Identification of Bradykinins in Solitary Wasp Venoms
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Toxicon 40 82002) 309±312 www.elsevier.com/locate/toxicon Identi®cation of bradykinins in solitary wasp venoms Katsuhiro Konnoa,b,*, Mario Sergio Palmaa,b, Izaura Yoshico Hitarab,c, Maria Aparecida Julianob,c, Luiz Julianob,c, Tadashi Yasuharad aCenter of Study of Social Insects, Department of Biology, Institute of Biosciences of Rio Claro, SaÄo Paulo State University, Rio Claro, SP 13506-900, Brazil bCenter for Applied Toxinology, CEPID/FAPESP, SaÄo Paulo, SP 05468-901, Brazil cDepartment of Biophysics, Paulista Medical School, Federal University of SaÄo Paulo, SaÄo Paulo, SP 04044-020, Brazil dDepartment of Nutrition, Junior College of Agriculture, Tokyo University of Agriculture, Setagaya, Tokyo 156-8502, Japan Received 17 July 2001; accepted 7 September 2001 Abstract Bradykinins were identi®ed in three solitary wasp venoms. Puri®cation and characterization of the venom extract of the scoliid wasp Megacampsomeris prismatica led to the identi®cation of bradykinin and threonine6-bradykinin as the major peptide components. The survey of a number of extracts from solitary wasp venom by MALDI±TOF MS revealed that the venoms of two other scoliid wasps, Campsomeriella annulata annulata and Carinoscolia melanosoma fascinata, also contained Thr6-BK as one of the major components. Thus, this study showed the presence of bradykinins in some of the solitary wasp venoms. Moreover, it indicated that these peptides play a major role in their paralyzing action for prey capture because these bradykinins have been shown to block the synaptic transmission of the nicotinic acetylcholine receptor in the insect central nervous system. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Bradykinin; Solitary wasp; Venom; MALDI±TOF MS The venoms of the Hymenoptera are a rich source of Thr6-BK was also isolated from the venom of another various bioactive substances 8Piek, 1986). For example, the scoliid wasp Colpa interrupta 8Piek et al., 1990). These venoms of the honeybee, hornets and paper wasps have been kinins irreversibly block the synaptic transmission of the well documented both chemically and pharmacologically, nicotinic acetylcholine receptor in the insect central nervous which revealed that a variety of bioactive peptides and enzy- system 8Piek et al., 1987a,b; Hue and Piek, 1989; Piek, matic proteins act together to produce various biological 1991). Shortly thereafter, philanthotoxins were found in effects. In contrast, only little is known about the chemical the venom of the sphecid wasp Philanthus triangulum components and biological properties of solitary wasp 8Eldefrawi et al., 1988; Piek et al., 1988). They are acylpo- venoms despite thousands of species inhabiting the planet. lyamine toxins and non-competitive antagonists of the Solitary wasps paralyze insects or spiders with their stinging postsynaptic glutamate and nicotinic receptors 8Nakanishi venoms, and feed the paralyzed prey to their larvae. There- et al., 1990; Piek and Hue, 1989). We have recently fore, the solitary wasp venoms may contain various bioactive surveyed the bioactive substances in solitary wasp venoms substances, in particular, neurotoxins. and found novel peptide neurotoxins, pompilidotoxins The ®rst neurotoxic component characterized in solitary 8PMTXs), from the venoms of the pompilid wasps Anoplius wasp venom was kinins. Piek et al. 81987a,b) isolated samariensis and Batozonellus maculifrons 8Konno et al., threonine6-bradykinin 8Thr6-BK) and megascoliakinin 1997, 1998). PMTXs affect both vertebrate and invertebrate 8Thr6-BK±Lys±Ala) from the venom of the European nervous systems, which is due to the slowing or blocking of scoliid wasp Megascolia ¯avifrons 8Yasuhara et al., 1987). sodium channel inactivation 8Sahara et al., 2000). This activity is similar to those of the a-scorpion and sea anemone toxins, but PMTX can discriminate the neuronal * Corresponding author. Tel: 155-19-534-8523; fax: 155-19- 534-0009. and cardiac sodium channels 8Kinoshita et al., 2001). A E-mail address: [email protected] 8K. Konno). further survey of solitary wasp venom components led to 0041-0101/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S0041-0101801)00230-6 310 K. Konno et al. / Toxicon 40 82002) 309±312 Table 1 TFA over 30 min at a ¯ow rate of 2.5 ml/min 8Fig. 1A). The solitary wasp species examined for the presence of BK and The major peak eluted at 17.3 min was collected and further Thr6-BK in their venoms. The presence of the peaks due to BK puri®ed by reverse phase HPLC using CAPCELL PAK C18, 8m/z 1059.5) and Thr6-BK 8m/z 1074.5) were analyzed by 6 £ 150 mm with isocratic elution of 18% CH3CN/H2O/ MALDI±TOF MS 0.1% TFA at ¯ow rate of 1 ml/min. An aliquot of the Solitary wasp species BK Thr6-BK main fraction eluted at 26.5 min was taken for sequence analysis by Edman degradation. This was shown to be Pompilidae 8pompilid wasps) about a 1:2 mixture of BK and Thr6-BK since the sequence Episyron arrogans 22 of nine amino acids was shown as Arg±Pro±Pro±Gly±Phe± Anoplius samariensis 22 Ser/Thr±Pro±Phe±Arg, where the 6 position appeared as an Batozonellus annulatus 22 approximately 1:2 mixture of Ser and Thr. The MALDI± Batozonellus maculifrons 22 TOF MS 8Micromass TofSpec-2, positive ion mode, a- Tachypompilus analis 22 cyano-4-hydroxycinnamic acid as the matrix) of this Cyphononyx dorsalis 21 Leptodialepis sugiharai 22 fraction was consistent with this result; the peaks at m/z 1 Eumenidae 8eumenid wasps) 1059.5 and 1074.5 8M 1 H) appeared at about a 1:2 inten- 6 Anterhynchium ¯avomarginatum micado 22 sity, corresponding to BK and Thr -BK, respectively. Orancistrocerus drewseni drewseni 22 Comparison of these peptides with the authentic specimens Oreumenes decoratus 22 by HPLC ®nally con®rmed the presence of these peptides. Eumenes fraterculus 22 We further surveyed the solitary wasp venoms for the Eumenes micado 22 presence of these kinins using MALDI±TOF MS. The Eumenes rubronotatum rubronotatum 22 venom extracts of 26 species from four families were Eumenes rubrofemoratus 22 prepared in the same manner as described above and Sphecidae 8sphecid wasps) analyzed by MALDI±TOF MS, `screening' the peaks due Ammophila subulosa 22 6 Hoplammophila aemulans 22 to BK 8m/z 1059.5) and Thr -BK 8m/z1074.5). The exam- Sceliphron caementarium 22 ined species are listed in Table 1. Most of them are dominant 6 Sphex argentatus argentatus 22 species in Japan. As a consequence, the Thr -BK peak was Isodontia nigella 22 found in the extracts from three species, the scoliid wasps Isodontia harmandi 22 Campsomeriella annulata annulata and Carinoscolia Tachytes sinensis sinensis 22 melanosoma fascinata and the spider wasp Cyphononyx Scoliidae 8scoliid wasps) dorsalis, whereas the BK peak was never found in any Megacampsomeris prismatica 11 venom extract. Of these, the isolation and identi®cation of Campsomeriella annulata annulata 21 Thr6-BK from the venom of the spider wasp Cyphononyx Scolia histrionica japonica 22 dorsalis has already been reported 8Konno et al., 2001). The Scolia decorata ventralis 22 Scolia oculata 22 HPLC pro®les of the venom extracts from Campsomeriella Carinoscolia melanosoma fascinata 21 annulata annulata and Carinoscolia melanosoma fascinata are shown in Fig. 1B and C, respectively. Puri®cation in the manner similar to that described above, followed by the identi®cation of bradykinins in three scoliid wasp comparison with the synthetic authentic peptide by HPLC venoms. Since bradykinins have been reported to be con®rmed the presence of Thr6-BK in these venoms. As neurotoxic in the insect central nervous systems, they may seen in their HPLC pro®les, this peptide is one of the play a major role in their paralyzing action for prey capture. major peptide components in these scoliid wasp venoms. Reported herein are the isolation and identi®cation of these Bradykinin related peptides have been found in many neurotoxic peptides in solitary wasp venoms. venoms of Hymenoptera such as ants and social wasps First, bradykinin 8BK) and Thr6-BK were found in the 8Nakajima et al., 1985; Piek et al., 1989; Piek, 1991). In venom extract of the scoliid wasp Megacampsomeris particular, those found in the Vespid wasp venoms are prismatica. Twenty-four lyophilized venom sacs of M. collectively called wasp kinins. They have the sequence of prismatica, collected in Kanagawa, Ibaraki and Kyoto, BK or Thr6-BK in their 11±18 amino acid chain and exhibit Japan, were extracted with 1:1 CH3CN/H2O/0.1% TFA similar pharmacological activities to BK. When injected 80.2 ml £ 4) and the extracts were subjected to reverse- into vertebrate predators by stinging, they produce severe phase HPLC using CAPCELL PAK C18,10£ 250 mm pain, thus playing a signi®cant role in their defense system. with a linear gradient from 5 to 65% CH3CN/H2O/0.1% On the other hand, kinins may play a different role in Fig. 1. The HPLC pro®les of the extracts of the solitary wasp venoms. Each venom extract was injected to the reverse-phase HPLC column using CAPCELL PAK C18,10£ 250 mm, with a linear gradient from 5 to 65% Me3CN±H2O containing 0.1% TFA for 30 min, monitored by UV 214 nm. 8A) Megacampsomeris prismatica, 8B) Campsomeriella annulata annulata, 8C) Carinoscolia melanosoma fascinata. K. Konno et al. / Toxicon 40 82002) 309±312 311 312 K. Konno et al. / Toxicon 40 82002) 309±312 solitary wasp venoms. Piek and co-workers isolated Thr6- Shimizu, E., Nakayama, H., Seyama, I., 2001. Novel wasp toxin BK and megascoliakinin 8Thr6-BK±Lys±Ala) from discriminates between neuronal and cardiac sodium channels. European scoliid wasp venoms 8Piek et al., 1987a,b; Yasu- Mol. Pharmacol. 59, 1457±1463. hara et al. 1987), and investigated the effects of these Konno, K., Miwa, A., Takayama, H., Hisada, M., Itagaki, Y., Naoki, peptides on the insect central nervous system because H., Yasuhara, T., Kawai, N., 1997.