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IsCT, a Novel Cytotoxic Linear Peptide from Scorpion Opisthacanthus madagascariensis
Biochemical and Biophysical Research Communications 286, 820 – 825 (2001) doi:10.1006/bbrc.2001.5472, available online at http://www.idealibrary.com on
IsCT, a Novel Cytotoxic Linear Peptide from Scorpion Opisthacanthus madagascariensis Li Dai,* ,1 Akikazu Yasuda,* Hideo Naoki,* Gerardo Corzo,* Marta Andriantsiferana,† and Terumi Nakajima* *Suntory Institute for Bioorganic Research, Shimamoto, Osaka 618-8503, Japan; and †Faculty of Science, University of Antananarivo, Antananarivo, Madagascar
Received July 24, 2001
A novel cytotoxic linear peptide, IsCT, was characterized from scorpion Opisthacanthus madagascariensis. It is a linear peptide with a molecular weight of 1501.9 Da composed of 13 amino acid residues without cysteines. MS/MS analysis showed that its C-terminal is amidated. The identity of IsCT is re-confirmed by comparing the chemical synthesized peptide with the natural one. IsCT demonstrated antimicrobial activity against both gram-positive and gram-negative bacteria and hemolytic activity to sheep red blood cells. Also, it can release histamine from rat peritoneal mast cells. The CD absorption suggested that IsCT had an ␣-helix configuration in aqueous TFE. IsCT is one of the shortest natural cytotoxic peptides described, and it will be a suitable model for studying peptide-lipid interactions. © 2001 Academic Press Key Words: scorpion venom; cytotoxic peptide; antimicrobial; hemolytic; histamine.
Cytotoxic peptides are relatively small cationic molecules found in venoms (1–5), blood (6, 7), tissue surface secretions (8 –10), or even as autocytotoxicity peptides (11) from a broad range of organisms. They interact with lipid bilayers resulting in an alteration of the membrane permeability of the cells through the formation of new ion channels in the cell membrane and/or by changing the activity of existing channels (12, 13). Recently, some cytotoxic peptides which act against bacterial and fungal cells via a specific, but not receptor-mediated, permeabilization of microbial membranes have aroused intense interest because they may represent a potential source of novel antibiotic drugs (14, 15). The new respect of this innate immunity led considerable effort to elucidate the mode of action and the production pathway of these peptides (16 –18). However, it is still not clear how these peptides work To whom correspondence should be addressed. Fax: ⫹75-9622115. E-mail: [email protected]. 1
0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
and what are the basis of their selectivity against different cell membranes. Exploring new natural antimicrobial peptides might be helpful to screen possible candidates and make clear the relationship between peptides and membranes. Until now, several of such peptides have been identified and they can be divided into two groups according to their structure: some are linear, mostly helical, without cysteines while others contain one or more disulfide bonds, forming -sheet or both -sheet and ␣-helix structures (19). Cytotoxic peptides have been described in bee venom, wasp venom, and spider venom (1–3). In the latest decades, the neurotoxic peptides in the scorpion venom that affect Na ⫹, K ⫹, Ca 2⫹, Cl ⫺ channels of excitable or nonexcitable cells tend to get all the attention (20 –23). However, little is known about other biological active components in the crude venom of scorpions. Besides defensins which have long ago been found in scorpion hemolymph (7), two antimicrobial peptides have been recently found in scorpion venom (4, 5). Here, we describe the isolation and chemical and biological characterization of a novel peptide with cytotoxic activity from the scorpion Opisthacanthus madagascariensis. We named it IsCT because the scorpion was found in Isalo, Madagascar and the peptide demonstrated cytotoxic activity. The low molecular weight, novel amino acid sequence which shared no homology with any other known scorpion venom component and cytotoxic activity of this linear peptide suggested a new class of scorpion venom constitutes. MATERIALS AND METHODS Materials. Scorpion Opisthacanthus madagascariensis was collected in Isalo, Madagascar. The crude venom was accumulated by electrical stimulation of the telson and immediately frozen in ice and stored in the freezer under ⫺75°C. The crude venom was extracted with water and the supernatant was stored in the freezer under ⫺20°C. Capillary reversed-phase HPLC/Q-Tof MS. Supernatant (0.1%) was directly injected to capillary RP-HPLC (24) using a PEEK tube
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(0.25 ⫻ 100 mm) packed with inhouse TSK gel ODS 120T (Tosoh, 5-m particle size) in the Hewlett–Packard HP1100 liquid chromatography system with a 60 min linear gradient of 5–70% acetonitrile/ H 2O in 0.1% TFA. The flow from the pump (100 l/min) was split by a T-connector and the flow toward the HPLC column was adjusted to 2 l/min. The elute was monitored at 220 nm using a UV detector equipped with a U-shaped cell (LC Packings; Model UZ-HP11-CAP). The outlet of the UV detector was connected to the electrospray interface of the mass spectrometer (Micromass, Manchester, UK). Typically, the spraying capillary voltage was used for 2.8 kV and the sample cone voltage was set at 50 V. The range of the total ion current was set at m/z 100 –2000. Purification of the peptide. The supernatant was applied to a 1.3 ⫻ 50 cm column containing Sephadex G-25 equilibrated with 50 mM acetic acid buffer, at a flow rate of 0.3 ml/min. The optical absorbance of the eluent was monitored at 220 nm. Sub-fractions were checked by MALDI-TOF mass spectrometer. Sub-fraction C was further separated with C 18 HPLC (Tosoh, TSK gel, ODS 120T, 0.46 ⫻ 25 cm) at 40°C with a 40 min linear gradient of 12.5– 65% actonitrile/H 2O in 0.1% TFA. The flow rate was set at 1 ml/min and the absorbance was monitored at 220 nm. Amino acid composition analysis and sequencing. The amino acid composition of IsCT was determined after acid hydrolysis [6 M HCl, 1% (mass/vol.) phenol], under vacuum at 110°C for 24 h. Dried hydrolyzed sample was derivatized with phenyl isothiocyanate and the subsequent phenyl thiocarbamy amino acids were separated at 40°C on a C 18 HPLC column (Tosoh, TSK gel, ODS 80T, 0.46 ⫻ 25 cm), by a linear gradient of 2–70% buffer B (buffer A: 0.05 M sodium acetic acid; buffer B: 60% actonitrile/H 2O) in 20 min. The absorbance was monitored at 254 nm. Amino acid standards derivatized with phenyl isothiocyanate were used for calibration. The amino acid sequence was analyzed by a gas-phase sequencer PPSQ-10 (Shimadzu, Kyoto, Japan) based on automated Edman degradation. Mass spectrometry analysis. Sub-fractions and purified peptides were vacuum dried and suspended in 0.1% TFA. The matrix for the measurement of MALDI-TOF-MS was prepared as follows (25). ␣-Cyanohydroxycinnamic acid was dissolved in 1:1 acetonitrile/H 2O in 0.1% TFA to obtain a saturated solution. Samples mixed with the matrix solution were placed on the sample plate and dried for 5 min. MS experiments were performed on a Voyager Elite MALDI-TOF mass spectrometer (PerSeptive Biosystems) using a positive mode. Sub-fractions were recorded in linear mode while purified natural and synthetic peptides were obtained in reflector mode. The amino acid sequence was also analyzed by collision-induced dissociation MS/MS experiments. Chemical synthesis. Peptides were synthesized by an automated solid-phase peptide synthesizer 433-A (Applied Biosystems, USA) based on the Fmoc-strategy. Purification to homogeneity of crude peptides was carried out on a preparative C 18 RP-HPLC column, by a 30 min linear gradient of 24 –54% acetonitrile/H 2O in 0.1% TFA. The identity of synthetic and natural products was confirmed by mass spectrometry analysis, C 18 HPLC coinjection experiment, and amino acid composition analysis. Anti-bacterial assay. Anti-bacterial activity was assayed by suppression of bacterial growth dependent on application of peptides to the top agar of antibiotic 3 medium (DIFCO). Bacteria were grown in liquid antibiotic 3 medium (DIFCO) for 18 h. Then, 1 ml of bacteria suspension, OD 600nm ⫽ 0.7– 0.8, was added to 9 ml of sterile antibiotic 3 medium to make a dilution 1:10. 1 ml of such a dilution was added to 9 ml of warm 1.5% agar in antibiotic 3 medium and was poured into 100 ⫻ 200 mm sterile petri dish. Five microliters of suspended synthetic IsCT in d H 2O with different concentrations were supplied to the solidified plate surface. Bacteria were incubated at 37°C for 12–14 h. Growth inhibition was detected as clear spots on the plate surface.
Hemolysis of sheep red blood cells. Hemolytic activity of the synthetic IsCT was tested against sheep red blood cells. Fresh sheep red blood cells were rinsed several times in PBS by centrifugation for 3 min at 3000g until the OD of the supernatant reached the OD of the blank (PBS). Then they were incubated at room temperature for 1 h in deionized water (positive control), in PBS (blank), or with different concentrations of synthetic IsCT. The samples were centrifuged at 10,000g for 5 min and the absorbance of supernatant was measured at 570 nm. The relative optical density compared to the one treated with deionized water defined the percentage of hemolysis. Melittin was used as positive control. Histamine release from mast cells. The mast cells were obtained from the peritoneal fluid of male, 11-week-old rats (Wistar, Clea Japan). Tyrode’s-Hepes solution (NaCl 137 mM, KCl 2.7 mM, CaCl 2 1.8 mM, MgCl 2 1 mM, NaH 2PO 4 0.4 mM, Hepes 20 mM, D-glucose 5.6 mM, 0.1% (w/v) gelatin, pH 7.4) was used for the isolation of all cells from peritoneal fluid. Then the mast cells were separated from other cells by centrifuging at 2000 rpm for 20 min in 30% Ficoll solution (30 g/100 ml Tyrode’s Hepes solution). Resuspended the mast cells in Tyrode’s-Hepes solution and after washing, adjusted the mast cell concentration to 10 5/ml. The mast cells were incubated with various concentrations of synthetic IsCT at 37°C for 10 min. The reaction was stopped by placing on ice. After centrifuging at 1500 rpm for 3 min, the histamine concentration in the supernatant was determined with ELISA kit. The total histamine content was obtained by freezing–thawing cell pellet and determining histamine concentration in the supernatant as described above. Mastoparan was used as positive control. CD spectra. The CD spectra of the peptides were measured with a JASCO J-725 spectropolarimeter. The spectra were scanned at room temperature in a capped, round optical cell with a 1-mm path length. Spectra were obtained at wavelengths of 260 –180 nm. Sixteen scans were accumulated for each sample at a scan rate of 50 nm/min. The synthetic IsCT was measured at concentration of 0.1% (w/v) in water, methanol, 40% TFE/H 2O or 70% TFE/H 2O. Data were analyzed with CD Deconvolution software to obtain the peptide configuration (26).
RESULTS AND DISCUSSION Scorpion venoms contain a variety of biologically active components: enzymes, peptides, nucleotides, lipids, mucoproteins, biogenic amines, and other unknown substances (27). The best-studied groups are the long-chain peptides (MW 6 – 8 kDa) to be mainly active on Na ⫹ channels and short-chain peptides (MW 3– 4 kDa) on K ⫹ channels respectively (20, 21). However, according to the results of LC-MS, the crude venom extract of scorpion Opisthacanthus madagascariensis also contained some peptides eluting at late retention time with molecular weight around 1500 Da which haven’t been reported before. Usually, investigation of crude and fractionated venom is monitored by various bioassay-guided methods. However, these methods need relatively large amounts of natural compounds in the bioassay especially in vivo assay. This time, we describe here a nonconventional approach for the identification of these compounds, that is, we isolated the main components and determined their structure first, then tried to characterize their biological activities with the synthetic products. The crude venom extract showed a complex profile of various components with a wide distribution between
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Minimal Inhibitory Concentration of IsCT against Bacteria Microorganisms Gram (⫹) bacteria Staphylococcus aureus ATCC 25293 Staphylococcus aureus ATCC 6538 Staphylococcus saprophyticus* Bacillus thuringensis** Bacillus subtillis CCT 2471 Gram (⫺) bacteria Pseudomonas aeroginosa ATCC 15442 Pseudomonas aeroginosa ATCC 27853 Escherichia coli CCT 1371 Escherichia coli ATCC 25922 Proteus mirabilis*
MIC (g/ml) 1 5 1 25 10 ⬎200 100 5 10 ⬎200
* Clinical strain. ** Wild strain (isolated from the nature).
gave one mass unit lower than that of isolated IsCT, and it was suggested to be caused by C-terminal amidation. To confirm this amidation, it was performed on
FIG. 1. Purification IsCT from scorpion Opisthacanthus madagascariensis. (A) The crude venom extract from one gland was applied to a Sephadex G-25 gel-filtration column (1.3 ⫻ 50 cm) equilibrated and run with 50 mM acetic acid buffer, at a flow rate of 21 ml/h. Four distinct subfractions (horizontal bars Fa-Fd) were obtained. (B) Subfraction Fc was further separated in a TSK gel ODS 120-T reverse-phase column (0.46 ⫻ 25 cm) of HPLC system, using a linear gradient from 90% A (5% acetonitrile/water in 0.1% TFA) to 80% B (80% acetonitrile/water in 0.08% TFA) in 40 min. Fraction IsCT was obtained.
1–10 kDa. Among them, we focused on a main constituent with low molecular weight of 1501.9 Da based on the LC-MS results. To isolate this hydrophobic and short peptide, the crude venom extract was first separated by gel filtration (sephadex G-25) (Fig. 1A) and the subfraction C containing low molecular weight components (MW 1500 – 4300 Da) was further separated with reverse phase C18 HPLC (Fig. 1B). The component named IsCT later was collected and the MALDI-TOF mass spectrum in reflector mode indicated molecular related peak (M ⫹ H) ⫹ at m/z 1502.9, corresponding to a monoisotopic mass. The amino acid composition analysis result showed that it was composed of 13 amino acids, enriched in hydrophobic amino acids (3 Ile, 2 Leu) and basic amino acids (2 Lys). The full amino acid sequence was obtained by Edman degradation sequencing. However, the calculated molecular weight
FIG. 2. Cytotoxic activity of IsCT. (A) Hemolysis assay. The hemolytic activity was estimated by monitoring the increase in the absorbance at 570 nm, after incubating the sheep red blood cells with different peptide concentrations at 37°C for 1 h. Positive control was deionized water, and PBS buffer as blank. Melittin was used as comparison. (B) The release of histamine from rat peritoneal mast cells induced by IsCT and Mastoparan. The mast cells were incubated with various concentrations of IsCT and Mastoparan at 37°C for 10 min and the histamine concentration in the supernatant was determined with ELISA kit. The total histamine concentration was obtained by freezing–thawing cell pellet.
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FIG. 3. (A) Sequence comparison of IsCT with other cytotoxic peptides. (B) Circular dichroism spectra of 0.1 g/l IsCT in water, methanol, 40 and 70% aqueous TFE. (C) Projections of the amphipathic ␣-helical structure of IsCT. In this view, the charged residues (F) and neutral polar residues (O) are located on one side, while the hydrophobic residues (E) are on the other side of the helix.
MS/MS and its spectrum clearly indicated the amidation at C-terminal (data not shown). To reconfirm the sequence and further characterize its biological properties, the chemical synthesis of IsCT was undertaken using optimized Fmoc/tert-butyl chemistry on preloaded resins, to give C-terminal amidated peptides. The MS/MS data for the synthetic IsCT
completely correspond to that of naturally occurring peptide. RP-HPLC of coinjection with synthetic and natural IsCT also gave in a sharp peak with the same retention time. All these data supported that IsCT is modified to the amidation at the C-terminal Compared with other known peptides, the amino acid sequence of IsCT showed low homology with any
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other scorpion toxic peptides, but shared higher homology with several cytotoxic peptides from wasp venoms (2). In fact, there was no obvious toxic effect to mice by icv injection or to insects by micro-injection and topical application. However, it was found to inhibit the growth of both gram-positive and gram-negative bacteria at micromolar concentrations (Table 1). Also, it induced hemolysis of sheep red blood cells (Fig. 2A). In addition, degranulation activity from rat peritoneal mast cells was examined with mastoparan as control. The synthetic IsCT showed almost twice-high potency as mastoparan at the same concentration (Fig. 2B). All these data suggested the short peptide to be a cytotoxic peptide that might be used as both defensive and offensive purposes in scorpion venom. Such economical utilization is also found in melittins from bee venom and lycotoxins from spider venom (1, 3). Melittin’s secondary structure is well established to be amphipathic ␣-helical in its crystalline state (28). Usually, natural antimicrobial peptides with such structure are considered to lyse cell membrane through pore formation or through destabilization of membrane phospholipid packing (12, 13). Some of these peptides form channels by self-aggregation of peptide monomers, whereby hydrophilic residues on one side of the helix face inward and hydrophobic residues on the opposite side of the helix interact with fatty acid side chains of the lipid bilayer. Synthetic IsCT showed high percentage of ␣-helix in aqueous TFE (Fig. 3B). When plotted as helical wheel projection (Fig. 3C), IsCT adapted an amphipathic ␣-helix conformation that may suggest IsCT might interact directly with microbial membranes. However, IsCT is one short natural cytotoxic peptide composed of only 13 amino acid residues less than 20 which is required for ␣-helices to span lipid bilayers. Whether it induced lipid-mediated channel based on the curvature strain imposed on lipid membranes in the presence of intercalated amphipathic peptides such as mastoparan, cecropin-melittin hybrids (29 –31), or by other mechanisms is still unknown. Since IsCT is a short peptide with relatively high antimicrobial activity, it will be a suitable model to study lipid-peptide reaction. Although at high peptide concentration, mastoparan can also increase permeability of ions and small molecules through membranes by forming pores, investigations showed that mastoparan induce exocytosis mainly through activating G proteins (32, 33). Whether IsCT effects G protein or not needs further study. Until now, only two other antimicrobial peptides isolated from scorpion venoms, Scorpine and Hadrurin (4, 5) have been characterized. Although their molecular size and amino sequence are quite different, they all demonstrated antimicrobial activity. It proposed that they define a new subclass in the cocktail of scorpion venom. Although their MIC against bacteria are relatively higher than conventional antibiotics and not spe-
cific selective against bacterial cells, they can still be useful models to study peptide-lipid interactions. ACKNOWLEDGMENTS We gratefully acknowledge Dr. M. Matsui (Suntory Biomedical Research LTD., Japan) for his help in histamine release assay and Professor Mario Sergio Palma (Laboratory of Structure Biology and Zoochemistry, Sao Paulo State University, Brazil) for his help in antimicrobial assay. This work was supported by a grant from the Research for the Future Program from the Japan Society for the Promotion of Science (JSPS).
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25. Romi-Lebrun, R., Martin-Eauclaire, M. F., Escoubas, P., Wu, F. Q., Lebrun, B., Hisada, M., and Nakajima, T. (1997) Characterization of four toxins from Buthus martensi scorpion venom, which act on apamin-sensitive Ca 2⫹-activated K ⫹ channels. Eur. J. Biochem. 245, 457– 464. 26. Bohm, G., Mohr, R., and Jaenicke, R. (1992) Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Prot. Eng. 5, 191–195. 27. Possani, L. D. (1984) Structure of scorpion toxins in Handbook of Natural Toxins, Vol. 2 (Tu, A. T., Ed.),pp. 513–550, Marcel Dekker, New York. 28. Terwilliger, T. C., and Eisenberg, D. (1982) The structure of melittin. I. Structure determination and partial refinement. J. Biol. Chem. 257, 6010 – 6015. 29. Higashijima, T., Uzu, S., Nakajima, T., and Ross, E. M. (1988) Mastoparan, a peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins (G proteins). J. Biol. Chem. 263, 6491– 6494. 30. Katsu, T., Kuroko, M., Morikawa, T., Sanchika, K., Yamanaka, H., Shimoda, S., and Fujita, Y. (1990) Interaction of wasp venom mastoparan with biomembranes. Biochim. Biophys. Acta 1027, 185–190. 31. Bechinger, B. (1999) The structure, dynamics, and orientation of antimicrobial peptides in membranes by multidimensional solid-state NMR spectroscopy. Biochim. Biophys. Acta 1462, 157–183. 32. Matsuzaki, K., Yoneyama, S., Murase, O., and Miyajima, K. (1996) Transbilayer transport of ions and lipids coupled with mastoparan X translocation. Biochemistry 35, 8450 – 8456. 33. Nakajima, T., Wakamatsu, K., and Mkai, H. (1999) Mastoparan as a G protein activator in Animal Toxins (Rochat, MartinEauclaire, Eds.), pp. 116 –126, Birkhauser.
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IsCT, a Novel Cytotoxic Linear Peptide from Scorpion Opisthacanthus madagascariensis Li Dai,* ,1 Akikazu Yasuda,* Hideo Naoki,* Gerardo Corzo,* Marta Andriantsiferana,† and Terumi Nakajima* *Suntory Institute for Bioorganic Research, Shimamoto, Osaka 618-8503, Japan; and †Faculty of Science, University of Antananarivo, Antananarivo, Madagascar
Received July 24, 2001
A novel cytotoxic linear peptide, IsCT, was characterized from scorpion Opisthacanthus madagascariensis. It is a linear peptide with a molecular weight of 1501.9 Da composed of 13 amino acid residues without cysteines. MS/MS analysis showed that its C-terminal is amidated. The identity of IsCT is re-confirmed by comparing the chemical synthesized peptide with the natural one. IsCT demonstrated antimicrobial activity against both gram-positive and gram-negative bacteria and hemolytic activity to sheep red blood cells. Also, it can release histamine from rat peritoneal mast cells. The CD absorption suggested that IsCT had an ␣-helix configuration in aqueous TFE. IsCT is one of the shortest natural cytotoxic peptides described, and it will be a suitable model for studying peptide-lipid interactions. © 2001 Academic Press Key Words: scorpion venom; cytotoxic peptide; antimicrobial; hemolytic; histamine.
Cytotoxic peptides are relatively small cationic molecules found in venoms (1–5), blood (6, 7), tissue surface secretions (8 –10), or even as autocytotoxicity peptides (11) from a broad range of organisms. They interact with lipid bilayers resulting in an alteration of the membrane permeability of the cells through the formation of new ion channels in the cell membrane and/or by changing the activity of existing channels (12, 13). Recently, some cytotoxic peptides which act against bacterial and fungal cells via a specific, but not receptor-mediated, permeabilization of microbial membranes have aroused intense interest because they may represent a potential source of novel antibiotic drugs (14, 15). The new respect of this innate immunity led considerable effort to elucidate the mode of action and the production pathway of these peptides (16 –18). However, it is still not clear how these peptides work To whom correspondence should be addressed. Fax: ⫹75-9622115. E-mail: [email protected]. 1
0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
and what are the basis of their selectivity against different cell membranes. Exploring new natural antimicrobial peptides might be helpful to screen possible candidates and make clear the relationship between peptides and membranes. Until now, several of such peptides have been identified and they can be divided into two groups according to their structure: some are linear, mostly helical, without cysteines while others contain one or more disulfide bonds, forming -sheet or both -sheet and ␣-helix structures (19). Cytotoxic peptides have been described in bee venom, wasp venom, and spider venom (1–3). In the latest decades, the neurotoxic peptides in the scorpion venom that affect Na ⫹, K ⫹, Ca 2⫹, Cl ⫺ channels of excitable or nonexcitable cells tend to get all the attention (20 –23). However, little is known about other biological active components in the crude venom of scorpions. Besides defensins which have long ago been found in scorpion hemolymph (7), two antimicrobial peptides have been recently found in scorpion venom (4, 5). Here, we describe the isolation and chemical and biological characterization of a novel peptide with cytotoxic activity from the scorpion Opisthacanthus madagascariensis. We named it IsCT because the scorpion was found in Isalo, Madagascar and the peptide demonstrated cytotoxic activity. The low molecular weight, novel amino acid sequence which shared no homology with any other known scorpion venom component and cytotoxic activity of this linear peptide suggested a new class of scorpion venom constitutes. MATERIALS AND METHODS Materials. Scorpion Opisthacanthus madagascariensis was collected in Isalo, Madagascar. The crude venom was accumulated by electrical stimulation of the telson and immediately frozen in ice and stored in the freezer under ⫺75°C. The crude venom was extracted with water and the supernatant was stored in the freezer under ⫺20°C. Capillary reversed-phase HPLC/Q-Tof MS. Supernatant (0.1%) was directly injected to capillary RP-HPLC (24) using a PEEK tube
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(0.25 ⫻ 100 mm) packed with inhouse TSK gel ODS 120T (Tosoh, 5-m particle size) in the Hewlett–Packard HP1100 liquid chromatography system with a 60 min linear gradient of 5–70% acetonitrile/ H 2O in 0.1% TFA. The flow from the pump (100 l/min) was split by a T-connector and the flow toward the HPLC column was adjusted to 2 l/min. The elute was monitored at 220 nm using a UV detector equipped with a U-shaped cell (LC Packings; Model UZ-HP11-CAP). The outlet of the UV detector was connected to the electrospray interface of the mass spectrometer (Micromass, Manchester, UK). Typically, the spraying capillary voltage was used for 2.8 kV and the sample cone voltage was set at 50 V. The range of the total ion current was set at m/z 100 –2000. Purification of the peptide. The supernatant was applied to a 1.3 ⫻ 50 cm column containing Sephadex G-25 equilibrated with 50 mM acetic acid buffer, at a flow rate of 0.3 ml/min. The optical absorbance of the eluent was monitored at 220 nm. Sub-fractions were checked by MALDI-TOF mass spectrometer. Sub-fraction C was further separated with C 18 HPLC (Tosoh, TSK gel, ODS 120T, 0.46 ⫻ 25 cm) at 40°C with a 40 min linear gradient of 12.5– 65% actonitrile/H 2O in 0.1% TFA. The flow rate was set at 1 ml/min and the absorbance was monitored at 220 nm. Amino acid composition analysis and sequencing. The amino acid composition of IsCT was determined after acid hydrolysis [6 M HCl, 1% (mass/vol.) phenol], under vacuum at 110°C for 24 h. Dried hydrolyzed sample was derivatized with phenyl isothiocyanate and the subsequent phenyl thiocarbamy amino acids were separated at 40°C on a C 18 HPLC column (Tosoh, TSK gel, ODS 80T, 0.46 ⫻ 25 cm), by a linear gradient of 2–70% buffer B (buffer A: 0.05 M sodium acetic acid; buffer B: 60% actonitrile/H 2O) in 20 min. The absorbance was monitored at 254 nm. Amino acid standards derivatized with phenyl isothiocyanate were used for calibration. The amino acid sequence was analyzed by a gas-phase sequencer PPSQ-10 (Shimadzu, Kyoto, Japan) based on automated Edman degradation. Mass spectrometry analysis. Sub-fractions and purified peptides were vacuum dried and suspended in 0.1% TFA. The matrix for the measurement of MALDI-TOF-MS was prepared as follows (25). ␣-Cyanohydroxycinnamic acid was dissolved in 1:1 acetonitrile/H 2O in 0.1% TFA to obtain a saturated solution. Samples mixed with the matrix solution were placed on the sample plate and dried for 5 min. MS experiments were performed on a Voyager Elite MALDI-TOF mass spectrometer (PerSeptive Biosystems) using a positive mode. Sub-fractions were recorded in linear mode while purified natural and synthetic peptides were obtained in reflector mode. The amino acid sequence was also analyzed by collision-induced dissociation MS/MS experiments. Chemical synthesis. Peptides were synthesized by an automated solid-phase peptide synthesizer 433-A (Applied Biosystems, USA) based on the Fmoc-strategy. Purification to homogeneity of crude peptides was carried out on a preparative C 18 RP-HPLC column, by a 30 min linear gradient of 24 –54% acetonitrile/H 2O in 0.1% TFA. The identity of synthetic and natural products was confirmed by mass spectrometry analysis, C 18 HPLC coinjection experiment, and amino acid composition analysis. Anti-bacterial assay. Anti-bacterial activity was assayed by suppression of bacterial growth dependent on application of peptides to the top agar of antibiotic 3 medium (DIFCO). Bacteria were grown in liquid antibiotic 3 medium (DIFCO) for 18 h. Then, 1 ml of bacteria suspension, OD 600nm ⫽ 0.7– 0.8, was added to 9 ml of sterile antibiotic 3 medium to make a dilution 1:10. 1 ml of such a dilution was added to 9 ml of warm 1.5% agar in antibiotic 3 medium and was poured into 100 ⫻ 200 mm sterile petri dish. Five microliters of suspended synthetic IsCT in d H 2O with different concentrations were supplied to the solidified plate surface. Bacteria were incubated at 37°C for 12–14 h. Growth inhibition was detected as clear spots on the plate surface.
Hemolysis of sheep red blood cells. Hemolytic activity of the synthetic IsCT was tested against sheep red blood cells. Fresh sheep red blood cells were rinsed several times in PBS by centrifugation for 3 min at 3000g until the OD of the supernatant reached the OD of the blank (PBS). Then they were incubated at room temperature for 1 h in deionized water (positive control), in PBS (blank), or with different concentrations of synthetic IsCT. The samples were centrifuged at 10,000g for 5 min and the absorbance of supernatant was measured at 570 nm. The relative optical density compared to the one treated with deionized water defined the percentage of hemolysis. Melittin was used as positive control. Histamine release from mast cells. The mast cells were obtained from the peritoneal fluid of male, 11-week-old rats (Wistar, Clea Japan). Tyrode’s-Hepes solution (NaCl 137 mM, KCl 2.7 mM, CaCl 2 1.8 mM, MgCl 2 1 mM, NaH 2PO 4 0.4 mM, Hepes 20 mM, D-glucose 5.6 mM, 0.1% (w/v) gelatin, pH 7.4) was used for the isolation of all cells from peritoneal fluid. Then the mast cells were separated from other cells by centrifuging at 2000 rpm for 20 min in 30% Ficoll solution (30 g/100 ml Tyrode’s Hepes solution). Resuspended the mast cells in Tyrode’s-Hepes solution and after washing, adjusted the mast cell concentration to 10 5/ml. The mast cells were incubated with various concentrations of synthetic IsCT at 37°C for 10 min. The reaction was stopped by placing on ice. After centrifuging at 1500 rpm for 3 min, the histamine concentration in the supernatant was determined with ELISA kit. The total histamine content was obtained by freezing–thawing cell pellet and determining histamine concentration in the supernatant as described above. Mastoparan was used as positive control. CD spectra. The CD spectra of the peptides were measured with a JASCO J-725 spectropolarimeter. The spectra were scanned at room temperature in a capped, round optical cell with a 1-mm path length. Spectra were obtained at wavelengths of 260 –180 nm. Sixteen scans were accumulated for each sample at a scan rate of 50 nm/min. The synthetic IsCT was measured at concentration of 0.1% (w/v) in water, methanol, 40% TFE/H 2O or 70% TFE/H 2O. Data were analyzed with CD Deconvolution software to obtain the peptide configuration (26).
RESULTS AND DISCUSSION Scorpion venoms contain a variety of biologically active components: enzymes, peptides, nucleotides, lipids, mucoproteins, biogenic amines, and other unknown substances (27). The best-studied groups are the long-chain peptides (MW 6 – 8 kDa) to be mainly active on Na ⫹ channels and short-chain peptides (MW 3– 4 kDa) on K ⫹ channels respectively (20, 21). However, according to the results of LC-MS, the crude venom extract of scorpion Opisthacanthus madagascariensis also contained some peptides eluting at late retention time with molecular weight around 1500 Da which haven’t been reported before. Usually, investigation of crude and fractionated venom is monitored by various bioassay-guided methods. However, these methods need relatively large amounts of natural compounds in the bioassay especially in vivo assay. This time, we describe here a nonconventional approach for the identification of these compounds, that is, we isolated the main components and determined their structure first, then tried to characterize their biological activities with the synthetic products. The crude venom extract showed a complex profile of various components with a wide distribution between
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Minimal Inhibitory Concentration of IsCT against Bacteria Microorganisms Gram (⫹) bacteria Staphylococcus aureus ATCC 25293 Staphylococcus aureus ATCC 6538 Staphylococcus saprophyticus* Bacillus thuringensis** Bacillus subtillis CCT 2471 Gram (⫺) bacteria Pseudomonas aeroginosa ATCC 15442 Pseudomonas aeroginosa ATCC 27853 Escherichia coli CCT 1371 Escherichia coli ATCC 25922 Proteus mirabilis*
MIC (g/ml) 1 5 1 25 10 ⬎200 100 5 10 ⬎200
* Clinical strain. ** Wild strain (isolated from the nature).
gave one mass unit lower than that of isolated IsCT, and it was suggested to be caused by C-terminal amidation. To confirm this amidation, it was performed on
FIG. 1. Purification IsCT from scorpion Opisthacanthus madagascariensis. (A) The crude venom extract from one gland was applied to a Sephadex G-25 gel-filtration column (1.3 ⫻ 50 cm) equilibrated and run with 50 mM acetic acid buffer, at a flow rate of 21 ml/h. Four distinct subfractions (horizontal bars Fa-Fd) were obtained. (B) Subfraction Fc was further separated in a TSK gel ODS 120-T reverse-phase column (0.46 ⫻ 25 cm) of HPLC system, using a linear gradient from 90% A (5% acetonitrile/water in 0.1% TFA) to 80% B (80% acetonitrile/water in 0.08% TFA) in 40 min. Fraction IsCT was obtained.
1–10 kDa. Among them, we focused on a main constituent with low molecular weight of 1501.9 Da based on the LC-MS results. To isolate this hydrophobic and short peptide, the crude venom extract was first separated by gel filtration (sephadex G-25) (Fig. 1A) and the subfraction C containing low molecular weight components (MW 1500 – 4300 Da) was further separated with reverse phase C18 HPLC (Fig. 1B). The component named IsCT later was collected and the MALDI-TOF mass spectrum in reflector mode indicated molecular related peak (M ⫹ H) ⫹ at m/z 1502.9, corresponding to a monoisotopic mass. The amino acid composition analysis result showed that it was composed of 13 amino acids, enriched in hydrophobic amino acids (3 Ile, 2 Leu) and basic amino acids (2 Lys). The full amino acid sequence was obtained by Edman degradation sequencing. However, the calculated molecular weight
FIG. 2. Cytotoxic activity of IsCT. (A) Hemolysis assay. The hemolytic activity was estimated by monitoring the increase in the absorbance at 570 nm, after incubating the sheep red blood cells with different peptide concentrations at 37°C for 1 h. Positive control was deionized water, and PBS buffer as blank. Melittin was used as comparison. (B) The release of histamine from rat peritoneal mast cells induced by IsCT and Mastoparan. The mast cells were incubated with various concentrations of IsCT and Mastoparan at 37°C for 10 min and the histamine concentration in the supernatant was determined with ELISA kit. The total histamine concentration was obtained by freezing–thawing cell pellet.
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FIG. 3. (A) Sequence comparison of IsCT with other cytotoxic peptides. (B) Circular dichroism spectra of 0.1 g/l IsCT in water, methanol, 40 and 70% aqueous TFE. (C) Projections of the amphipathic ␣-helical structure of IsCT. In this view, the charged residues (F) and neutral polar residues (O) are located on one side, while the hydrophobic residues (E) are on the other side of the helix.
MS/MS and its spectrum clearly indicated the amidation at C-terminal (data not shown). To reconfirm the sequence and further characterize its biological properties, the chemical synthesis of IsCT was undertaken using optimized Fmoc/tert-butyl chemistry on preloaded resins, to give C-terminal amidated peptides. The MS/MS data for the synthetic IsCT
completely correspond to that of naturally occurring peptide. RP-HPLC of coinjection with synthetic and natural IsCT also gave in a sharp peak with the same retention time. All these data supported that IsCT is modified to the amidation at the C-terminal Compared with other known peptides, the amino acid sequence of IsCT showed low homology with any
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other scorpion toxic peptides, but shared higher homology with several cytotoxic peptides from wasp venoms (2). In fact, there was no obvious toxic effect to mice by icv injection or to insects by micro-injection and topical application. However, it was found to inhibit the growth of both gram-positive and gram-negative bacteria at micromolar concentrations (Table 1). Also, it induced hemolysis of sheep red blood cells (Fig. 2A). In addition, degranulation activity from rat peritoneal mast cells was examined with mastoparan as control. The synthetic IsCT showed almost twice-high potency as mastoparan at the same concentration (Fig. 2B). All these data suggested the short peptide to be a cytotoxic peptide that might be used as both defensive and offensive purposes in scorpion venom. Such economical utilization is also found in melittins from bee venom and lycotoxins from spider venom (1, 3). Melittin’s secondary structure is well established to be amphipathic ␣-helical in its crystalline state (28). Usually, natural antimicrobial peptides with such structure are considered to lyse cell membrane through pore formation or through destabilization of membrane phospholipid packing (12, 13). Some of these peptides form channels by self-aggregation of peptide monomers, whereby hydrophilic residues on one side of the helix face inward and hydrophobic residues on the opposite side of the helix interact with fatty acid side chains of the lipid bilayer. Synthetic IsCT showed high percentage of ␣-helix in aqueous TFE (Fig. 3B). When plotted as helical wheel projection (Fig. 3C), IsCT adapted an amphipathic ␣-helix conformation that may suggest IsCT might interact directly with microbial membranes. However, IsCT is one short natural cytotoxic peptide composed of only 13 amino acid residues less than 20 which is required for ␣-helices to span lipid bilayers. Whether it induced lipid-mediated channel based on the curvature strain imposed on lipid membranes in the presence of intercalated amphipathic peptides such as mastoparan, cecropin-melittin hybrids (29 –31), or by other mechanisms is still unknown. Since IsCT is a short peptide with relatively high antimicrobial activity, it will be a suitable model to study lipid-peptide reaction. Although at high peptide concentration, mastoparan can also increase permeability of ions and small molecules through membranes by forming pores, investigations showed that mastoparan induce exocytosis mainly through activating G proteins (32, 33). Whether IsCT effects G protein or not needs further study. Until now, only two other antimicrobial peptides isolated from scorpion venoms, Scorpine and Hadrurin (4, 5) have been characterized. Although their molecular size and amino sequence are quite different, they all demonstrated antimicrobial activity. It proposed that they define a new subclass in the cocktail of scorpion venom. Although their MIC against bacteria are relatively higher than conventional antibiotics and not spe-
cific selective against bacterial cells, they can still be useful models to study peptide-lipid interactions. ACKNOWLEDGMENTS We gratefully acknowledge Dr. M. Matsui (Suntory Biomedical Research LTD., Japan) for his help in histamine release assay and Professor Mario Sergio Palma (Laboratory of Structure Biology and Zoochemistry, Sao Paulo State University, Brazil) for his help in antimicrobial assay. This work was supported by a grant from the Research for the Future Program from the Japan Society for the Promotion of Science (JSPS).
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