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Toxicon 48 (2006) 536–542

www.elsevier.com/locate/toxicon

Isolation and cDNA cloning of a potassium channel peptidetoxin from the sea anemone Anemonia erythraea

Yuichi Hasegawaa, Tomohiro Honmaa, Hiroshi Nagaib, Masami Ishidab,Yuji Nagashimaa, Kazuo Shiomia,�

aDepartment of Food Science and Technology, Tokyo University of Marine Science and Technology,

Konan-4, Minato-ku, Tokyo 108-8477, JapanbDepartment of Ocean Sciences, Tokyo University of Marine Science and Technology, Konan-4, Minato-ku, Tokyo 108-8477, Japan

Received 11 May 2006; received in revised form 26 June 2006; accepted 3 July 2006

Available online 7 July 2006

Abstract

A potassium channel peptide toxin (AETX K) was isolated from the sea anemone Anemonia erythraea by gel filtration

on Sephadex G-50, reverse-phase HPLC on TSKgel ODS-120T and anion-exchange HPLC on Mono Q. AETX K

inhibited the binding of 125I-a-dendrotoxin to rat synaptosomal membranes, although much less potently than

a-dendrotoxin. Based on the determined N-terminal amino acid sequence, the nucleotide sequence of the full-length cDNA

(609 bp) encoding AETX K was elucidated by a combination of degenerate RT-PCR, 30RACE and 50RACE. The

precursor protein of AETX K is composed of a signal peptide (22 residues), a propart (27 residues) ended with a pair of

basic residues (Lys–Arg) and a mature peptide (34 residues). AETX K is the sixth member of the type 1 potassium channel

toxins from sea anemones, showing especially high sequence identities with HmK from Heteractis magnifica and ShK from

Stichodactyla helianthus. It has six Cys residues at the same position as the known type 1 toxins. In addition, the dyad

comprising Lys and Tyr, which is considered to be essential for the binding of the known type 1 toxins to potassium

channels, is also conserved in AETX K.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Anemonia erythraea; CDNA cloning; Potassium channel peptide toxin; Sea anemone

1. Introduction

Sea anemones are a rich source of 3–5kDaneurotoxins acting on sodium channels. Since the firstdiscovery of three toxins in Anemonia sulcata (Beresset al., 1975), as many as about 50 sodium channelpeptide toxins have been isolated from various speciesof sea anemones and classified into three types based

e front matter r 2006 Elsevier Ltd. All rights reserved

xicon.2006.07.002

ng author. Tel.: +81 3 5463 0601;

0669.

ss: shiomi@s.kaiyodai.ac.jp (K. Shiomi).

on the determined amino acid sequences (Norton,1991; Honma and Shiomi, 2006). Irrespective of thetypes, they bind to the receptor site 3 of sodiumchannels, thereby delaying channel inactivation duringthe depolization procedure. Because of this uniquebiological activity, some of the known sea anemonesodium channel toxins, such as ATX II fromAnemonia sulcata (Wunderer et al., 1976) andanthopleurin A from Anthopleura xanthogrammica

(Tanaka et al., 1977), have been used as valuablepharmacological reagents in studying the structureand function of sodium channels.

.

ARTICLE IN PRESSY. Hasegawa et al. / Toxicon 48 (2006) 536–542 537

Besides the sodium channel peptide toxins,3.5–6.5 kDa neurotoxins acting on potassium chan-nels were also discovered in the middle 1990s. The11 potassium channel toxins so far isolated fromsome sea anemones are divided into three typesbased on the structural and functional features(Honma and Shiomi, 2006). Type 1 toxins (com-posed of 35–37 amino acid residues) include ShKfrom Stichodactyla helianthus (Castaneda et al.,1995), AsKS (kaliseptine) from Anemonia sulcata

(Schweitz et al., 1995), BgK from Bunodosoma

granulifera (Cotton et al., 1997), HmK fromHeteractis magnifica (Gendeh et al., 1997a) andAeK from Actinia equina (Minagawa et al., 1998)and block Kv1 (Shaker) potassium channels. Type 2toxins (composed of 58 or 59 amino acid residues)including AsKC 1–3 (kalicludines 1–3) are homo-logous to Kunitz-type protease inhibitors (e.g.bovine pancreatic trypsin inhibitor) and exhibitblocking of Kv1 potassium channels as well asprotease inhibitory activity (Schweitz et al., 1995).Three toxins (composed of 42 or 43 amino acidresidues), BDS-I and II from Anemonia sulcata

(Diochot et al., 1998) and APETx1 from Antho-

pleura elegantissima (Diochot et al., 2003), aremembers of type 3 potassium channel toxins.BDS-I and II are the first specific blockers ofKv3.4 potassium channels. APETx1 targets notKv3.4 channels but human ether-a-go-go-relatedgene potassium channels although it has 42%sequence identities with BDS-I and II.

In the course of our previous screening for toxinsin sea anemones, we found potassium channeltoxicity in the crude extracts from Actinia equina

and Anemonia erythraea (Shiomi et al., 1998). Apotassium channel peptide toxin (AeK) has alreadybeen isolated from Actinia equina and established tobe one of the type 1 potassium channel toxins(Minagawa et al., 1998). We report here the isolationof a type 1 potassium channel peptide toxin (namedAETX K) from Anemonia erythraea and its structur-al elucidation by cDNA cloning. A type 1 sodiumchannel toxin (AETX I) and two novel peptide toxins(AETX II and III) have previously been isolatedfrom this species (Shiomi et al., 1997).

2. Materials and methods

2.1. Sea anemone

Specimens of Anemonia erythraea used for toxinisolation were collected at Banda, Chiba Prefecture,

in May 2002 and stored at �20 1C until used. Aspecimen used for cDNA cloning was collected atChojagasaki, Kanagawa Prefecture, in June 2003,transported alive to our laboratory, frozen in liquidnitrogen and stored at �80 1C until used.

2.2. Isolation procedure

Frozen specimens (40 specimens, 220 g) werethawed and well macerated in a motor. A smallaliquot (usually 5 g) of the macerate was homo-genized in five volumes of distilled water andcentrifuged at 18 800g for 15min. The supernatantobtained was layered onto a Sephadex G-50 column(2.5� 90 cm; Amersham Biosciences, Buckingham-shire, UK), which was eluted with 0.15M NaCl in0.01M phosphate buffer (pH 7.0). Fractions of 8mlwere collected and measured for absorbance at280 nm and potassium channel toxicity. Toxicfractions were pooled and subjected to reverse-phase HPLC on a TSK gel ODS-120T column(0.46� 25 cm; Tosoh, Tokyo, Japan). The columnwas washed with 0.1% trifluoroacetic acid (TFA)and then eluted at a flow rate of 1ml/min with alinear gradient of acetonitrile (0–54.6% in 70min) in0.1% TFA. Peptides were monitored by absorbanceat 220 nm with a UV detector. The eluate containingAETX K was manually collected and lyophilized.The dried material was dissolved in 0.01M phos-phate buffer (pH 6.0) and subjected to anion-exchange HPLC on a Mono Q HR5/5 column(0.5� 5 cm; Amersham Biosciences). Elution of thecolumn was achieved by a linear gradient of NaCl(0.0–0.4M in 50min and 0.4–1.0M in 5min) in0.01M phosphate buffer (pH 6.0) at a flow rate of1ml/min, with monitoring at 220 nm. The eluatecorresponding to AETX K was collected and finallydesalted by reverse-phase HPLC on TSKgel ODS-120T, which was performed as described above.

2.3. Assay of potassium channel toxicity

Potassium channel toxicity was indirectly assayedby competitive inhibition of the binding of 125I-a-dendrotoxin, a potent potassium channel toxin fromthe green mamba Dendroaspis angusticeps, to ratsynaptosomal membranes, as reported previously(Harvey et al., 1989; Minagawa et al., 1998).Labeling of a-dendrotoxin (Sigma, St. Louis, CA,USA) with 125I was performed by the chloramine-T(N-chloro-p-toluenesulfonamide) method accordingto the instructions of Amersham Biosciences and

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125I-a-dendrotoxin (95Ci/mmol) was purified by gelfiltration on a Sephadex G-10 column (1.2� 2.5 cm;Amersham Biosciences). Synaptosomal membranesuspension (0.4mg protein/ml) was prepared fromrat brains (Funakoshi, Tokyo, Japan). For compe-titive binding experiments, 0.2ml of the synaptoso-mal membrane suspension was incubated with0.04ml of sample solution and 0.01ml of 606 pM125I-a-dendrotoxin at room temperature for 30min.The membranes were then collected by centrifuga-tion and the radioactivity bound to the membraneswas measured on a COBRA II gamma counter(Packard, Meriden, CT, USA). Non-specific bind-ing (about 20%) was determined by replacingsample solution with 1.42 mM a-dendrotoxin andsubtracted from each datum.

2.4. Analytical methods

Peptides were determined by the method ofLowry et al. (1951) using bovine serum albumin asa standard. Amino acid sequencing was performedwith an automatic gas-phase protein sequencer (LF-3400D TriCart with high sensitivity chemistry;Beckman Coulter, Fullerton, CA, USA). Molecularweight determination was carried out by matrixassisted laser desorption ionization/time of flightmass spectrometry (MALDI/TOFMS) with a Shi-madzu/Kratos Kompact MALDI I instrument(Shimadzu, Kyoto, Japan). Sinapinic acid was usedas a matrix.

2.5. Reverse transcriptase-polymerase chain reaction

(RT-PCR)

Total RNA was extracted from 1g of the frozensea anemone sample with the TRIzol reagent(Invitrogen, Carlsbad, CA, USA). First strandcDNA was synthesized from 5mg of total RNAusing the 30RACE System for Rapid Amplificationof cDNA Ends (Invitrogen) according to themanufacturer’s instructions and subjected to RT-PCR as a template. The following degenerate primerswere designed based on the determined N-terminalamino acid sequence of AETX K: a forward primer50-CITGYAARGAYTAYYTICCIAARWSIG-30

(corresponding to 1ACKDYLPKSE10) and a reverseprimer 50-GTICCRCAIGTYTTYTTRCARTTIGT-30 (corresponding to 25TNCKKTCGT33) for the firstRT-PCR and a forward primer 50-GARTGYACICARTTYMGITGYMGIAC-30 (corresponding to10ECTQFRCRT18) and the above reverse primer

for the second RT-PCR. Amplification was carriedout using Ex Taq polymerase (Takara, Otsu, Japan)under the following conditions: 94 1C for 5min; 35cycles of 94 1C for 30 s, 55 1C for 30 s and 72 1C for1min; and 72 1C for 5min. Amplified products by thesecond RT-PCR were subcloned into the pT7BlueT-vector (Novagen, Darmstadt, Germany) and se-quenced using a Thermo Sequence Cy5 Dye Termi-nator kit (Amersham Biosciences) and a Long-ReadTower DNA sequencer (Amersham Biosciences).

2.6. Rapid amplification of cDNA ends (RACE)

30RACE was performed using the 30RACESystem for Rapid Amplification of cDNA Ends(Invitrogen) as recommended by the manufacturer.The first strand cDNA used as a template was thesame as synthesized for RT-PCR. The followingprimers were used: the gene-specific primer50-ATTTCGTTGTAGGACGTCAAT-30 (corre-sponding to 13QFRCRTSM20) and the abridgeduniversal amplification primer (AUAP) for the firstPCR and the gene-specific primer 50-GAAATACAAATATACCAACTGTA-30 (corresponding to20MKYKYTNCK28) and AUAP for the secondPCR. Amplification conditions were the same asadopted for RT-PCR. The second PCR productswere subcloned into the pT7Blue T-vector andsequenced as described above. 50RACE was carriedout using the 50RACE System for Rapid Amplifica-tion of cDNA Ends (Invitrogen) following themanufacturer’s instructions. First strand cDNAwas synthesized from 5 mg of total RNA using thegene-specific primer 50-GTTCCACAAGTTTTTTTACA-30 (corresponding to 27CKKTCGT33). Thefirst 50RACE reaction using the gene-specific primer50-ACAGTTGGTATATTTGTATTTCA-30 (corre-sponding to 20MKYKYTNC27) and the abridgedanchor primer and the second 50RACE reactionusing the gene-specific primer 50-CATTGACGTCCTACAACGAAA-30 (corresponding to14FRCRTSM20) and AUAP were completed underthe same conditions as adopted for RT-PCR. Thesecond PCR products were subcloned into thepT7Blue T-vector and sequenced.

3. Results and discussion

3.1. Isolation of AETX K

When the crude extract was subjected to gelfiltration on Sephadex G-50, AETX K appeared

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0

20

40

60

80

100

0.01 0.1 1 10 100 1000 10000Concentration (nM)

Fig. 2. Inhibition of the binding of 125I-a-dendrotoxin to rat

synaptosomal membranes by AETX K (closed circle) and a-dendrotoxin (open circle). Bars indicate the standard deviation

(n ¼ 4).

Y. Hasegawa et al. / Toxicon 48 (2006) 536–542 539

between fractions 52 and 60 (Fig. 1A). In reverse-phase HPLC on TSKgel ODS-120T, AETX K waseluted in a symmetrical peak at a retention time of41min (Fig. 1B). Since protein sequencing revealedthe presence of some impurities in the peak, furtherpurification of AETX K was performed by anion-exchange HPLC on Mono Q HR5/5 (Fig. 1C). In atypical run, 29 mg of AETX K was obtained from 5 gof the starting material. As shown in Fig. 2, AETXK as well as a-dendrotoxin blocked the binding of125I-a-dendrotoxin to rat synaptosomal membranesin a dose-dependent manner. The IC50 (50%inhibitory concentration) values were estimated tobe 91 nM for AETX K and 0.43 nM for a-dendrotoxin, implying that AETX K is about 210times less potent than a-dendrotoxin. As discussedbelow in more detail, AETX K is a member of thetype 1 potassium channel toxins from sea anemones.Of the known type 1 potassium channel toxins,AsKS (Schweitz et al., 1995) and AeK (Minagawa etal., 1998), like AETX K, are considerably less toxicthan dendrotoxins, while ShK (Castaneda et al.,1995) and BgK (Cotton et al., 1997) are comparablein toxicity to dendrotoxins. The marked differencein toxicity among the type 1 potassium channeltoxins from sea anemones remains to be clarified atthe molecular level.

3.2. Nucleotide sequence of the cDNA encoding

AETX K

Analysis by a protein sequencer identified 33amino acid residues from the N-terminus of AETX

Fig. 1. Isolation of AETX K from Anemonia erythraea. (A) Gel filtra

solvent, 0.15M NaCl in 0.01M phosphate buffer (pH 7.0); volume per

phase HPLC of the toxic fractions obtained by gel filtration. Colum

acetonitrile in 0.1%TFA; flow rate, 1ml/min. AETX K was eluted in th

AETX K fraction obtained by reverse-phase HPLC. Column, Mono Q

phosphate buffer (pH 6.0); flow rate, 1ml/min. AETX K was eluted in

K. Judging from the difference between themolecular weight (3883.0) calculated from thedetermined N-terminal amino acid sequence andthat (3999.3) of AETX K estimated by MALDI/TOFMS, it is apparent that additional one residue ispresent at the C-terminus of AETX K. Since AETXK is a type 1 potassium channel toxin, its C-terminalresidue is easily expected to be Cys.

To elucidate the complete amino acid sequence ofAETX K, a cDNA cloning technique was adopted

tion of the crude extract. Column, Sephadex G-50 (2.5� 90 cm);

fraction, 8ml. Toxic fractions are indicated by a bar. (B) Reverse-

n, TSKgel ODS-120T (0.46� 25 cm); elution, linear gradient of

e peak indicated by an arrow. (C) Anion-exchange HPLC of the

HR5/5 (0.5� 5 cm); elution, linear gradient of NaCl in 0.01M

the peak indicated by an arrow.

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in this study since it provides information onstructures of precursor proteins as well as those ofmature proteins. A nucleotide fragment (71 bp) wassuccessfully amplified by the second RT-PCR.Based on its nucleotide sequence, the remainingsequence was analyzed by both 30RACE and50RACE. Thus, the nucleotide sequence of thefull-length cDNA (609 bp) encoding AETX K wasdetermined as shown in Fig. 3. A stop codon (TGA)is observed in the 50-untranslated region upstreamthe putative initiating methionine and a poly(A)signal (AATAAA) and a poly(A) tail in the30-untranslated region. An open reading frame iscomposed of 249 bp, which codes for a precursorprotein of 83 amino acid residues from the putativeinitiating methionine to the putative last cysteine.

3.3. Amino acid sequence of AETX K and its

precursor

The mature peptide of AETX K was judged tocorrespond to the segment 50–83 (composed of 34amino acid residues) of the precursor protein fromthe following criteria: (1) the N-terminal amino acid

Fig. 3. Nucleotide sequence of the cDNA encoding AETX K. The dedu

Nucleotide and amino acid numbers are shown at the right. In-fram

background. Putative signal peptide and propart portions are singly and

Asterisks denote a poly (A) signal. The determined nucleotide sequen

under the accession number AB259113.

sequence determined by protein sequencing iscompletely consistent with the segment 50–82 and(2) the molecular weight (3990.6) calculated fromthe sequence of the segment 50–83 is close to that(3999.3) estimated by MALDI/TOFMS. As ex-pected, the C-terminal residue, which was notidentified by protein sequencing, is Cys. Analysisby SignalP V3.0 (http://www.cbs.dtu.dk/services/SignalP) suggested that the N-terminal segment upto the 22nd residue of the precursor protein is asignal peptide. It is thus assumed that an insertionof 27 residues (segment 23–49) between the signalpeptide and the mature peptide should be a propart.

The same structural framework as the AETX Kprecursor composed of signal peptide, propart andmature peptide portions is known for the precursorof HmK from Heteractis magnifica (Gendeh et al.,1997b), the only sea anemone potassium channeltoxin previously cloned. Although limited in num-ber, the following sea anemone peptide toxins,which act on sodium channels or have not beenidentified for the target, have been elucidated fortheir precursor structures: calitoxin 1 and 2 fromCalliactis parasitica (Spagnuolo et al., 1994), AeNa

ced amino acid sequence is aligned below the nucleotide sequence.

e stop codons (TGA and TAA) are shown in white on a black

doubly underlined, respectively, and the mature peptide is boxed.

ce has been deposited in the DDBJ/EMBL/GenBank databases

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Fig. 4. Alignment of the amino acid sequence of AETX K with those of the known type 1 potassium channel toxins from sea anemones.

HmK was isolated from Heteractis magnifica (Gendeh et al., 1997a), ShK from Stichodactyla helianthus (Castaneda et al., 1995), AeK

from Actinia equina (Minagawa et al., 1998), AsKS from Anemonia sulcata (Schweitz et al., 1995) and BgK from Bunodosoma granulifera

(Cotton et al., 1997). The residues identical with AETX K are boxed. Asterisks represent the dyad (Lys–Tyr) that is crucial for the binding

to potassium channels.

Y. Hasegawa et al. / Toxicon 48 (2006) 536–542 541

( ¼ Ae I) from Actinia equina (Anderluh et al.,2000), Hk2a from Anthopleura sp. (Liu et al., 2003),gigantoxin I–III from Stichodactyla gigantea (Hon-ma et al., 2003), Am I–III from Antheopsis maculata

(Honma et al., 2005a) and acrorhagin I and II fromacrorhagi (aggressive organs) of Actinia equina

(Honma et al., 2005b). The precursor of Hk2a hasneither signal peptide nor propart and that ofacrorhagin II is devoid of propart. However, all theremaining precursors share the same framework asthat of AETX K, although as many as six repeats ofmature peptide are contained in the precursor ofAm I. Interestingly, the propart is ended with a pairof basic residues (Lys–Arg), a cleavage site forsubtilisin-like proteases, regardless of the precursorsincluding that of AETX K. It should be also notedthat the propart has been proposed to function as asignal directing a toxin to a maturing nematocyst(Anderluh et al., 2000). We thus tentatively assumethat AETX K is derived from nematocysts.

As shown in Fig. 4, AETX K is a new member ofthe type 1 potassium channel toxins from seaanemones; it shares 65% and 59% sequenceidentities with HmK and ShK, respectively, whilethe identities with AeK, AsKS and BgK are ratherlow (41–44%). AETX K has six Cys residues at thesame position as the known type 1 toxins. Thissuggests that the three disulfide bridges are locatedbetween 2Cys and 34Cys, 11Cys and 27Cys and 16Cysand 31Cys as demonstrated for HmK (Gendeh et al.,1997a), ShK (Pohl et al., 1995) and BgK (Cotton etal., 1997), although future experimental evidence isneeded. Experiments with ShK (Pennington et al.,1996) and BgK (Dauplais et al., 1997; Alessandri-Haber et al., 1999; Gilquin et al., 2002) haveconfirmed that three residues (20Ser, 22Lys and23Tyr in ShK and 23Ser, 25Lys and 26Tyr in BgK),especially the dyad comprising Lys and Tyr, arecrucial for the toxin binding to potassium channels.

These three residues are completely conserved inAETX K as well as in the other type 1 potassiumchannel toxins.

Further insight into the amino acid sequencesallows us to assume that the type 1 potassiumchannel toxins are divided into the following twosubtypes: subtype 1 toxins (AETX K, HmK andShK) having four amino acid residues between thesecond and third Cys residues (11Cys and 16Cys inAETX K) from the N-terminus and subtype 2toxins (AeK, AsKS and BgK) having eight residuesin the corresponding region. It is worth mentioningthat the distribution of subtype 1 and 2 toxins maybe related to the taxonomical position of seaanemones. Two subtype 1 toxins, HmK and ShK,are from members of the family Stichodactylidaeand all the subtype 2 toxins from those of the familyActiniidae. At present, AETX K, a subtype 1 toxinfrom the member (Anemonia erythraea) of thefamily Actiniidae, is the only exception.

In conclusion, we isolated a potassium channeltoxin (AETX K) from Anemonia erythraea andshowed that it is the sixth member of the type 1potassium channel toxins from sea anemones. Seaanemone sodium channel toxins have been thor-oughly characterized, whereas information on seaanemone potassium channel toxins is still limited.Since a wide distribution of potassium channeltoxins in sea anemones is suggested by our recentscreening (data not shown), structurally and/orfunctionally unique potassium channel toxins areexpected to emerge from sea anemones.

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