A novel proteinaceous cytotoxin from the northern Scyphozoa Cyanea capillata (L.) with structural homology to cubozoan haemolysins

Toxicon 57 (2011) 721–729

Contents lists available at ScienceDirect

Toxicon journal homepage: www.elsevier.com/locate/toxicon

A novel proteinaceous cytotoxin from the northern Scyphozoa Cyanea capillata (L.) with structural homology to cubozoan haemolysins Stephan Lassen*, Heike Helmholz, Christiane Ruhnau, Andreas Prange Helmholtz-Zentrum Geesthacht, Centre for Materials and Coastal Research, Institute of Coastal Research, Department for Marine Bioanalytical Chemistry, Max-Planck-St. 1, 21502 Geesthacht, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 December 2010 Received in revised form 1 February 2011 Accepted 8 February 2011 Available online 17 February 2011

It is well known that jellyfish are producers of complex mixtures of proteinaceous toxins for prey capture and defence. Nevertheless, studies on boreal scyphozoans concerning venom composition and toxic effects are rare. Here the isolation of a novel cytotoxic protein from the fishing tentacle venom of Cyanea capillata (L. 1758) using bioactivityguided, multidimensional liquid chromatography is described. The crude venom was purified utilising preparative size-exclusion, ion-exchange, and reversed-phase chromatography. The cytotoxicity of resulting chromatographic fractions has been proven by a dye-uptake assay with the human hepatocyte cell line HepG2. The final purification step yielded, among other fractions, a fraction containing a single protein (named CcTX-1) with a molecular weight of its main isoform of 31.17 kDa The purification process leads to an increased cytotoxic activity per protein equivalents and the finally isolated CcTX-1 caused a nearly total loss of cell viability at a protein concentration of 1.3 mg mL
1 corresponding to 0.4 mg/105 cells. De novo sequencing of CcTX-1 was conducted after enzymatic digestion and subsequent matrix-assisted laser desorption ionisation time-of-flight/time-of-flight mass spectrometry (MALDI-ToF/ToF MS/MS). The obtained sequence data provide an approximate 85% description of the amino acid sequence. This sequence information partially matched that of two known haemolytic proteins of two cubozoan species: CaTX-1 from Carybdea alata Reynaud, 1830 and CrTX-1 from Carybdea rastonii Haacke, 1886. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Jellyfish Cyanea Venom Toxin Mass spectrometry Protein sequencing

1. Introduction The lion’s mane jellyfish Cyanea capillata (Scyphozoa), widespread in northern temperate waters, is among the most abundant jellyfish species in the North and Baltic Sea (Gröndahl, 1988; Båmstedt et al., 1994; Holst and Jarms, 2010). It is well known to humans due to their painful stinging effects. An early report of C. capillata envenomation describes the pathophysiological effects of the venom on human skin (Kristenson, 1949). These painful effects are caused by venomous substances delivered by subcellular stinging organelles, the nematocysts, by which jellyfish

* Corresponding author. E-mail address: [email protected] (S. Lassen). 0041-0101/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2011.02.004

capture prey and defend themselves against predators (Heeger et al., 1992; Lotan et al., 1995; Fautin, 2009). Crude or partially purified C. capillata venoms, mainly derived from tentacle nematocysts, express various biological activities. Lytic, including haemolytic, cardiovascular, enzymatic, and neurotoxic properties have been demonstrated by in vivo and in vitro studies (Walker, 1977a,b; Walker et al., 1977; Burnett and Calton, 1987; Long and Burnett, 1989; Helmholz et al., 2007; Lassen et al., 2010; Xiao et al., 2010a, 2010b). According to Rice and Powell (1972) the venom in the nematocysts of C. capillata basically consists of proteinaceous compounds. A protein with approximately 70 kDa molecular weight was found to be associated with cardiotoxicity (Walker, 1977a,b) while a haemolytic activity is possibly attributed to smaller polypeptides (Long and Burnett, 1989). However,

722

S. Lassen et al. / Toxicon 57 (2011) 721–729

effective proteins from C. capillata venoms were neither further isolated, nor characterised or sequenced. Purification of cnidarian toxins turned out to be difficult due to their complexity, stated high thermal instability, and their tendency to aggregate or disaggregate (Endean et al., 1993; Gusmani et al., 1997; Anderluh and Macek, 2002; Turk and Kem, 2009). Burnett and Calton (1987) described an increasing thermolability of a biologically active material from C. capillata during the purification process. However, the isolation and characterisation of venomous polypeptides from box jellyfish (Cubozoa) and from Scyphozoa, e.g. the Atlantic sea nettle Chrysaora quinquecirrha (Desor, 1848) has been achieved in recent  years (Suput, 2009). Several bioactive proteins from various Cubomedusae, e.g. Carybdea marsupialis (L. 1758) (Rottini et al., 1995), C. rastoni (Nagai et al., 2000a) and C. alata (Nagai et al., 2000b; Chung et al., 2001) as well as Chironex fleckeri Southcott, 1956 (Brinkman and Burnell, 2007) have been purified and structurally characterised. A current study regarding the venom of the Scyphomedusa Cyanea nozakii Kishinouye, 1891, a related species to C. capillata, depicts the isolation and characterisation of two lethal proteins (50 and 60 kDa mol wt.) (Feng et al., 2010). Due to the complexity of crude jellyfish venoms, the minute quantities of active compounds and the multitude of toxic effects, the integration of specific in vitro assays during the purification process is a possible strategy to track compounds with a certain activity (Aneiros and Garateix, 2004; Brinkman and Burnell, 2008). We present a novel cytotoxic protein from the nematocyst-derived tentacle venom of C. capillata isolated by a cytotoxicity assay-guided multidimensional separation procedure. Analogous to the naming of certain similar cubozoan toxins the C. capillata cytotoxin was termed CcTX-1. The investigation of its primary structure by matrix-assisted laser desorption ionisation time-of-flight/ time-of-flight mass spectrometry (MALDI ToF/ToF MS) and subsequent sequence alignment with common cubozoan haemolysins led to the assumption that CcTX-1 may also possess lytic properties. This study contributes to the biochemical and toxicological characterisation of jellyfish and enhances the knowledge on isolated and sequenced toxins derived from northern Scyphozoa. 2. Materials and methods 2.1. Preparation of nematocysts Cyanea capillata medusae were collected at the Isle of Lewis, Western Isles, Scotland in 2005. Fishing tentacles of eight medusae with umbrella diameters larger than 30 cm (mean umbrella diameter 36 cm, range 30–42 cm) were sliced immediately after the animals were caught. The tentacles were pooled and further prepared for nematocysts extraction on board of the RV “Heincke” in a thermostated laboratory. The tentacle material was mixed with ice-cold distilled water (volume ratio tissue to liquid 1:5) and gently stirred for 10 h on ice. The resulting mixture was filtered through a nylon sieve (mesh size 500 mm) and the filtrate was centrifuged at 4  C and 1100g for 5 min. The supernatants were discarded and the residues were washed three

times with sterile filtered seawater. The nematocyst suspensions were immediately stored at
80  C. 2.2. Venom preparation, purification and protein isolation Thawed nematocyst suspensions were diluted ten-fold in ice-cold 10 mM ammonium acetate (Merck, Germany) buffer, pH 5.5. The nematocysts were discharged by sonication in a cooled, bath-type sonicator (Branson Sonifier 450, Heinemann Ultraschall-und Labortechnik, Germany) according to Radwan et al. (2000). A subsequent centrifugation step at 4  C and 14400g for 5 min yielded the crude tentacle venom. The venom was fractionated by sizeexclusion chromatography (SEC) using a High Load 16/60 Superdex 75 prep column (bed volume 120 mL, Amersham Biosciences, Germany) at 10  C connected to a fast protein liquid chromatography (FPLC) system (Äkta, Amersham Biosciences, Germany) with 10 mM ammonium acetate buffer, pH 5.5 at a flow rate of 1 mL min
1 (UV detection at 280 nm, injection volume 5 mL). The obtained fractions were immediately stored on ice. Corresponding venom fractions from several SEC runs were combined and their cytotoxic activity investigated (see Section 2.4). The fractions were concentrated by lyophilisation and stored at
80  C until further use. Corresponding lyophilisates of cytotoxic SEC fractions were redissolved in 50 mM ammonium acetate buffer (pH 4.8) and further purified by cation-exchange chromatography (CEX) using a High Prep 16/10 SP FF column (bed volume 20 mL; Amersham Biosciences, Germany) at 10  C connected to the FPLC system described above. The venom compounds were eluted using a linear ion strength gradient. The initial elution buffer concentration was 100% mobile phase A (A: 50 mM ammonium acetate buffer, pH 4.8) and the gradient increased to 100% mobile phase B (B: 1.0 M sodium chloride in 50 mM ammonium acetate buffer, pH 4.8) in 40 min. The flow rate was set to 4.0 mL min
1 and 1.0 mL of cytotoxic SEC fractions were injected into the FPLC system (UV detection at 280 nm). The corresponding CEX fractions were stored on ice, combined, and subsequently ultrafiltrated with Vivaspin 2 cartridges (MWCO 3 kDa; Vivascience, Germany) at 4  C and 12,000g. The concentrated and desalted fractions were directly used for the cell assay (Section 2.4) or stored at
80  C for further isolation. A final purification step of cytotoxic CEX fractions was carried out by reversed-phase (RP) liquid chromatography on a HPLC system (1100 series, Agilent, Germany) equipped with a cooled autosampler and fraction collector bothtemperature-controlled at 4  C. A volume of 75 mL of the active fractions was chromatographed using a Jupiter C4 (5 mm, 300 Å; 150  4.6 mm) analytical column (Phenomenex, Germany) at 30  C and a flow rate of 0.75 mL min
1. The applied gradient with solvents A and B described below (Section 2.5), had to be slightly adjusted with respect to the upscaling of the injection volume, column diameter, and flow rate. Chromatograms were recorded at 280 nm with a diode array detector (Agilent). Corresponding RP fractions were combined, concentrated in a vacuum centrifuge evaporator (Thermo, Germany), and stored at
80  C for subsequent cytotoxicity tests and mass spectrometric analysis.

S. Lassen et al. / Toxicon 57 (2011) 721–729

2.3. Determination of protein contents The protein content, as a reference value for the venom concentration in all investigated chromatographic fractions, was determined using Bradford’s Reagent (Sigma) in a 96-well microtiter plate assay with bovine serum albumin as standard protein (Bradford, 1976). The compound concentrations mentioned throughout the manuscript refer to protein equivalents. 2.4. Dye-uptake cytotoxicity assay A Neutral Red dye-uptake cell assay was used to detect the cytotoxicity of venom fractions. The human heptocarcinoma cell line HepG2 (CCS Cell Culture Servive, Germany) was cultivated in Roswell Park Memorial Institute (RPMI) 1640 cell culture medium (Invitrogen, Germany) supplemented with 10% foetal calf serum (PAA Laboratories, Germany) and penicillin/streptomycin 100 units/100 mg per mL medium (Sigma, Germany) at 37  C in a humidified 5% carbon dioxide (CO2) atmosphere. Cells from a continuous culture were seeded into 96-well microtiter plates (150 mL per well equivalent to 105 cells) and allowed to settle for 24 h. Subsequently, culture wells were inoculated with 150 mL of SEC, CEX, or RP fractions in dilution series of accordant lyophilised and desalted concentrates. For the detection of the cytotoxicity, the following controls were prepared: a cell positive control growing in RPMI medium only and a negative control without cells. Controls and dilution series of chromatographic fractions were tested in eight replicates. After an incubation time of 48 h in a final volume of 300 mL, the cells were washed with phosphate buffered saline (PBS; Invitrogen, Germany). Subsequently, 200 mL of a freshly prepared Neutral Red solution (0.02%) in RPMI medium without supplements was added. After incubation for 4 h, the cells were washed again with PBS to remove unspecifically adsorbed dye and lysed (lysis reagent: 1% glacial acid, 50% ethanol), The Neutral Red, released from vital cells, was measured in a microtiter plate reader (Spectra, Tecan, Switzerland) at 550 nm. The toxicity of the fraction was calculated in relation to the positive cell control set as 100% cell viability. For experimental details and calculation of relative toxicities refer to Helmholz et al. (2007). 2.5. Protein analysis by capLC–ESI–ToF mass spectrometry Concentrated CEX fractions were analysed utilising a capillary liquid chromatography (capLC) system (1100 series, Agilent, Germany) equipped with a Jupiter C4 (5 mm, 300 Å, 150  0.32 mm) capillary column (Phenomenex, Germany) at 30  C and a constant flow rate of 10 mL min
1. The proteins were eluted isocratically with 10% mobile phase B (B: acetonitrile (Merck, Germany) with 0.09% formic acid (Merck)/0.01% trifluoroacetic acid (TFA, Fluka, Germany)) for 5 min, followed by a linear gradient to 90% mobile phase B for 25 min and a second isocratic step for 10 min at 90% B (Mobile phase A: ultrapure water with 0.09% formic acid/0.01% TFA). 2 mL volumes of CEX fractions were injected into the capLC system which was connected to an API QStar Pulsar i hybrid quadrupole time-of-flight

723

mass spectrometer (QToF MS) fitted with an IonSpray source (Applied Biosystems, Germany) under positive ionisation conditions. External calibration was performed in the ToF mode with an electrospray tuning mix (Agilent, Germany) over a mass range of 300–2700 m/z. Quasimolecular masses [M þ H]þ of the separated proteins could be deduced by deconvolution of the resulting high resolution ToF MS data using the BioTools extension of the Analyst QS software (Applied Biosystems). 2.6. Protein structure elucidation by MALDI-ToF and ToF/ToF mass spectrometry Purified RP protein fractions (Section 2.2) were further concentrated to 0.2 mg mL
1 50 mL volumes of the RP concentrates were adjusted to pH 8 with 25% ammonium hydroxide (Merck, Germany) and diluted with 100 mM ammonium hydrogen carbonate (Merck) solution (1:1, v/v). After reduction of the proteins with 5 mL of 200 mM dithiothreitol (DTT, Sigma, Germany) at 65  C for 15 min, the solution was allowed to cool down. An alkylation step with 5 mL of 1 M iodoacetamide (IAA, Sigma) was carried out in the dark for 1 h at room temperature. Remaining IAA was neutralised by incubation with 20 mL of DTT solution for 45 min. The proteins in the resulting solutions were digested over night at 37  C with a trypsin/protein ratio of 1:20 (w/w). Trypsin (Sigma, proteomics grade) was prepared as 0.5 mg mL
1 in 1 mM HCl. The tryptic peptide mixture was then spotted on an 800 mm AnchorChipÔ target (Bruker, Germany) according to Rosenfeld et al. (2009) with a-cyano-4-hydroxycinamic acid (CHCA, Bruker) as matrix. MALDI-ToF MS and MALDI–ToF/ToF MS/ MS data were acquired using an Ultraflex II mass spectrometer (Bruker). The digests were first analysed in the Tof reflectron mode, followed by ToF/ToF analysis in the LIFT mode. Prior to MS and MS/MS experiments, the Ultraflex II was externally calibrated with a mixture of bradykinin (1–7), angiotensin I and II, substance P, bombesin, renin, ACTH (1–17), ACTH (18–39), somatostatin and oxidised insulin b-chain. The Ultraflex II was controlled by the FlexControl 3.0 software and the mass signals (m/z) were annotated using the FlexAnalysis 3.0 software (Bruker). Labelled ToF/ToF mass spectra were further processed with the BioTools 3.1 and the RapiDeNovo software packages (Bruker). Potential disulphide bonds were analysed according to Svoboda et al. (1995). 2.7. Data base homology search and sequence alignment De novo peptide sequences were deduced from the fragment spectra of appropriate precursor ions using RapiDeNovo (precursor mass tolerance adjustment: 50–100 ppm, fragment mass tolerance adjustment: 0.2–1.0 Da; variable modification: oxidation of methionine, fixed modification: carbamidomethylation of cysteine). The sequences were edited according to the rules published by Shevchenko et al. (2001). Homology searches were executed with the MS BLAST search engine (http://genetics.bwh.harvard.edu/msblast) and performed with the following settings: Program, blast2p; Database, Swiss-Prot; Matrix, PAM30MS; Expect, 100; other advanced options, nogap-hspmax100-sort_by_totalscore-span1. The

724

S. Lassen et al. / Toxicon 57 (2011) 721–729

obtained sequence data were further analysed utilising the multiple sequence alignment program ClustalW2 (Thompson et al., 1994; Larkin et al., 2007) that can be run online from the web server of the European Bioinformatics Institute (EBI; http://www.ebi.ac.uk/Tools/clustalw2). With this program the best matches for selected partial or complete sequences were computable. The sequences were lined up in order to make identities, similarities and differences apparent. The following settings were used: Matrix, GONNET 250; Gapopen, 10; No endgap, yes; Gapext; 0.2; Gapdist; 4. 3. Results 3.1. Venom purification guided by the cytotoxicity assay The crude tentacle venom expressed a concentrationdependent cytotoxicity in the dye-uptake HepG2 cell assay. A protein equivalent of 10 mg per well induced a 43% cell death. The crude venom was initially separated by SEC into eight consecutive fractions (Fig. 1, SE1–SE8). Corresponding fractions of ten chromatographic runs were combined and examined with regard to their cytotoxic activity. SE1 turned out to be the most active fraction. It exhibited a dosedependent cytotoxicity in a tested concentration range of 0.5–10 mg protein equivalent per well corresponding to 1.7– 33 mg mL
1. Compared to the crude venom, in SE1 an increased relative toxicity of 68% at 10 mg protein equivalent per well was observed. SE1 was further fractionated by CEX, yielding four fractions (Fig. 2, CE1–CE4). Fraction CE1, the most active CEX fraction, also showed a dose-dependent cytotoxic effect in a tested concentration range from 0.5 to 3 mg protein equivalent per well (1.7–10 mg mL
1) with a further increased relative toxicity of 83% at a concentration of 3 mg protein equivalent per well. CE1 was analysed by capLC–ESI–ToF mass spectrometry in order to get an overview on present proteins, their molecular masses (Section 3.3) and estimate suitable retention ranges for a further fractionation by RP-chromatography. Three proteins could be separated, representing three RP fractions (Fig. 3, RP1–RP3). In order to yield a sufficient amount of material for further analytical and toxicological investigations, the fractionation of CE1 was carried out on

Fig. 1. Typical size-exclusion chromatogram of the crude nematocystderived tentacle venom of Cyanea capillata medusae. Collected fractions SE1
SE8 are indicated and were investigated with the HepG2 cell assay.

Fig. 2. Typical cation-exchange chromatogram of the cytotoxic fraction SE1. Collected fractions CE1–CE4 are indicated and were investigated with the HepG2 cell assay.

a semipreparative LC system equipped with a cooled fraction collector. The performance and reproducibility of the chromatographic process allowed the combination of corresponding fractions of twelve consecutive LC runs. The purified protein in the RP1 fraction was tested regarding a preserved bioactivity in the HepG2 cell assay in a concentration range measuring 0.2–1 mg protein equivalent per well (0.7–3.3 mg mL
1). A maximum of 75% loss of cell viability has been detected at a concentration of 0.4 mg protein equivalent per well. During the chromatographic stages of the purification process (SEC, CEX, RP) a more than 25-fold increase of the relative toxicity per protein equivalent has been achieved (crude venom 43% relative toxicity per 10 mg protein equivalent, RP1 75% relative toxicity per 0.4 mg protein equivalent). 3.2. Structural toxin analysis by mass spectrometry The cytotoxic protein in the reversed-phase chromatography fraction 1 (RP1) was named CcTX-1. At first, the determination of its molecular weight was carried out. Deconvolution of the multi-charge electrospray mass spectrum obtained by capLC–ESI–ToF MS, which is shown in Fig. 4, revealed a quasi-molecular mass of 31173 Da.

Fig. 3. Total ion chromatogram of the cytotoxic CEX fraction CE1. Collected fractions RP1–RP3 are indicated and were investigated with the HepG2 cell assay.

S. Lassen et al. / Toxicon 57 (2011) 721–729

CcTX-1 is accompanied by two isoforms, I2 and I3, with the molecular masses 31287 Da and 31488 Da, respectively. The molecular masses of the proteins representing RP2 and RP3, were deduced from their mass spectra (data not shown) to be around 10.9 kDa and 11.7 kDa, respectively. In order to elucidate the primary structure of CcTX-1 by mass spectrometry, the protein was initially digested insolution with trypsin. The resulting peptide mixture was analysed by MALDI-ToF MS in the positive mode. The mass spectrum exhibited a characteristic peptide mass fingerprint (PMF) comprising more than 20 abundant tryptic peptides (Fig. 5) of which 15 could be analysed subsequently by MALDI-ToF/ToF MS/MS. Fragment spectra of adequate quality were recorded. As an example, the fragment spectrum of the protonated peptide ([M þ H]þ) 1812.748 m/z is shown in Fig. 6. The amino acid sequence could be derived as FGLCHYLGSMMDHGR from a complete b- and y-fragment ion series. De novo sequences of all fragmented tryptic peptides are summarised in Table 1. Corresponding MS/MS spectra are provided in the supplementary data. Arginine (R) or lysine (K) residues were present somewhere else in the sequences (refer to Table 1) because tryptic cleavages in most of the investigated peptides were not complete. Only four peptides, 1001.468 m/z, 1197.688 m/z, 1312.712 m/z and 1812.748 m/z exhibited no missed cleavages (mc ¼ 0). Sequence EKTLVGSGKDSTNFVYR of peptide 1900.983 m/z (mc ¼ 2) could be confirmed by two overlapping sequences, DSTNFVYR of peptide 1001.468 m/z (mc ¼ 0) and TLVGDSGKDSTNFVYR of peptide 1643.836 m/z (mc ¼ 1). The obtained de novo sequence data of 13 sequenced peptides (15 sequenced peptides minus the two overlapping sequences) comprised 242 amino acids (AA). These provide an approximate 85% description of the primary sequence of CcTX-1, which is theoretically composed of 280 amino acids (31172 Da/111.1254 Da ¼ 280 AA; 111.1254 ¼ average amino acid residue mass). 3.3. Data base homology search and sequence alignment

725

Fig. 5. Exemplary peptide mass fingerprint of a tryptic digest of CcTX-1. The peptides marked by an asterisk were further fragmented to elucidate their de novo sequences. (m/z: mass-to-charge ratio; a. u.: arbitary units).

3313.554 m/z, significantly matched that of a haemolytic protein, identified as CaTX-A (Q9GNN8) from the box jellyfish Carybdea alata. The MS BLAST result (provided in the Supplementary data) also contained a reference to another haemolytic protein, namely CrTX-A (Q9GV72) from Carybdea rastonii. There were hints that peptide 1912.881 m/z and peptide 3313.554 m/z as well, shared partial homology with the C. rastonii haemolysin, but the total score for the calculated two high scoring pairs was not sufficient for an identification. These two resulting cubozoan protein sequences formed the basis for a comparative analysis in terms of further identities and similarities with the acquired entire sequence data of CcTX-1. An improved sequence alignment was conducted with the program ClustalW2. The best matches were calculated and the alignment results are represented in Fig. 7. In summary, 39 AA are identical in all three sequences. Referred to the 242 AA of the CcTX-1 sequence the percentage of overall identity was 16.1%. The

A query of the 13 de novo peptide sequences was subjected to a MS BLAST search within the Swiss-Prot database. Two partial sequences of two peptides, 2271.164 m/z and

Fig. 4. Electrospray mass spectrum of the purified cytotoxic protein in fraction RP1. The molecular mass of the main isoform of CcTX-1 (31172 Da) was deduced from the exemplarily annotated multi-charge spectrum. (m/z: mass-to-charge ratio; cps: counts per second).

Fig. 6. Fragment spectrum of the protonated peptide m/z 1812.748. The sequence was annotated as FGLCHYLGSMMDHGR from a complete b- and y-fragment ion series. (m/z: mass-to-charge ratio; b: b-fragment ions; y: y-fragment ions; M*: oxidised methionine).

726

S. Lassen et al. / Toxicon 57 (2011) 721–729

Table 1 Parent masses and de novo sequences of the fragmented tryptic peptides of CcTX-1. Peptide Parent number mass [m/z]

Sequence

Mass error [Da]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

DSTNFVYR LLTEPLLNER DDPEPNGNLEGGV TLVGSGKDSTNFVYR FGLCHYLGSMMDHGR EKTLVGSGKDSTNFVYR SSAKNSGNGYGCVDVNAGR EYVMHSGGLYGDLTDKR KDGKSHWYDSMLKHRSPR EPLTGPGKYPSSFLLLLEHR KPPDKDRDRWDNVKGCER EECKFYWCWSGSEERAGGMGR SSPEKKNDMSKPGRMRFDNKKEPR AGCKVVDMWYSLSYWMDVVNKR WNDYSLCKDSYAGKHKPAGGPPTPRLKNR


0.001 0.004 0.146
0.003
0.014 0.006 0.024 0.037
0.053
0.017 0.059 0.030
0.281 0.191
0.111

1001.468 1197.688 1312.712 1643.836 1812.748 1900.983 1912.881 1940.955 2244.057 2254.207 2271.164 2582.095 2878.161 2979.363 3313.554

Mass errors express the mass accuracy between the measured parent mass and the resulting monoisotopic mass of the deduced sequence.

percentages of conserved and semi-conserved substitutions were 18.2% and 15.3%, respectively. If only CcTX-1 and CaTX-A were taken into account, the homology increased to 25.2% (26.9% similarity). Two regions of highly conserved and consecutive amino acid residues in the sequences of CrTX-A and CaTX-A are indicated as underlined regions in Fig. 7. These sequence segments are predicted as functional transmembrane-spanning region (TSR1), spanning amino acids 68 to 115 and 80 to 126, respectively and as transmembrane a-helix (TH) outside TSR1, spanning amino acids 205–223 and 212–239, respectively (Brinkman and Burnell, 2007). In both segments, the CcTX-1 sequence coincided with several amino acids and multiple homologue substitutions were observed as well. Compared to the two cubozoan sequences, its percentage of identical and functional related AA in TSR1, was 22.9% and 25.7%, respectively and in TH 20% and 40% (in case of CaTX-A)/ 28.6% and 42.9% (in case of CrTX-A), respectively. The identity in both regions was approximately 4–13% higher as the overall identity mentioned above. Additionally, a putative amphiphilic a-helix at the N-terminus of CaTX-A (Brinkman and Burnell, 2007), spanning amino acids 38 to 54 (underlined in Fig. 7), had a 23.5% identity and 17.6% similarity to the aligned sequence segment of CcTX-1. In contrast to four cysteine residues (Cys) in both cubozoan haemolysins, CcTX-1 contained seven Cys, of which Cys35 matched the Cys in TSR1 and Cys169 matched another Cys at position 333 of the CaTX-A sequence (Cys319 of CrTX-A). A third cysteine residue (Cys150) of CcTX-1 coincided with a Cys at position 282 of CaTX-A. 4. Discussion A toxic protein, isolated by a multidimensional purification process from the venom of C. capillata with proven cytotoxicological effects is structurally characterised and described for the first time. This toxin was denoted CcTX-1 according to the naming used for known cubozoan toxins. The cytotoxicity of crude nematocyst-derived tentacle

venom of C. capillata on human HepG2 cells was demonstrated by Helmholz et al. (2007). This Neutral Red dye-uptake cell-based assay was utilised for the bioassayguided purification of CcTX-1. The assay was suitable for the detection of cytotoxicity in a concentration range of 10 mg protein equivalent per well (33 mg mL
1) in case of partially purified venom fractions down to 200 ng per well (670 ng mL
1) in case of purified CcTX-1. The cytotoxicity, attributed to CcTX-1, could be maintained during the whole sample treatment, the venom preparation and throughout the isolation procedure. It is known that jellyfish toxins can be instable and/or thermolabile (Burnett and Calton, 1987; Anderluh and Macek, 2002). However, a thermolability of jellyfish toxins cannot be generalised. Kang et al. (2009) observed a relatively good stability at temperatures below 20  C for the venom of Nemopilema nomurai Kishinouye, 1922 and the crude venom of nematocysts from the Mediterranean jellyfish Pelagia noctiluca (Forskål, 1775) expressed a remarkable stability at increased temperatures (Marino et al., 2007). In order to minimise potential degradation of the toxins, the sample treatment and venom purification was carried out at low temperatures (between 0  C and 10  C) wherever applicable and resulting nematocyst suspensions and purified venom samples were stored at
80  C. Nevertheless, a partial decomposition of CcTX-1 possibly occurred, because the proteins representing RP2 and RP3 are supposed to be degradation products of CcTX-1. Their peptide mass fingerprints obtained by tryptic digestions and subsequent mass spectrometry were almost identical to the PMF exhibited by CcTX-1. Two accompanying isoforms (I2 and I3) of the purified CcTX-1 could be deduced from the mass spectometric data with minor variations of their corresponding molecular weights (CcTX-1: 31173 Da, I2: 31287 Da, I3: 31488 Da). The amino acid sequences of CcTX-1 and I2 differ only in Dm ¼ 114 Da, representing the amino acid asparagine (Asn). The mass difference of 201 Da between I2 and I3 probably indicates an elongation of the sequence of I3 with the dipeptide asparagine–serine (Asn–Ser). This result cannot be considered as a variation of the toxin within a single individuum of the species C. capillata due to the origin of the investigated pooled sample from different medusae. It can be assumed that this part belongs to an imprecisely sliced signal sequence or it can also be a hint for protein degradation by exoproteinases. An extensive set of de novo peptide sequences from CcTX-1 was achieved. A 85% description of its amino acid sequence could be provided, although its tryptic digestion was not complete. However, it could not be excluded, that deduced lysine residues somewhere in the peptide sequences were glutamine (Q) residues, because their almost identical masses (K ¼ 128.095 Da; Q ¼ 128.059 Da) were spectrometrically not resolvable by Tof MS. Based on these structural data, it could be excluded that the isolated isoforms of CcTX-1 are minicollagens that are known to occur in the molecular weight range 30–40 kDa. Minicollagens represent the major component of the nematocyst wall in Hydra and other Cnidaria. They are composed of collagen helices with up to 16 Gly-X-Y repeats flanked by polyproline stretches and terminal Cys-rich domains with a conserved pattern

S. Lassen et al. / Toxicon 57 (2011) 721–729

727

Fig. 7. Comparison of the detected amino acid sequence of the novel Cyanea capillata toxin CcTX-1 with two cubozoan protein haemolysins utilising ClustalW2 multiple amino acid sequence alignment: CrTX-A (Carybdea rastoni), CaTX-A (Carybdea alata), CcTX-1 (C. capillata). Identical amino acids are marked by an asterisk. Conserved and semi-conserved substitutions are denoted by colons and stops, respectively. Cysteine residues (C) are highlighted by grey boxes. Amino acids in black indicate additional identity between CaTX-A and CcTX-1. Dashes indicate gaps inserted for a better alignment. Grey lines below and numbers above the sequences of the cubozoan haemolysins mark the positions of putative transmembrane and amphiphilic helices (number in parentheses: position in the CaTX-A sequence). (38)–(54): amphiphilic a-helix; 68/(80)–115/(126): transmembrane-spanning region (TSR1); 205–223 and (212)–(239): transmembrane helices outside TSR1.

(CXXXCXXXCXXXCXXXCC) (Engel et al., 2001; Pokidysheva et al., 2004; Özbek et al., 2004). None of these structural features have been observed in the deduced de novo sequences. A special structural and stabilising feature of proteins are residues of the sulphurous-containing amino acid cystein providing the opportunity for inter and intramolecular disulphide bonds. In the primary structure of Cctx-1 seven cystein residues could be detected whereas four Cys are known to be present in CaTX-A and CrTX-A. Two of these seven Cys of CcTX-1 coincided with two conserved Cys in CaTX-A and CrTX-A. Disulphide bridges are often

conserved, because they are important for the development and preservation of the secondary structure of proteins. Due to the lack of information on the conformation and the number of disulphide bonds in jellyfish toxins it is difficult to discuss a general Cys containing sequence motif. Brinkman and Burnell (2007) investigated two major venom proteins from Chironex fleckeri under reducing and non-reducing conditions and suggested any disulphide bond formation to be intramolecular. We analysed CcTX-1 after reduction (data not shown) with mercaptoethanol by MALDI-ToF MS (Svoboda et al., 1995). Up to four possible intramolecular disulphide bridges were figured out due to

728

S. Lassen et al. / Toxicon 57 (2011) 721–729

the observed increase of the molecular weight by the addition reaction with mercaptoethanol. Theoretically, the determined seven Cys of CcTX-1 permit a number of at most three disulphide bonds. An additional Cys to complete the four intramolecular bridges may be hidden in the remaining 15% of the protein sequence. The aligned sequence data revealed a remarkable high degree of similarity between CcTX-1 and CaTX-A: 25.2% homology and an almost equal amount of conserved and semi-conserved substitutions of 26.9%. Another isolated and characterised Cubozoa toxin, the CqTX-A from Chiropsalmus quadrigatus Haeckel, 1880 showed only a 21.6% sequence homology to CaTX-A although these species are phylogenetically closely related (Carybdea alata family Carybdeidae, Chiropsalmus quadrigatus family Chirodropidae, both order cubomedusa) (Nagai et al., 2002). The high sequence homology between CcTX-1 and CaTx-A is also remarkable because the number of amino acids in the CcTX-1 sequence (w280) is compared to 442 AA in CqTX-A about 40% shorter. The overall similarity between CcTX-1 and the two cubozoan haemolysins, leads to the hypothesis that the cytotoxic CcTX-1 acts as lysin as well. The general cytotoxic activity was proven by an in vitro assay but this bioassay is not suited to clarify the mode of toxic action. The high degree of homology of CcTX-1 to a transmembrane-spanning region (TSR1) and a transmembrane a-helix (TH), which were predicted for CaTX-A and CrTX-A (Brinkman and Burnell, 2007), support the thesis that the isolated C. capillata toxin express a lytic, cell membrane destroying activity. These structural properties are discussed to be involved in a pore-forming mechanism and may responsible for the potent cytolytic activity of the two box jellyfish toxins. Partial sequence homology of CcTX-1 to a predicted N-terminal amphiphilic a-helix in CaTX-A additionally indicates a possible potent haemolytic activity of CcTX-1 as described for CaTX-A (Nagai et al., 2000b). Nterminal amphiphilic a-helices in cytolysins are discussed as structures that initiate the interaction with susceptible lipid membranes and are also involved in pore formation (Belmonte et al., 1994; Ma cek et al., 1994). In the crude venom of C. capillata lytic activity towards human, sheep, and rabbit erythrocytes, binding activity towards cell membrane lipids immobilised as liposomes, as well as phospholipase A activity, could be demonstrated (Helmholz et al., 2007; Helmholz, 2010). CcTX-1, isolated and characterised in this study according to its activity in the dye-uptake cell assay and revealed structural motifs, can be one of the active compounds in the crude venom responsible for lytic and/or other cell membrane related activities. 5. Conclusion The developed multidimensional isolation strategy in combination with highly sensitive mass spectrometry has led to the purification and characterisation of a new proteinaceous toxin from the C. capillata venom. Characteristic amino acid sequence data from CcTX-1 significantly match that of known haemolysins from two Cubozoa species. The present bioassay-guided proteomic study for a common northern Scyphozoa contributes to

the enlargement of the spectrum of known jellyfish toxins and could be a useful tool for interspecies comparisons within the Medusozoa. Acknowledgements The authors would like to thank Dr. C. Schuett and his colleagues from the Alfred-Wegener-Institute Helgoland and the crew of the RV “Heincke” for their substantial help and support and Dr. J. Gandrass (Institute of Coastal Research, Department for Marine Bioanalytical Chemistry) for critically reading the manuscript. Special gratitude appertain to our former colleague Dr. R. Pepelnik (Institute of Coastal Research, Department for Marine Bioanalytical Chemistry). Ethical statement The authors understood and adhered to the ethical guidelines for journal publication during preparation of the manuscript. Conflict of interest The authors declare that there are no conflicts of interest. Appendix. Supplementary data Supplementary data related to this article can be found online at doi:10.1016/j.toxicon.2011.02.004. References Anderluh, G., Macek, P., 2002. Cytolytic peptide and protein toxins from sea anemones (Anthozoa: Actiniaria). Toxicon 40, 111–124. Aneiros, A., Garateix, A., 2004. Bioactive peptides from marine sources: pharmacological properties and isolation procedures. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 803, 41–53. Båmstedt, U., Martinussen, M.B., Matsakis, S., 1994. Trophodynamics of the two scyphozoan jellyfishes, Aurelia aurita and Cyanea capillata, in western Norway. ICES J. Mar. Sci. 51, 369–382. Belmonte, G., Menestrina, G., Pederzolli, C., Kri zaj, I., Gubensek, F., Turk, T., Ma cek, P., 1994. Primary and secondary structure of a pore-forming toxin from the sea anemone, Actinia equina (L.), and its association with lipid vesicles. Biochim. Biophy. Acta 1192, 197–204. Bradford, M.M., 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brinkman, D., Burnell, J., 2007. Identification, cloning and sequencing of two major venom proteins from the box jellyfish, Chironex fleckeri. Toxicon 50, 850–860. Brinkman, D., Burnell, J., 2008. Partial purification of cytolytic venom proteins from the box jellyfish, Chironex fleckeri. Toxicon 51, 853–863. Burnett, J.W., Calton, G.J., 1987. Venomous pelagic coelenterates – chemistry, toxicology, immunology and treatment of their stings. Toxicon 25, 581–602. Chung, J.J., Ratnapala, L.A., Cooke, I.M., Yanagihara, A.A., 2001. Partial purification and characterization of a hemolysin (CAH1) from Hawaiian box jellyfish (Carybdea alata) venom. Toxicon 39, 981–990. Endean, R., Monks, S.A., Cameron, A.M., 1993. Toxins from the box jellyfish Chironex fleckeri. Toxicon 31, 397–410. Engel, U., Pertz, O., Fauser, C., Engel, J., David, C.N., Holstein, T.W., 2001. A switch in disulfide linkage during minicollagen assembly in Hydra nematocysts. EMBO J. 20, 3063–3073. Fautin, D.G., 2009. Structural diversity, systematics, and evolution of cnidae. Toxicon 54, 1054–1064. Feng, J.H., Yu, H.H., Li, C.P., Xing, R.G., Liu, S., Wang, L., Cai, S.B., Li, P.C., 2010. Isolation and characterization of lethal proteins in nematocyst

S. Lassen et al. / Toxicon 57 (2011) 721–729 venom of the jellyfish Cyanea nozakii Kishinouye. Toxicon 55, 118– 125. Gröndahl, F., 1988. A comparative ecological study on the scyphozoans Aurelia aurita, Cyanea capillata and C. lamarckii in the Gullmar Fjord, western Sweden, 1982 to 1986. Mar. Biol. 97, 541–550. Gusmani, L., Avian, M., Galil, B., Patriarca, P., Rottini, G., 1997. Biologically active polypeptides in the venom of the jellyfish Rhopilema nomadica. Toxicon 35, 637–648. Heeger, T., Møller, H., Mrowietz, U., 1992. Protection of human skin against jellyfish (Cyanea capillata) stings. Mar. Biol. 113, 669–678. Helmholz, H., 2010. Selective toxin-lipid membrane interactions of natural, haemolytic scyphozoan toxins analyzed by surface plasmon resonance. Biochim. Biophy. Acta Biomembr. 1798, 1944–1952. Helmholz, H., Ruhnau, C., Schütt, C., Prange, A., 2007. Comparative study on the cell toxicity and enzymatic activity of two northern scyphozoan species Cyanea capillata (L.) and Cyanea lamarckii (Peron & Leslieur). Toxicon 50, 53–64. Holst, S., Jarms, G., 2010. Effects of low salinity on settlement and strobilation of scyphozoa (Cnidaria): is the lion’s mane Cyanea capillata (L.) able to reproduce in the brackish Baltic Sea? Hydrobiologia 645, 53–68. Kang, C., Munawir, A., Cha, M.J., Sohn, E.T., Lee, H., Kim, J.S., Yoon, W.D., Lim, D., Kim, E., 2009. Cytotoxicity and hemolytic activity of jellyfish Nemopilema nomurai (Scyphozoa: Rhizostomeae) venom. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 150, 85–90. Kristenson, A., 1949. Some observations concerning the pathophysiological effects on the human skin caused by the stinging jellyfish (Cyanea capillata). Acta Physiol. Scand 18, 151–156. Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., Higgins, D.G., 2007. Clustal W and clustal X version 2.0. Bioinformatics 23, 2947–2948. Lassen, S., Helmholz, H., Ruhnau, C., Prange, A., 2010. Characterisation of neurotoxic polypeptides from Cyanea capillata medusae (Scyphozoa). Hydrobiologia 645, 213–221. Long, K.O., Burnett, J.W., 1989. Isolation, characterization, and comparison of hemolytic peptides in nematocyst venoms of two species of jellyfish (Chrysaora quinquecirrha and Cyanea capillata). Comp. Biochem. Physiol. 94B, 641–646. Lotan, A., Fishman, L., Loya, Y., Zlotkin, E., 1995. Delivery of a nematocyst toxin. Nature 375, 456. Ma cek, P., Belmonte, G., Pederzolli, C., Menestrina, G., 1994. Mechanism of a action of equinatoxin-II, a cytolysin from the sea anemone Actinia equina (L.) belonging to the family of actinoporins. Toxicology 87, 205–227. Marino, A., Crupi, R., Rizzo, G., Morabito, R., Musci, G., La Spada, G., 2007. The unusual toxicity and stability properties of crude venom from isolated nematocysts of Pelagia Noctiluca (Cnidaria, Scyphozoa). Cell. Mol. Biol. 53, Ol994–Ol1002. Nagai, H., Takuwa, K., Nakao, M., Ito, E., Miyake, M., Noda, M., Nakajima, T., 2000a. Novel proteinaceous toxins from the box jellyfish (sea wasp) Carybdea rastoni. Biochem. Biophys. Res. Commun. 275, 582–588. Nagai, H., Takuwa, K., Nakao, M., Sakamoto, B., Crow, G.L., Nakajima, T., 2000b. Isolation and characterization of a novel protein toxin from the Hawaiian box jellyfish (sea wasp) Carybdea alata. Biochem. Biophys. Res. Commun. 275, 589–594.

729

Nagai, H., Takuwa-Kuroda, K., Nakao, M., Oshiro, N., Iwanaga, S., Nakajima, T., 2002. A novel protein toxin from the deadly box jellyfish (sea wasp, habu-kurage) Chiropsalmus quadrigatus. Biosci. Biotechnol. Biochem. 66, 97–102. Özbek, S., Pokidysheva, E., Schwager, M., Schulthess, T., Tariq, N., Barth, D., Milbradt, A.G., Moroder, L., Engel, J., Holstein, T.W., 2004. The glycoprotein NOWA and minicollagens are part of a disulfide-linked polymer that forms the cnidarian nematocyst wall. J. Biol. Chem. 279, 52016–52023. Pokidysheva, E., Milbradt, A.G., Meier, S., Renner, C., Haussinger, D., Bachinger, H.P., Moroder, L., Grzesiek, S., Holstein, T.W., Özbek, S., Engel, J., 2004. The structure of the cys-rich terminal domain of hydra minicollagen, which is involved in disulfide networks of the nematocyst wall. J. Biol. Chem. 279, 30395–30401. Radwan, F.F.Y., Gershwin, L.A., Burnett, J.W., 2000. Toxinological studies on the nematocyst venom of Chrysaora achlyos. Toxicon 38, 1581–1591. Rice, N.E., Powell, W.A., 1972. Observations on three species of jellyfishes from Chesapeake Bay with special reference to their toxins. II. Cyanea capillata. Biol. Bull. 143, 617–622. Rosenfeld, H., Lassen, S., Prange, A., 2009. Characterization of haptoglobin in the blood plasma of harbor seals (Phoca vitulina). J. Proteome Res. 8, 2923–2932. Rottini, G., Gusmani, L., Parovel, E., Avian, M., Patriarca, P., 1995. Purification and properties of a cytolytic toxin in venom of the jellyfish Carybdea marsupialis. Toxicon 33, 315–326. Shevchenko, A., Sunyaev, S., Loboda, A., Shevehenko, A., Bork, P., Ens, W., Standing, K.G., 2001. Charting the proteomes of organisms with unsequenced genomes by MALDI-quadrupole time of flight mass spectrometry and BLAST homology searching. Anal. Chem. 73, 1917– 1926. Svoboda, M., Meister, W., Vetter, W., 1995. A method for counting disulfide bridges in small proteins by reduction with mercaptoethanol and electrospray mass-spectrometry. J. Mass Spectrom. 30, 1562–1566.  Suput, D., 2009. In vivo effects of cnidarian toxins and venoms. Toxicon 54, 1190–1200. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. Clustal-W – improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Turk, T., Kem, W.R., 2009. The phylum cnidaria and investigations of its toxins and venoms until 1990. Toxicon 54, 1031–1037. Walker, M.J.A., 1977a. Cardiac actions of a toxin-containing material from jellyfish, Cyanea capillata. Toxicon 15, 15–27. Walker, M.J.A., 1977b. Pharmacological and biochemical properties of a toxin containing material from jellyfish, Cyanea capillata. Toxicon 15, 3–14. Walker, M.J.A., Martinez, T.T., Godin, D.V., 1977. Investigations into cardiotoxicity of a toxin from nematocysts of jellyfish, Cyanea capillata. Toxicon 15, 339–346. Xiao, L., Liu, G.S., Wang, Q.Q., He, Q., Liu, S.H., Li, Y., Zhang, J., Zhang, L.M., 2010a. The lethality of tentacle-only extract from jellyfish Cyanea capillata is primarily attributed to cardiotoxicity in anaesthetized SD rats. Toxicon 55, 838–845. Xiao, L., Zhang, J., Wang, Q.Q., He, Q.A., Liu, S.H., Li, Y., Zhang, L.M., 2010b. In vitro and in vivo haemolytic studies of tentacle-only extract from jellyfish Cyanea capillata. Toxicol. Vitro 24, 1203–1207.