Proteomic View of the Venom from the Fire Ant Solenopsis invicta

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Proteomic View of the Venom from the Fire Ant Solenopsis invicta Buren José R. A. dos Santos Pinto,† Eduardo G. P. Fox,‡ Daniel M. Saidemberg,† Lucilene D. Santos,† Anally R. da Silva Menegasso,† Eliúde Costa-Manso,§ Ednildo A. Machado,‡ Odair C. Bueno,† and Mario S. Palma*,† †

Institute of Biosciences, Center of the Study of Social Insects/Department of Biology, University of São Paulo State (UNESP), Rio Claro, SP, Brazil ‡ Laboratório de Entomologia Médica e Molecular, Instituto de Biofísica Carlos Chagas Filho, Federal University of Rio de Janeiro (IBCCF/UFRJ), Rio de Janeiro, Brazil § Clinic of Allergology, Pouso Alegre, MG, Brazil S Supporting Information *

ABSTRACT: Fire ants are well-known by their aggressive stinging behavior, causing many stinging incidents of medical importance. The limited availability of fire ant venom for scientific and clinical uses has restricted, up to now, the knowledge about the biochemistry, immunology, and pharmacology of these venoms. For this study, S. invicta venom was obtained commercially and used for proteomic characterization. For this purpose, the combination of gel-based and gel-free proteomic strategies was used to assign the proteomic profile of the venom from the fire ant S. invicta. This experimental approach permitted the identification of 46 proteins, which were organized into four different groups according to their potential role in fire ant venom: true venom components, housekeeping proteins, body muscle proteins, and proteins involved in chemical communication. The active venom components that may not present toxic roles were classified into three subgroups according to their potential functions: self-venom protection, colony asepsis, and chemical communication. Meanwhile, the proteins classified as true toxins, based on their functions after being injected into the victims’ bodies by the fire ants, were classified in five other subgroups: proteins influencing the homeostasis of the victims, neurotoxins, proteins that promote venom diffusion, proteins that cause tissue damage/inflammation, and allergens. KEYWORDS: Hymenoptera, allergen, toxin, proteomic analysis, mass spectrometry, shotgun proteomics, envenoming mechanism



pustules at the stung area, may lead to secondary infections.5,7 Moreover, sensitized people and victims of accidents with multiple stings can develop more serious allergic reactions, which sometimes may culminate in anaphylactic shock, followed by coma and even death.6,8−10 Other common symptoms occurring during the envenoming with fire ant venom are: severe anxiety, urticarial skin rash, breathlessness, vomiting, tachycardia, hypotension, and angioedema.11 In Brazil, ants are responsible for 26.8% of the stinging incidents caused by Hymenoptera insects;12 possibly most of these incidents are caused by the fire ant. In some endemic regions of the United States, from 30 to 50% of the population is sensitized by fire ant venom, revealing the presence of specificIgE against the major proteins from fire ant venom.13 The venom of fire ants is constituted by over 90% of piperidinic alkaloids, while the remaining fraction is composed of an aqueous solution of proteins.7 The venom proteins of S.

INTRODUCTION Two Brazilian fire ants species, Solenopsis invicta Buren and Solenopsis richteri Forel, were accidently introduced in the United States and later in farther countries like Australia and Vietnam.1,2 The fire ants comprise about 20 American species of Solenopsis, which construct fragile earthen mounds in open sunny areas and are frequently found in places like lawns, highways, and city sidewalks; the fire ants are resistant to chemical and biological control methods and currently are infesting more than 310 million acres of land.3 These ants are omnivorous, feeding on almost any plant or animal material; they can damage farm equipment, irrigation systems, and even agriculturable lands.4 The ants’ attack is characterized by bites with their mandibles, followed by a painful sting; a single fire ant worker can sting repeatedly, until its venom sac has been depleted.5 Following the sting, a localized and intense burning sensation caused by the injected alkaloids in the sting site of the victims’ skin, followed by local erithrema and the formation of white © XXXX American Chemical Society

Received: May 18, 2012

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invicta have been the focus of a series of studies,14−18 in which only four proteins were identified and sequenced up to now:16 Sol i 1, a phospholipase A/B similar to those reported in wasp venoms; Sol i 2, which apparently seems to be a pheromone binding protein; Sol i 3, a member of the antigen 5/ pathogenesis-related protein; and Sol i 4, a member of a unique protein family of unknown function. All of these proteins were characterized as potent allergens.19 Despite the importance of the venom of this insect as a chemical tool used as part of the strategies for colony defense, prey capture and territory dominance against other animals, very little is known about its venom protein composition. The knowledge about the allergenicity against humans and pet animals is currently limited to the four proteins described above. Fire ant venom is not easily available, even for immunotherapeutical use; currently, the immunotherapy protocols for fireant-sensitive patients employ whole-body ant extracts, which may not be efficient.20 The tiny amount of venom existing in each worker (about 500 ng), the laborious protocol necessary for its milking, and the difficulty of obtaining it free from the alkaloids have been considered serious obstacles for venom obtainment by the venom suppliers and scientists abroad.21 The very limited availability of fire ants venom for scientific and clinical uses has restricted our knowledge about the biochemistry, immunology and pharmacology of these venoms. However, in response to a special request, the venom supplier company obtained a few milligrams of pure S. invicta venom in absence of alkaloids, which was used in the present investigation for proteomic characterization. For this purpose both a gel-dependent and gel-free proteomic approaches were applied.



tion. Images were analyzed using Image Master Platinum software v.7 (GE Healthcare). Samples Digestion

The protocol for in-gel digestion was based on a protocol described as follows: gel pieces were destained twice for 30 min at 25 °C with 50 mM ammonium bicarbonate/50% acetonitrile (ACN), dehydrated in ACN, dried, treated with trypsin (20 μg/mL, Promega, Madison, WI) in 50 mM ammonium bicarbonate pH 7.9 at 37 °C, during 18 h. Digests were extracted from gel pieces with 60% (v/v) ACN/water and 0.1% (v/v) formic acid, combined, desalted and cleaned with PerfectPure C18 pipet tips (Eppendorf, Hamburg, Germany) according to the manufacturer’s instructions and vacuum-dried. The concentrated digests were mixed with 0.5 μL of matrix (10 mg/mL α-cyano-4-hydroxycinnamic acid in methanol/acetonitrile (1:1, v/v) mixed with an equal volume of 0.2% (v/v) aqueous TFA) and spotted onto a MALDI plate target and submitted to MALDI-TOF-TOF analysis. In solution digestion was also used for a shotgun strategy with the soluble venom. For this purpose, S. invicta crude venom (50 μg) proteins was solubilized in 50 mM ammonium bicarbonate, pH 7.9 containing 7.5 M urea, during for 60 min at 37 °C for denaturation, and then reduced with 10 mM DTT at 37 °C during 60 min. After this treatment the proteins were alkylated with 40 mM iodoacetamide at 25 °C, during for 60 min in the absence of light. Samples were diluted 5-fold with 100 mM ammonium bicarbonate, pH 7.8, and sufficient amount of 1 M calcium chloride was added to the samples until the concentration of 1 mM concentration in the sample. Nonautolytic trypsin (Promega) was added to the denatured protein solution (with 1:50 w/w trypsin/venom protein) during 18 hs at 37 °C. Rapid freezing of the samples in liquid nitrogen quenched the enzymatic digestion. Digested samples were desalted by using a SPE C18 column (Discovery DSC-18, SUPELCO, Bellefonte, PA) conditioned with MeOH and rinsed with 1 mL 0.1% TFA, and washed with 4 mL of 0.1% TFA/5% ACN. Peptides were eluted from the SPE column with 1 mL of 0.1% TFA/80% ACN and concentrated in a Speed-Vac (Heto, Dry Winner 3) until completely dry. Digested samples were stored at −80 °C until needed for analysis; the tryptic peptides were solubilized in 50% ACN and submitted to LC−IT-TOF/MS and MSn analysis.

EXPERIMENTAL SECTION

Venom

S. invicta venom completely free from alkaloids was obtained from Vespa Laboratories Inc. (Spring Mills, PA). The crude venom was maintained at −80 °C until use. Protein Assay

Protein quantification was determined by the method reported by Bradford (1976),22 using bovine serum albumin (BSA) as standard.

Mass Spectrometry Analysis

Two-dimensional Gel Electrophoresis

MALDI-TOF/TOF Mass Spectrometry Data. Mass spectrometric analysis was performed by MALDI-TOF/TOF MS (matrix-assisted laser desorption ionization time-of-flight/ time-of-flight mass spectrometry) instrument (Shimadzu, Axima Performance). Both MS and MS/MS spectra were acquired in the positive ion reflectron mode using a N2 laser. Typically, 250−500 laser shots were acquired for MS and MS/ MS mode, and spectra were obtained at a laser power, maximized for the highest possible resolution and peak intensity in each analytical situation. CID spectra were acquired using a dual-timed ion gate at a laser power approximately 20% higher than MS acquisition. MS data were acquired in the m/z range 700−3500, with an accelerating voltage of 20 kV and delayed extraction, peak density of maximum 50 peaks per 200 Da, minimal S/N ratio of 10 and maximum peak at 60. MS/MS data were acquired in the mass range from 60 Da until each precursor mass, with a minimum S/N ratio of 10; a maximum number of peak set at 65 and peak density of maximum 50 peaks per 200 Da.

Samples (250 μg protein) were applied by rehydration to 7 cm IPG strips, pH 3−10. Isoelectric focusing (IEF) was carried out on a Multiphor II System (GE Healthcare) at 3500 V for 7100 Vh. IPG strips were incubated in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS) containing 0.5% (w/v) DTT for 15 min, followed by equilibration buffer containing 4% (w/v) iodoacetamide for 15 min. The second dimension was run on self-cast SDS-PAGE gels [15% (w/v) polyacrylamide and 0.8% (w/v) bis (N,N′methylenebisacrylamide)] at 15 mA/gel for 15 min and 30 mA/gel for 1 h, at 10 °C in a Mini VE system (GE Healthcare). Gels were stained overnight with Coomassie Brilliant Blue R250 (CBB) and stored at 21 °C in preserving solution [7% (v/ v) acetic acid]. Image Acquisition

2-DE gels stained with CBB were scanned and digitized (BioImage, GE Healthcare) in the transparency mode at 24-bit red-green-blue colors and 400 dpi resolutions for documentaB

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Figure 1. MALDI-TOF mass spectrum of the whole venom (free from alkaloids) from the fire ant Solenopsis invicta, in the positive mode.

Ion Trap/TOF MS. MS and MSn analyses were conducted on a hybrid system, where a UFLC (Prominence) was online connected to an IT/TOF MS (ion trap/time-of-flight mass spectrometer) instrument (Shimadzu, Kyoto, Japan), equipped with an electrospray ionization source. The UFLC system was constituted of two LC20AD pumps, an automatic injector SIL 20 AHT, SPD M20A diode array detector and column oven CTO 20A. The LC system was connected to a Shimpac XR ODS C-18 column (100 × 3 mm, i.d; 130 μm), and submitted to a gradient of acetonitrile from 5% (v/v) to 85% (v/v) during 40 min at 30 °C. The exit of column was directly connected to the ionization system of the mass spectrometer, which in turn was set to permit the accumulation of all ions in the octapole, followed by rapid pulsing into the IT for MSn analysis, and then the introduction into the TOF sector for accurate mass determinations. The setting conditions for optimized operations were: positive mode, electrospray voltage 4.5 kV, CDL temperature 200 °C, block heater temperature 200 °C, nebulizer gas (N2) flow of 1.5 L/min, trap cooling gas (Ar) flow of 95 mL/min, ion trap pressure 1.7 × 10−2 Pa, TOF region pressure 1.5 × 10−4 Pa, ion accumulation time 50 ms, collision energy set at 35% both for MS2 and MS3, and collision gas set to 20%. Autotuning of the instrument was performed with a Na-TFA solution using the following parameters: for the positive mode, error 3.08 ppm and resolution 15.414; and for the negative mode, error 2.69 ppm and resolution 13.212. Protein Identification. Launchpad 2.8 (Shimadzu) was used to submit the combined MS and MS/MS data to the MASCOT protein search engine v. 2.2 (http://www. matrixscience.com) against publicly available ants’ protein sequences deposited in the National Center for Biotechnology Information nonredundant protein database (NCBInr) (http://blast.ncbi.nlm.nih.gov/Blast.cgi); for this purpose. we selected all 76281 entries contained in the taxa “ant”, which contains 208 genera. For those proteins that could not be identified within this databank, the searches were performed against “proteins from animal venoms”, which includes snakes, spiders, wasps and bees. The databanks mentioned above were appended with common external contaminants from cRAP, a maintained list

of contaminants, laboratory proteins and protein standards provided through the Global Proteome Machine Organization (http://www.thegpm.org/crap/index.html). The following search parameters were used: no restrictions on protein molecular weight, one tryptic missed cleavage allowed; peptide mass tolerance in the searches was 0.8 Da for MS spectra and 0.5 Da for MS/MS spectra. Iodoacetamide derivative of cysteine and oxidation of methionine were specified in MASCOT as fixed and variable modifications, respectively. Proteins identified after database search were subjected to additional filtering using Scaffold (version 2.04.00, Proteome Software Inc., Portland, OR) to validate peptide identification and to obtain a false discovery rate (FDR) of less than 1%; FDR was calculated from the forward and decoy matches. Peptide identifications were accepted if they could be established at ≥95% probability as specified by the Peptide Prophet algorithm. Protein probabilities were assigned by the Protein Prophet algorithm, and the identifications were accepted if they could be established at ≥99.0% probability and contained at least two identified peptides.



RESULTS Since this is the first proteomic characterization of a fire-ant venom, we decided to include MALDI-TOF MS spectrum of the whole venom to show a molecular range of proteins existing in the composition of this venom. Figure 1 shows a series of small proteins (or large peptides) that occurs at molecular weights smaller than 10 kDa, while another series of protein components may be observed between 15 and 44 kDa. Patterns of venoms from three different 2-DE gels were analyzed, showing a high similarity between each other, reflected by the high scatter plot correlation coefficient (>97%) between the three gels. Figure 2 shows a representative 2-DE gel, which revealed 25 spots in S. invicta venom, in the MW range from 15.2 to 70.1 kDa and pI from 5.27 to 9.45. Protein identification by mass spectrometry was performed for all 25 spots observed in Figure 2. It must be considered that the genomic information available in databanks for S. invicta is not complete; thus, the identifications were initially performed through cross-species data, using a combination of incomplete protein databanks (DBs) of different ant species. The proteins C

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identified in S. invicta DB were: allergenic proteins (spots 15, 16, 17, 21, 23, and 24), phospholipase A2 (spots 9 and 11) and a growth factor (spot 12); other proteins were identified as similar to snakes venom toxins, such as myotoxins (spots 3 to 8), phospholipase A2 inhibitor (spot 19) and thioredoxin peroxidase (spot 20), or even as arthropod-like neurotoxins (spots 10, 14 and 18) and the anemone cytolytic toxin (spot 13). The spots 13 (PsTx-like protein), 14 (Scolopendra toxinlike protein), 15−17 (different forms of venom allergen-3) and 21 (venom allergen-1) were apparently the most abundant fire ant venom components; although these spots were large (Figure 2), their identification indicated the presence of only one protein even when different pieces of each spot were excised and individually analyzed. Considering that the number of proteins present in S. invicta venom is very reduced when compared to those already reported for the venoms from other social Hymenoptera, such as honeybees23,24 and wasps,25 we decided also to focus the proteomic analysis of the S. invicta venom using a gel-free approach. Thus, the venom from S. invicta was reduced, alkylated, digested with trypsin and analyzed using a LC−IT/ TOF MS and MSn system, according to the protocol shown in Figure 3. Depending on the population of the constituent peptides, up to 650 MS/MS spectra were collected for each injection of S. invicta venom. After acquisition of MS/MS spectra, the proteins and peptides were identified by the software algorithm Mascot; all proteins were considered as identified when at least two peptides matched. About 22% of the mass spectra resulted in reliable candidate peptides following searches of the NCBI

Figure 2. Representative 2-DE profile of Solenopsis invicta venom, stained with Coomassie Brilliant Blue G-250.

not identified with this initial approach were searched against a DB constituted by “proteins from animal venoms”, which included snakes, spiders, wasps, and bees, among others. Table 1 shows the identification of 21 out of the 25 protein spots observed in Figure 2. The only proteins not identified are those from spots 1, 2, 22, and 25. The identified proteins (Table 1) belong to different functional classes, typical of animal venoms. The proteins

Table 1. Protein Identification in 2-DE Gels of Solenopsis invicta Venom by Using MALDI-TOF/TOF Analysis spot

a

accession code

3

P24332

4 5 6 7 8 9 10

P24332 P24332 P24332 P24332 P24332 EFZ20207 P84062

11

EFZ10421

12

P67862

13

P58911

14

P0C8C2

15 16 17 18 19

P35778 P35778 P35778 P60212 B1A4N2

20

Q98TX1

21 23 24

Q68KK0 P35775 P35775

protein Myotoxin 2-like protein precursorb Myotoxin 2-like proteinb Myotoxin 2-like proteinb Myotoxin 2-like proteinb Myotoxin 2-like proteinb Myotoxin 2-like proteinb Phospholipase A2a U5-ctenitoxin Pk1a precursorb Phospholipase A2precursora VEGF-like protein precursorb Toxin PsTX-60-like proteinb Scolopendra toxin-like precursorb Venom allergen 3a Venom allergen 3a Venom allergen 3a Alpha-toxin Tc48ab Phospholipase A2 inhibitorb Thioredoxin peroxidase precursorb Venom allergen 1a Venom allergen 2a Venom allergen 2a

exp. MWc/ pI

calculated MWc/pI

43.0/5.27

7.30/9.79

ICIPPSSDFGK (42), GGHCFPK (25) ILYLLFAFLFLAFLSEPGNAYK (63)

42.9/5.45 43.5/5.52 43.1/5.64 42.9/5.86 43.1/6.01 28.5/6.21 27.6/6.73

7.30/9.79 7.30/9.79 7.30/9.79 7.30/9.79 7.30/9.79 29.7/8.22 8.75/7.58

ICIPPSSDFGK (42), GGHCFPK (25) ILWLLFAFLFLAFLSEPGNAYK (64) ICIPPSSDFGK (40), MDCPWR (26), GGHCFPK (25) MDCPWR (26), ICIPPSSDFGK (42), ILYLLFAFLFLAFLSEPGNAYK (63) MDCPWR (25), ICIPPSSDFGK (43), ILFLLFAFLFLAFLSEPGNAYK (63) ICIPPSSDFGK (54), MDCPYR (26), ILYLLFAFLFLAFLSEPGNAYK (62) GLSGLTPSAIALMK (68), SHCTCDQLLYQCLK (61) HLWPAKECK (37), VADQSYAYGICKDK (56)

27.2/7.13

21.7/5.25

TRLSPIESGLK (35), SSCTCDDK (38)

27.3/7.78

16.1/8.09

VDPHKGTSKMEVMQFK (45), NPEEGGPR (32)

28.6/7.90

56.0/7.16

18.3/7.93

3.70/9.58

YKVLLEKYGSRIVK (41), KLVVRGGK (23), FEPIWTILKTR (27), VAEMQK (21) AAAAAFTGGDK (45), EQLIHTDVK (51)

26.3/8.10 27.6/8,40 26.8/6.45 18.0/591 17.9/6.68

26.4/8.18 26.4/8.18 26.4/8.18 7.4/6.30 18.4/6.58

17.3/8.05

12.0/5.30

VGHYTQIVWAK (35), WANQCTFEHDACR (38) VGHYTQIVWAK (34), WANQCTFEHDACR (37) VGHYTQIVWAK (34), WANQCTFEHDACR (36) DGYLMEGDGCK (63), MGCLTR (29) LRGAFLTVYK (34), SFGNGSER (30), LYVTNK (28) NFEALR (29), VLNSLIDALMHLQR (62), EFAKLR (30) CMPTFQFYK (38), PFFHSMVEK (36)

29.3/9.32 16.2/6.23 15.2/7.82

38.4/8.57 20.5/9.06 20.5/9.06

AEPDPGVVEYLK (37), DLANAFVQK (30), INPIQWKFWR (32) AVNIIGCALR (20), DVAECLR (23), AGVAETTVLAR (26) AVNIIGCALR (20), AGVAETTVLAR (25) DVAECLR (22)

peptides sequences (ion score)

Protein identified in ants database. bProteins identified in “animal venoms” database. cMW expressed in kDa. D

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allergens 1 to 3), and venom self-protection (transferrin, glutathione-S-transferase, cytochrome c oxidase, thioredoxin and growth hormone). The second group of proteins shown in Table 2 probably originated from the cells of venom glands and might not constitute true venom toxins. They belong to the group of housekeeping proteins, playing metabolic functions in secretory cells of venom glands and/or in the associated Dufour gland; such proteins may be contaminants from the neighboring tissues that leaked to the venom during its milking (Vespa Laboratories did not disclose how the venom extract was prepared). Apparently, this group of proteins does not play any functional role in the envenoming mechanism. Proteins included in this group were vitellogenins, ribosomal proteins, nuclear proteins, hormone receptors and regulatory proteins. The third group included those proteins related to insect body muscles. The proteins identified were prominin-like protein and troponin-C; generally these proteins are structures of striated muscles. Surrounding muscles probably might have been disrupted during the venom extraction or dissections, leaking part of myocytes contents into the venom reservoir. The fourth group is constituted basically by proteins related to the transport of pheromone, and their roles will be discussed later in this manuscript.



DISCUSSION Considering the importance of S. invicta as one of the most representative species of the fire ants in the Americas, we decided to make a characterization of the venom of this insect, using the combination of the classical bottom-up approach of gel-based proteomics, with the gel-free proteomic strategy. The combination of both experimental approaches resulted in the identification of 46 proteins in the commercial venom extract of the fire ant; it is important to emphasize that the 21 proteins identified in the 2-DE, which apparently represent the most abundant S. invicta venom proteins, were also identified in the shotgun approach. The 25 proteins identified exclusively in the gel-free approach (not visualized in Figure 2) probably correspond to few abundant venom components, not detected by CBB staining; these proteins were detected using a HPLC− MS/MS approach due to the high sensitivity and resolution of this mass spectrometric system. The MW of protein spots observed in the 2-DE (Figure 2; Table 1) fit well to the MW range observed in the MALDI-TOF mass spectrum obtained for the whole venom (Figure 1). The 46 proteins were classified into four different groups, according their potential role in fire ants venom based on their currently described roles: true venom components, housekeeping proteins, muscle proteins, and proteins involved in chemical communication (Table 2). The proteins classified as housekeeping components as well those from the muscles of insect bodies are not true venom components and apparently do not play any role in the envenoming mechanism or in any other biological function played by the venom. The group of proteins classified as “true venom components” is composed of proteins presenting similar roles in other animal venoms, especially social Hymenoptera insects. This group contains 21 different proteins, which may be organized according their different potential role in the envenoming process and/or venom self-protection. Thus, the most wellknown proteins in S. invicta venom are those considered potent allergens, that is, Sol i 1 to Sol i 4.26 Table 1 shows one form of PLA1, also known as venom allergen 1 (spot 21), and two different forms of PLA2 (spots 9

Figure 3. Representative protocol schematized for gel-free proteomic strategy of Solenopsis invicta venom using a LC−IT/TOF MS and MSn system.

protein databases (mainly by filtering for venom components). A total of 46 proteins were identified, from which 26 were from S. invicta and 13 were from other ant species (Acromyrmex echinatior, Pachychondyla goeldii, and Camponotus f loridanus); 7 proteins were identified in DBs composed of venom proteins from snakes, spiders, centipedes, anemones, and scorpions (Tables S1 and S2 in Supporting Information). All of the proteins identified in 2-DE were also identified through gel-free approach. The proteins identified were organized and presented in Table 2, classified into four different groups according to their general function: (i) true venom toxins, (ii) housekeeping proteins, (iii) muscle proteins from insect body, and iv) chemical communication. The venom toxins group is constituted of typical proteins from the animal venoms, involved with tissue damage (phospholipases A1 and A2, disintegrin/metalloproteinase, and myotoxins), neurotoxicity (toxin PsTX-60, U5-ctenotoxins Pk1a, alpha-toxins Tc48a, and Scolopendra toxin), antibiosis (ponericin-like peptides), vasodilation (atrial natriuretic peptide), allergenicity (venom E

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Table 2. Protein Identification of Solenopsis invicta Venom by Using HPLC−ESI−MS/MS no.

a

accession code

protein

peptide sequences (ion score) True venom components TRLSPIESGLK (31), SHCTCDQLLYQCLK (53) VLNSLIDALMHLQR (55), EFAKLR (27) AEPDPGVVEYLK (45), INPIQWKFWR (36) DVAECLR (20), AGVAETTVLAR (24) MPNLTWDPELATIAQR (64), GTNGPQPPAVK (25) ETILKVHNDERQK (43), INTAIK (25) LLKELWTK (29), AVLGKIKGL (32) ELWTKMK(36), AVLGKIKGLL (33) DFKDWMK (30), TAGEWLK (34) YAEGTDVCISGECMK (62), SADESGRPSVK (59)

Phospholipase A2a Phospholipase A2 inhibitorb Venom allergen 1a Venom allergen 2a Venom allergen 3a Pac c 3 allergen-like proteina Ponericin L2-like peptidea Ponericin L1-like peptidea Ponericin G4-like peptidea Disintegrin and metalloproteinasea Transferrina Glutathione-S-transferasea Cytochrome c oxidasea Thioredoxinb VEGF-like proteina Atrial natriuretic peptidea Toxin PsTX-60b Myotoxin 2-like proteinb U5-ctenitoxin Pk1ab Scolopendra toxinb Alpha-toxin Tc48ab

1 2 3 4 5 6 7 8 9 10

EFZ10421 B1A4N2 Q68KK0 P35775 P35778 C0ITL3 P82422 P82421 P82417 F4X7M3

11 12 13 14 15 16 17 18 19 20 21

Q3MJL5 Q6X4T7 Q85AX5 Q98TX1 P67862 GL888002.1 P58911 P24332 P84062 P0C8C2 P60212

22 23 24 25 26 27 28 29 30 31 32

Q7Z1M0 Q2VQM6 Q2VQM5 D3KCZ8 D3KD00 A3RI43 D3KCZ8 E9IKA2 GL888719.1 GL888033.1 GL767586.1

33

GL888243.1

34

F4WVX1

35 36 37 38 39

E2B9V6 GL888525.1 DQ026281.1 GL765434.1 GL887707.1

Vitellogenin-1a Vitellogenin-2a Vitellogenin-3a Ribosomal proteina Ribosomal proteina Ribosomal proteina Ribosomal proteina Pescadillo proteina Telomere-associated proteina ATP-dependent helicasea Tyrosyl-DNA phosphodiesterasea Vigilin lipoprotein binding proteina Potassium voltage-gated channela Insulin receptor substrate 1-Ba Integrator complex subunit 10a Short neuropeptide F receptora Neurofibromina Phospholipase D1a

40 41

GL888090.1 AF432912.1

Prominin-like proteina Troponin Ca

42 43 44 45 46

FJ215318.1 FJ215317.1 FJ215315.1 FJ215314.1 FJ387502.1

Odorant Odorant Odorant Odorant Odorant

binding binding binding binding binding

proteina proteina proteina proteina proteina

HAINDYNAK (54), MMEDSASK (48) PNMPMGQMPILEIDGK (73), NGGYFVGGK (55) IFSWISTLHGMK (38), AYFTSATMIIAIPTGIK (48) GCLEFWWK (36), CNPNDDK (40) VDPHKGTSKMEVMQFK (41), NPEEGGPR (31) LVGEALR (26), VTSYWLTGCSEPDSR (78), KVQGSYPTCSGAR (67) FEPIWTILKTR (37), VAEMQKFDFAK (51) MDCPWR (34), PPSSDFGKMD (42) VADQSYAYGICK (50), VNCPNR (37), HLWPAK (35) SAECVR (32), AAAAAFTGGDK (57) MEGDGCK (33), MGCLTR (25) Housekeeping proteins of the venom gland QGNILLPACQK (49), GGLTIQVKS (29) LLHYSVVPFTTQQDLLDLK (76), SPELLQAK (29) GEEAAELLSK (40), PDRVPMSSVPSK (64) QELLQSYQR (35), LAASVMRCGK (46) LTLDFHTNK (37), YYPRLTLDFHTNK (42) FVNVVQTFGR (36), VHGSLARAGK (38) LTLDFHTNK (38), GRHCGFGKR (36) DIQFLMHEPIIWK (64), YYVQPQWIFDSVNAR (72) DNNNTR (36), DALLYITSQARK (47) SSFEEGR (45), APCRGK (42) VEELLIDQLDGVIK (65), ALVNEECDSSGSVK (70) KGVNIK (24), TITQMFR (37) GATILIIDYCNR (58), STNQAMSLAILR (63)

DECFCLILDNEK (56), MDGTAEDWSHSEVSPK (75) STVIGMLEK (17), PSGTDNQLSK (63) NQLNQPSCSK (65), LNENFRK (50) CLIEISRYR (31), VNELTPSTNSQR (57) EDTECIDNKET (58), DIWCARSK (48), QNTEIYEEVFHCIPTDK (82), LRGAFLTVYK (33), SFGNGSER (31) Proteins from the muscles involving the insect body SQPFDK (35), LEIDNLLK (32) MDDLTK (31), AFDAFDHEK (40), Chemical communication RDITQECAK (42), FSIFECIIQTMDK (61) VHTESVNAER (65), INELFEGR (39) IYVCIIK (40), IHAVNEER (43) ILYNVAK (42), CAVQDGADK (68) TQICTPK (49), QQIQLITDWYK (68)

Protein identified in ants database. bProteins identified in “animal venoms” database.

and 11 in Figure 1; Table 1); apparently, the PLA1 corresponds to the allergen Sol i1.18 Table 2 shows one PLD1 (which apparently is not known as component from any animal venom). Phospholipases are relatively common in Hymenoptera venoms, occurring mainly as -A and -B types.27 The PLA1

and PLA2 catalyze the specific hydrolysis of ester bonds of 1,2diacyl-3-sn glycerophospholipids, at the positions sn-1 and sn-2, respectively, converting these substrates into their corresponding lyso compounds with the release of fatty acids.28 Thus, the PLAs are able to disrupt the phospholipidic wrappings of F

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involved with the occurrence of myonecrosis, skin damage, edema, among other inflammation related reactions.39 Some of these manifestations have already been reported in patients who suffered massive attacks of social Hymenoptera.40 The typical symptoms caused by the action of disintegrins and metalloproteinases in the envenoming process are hemorrhage and edema around the site of the bite/stinging, which lead to local necrosis and tissue damage, hemorrhage, shock, and some types of coagulopathies.41 In addition to this, some metalloproteinases may degrade the extracellular matrix and capillary basement membranes, contributing to the disruption of local capillary networks, resulting in hemorrhage and edema.42 Systemic toxic reactions are frequently observed in victims of massive attacks by fire ants, which result in hemolysis, coagulopathy, rhabdomyolysis, acute renal failure and hypovolemia.41 Thus, some of the characteristic effects caused by the presence of zinc metalloproteinases-disintegrins are also observed in the victims of fire ant stinging envenomation. This is the first report about the presence of disintegrin and metalloproteinase-like proteins in fire ant venom; therefore, this enzyme must be individually characterized, and the clinical manifestations caused by its action would be observed in more details. Since the presence of this protein in fire ant venom represents a novelty in the literature, its occurrence was validated in the present investigation through the use of the of gelatin zymography, in presence and absence of selective inhibitors of metalloproteinases (Figure S1 in the Supporting Information). In addition to the myotoxins discussed above, another type of cytolytic protein identified in fire ant venom was the toxin similar to the PSTx-60 from sea anemone (Table 2); this protein causes a pronounced hemolysis.43 An interesting observation in the proteomic composition of S. invicta venom was the presence of the atrial natriuretic peptide (ANP) (Table 2), which plays an important role in blood pressure regulation, causing a decrease in the mean arterial pressure;44 thus, it influences significantly the regulation of homeotasis. As far as we know, this is the first report about the presence of an ANP in Hymenoptera venom. Neurotoxins are characteristic proteins from the venoms of solitary predator arthropods such as scorpions, spiders and solitary wasps, which use these toxins for prey killing/ paralysis.45 The presence of neurotoxins in the venoms of social insects in general is not common, due to the defensive nature their venoms.46 However, fire ants are aggressive predators that use their venoms both for nest defense as well as for prey capture.47 The present study identified three different types of venom neurotoxins: a protein similar to U5ctenotoxin Pk 1a from the spider Phoneutria keyserlingi, a protein similar to the Scolopendra toxin from the venom of the centipede Scolopendra angulata, and a protein similar to the alpha-toxin Tc48a from the scorpion Tytius cambridgei (spots 10, 14 and 18, respectively, in Figure 2; Table 1). The U5ctenotoxin Pk 1a causes spastic paralysis and death in mice;48 the alpha-toxin Tc48a is Na+ channel blocker,49 while the Scolopendra toxin seems to be an arthropod-toxic component.50 Thus, the presence of these neurotoxins in S. invicta venom may be related to their use in prey capture. A group of antimicrobial components was detected in S. invicta venom, constituted of three different ponericin-like peptides (-L1, -L2, and -L4) (Table 2); these peptides present a broad spectrum of activity both against Gram-positive and Gram-negative bacteria51 and probably are used by the fire ants

biological membranes, leading to pore formation, cell lysis, inflammation and tissue damage.29 When compared to each other, PLA1s and PLA2s have no sequence homology; apparently, these proteins have distinct functions. PLA2 was previously reported in social Hymenoptera venoms and described as highly hemolytic;30 meanwhile, PLA1 has been partially characterized in the venoms of some wasp species from the Northern hemisphere,31 in ants,32 and in the neotropical social wasp Polybia paulista.30 Generally, PLA2 from wasp and bee venoms are reported as allergenic proteins; meanwhile, the enzyme from fire ant venoms is not described as an allergen.33 These enzymes probably are also involved with tissue damage and venom diffusion around the site of stinging, as previously reported in wasp venoms.25 The occurrence of PLA1 and PLA2 in fire ant venom was validated through the use of TLC analysis of the products of digestion of the phospholipid substrates 1stearoyl-2-oleoyl-3-sn-glycerophosphorylcholine and 1-oleoyl2-stearoyl-3-sn-glicerophosphorylcholine by S. invicta venom, as reported in the Supporting Information. The well-known allergens from fire ant venoms, the allergen 2 (Sol i 2) (spots 23 and 24 in Figure 2; Tables 1 and 2) and venom allergen 3 (Sol i 3) (spots 15, 16, and 17 in Figure 1), as well the allergen identified as similar to another ant’s venom allergen in a shotgun approach, known as Pac c 3 (Table 2), were identified in the present study. The allergen Sol i 2 corresponds to a major allergen from fire ant venoms, which can induce serious systemic IgE-mediated anaphylactic reactions in the victims of fire ant stings.13 This allergen was identified in S. invicta venom under two different forms: spot 23, presenting MW 16.2 kDa and pI 6.23, and spot 24, with MW 15.2 kDa and pI 7.82. Until recently, the biological function of the allergen 2 in fire ant venom was unknown; however, a careful structural and functional analysis of this protein demonstrated that it is a capturer/transporter of small hydrophobic ligands such as pheromones, odors, fatty acids, or short-living hydrophobic primers. 34 It is interesting to emphasize that a series of other pheromone/odor transporters were also detected in the present study, using the gel-free approach (Table 2). These might be proved also as allergens in future investigations. The different forms of the allergen 3 (Sol i 3 and Pac c 3) are related to the antigen 5 from wasp venoms and are members of the cysteine-rich secretory proteins (CRISP) family, which are characterized by the presence of many cysteine residues in their sequences, generally forming several disulfide bridges.35 The CRISP proteins are structurally similar to a series of cancerrelated antigens and potent inflammatory proteins from sandflly saliva.36,37 The fire ant allergen 4 (Sol i 4) was not detected in the present investigation. Another type of protein identified in S. invicta venom is related to the presence of the myotoxin 2-like protein (spots 3−8 in Figure 2; Table 1); these proteins are similar to the basic PLA2 from crotalid snake venoms.38 This class of myotoxins is divided into two groups, based on the presence of an aspartic acid residue or lysine residue at position 49 (Asp49/Lys49); in Lys49, PLA2s lack the catalytic activity (or it is extremely low) but present myotoxic activity similar to that of Asp49 counterparts. These proteins are directly involved with skeletal muscle necrosis around the site of venom injection, with an immediate increase of local microvascular permeability and cytolysis.38 The only proteinase detected in S. invicta venom was the disintegrin and metalloproteinase (Table 2); this enzyme is G

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Figure 4. Representation of general mechanisms of actions proposed for the venom of Solenopsis invicta involving of the proteins identified in the present investigation.

The fourth group of proteins identified in the S. invicta venom is related to the “chemical communication”; this group is constituted of odorant binding proteins, chemosensory proteins, and pheromone binding proteins57 (Table 2). These proteins are secreted in the sensillar lymph of chemosensory sensilla of insects, as well in the venom of some Hymenoptera insects;58 their physiological function has not been clearly defined up to now. The odors/pheromones generally are low molecular mass hydrophobic compounds that must cross the aqueous sensillar lymph before reaching the neuronal membrane, where the olfactory receptor proteins are believed to be localized on. The transport of the odors/pheromones until their specific receptors is done by the odorant binding protein/pheromone binding proteins.59 The occurrence of these proteins in S. invicta bodies was inferred as related to their role in the regulation of complex social behavior.60 Considering that fire ants were reported to disperse venom around their nests and/or trails,52 the presence of these types of proteins in fire ant venom may be related to guiding the ants while foraging underground. To reinforce this potential role, it must be emphasized that the venom of fire ants also contains a series of different types of pheromones, such as alarm, aggregation and trail marking.60 One of the major odorant binding proteins from S. invicta venom is the well-known allergen 2 (Sol i 2),34 previously discussed.

to promote colony asepsis, as previously reported for other social insects.46,52 The presence of transferrin was also reported in the present investigation (Table 2); this protein was previously identified in the venoms from honeybees and wasps.24,25 Transferrin is an iron-binding glycoprotein that controls the levels of free iron ions in biological fluids; because of this, it plays a biocide effect on bacteria, since it makes Fe3+ ions unavailable for the microorganism survival.53 Thus, its presence in animal venoms may be related to an antibacterial role.54 Recently it was reported that the venoms from honeybees and wasps contains a series of proteins involved with the protection of venom toxins against natural oxidative stress (superoxide dismutase, glutathione-S-transferase, peroxiredoxin and thioredoxin peroxidase).24,25 Among these proteins, thioredoxin peroxidase (spot 20 in Figure 2; Table 1), and glutathione-S-transferase (Table 2) were identified in the present study. It was also identified the protein cytochrome c oxidase (Table 2) that acts in the oxidation−reduction processes.55 Thus, it is reasonable to consider that these proteins play the same role in S. invicta venom. A vascular endothelial growth factor (VEGF) was identified in S. invicta venom (spot 12 in Figure 2); this protein was also reported in honeybee and wasp venoms.24,25 The VEGF is wellknown to promote vascular permeability, which may contribute to venom diffusion.56 In addition, S. invicta venom contains a series of cytolytic proteins (such as myotoxins and a PSTx 60like protein), which may damage the venom glands and reservoir. The presence of VEGF could contribute to stimulating the growth of cells from the venom glands and reservoir, preventing the rupture of the tissues due to the action of cytolytic components. The venom also showed the presence of PLA2 inhibitor (spot 19 in Figure 2; Table 1); this protein could act as an inhibitor of stored venom PLA2 to prevent the self-hydrolysis of the venom glands/reservoir cells, preventing the tissues from rupturing.



CONCLUDING REMARKS The combination of gel-based and gel-free proteomics was used to assign the proteomic profile of the venom from the fire ant S. invicta. This experimental approach permitted the identification of 46 proteins that were organized into four different groups, according their potential role in fire ants venom: true venom components, housekeeping proteins, body muscle proteins, and proteins involved in chemical communication. The housekeeping proteins and the body muscle proteins apparently do not present any role in fire ant venom and thus were not the subject of discussion in the present manuscript. However, the H

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proteins from the two other groups (true venom protein components and proteins involved in chemical communication) were apparently relevant for the use of venom as instrument attack/defense, nest maintenance, trail marker, and elicitor of social behavior. The active venom components that do not present toxic roles were classified into three subgroups according to their potential functions (Figure 4): (i) self-venom protection − proteins related to the preservation of the integrity of the toxic proteins, preventing oxidation and structural damage to the tissue of venom glands and reservoir (cytochrome C oxidase, glutathione-S-transferase, PLA2 inhibitor, thioredoxin, and VEGF-like protein); (ii) colony asepsis − constituted of proteins/peptides related to the antimicrobial action inside the colony, preventing the infection of the stored food, adults and the brood; (iii) chemical communication − constituted of odorant binding proteins and chemosensory proteins, which are responsible by promptly activating the mechanisms of chemical communication of alarm, aggression, trail marking/following, among other social behaviors, generally elicited by pheromones existing in the venom composition. Meanwhile, the proteins classified as toxins play their functions after injected into the victims’ bodies by the fire ants; these proteins, in turn, were classified in five other subgroups as shown in Figure 4: (iv) proteins influencing the homeostasis of the victims − constituted of proteins that cause changes in cells permeability and alter the blood pressure of the victims of fire ant stinging, (atrial natriuretic peptide, ponericinlike peptides and PLA2); (v) neurotoxins − constituted of proteins that cause neurotoxicity, leading to prey paralysis/killing (U5ctenotoxin Pk1a-like protein, alpha-toxin Tc48a-like protein, and Scolopendra toxin-like protein); (vi) proteins that promote venom diffusion − constituted of toxins that cause tissues lesion, permitting the venom diffusion (PLA2, myotoxin 2-like proteins, and PSTx 60like protein); (vii) proteins that cause tissue damage and inflammation − constituted of proteins responsible by serious cytolytic effects of cells, followed by large damage in different types of tissues (PLA1, PLA2, disintegrin and metalloproteinase, myotoxin 2-like protein, and PSTx 60-like protein); (viii) allergens − constituted of proteins immunoreactive to specific-IgE, which are potentially allergenic (allergen 1, allergen 2, allergen 3, pac c 3-like protein. Thus, results of the proteomic analysis from the S. invicta venom will contribute to a better understanding of the general envenoming mechanism caused by this insect. Also, the identification of the whole allergen range in this venom may improve the diagnostics of allergies in the near future, mainly by identifying novel protein targets, to build a more complete microarray of allergenic proteins or even for the preparation of suitable recombinant proteins to be used in immunotherapy of patients sensitive to ant venom.

Article

ASSOCIATED CONTENT

S Supporting Information *

Table S1. Protein identification on the 2-DE gels of S. invicta venom showing the biological reference of each cross-species data. Table S2. Protein identification in by using HPLC−ESI− MS/MS analysis of S. invicta venom showing the biological reference of each cross-species data. Figure S1. (Lane A) Molecular weight markers. (Lane B) Gelatin zymography of fire-ant venom. Inhibition of metalloproteinase activity with (Lane C) EDTA and (Lane D) CT1166 prevented lysis of gelatin, indicating that the lysis bands from the fire-ant venom are due to metalloproteinase activity. The use of (Lane E) pepstatin, (Lane F) PMSF and (Lane G) E-64 did not inhibited the intensity of metalloproteinase band. Figure S2. Thin-layer chromatography of lipids developed on silica gel plates using chloroform−methanol−0.1 N HCl (60:35:5) as mobile phase. About 2.25 mg of each lipid was used: Lane A, standard oleic acid; Lane B, standard stearic acid; Lane C, products of incubation of SOGPC with fire-ant venom; Lane D, products of incubation of OSGPC with fire-ant venom. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*Prof. Mario Sergio Palma. CEIS-IBRC- UNESP, Av. 24A no 1515, Bela Vista - Rio Claro, SP, Brazil, CEP 13506-900. Phone: 55-(19)- 3526 4163. Fax: 55-(19)-35348523. E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by grants from FAPESP (Proc. 05/ 00982-1 and 06/02115-6) and BIOprospecTA/FAPESP program (Proc. 11/51684-1), INCT/CNPq-Instituto de Investigacões em Imunologia (iii), CNPq and CAPES. M.S.P. and O.C.B. are researchers awardees from the National Research Council of Brazil-CNPq; L.D.S. is a postdoctoral fellow from FAPESP, E.G.P.F. is a postdoc researcher from CAPES, J.R.A.S.P. is a Ph.D student fellow from FAPESP and A.R.S.M. is a MSc fellow from CAPES.



ABBREVIATIONS ACN, acetonitrile; ANP, Atrial Natriuretic Peptide; CBB, Coomassie Brilliant Blue; DTT, dithiothreitol; MMP, metalloproteinase; PLA1/PLA2, phospholipase A1/phospholipase A2; SPE, solid phase extraction; TFA, trifluoracetic Acid; TOF, time-of-flight; VEGF, Vascular Endothelia Growth Factor.



REFERENCES

(1) Lofgren, C. S. The economic importance and control of imported fire ants in the United States. In Economic impact and control of social insects.; Vinson, S. B., Ed.; Praeger: New York, 1986; pp 227−256. (2) Luo, L. Z. Considerations and suggestions on managing the red imported fire ant, Solenopsis invicta Buren in China. Plant Protection 2005, 31, 25−28. (3) Collins, H. C. 1992. Control of imported fire ants: a review of current knowledge. USDA-APHIS Tech. Bull. 1982, 27.

I

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(4) Allen, H. R.; Valles, S. M.; Miller, D. M. Characterization of Solenopsis invicta (Hymenoptera: Formicidae) populations in Virginia: social form genotyping and pathogen/parasitoid detection. Florida Entomol. 2010, 93 (1), 80−88. (5) Adams, C. T. Agricultural and medical impact of the imported fire ant. In Fire Ants and Leaf-Cutting Ants. Biology and Management; Lofgren, C. S., vander Meer, R. K., Eds.; Westview Press: Boulder, CO, 1986; pp 48−57. (6) Stafford, C. T. Hypersensitivity to fire ant venom. Ann. Allergy Asthma Immunol. 1996, 77, 87−95. (7) Hoffman, D. R. Fire ant and venom allergy. Allergy 1995, 50, 535−544. (8) Stablein, J. J.; Lockey, R. F.; Hensel, A. E. Death from imported fire ant stings. Immunol. Allergy Practice 1985, 7, 279−282. (9) Rhoades, R. B.; Stafford, C. T.; James, F. K. Survey of fatal anaphylactic reactions to imported fire ants stings. J. Allergy Clin. Immunol. 1989, 84, 159−162. (10) Prahlow, J. A.; Barnard, J. J. Fatal anaphylaxis due to fire ant stings. Am. J. Forensic Med. Pathol. 1989, 19, 137−142. (11) Khan, S. A.; Shelleh, H. H.; Khan, L. A.; Shah, H. Black fire ant (Solenopsis richteri) sting producing anaphylaxis: a report of 10 cases from najran. Ann. Saudi Med. 1999, 19, 462−464. (12) Esher, S. H.; Castro, A. P. B.; Croce, J; Palma, M. S.; Malaspina, O; Kalil, J. E.; Castro, F. F. M. Study of laboratorial methods for Hymenoptera allergy diagnosis: a critical analysis. J. Allergol. Clin. Immunol. 2001, 107 (2), 375 Suppl. SB. (13) Partridge, M. E.; Blackwood, W.; Hamilton, R. G.; Ford, J.; Young, P.; Ownby, D. R. Prevalence of allergic sensitization to imported fire ants in children living in an endemic region of the southeastern United States. Ann. Allergy Asthma Immunol. 2008, 100, 54−58. (14) Baer, H.; Liu, T. Y.; Anderson, M. C.; Blum, M.; Schmidt, W. H.; James, F. J. Protein components of fire any venom. Toxicon 1979, 17, 397−405. (15) Hoffman, D. R.; Dove, D. E.; Jacobson, R. S. Allergens in Hymenoptera venom XX: isolation of four allergens from imported fire ant (Solenopsis invicta) venom. J. Allergy Clin. Immunol. 1988, 82, 818−827. (16) Hoffman, D. R.; Smith, A. M.; Schmidt, M.; Moffitt, J. E.; Guralnick, M. Allergens in Hymenoptera venom XXII: comparison of venoms from two species of imported fire ants, Solenopsis invicta and richteri. J. Allergy Clin. Immunol. 1990, 85, 988−996. (17) Hoffman, D. R. Allergens in Hymenoptera venom XXIV: the amino acid sequences of imported fire ant venom allergens Sol i II, Sol i III and Sol i IV. J. Allergy Clin. Immunol. 1993, 91, 71−78. (18) Hoffman, D. R.; Sakell, R. H.; Schmidt, M. Sol i 1, the phospholipase allergen of imported fire ant venom. J. Allergy Clin. Immunol. 2005, 115, 611−616. (19) Hoffman, D. R. Reactions to less common species of fire ants. J Allergy Clin Immunol 1997, 100, 679−683. (20) Golden, D. B.; Moffitt, J.; Nicklas, R. A.; Freeman, T.; Graft, D. F.; Reisman, R. E.; Tracy, J. M.; Bernstein, D.; Blessing-Moore, J.; Cox, L.; David, A.; Khan, D. A.; Lang, D. M.; Oppenheimer, J.; Portnoy, J. M.; Randolph, C.; Schuller, D. E.; Spector, S. L.; Tilles, S. A.; Wallace, D. Stinging insect hypersensitivity: a practice parameter update 2011. J. Allergy Clin. Immunol. 2011, 127, 852−871. (21) Haight, K. L.; Tschinkel, W. R. Patterns of venom synthesis and use in the fire ant, Solenopsis invicta. Toxicon 2003, 42, 673−682. (22) Bradford, M. M. A rapid and sensitive for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248−254. (23) Peiren, N.; Vanrobaeys, F.; Graaf, D.; Devreese, B.; Beeumen, J. V.; Jacobs, F. J. The protein composition of honeybee venom reconsidered by a proteomic approach. Biochim. Biophys. Acta 2005, 1752, 1−5. (24) Peiren, N.; De Graaf, D. C.; Vanrobaeys, F.; Danneels, E. L.; Devreese, B.; Van Beeumen, J.; Jacobs, F. J. Proteomic analysis of the honey bee worker venom gland focusing on the mechanisms of protection against tissue damage. Toxicon 2008, 52, 72−83.

(25) Santos, L. D.; Santos, K. S.; Pinto, J. R. A. S.; Dias, N. B.; Souza, B. M.; Dos Santos, M. F.; Perales, J.; Domont, G. B.; Castro, F. M.; Kalil, J. E.; Palma, M. S. Profiling the proteome of the venom from the social wasp Polybia paulista: a clue to understand the envenoming mechanism. J. Proteome Res. 2010, 9, 3867−3877. (26) Padavattan, S; Schmidt, M.; Hoffman, D. R.; Marković-Housley, Z. Crystal structure of the major allergen from fire ant venom, Sol i 3. J. Mol. Biol. 2008, 383, 178−185. (27) Santos, L. D.; Souza, K. S.; de Souza, B. M.; Arcuri, H. A.; Cunha- Neto, E.; Castro, F. F. M.; Kalil, J. E.; Palma, M. S. Purification, sequencing and structural characterization of the phospholipase A1 from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae). Toxicon 2007, 50, 923−937. (28) Santos, L. D.; Menegasso, A. R. S.; Pinto, J. R. A. S.; Santos, K. S.; Castro, F. M.; Kalil, J. E.; Palma, M. S. Proteomic characterization of the multiple forms of the PLAs from the venom of the social wasp Polybia paulista. Proteomics 2011, 11, 1403−1412. (29) Dotimas, E.; Hider, R. C. Honeybee venom. Bee World 1987, 68, 51−71. (30) Oliveira, M. R.; Palma, M. S. Polybitoxins: a group of phospholipases A2 from the venom of the neotropical social wasp paulistinha (Polybia paulista). Toxicon 1998, 36, 189−199. (31) King, T. P.; Jim, S. Y.; Wittkowski, K. M. Inflammatory role of two venom components of yellow jackets (Vespula vulgaris): a mast cell degranulating peptide mastoparan and phospholipase A1. Int. Arch. Allergy Immunol. 2003, 13, 25−32. (32) Hoffman, D. R. Hymenoptera venom allergens. Clin. Rev. Allergy Immunol. 2006, 30, 109−128. (33) King, T. P.; Spangfort, M. D. Structure and biology of stinging insect venom allergens. Int. Arch. Allergy Immunol. 2000, 123, 99−106. (34) Borer, A. S.; Wassmann, P.; Schmidt, M.; Hoffman, D. R.; Zhou, J. J.; Wright, C.; Schirmer, T.; Marković-Housley, Z. Crystal structure of Sol i 2: a major allergen from fire ant venom. J. Mol. Biol. 2012, 415, 635−648. (35) Lu, G.; Kochoumian, L.; King, T. P. Sequence identity and antigenic cross -reactivity of white face hornet venom allergy, also a hyaluronidase with other proteins. J. Biol. Chem. 1995, 270, 4457− 4465. (36) Anderson, J. M.; Oliveira, F.; Kamhawi, S.; Mans, B. J.; Reynoso, D.; Seitz, A. E.; Lawyer, P.; Garfield, M.; Pham, M.; Valenzuela, J. G. Comparative salivary gland transcriptomics of sandfly vectors of visceral leishmaniasis. BMC Genomics 2006, 7, 52. (37) Stafford, C. T.; Wise, S. L.; Robinson, D. A.; Crosby, B. L.; Hoffman, D. R. Safety and efficacy of fire ant venom in the diagnosis of fire ant allergy. J. Allergy Clin. Immunol. 1992, 90, 653−661. (38) Angulo, Y.; Olamendi-Portugal, T.; Possani, L. D.; Lomonte, B. Isolation and characterization of myotoxin II from Atropoides (Bothrops) nummifer snake venom, a new Lys49 phospholipase A2 homologue. Int. J. Biochem. Cell Biol. 2000, 32, 63−71. (39) Gutiérrez, J. M.; Rucavado, A. Snake venom metalloproteinases: their role in the pathogenesis of local tissue damage. Biochimie 2000, 82, 841−850. (40) Stiprija, V.; Boonpuknavig, V. Renal failure and myonecrosis following wasp sting. Lancet 1972, 1, 749. (41) Koya, S.; Crenshaw, D.; Agarwal, A. Rhabdomyolysis and acute renal failure after fire ant bites. J. Gen. Intern. Med. 2007, 22, 145−147. (42) Gallagher, P.; Yongde Bao, Y.; Serrano, S. M. T.; Laing, G. D.; Theakston, R. D.; Gutiérrez, J. M.; Escalante, T.; Zigrino, P.; MouradaSilva, A. M.; Nischt, R.; Mauch, C.; Moskaluk, C.; Fox, J. W. Role of the snake venom toxin jararhagin in proinflammatory pathogenesis: in vitro and in vivo gene expression analysis of the effects of the toxin. Arch. Biochem. Biophys. 2005, 441, 1−15. (43) Satoh, H.; Oshiro, N.; Iwanaga, S.; Namikoshi, M.; Nagai, H. Characterization of PsTX-60B, a new membrane-attack complex/ perforin (MACPF) family toxin, from the venomous sea anemone Phyllodiscus semoni. Toxicon 2007, 49, 1208−1210. (44) Schweitz, H.; P Vigne, P.; Moinier, D.; Frelin, C.; Lazdunski, M. A new member of the natriuretic peptide family is present in the J

dx.doi.org/10.1021/pr300451g | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

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venom of the green mamba (Dendroaspis angusticeps). J. Biol. Chem. 1992, 267, 13928−13932. (45) Palma, M. S. Peptides as toxins/defensins. Amino Acids 2011, 40 (1), 1−4. (46) Palma, M. S. Insect Venom Peptides. In Handbook of Biologically Active Peptides; Kastin, A., Ed.; Academic Press: San Diego, 2006; Chapter 56, pp 409−417, p 1545. (47) Allen, C. R.; Forys, E. A.; Rice, K. G.; Wojcik, D. P. Effects of fire ants (Hymenoptera: Formicidae) on hatching turtles and prevalence of fire ants on sea turtle nesting beaches in Florida. Florida Entomol. 2001, 84, 250−253. (48) Richardson, M.; Pimenta, A. M.; Bemquerer, M. P.; Santoro, M. M.; Beirao, P. S.; Lima, M. E.; Figueiredo, S. G.; Bloch, C., Jr; Vasconcelos, E. A.; Campos, F. A.; Gomes, P. C.; Cordeiro, M. N. Comparison of the partial proteomes of the venoms of Brazilian spiders of the genus Phoneutria. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2006, 142, 173−187. (49) Batista, C. V.; Zamudio, F. Z.; Lucas, S.; Fox, J. W.; Frau, A.; Prestipino, G.; Possani, L. D. Scorpion toxins from Tityus cambridgei that affect Na+-channels. Toxicon 2002, 40, 557−562. (50) Rates, B.; Bemquerer, M. P.; Richardson, M.; Borges, M. H.; Morales, R. A. V.; De Lima, M. E.; Pimenta, A. M. C. Venomic analyses of Scolopendra viridicornis nigra and Scolopendra angulata (Centipede, Scolopendromorpha): shedding light on venoms from a neglected group. Toxicon 2007, 49, 810−826. (51) Orivel, J.; Redeker, V.; Le Caer, J. P.; Krier, F.; Revol-Junelles, A. M.; Longeon, A.; Chaffotte, A.; Dejean, A.; Rossier, J. Ponericins, new antibacterial and insecticidal peptides from the venom of the ant Pachycondyla goeldii. J. Biol. Chem. 2001, 276, 17823−17829. (52) Obin, M. S.; Vander Meer, R. K. Gaster flagging by fire ants (Solenopsis spp.): functional significance of venom dispersal behavior. J. Chem. Ecol. 1985, 11, 1757−1768. (53) Crichton, R. R.; Charloteaux-Wauters, M. Iron transport and storage. Eur. J. Biochem. 1987, 164, 485−506. (54) Georgieva, D.; Seifert, J.; Ö hler, M.; von Bergen, M.; Spencer, P.; Arni, R. K.; Genov, N.; Betzel, C. Pseudechis australis venomics: adaptation for a defense against microbial pathogens and recruitment of body transferrin. J. Proteome Res. 2011, 10, 2440−2464. (55) Tian, H.; Vinson, S. B.; Coates, C. J. Differential gene expression between alate and dealate queens in the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae). Insect Biochem. Mol. Biol. 2004, 34, 937−949. (56) Weis, S. M.; Cheresh, D. A. Pathophysiological consequences of VEGF-induced vascular permeability. Nature 2005, 437, 497−504. (57) Danneels, E. L.; Rivers, D. B.; Graaf, D. C. Venom proteins of the parasitoid wasp Nasonia vitripennis: recent discovery of an untapped Pharmacopee. Toxins 2010, 2, 494−516. (58) Pelosi, P.; Calvello, M.; Liping Ban, L. Diversity of odorantbinding proteins and chemosensory proteins in insects. Chem. Senses 2005, 30 (suppl 1), 291−292. (59) Pelosi, P.; Maida, R. Odorant-binding proteins in insects. Comp. Biochem. Physiol. 1995, 111B, 503−514. (60) Krieger, M. J. B.; Ross, K. G. Identification of a major gene regulating complex social behavior. Science 2002, 295, 328−332.

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dx.doi.org/10.1021/pr300451g | J. Proteome Res. XXXX, XXX, XXX−XXX