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Proteome and Allergenome of Asian Wasp, Vespa af finis, Venom and IgE Reactivity of the Venom Components Nitat Sookrung,*,† Siriporn Wong-din-dam,‡,§ Anchalee Tungtrongchitr,§ Onrapak Reamtong,⊥ Nitaya Indrawattana,# Yuwaporn Sakolvaree,§ Nualanong Visitsunthorn,∥ Wiparat Manuyakorn,▽ and Wanpen Chaicumpa§ †

Department of Research and Development, ‡Graduate Program in Immunology, Department of Immunology, §Department of Parasitology, and ∥Department of Pediatrics, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand ⊥ Department of Molecular Tropical Medicine and Genetics and #Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand ▽ Department of Pediatrics, Faculty of Medicine Ramathibodi Hispital, Mahidol University, Bangkok 10400, Thailand S Supporting Information *

ABSTRACT: Vespa af f inis (Asian wasp, Thai banded tiger wasp, or local name: Tor Hua Seua) causes the most frequent incidence of medically important Hymenoptera sting in South and Southeast Asia. However, data on the venom components attributable to the sting derived-clinical manifestations (local reactions, IgE mediated-anaphylaxis, or systemic envenomation) are lacking. This study provides the first set information on V. af finis venom proteome, allergenome, and IgE reactivity of individual venom components. From 2DE-gel basedproteomics, the venom revealed 93 protein spots, of which proteins in 51 spots could be identified and classified into three groups: typical venom components and structural and housekeeping proteins. Venom proteins in 32 spots reacted with serum IgE of wasp allergic patients. Major allergenic proteins that reacted to IgE of >50% of the wasp allergic patients included PLA1 (100%), arginine kinase (73%), heat shock 70 kDa protein (73.3%), venom allergen-5 (66.7%), enolase (66.7%), PLA1 magnifin (60%), glyceraldehyde-3-phosphate dehydrogenase (60%), hyaluronidase (53.3%), and fructose-bisphosphate aldolase (53.3%). The venom minor allergens were GB17876 transcript (40%), GB17291 transcript (20%), malic enzyme (13.3%), aconitate hydratase (6.7%), and phosphoglucomutase (6.7%). The information has diagnostic and clinical implications for future improvement of case diagnostic sensitivity and specificity, component-resolve diagnosis, and design of specific Hymenoptera venom immunotherapy. KEYWORDS: allergen, allergenome, IgE immunoblotting, proteomics, Vespa affinis

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INTRODUCTION Members of the order Hymenoptera are classified into three families including Apidae (bee), Vespidae (wasp), and Formicidae (ant).1 Hymenoptera venoms contain a mixture of vasoactive amines, acetylcholine, kinins, and proteins, many of which have been recognized as important allergens, that is, phospholipase A1 (PLA1), phospholipase A2 (PLA2), hyarulonidase, acid phosphatase, and other proteins with unknown functions.2 The venom serves as a chemical arsenal for the defense of the insect themselves, their colony, and territory.3,4 Clinical manifestations of the Hymenoptera stings in human can be broadly classified into six types: (1) small local reaction characterized by redness, pain, and swelling (about 4 to 5 cm), which improves within ∼20 min and disappeared in ∼24 h without complication or sequel; (2) large local swelling (>10 cm) with fever, malaise, headache, and chill, which usually occurs after repeated stinging and lasts longer than 24 h; (3) © 2014 American Chemical Society

generalized cutaneous reaction manifested as urticaria, itch, and angioedema, which appears some minutes after stinging; (4) systemic anaphylactic reaction (type-1/IgE-mediated hypersensitivity), which involves not only cutaneous tissue but also gastrointestinal, respiratory, cardiovascular, and neurological systems and can be fatal if not treated promptly and properly; (5) systemic toxic reaction (non-IgE mediated) due to large injected venom amount that causes multiorgan dysfunction, which may also be fatal; and (6) unusual reaction that occurs several hours after stinging including serum sickness-like syndrome, nephritic syndrome, myocarditis, vasculitis, neuritis, and encephalopathy. In Thailand and neighboring countries, the most frequent incidence of medically important Hymenoptera sting is caused Received: September 7, 2013 Published: January 20, 2014 1336

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Table 1. Background Information of the 15 Hymenoptera Allergic Patients and 9 Normal Controlsa,b subject

sex

age (years)

symptoms

skin testc

clinical diagnosis

serum specific IgE in KAU/L to Hymenoptera venom (insect species)

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 N1 N2 N3 N4 N5 N6 N7 N8 N9

M M M M M M M M M M F M M F M F F F F F M M M M

15 9 9 7 9 14 10 7 10 10 14 15 14 15 15 25 25 25 30 35 18 15 24 30

S, CVS, RS S, CVS, RS S, RS S, CVS, RS S, RS S, RS S, RS S, CVS, RS S, RS, NS, GI S, CVS, RS S, CVS, RS S, CVS, RS S, CVS, RS S, CVS, RS S, CVS, RS Nil Nil Nil Nil Nil Nil Nil Nil Nil

+ + + + + + + + + + + + + + + − − − − − − − − −

venom anaphylaxis venom anaphylaxis venom anaphylaxis venom anaphylaxis venom anaphylaxis venom anaphylaxis venom anaphylaxis venom anaphylaxis venom anaphylaxis venom anaphylaxis venom anaphylaxis venom anaphylaxis venom anaphylaxis venom anaphylaxis venom anaphylaxis normal normal normal normal normal normal normal normal normal

5.7 (HB), 0.55 (PW), 1.20 (WFH), 3.10 (YH), 3.67 (YJH) 3.26 (WFH), 3.10 (YH), 3.67 (YJH) 2.57 (YH), 3.03 (WFH), 0.46 (PW) 9.4 (HB), 1.92 (WFH), 1.17 (YJH), 0.41 (PW), 1.63 (YH) 2.9 (HB), 3.28 (WFH), 2.16 (YH), 0.96 (PW), 2.16 (YJH) 2.49 (WFH), 11.7 (HB) 4.17 (YH) 4.6 (HB) 56.3 (HB) 9.9 (HB) 1.8 (HB) 3.0 (HB) 3.2 (HB) 3.0 (HB) 60.40 (HB), 3.50 (WFH), 16.80 (YH), 8.24 (WYJ) 0.15 (HB), 0.85 (YH), 0.62 (WYJ), 1.58 (WFH) − − − − − − − − −

a

P, patient; N, normal; M, male; F, female; HB, honey bee (Apis mellifera); PW, paper wasp (Polistes spp.); WFH, white-faced hornet (Dolichovespula maculata); YJH, yellow jacket hornet (Vespula spp.); YH, yellow hornet (Dolichovespula arenaria); S, symptoms of urticaria, angioedema, and swelling of eyes and mouth; CVS, low blood pressure; NS, neurological signs (depress); RS, wheeze and bronchospasm; GI, gastrointestinal symptoms (vomiting). bSerum samples of P7−P13 were tested only against venom of honey bees (HB). cSkin test was performed using imported Hymenoptera venom preparations that were from species other than V. af f inis; +, positive; −, negative.

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by a highly aggressive Vespa af finis (Asian wasp or Thai banded tiger wasp). This wasp species is aerial; they nest ubiquitously on trees or under eaves of houses.5 Diagnosis of Hymenoptera allergy in Thailand is based on the stinging history, physical examination, and specific antibody detection by using in vivo skin prick/intradermal test and in vitro serum-specific IgE level determination.6 The diagnostic reagents are imported from abroad and contain venoms of five insect species including honey bee, yellow jacket, wasp, yellow hornet, and white-faced hornet, which are heterologous to the causative wasp species of the patients. Besides, the reagents are expensive, and the supply is limited or slow to come. By using the heterologous venom for diagnosis, it is not known what venom component(s) the patients are actually allergic to, thus making it difficult to design a proper specific immunotherapeutic plan. Efficacy of the treatment using heterologous venom preparation is unsatisfactory. Among the stinging Hymenoptera spp., only the venom of honey bee (Apis mellifera carnica), fire ant (Solenopis invicta), and social wasp, that is, Polybia paulista, which is predominant in Southeast Brazil, has been well-characterized.7−12 Information on the venom components of other wasps, particularly the Asian wasps, including their biological and chemical natures, physiological functions, role as allergens, as well as degrees of allergenicity of individual components, is negligible. Thus, in this study, proteome and allergenome of V. af finis were characterized by using gel-based-proteomics and 2D IgE immunoblotting, respectively. Allergenicity of the wasp allergenic components was also studied using sera of wasp allergic patients.

MATERIALS AND METHODS

Serum Samples

This study was approved by Ethical Committee of the Faculty of Medicine Siriraj Hospital Siriraj (COA no. SI550/2011) and the Faculty of Medicine Ramathibodi Hospital (COA no. 2555/179), Mahidol University, Bangkok, Thailand. Serum samples were collected from 15 patients who were stung by the V. aff inis (this wasp was common, and the patients could recognize them through their unique appearance and colors) and positive by skin prick test to imported wasp venom extract. Sera from nine nonallergic subjects served as controls. Background information of all subjects is shown in Table 1. Amounts of serum-specific IgE of the subjects were determined using reagents containing venoms of various Hymenoptera spp. (ImmunoCAP, Thermo Scientific, USA). The cutoff levels between positive and negative specific IgE were 0.35 kiloallergy units (kAU)/L. All serum samples were stored at −80 °C until use. Preparation of V. aff inis Venom

Nests of the aerial wasps collected from Nakhon-Ratchasima province located 260 km northeast of Bangkok were placed in plastic bags and immediately transported to Bangkok where adult wasps were verified as V. af f inis by an entomologist. The freshly collected mature wasps were kept frozen. The venom sac was removed from each insect by pulling it out of the body using forceps and small scissors, washed with PBS, and placed in small volume of buffer containing a cocktail of protease inhibitors (Roche, USA). Venom was collected from each sac, pooled, and centrifuged at 10 000g, 4 °C for 10 min. The 1337

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Two DE-IgE Immunoblotting

supernatant was dialyzed in distilled water, lyophilized, and kept at −80 °C until use.

The 2DE separated V. af f inis venom was blotted onto nitrocellulose (NC) membranes (Pall, Mexico), and the NC was blocked with 3% bovine serum albumin (BSA) in PBS. After washing with PBS containing 0.05% Tween-20 (PBS-T), the NCs were reacted with individual serum samples (diluted 1:4) at 4 °C overnight. Excess serum was discarded, and the NC was washed with the PBS-T before incubating with 1:2000 diluted mouse antihuman IgE-biotin conjugate (Southern Biotech, USA) for 3 h, washed, and incubated with 1:5000 diluted streptavidin-labeled horseradish peroxidase (HRP) (Southern Biotech) for 30 min. Protein spots were detected by using chemiluminescence (LuminataClassico Western HRP substrate, Millipore, Germany) and autoradiography (Syngene, U.K.).

Sodium Dodecyl Sulfate Polyacrilamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE was carried out in 8.0 × 9.5 cm gel by using the Hoefer SE 260 casting apparatus (GE Healthcare, USA). A 4% stacking gel and a 13% separating gel were used. Two-Dimensional Gel Electrophoresis and Protein Identification by LC−MS/MS

The wasp venom two-dimensional gel electrophoresis (2DE) was performed as previously described.13 The venom was cleaned to eliminate nonprotein substances, and the protein content was determined using 2D-Quant kit (GE Healthcare). Sixty micrograms of the venom was added to DeStreak Rehydration Solution containing IPG (0.5% IPG buffer pH 3− 11 nonlinear; GE Healthcare) and transferred to the IPG strip holder. An IPG strip was placed right-side-down into the strip holder containing the venom, and 1 mL of the dry strip cover fluid was added. The strip was allowed to rehydrate at 20 °C for 12 h. Electrophoresis was performed initially at 0.2 kV/h for 30 min, followed by 0.3 kV/h gradient for 30 min, 4.0 kV/h for 80 min, and step and hold for 15 min. The focused IPG strip was equilibrated in a reduction buffer [50 mM Tris-HCl, pH 8.8; 6 M urea; 30% (v/v) glycerol; 2% (w/v) SDS; 0.002% bromophenol blue; and 1% (w/v) dithiothreitol (DTT)] at 25 °C for 15 min; it was then placed in an alkylation buffer [50 mM Tris-HCl, pH 8.8; 6 M urea; 30% (v/v) glycerol; 2% (w/v) SDS; 0.002% bromophenol blue; and 2.5% (w/v) iodoacetamide] at 25 °C for 15 min. SDS-PAGE was carried out, and the protein spots were revealed by staining with Coomassie Brilliant Blue G-250 (CBB) dye. Gel plugs containing proteins in the CBB-stained-2DE gel were excised out and destained with a wash solution (50% methanol and 5% acetic acid in ultrapure distilled water) at 25 °C overnight. Individual gel plugs were rinsed with the wash solution, and proteins in the plugs were digested with trypsin. nLC−MS/MS model MicroToF Q Mass Spectrometer (Bruker Daltonics, Germany) was used for peptide analysis. The nLC system was an ultimate 3000 (Dionex, U.K.), and the flow rate was 300 nL/min. The two mobile phases were: (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile. After washing to remove unbound peptides with A, the following gradients of B were applied to the column: 0−60% B in A for 30 min, 65−80% B for 5 min, and 80−0% B for 2 min. Peptides in the eluates were analyzed by mass spectrometry. NanoSpray with positive ions in ionization mode at a capillary temperature of 85 °C and a 22 kV spray needle were used. The scan sequence was scanned MS with a mass range of 400−2000 m/z and MS−MS scan with a mass range of 50−1500 m/z. The ion spectra of peptides generated by the mass spectrometry were interpreted using MASCOT version 2.2. The SwissProt database was set (UniProtKB/SwissProt Release of May 16th, 2012 contained 536 029 sequence entries). Protein search parameter included a precursor peptide mass tolerance of 200 ppm, fragment mass tolerance of 0.6 Da, variable modification of methionine (M) oxidation, and cysteine (C) carbamidomethylation. For the tryptic status requirement, missed cleavage was set to 1. Only peptides identified above 95% confidence were reported in this study.

Purification of V. af finis Phospholipase A1 (PLA1) and the PLA1 Enzymatic Activity

The lyophilized wasp venom (10 mg) was solubilized in 0.05 mM phosphate buffer, pH 7.0, and loaded to a pre-equilibrated Hiprep 16/60 Sephacryl S-300 column (120 mL) (GE Healthcare) connected to an AKTA-FPLC system. The proteins were eluted out with the buffer at 0.5 mL/min, and 1 mL fractions were collected. Fractions of the same protein peak were pooled. Phospholipase activity of each pool was determined by using fluorometric EnzCheck phospholipase A1 assay kit (Invitrogen, USA). The PED-A1 substrate provided in the kit (a glycerophosphoethanolamine with BODIPY FL dyelabeled acyl chain at the sn-1 position and dinitrophenyl quencher modified headgroup) was specific for PLA1 . Quenching efficiency was decreased after cleaving the BODIPY FL pentanoic acid substituent at the sn-1 position, and the result was a PLA1-dependent increase in fluorescence emission detectable at ∼515 nm (OD515nm). Fluorescence was measured on a Fluoroskan Ascent FL microplate fluorometer and luminometer (Thermo Scientific, USA) and subtracted by the background fluorescence control of the negative PLA1 enzyme. The lowest limit of the assay was 0.04 PLA1 units/mL. In the assay, a standard curve was constructed by plotting the fluorescence emission intensities versus known PLA1 concentrations. A selected pool with PLA1 activity was dialyzed against distilled water, lyophilized, and subjected to SDS-PAGE, and the proteins in the stained bands were identified by LC−MS/ MS. Dot Blot-ELISA for Determining Serum IgE Binding to V. af finis PLA1

Squares (10 × 10 mm) were made on a 0.2 μm NC (Bio-RAD, USA). Onto each square, purified PLA1 (1 μg in 100 μL of PBS) was dotted using a slot blot machine (Bio-Dot and BioDot SF Microfiltration Apparatus) connected to a suction pump. The NC was air-dried and blocked with 3% (w/v) BSA in PBS for 1 h. After washing with PBST, the NC was cut into small squares (each square contained one PLA1 dot), and individual NC pieces were reacted separately with individual serum samples at 4 °C overnight. The NC pieces were washed and incubated with 1:2000 diluted mouse antihuman IgE-biotin conjugate (Southern Biotech) and HRP-labeled streptavidin (1:5000 in PBS), respectively, with washing between the steps. The PLA1-IgE reactive spots were revealed by chemiluminescence as previously described. 1338

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Basophil Degranulation (Histamine Release) Assay

RBL-2H3 cells (Wistar rat basophilic leukemic cell line), which displayed abundant high affinity IgE receptors (FcεRI) on their surface, were used. These cells could be activated to secrete histamine and other mediators by allergen that cross-links their surface IgE or by calcium ionophores.14 The cells were cultured in a 24-well tissue culture plate (105 cells/well) in 100 μL of DMEM containing 15% heat-inactivated fetal calf serum (Hyclone, USA) at 37 °C in 5% CO2 atmosphere. Individual serum samples (diluted 1:4) were added to triplicate wells and incubated overnight. The culture medium was removed, and the cells were washed with fresh medium before the addition of either 100 μL containing 5 ng of purified wasp PLA1 (test) or medium (control), and the plate was kept further for 1 h. Culture supernatant (50 μL) was collected from each well for histamine quantification by using a histamine competitive ELISA kit (Genway, USA). In brief, each culture supernatant was added to a well coated with fixed amount of antibody, incubated, and washed. Fixed amount of enzyme-labeled histamine was added to the well, and the color reaction was developed. The intensity of the color was inversely proportional to the amount of histamine in the test sample; the exact amount could be extrapolated from a standard curve constructed from the reagents provided with the test kit.

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Figure 2. Representative of 2DE-gels of V. af f inis venom stained with CBB dye.

ranged in molecular masses from 10 to 93 kDa and pI from 3 to 11. Among them, proteins in 51 spots could be identified by LC−MS/MS (Table S1 in the Supporting Information). No peptides of the database matched with peptides derived from venom proteins of the other 42 spots. The identified wasp venom proteins were classified into three groups (Figure 3 and Table S2 in the Supporting Information): (1) typical venom proteins that included PLA1, arginine kinase, venom allergen-5, enolase, hyaluronidases, and heat shock proteins; (2) structural proteins, that is, actin, muscle specific protein-20, and troponint; and (3) housekeeping proteins including aconitate hydratase, δ-1-pyrroline-5-carboxylate dehydrogenase, exonuclease SbcC, fructose-biphosphate aldolase, malic enzyme, NADH dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglucomutase, pyruvate kinase, triosephosphate isomerase, GB transcription factors, ENSAPMG transcription factor, putative uncharacterized protein, 12 kDa FK506-binding protein, and ubiquitin.

RESULTS

SDS-PAGE Patterns of the V. af finis Venom

The protein amounts in venom collected from individual V. af finis venom sacs ranged from 11 to 14 μg. Patterns of SDSPAGE-separated V. af f inis venom proteins after staining with CBB and Silver dyes are shown in Figure 1A,B, respectively. The protein molecular masses ranged from ∼10 to 130 kDa. 2DE Pattern of V. aff inis Venom

Representative 2DE pattern of V. af f inis venom after staining with CBB dye is shown in Figure 2. The 2DE gels from different runs showed a high degree of identity (correlation coefficient >95%). There were 93 visually stained protein spots

Allergenome of V. aff inis Venom Identified by 2DE-IgE Immunoblotting

After blotting the 2DE separated V. af f inis venom onto the NC and probed with individual serum samples for detecting protein spots bound by the serum IgE, it was found that no spot appeared after probing the blots with the normal sera, but there were altogether 32 spots reactive to the IgE in the sera of the 15 allergic patients. They were spot nos. 3, 7, 8, 9, 11, 12, 14, 20− 29, 32−35, 37−44, 47, 51, and 60 (Table 2). Sera of different patients reacted with different protein spots. Example of the 2DE-IgE immunoblot result (patient no. 11) is shown in Figure 4. Twenty-six V. af finis protein spots reacted with IgE in >50% of the patient sera, implying that the proteins in these spots are major allergens. The protein components in these spots were: PLA1 (100%), arginine kinase (73%), heat shock 70 kDa protein (73.3%), venom allergen-5 (66.7%), enolase (66.7%), PLA1 magnifin (60%), glyceraldehyde-3-phosphate dehydrogenase (60%), hyaluronidase (53.3%), and fructose-bisphosphate aldolase (53.3%). The minor allergens that reacted with serum IgE of 6, 3, 2, 1, and 1 patient(s) were GB17876

Figure 1. Proteins in V. af f inis venom revealed by SDS-PAGE. SDSPAGE separated venom stained with CBB dye (lane 1 of A) and Silver stain (lane 1 of B). Lane M of panels A and B is prestained broad range protein standard. Numbers at the left of both (A) and (B) are protein masses in kilodaltons. 1339

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Figure 3. Three groups of the identified wasp venom proteins.

normal controls (background), which was 407.93 ng/mL. The cells sensitized with all patient sera released significant amounts of histamine above the background after incubating with V. af f inis PLA1 (Figure 8). Overall results indicated that V. af f inis PLA1 is a major allergen that could bind to IgE in 100% of the wasp allergic patients and caused histamine release from the IgE-fixed cells.

transcript (40%), GB17291 transcript (20%), malic enzyme (13.3%), aconitate hydratase (6.7%), and phosphoglucomutase (6.7%), respectively (Table 2). Native V. aff inis PLA1 and Enzymatic Activity

Five protein profiles of V. af f inis venom were eluted from the Sephacryl S-300 column chromatography (Figure 5), that is, pool 1 (fraction nos. 21−26), pool 2 (fraction nos. 27−30), pool 3 (fraction nos. 31−37), pool 4 (fraction nos. 38−51), and pool 5 (fraction nos. 52−65). SDS-PAGE and CBB staining revealed that the pool 4 contained only two protein bands, while the other pools (pools 1−3) revealed multiple bands and pool 5 had no band (the proteins in this pool might be 90% of the mold allergic patients.35 In this study, the V. af f inis GAPDH reacted with IgE in 60% of the Hymenoptera allergic patients. Fructose-bisphosphate aldolase is a tetrameric glycolytic enzyme that catalyzes the reversible conversion of fructose-1,6bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. The aldolase from Candida albicans was identified as allergen.36 This study reports for the first time the allergenic role of Hymenoptera fructose-bisphosphate aldolase V. af f inis. The Hymenoptera allergens that have been reported previously but were not found in V. af finis venom in this study included dipeptideylpeptidase IV of Vespula vulgaris (Ves v 3) and Apis mellifera (Api m 5), PLA2 (Api m 1), acid phosphatase (Api m 3), CUB serine protease (Api m 7), carboxylesterase (Api m 8) and icarapin, Apis cerana cerana melittin (Api c 4), and Myrmecia pilosula pilosulin-3 (Myr p 2).20

Two-DE-based-proteome of V. aff inis venom identified by LC−MS/MS. LC−MS/MS Mascot results of Vespa af f inis ingel trypsin digestions. Standard curve of PLA1 fluorescence intensity excitation at absorbance 505 nm and emission at 515 nm as measured by using a Fluoroskan Ascent FL Microplate Fluorometer and Luminometer. A standard curve of histamine concentrations constructed from the standard histamine provided in the assay kit. Annotated tandem mass spectra of single high-confidence peptide assignments. This material is available free of charge via the Internet at http://pubs.acs.org.

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Corresponding Author

*Tel./Fax: +66-4196497. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was supported by the Thailand Research Fund (DPG5380001 and MRG5308080), the National Research University (NRU) project of the Office of Higher Education Commission (CHE), and the Faculty of Medicine Siriraj Hospital, Mahidol University. We thank all patients and control subjects and Dr. Suwat Benjapolpitak, Ramathibodi Hospital, for his encouragement and support.

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ABBREVIATIONS BSA, bovine serum albumin; CBB, Coomassie Brilliant Blue G250 dye; DMEM, Dulbecco’s modified Eagle’s medium; 2DE, two-dimensional gel electrophoresis; DTT, dithiothreitol; IPG, Immobiline DryStrip gel; IgE, immunoglobulin E; kV, kilovolts; LC−MS/MS, liquid chromatography/tandem mass spectrometry; NC, nitrocellulose membrane; PBS, phosphate-buffered saline, pH 7.4; PBST, PBS containing 0.05% Tween-20; PLA1, phospholipase A1; PLA2, phospholipase A2; SDS-PAGE, sodium dodecyl sulfate polyacrilamide gel electrophoresis; V. af f inis, Vespa af f inis

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REFERENCES

(1) Fitzgerald, K. T.; Flood, A. A. Hymenoptera stings. Clin. Tech. Small Anim. Pract. 2006, 21, 194−204. (2) Dongol, Y.; Shrestha, R. K.; Aryal, G.; Lakkappa, D. B. Hymenoptera stings and the acute kidney injury. EMJ Nephrol. 2013, 1, 68−75. (3) Palma, M. S. Insect Venom Peptides. In Handbook of Biologically Active Peptides; Kastin, A., Ed.; Academic Press: San Diego, 2006; pp 409−417. (4) dos Santos, L. D.; de Silva Menegasso, A. R.; dos Santos Pinto, J. R.; 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. (5) Seeley, T. D.; Seeley, R. H. A nest of a social wasp, Vespa af finis, in Thailand (Hymenoptera: Vespidae). Psyche 1980, 87, 299−304. (6) Lertnawapan, R.; Maek-a-nantawat, W. Anaphylaxis and biphasic phase in Thailand: 4-year observation. Allergol. Int. 2011, 60, 283−289. (7) Peiren, N.; Vanrobaeys, F.; de Graaf, D. C.; Devreese, B.; Van, B. J.; Jacobs, F. J. The protein composition of honeybee venom reconsidered by a proteomic approach. Biochim. Biophys. Acta 2005, 1752, 1−5. (8) Peiren, N.; de Graaf, D. C.; Evans, J. D.; Jacobs, F. J. Genomic and transcriptional analysis of protein heterogeneity of the honeybee venom allergen Api m 6. Insect Mol. Biol. 2006, 15, 577−581. (9) Peiren, N.; de Graaf, D. C.; Vanrobaeys, F.; Danneels, B.; Devreese, B.; Beeumen, J. V.; Jacobs, F. J. Proteomic analysis of the

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CONCLUSIONS The first data set on proteome and allergenome of Asian wasp, V. af finis, which is the predominant wasp species in Thailand and South and Southeast Asia is reported herein. Major and minor allergens of the wasp venom components were identified, of which many components are novel Hymenoptera allergens. Allergenicity (IgE reactivity) of each V. af f inis allergenic component was determined. Information provided in this study has diagnostic and clinical implications in future improvement of case diagnostic sensitivity and specificity, CRD, and design of specific Hymenoptera venom immunotherapy.

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AUTHOR INFORMATION

ASSOCIATED CONTENT

S Supporting Information *

LC−MS/MS Mascot results of in-gel tryptic digestion of V. af finis venom sac protein searching against NCBInr database. 1343

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Journal of Proteome Research

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dx.doi.org/10.1021/pr4009139 | J. Proteome Res. 2014, 13, 1336−1344