, PsTX-60B should also be in the same family.
Acknowledgments The authors thank Dr. T. Koyama of Nagoya University and Mr. T. Maenosono of Okinawa Prefectural Institute of Health and Environment for helping with sample collection; Dr. H. Uchida of Kushimoto Marine Park Center for sample identification and biological information; Mr. Y. Araki, Okinawa Prefectural Central Health Center for epidemiological and biological information; Ms. E. Iwakoshi of Suntory Institute for Bioorganic Research (SUNBOR) for assistance in peptide sequencing and also Dr. H. Minakata of SUNBOR; and Dr. M. Nakao of Suntory Limited for their insightful discussions. N.O. thanks Okinawa International Exchange and Human Resources Development Foundation for scholarships. This research was supported by Grants-in-Aid from The Ministry of Education, Science, Sports and Culture of Japan (Nos. 11660210 and 12045267 to H.N.).
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