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Novel peptide toxins from the sea anemone Stichodactyla haddoni
peptides 29 (2008) 536–544
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Novel peptide toxins from the sea anemone Stichodactyla haddoni Tomohiro Honma a, Shino Kawahata a, Masami Ishida b, Hiroshi Nagai b, Yuji Nagashima a, Kazuo Shiomi a,* a
Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Konan-4, Minato-ku, Tokyo 108-8477, Japan b Department of Ocean Sciences, Tokyo University of Marine Science and Technology, Konan-4, Minato-ku, Tokyo 108-8477, Japan
article info
abstract
Article history:
Four peptide toxins, SHTX I–III with crab-paralyzing activity and SHTX IV with crab lethality,
Received 3 October 2007
were isolated from the sea anemone Stichodactyla haddoni and their primary structures
Received in revised form
elucidated by protein sequencing and cDNA cloning. SHTX I (new toxin, 28 residues), II
13 December 2007
(analogue of SHTX I, 28 residues) and III (Kunitz-type protease inhibitor, 62 residues) are
Accepted 14 December 2007
potassium channel toxins and SHTX IV (48 residues) is a member of the type 2 sea anemone
Published on line 19 February 2008
sodium channel toxins. The precursor protein of SHTX IV is composed of a signal peptide, propart and mature peptide, while the propart is missing in that of SHTX III. In addition to
Keywords:
these four toxins, an epidermal growth factor-like peptide was detected in S. haddoni by RT-
EGF-like peptide
PCR. # 2007 Elsevier Inc. All rights reserved.
Peptide toxin Potassium channel toxicity Sea anemone Stichodactyla haddoni
1.
Introduction
Sea anemones are known to be rich in peptide toxins, of which two classes of peptide toxins, site-3 sodium channel toxins [4,12,27,34] and Kv1 potassium channel toxins [8,12], have been particularly well characterized. So far, more than 50 site3 sodium channel toxins and nearly 10 Kv1 potassium channel toxins have been isolated from various species of sea anemones. Based on the determined amino acid sequences, the site-3 sodium channel toxins and Kv1 potassium channel toxins are further divided into three and two types, respectively [12]. Some of these peptide toxins have been used as valuable pharmacological reagents in studying the structure and function of ion channels because of their high affinity to the specific channel.
Besides the site-3 sodium channel toxins and Kv1 potassium channel toxins, structurally and/or functionally novel peptide toxins have recently emerged in some species of sea anemones. Among these toxins are BDS-I and II from Anemonia sulcata [5], APETx1 [6] and APETx2 [7] from Anthopleura elegantissima, BcIV from Bunodosoma caissarum [28], Am II from Antheopsis maculata [15], gigantoxin I from Stichodactyla gigantea [33] and acrorhagin I and II from Actinia equina [16]. Although six toxins (BDS-I and II, APETx1 and 2, BcIV and Am II) structurally constitute a new family of sea anemone peptide toxins, five of them have already been shown to target different ion channels; BDS-I and II act on Kv3 channels [5,36], APETx1 on human ether-a-go-go-related gene potassium channels [6,29,37], APETx2 on acid-sensitive ion channels [7] and BcIV on sodium channel toxins [28]. On the other hand,
* Corresponding author. Tel.: +81 3 5463 0601; fax: +81 3 5463 0601. E-mail address: [email protected] (K. Shiomi). 0196-9781/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2007.12.010
peptides 29 (2008) 536–544
gigantoxin I is quite unique in that it is the first epidermal growth factor (EGF)-like toxin from biological sources. It shares 31–33% sequence identities with mammalian EGFs and exhibits EGF-like activity although much more weakly than human EGF [33]. Acrorhagin I and II are also of particular interest in that they were isolated from specialized aggressive organs (acrorhagi) differing from other sea anemone peptide toxins derived from whole bodies, tentacles or secreted mucus and that they share little or no homologies with peptide toxins of natural origin [16]. It should be noted that BcIV [28], Am II [15] and gigantoxin I [33] are potently paralytic to crabs but substantially not lethal. Taking this into consideration, we surveyed peptide toxins in the sea anemone Stichodactyla haddoni, which is a relatively large species (up to 50 cm in diameter) inhabiting tropical and subtropical waters, by a careful observation of the symptoms induced in crabs by samples. As a result, four peptide toxins, SHTX I–III with crab-paralyzing activity and SHTX IV with crab lethality, were isolated and elucidated for their primary structures by protein sequencing and cDNA cloning. Although SHTX IV was a member of the type 2 sea anemone sodium channel toxin family, the other three toxins were found to be structurally and functionally novel; SHTX I and II were new toxins with potassium channel toxicity and SHTX III was a Kunitz-type protease inhibitor with potassium channel toxicity. We report here the isolation, primary structures (including precursor structures) and some biological activities of SHTX I–IV from S. haddoni.
2.
Materials and methods
2.1.
Sea anemone
A specimen of S. haddoni collected along the coast of Okinawa Prefecture was shipped frozen to our laboratory and stored at 20 8C until extraction. For cDNA cloning, a live specimen imported from the Philippines was purchased from a retail aquarium shop in Yokohama, Kanagawa Prefecture, and cut into small pieces, which were immediately frozen in liquid nitrogen and stored at 80 8C until use.
2.2.
Isolation method
The frozen specimen (47 g) was thawed and well macerated in a motor. A 5 g-portion of the macerate was homogenized in 25 ml of distilled water and centrifuged at 18,800 g for 15 min. The supernatant (crude extract) was applied to gel filtration on a Sephadex G-50 column (2.5 cm 90 cm; GE Healthcare Bio-Sciences, Piscataway, NJ), which was eluted with 150 mM NaCl in 10 mM phosphate buffer (pH 7.0). Fractions of 8 ml were collected and measured for absorbance at 280 nm and crab toxicity. Toxic fractions were combined and then subjected to reverse-phase HPLC using a TSKgel ODS-120T column (0.46 cm 25 cm; Tosoh, Tokyo, Japan). After being washed with 0.1% trifluoroacetic acid (TFA), the column was eluted with two steps of linear gradients of acetonitrile (0–14% in 5 min and 14–42% in 80 min) in 0.1% TFA at a flow rate of 1 ml/min. Peptides were monitored at 220 nm with a UV detector. The eluate corresponding to each peak was
537
manually collected and assayed for crab toxicity. Thus, four peptide toxins (SHTX I–IV) were isolated.
2.3.
Chemical analysis
Peptides were determined according to the method of Lowry et al. [22] using bovine serum albumin as a standard. Molecular weight determination was performed by matrix assisted laser desorption ionization/time of flight mass spectrometry (MALDI/TOFMS) in the positive ion mode, using a Shimadzu/Kratos Kompact MALDI I instrument (Shimadzu, Kyoto, Japan). Sinapinic acid was used as a matrix. For the calibration of the instrument, insulin and sinapinic acid were used. Amino acid sequencing based on the Edman degradation method was carried out using an automatic gas-phase protein sequencer (LF-3400D TriCart with high sensitivity chemistry; Beckman Coulter, Fullerton, CA).
2.4.
Tryptic digestion and isolation of peptide fragments
SHTX I (40 mg) was digested with 10 mg of trypsin (Worthington Biochemical Corp., Lakewood, NJ) in 300 ml of 50 mM ammonium hydrogen carbonate at 37 8C for 24 h. Isolation of peptide fragments from the digest was achieved by reverse-phase HPLC on a TSKgel ODS-120T column, which was eluted with a linear gradient of acetonitrile (0–35% in 80 min) in 0.1% TFA.
2.5.
cDNA cloning
Cloning of the cDNA encoding either SHTX III or IV was performed by 30 and 50 rapid amplification of cDNA ends (RACE) as described below. Designations and nucleotide sequences of the primers used are shown in Table 1. The degenerate primers used for 30 RACE were designed based on the partial amino acid sequences determined by protein sequencing and the gene-specific primers used for 50 RACE based on the partial nucleotide sequences determined by 30 RACE. Total RNA was extracted from 1 g of the frozen sample with TRIzol reagent (Invitrogen, Carlsbad, CA). For 30 RACE, firststranded cDNA was synthesized from 5 mg of total RNA using a 30 RACE System for Rapid Amplification of cDNA Ends Kit (Invitrogen) and the oligo dT-adapter primer. The first 30 RACE reaction was performed using a degenerate primer (III-30 -1 for SHTX III or IV-30 -1 for SHTX IV) and the abridged universal amplification primer (AUAP) and the second 30 RACE reaction using a degenerate primer (III-30 -2 for SHTX III or IV-30 -2 for SHTX IV) and AUAP. Polymerase chain reaction (PCR) was carried out using Ex Taq polymerase (Takara, Kyoto, Japan) under the following conditions: 94 8C for 5 min; 35 cycles of 94 8C for 0.5 min; 55 8C for 0.5 min and 72 8C for 1 min; 72 8C for 5 min. The secondary PCR products were subcloned into the pT7Blue T-vector (Novagen, Darmstadt, Germany) and nucleotide sequences were determined using a Cy5 Thermo Sequenase Dye Terminator Kit (GE Healthcare Bio-Sciences) and a Long-Read Tower DNA sequencer (GE Healthcare BioSciences). On the basis of the determined partial nucleotide sequences, the remaining 50 -terminal sequences were analyzed by 50 RACE as follows. First-stranded cDNA was synthesized from 5 mg of total RNA using a 50 RACE System for Rapid
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peptides 29 (2008) 536–544
Table 1 – Designations and nucleotide sequences of the primers used for cDNA cloning of SHTX III and IV Designation of primer III-30 -l III-30 -2 III-50 -cDNA III-50 -l III-50 -2 III-RT-f III-RT-r IV-30 -1 IV-30 -2 IV-50 -cDNA IV-50 -1 IV-50 -2 IV-RT-f IV-RT-r AUAP AAP a
Corresponding amino acid sequencea
Nucleotide sequence of primer 50 -CIGARGARATGCCIGCIYTITGYC-30 50 -GA YGTICCIAARTG YMGIGGITA YTT-30 50 -GTTTCCTCC AC ATCCTCCG-30 50 -CTCCGTAGATAAACTGCTC AC-30 50 -GTAATACCTTGGGAAGTACCC-30 50 -CTT CAAAACAGACTCCCAAGA-30 50 -C ATTCC ATAACTGCTTTTATTTAT-30 50 -CIGCITGYAARTGYGAYGAYGAYG-30 50 -GGICCIGA YATHMGIWSIGCIACIYT-30 50 -TCCCTCGTTGC AGTTCC AG-30 50 -AGTAGC ACTGCGAATGTC AG-30 50 -GTC ATC ATCGC ACTTAC ACG-30 50 -AACTTATGCTTGATAAGAC AGTG-30 50 -GTAGTCTTATACCCGATCTTTC-30 50 -GGCC ACGCGTCGACTAGTAC-30 50 -GGCC ACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-30
1-TEEMPALCH-9 13-DVPKCRGYF-21 38-YGGCGGN-44 33-CEQFIYGG-40 19-GYFPRYY-25 None (50 -untranslated) None (30 -untranslated) 1-AACKCDDDG-9 9-GPDIRSATL-17 23-FWNCNEG-29 10-PDIRSATL-17 2-ACKCDDD-8 None (50 -untranslated) None (30 -untranslated)
Refer to Fig. 4 or Fig. 6 for amino acid sequences.
Amplification of cDNA Ends Kit (Invitrogen) and a gene-specific primer (III-50 -cDNA for SHTX III or IV-50 -cDNA for SHTX IV). The first 50 RACE reaction was completed using a gene-specific primer (III-50 -1 for SHTX III or IV-50 -1 for SHTX IV) and the abridged anchor primer (AAP), followed by reamplification of the PCR products using a gene-specific primer (III-50 -2 for SHTX III or IV-50 -2 for SHTX IV) and AUAP. Amplification conditions were the same as in 30 RACE experiments. The secondary PCR products were subcloned into the pT7Blue T-vector and sequenced. Finally, reverse transcriptase (RT)-PCR was carried out to confirm the nucleotide sequences determined by both 30 and 50 RACE, using the first-stranded cDNA for 30 RACE as a template and the following primer combinations: III-RT-f and -r for SHTX III and IV-RT-f and -r for SHTX IV. In a separate set of cloning experiments, amplification of cDNAs encoding EGF-like peptides such as gigantoxin I previously found in S. gigantea [33] was attempted by RT-PCR. The first-stranded cDNA synthesized for 30 RACE was used as a template. Gene-specific forward (50 -TGTGAGAAGCAAATTGACCAG-30 ) and reverse (50 -GCCTTTTATTTGAAATCCATGTT-30 ) primers were designed from the nucleotide sequences in the 50 - and 30 -untranslated regions of the gigantoxin I cDNA (DDBJ/ EMBL/GenBank accession no. AB110014), respectively.
2.6.
Assay of crab toxicity
Crab toxicity was assayed using freshwater crabs (Potamon dehaani) weighing about 5 g purchased from the Tokyo Central Wholesale Market. Sample solutions were injected into crabs at 10 ml/g of crab body weight at the junction between the body and the leg. The symptoms induced in crabs were carefully observed up to 2 h after injection. To calculate LD50 (for lethal activity) or ED50 (for paralytic activity) against crabs by the method of Litchfield and Wilcoxon [20], groups of five crabs were administered with various doses of toxin.
2.7.
Assay of antitryptic activity
Antitryptic activity of SHTX III was estimated using a microtiter plate with 96-wells, as described previously [25].
In brief, trypsin solution (containing 0.15 unit) was preincubated with SHTX III solution at 37 8C for 10 min and the residual enzymatic activity was determined using the synthetic substrate, benzoyl-D,L-4-arginine-p-nitroanilide (Funakoshi, Tokyo, Japan). Antitryptic activity was expressed in terms of inhibitory units (IU), where 1 IU was defined as the amount of SHTX III required to inhibit one unit of trypsin.
2.8.
Assay of potassium channel toxicity
Potassium channel toxicity of SHTX II and III was indirectly assayed by competitive inhibition of the binding of 125I-adendrotoxin to rat synaptosomal membranes, as reported previously [10,24]. For comparison, a-dendrotoxin (Sigma, St. Louis, CA) and bovine pancreatic trypsin inhibitor (Sigma) were also evaluated for potassium channel toxicity. Labeling of a-dendrotoxin with 125I was performed by the chloramine-T (N-chloro-p-toluenesulphonamide) method according to the instructions of GE Healthcare Bio-Sciences and the labeled toxin was purified by gel filtration on a Sephadex G-10 column (1.2 cm 2.5 cm; GE Healthcare Bio-Sciences). Synaptosomal membrane suspension (0.4 mg protein/ml) was prepared from rat brains (Funakoshi, Tokyo, Japan). For competitive binding experiments, 200 ml of the synaptosomal membrane suspension was incubated with 40 ml of sample solution and 10 ml of 337 pM 125I-a-dendrotoxin (66.6 TBq/mmol) at room temperature for 30 min. The membranes were then collected by centrifugation and the radioactivity bound to the membranes was measured on a COBRA II gamma counter (Packard, Meriden, CT). Non-specific binding (about 7%) was determined by replacing sample solution with 450 nM a-dendrotoxin and subtracted from each datum.
3.
Results
3.1.
Isolation of toxins
When serial twofold dilutions of the crude extract were tested for crab toxicity, lethality was detected up to 32-fold dilution.
peptides 29 (2008) 536–544
539
Fig. 1 – Isolation of SHTX I–IV from Stichodactyla haddoni. (A) Gel filtration. Sample, crude extract; column, Sephadex G-50 (2.5 cm T 90 cm); solvent, 0.15 M NaCl in 0.01 M phosphate buffer (pH 7.0); volume per fraction, 8 ml. Toxic fractions are indicated by a bar. (B) Reverse-phase HPLC. Sample, toxic fractions obtained by gel filtration; column, TSKgel ODS-120T (0.46 cm T 25 cm); elution, linear gradient of acetonitrile in 0.1% TFA; flow rate, 1 ml/min. SHTX I–IV were eluted in labeled peaks.
In gel filtration of the crude extract on a Sephadex G-50 column, crab lethality appeared between fractions 42 and 58 (Fig. 1A). The toxic fraction obtained by gel filtration was then subjected to reverse-phase HPLC on a TSKgel ODS-120T column. Crab lethality was found only in a peak at a retention time of 67 min and crab paralysis in three peaks at 42, 43 and 54 min (Fig. 1B). Thus, four toxins (named SHTX I, II, III and IV in the order of elution) were successfully isolated. The purity of the four toxins was supported by protein sequencing, in which they gave only one amino acid in each cycle. The yields from 5 g of the starting sample were as follows: 144 mg of SHTX I, 104 mg of SHTX II, 200 mg of SHTX III and 320 mg of SHTX IV. The LD50 against crabs of SHTX IV was estimated to be 93 mg/ kg. On the other hand, SHTX I, II and III induced paralysis in crabs with ED50 of 430, 430 and 183 mg/kg, respectively, but none of them exhibited lethality even at 1000 mg/kg.
3.2.
Amino acid sequences of SHTX I and II
In the first cycle of protein sequencing, no amino acid was detected for either SHTX I or II. However, the Edman degradation reaction proceeded in the subsequent cycles, suggesting that the N-terminal residue of both toxins has a free amino group but its side chain is modified. As shown in Fig. 2, the same amino acid sequence ranging from the second position (Ile-2) to the C-terminal position (Gln-28) was
elucidated for SHTX I and II, except for only one difference at position 6 (hydroxy Pro for SHTX I and Pro for SHTX II). A search by protein databases revealed that SHTX I and II are homologous only to Am I [15]. However, SHTX I and II show considerable sequence differences from Am I, except for the four Cys residues (Fig. 2). In the next step, we attempted to assign the location of two disulfide bridges in SHTX I by sequencing of peptide fragments produced upon tryptic digestion. Two tryptic peptides (peptides 1 and 2), isolated by reverse-phase HPLC, were sequenced as follows: (unknown, Gly)-(Ile, Gly)-(Ile, Cys)-(Gly, Val)-(Ala, Arg)-hydroxy Pro-Cys-Arg for peptide 1; (Cys, Asp)-(Tyr, Trp)-(His, Ser)-(Ser, Cys)-(Asp, Gly)-(Gly, Gln)(Lys, Gln) for peptide 2. Comparison of these sequencing results with the entire amino acid sequence demonstrated that the two disulfide bridges of SHTX I are formed between Cys-7 and -19 and between Cys-10 and -25. It is reasonable to infer that SHTX II and Am I have the same disulfide bridge pattern as SHTX I.
3.3.
Amino acid sequence of SHTX III and its precursor
Protein sequencing identified N-terminal 54 amino acid residues of SHTX III. To determine the remaining C-terminal sequence, we employed a cDNA cloning technique. As a result, a full-length cDNA (503 bp; deposited in the DDBJ/
Fig. 2 – Amino acid sequences of SHTX I, SHTX II and Am I. Am I was isolated from Antheopsis maculata [15]. Identical residues with SHTX I are boxed. Two disulfide bridges are indicated above the sequence of SHTX I. Unidentified N-terminal residues (for SHTX I and II) and hydroxy Pro (for position 6 of SHTX I and Am I) are denoted by ‘X’ and ‘O’, respectively.
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Fig. 3 – Nucleotide sequence of the cDNA encoding SHTX III. The deduced amino acid sequence is aligned below the nucleotide sequence. Nucleotide and amino acid numbers are shown at the right. Asterisks indicate a stop codon (TGA). A putative signal sequence is underlined. The determined nucleotide sequence has been deposited in the DDBJ/EMBL/ GenBank databases under the accession no. AB362569.
EMBL/GenBank databases under the accession no. AB362569) encoding SHTX III was successfully obtained by a combination of 30 and 50 RACE. The open reading frame (243 bp) codes for the precursor protein of 81 amino acid residues (Fig. 3). Analysis by the SignalP program [3] suggested that the region 1–19 is a signal peptide. The remaining region 20–81 was judged to represent the mature peptide of SHTX III from the following two criteria: (1) the region 20–73 completely matches the N-terminal 54 residues determined for SHTX III by protein sequencing and (2) the molecular weight (7034.0) calculated for the region 20–81 is close to that (7035.0) determined for SHTX III by MALDI/TOFMS. A database search proved that SHTX III (62 residues) is a new member of the Kunitz-type protease inhibitor family. For comparison, the amino acid sequence of SHTX III is aligned in Fig. 4 with those of some sea anemone Kunitz-type protease inhibitors (ShPI from Stichodactyla helianthus [2], AEPI-I from A. equina [17], AXPI-I from Anthopleura aff. xanthogrammica [23] and AsKC 1-3 from A. sulcata [31]) and bovine pancreatic trypsin inhibitor (representative Kunitz-type protease inhibitor). The sequence identity of SHTX III with the above protease inhibitors ranges from 37% (with bovine pancreatic trypsin inhibitor) to 61% (with AEPI-I). In accordance with the sequence similarity to Kunitz-type protease inhibitors, SHTX III was confirmed to inhibit trypsin with an antitryptic activity of 203 IU/mg.
3.4.
Amino acid sequence of SHTX IV and its precursor
Similar to the case of SHTX III, the amino acid sequence of SHTX IV was established by both protein sequencing and molecular cloning. Analysis by a protein sequencer elucidated the N-terminal sequence (up to the 44th residue) of SHTX IV. A full-length SHTX IV cDNA (581 bp; deposited in the DDBJ/EMBL/GenBank databases under the accession no. AB362570) cloned by 30 and 50 RACE contained an open reading frame (252 bp) coding for the precursor protein of 84 amino acid residues (Fig. 5). The N-terminal region up to the 19th residue was predicted to be a signal peptide. The succeeding region 20–35 is featured by ending with a pair of basic residues (Lys-Arg). Since this feature is observed for the propart in most precursor proteins of sea anemone peptide toxins [1,9,11,14–16,21,35], the region 20–35 was assumed to be a propart. The N-terminal sequence determined for SHTX IV was completely recognized in the region 36–79. On the basis of the molecular weight (5229.5) determined for SHTX IV by MALDI/TOFMS, the region 36–83 with a calculated molecular weight of 5226.8 was judged to be the mature peptide of SHTX IV. Interestingly, the C-terminal residue (Gly) of the precursor protein was not involved in the mature peptide. It is known that peptidylglycines undergo removal of glycine and subsequent amidation of the penultimate residue by sequential actions with peptidylglycine a-ami-
Fig. 4 – Amino acid sequence alignment of SHTX III with Kunitz-type protease inhibitors. Inhibitors: ShPI from Stichodactyla helianthus [2]; AEPI-I from Actinia equina [17]; AXPI-I from Anthopleura aff. xanthogrammica [23]; AsKC 1–3 (kalicludines 1–3) from Anemonia sulcata [31]; BPTI (bovine pancreatic trypsin inhibitor) from bovine pancreas. Identical residues with SHTX III are boxed.
peptides 29 (2008) 536–544
541
Fig. 5 – Nucleotide sequence of the cDNA encoding SHTX IV. The deduced amino acid sequence is aligned below the nucleotide sequence. Nucleotide and amino acid numbers are shown at the right. Asterisks indicate a stop codon (TGA or TAA). A putative signal peptide and propart are singly and doubly underlined, respectively. The determined nucleotide sequence has been deposited in the DDBJ/EMBL/GenBank databases under the accession no. AB362570.
Fig. 6 – Amino acid sequence alignment of SHTX IV with type 2 sea anemone sodium channel toxins. Rp II was isolated from Radianthus (Heteractis) paumotensis [30]; RTX I from Radianthus (Heteractis) macrodactylus [38]; gigantoxin III from Stichodactyla gigantea [32]; Sh I from S. helianthus [19]. Identical residues with SHTX IV are boxed.
dating monooxygenase and peptidylamidoglycolate lyase [18]. Therefore, we can conclude that the Gly residue at the Cterminus of the precursor protein is removed after translation and that SHTX IV has an amidated residue (Lys-NH2) at the C-terminus. SHTX IV (48 residues) obviously belongs to the type 2 sea anemone sodium channel toxin family, as aligned in Fig. 6 with four type 2 sea anemone sodium channel toxins (Rp II from Radianthus (Heteractis) paumotensis [30], RTX I from Radianthus (Heteractis) macrodactylus [38], gigantoxin III from S. gigantea [33] and Sh I from S. helianthus [19]). Particularly, SHTX IV shows as high as 91% sequence identity with Rp II.
3.5.
270, 650 and 5.7 nM, respectively, indicating that SHTX II and III are about 50 and 110 times less potent than a-dendrotoxin. The binding between 125I-a-dendrotxin and the synaptosomal membranes was not affected by bovine pancreatic trypsin inhibitor even at 16,000 nM. Although not examined in this study, SHTX I can be considered to exhibit the same degree of potassium channel toxicity as SHTX II based on no substantial difference in both sequence and crab-paralyzing activity between the two toxins.
Potassium channel toxicity of SHTX II and III
SHTX IV is considered to act on sodium channels because of its high sequence identity with the type 2 sea anemone sodium channel toxins. On the other hand, SHTX III is expected to target potassium channels, similar to the type 2 sea anemone potassium channel toxins, AsKC 1–3 (kalicludines 1–3) that were isolated as potassium channel toxins from A. sulcata and confirmed to be Kunitz-type protease inhibitors [31]. SHTX I and II may also have potassium channel toxicity. In this study, therefore, two toxins, SHTX II and III, were evaluated for potassium channel toxicity by competitive binding assay using rat synaptosomal membranes. As depicted in Fig. 7, both SHTX II and III inhibited the binding of 125I-a-dendrotxin to synaptosomal membranes. IC50 of SHTX II, III and a-dendrotoxin were estimated to be
Fig. 7 – Inhibition of the binding of 125I-a-dendrotoxin to rat synaptosomal membranes by SHTX II (open circle), SHTX III (open triangle) and a-dendrotoxin (closed circle).
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Fig. 8 – Nucleotide sequence of the cDNA encoding the EGF-like peptide (SHTX IV). The deduced amino acid sequence is aligned below the nucleotide sequence. Nucleotide and amino acid numbers are shown at the right. Asterisks indicate a stop codon (TAA). A putative signal peptide and propart are singly and doubly underlined, respectively. Different nucleotides from the gigantoxin I cDNA (DDBJ/EMBL/GenBank accession no. AB110014) are shaded. Amino acid residues of gigantoxin I differing from SHTX V are indicated below the deduced amino acid sequence. The determined nucleotide sequence has been deposited in the DDBJ/EMBL/GenBank databases under the accession no. AB362571.
3.6.
Detection of an EGF-like peptide by RT-PCR
In this study, any EGF-like peptides were not isolated from S. haddoni. However, a cDNA (581 bp; deposited in the DDBJ/ EMBL/GenBank databases under the accession no. AB362571) encoding an EGF-like peptide (named SHTX V) was amplified by RT-PCR using the primers designed from the nucleotide sequence of the gigantoxin I cDNA. The precursor protein of SHTX V (86 residues) was assumed to comprise a signal peptide (23 residues), propart (15 residues) and mature peptide (48 residues), as in the case of the gigantoxin I precursor [33] (Fig. 8). As compared to the nucleotide sequence of the gigantoxin I cDNA, the SHTX V cDNA has 16 alterations, 14 of which are recognized in the mature peptide region. In reflection of this, SHTX V has the same amino acid sequence as gigantoxin I in both signal peptide and propart regions but differs from gigantoxin I at nine positions in the mature region.
4.
Discussion
Four peptide toxins (SHTX I–IV) with crab toxicity were isolated from S. haddoni by gel filtration and reverse-phase HPLC and their primary structures elucidated by protein sequencing and cDNA cloning techniques. Importantly, SHTX I–III were not lethal but only paralytic to crabs and found to be unique from both structural and functional points of view, while SHTX IV with crab lethality was a member of the wellknown type 2 sea anemone sodium channel toxin family. Previously, three novel peptide toxins, gigantoxin I [33], Am II [15] and BcIV [28], have been isolated as crab-paralyzing factors from S. gigantea, A. maculata and Bunodosoma caissarum, respectively. This study again confirmed that the crab assay, if only carefully performed, is very useful to discover novel peptide toxins in sea anemones. The most significant finding of this study is that SHTX I and II are structurally new peptide toxins displaying potassium channel toxicity, although their modified N-terminal residue remains to be clarified. Both toxins are relatively short
peptides and are cross-linked by two disulfide bridges (7–19 and 10–25). Sea anemone peptide toxins are mostly crosslinked by three disulfide bridges. Exceptionally, four disulfide bridges are known for members of the type 3 sea anemone sodium channel toxins, PaTX from Entacmaea actinostoloides [26] and its related peptide toxins (Da I and II from Dofleina armata [13] and Er I from Entacmaea ramsayi [13]), and even five disulfide bridges for two novel toxins (AETX II and III from Anemonia erythraea [32]). Of the known sea anemone peptide toxins, Am I from A. maculata has been the sole toxin that contains two disulfide bridges similar to SHTX I and II [15]. SHTX I, SHTX II and Am I have four Cys residues at the same positions, suggesting the same structural architecture for the three toxins. However, SHTX I and II share a considerably low sequence identity (32% identity) with Am I. This low sequence identity may account for the difference in toxicity; SHTX I and II are not lethal to crabs at 1000 mg/kg, while Am I shows crab lethality with an LD50 of 830 mg/kg [15]. Another significant finding is that SHTX III is a Kunitz-type protease inhibitor showing potassium channel toxicity as well as antitryptic activity. Of a number of Kunitz-type protease inhibitors so far isolated from sea anemones (e.g., ShPI, AEPI-I, AXPI-I and AsKC 1–3 shown in Fig. 4), only AsKC 1–3 from A. sulcata have been experimentally demonstrated to be potassium channel blockers [31]. In general, sea anemone protease inhibitors are assumed to function to inhibit endogenous proteases in animal themselves and/or to protect the peptide toxins injected into prey animals or potential predators from rapid destruction. However, our finding of a Kunitz-type protease inhibitor with potassium channel toxicity in S. haddoni may reinforce the previous hypothesis that sea anemone protease inhibitors have an aggressive function in that they contribute to the paralysis of prey animals [31]. Further survey for potassium channel toxicity of sea anemone protease inhibitors is needed to discuss their biological functions in more detail. Previous cloning studies have established the precursor structures of the following sea anemone peptide toxins: calitoxin 1 and 2 from Calliactis parasitica [35], Ae I (=AeNa) from A. equina [1], HK2a from Anthopleura sp. [21], gigantoxin I–
peptides 29 (2008) 536–544
III from S. gigantea [33], Am I–III from A. maculata [15], acrorhagin I and II from acrorhagi of A. equina [16], HmK from Heteractis magnifica [9] and AETX K from Anemonia erythraea [11]. It is generally considered from the accumulated knowledge that the precursors of sea anemone peptide toxins are commonly composed of a signal peptide, propart and mature peptide; two exceptions are the precursor of HK2a devoid of both signal peptide and propart and that of acrorhagin II devoid of propart. This study showed that the precursor structure of SHTX IV conform well to the above generalization, while the propart is missing in the precursor of SHTX III as in the case of acrorhagin II. Anderluh et al. [1] previously proposed that the propart with two basic residues at the end (cleavage site of subtilisin-like proteases) serves as a signal directing toxins into nematocysts, stinging organelles peculiar to cnidarians. According to this proposal, we tentatively assume that SHTX IV is contained in nematocysts but SHTX III is not. To verify the function of the propart, it is essential to examine whether sea anemone peptide toxins are localized in nematocysts. Finally, it should be noted that, next to gigantoxin I from S. gigantea [33], an EGF-like peptide (SHTX V) was detected in S. haddoni by RT-PCR, suggesting a wide distribution of EGF-like peptides in sea anemones. SHTX V was not isolated from S. haddoni in this study, probably because its concentration in the sea anemone is very low or because it has no toxic activity to crabs. However, our results will facilitate future study on sea anemone EGF-like peptides as well as other classes of sea anemone peptide toxins.
references
[1] Anderluh G, Podlesek Z, Macˇek P. A common motif in proparts of Cnidarian toxins and nematocyst collagens and its putative role. Biochim Biophys Acta 2000;1476:372–6. [2] Antuch W, Berndt KD, Chavez M, Delfin J, Wuethrich K. The NMR solution structure of a Kunitz-type protease inhibitor from the sea anemone Stichodactyla helianthus. Eur J Biochem 1993;212:675–84. [3] Bendtsen JD, Nielsen H, von Heijne G, Brunak S. Improved prediction of signal peptides: signalP 3.0. J Mol Biol 2004;340:783–95. [4] Bosmans F, Tytgat J. Sea anemone venom as a source of insecticidal peptides acting on voltage-gated Na+ channels. Toxicon 2007;49:550–60. [5] Diochot S, Schweitz H, Be´ress L, Lazdunski M. Sea anemone peptides with a specific blocking activity against the fast inactivating potassium channel Kv3.4. J Biol Chem 1998;273:6744–9. [6] Diochot S, Loret E, Bruhn T, Be´ress L, Lazdunski M. APETx1, a new toxin from the sea anemone Anthopleura elegantissima, blocks voltage-gated human ether-a-go-gorelated gene potassium channels. Mol Pharmacol 2003;64:59–69. [7] Diochot S, Baron A, Rash LD, Deval E, Escoubas P, Scarzello S, Salinas M, Lazdunski M. A new sea anemone peptide, APETx2, inhibits ASIC3, a major acid-sensitive channel in sensory neurons. EMBO J 2004;23:1516–25. [8] Gasparini S, Gilquin B, Menez A. Comparison of sea anemone and scorpion toxins binding to Kv1 channels: an example of convergent evolution. Toxicon 2004;43:901–8.
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[9] Gendeh GS, Young LC, de Medeiros LC, Jeyaseelan K, Harvey AL, Chung MCM. A new potassium channel toxin from the sea anemone Heteractis magnifica: isolation, cDNA cloning, and functional expression. Biochemistry 1997;36:11461–7. [10] Harvey AL, Marshall DL, De-Allie FA, Strong PN. Interactions between dendrotoxin, a blocker of voltagedependent potassium channels, and charybdotoxin, a blocker of calcium-activated potassium channels, at binding sites on neuronal membranes. Biochem Biophys Res Commun 1989;163:394–7. [11] Hasegawa Y, Honma T, Nagai H, Ishida M, Nagashima Y, Shiomi K. Isolation and cDNA cloning of a potassium channel peptide toxin from the sea anemone Anemonia erythraea. Toxicon 2006;48:536–42. [12] Honma T, Shiomi K. Peptide toxins in sea anemones: structural and functional aspects. Mar Biotechnol 2006;8:1– 10. [13] Honma T, Iso T, Ishida M, Nagashima Y, Shiomi K. Occurrence of type 3 sodium channel peptide toxins in two species of sea anemones (Dofleinia armata and Entacmaea ramsayi). Toxicon 2003;41:637–9. [14] Honma T, Nagai H, Nagashima Y, Shiomi K. Molecular cloning of an epidermal growth factor-like toxin and two sodium channel toxins from the sea anemone Stichodactyla gigantea. Biochim Biophys Acta 2003;1652:103–6. [15] Honma T, Hasegawa Y, Ishida M, Nagai H, Nagashima Y, Shiomi K. Isolation and molecular cloning of novel peptide toxins from the sea anemone Antheopsis maculata. Toxicon 2005;45:33–41. [16] Honma T, Minagawa S, Nagai H, Ishida M, Nagashima Y, Shiomi K. Novel peptide toxins from acrorhagi, aggressive organs of the sea anemone Actinia equina. Toxicon 2005;46:768–74. [17] Ishida M, Minagawa S, Miyauchi K, Shimakura K, Nagashima Y, Shiomi K. Amino acid sequences of Kunitztype protease inhibitors from the sea anemone Actinia equina. Fish Sci 1997;63:794–8. [18] Katapodis AG, Ping D, May SW. A novel enzyme from bovine neurointermediate pituitary catalyzes dealkylation of a-hydroxyglycine derivatives, thereby functioning sequentially with peptidylglycine a-amidating monooxygenase in peptide amidation. Biochemistry 1990;29:6115–20. [19] Kem WR, Parten B, Pennington MW, Price DA, Dunn BM. Isolation, characterization, and amino acid sequence of a polypeptide neurotoxin occurring in the sea anemone Stichodactyla helianthus. Biochemistry 1989;28:3483–9. [20] Litchfield Jr JT, Wilcoxon F. A simplified method of evaluating dose-effect experiments. J Pharmacol Exp Ther 1949;96:99–113. [21] Liu WH, Wang L, Wang YL, Peng LS, Wu WY, Peng WL, Jiang XY, Tu HB, Chen HP, Ou-Yang P, Xu AL. Cloning and characterization of a novel neurotoxin from the sea anemone Anthopleura sp. Toxicon 2003;41:793–801. [22] Lowry OH, Rosebrough AL, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75. [23] Minagawa S, Ishida M, Shimakura K, Nagashima Y, Shiomi K. Isolation and amino acid sequences of two Kunitz-type protease inhibitors from the sea anemone Anthopleura aff. xanthogrammica. Comp Biochem Physiol 1997;118B:381–6. [24] Minagawa S, Ishida M, Nagashima Y, Shiomi K. Primary structure of a potassium channel toxin from the sea anemone Actinia equina. FEBS Lett 1998;427:149–51. [25] Nagashima Y, Takeda M, Ohta I, Shimakura K, Shiomi K. Purification and properties of proteinaceous trypsin inhibitors in the skin secretion of puffer Takifugu pardalis. Comp Biochem Physiol 2004;138B:103–10.
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[26] Nishida S, Fujita S, Warashina A, Satake M, Tamiya N. Amino acid sequence of a sea anemone toxin from Parasicyonis actinostoloides. Eur J Biochem 1985;150:171–3. [27] Norton RS. Structure and structure–function relationships of sea anemone proteins that interact with the sodium channel. Toxicon 1991;29:1051–84. [28] Oliveira JS, Zaharenko AJ, Ferreira Jr WA, Konno K, Shida CS, Richardson M, Lucio AD, Beirao PS, de Freitas JC. BcIV, a new paralyzing peptide obtained from the venom of the sea anemone Bunodosoma caissarum. A comparison with the Na+ channel toxin BcIII. Biochim Biophys Acta 2006;1764:1592–600. [29] Restano-Cassulini R, Korolkova YV, Diochot S, Gurrola G, Guasti L, Possani LD, Lazdunski M, Grishin EV, Arcangeli A, Wanke E. Species diversity and peptide toxins blocking selectivity of ether-a-go-go-related gene subfamily K+ channels in the central nervous system. Mol Pharmacol 2006;69:1673–83. [30] Schweitz H, Bidard JN, Frelin C, Pauron D, Vijverberg HPM, Mahasneh DM, Lazdunski M, Vilbois F, Tsugita A. Purification, sequence, and pharmacological properties of sea anemone toxins from Radianthus paumotensis. A new class of sea anemone toxins acting on the sodium channel. Biochemistry 1985;24:3554–61. [31] Schweitz H, Bruhn T, Guillemare E, Moinier D, Lancelin JM, Be´ress L, Lazdunski M. Kalicludines and kaliseptine. Two different classes of sea anemone toxins for voltagesensitive K+ channels. J Biol Chem 1995;270:25121–6.
[32] Shiomi K, Qian WH, Lin XY, Shimakura K, Nagashima Y, Ishida M. Novel polypeptide toxins with crab toxicity from the sea anemone Anemonia erythraea. Biochim Biophys Acta 1997;1335:191–8. [33] Shiomi K, Honma T, Ide M, Nagashima Y, Ishida M, Chino M. An epidermal growth factor-like toxin and two sodium channel toxins from the sea anemone Stichodactyla gigantea. Toxicon 2003;41:229–36. [34] Smith JJ, Blumenthal KM. Site-3 sea anemone toxins: molecular probes of gating mechanisms in voltagedependent sodium channels. Toxicon 2007;49: 159–70. [35] Spagnuolo A, Zanetti L, Cariello L, Piccoli R. Isolation and characterization of two genes encoding calitoxins, neurotoxic peptides from Calliactis parasitica (Cnidaria). Gene 1994;138:187–91. [36] Yeung SY, Thompson D, Wang Z, Fedida D, Robertson B. Modulation of Kv3 subfamily potassium currents by the sea anemone toxin BDS: significance for CNS and biophysical studies. J Neurosci 2005;25:8735–45. [37] Zhang M, Liu XS, Diochot S, Lazdunski M, Tseng GN. APETx1 from sea anemone Anthopleura elegantissima is a gating modifier peptide toxin of the human ether-a-go-gorelated (hERG) potassium channel. Mol Pharmacol 2007;72:259–68. [38] Zykova TA, Kozlovskaya EP. Amino acid sequence of neurotoxin I from the sea anemone Radianthus macrodactylus. Bioorg Khim 1989;15:1301–6.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
Novel peptide toxins from the sea anemone Stichodactyla haddoni Tomohiro Honma a, Shino Kawahata a, Masami Ishida b, Hiroshi Nagai b, Yuji Nagashima a, Kazuo Shiomi a,* a
Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Konan-4, Minato-ku, Tokyo 108-8477, Japan b Department of Ocean Sciences, Tokyo University of Marine Science and Technology, Konan-4, Minato-ku, Tokyo 108-8477, Japan
article info
abstract
Article history:
Four peptide toxins, SHTX I–III with crab-paralyzing activity and SHTX IV with crab lethality,
Received 3 October 2007
were isolated from the sea anemone Stichodactyla haddoni and their primary structures
Received in revised form
elucidated by protein sequencing and cDNA cloning. SHTX I (new toxin, 28 residues), II
13 December 2007
(analogue of SHTX I, 28 residues) and III (Kunitz-type protease inhibitor, 62 residues) are
Accepted 14 December 2007
potassium channel toxins and SHTX IV (48 residues) is a member of the type 2 sea anemone
Published on line 19 February 2008
sodium channel toxins. The precursor protein of SHTX IV is composed of a signal peptide, propart and mature peptide, while the propart is missing in that of SHTX III. In addition to
Keywords:
these four toxins, an epidermal growth factor-like peptide was detected in S. haddoni by RT-
EGF-like peptide
PCR. # 2007 Elsevier Inc. All rights reserved.
Peptide toxin Potassium channel toxicity Sea anemone Stichodactyla haddoni
1.
Introduction
Sea anemones are known to be rich in peptide toxins, of which two classes of peptide toxins, site-3 sodium channel toxins [4,12,27,34] and Kv1 potassium channel toxins [8,12], have been particularly well characterized. So far, more than 50 site3 sodium channel toxins and nearly 10 Kv1 potassium channel toxins have been isolated from various species of sea anemones. Based on the determined amino acid sequences, the site-3 sodium channel toxins and Kv1 potassium channel toxins are further divided into three and two types, respectively [12]. Some of these peptide toxins have been used as valuable pharmacological reagents in studying the structure and function of ion channels because of their high affinity to the specific channel.
Besides the site-3 sodium channel toxins and Kv1 potassium channel toxins, structurally and/or functionally novel peptide toxins have recently emerged in some species of sea anemones. Among these toxins are BDS-I and II from Anemonia sulcata [5], APETx1 [6] and APETx2 [7] from Anthopleura elegantissima, BcIV from Bunodosoma caissarum [28], Am II from Antheopsis maculata [15], gigantoxin I from Stichodactyla gigantea [33] and acrorhagin I and II from Actinia equina [16]. Although six toxins (BDS-I and II, APETx1 and 2, BcIV and Am II) structurally constitute a new family of sea anemone peptide toxins, five of them have already been shown to target different ion channels; BDS-I and II act on Kv3 channels [5,36], APETx1 on human ether-a-go-go-related gene potassium channels [6,29,37], APETx2 on acid-sensitive ion channels [7] and BcIV on sodium channel toxins [28]. On the other hand,
* Corresponding author. Tel.: +81 3 5463 0601; fax: +81 3 5463 0601. E-mail address: [email protected] (K. Shiomi). 0196-9781/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2007.12.010
peptides 29 (2008) 536–544
gigantoxin I is quite unique in that it is the first epidermal growth factor (EGF)-like toxin from biological sources. It shares 31–33% sequence identities with mammalian EGFs and exhibits EGF-like activity although much more weakly than human EGF [33]. Acrorhagin I and II are also of particular interest in that they were isolated from specialized aggressive organs (acrorhagi) differing from other sea anemone peptide toxins derived from whole bodies, tentacles or secreted mucus and that they share little or no homologies with peptide toxins of natural origin [16]. It should be noted that BcIV [28], Am II [15] and gigantoxin I [33] are potently paralytic to crabs but substantially not lethal. Taking this into consideration, we surveyed peptide toxins in the sea anemone Stichodactyla haddoni, which is a relatively large species (up to 50 cm in diameter) inhabiting tropical and subtropical waters, by a careful observation of the symptoms induced in crabs by samples. As a result, four peptide toxins, SHTX I–III with crab-paralyzing activity and SHTX IV with crab lethality, were isolated and elucidated for their primary structures by protein sequencing and cDNA cloning. Although SHTX IV was a member of the type 2 sea anemone sodium channel toxin family, the other three toxins were found to be structurally and functionally novel; SHTX I and II were new toxins with potassium channel toxicity and SHTX III was a Kunitz-type protease inhibitor with potassium channel toxicity. We report here the isolation, primary structures (including precursor structures) and some biological activities of SHTX I–IV from S. haddoni.
2.
Materials and methods
2.1.
Sea anemone
A specimen of S. haddoni collected along the coast of Okinawa Prefecture was shipped frozen to our laboratory and stored at 20 8C until extraction. For cDNA cloning, a live specimen imported from the Philippines was purchased from a retail aquarium shop in Yokohama, Kanagawa Prefecture, and cut into small pieces, which were immediately frozen in liquid nitrogen and stored at 80 8C until use.
2.2.
Isolation method
The frozen specimen (47 g) was thawed and well macerated in a motor. A 5 g-portion of the macerate was homogenized in 25 ml of distilled water and centrifuged at 18,800 g for 15 min. The supernatant (crude extract) was applied to gel filtration on a Sephadex G-50 column (2.5 cm 90 cm; GE Healthcare Bio-Sciences, Piscataway, NJ), which was eluted with 150 mM NaCl in 10 mM phosphate buffer (pH 7.0). Fractions of 8 ml were collected and measured for absorbance at 280 nm and crab toxicity. Toxic fractions were combined and then subjected to reverse-phase HPLC using a TSKgel ODS-120T column (0.46 cm 25 cm; Tosoh, Tokyo, Japan). After being washed with 0.1% trifluoroacetic acid (TFA), the column was eluted with two steps of linear gradients of acetonitrile (0–14% in 5 min and 14–42% in 80 min) in 0.1% TFA at a flow rate of 1 ml/min. Peptides were monitored at 220 nm with a UV detector. The eluate corresponding to each peak was
537
manually collected and assayed for crab toxicity. Thus, four peptide toxins (SHTX I–IV) were isolated.
2.3.
Chemical analysis
Peptides were determined according to the method of Lowry et al. [22] using bovine serum albumin as a standard. Molecular weight determination was performed by matrix assisted laser desorption ionization/time of flight mass spectrometry (MALDI/TOFMS) in the positive ion mode, using a Shimadzu/Kratos Kompact MALDI I instrument (Shimadzu, Kyoto, Japan). Sinapinic acid was used as a matrix. For the calibration of the instrument, insulin and sinapinic acid were used. Amino acid sequencing based on the Edman degradation method was carried out using an automatic gas-phase protein sequencer (LF-3400D TriCart with high sensitivity chemistry; Beckman Coulter, Fullerton, CA).
2.4.
Tryptic digestion and isolation of peptide fragments
SHTX I (40 mg) was digested with 10 mg of trypsin (Worthington Biochemical Corp., Lakewood, NJ) in 300 ml of 50 mM ammonium hydrogen carbonate at 37 8C for 24 h. Isolation of peptide fragments from the digest was achieved by reverse-phase HPLC on a TSKgel ODS-120T column, which was eluted with a linear gradient of acetonitrile (0–35% in 80 min) in 0.1% TFA.
2.5.
cDNA cloning
Cloning of the cDNA encoding either SHTX III or IV was performed by 30 and 50 rapid amplification of cDNA ends (RACE) as described below. Designations and nucleotide sequences of the primers used are shown in Table 1. The degenerate primers used for 30 RACE were designed based on the partial amino acid sequences determined by protein sequencing and the gene-specific primers used for 50 RACE based on the partial nucleotide sequences determined by 30 RACE. Total RNA was extracted from 1 g of the frozen sample with TRIzol reagent (Invitrogen, Carlsbad, CA). For 30 RACE, firststranded cDNA was synthesized from 5 mg of total RNA using a 30 RACE System for Rapid Amplification of cDNA Ends Kit (Invitrogen) and the oligo dT-adapter primer. The first 30 RACE reaction was performed using a degenerate primer (III-30 -1 for SHTX III or IV-30 -1 for SHTX IV) and the abridged universal amplification primer (AUAP) and the second 30 RACE reaction using a degenerate primer (III-30 -2 for SHTX III or IV-30 -2 for SHTX IV) and AUAP. Polymerase chain reaction (PCR) was carried out using Ex Taq polymerase (Takara, Kyoto, Japan) under the following conditions: 94 8C for 5 min; 35 cycles of 94 8C for 0.5 min; 55 8C for 0.5 min and 72 8C for 1 min; 72 8C for 5 min. The secondary PCR products were subcloned into the pT7Blue T-vector (Novagen, Darmstadt, Germany) and nucleotide sequences were determined using a Cy5 Thermo Sequenase Dye Terminator Kit (GE Healthcare Bio-Sciences) and a Long-Read Tower DNA sequencer (GE Healthcare BioSciences). On the basis of the determined partial nucleotide sequences, the remaining 50 -terminal sequences were analyzed by 50 RACE as follows. First-stranded cDNA was synthesized from 5 mg of total RNA using a 50 RACE System for Rapid
538
peptides 29 (2008) 536–544
Table 1 – Designations and nucleotide sequences of the primers used for cDNA cloning of SHTX III and IV Designation of primer III-30 -l III-30 -2 III-50 -cDNA III-50 -l III-50 -2 III-RT-f III-RT-r IV-30 -1 IV-30 -2 IV-50 -cDNA IV-50 -1 IV-50 -2 IV-RT-f IV-RT-r AUAP AAP a
Corresponding amino acid sequencea
Nucleotide sequence of primer 50 -CIGARGARATGCCIGCIYTITGYC-30 50 -GA YGTICCIAARTG YMGIGGITA YTT-30 50 -GTTTCCTCC AC ATCCTCCG-30 50 -CTCCGTAGATAAACTGCTC AC-30 50 -GTAATACCTTGGGAAGTACCC-30 50 -CTT CAAAACAGACTCCCAAGA-30 50 -C ATTCC ATAACTGCTTTTATTTAT-30 50 -CIGCITGYAARTGYGAYGAYGAYG-30 50 -GGICCIGA YATHMGIWSIGCIACIYT-30 50 -TCCCTCGTTGC AGTTCC AG-30 50 -AGTAGC ACTGCGAATGTC AG-30 50 -GTC ATC ATCGC ACTTAC ACG-30 50 -AACTTATGCTTGATAAGAC AGTG-30 50 -GTAGTCTTATACCCGATCTTTC-30 50 -GGCC ACGCGTCGACTAGTAC-30 50 -GGCC ACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-30
1-TEEMPALCH-9 13-DVPKCRGYF-21 38-YGGCGGN-44 33-CEQFIYGG-40 19-GYFPRYY-25 None (50 -untranslated) None (30 -untranslated) 1-AACKCDDDG-9 9-GPDIRSATL-17 23-FWNCNEG-29 10-PDIRSATL-17 2-ACKCDDD-8 None (50 -untranslated) None (30 -untranslated)
Refer to Fig. 4 or Fig. 6 for amino acid sequences.
Amplification of cDNA Ends Kit (Invitrogen) and a gene-specific primer (III-50 -cDNA for SHTX III or IV-50 -cDNA for SHTX IV). The first 50 RACE reaction was completed using a gene-specific primer (III-50 -1 for SHTX III or IV-50 -1 for SHTX IV) and the abridged anchor primer (AAP), followed by reamplification of the PCR products using a gene-specific primer (III-50 -2 for SHTX III or IV-50 -2 for SHTX IV) and AUAP. Amplification conditions were the same as in 30 RACE experiments. The secondary PCR products were subcloned into the pT7Blue T-vector and sequenced. Finally, reverse transcriptase (RT)-PCR was carried out to confirm the nucleotide sequences determined by both 30 and 50 RACE, using the first-stranded cDNA for 30 RACE as a template and the following primer combinations: III-RT-f and -r for SHTX III and IV-RT-f and -r for SHTX IV. In a separate set of cloning experiments, amplification of cDNAs encoding EGF-like peptides such as gigantoxin I previously found in S. gigantea [33] was attempted by RT-PCR. The first-stranded cDNA synthesized for 30 RACE was used as a template. Gene-specific forward (50 -TGTGAGAAGCAAATTGACCAG-30 ) and reverse (50 -GCCTTTTATTTGAAATCCATGTT-30 ) primers were designed from the nucleotide sequences in the 50 - and 30 -untranslated regions of the gigantoxin I cDNA (DDBJ/ EMBL/GenBank accession no. AB110014), respectively.
2.6.
Assay of crab toxicity
Crab toxicity was assayed using freshwater crabs (Potamon dehaani) weighing about 5 g purchased from the Tokyo Central Wholesale Market. Sample solutions were injected into crabs at 10 ml/g of crab body weight at the junction between the body and the leg. The symptoms induced in crabs were carefully observed up to 2 h after injection. To calculate LD50 (for lethal activity) or ED50 (for paralytic activity) against crabs by the method of Litchfield and Wilcoxon [20], groups of five crabs were administered with various doses of toxin.
2.7.
Assay of antitryptic activity
Antitryptic activity of SHTX III was estimated using a microtiter plate with 96-wells, as described previously [25].
In brief, trypsin solution (containing 0.15 unit) was preincubated with SHTX III solution at 37 8C for 10 min and the residual enzymatic activity was determined using the synthetic substrate, benzoyl-D,L-4-arginine-p-nitroanilide (Funakoshi, Tokyo, Japan). Antitryptic activity was expressed in terms of inhibitory units (IU), where 1 IU was defined as the amount of SHTX III required to inhibit one unit of trypsin.
2.8.
Assay of potassium channel toxicity
Potassium channel toxicity of SHTX II and III was indirectly assayed by competitive inhibition of the binding of 125I-adendrotoxin to rat synaptosomal membranes, as reported previously [10,24]. For comparison, a-dendrotoxin (Sigma, St. Louis, CA) and bovine pancreatic trypsin inhibitor (Sigma) were also evaluated for potassium channel toxicity. Labeling of a-dendrotoxin with 125I was performed by the chloramine-T (N-chloro-p-toluenesulphonamide) method according to the instructions of GE Healthcare Bio-Sciences and the labeled toxin was purified by gel filtration on a Sephadex G-10 column (1.2 cm 2.5 cm; GE Healthcare Bio-Sciences). Synaptosomal membrane suspension (0.4 mg protein/ml) was prepared from rat brains (Funakoshi, Tokyo, Japan). For competitive binding experiments, 200 ml of the synaptosomal membrane suspension was incubated with 40 ml of sample solution and 10 ml of 337 pM 125I-a-dendrotoxin (66.6 TBq/mmol) at room temperature for 30 min. The membranes were then collected by centrifugation and the radioactivity bound to the membranes was measured on a COBRA II gamma counter (Packard, Meriden, CT). Non-specific binding (about 7%) was determined by replacing sample solution with 450 nM a-dendrotoxin and subtracted from each datum.
3.
Results
3.1.
Isolation of toxins
When serial twofold dilutions of the crude extract were tested for crab toxicity, lethality was detected up to 32-fold dilution.
peptides 29 (2008) 536–544
539
Fig. 1 – Isolation of SHTX I–IV from Stichodactyla haddoni. (A) Gel filtration. Sample, crude extract; column, Sephadex G-50 (2.5 cm T 90 cm); solvent, 0.15 M NaCl in 0.01 M phosphate buffer (pH 7.0); volume per fraction, 8 ml. Toxic fractions are indicated by a bar. (B) Reverse-phase HPLC. Sample, toxic fractions obtained by gel filtration; column, TSKgel ODS-120T (0.46 cm T 25 cm); elution, linear gradient of acetonitrile in 0.1% TFA; flow rate, 1 ml/min. SHTX I–IV were eluted in labeled peaks.
In gel filtration of the crude extract on a Sephadex G-50 column, crab lethality appeared between fractions 42 and 58 (Fig. 1A). The toxic fraction obtained by gel filtration was then subjected to reverse-phase HPLC on a TSKgel ODS-120T column. Crab lethality was found only in a peak at a retention time of 67 min and crab paralysis in three peaks at 42, 43 and 54 min (Fig. 1B). Thus, four toxins (named SHTX I, II, III and IV in the order of elution) were successfully isolated. The purity of the four toxins was supported by protein sequencing, in which they gave only one amino acid in each cycle. The yields from 5 g of the starting sample were as follows: 144 mg of SHTX I, 104 mg of SHTX II, 200 mg of SHTX III and 320 mg of SHTX IV. The LD50 against crabs of SHTX IV was estimated to be 93 mg/ kg. On the other hand, SHTX I, II and III induced paralysis in crabs with ED50 of 430, 430 and 183 mg/kg, respectively, but none of them exhibited lethality even at 1000 mg/kg.
3.2.
Amino acid sequences of SHTX I and II
In the first cycle of protein sequencing, no amino acid was detected for either SHTX I or II. However, the Edman degradation reaction proceeded in the subsequent cycles, suggesting that the N-terminal residue of both toxins has a free amino group but its side chain is modified. As shown in Fig. 2, the same amino acid sequence ranging from the second position (Ile-2) to the C-terminal position (Gln-28) was
elucidated for SHTX I and II, except for only one difference at position 6 (hydroxy Pro for SHTX I and Pro for SHTX II). A search by protein databases revealed that SHTX I and II are homologous only to Am I [15]. However, SHTX I and II show considerable sequence differences from Am I, except for the four Cys residues (Fig. 2). In the next step, we attempted to assign the location of two disulfide bridges in SHTX I by sequencing of peptide fragments produced upon tryptic digestion. Two tryptic peptides (peptides 1 and 2), isolated by reverse-phase HPLC, were sequenced as follows: (unknown, Gly)-(Ile, Gly)-(Ile, Cys)-(Gly, Val)-(Ala, Arg)-hydroxy Pro-Cys-Arg for peptide 1; (Cys, Asp)-(Tyr, Trp)-(His, Ser)-(Ser, Cys)-(Asp, Gly)-(Gly, Gln)(Lys, Gln) for peptide 2. Comparison of these sequencing results with the entire amino acid sequence demonstrated that the two disulfide bridges of SHTX I are formed between Cys-7 and -19 and between Cys-10 and -25. It is reasonable to infer that SHTX II and Am I have the same disulfide bridge pattern as SHTX I.
3.3.
Amino acid sequence of SHTX III and its precursor
Protein sequencing identified N-terminal 54 amino acid residues of SHTX III. To determine the remaining C-terminal sequence, we employed a cDNA cloning technique. As a result, a full-length cDNA (503 bp; deposited in the DDBJ/
Fig. 2 – Amino acid sequences of SHTX I, SHTX II and Am I. Am I was isolated from Antheopsis maculata [15]. Identical residues with SHTX I are boxed. Two disulfide bridges are indicated above the sequence of SHTX I. Unidentified N-terminal residues (for SHTX I and II) and hydroxy Pro (for position 6 of SHTX I and Am I) are denoted by ‘X’ and ‘O’, respectively.
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Fig. 3 – Nucleotide sequence of the cDNA encoding SHTX III. The deduced amino acid sequence is aligned below the nucleotide sequence. Nucleotide and amino acid numbers are shown at the right. Asterisks indicate a stop codon (TGA). A putative signal sequence is underlined. The determined nucleotide sequence has been deposited in the DDBJ/EMBL/ GenBank databases under the accession no. AB362569.
EMBL/GenBank databases under the accession no. AB362569) encoding SHTX III was successfully obtained by a combination of 30 and 50 RACE. The open reading frame (243 bp) codes for the precursor protein of 81 amino acid residues (Fig. 3). Analysis by the SignalP program [3] suggested that the region 1–19 is a signal peptide. The remaining region 20–81 was judged to represent the mature peptide of SHTX III from the following two criteria: (1) the region 20–73 completely matches the N-terminal 54 residues determined for SHTX III by protein sequencing and (2) the molecular weight (7034.0) calculated for the region 20–81 is close to that (7035.0) determined for SHTX III by MALDI/TOFMS. A database search proved that SHTX III (62 residues) is a new member of the Kunitz-type protease inhibitor family. For comparison, the amino acid sequence of SHTX III is aligned in Fig. 4 with those of some sea anemone Kunitz-type protease inhibitors (ShPI from Stichodactyla helianthus [2], AEPI-I from A. equina [17], AXPI-I from Anthopleura aff. xanthogrammica [23] and AsKC 1-3 from A. sulcata [31]) and bovine pancreatic trypsin inhibitor (representative Kunitz-type protease inhibitor). The sequence identity of SHTX III with the above protease inhibitors ranges from 37% (with bovine pancreatic trypsin inhibitor) to 61% (with AEPI-I). In accordance with the sequence similarity to Kunitz-type protease inhibitors, SHTX III was confirmed to inhibit trypsin with an antitryptic activity of 203 IU/mg.
3.4.
Amino acid sequence of SHTX IV and its precursor
Similar to the case of SHTX III, the amino acid sequence of SHTX IV was established by both protein sequencing and molecular cloning. Analysis by a protein sequencer elucidated the N-terminal sequence (up to the 44th residue) of SHTX IV. A full-length SHTX IV cDNA (581 bp; deposited in the DDBJ/EMBL/GenBank databases under the accession no. AB362570) cloned by 30 and 50 RACE contained an open reading frame (252 bp) coding for the precursor protein of 84 amino acid residues (Fig. 5). The N-terminal region up to the 19th residue was predicted to be a signal peptide. The succeeding region 20–35 is featured by ending with a pair of basic residues (Lys-Arg). Since this feature is observed for the propart in most precursor proteins of sea anemone peptide toxins [1,9,11,14–16,21,35], the region 20–35 was assumed to be a propart. The N-terminal sequence determined for SHTX IV was completely recognized in the region 36–79. On the basis of the molecular weight (5229.5) determined for SHTX IV by MALDI/TOFMS, the region 36–83 with a calculated molecular weight of 5226.8 was judged to be the mature peptide of SHTX IV. Interestingly, the C-terminal residue (Gly) of the precursor protein was not involved in the mature peptide. It is known that peptidylglycines undergo removal of glycine and subsequent amidation of the penultimate residue by sequential actions with peptidylglycine a-ami-
Fig. 4 – Amino acid sequence alignment of SHTX III with Kunitz-type protease inhibitors. Inhibitors: ShPI from Stichodactyla helianthus [2]; AEPI-I from Actinia equina [17]; AXPI-I from Anthopleura aff. xanthogrammica [23]; AsKC 1–3 (kalicludines 1–3) from Anemonia sulcata [31]; BPTI (bovine pancreatic trypsin inhibitor) from bovine pancreas. Identical residues with SHTX III are boxed.
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541
Fig. 5 – Nucleotide sequence of the cDNA encoding SHTX IV. The deduced amino acid sequence is aligned below the nucleotide sequence. Nucleotide and amino acid numbers are shown at the right. Asterisks indicate a stop codon (TGA or TAA). A putative signal peptide and propart are singly and doubly underlined, respectively. The determined nucleotide sequence has been deposited in the DDBJ/EMBL/GenBank databases under the accession no. AB362570.
Fig. 6 – Amino acid sequence alignment of SHTX IV with type 2 sea anemone sodium channel toxins. Rp II was isolated from Radianthus (Heteractis) paumotensis [30]; RTX I from Radianthus (Heteractis) macrodactylus [38]; gigantoxin III from Stichodactyla gigantea [32]; Sh I from S. helianthus [19]. Identical residues with SHTX IV are boxed.
dating monooxygenase and peptidylamidoglycolate lyase [18]. Therefore, we can conclude that the Gly residue at the Cterminus of the precursor protein is removed after translation and that SHTX IV has an amidated residue (Lys-NH2) at the C-terminus. SHTX IV (48 residues) obviously belongs to the type 2 sea anemone sodium channel toxin family, as aligned in Fig. 6 with four type 2 sea anemone sodium channel toxins (Rp II from Radianthus (Heteractis) paumotensis [30], RTX I from Radianthus (Heteractis) macrodactylus [38], gigantoxin III from S. gigantea [33] and Sh I from S. helianthus [19]). Particularly, SHTX IV shows as high as 91% sequence identity with Rp II.
3.5.
270, 650 and 5.7 nM, respectively, indicating that SHTX II and III are about 50 and 110 times less potent than a-dendrotoxin. The binding between 125I-a-dendrotxin and the synaptosomal membranes was not affected by bovine pancreatic trypsin inhibitor even at 16,000 nM. Although not examined in this study, SHTX I can be considered to exhibit the same degree of potassium channel toxicity as SHTX II based on no substantial difference in both sequence and crab-paralyzing activity between the two toxins.
Potassium channel toxicity of SHTX II and III
SHTX IV is considered to act on sodium channels because of its high sequence identity with the type 2 sea anemone sodium channel toxins. On the other hand, SHTX III is expected to target potassium channels, similar to the type 2 sea anemone potassium channel toxins, AsKC 1–3 (kalicludines 1–3) that were isolated as potassium channel toxins from A. sulcata and confirmed to be Kunitz-type protease inhibitors [31]. SHTX I and II may also have potassium channel toxicity. In this study, therefore, two toxins, SHTX II and III, were evaluated for potassium channel toxicity by competitive binding assay using rat synaptosomal membranes. As depicted in Fig. 7, both SHTX II and III inhibited the binding of 125I-a-dendrotxin to synaptosomal membranes. IC50 of SHTX II, III and a-dendrotoxin were estimated to be
Fig. 7 – Inhibition of the binding of 125I-a-dendrotoxin to rat synaptosomal membranes by SHTX II (open circle), SHTX III (open triangle) and a-dendrotoxin (closed circle).
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Fig. 8 – Nucleotide sequence of the cDNA encoding the EGF-like peptide (SHTX IV). The deduced amino acid sequence is aligned below the nucleotide sequence. Nucleotide and amino acid numbers are shown at the right. Asterisks indicate a stop codon (TAA). A putative signal peptide and propart are singly and doubly underlined, respectively. Different nucleotides from the gigantoxin I cDNA (DDBJ/EMBL/GenBank accession no. AB110014) are shaded. Amino acid residues of gigantoxin I differing from SHTX V are indicated below the deduced amino acid sequence. The determined nucleotide sequence has been deposited in the DDBJ/EMBL/GenBank databases under the accession no. AB362571.
3.6.
Detection of an EGF-like peptide by RT-PCR
In this study, any EGF-like peptides were not isolated from S. haddoni. However, a cDNA (581 bp; deposited in the DDBJ/ EMBL/GenBank databases under the accession no. AB362571) encoding an EGF-like peptide (named SHTX V) was amplified by RT-PCR using the primers designed from the nucleotide sequence of the gigantoxin I cDNA. The precursor protein of SHTX V (86 residues) was assumed to comprise a signal peptide (23 residues), propart (15 residues) and mature peptide (48 residues), as in the case of the gigantoxin I precursor [33] (Fig. 8). As compared to the nucleotide sequence of the gigantoxin I cDNA, the SHTX V cDNA has 16 alterations, 14 of which are recognized in the mature peptide region. In reflection of this, SHTX V has the same amino acid sequence as gigantoxin I in both signal peptide and propart regions but differs from gigantoxin I at nine positions in the mature region.
4.
Discussion
Four peptide toxins (SHTX I–IV) with crab toxicity were isolated from S. haddoni by gel filtration and reverse-phase HPLC and their primary structures elucidated by protein sequencing and cDNA cloning techniques. Importantly, SHTX I–III were not lethal but only paralytic to crabs and found to be unique from both structural and functional points of view, while SHTX IV with crab lethality was a member of the wellknown type 2 sea anemone sodium channel toxin family. Previously, three novel peptide toxins, gigantoxin I [33], Am II [15] and BcIV [28], have been isolated as crab-paralyzing factors from S. gigantea, A. maculata and Bunodosoma caissarum, respectively. This study again confirmed that the crab assay, if only carefully performed, is very useful to discover novel peptide toxins in sea anemones. The most significant finding of this study is that SHTX I and II are structurally new peptide toxins displaying potassium channel toxicity, although their modified N-terminal residue remains to be clarified. Both toxins are relatively short
peptides and are cross-linked by two disulfide bridges (7–19 and 10–25). Sea anemone peptide toxins are mostly crosslinked by three disulfide bridges. Exceptionally, four disulfide bridges are known for members of the type 3 sea anemone sodium channel toxins, PaTX from Entacmaea actinostoloides [26] and its related peptide toxins (Da I and II from Dofleina armata [13] and Er I from Entacmaea ramsayi [13]), and even five disulfide bridges for two novel toxins (AETX II and III from Anemonia erythraea [32]). Of the known sea anemone peptide toxins, Am I from A. maculata has been the sole toxin that contains two disulfide bridges similar to SHTX I and II [15]. SHTX I, SHTX II and Am I have four Cys residues at the same positions, suggesting the same structural architecture for the three toxins. However, SHTX I and II share a considerably low sequence identity (32% identity) with Am I. This low sequence identity may account for the difference in toxicity; SHTX I and II are not lethal to crabs at 1000 mg/kg, while Am I shows crab lethality with an LD50 of 830 mg/kg [15]. Another significant finding is that SHTX III is a Kunitz-type protease inhibitor showing potassium channel toxicity as well as antitryptic activity. Of a number of Kunitz-type protease inhibitors so far isolated from sea anemones (e.g., ShPI, AEPI-I, AXPI-I and AsKC 1–3 shown in Fig. 4), only AsKC 1–3 from A. sulcata have been experimentally demonstrated to be potassium channel blockers [31]. In general, sea anemone protease inhibitors are assumed to function to inhibit endogenous proteases in animal themselves and/or to protect the peptide toxins injected into prey animals or potential predators from rapid destruction. However, our finding of a Kunitz-type protease inhibitor with potassium channel toxicity in S. haddoni may reinforce the previous hypothesis that sea anemone protease inhibitors have an aggressive function in that they contribute to the paralysis of prey animals [31]. Further survey for potassium channel toxicity of sea anemone protease inhibitors is needed to discuss their biological functions in more detail. Previous cloning studies have established the precursor structures of the following sea anemone peptide toxins: calitoxin 1 and 2 from Calliactis parasitica [35], Ae I (=AeNa) from A. equina [1], HK2a from Anthopleura sp. [21], gigantoxin I–
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III from S. gigantea [33], Am I–III from A. maculata [15], acrorhagin I and II from acrorhagi of A. equina [16], HmK from Heteractis magnifica [9] and AETX K from Anemonia erythraea [11]. It is generally considered from the accumulated knowledge that the precursors of sea anemone peptide toxins are commonly composed of a signal peptide, propart and mature peptide; two exceptions are the precursor of HK2a devoid of both signal peptide and propart and that of acrorhagin II devoid of propart. This study showed that the precursor structure of SHTX IV conform well to the above generalization, while the propart is missing in the precursor of SHTX III as in the case of acrorhagin II. Anderluh et al. [1] previously proposed that the propart with two basic residues at the end (cleavage site of subtilisin-like proteases) serves as a signal directing toxins into nematocysts, stinging organelles peculiar to cnidarians. According to this proposal, we tentatively assume that SHTX IV is contained in nematocysts but SHTX III is not. To verify the function of the propart, it is essential to examine whether sea anemone peptide toxins are localized in nematocysts. Finally, it should be noted that, next to gigantoxin I from S. gigantea [33], an EGF-like peptide (SHTX V) was detected in S. haddoni by RT-PCR, suggesting a wide distribution of EGF-like peptides in sea anemones. SHTX V was not isolated from S. haddoni in this study, probably because its concentration in the sea anemone is very low or because it has no toxic activity to crabs. However, our results will facilitate future study on sea anemone EGF-like peptides as well as other classes of sea anemone peptide toxins.
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