Toxicon 71 (2013) 105–112 Contents lists available at SciVerse ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon Top-down sequencing of Apis dorsata apamin by MALDI-TOF MS and evidence of its inactivity against microorganisms D. Baracchi a,*, G. Mazza a, E. Michelucci b, G. Pieraccini b, S. Turillazzi a,b, G. Moneti b a Department of Biologia Evoluzionistica “Leo Pardi”, University of Firenze, Via Romana 17, 50125 Firenze, Italy b Mass Spectrometry Centre, University of Firenze, Viale G. Pieraccini 6, 50139 Firenze, Italy article info abstract Article history: Apis mellifera venom is one of the best characterized venoms among Hymenoptera, yet Received 13 March 2013 relatively little is known about venom belonging to other species in the genus Apis. Received in revised form 18 May 2013 Melittin, one of the most important bioactive peptides, has been isolated and characterized Accepted 22 May 2013 in A. mellifera, Apis cerana, Apis dorsata and Apis florea, while apamin has been only Available online 7 June 2013 characterized in A. mellifera and A. cerana. At present, no information is available about the sequence of A. dorsata apamin. Moreover, while the antiseptic properties of melittin and Keywords: MCD peptides are well documented, the antimicrobial activity of apamin has never been MALDI-TOF Venom tested. In the present study, we isolated and characterized apamin from the venom of the Honeybee giant honeybee A. dorsata. We tested the activity of apamin against bacteria and yeasts in a Antimicrobial peptides microbiological assay to gain a more complete understanding of the antimicrobial Peptide sequencing competence of the medium molecular weight venom fraction. We show that A. dorsata apamin toxin has the same primary sequence as apamin in A. mellifera and A. cerana,yet with a different C-terminal amidation. We did not find any antiseptic activity of apamin against any of the tested microorganisms. We discuss the evolutionary processes con- nected to the ecological context of venom use that drove the generation of Apis venom complexity. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction 2006; Nascimento et al., 2006) that target a myriad of re- ceptors and ion channels, making them ideal for pharma- Arthropods such as scorpions, spiders and stinging hy- ceutical and agrochemical research (King and Hardy, 2013; menopterans have developed venom glands that mainly Morgenstern and King, 2013). Many studies have been produce proteins including several enzymes, peptides, conducted to understand the evolutionary processes that several neurotoxic, antimicrobial and cytolytic compounds drove the generation of this venom complexity (for reviews and low-molecular mass substances such as ionic salts, see Fry et al., 2009). biogenic amines, amino acids and alkaloids (Bettini, 1978; Apis mellifera venom, one of the best characterized Piek, 1986; Kuhn-Nentwig, 2003). Venoms are extremely venoms among Hymenoptera, is composed of a wide spec- complex blends of diverse substances (Escoubas et al., trum of biomolecules, the structure and function of which have been determined in great detail (Kreil, 1973; Hoffman, 1996). Recently, a straightforward characterization of the * Corresponding author. Current address: Queen Mary University of peptidic fraction ranging from 1000 to 4000 Da of the pure London, Research Centre for Psychology, School of Biological and Chem- venom by MALDI-TOF MS allowed the detection of the three ical Sciences, Mile End Road, London E1 4NS, UK. Tel.: þ44 (0)20 7882 4807. major types; apamin, mast cell degranulating peptide E-mail address: [email protected] (D. Baracchi). (MCD) and melittin (Francese et al., 2009). Furthermore, 0041-0101/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxicon.2013.05.020 106 D. Baracchi et al. / Toxicon 71 (2013) 105–112 other minor compounds were detected in the honeybee Hence, this study focuses on the chemical character- venom and they still remain to be identified (Baracchi and ization of apamin isolated from the venom of the giant Turillazzi, 2010; Baracchi et al., 2011). Melittin is a basic honeybee A. dorsata. The peptide sequence was entirely peptide of 26 residues (MWw2.8 kDa), accounts for 45%– obtained by means of top down MALDI-TOF MS experi- 50% of the venom dry weight (de Lima and Brochetto-Braga, ments. Additionally, to complete the framework of the 2003; Ownby et al., 1997) and exhibits an amphipathic antimicrobial competence of the medium MW venom structure; its polar and non-polar ends allow it to interact fraction, a microbiological assay was used to test the ac- with lipid membranes ultimately increasing its perme- tivity of apamin against Gram-positive and Gram-negative ability. Two other peptides are considered major venom bacteria and yeasts. toxins: MCD and apamin. The former causes mast cell break down, accounts for about 2% of the venom dry weight (de 2. Material and methods Lima and Brochetto-Braga, 2003) and comprises 22 amino acid residues (MWw2.6 kDa). Apamin is a well character- 2.1. Venom collection ized small peptide (18 amino acids, MWw2.0 kDa) with two disulphide bridges connecting the four cysteine residues in Sixty adult worker bees were collected from a wild an overlapping pattern that recurs in at least one other colony of A. dorsata located in Bukit Katil (Melaka State, venom peptide component (Gauldie et al., 1978). The sec- Malaysia) and killed by freezing. ondary or tertiary structure of apamin is exceptionally sta- All specimens were dissected and drops of their venom ble with respect to pH, temperature, and denaturants were extracted directly from the tip of the sting with a (Miroshnikov et al., 1978; Pease and Wemmer, 1988). Apa- small capillary glass tube after gently squeezing the venom min accounts for less than 2% of venom dry weight, presents sac with a glass plate. All the collected venom were a neurotoxic action (de Lima and Brochetto-Braga, 2003) reunited in one single tube and 2 ml methanol added. The and possesses unusual functional as well as structural tube with the sample was transported to the Italian mass properties (Habermann, 1972). It is remarkable among spectrometry centre at the university of Firenze, Italy, and peptides in its ability to cross the blood–brain barrier and stored at À20 C until MS analyses. act on the central nervous system. Apamin is known to block þ calcium dependent potassium fluxes by binding to a Ca2 - 2.2. Chemical analysis dependent potassium channel (Banks et al., 1979; Castle et al., 1989; Labbé-Jullié et al., 1991; Ishii et al., 1997). 2.2.1. Chemicals While the antiseptic properties of melittin (from A. melli- Methanol, acetonitrile (ACN), and n-pentane were of fera) and many MCD peptides (from other arthropods) are chromatography grade and purchased of Riedel de Haen well documented (Kuhn-Nentwig, 2003; Konno et al., 2001, (Sigma Aldrich Italia, Milan, Italy). Purified and deionized 2006; Mendes et al., 2004; Souza et al., 2005; Turillazzi et al., water was prepared using a Milli-Q system (Millipore, 2006; Xu et al., 2006), the antimicrobial action for apamin Bedford, MA, USA). Formic acid and trifluoroacetic acid has never been tested. Although apamin lacks the surfactant (TFA) were purchased from Fluka (Sigma Aldrich Italia). character of melittin (Bettini, 1978), this peptide shares at The a-cyano-4-hydroxycinnamic acid (a-CHCA) and least three similar characteristics with a group of membrane 1,5-diaminonaphthalene (1,5-DAN) were obtained from modulators and molecules implicated in defence against Bruker Daltonics (Bremen, Germany). Unless stated other- pathogens, including: scorpion toxin, insects and scorpion wise, all other reagents were of analytical grade and used as defensins, snake sarafotoxins, plant thionins and human supplied. endothelins (Froy and Gurevitz, 1998). In particular, they have an effect on the membrane potential, have similar gene 2.2.2. General procedure organization, and they share a similar cysteine-stabilized a- Apamin was isolated from the crude venom by HPLC helical (CSH) motif, which involves a Cys-X-X-X-Cys stretch and detected by ESI-TOF. To confirm the presence of cystins, of the a-helix bonded through two disulphide bridges to a we compared alkylated apamin to native apamin using Cys-X-Cys triplet of a b-strand (Kobayashi et al., 1991; MALDI-TOF MS. Top-down sequencing of apamin was done Bonmatin et al., 1992; Bruix et al., 1993; Bulet et al., 2004). with MALDI-TOF instruments using Anchor–chips plate. In contrast to the A. mellifera venom, which has one of the We measured the exact mass of apamin using LTQ- best characterized venoms amongst all Hymenoptera, rela- Orbitrap. tively little is known about the venom composition of other Apis species (reviewed in Schmidt, 1995). The three main 2.2.3. Peptide fractionation venom peptides melittin, apamin and MCD are also present in Extracts were taken to dryness and then resuspended in the venom of Apis cerana, Apis dorsata,andApis andreniformis 3 ml of a solution of water and ACN (70:30 v/v containing (Schmidt, 1995; Baracchi et al., 2011). In a pioneering study 0.5% formic acid). The peptide was separated from the other Kreil (1973) showed that melittin has the same amino acid components of the venom by HPLC using a Series 200 sequence in A. mellifera and A. cerana; melittin of A. dorsata (Perkin–Elmer, Boston, MA, USA) HPLC system including an differs in three amino acid residues (Kreil, 1975). autosampler, a quaternary pump, and an UV–VIS detector Apamin also has the same sequence in A. mellifera and A. coupled with a fraction collector (Biologic BioFrac, BioRad, cerana (Haux et al., 1967; Zhang et al., 2003). However, no Hercules, CA, USA). The RP-HPLC column was a Luna C8, information is available on the sequence of apamin in other 150 Â 4.6 mm, 5 mm (Phenomenex, Torrance, CA, USA), Apis species.