Proteomics of the neurotoxic fraction from the sea anemone Bunodosoma cangicum venom: Novel peptides belonging to new classes of toxins

Comparative Biochemistry and Physiology, Part D 3 (2008) 219–225

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Comparative Biochemistry and Physiology, Part D j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p d

Proteomics of the neurotoxic fraction from the sea anemone Bunodosoma cangicum venom: Novel peptides belonging to new classes of toxins André Junqueira Zaharenko a,⁎, Wilson Alves Ferreira Jr. a, Joacir Stolarz Oliveira a, Michael Richardson b, Daniel Carvalho Pimenta d, Katsuhiro Konno e, Fernanda C.V. Portaro c, José Carlos de Freitas a a

Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, Travessa 14, 321, CEP 05508-900 São Paulo-SP, Brazil Fundação Ezequiel Dias—FUNED, Rua Conde Pereira Carneiro 80, CEP 30510-010 Belo Horizonte-MG, Brazil Laboratório de Imunoquímica, Instituto Butantan, Av. Vital Brazil 1500, CEP 05503-900 São Paulo-SP, Brazil d Laboratório de Bioquímica e Biofísica, Instituto Butantan, Av. Vital Brazil 1500, CEP 05503-900 São Paulo-SP, Brazil e Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama-shi, Toyama 930-0194, Japan b c

A R T I C L E

I N F O

Article history: Received 10 March 2008 Received in revised form 18 April 2008 Accepted 19 April 2008 Available online 26 April 2008 Keywords: Bunodosoma cangicum Sea anemone Mass spectrometry Peptide mass fingerprint HPLC Neurotoxins Ion channels

A B S T R A C T In contrast to the many studies on the venoms of scorpions, spiders, snakes and cone snails, up to now there has been no report of the proteomic analysis of sea anemones venoms. In this work we report for the first time the peptide mass fingerprint and some novel peptides in the neurotoxic fraction (Fr III) of the sea anemone Bunodosoma cangicum venom. Fr III is neurotoxic to crabs and was purified by rp-HPLC in a C-18 column, yielding 41 fractions. By checking their molecular masses by ESI-Q-Tof and MALDI-Tof MS we found 81 components ranging from near 250 amu to approximately 6000 amu. Some of the peptidic molecules were partially sequenced through the automated Edman technique. Three of them are peptides with near 4500 amu belonging to the class of the BcIV, BDS-I, BDS-II, APETx1, APETx2 and Am-II toxins. Another three peptides represent a novel group of toxins (~ 3200 amu). A further three molecules (~∼ 4900 amu) belong to the group of type 1 sodium channel neurotoxins. When assayed over the crab leg nerve compound action potentials, one of the BcIV- and APETx-like peptides exhibits an action similar to the type 1 sodium channel toxins in this preparation, suggesting the same target in this assay. On the other hand one of the novel peptides, with 3176 amu, displayed an action similar to potassium channel blockage in this experiment. In summary, the proteomic analysis and mass fingerprint of fractions from sea anemone venoms through MS are valuable tools, allowing us to rapidly predict the occurrence of different groups of toxins and facilitating the search and characterization of novel molecules without the need of full characterization of individual components by broader assays and bioassay-guided purifications. It also shows that sea anemones employ dozens of components for prey capture and defense. © 2008 Elsevier Inc. All rights reserved.

1. Introduction Living exclusively in aquatic environments, cnidarians represent the first group of venomous animals. Among the 5 classes of the phylum Cnidaria, the class Anthozoa is represented by the sea anemones, sessile animals that employ a variety of proteic (peptides, proteins, enzymes and proteinase inhibitors) substances as natural weapons (Malpezzi et al., 1993; Schweitz et al., 1995; Grotendorst and Hessinger, 2000; Anderluh and Macek, 2002). These animals are characterized by the

Abbreviations: amu, atomic mass unit; ASIC, acid-sensing ion channel; CAPs, compound action potentials; CH3CN, acetonitrile; ESI-Q-TOF MS/MS, Electrospray ionisation quadrupole tandem mass spectrometry; HERG, human ether-a-gogo related gene potassium channel; Kv, voltage-gated potassium channel; LC, liquid chromatography; MALDI-Tof MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MS, mass spectrometry; Nav, voltage-gated sodium channel; rp-HPLC, reversed-phase high performance liquid chromatography; TFA, trifluoroacetic acid. ⁎ Corresponding author. Tel.: +55 11 30917522; fax: +55 11 3091 7568. E-mail address: [email protected] (A.J. Zaharenko). 1744-117X/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbd.2008.04.002

presence of stinging organelles, the nematocysts, employed for defense against predators, in competition for substrate and also in prey capture. Nematocysts possess a high concentration of polypeptides and proteins that act as neurotoxins, hemolysins and phospholipase A2 (PLA2) enzymes, and are responsible for a variety of pathological responses (cardiotoxicity, dermatitis, local itching, swelling, erythema, paralysis, pain and necrosis) (Norton, 1991; Anderluh and Macek, 2002; Nevalainen et al., 2004; Watters and Stommel, 2004). Despite the fact that sea anemones produce their toxins in the restricted and specialized nematocysts, it is interesting to note that most of the papers that describe the isolation and characterization of these molecules follow bioassay-guided purifications from total body extracts (Beress and Beress, 1975; Barhanin et al., 1981; Castañeda et al., 1995; Bruhn et al., 2001; Salceda et al., 2002). Recently, the novel sea anemone toxins APETx1 and APETx2, a “Human Ether-a-gogo Related Gene” (HERG) modulator and an “Acid-Sensing Ion Channel 3” (ASIC3) blocker, respectively, were reported by this approach (Diochot et al., 2003; Diochot et al., 2004).

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On the other hand, our group developed a technique to milk the sea anemone Bunodosoma caissarum by electric stimuli and to obtain a rich polypeptidic mixture from its nematocyst venom (Malpezzi et al., 1993). Further purifications of this venom by gel-filtration yielded some fractions with hemolytic and neurotoxic activities (Oliveira et al., 2004; de Oliveira et al., 2006; Oliveira et al., 2006). Applying the same technique to the sea anemone Bunodosoma cangicum, other work (Lagos et al., 2001) reported that the so-called fraction III (Fr III) eluted from Sephadex G50 gel-filtration chromatography contained neurotoxins that target voltage-gated sodium and potassium channels. This methodology ensures that the active components are coming directly from the electrically-discharged nematocysts and not from other animal body parts. In order to verify and partially characterize the composition of the toxins presented in the neurotoxic fraction of B. cangicum species, we performed two steps of fractionation by chromatography. The venom was initially submitted to molecular sieving by Sephadex G-50 and the neurotoxic fraction (Fr III) was further fractionated by reversed-phase high performance liquid chromatography (rp-HPLC). The peptide mass fingerprint of Fr III was carried out by ESI-Q-Tof and MALDI-Tof MS analyses offline. This proteomic approach, either through nanoLC-MS or LC-MS and MALDI-Tof offline has previously been done with many venomous organisms, such as cone snails (Vianna Braga et al., 2005; Jakubowski et al., 2006; Quinton et al., 2006), spiders (Machado et al., 2005; Guette et al., 2006; Wilson and Alewood, 2006), snakes (Nawarak et al., 2003; Li et al., 2004; Fox et al., 2006) and scorpions (Pimenta et al., 2003; Batista et al., 2004; Barona et al., 2006; Batista et al., 2006; Favreau et al., 2006; Nascimento et al., 2006), leading to the characterization of many novel components and also to the mass profile of their venoms. As the search for lead compounds and novel pharmacological tools are increasingly based on the naturally evolved venom molecules (Lewis and Garcia, 2003; Terlau and Olivera, 2004), we believe it is extremely important to survey a sea anemone venom by using the proteomic approach, combining the use of venom fractionation by rpHPLC with the employment of ESI and MALDI-Tof mass spectrometry. Our findings lead to the discovery of 9 novel peptides, including 6 novel ones from 2 new classes of toxins. Another 3 toxins are from the group of type 1 sodium channel toxins. Furthermore, our data shows that at least 81 molecules are eluted in the neurotoxic fraction of the sea anemone B. cangicum venom and may be employed as active peptides during stings. 2. Materials and methods 2.1. Venom collection and fractionation Twenty B. cangicum specimens were collected on the northern coast of São Paulo State, Brazil, and venom was obtained by electrical stimulation of animals as previously described (Malpezzi et al., 1993). The B. cangicum venom (approximately 70 mg) was fractionated by gel-filtration chromatography using a Sephadex G-50 column (1.9 × 131 cm, Amersham Biosciences, Uppsala, Sweden), according to Lagos et al. (2001) and Oliveira et al. (2004). The neurotoxic fraction (1 mg), eluted in the third peak (Fr III), was submitted to rp-HPLC chromatography in an ÄKTA Purifier machine (Amersham Biosciences, Uppsala, Sweden) using a semipreparative CAPCELL PAK C-18, 10 × 250 mm (Shiseido Corp., Kyoto, Japan) column. The HPLC conditions used were: 0.1% trifluoroacetic acid (TFA) in water (solvent A) and acetonitrile containing 0.1% TFA (solvent B). The separations were performed at a flow rate of 2.5 mL/min and a 10–60% gradient of solvent B over 40 min and the peptides were monitored at UV 214 nm. Each of the individual sub-fractions from Fr III were manually collected and lyophilized or concentrated for further molecular mass assessments by either ESI-Q-Tof or MALDI-Tof mass spectrometry. In the case of individual peak repurification, different gradients of

the solvents A and B described above and flow rate conditions were adjusted to best fit the purified peak to a proper symmetry. The protein content of either the neurotoxic fraction or the pure peptide samples was estimated by the bicinchoninic acid (BCA) method (Pierce, Rockford, IL, USA) following the manufacturer's instructions. 2.2. Mass spectrometry analyses Analyses of rp-HPLC fractions from the venom Fr III were performed on an Ettan MALDI-TOF/Pro (Amersham Biosciences, Uppsala, Sweden) equipped with 337 nm pulsed nitrogen laser under reflectron mode. The accelerating voltage was 20 kV. Matrix, α-cyano-4hydroxycinnamic acid (Sigma-Aldrich Co., USA), was prepared at a concentration of 10 mg/mL in 1:1 CH3CN/0.1% TFA. External calibration was performed with [Ile7]-angiotensin III (m/z 897.51, monoisotopic, Sigma) and human ACTH fragment 18–39 (m/z 2465.19, monoisotopic, Sigma). The sample solution (0.5 μL) dropped onto the MALDI sample plate was added to the matrix solution (0.5 μL) and allowed to dry at room temperature. The same samples were also analysed on a Q-TOF Ultima API instrument fitted with an electrospray ion source (Micromass, Manchester, UK) under positive ionization mode. The aqueous sample solutions (2 μL) were directly injected using a Rheodyne 7010 sample loop coupled to a LC-10A VP Shimadzu pump at 20 μL/min constant flow rate. External calibration was performed with NaI (Fluka) over m/z 100–2000. 2.3. Partial primary structure determination of B. cangicum Fr III peptides The partial amino acid sequences of some pure native peptides were determined by Automated Edman degradation through a gasphase sequencer PPSQ-10 (Shimadzu Corp., Kyoto, Japan). Cysteines' positions were considered based on blank cycles. 2.4. Crab nerve assay (sucrose-gap) The nerve preparation was obtained from the crab leg sensory nerve, as described previously (Lagos et al., 2001). A walking leg was isolated from an adult blue crab Callinectes danae and its nerve exposed by cutting the membranes and articulations of the leg as described by Malpezzi et al. (1993). The nerve was placed in a groove of an acrylic chamber across five interconnected compartments, each one isolated with vaseline plugs. The electrodes for stimulation (platinum–iridium) were connected to compartments 1 (positive) and 2 (negative), and the recording electrode (silver chloride) to compartments 3 and 5. Compartments 1, 2, 3 and 5 contained physiological solution for crabs, while compartment 4 contained 1 M of sucrose. The test substance was added to compartment 3, which contained 100 μL of physiological solution. Extracellular compound action potentials were evoked by single supra-maximal stimuli (18– 20 V) at 0.1 Hz and lasting 0.05 ms (Grass S8800 Stimulator, Grass Instruments, Warwick, USA). The action potentials of the nerve were amplified with a pre-amplifier (CP-511 AC, Grass Instruments, Warwick, USA), and cut-off frequency 3 to30 Hz. The data were recorded and saved on a PC hard disk using the WinWCP V 3.1.6 software (Whole Cell Analysis Program, version 3.1.6, Strathclyde Electrophysiology Software, John Dempster, University of Strathclyde, Scotland), controlling an Analogical/Digital board (National Instruments—NIDAQ, model Lab-PC+ 28). The sampling interval was 0.2 ms and the record size was 512 samples within each record. Action potentials measured before each treatment were used as controls. The effect of 1 μM of synthetic ShK peptide (Peptides International, Louisville, USA) was studied as a control on action potentials in order to verify the effect over voltage-gated potassium channels blockage in this preparation.

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3. Results and discussion 3.1. Venom fractionation and purification of neurotoxic fraction (Fr III) Taking into account that our method of venom collection provides a toxic mixture coming directly from the nematocysts (Malpezzi et al., 1993), the purification is facilitated and free of animal body contaminants that could interfere in our analyses. The total B. cangicum venom fractionated by the Sephadex G-50 gel-filtration chromatography yielded 5 fractions (data not shown), confirming the data obtained by a previous work (Lagos et al., 2001). The neurotoxic fraction (Fr III), the main object of our investigation, was pooled, lyophilized and further re-suspended in Milli-Q water. As most of the neurotoxic fractions from sea anemone venoms and extracts were previously reported to contain peptides in the mass range from 3000 to 5000 amu (Sunahara et al., 1987; Shiomi et al., 1997; Bruhn et al., 2001; Honma et al., 2003), the C-18 column was our first choice to purify components averaging these masses. In spite of the advantages of a direct infusion of complex mixture samples in an ESI-Q-Tof instrument for either an LC-MS or nano-LC-MS experiments, our preference was to directly purify the Fr III through rp-HPLC. Each individual sub-fractions were collected and further analyzed by offline mass spectrometry. This strategy was performed in order to avoid underestimations on the total number of components in the fraction that may happen in direct infusion of the whole fraction in an ESI-QTof instrument or during LC-MS acquisition. In the case of cone snails venom proteomic approaches, such a drawback was already reported (Quinton et al., 2006). Also, other authors demonstrated that experimentally determined chromatographic conditions in LC-MS experiments are essential in recovering all the molecules present in spider venom (Taggi et al., 2004). The chromatogram profile of the rp-HPLC purification of 1 mg of Fr III in a reversed-phase C-18 semi-preparative column is shown in Fig. 1. We observed a complex profile containing at least 41 main peaks. It is interesting that our HPLC purification yielded more peaks than reported in previous papers on the isolation and characterization of neurotoxins from extracts or mucous exudates of other sea anemone species (Shiomi et al., 1997; Shiomi et al., 2003; Cunha et al., 2005; Honma et al., 2005b). Possibly this increase in peak number might be due to the massive release of neurotoxins from the nematocysts during our venom extraction by electric shock. In addition, we suppose that the low levels of neurotoxins in the sea anemone extracts reported in the above mentioned papers could be

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Table 1 Mass fingerprinting of the neurotoxic fraction (Fr III) of the sea anemone B. cangicum venom by MALDI-TOF MS and ESI-Q-TOF MS offline Peak Retention number time

Molecular mass [average]

Peak Retention Molecular mass number time [average]

1 2

7.50 7.60–9.30

21a 22

23.41 23.84

3176.4b; 5001 6316.1; 6356.1

3 4 5 6 7 8

387.3; 405.3 280.1; 1437.4; 1566.4; 1835.7; 1950.8 1801.4; 2221.4 1841.1; 2192.4 1856.3

23 24a 25a 26 27 28

24.40 25.52 25.96 26.12 26.64 27.00

9

11.43 12.34 13.00 14.41–15.00 c 15.67 5416.8; 5439.3 16.00–17.00 1328.3; 1517.6; 1560.9; 1576.9 17.48 4931.1

29

27.47

10

17.74

4709.7; 4915; 4930.5 3948.7; 3957.8

30a

28.19

a

28.78

4577.1; 4595.8; 4647.7 5571.6; 6134

32a

29.21

33 34

29.67 29.85

35

30.24

3257.4 4158.4b; 4172.7 4972.4 4941.1; 4958 4470.4; 4486.1 2462.2; 4892.1; 4947.7 4415.4; 4481.9; 4798.4; 4925.3 4768.8; 4888.4; 4907b 4326; 4342.6b; 4669.1 4286; 4303.3b; 4399.3; 4626.7 4128.4; 4223 4499.1; 4779.6b; 4641.4 4762.8; 4781.3

36a 37a 38

30.57 31.16 31.46

4762.8; 4781.3b 4235.7; 4371.1b 4745.3; 4762.3

39 40 41

32.55 32.97 33.27

4245.1; 4594.1 4536.8 4715.2

11

18.05

12

18.44

13 14

18.73 19.43

15

20.20

16 17 a 18

20.64 21.00 21.55

19 a 20

21.75 22.10–23.10

c

1170.4; 4007.5; 4658.5 3933.5 3215.2 3684.7; 3813.4; 4662 3181 c

31

a

Peak containing a peptide that was partially sequenced by Edman degradation technique. Indicate the peptide which was sequenced by Edman degradation technique, in the referred peak. c Not determined. b

due to contamination of the samples with body (endogenous) proteins and peptides. It is reasonable that if a whole animal is macerated, most of its extract content will come from body structures, with the nematocysts content being only a small portion of it. Thus, our sea anemone milking procedure would have an extra advantage in the survey of neurotoxins. It is worth noting that the sea anemone B. caissarum Fr III yielded a very similar profile to B. cangicum Fr III (Oliveira et al., 2004)

Fig. 1. RP-HPLC purification of 1 mg of neurotoxic fraction (Fr III) was injected onto semipreparative CAPCELL PAK C18 (10 × 250 mm) column and eluted using a linear gradient from 10% to 60% of buffer B (CH3CN/H2O/0.1% TFA), under monitoring at 214 nm, and a flow rate of 2.5 mL/min. Each collected peak was sequentially numbered by Arabic numerals and had their molecular masses assessed (see Table 1). When necessary, pure components were obtained by further steps of repurification using the same column.

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during rp-HPLC, suggesting that both species express a similar pattern of toxins in their neurotoxic fractions. 3.2. Fr III mass fingerprinting and partial sequence determination of novel peptides Table 1 summarizes all the mass signals observed by offline measurements in ESI-Q-Tof and MALDI-Tof instruments. All the Na+ and K+ adducts, as well as oxidation signals, were omitted. Overall we observed that a total number of 81 different components were present in the 41 fractions of Fr III. In the first measured peak, at the retention time (RT) of 7.50 min, we observed the presence of a low molecular weight component possibly containing a bromine atom (405/407 amu doublets), as observed during the ESI-Q-Tof acquisitions (data not shown). This typical mass signal observation was easily detected by ESI-MS during the characterization of the post-translationally modified peptides of cone snails venom which contain 6-Br tryptophan (Jakubowski et al., 2006), because of the bromine isotopes doublets 79/81 which occur in 50.7% and 49.3%, respectively. Also, in the next fraction at RT: 7.60–9.30, a component of 280.1 amu is present. The functional role of these molecules in the venom and their structures remain to be determined. In checking the subsequent peaks, eluted up to the RT 16.00 min mostly only low molecular weight peptides were observed. The only exception is for the hydrophilic peptides at RT 15.67 min, which have 5416.8 and 5439.3 amu. Under the chromatographic conditions employed during the rp-HPLC, a buffer B (CH3CN/0.1% TFA) concentration of only around 25% was required to elute them from the column.

Generally, the eluted components of animal venoms show higher molecular masses as the acetonitrile concentration gradient increases. This was previously observed for example in the case of rp-HPLC of total spider venoms, which contain the very hydrophilic low molecular weight acylpolyamines and the more hydrophobic peptides (Kawai et al., 1991; Pimenta et al., 2005), and also in the case of rpHPLC (in C-18 columns) purification of scorpion venoms (Batista et al., 2004; Batista et al., 2006). In scorpion venoms, particularly, the K+ channel blockers (molecular masses around 3000 amu) are eluted before the Na+ channel modulators (molecular masses around 7000 amu). Very characteristic is the distribution of the peptides from the RT 25.00 min towards the end of the chromatogram. They are all in the size range from 4000 to 5000 amu and are clearly the most abundant components in this fraction. In order to verify the predominant type of molecules along this mass range, some peaks were chosen based on their intensity and ease of purification. They were manually collected and subjected to rechromatography. All the pure components were checked by MALDI-Tof to ensure their purity and molecular masses, and the native molecules were partially sequenced by the automated Edman degradation technique. Fig. 2 shows a compilation of the different components found by this approach and their respective classes of toxins. The molecules named in bold as Bcg (abbreviation of B. cangicum) letters indicate those that were obtained in this work and the following numbers represent their retention times based on the peaks from Fig. 1. Thus, as shown in Fig. 2, nine novel peptides were discovered in this study. In Fig. 2A there is an alignment of many previously reported type 1 sodium channel toxins. These peptides typically delay the inactivation

Fig. 2. Primary sequence alignment of different types of sea anemone toxins. The sequences identified by bold letters are the peptides sequenced in this work (Bcg, abbreviation of B. cangicum), followed by their retention times according to Fig. 1 and Table 1. Conservative cysteines are shown in bold C. (A) Alignment of the sodium channel toxins. BcIII is from Bunodosoma caissarum, BgII and BgIII from B. granulifera, ATX-I and II and BDS-I and II from Anemonia sulcata, AP-B and AP-C from Anthopleura xanthogrammica and AFT-II from A. fuscoviridis. (B) New class of the BcIV- and APETx-like peptides. BcIV is from B. caissarum, APETx1 and 2 are from Anthopleura elegantissima and Am-II from Antheopsis maculata. (C) The novel class of near 3000 amu peptides reported in this work. In all figures, the three dots in the end of the sequences mean that the full sequence determinations remain to be determined.

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process of the voltage-gated sodium channels (Nav), dramatically increasing the sodium currents and prolonging the axonal and muscular action potentials (Norton, 1991; Goudet et al., 2001; Oliveira et al., 2004; Hanck and Sheets, 2007). The component Bcg25.96 apparently is the toxin BcIII (accession number Q7M425) itself, as its molecular mass exactly matches the mass of the latter. In addition, Bcg25.96 is eluted in the same RT as BcIII, following the same repurification conditions. If Bcg25.96 is confirmed as BcIII, it would be the first occurrence of a same neurotoxin in two distinct sea anemone species. On the other hand, isolation of identical primary structure of sea anemone cytolysins was reported previously. Tenebrosin-C, from sea anemone Actinia tenebrosa, has exactly the same sequence as equinatoxin II from the species Actinia equina (Simpson et al., 1990; Belmonte et al., 1994). In this work, the mass fingerprint of the neurotoxic fraction shows that one more peptide is already known. This is the one eluted at RT 26.12 min (peak number 26, in Fig. 1) and having 4958 amu, corresponding to cangitoxin-II, previously isolated by our group (Zaharenko et al., in press). Cangitoxin-II differs from its isoform cangitoxin (Cunha et al., 2005) only by the N16D substitution. One other new toxin obtained in this work, Bcg28.19, is also very interesting, as it contains the substitution N16D which is observed in the peptide BgIII (from Bunodosoma granulifera) and in cangitoxins-II and III isolated previously from this species (Zaharenko et al., in press). The asparagine at position 16 was supposed to be very critical for this group of toxins, as the single N16D substitution makes BgIII nearly 100 times less potent than BgII, even though they differ only in this residue (see Fig. 2A) (Bosmans et al., 2002; Salceda et al., 2002). On the other hand, Zaharenko et al. (in press) demonstrated that the N16D substitution is not critical for the activity of cangitoxin-II over Nav 1.1. Consequently, we can surmise that Bcg28.19 is a good candidate to expand the characterization of type 1 toxins over sodium channels. In respect to Bcg30.24, another type 1 sodium channel toxin, an intriguing and interesting negatively charged patch is observed at the positions 36–38 (TSD). Normally in most of the peptides of this group (Fig. 2A), this region is composed by positively charged and polar amino acids. Oliveira et al. (2004) confirmed the critical role of the

Fig. 3. Compound action potential (CAP) recordings from crab leg nerve bioassay employing different B. cangicum peptides. C means control recording and number 1 means the toxin application effect in each experiment. (A) Effect of Bcg31.16 peptide (see Fig. 2B) at 50 nM concentration 1 min after application over the crab leg nerve. N = 5. (B) Effect of Bcg30.24 peptide (see Fig. 2A) at 40 nM concentration 1 min after application. N = 5. (C) Effect of Bcg23.41 peptide (see Fig. 2C) at 10 μM concentration 15 min after application. N = 4. (D) Effect of the synthetic ShK peptide (Peptides Intl., Louisville, USA) at 1 μM concentration 5 min after application. N = 5.

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KKH patch of the ATX-II peptide in discriminating and binding with high potency to human Nav 1.1–1.6. Rogers et al. (1996) had already proposed the importance of this surface in ATX-II for the overlapping binding site of sea anemone type 1 sodium channel toxins and the αscorpion toxins in Nav clones. In the case of Bcg30.24 it contains two polar and a negatively charged amino acid in the corresponding positions (TSD). Surprisingly, it is a highly active peptide disturbing the extracellular compound action potentials of crab leg nerve, as shown in Fig. 3B and discussed later on. On examination of the peptides described in Fig. 2B, a striking variety of sequences between the conserved cysteines is observed. It is very interesting that these molecules appear to be eluted in the last third of the chromatogram, representing the most hydrophobic components and averaging from 4000 to 4600 amu. Considering the high variability of the primary sequences, it may be also suggested that most of these peptides might target different types or subtypes of ion channels. The recently isolated toxins APETx1 (accession number P61541) and APETx2 (accession number P61542) target the human cardiac potassium channel HERG and the human acid-sensing ion channel ASIC3, respectively (Diochot et al., 2003, 2004). It is noteworthy that both peptides were isolated from extracts of Anthopleura elegantissima specimens only by bioassay-guided purifications. Therefore, the question arises as to whether these molecules may have some type of toxicity towards fishes or crustaceans, the most likely potential preys of sea anemones. This remains to be determined, as both were characterized mainly over their screened targets (the channels HERG and ASIC). However, in the case of the toxin Am-II (accession number P69930), it was initially characterized over crustaceans by in vivo assay, demonstrating paralyzing activity (Honma et al., 2005a). On the other hand, there is no information available yet about the potential targeting of mammalian transfected channels by Am-II. The other known toxins presented in Fig. 2B, BDS-I and BDS-II (accession numbers P11494 and P59084, respectively), were the first members of this family to be characterized, representing Kv3.4 channel blockers (Diochot et al., 1998). The last example of this family is the toxin BcIV (accession number P84919), which was isolated from B. caissarum and is also paralytic to crabs. BcIV, which is inactive over Na+ channels of mammalian GH3 cells, also displays some effect on the extracellular compound action potentials (CAPs) of the crab leg nerve, suggesting a specific effect on the sodium channels of crustaceans in a type 1 sodium channel toxin-like fashion (Oliveira et al., 2006). In this way, the peptides Bcg25.52, Bcg28.78, Bcg29.21 and Bcg31.16 are novel members of this recently discovered family of neurotoxins, but their exact molecular targets remain to be determined and must await further studies. The last one (Bcg31.16), with a molecular mass of 4371 amu, was assayed over the crab leg nerve extracellular CAPs and its effects are discussed below. Another group of toxins that attracted our attention is the group of peptides with masses of approximately 3000 amu. They were eluted with RTs between 21.00 and 23.00 min and apparently should be considered as novel peptides because they do not share any similarity with the sea anemone toxins reported so far having this molecular mass range, such as ATX-III (accession number P01535) from Anemonia sulcata, Am-I (accession number P69929) from Antheopsis maculata, Da-I, Da-II from Dofleinia armata and Er-I from Entacmea ramsayi (Beress et al., 1977; Honma et al., 2003; Honma et al., 2005a). Surprisingly, sequencing of the peptides Bcg21.00, Bcg21.75 and Bcg23.41 revealed that they belong to a totally new group of toxins (Fig. 2C). The three molecules have conservative amino acid residues but the cysteines' positions are different among them. From the number of C residues, it is suggested that Bcg21.00 and Bcg23.41 have 2 C–C bonds while Bcg21.75 has only one. We were able to determine only the full sequence of the peptide Bcg21.75 by direct automated Edman sequencing. Checking the experimentally determined sequence using the University of California in San Francisco/Protein Prospector homepage (http://prospector.ucsf.edu/) (Clauser et al.,

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1999) the theoretical MALDI-Tof molecular mass matches exactly the experimentally determined molecular mass (3181 amu). However the residues located close to the C-terminal of the peptides Bcg21.00 and Bcg23.41 did not yield clear results and some final positions remain to be determined. One of these peptides (Bcg23.41) was assayed in the extracellular crab leg nerve CAPs and its effects are discussed in the next section. 3.3. Biological activity of some toxins over the extracellular crab leg nerve compound action potentials (caps) In order to verify if some of the novel peptides could be acting in crustaceans ion channels, their activities over the extracellular crab leg nerve CAPs were evaluated. Fig. 3A shows the effect of the peptide Bcg31.16 and Fig. 3B the effect of the type 1 sodium channel toxin Bcg30.24. Both peptides rapidly diminished the amplitude of the CAPs and increased its duration, when administered at 50 nM and 40 nM, respectively. Curiously, even though it belongs to a different family of toxins, Bcg31.16 evoked the same effect as Bcg30.24 at a very low concentration. These effects are very similar to those observed with the peptides BcIV and BcIII in the same preparation (Oliveira et al., 2006). In conclusion, we may see that both Bcg31.16 (similar to BcIV) and Bcg30.24 (similar to BcIII) act in a similar fashion but with a higher potency. As BcIII acts over human Nav 1.1–1.6 and delays their inactivation processes (Oliveira et al., 2004) and Bcg31.16 and Bcg30.24 also evoke similar effects on the crab leg nerve CAPs, we may suggest that these two peptides are targeting sodium channels in the crab nerve. Consequently, the Bcg30.24 and Bcg31.16 peptides may be very useful for assays over voltage-gated sodium channels and could be used as pharmacological tools in biophysical studies on the peptide–channel interactions. This leads us to suggest that sea anemone peptides from different toxin groups (BcIV, the APETx-like, and the type 1 sodium channel toxins) act probably by targeting the voltage-gated sodium channels of this preparation. The toxin Bcg23.41 was chosen from the group of the near 3000 amu peptides (see Fig. 3C) to be tested over the crab leg nerve CAPs. Fig. 3C shows that 15 min after the administration of this peptide at 10 μM, a marked increase in the CAP amplitude and duration could be observed. This effect is similar to a potassium channels blockage in this preparation, as the administration of ShK peptide (1 μM, see Fig. 3D), a typical voltage-gated potassium channel blocker, increased the CAPs amplitude and duration in this assay. On the other hand, Bcg23.41 peptide apparently evokes this effect at a higher concentration (10 μM). Thus a new class of potassium channel blockers may now be available. Further studies are in progress in order to define the proper target and affinity of these novel toxins. Acknowledgements We thank Dr. Maria L. Garcia from the Merck Laboratories (Rahway, USA) for her generous gift of synthetic ShK peptide, and Mr. Vagner Alberto of the Biosciences Institute at USP for his technical assistance. This study was supported by CNPq (563874/2005-8), FAPESP (01/ 14243-5) and CAT/CEPID grants. AJZ is a post-doctoral fellow and WAF-Jr. is an undergraduate student. References Anderluh, G., Macek, P., 2002. Cytolytic peptide and protein toxins from sea anemones (Anthozoa: Actiniaria). Toxicon 40, 111–124. Barhanin, J., Hugues, M., Schweitz, H., Vincent, J.P., Lazdunski, M., 1981. Structure– function relationships of sea anemone toxin II from Anemonia sulcata. J. Biol. Chem. 256, 5764–5769. Barona, J., Batista, C.V., Zamudio, F.Z., Gomez-Lagunas, F., Wanke, E., Otero, R., Possani, L.D., 2006. Proteomic analysis of the venom and characterization of toxins specific for Na+and K+ -channels from the Colombian scorpion Tityus pachyurus. Biochim. Biophys. Acta. 1764, 76–84.

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