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Molecular components and toxicity of the venom of the solitary wasp, Anoplius samariensis
BBRC Biochemical and Biophysical Research Communications 330 (2005) 1048–1054 www.elsevier.com/locate/ybbrc
Molecular components and toxicity of the venom of the solitary wasp, Anoplius samariensis Miki Hisada a,*, Honoo Satake a, Katsuyoshi Masuda a, Masato Aoyama a, Kazuya Murata b, Testuro Shinada c, Takashi Iwashita a, Yasufumi Ohfune c, Terumi Nakajima d a
Suntory Institute for Bioorganic Research, 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan Sakai Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 1-1-53 Takasu-cho, Sakai-shi, Osaka 590-0003, Japan c Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan d Hoshi University, 2-4-41 Ebara, Shinagawa, Tokyo 142-8501, Japan
b
Received 15 March 2005 Available online 23 March 2005
Abstract The solitary spider wasp, Anoplius samariensis, is known to exhibit a unique long-term, non-lethal paralysis in spiders that it uses as a food source for its larvae. However, neither detailed venom components nor paralytic compounds have ever been characterized. In this study, we examined the components in the low molecular weight fraction of the venom and the paralytic activity of the high molecular weight fraction. The major low molecular weight components of the venom were identified as c-aminobutyric acid and glutamic acid by micro-liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance spectrometry analysis. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis and mass analysis revealed that the A. samariensis venom contained the various proteins with weights of 4–100 kDa. A biological assay using Joro spiders (Nephila clavata) clearly showed that the high molecular weight fraction of the venom prepared by ultrafiltration exerted as potent non-lethal long-term paralysis as the whole venom, whereas the low molecular weight fraction was devoid of any paralytic activity. These results indicated that several venomous proteins in the high molecular weight fraction are responsible for the paralytic activity. Furthermore, we determined the primary structure of one component designated As-fr-19, which was a novel multiple-cysteine peptide with high sequence similarity to several sea anemone and snake toxins including dendrotoxins, rather than any insect toxic peptides identified so far. Taken together, our data showed the unprecedented molecular and toxicological profiles of wasp venoms. 2005 Elsevier Inc. All rights reserved. Keywords: Solitary wasp; Venom; Toxin; Paralysis; Channel blocker
The venoms of arthropods have attracted considerable interest as a potential source of bioactive substances. Venoms of solitary wasps are known to possess long-term, non-lethal paralytic effects on their preys (insects or spiders). The immobilized preys are then used to feed the waspsÕ larvae [1,2]. Such unique paralytic activity suggests the presence of novel neurotoxic compounds in the venom of solitary wasps [3–5]. To date, *
Corresponding author. Fax: +81 75 962 2115. E-mail address: [email protected] (M. Hisada).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.087
several neurotoxic components have been characterized from solitary wasp venoms. Philanthotoxins that inhibit neuromuscular transmission by blocking post-synaptic glutamate receptors have been found in the venom of Philanthus triangulum [3,6,7]. The venom of Megascolia flavifrons also contains two kinins (megascoliakinins) [8,9] that block nicotinic acetylcholine receptors [10]. In our previous study, the novel peptides such as pompilidotoxins and wasp kinins have been identified from the spider wasps Anoplius samariensis [11–14], Batozonellus maculiforns [12], and Cyphononyx dorsaris [15] by mass
M. Hisada et al. / Biochemical and Biophysical Research Communications 330 (2005) 1048–1054
spectrometry. However, none of the identified peptides were shown to be involved in the long-term, non-lethal activity. Solitary wasp venoms have also been found to contain diverse low molecular weight components and proteins, indicating the possibility that these components are responsible for the paralytic activity. To explore the compounds with the paralytic activity, the venom components of the spider wasp, A. samariensis was analyzed by micro-liquid chromatography/electrospray ionization mass spectrometry (l-LC/ESI-MS), nuclear magnetic resonance spectrometry (NMR), ultrafiltration, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). In this article, we present the identification of the low molecular weight compounds by comprehensive MS analysis of the venom, and the long-term non-lethal paralytic activity of the high molecular weight fractions. We also report the primary structure of a novel multiple-cysteine peptide, As-fr-19, which was identified from the high molecular weight fractions.
Materials and methods Wasps and spiders Three female wasps (A. samariensis) were collected in Kyoto and Osaka, Japan. Wasps were raised at 25 C in plastic cages and provided with abundant water and honey. For milking of the venom, wasps were immobilized with CO2 and confined on the stage to be provoked to sting a piece of Parafilm (Pechiney Plastic Packaging, IL, USA). The venom droplets were collected and immediately frozen at 80 C. Female spiders (Nephila clavata) for bioassay were collected in Osaka, Japan. Bioassay Fivefold diluted venom and ultrafiltrate fractions (filtrate and supernatant: see the section ‘‘Identification of the full-length As-fr-19 sequence’’) were tested for paralytic assay using spiders (N. clavata). The spiders were fixed on the stage by bands made from polystyrene, and the sample solution (0.2 ll) was injected from the side of the spidersÕ cephalothorax in the direction of its center. The syringe (SEG, Victoria, Australia) was sharpened to be less than 0.19 mm diameters of its sting under the microscope. Mass spectrometry l-LC/ESI-MS. Twenty-fivefold diluted venom (1 ll) was subjected to l-LC/ESI-MS analysis. A LC system Hewlett–Packard 1100 (Hewlett–Packard, Palo Alto, CA) was interfaced to a hybrid quadrupole orthogonal acceleration tandem mass spectrometer QTOF (Micromass, Manchester, UK) fitted with a Z-spray electrospray ion source. The LC separation was performed using a Wakosil II 3C18 RS (3 lm; 2 · 75 mm; Wako Pure Chemical Industries, Osaka, Japan) with a linear gradient of 0–100% methanol in 10 mM heptafluorobutyric acid (Tokyo Kasei Kogyo, Tokyo, Japan) delivered over 1 h at a flow rate of 80 ll/min. A cone voltage of 20 V and a capillary voltage of 2.8 kV were used in positive ionization mode. Nitrogen acted as both the nebulizing and desolvating gas. Leucine-enkephalin ([M + H]+ = 556.2771; 200 ng/ll; Peptide Institute, Osaka, Japan), used as a lock mass, was continuously infused post-column at 0.5 ll/min via a syringe pump. A standard mixture (1 ll containing 100 pmol each of alanine,
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c-aminobutyric acid [GABA], histamine, lysine, glutamic acid, arginine, putrescine, cadaverine, spermidine, spermine, tyramine, octopamine, dopamine, serotonin, acetylcholine, nicotine, adrenalin, noradrenalin, and adenosine; Nacalai Tesque, Kyoto, Japan) was used as standard for LC/ESI-MS. MALDI-TOFMS. The matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrum was acquired on a Voyager Elite MALDI-TOFMS (Applied Biosystems, Foster City, CA, USA), equipped with a delayed extraction source and 337 nm pulsed nitrogen laser. The accelerating voltage was 20 kV. A Matrix, sinapinic acid (Aldrich Chemical, WI, USA) was prepared at a concentration of 5 mg/ ml in 1:2 acetonitrile/0.1% trifluoroacetic acid (TFA). Twenty-fivefold diluted venom (0.2 ll) dropped onto the MALDI sample plate was added to the matrix (0.2 ll) and allowed to dry at room temperature. SDS–PAGE Tenfold diluted venom was analyzed by SDS–PAGE using a BioRad Mini-Protean II electrophoresis cell (Bio-Rad, Hercules, CA, USA) and the gel was stained using a silver satin kit (Wako Pure Chemical Industries, Osaka, Japan) according to the manufacturerÕs instructions. Identification of the full-length As-fr19 sequence Venom (0.8 ll) was dissolved in water (100 ll) and subjected to ultrafiltration using a Microcon YM-10 centrifugal filter unit (Millipore, Billerica, MA, USA). The sample was centrifuged twice for 25 min at 8000 rpm in a 45 angle rotor at 4 C. After the ultrafiltration, the filtrate and supernatant fractions were lyophilized and then dissolved in 5 ll water for paralysis assay, resulting in a 6.25-hold concentration compared to the original venom. The supernatant fraction was separated by reversed phase high performance liquid chromatography (HPLC) with a linear gradient of 2–80% acetonitrile in 0.1% TFA. The N-terminal amino acid sequence of the fraction designated As-fr-19 (VSFXLLPIVPGXTQYVIRAYAF where X is an unidentified amino acid) was determined by sequencing of the purified fraction on a model 491 cLC peptide sequencer (Applied Biosystems). Total RNA was extracted from the posterior part of the wasp using SepaZol-I reagent (Nacalai Tesque). The amplified whole cDNA was prepared employing the SMART PCR cDNA system (BD Science, Palo Alto, CA, USA) as previously described [16,17], and applied to cloning of an As-fr-19 cDNA as a template DNA. The partial As-fr-19 cDNA was obtained by PCR using first-round degenerative primers encoding FCLLPIVPG, followed by reamplification of the first-round PCR products using the second degenerative primers encoding VPGPCTQYV. The nested PCR products were subcloned and sequenced using SP6 and T7 primer sets, and Big-Dye sequencing kit version 1.1 on a model 373 DNA sequencer (Applied Biosystems). To determine the full-length sequence of the cDNA, we performed rapid amplification of 5 0 -ends using a primer complementary to nucleotides 288–307 for the first-round PCR and a primer complementary to nucleotides 257–277 for the second-round PCR. The final PCR products were sequenced as described above. The nucleotide sequence data are available in the DDBJ, EMBL, and GenBank Sequence databases under Accession No. AB186137.
Results and discussion l-LC/ESI-MS and NMR analysis of the venom from A. samariensis The low molecular weight components of the venom from A. samariensis were identified by l-LC/ESI-MS
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analysis. Nineteen authentic compounds that were expected to be present in the venom were used as standards. Glutamic acid, GABA, alanine, histamine, tyramine, spermine, and adenosine were identified by the accurate mass and the HPLC retention time (Fig. 1 and Table 1). NMR analysis of the low molecular weight components was in good agreement with the MS analysis (data not shown), and provided the quantitative data that GABA and glutamic acid were major components in the whole venom of A. samariensis. This low-molecular weight compound profile is in contrast with those of other wasps, given that pain-producing biogenic amines including histamine and serotonin, which are abundantly present in other waspsÕ venoms, were detected in much lower levels in the venom of A.
samariensis. For example, the venoms of Vespid wasps (social wasps) contain serotonin and histamine as the major active amines, and the venom of Eumenes wasps (solitary wasps) has histamine as major amine components, but lacks serotonin [18,19]. These findings suggested that these active amines are responsible for pain-producing by the venom, although the role of the active amines on the paralysis of the solitary waspsÕ preys is not clear. Effect of the venom injection into spider To examine the paralytic activity of the wasp venom, the venom of A. samariensis was injected into Joro spiders (N. clavata). The injection of the diluted venom
Fig. 1. l-LC/ESI-MS analysis of the venom from A. samariensis. Selected ion chromatograms of (A) adenosine (268), (B) spermine (203), (C) glutamic acid (148), (D) tyramine (138), (E) histamine (112), (F) GABA (104), and (G) alanine (90). Numbers in parentheses indicate the selected masses. The bottom (H) shows the total ion chromatogram (TIC).
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Table 1 Observed and calculated masses of low molecular weight components in the venom from A. samariensis Retention time
Mass (obs.)
Formula
Mass (calc.)
Error (mDa)
22.4 44.4 5.6 27.6 28 7.5 5.6
268.1042 203.2219 148.059 138.0906 112.0858 104.0672 90.0579
C10H14N5O4 (adenosine) C10H27N4 (spermine) C5H10NO4 (glutamic acid) C8H12NO (tyramine) C5H10N3 (histamine) C4H10NO2 (GABA) C3H8NO2 (alanine)
268.1046 203.2236 148.061 138.0919 112.0875 104.0712 90.0555
0.4 1.6 2 1.3 1.7 4 2.4
(0.2 ll, 1:5 with water) induced the long-term non-lethal paralysis of all the five spiders, which lasted until they died after 3–14 days post-injection. This biological assay confirmed the reproducibility of the paralytic activity of the venom. The high molecular weight fraction was prepared by ultrafiltration and applied to the same biological assay. As shown in Fig. 2, injection of the supernatant resulted in the equivalent paralysis for all spiders; the injected spiders were also paralyzed for 3–10 days post-injection
Fig. 3. SDS–PAGE of the venom from A. samariensis. Lane 1, molecular weight markers; lane 2, venom from A. samariensis (0.09 ll).
Fig. 2. (A) Paralytic assay of the A. samariensis venom. Spiders that recovered within 15 min were regarded as ‘‘not paralyzed.’’ (B) Spiders were injected with 0.2 ll of (a) water (control), (b) diluted venom, (c) supernatant, or (d) filtrate. The spiders in (a) and (d) recovered quickly, however, those in (b) and (c) were paralyzed until they died after 3–14 days post-injection. Their legs were stretching in contrast with those of dead spiders. Notably, such paralysis, which is featured by stretching of legs, was observed by administration of both whole venom (b) and high molecular components (c).
Fig. 4. MALDI-TOFMS spectrum of the venom from A. samariensis. The inserted table indicates the mass of the observed and calculated ions ([M + H]+).
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Fig. 5. Sequence alignment of As-fr-19 and other related toxins. All conserved half-cysteines are indicated in boldface.
followed by death. In contrast, the filtrate was devoid of any activity. These results provide evidence that the high molecular components are requisite for the paralytic effect. SDS–PAGE and MS analysis of the A. samariensis venom revealed that it contains a variety of large peptides and proteins with molecular weights ranging from 4 to 100 kDa (Figs. 3 and 4), indicating the possibility that diverse protein components are involved in the long-term paralysis. This result can be interpreted in two ways. First, some tissue-degrading enzymes are likely essential for the paralytic activity of the venom; indeed, degrading enzymes, such as hyaluronidase and phospholipase, are known to be contained in hornet [20–25] and honeybee [26,27] venoms. These findings imply the possibility that several high molecular weight components in the venom of A. samariensis are indirectly implicated with the long-term paralysis by degradation of tissues. Second, venomous proteins with high molecular weight may possess novel neurotoxic activities, which have a key role in the long-term paralysis. To date, latrotoxins, isolated from a spider species, Lactrodectus, have been identified as arthropod protein neurotoxins with the molecular weight of more than 100 KDa, which induces extraordinary oversecretion of excitatory neurotransmitters at the nerve terminus [28–30]. Consequently, the high molecular proteins in the A. samariensis venom are expected to exert novel neurotoxic actions responsible for the long-term paralysis. Sequence analysis of a high molecular weight component, As-fr-19 Sequence analysis of each component is currently in progress. We determined the sequences of a peptide and its cDNA corresponding to the fraction with a mass number of 6663.5 (Fig. 4). This peptide, designated Asfr-19, comprised 58 amino acids including six cysteine residues (Fig. 5). The predicted mass number of the cross-linked form is in good agreement with the observed mass of the fraction, confirming the presence of the folded form of As-fr-19 in the venom. All wasp and bee peptide toxins obtained so far were shown to comprise fewer than 26 amino acid residues [11,13–
15,19,31–33]. Also of interest is that As-fr-19 displays high sequence identity to several snake and sea anemone peptide toxins, whereas significant similarity was not found with any insect toxins except for a spider toxin (Toxin-1) [34]. These results indicate that As-fr-19 belongs to a novel insect peptide toxin family. Furthermore, as shown in Fig. 5, the cysteine locations all coincide with those of snake toxins (dendrotoxin I [35], K [36], and calcicludine [37]) and sea anemone toxins (Anemonia sulcata kalicludines :AsKC 1–3 [38]), suggesting that all these toxins share the essential tertiary structure. In addition, dendrotoxins, AsKCs, and calcicludine were shown to exert potent inhibitory effects on potassium and calcium channels, respectively [38–41]. These data support a notion that As-fr-19 also serves as a potassium and/or calcium blocker and then participates in the long-term non-lethal paralysis on the prey. Toxicological and pharmacological activities of As-fr-19 are now being examined. In summary, we show the novel component and toxicity of the venom of the solitary spider wasp, A. samariensis. Our data will contribute to the further understanding of the mechanism in the longterm non-lethal paralysis.
Acknowledgments We are grateful to Prof. Akira Endo (Ritsumeikan University) for the identification of wasps. A part of this study 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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Molecular components and toxicity of the venom of the solitary wasp, Anoplius samariensis Miki Hisada a,*, Honoo Satake a, Katsuyoshi Masuda a, Masato Aoyama a, Kazuya Murata b, Testuro Shinada c, Takashi Iwashita a, Yasufumi Ohfune c, Terumi Nakajima d a
Suntory Institute for Bioorganic Research, 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan Sakai Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 1-1-53 Takasu-cho, Sakai-shi, Osaka 590-0003, Japan c Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan d Hoshi University, 2-4-41 Ebara, Shinagawa, Tokyo 142-8501, Japan
b
Received 15 March 2005 Available online 23 March 2005
Abstract The solitary spider wasp, Anoplius samariensis, is known to exhibit a unique long-term, non-lethal paralysis in spiders that it uses as a food source for its larvae. However, neither detailed venom components nor paralytic compounds have ever been characterized. In this study, we examined the components in the low molecular weight fraction of the venom and the paralytic activity of the high molecular weight fraction. The major low molecular weight components of the venom were identified as c-aminobutyric acid and glutamic acid by micro-liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance spectrometry analysis. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis and mass analysis revealed that the A. samariensis venom contained the various proteins with weights of 4–100 kDa. A biological assay using Joro spiders (Nephila clavata) clearly showed that the high molecular weight fraction of the venom prepared by ultrafiltration exerted as potent non-lethal long-term paralysis as the whole venom, whereas the low molecular weight fraction was devoid of any paralytic activity. These results indicated that several venomous proteins in the high molecular weight fraction are responsible for the paralytic activity. Furthermore, we determined the primary structure of one component designated As-fr-19, which was a novel multiple-cysteine peptide with high sequence similarity to several sea anemone and snake toxins including dendrotoxins, rather than any insect toxic peptides identified so far. Taken together, our data showed the unprecedented molecular and toxicological profiles of wasp venoms. 2005 Elsevier Inc. All rights reserved. Keywords: Solitary wasp; Venom; Toxin; Paralysis; Channel blocker
The venoms of arthropods have attracted considerable interest as a potential source of bioactive substances. Venoms of solitary wasps are known to possess long-term, non-lethal paralytic effects on their preys (insects or spiders). The immobilized preys are then used to feed the waspsÕ larvae [1,2]. Such unique paralytic activity suggests the presence of novel neurotoxic compounds in the venom of solitary wasps [3–5]. To date, *
Corresponding author. Fax: +81 75 962 2115. E-mail address: [email protected] (M. Hisada).
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several neurotoxic components have been characterized from solitary wasp venoms. Philanthotoxins that inhibit neuromuscular transmission by blocking post-synaptic glutamate receptors have been found in the venom of Philanthus triangulum [3,6,7]. The venom of Megascolia flavifrons also contains two kinins (megascoliakinins) [8,9] that block nicotinic acetylcholine receptors [10]. In our previous study, the novel peptides such as pompilidotoxins and wasp kinins have been identified from the spider wasps Anoplius samariensis [11–14], Batozonellus maculiforns [12], and Cyphononyx dorsaris [15] by mass
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spectrometry. However, none of the identified peptides were shown to be involved in the long-term, non-lethal activity. Solitary wasp venoms have also been found to contain diverse low molecular weight components and proteins, indicating the possibility that these components are responsible for the paralytic activity. To explore the compounds with the paralytic activity, the venom components of the spider wasp, A. samariensis was analyzed by micro-liquid chromatography/electrospray ionization mass spectrometry (l-LC/ESI-MS), nuclear magnetic resonance spectrometry (NMR), ultrafiltration, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). In this article, we present the identification of the low molecular weight compounds by comprehensive MS analysis of the venom, and the long-term non-lethal paralytic activity of the high molecular weight fractions. We also report the primary structure of a novel multiple-cysteine peptide, As-fr-19, which was identified from the high molecular weight fractions.
Materials and methods Wasps and spiders Three female wasps (A. samariensis) were collected in Kyoto and Osaka, Japan. Wasps were raised at 25 C in plastic cages and provided with abundant water and honey. For milking of the venom, wasps were immobilized with CO2 and confined on the stage to be provoked to sting a piece of Parafilm (Pechiney Plastic Packaging, IL, USA). The venom droplets were collected and immediately frozen at 80 C. Female spiders (Nephila clavata) for bioassay were collected in Osaka, Japan. Bioassay Fivefold diluted venom and ultrafiltrate fractions (filtrate and supernatant: see the section ‘‘Identification of the full-length As-fr-19 sequence’’) were tested for paralytic assay using spiders (N. clavata). The spiders were fixed on the stage by bands made from polystyrene, and the sample solution (0.2 ll) was injected from the side of the spidersÕ cephalothorax in the direction of its center. The syringe (SEG, Victoria, Australia) was sharpened to be less than 0.19 mm diameters of its sting under the microscope. Mass spectrometry l-LC/ESI-MS. Twenty-fivefold diluted venom (1 ll) was subjected to l-LC/ESI-MS analysis. A LC system Hewlett–Packard 1100 (Hewlett–Packard, Palo Alto, CA) was interfaced to a hybrid quadrupole orthogonal acceleration tandem mass spectrometer QTOF (Micromass, Manchester, UK) fitted with a Z-spray electrospray ion source. The LC separation was performed using a Wakosil II 3C18 RS (3 lm; 2 · 75 mm; Wako Pure Chemical Industries, Osaka, Japan) with a linear gradient of 0–100% methanol in 10 mM heptafluorobutyric acid (Tokyo Kasei Kogyo, Tokyo, Japan) delivered over 1 h at a flow rate of 80 ll/min. A cone voltage of 20 V and a capillary voltage of 2.8 kV were used in positive ionization mode. Nitrogen acted as both the nebulizing and desolvating gas. Leucine-enkephalin ([M + H]+ = 556.2771; 200 ng/ll; Peptide Institute, Osaka, Japan), used as a lock mass, was continuously infused post-column at 0.5 ll/min via a syringe pump. A standard mixture (1 ll containing 100 pmol each of alanine,
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c-aminobutyric acid [GABA], histamine, lysine, glutamic acid, arginine, putrescine, cadaverine, spermidine, spermine, tyramine, octopamine, dopamine, serotonin, acetylcholine, nicotine, adrenalin, noradrenalin, and adenosine; Nacalai Tesque, Kyoto, Japan) was used as standard for LC/ESI-MS. MALDI-TOFMS. The matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrum was acquired on a Voyager Elite MALDI-TOFMS (Applied Biosystems, Foster City, CA, USA), equipped with a delayed extraction source and 337 nm pulsed nitrogen laser. The accelerating voltage was 20 kV. A Matrix, sinapinic acid (Aldrich Chemical, WI, USA) was prepared at a concentration of 5 mg/ ml in 1:2 acetonitrile/0.1% trifluoroacetic acid (TFA). Twenty-fivefold diluted venom (0.2 ll) dropped onto the MALDI sample plate was added to the matrix (0.2 ll) and allowed to dry at room temperature. SDS–PAGE Tenfold diluted venom was analyzed by SDS–PAGE using a BioRad Mini-Protean II electrophoresis cell (Bio-Rad, Hercules, CA, USA) and the gel was stained using a silver satin kit (Wako Pure Chemical Industries, Osaka, Japan) according to the manufacturerÕs instructions. Identification of the full-length As-fr19 sequence Venom (0.8 ll) was dissolved in water (100 ll) and subjected to ultrafiltration using a Microcon YM-10 centrifugal filter unit (Millipore, Billerica, MA, USA). The sample was centrifuged twice for 25 min at 8000 rpm in a 45 angle rotor at 4 C. After the ultrafiltration, the filtrate and supernatant fractions were lyophilized and then dissolved in 5 ll water for paralysis assay, resulting in a 6.25-hold concentration compared to the original venom. The supernatant fraction was separated by reversed phase high performance liquid chromatography (HPLC) with a linear gradient of 2–80% acetonitrile in 0.1% TFA. The N-terminal amino acid sequence of the fraction designated As-fr-19 (VSFXLLPIVPGXTQYVIRAYAF where X is an unidentified amino acid) was determined by sequencing of the purified fraction on a model 491 cLC peptide sequencer (Applied Biosystems). Total RNA was extracted from the posterior part of the wasp using SepaZol-I reagent (Nacalai Tesque). The amplified whole cDNA was prepared employing the SMART PCR cDNA system (BD Science, Palo Alto, CA, USA) as previously described [16,17], and applied to cloning of an As-fr-19 cDNA as a template DNA. The partial As-fr-19 cDNA was obtained by PCR using first-round degenerative primers encoding FCLLPIVPG, followed by reamplification of the first-round PCR products using the second degenerative primers encoding VPGPCTQYV. The nested PCR products were subcloned and sequenced using SP6 and T7 primer sets, and Big-Dye sequencing kit version 1.1 on a model 373 DNA sequencer (Applied Biosystems). To determine the full-length sequence of the cDNA, we performed rapid amplification of 5 0 -ends using a primer complementary to nucleotides 288–307 for the first-round PCR and a primer complementary to nucleotides 257–277 for the second-round PCR. The final PCR products were sequenced as described above. The nucleotide sequence data are available in the DDBJ, EMBL, and GenBank Sequence databases under Accession No. AB186137.
Results and discussion l-LC/ESI-MS and NMR analysis of the venom from A. samariensis The low molecular weight components of the venom from A. samariensis were identified by l-LC/ESI-MS
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analysis. Nineteen authentic compounds that were expected to be present in the venom were used as standards. Glutamic acid, GABA, alanine, histamine, tyramine, spermine, and adenosine were identified by the accurate mass and the HPLC retention time (Fig. 1 and Table 1). NMR analysis of the low molecular weight components was in good agreement with the MS analysis (data not shown), and provided the quantitative data that GABA and glutamic acid were major components in the whole venom of A. samariensis. This low-molecular weight compound profile is in contrast with those of other wasps, given that pain-producing biogenic amines including histamine and serotonin, which are abundantly present in other waspsÕ venoms, were detected in much lower levels in the venom of A.
samariensis. For example, the venoms of Vespid wasps (social wasps) contain serotonin and histamine as the major active amines, and the venom of Eumenes wasps (solitary wasps) has histamine as major amine components, but lacks serotonin [18,19]. These findings suggested that these active amines are responsible for pain-producing by the venom, although the role of the active amines on the paralysis of the solitary waspsÕ preys is not clear. Effect of the venom injection into spider To examine the paralytic activity of the wasp venom, the venom of A. samariensis was injected into Joro spiders (N. clavata). The injection of the diluted venom
Fig. 1. l-LC/ESI-MS analysis of the venom from A. samariensis. Selected ion chromatograms of (A) adenosine (268), (B) spermine (203), (C) glutamic acid (148), (D) tyramine (138), (E) histamine (112), (F) GABA (104), and (G) alanine (90). Numbers in parentheses indicate the selected masses. The bottom (H) shows the total ion chromatogram (TIC).
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Table 1 Observed and calculated masses of low molecular weight components in the venom from A. samariensis Retention time
Mass (obs.)
Formula
Mass (calc.)
Error (mDa)
22.4 44.4 5.6 27.6 28 7.5 5.6
268.1042 203.2219 148.059 138.0906 112.0858 104.0672 90.0579
C10H14N5O4 (adenosine) C10H27N4 (spermine) C5H10NO4 (glutamic acid) C8H12NO (tyramine) C5H10N3 (histamine) C4H10NO2 (GABA) C3H8NO2 (alanine)
268.1046 203.2236 148.061 138.0919 112.0875 104.0712 90.0555
0.4 1.6 2 1.3 1.7 4 2.4
(0.2 ll, 1:5 with water) induced the long-term non-lethal paralysis of all the five spiders, which lasted until they died after 3–14 days post-injection. This biological assay confirmed the reproducibility of the paralytic activity of the venom. The high molecular weight fraction was prepared by ultrafiltration and applied to the same biological assay. As shown in Fig. 2, injection of the supernatant resulted in the equivalent paralysis for all spiders; the injected spiders were also paralyzed for 3–10 days post-injection
Fig. 3. SDS–PAGE of the venom from A. samariensis. Lane 1, molecular weight markers; lane 2, venom from A. samariensis (0.09 ll).
Fig. 2. (A) Paralytic assay of the A. samariensis venom. Spiders that recovered within 15 min were regarded as ‘‘not paralyzed.’’ (B) Spiders were injected with 0.2 ll of (a) water (control), (b) diluted venom, (c) supernatant, or (d) filtrate. The spiders in (a) and (d) recovered quickly, however, those in (b) and (c) were paralyzed until they died after 3–14 days post-injection. Their legs were stretching in contrast with those of dead spiders. Notably, such paralysis, which is featured by stretching of legs, was observed by administration of both whole venom (b) and high molecular components (c).
Fig. 4. MALDI-TOFMS spectrum of the venom from A. samariensis. The inserted table indicates the mass of the observed and calculated ions ([M + H]+).
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Fig. 5. Sequence alignment of As-fr-19 and other related toxins. All conserved half-cysteines are indicated in boldface.
followed by death. In contrast, the filtrate was devoid of any activity. These results provide evidence that the high molecular components are requisite for the paralytic effect. SDS–PAGE and MS analysis of the A. samariensis venom revealed that it contains a variety of large peptides and proteins with molecular weights ranging from 4 to 100 kDa (Figs. 3 and 4), indicating the possibility that diverse protein components are involved in the long-term paralysis. This result can be interpreted in two ways. First, some tissue-degrading enzymes are likely essential for the paralytic activity of the venom; indeed, degrading enzymes, such as hyaluronidase and phospholipase, are known to be contained in hornet [20–25] and honeybee [26,27] venoms. These findings imply the possibility that several high molecular weight components in the venom of A. samariensis are indirectly implicated with the long-term paralysis by degradation of tissues. Second, venomous proteins with high molecular weight may possess novel neurotoxic activities, which have a key role in the long-term paralysis. To date, latrotoxins, isolated from a spider species, Lactrodectus, have been identified as arthropod protein neurotoxins with the molecular weight of more than 100 KDa, which induces extraordinary oversecretion of excitatory neurotransmitters at the nerve terminus [28–30]. Consequently, the high molecular proteins in the A. samariensis venom are expected to exert novel neurotoxic actions responsible for the long-term paralysis. Sequence analysis of a high molecular weight component, As-fr-19 Sequence analysis of each component is currently in progress. We determined the sequences of a peptide and its cDNA corresponding to the fraction with a mass number of 6663.5 (Fig. 4). This peptide, designated Asfr-19, comprised 58 amino acids including six cysteine residues (Fig. 5). The predicted mass number of the cross-linked form is in good agreement with the observed mass of the fraction, confirming the presence of the folded form of As-fr-19 in the venom. All wasp and bee peptide toxins obtained so far were shown to comprise fewer than 26 amino acid residues [11,13–
15,19,31–33]. Also of interest is that As-fr-19 displays high sequence identity to several snake and sea anemone peptide toxins, whereas significant similarity was not found with any insect toxins except for a spider toxin (Toxin-1) [34]. These results indicate that As-fr-19 belongs to a novel insect peptide toxin family. Furthermore, as shown in Fig. 5, the cysteine locations all coincide with those of snake toxins (dendrotoxin I [35], K [36], and calcicludine [37]) and sea anemone toxins (Anemonia sulcata kalicludines :AsKC 1–3 [38]), suggesting that all these toxins share the essential tertiary structure. In addition, dendrotoxins, AsKCs, and calcicludine were shown to exert potent inhibitory effects on potassium and calcium channels, respectively [38–41]. These data support a notion that As-fr-19 also serves as a potassium and/or calcium blocker and then participates in the long-term non-lethal paralysis on the prey. Toxicological and pharmacological activities of As-fr-19 are now being examined. In summary, we show the novel component and toxicity of the venom of the solitary spider wasp, A. samariensis. Our data will contribute to the further understanding of the mechanism in the longterm non-lethal paralysis.
Acknowledgments We are grateful to Prof. Akira Endo (Ritsumeikan University) for the identification of wasps. A part of this study 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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