Proteomic analysis identification of antigenic proteins in Gnathostoma spinigerum larvae

Experimental Parasitology 159 (2015) 53e58

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Proteomic analysis identification of antigenic proteins in Gnathostoma spinigerum larvae Penchom Janwan a, b, Pewpan M. Intapan b, c, Porntip Laummaunwai b, c, Rutchanee Rodpai b, c, Chaisiri Wongkham d, Tonkla Insawang e, Tongjit Thanchomnang b, f, Oranuch Sanpool b, f, Wanchai Maleewong b, c, * a

Department of Medical Technology, School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat 80161, Thailand Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand d Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand e Khon Kaen University Research Instrument Center, Research Affairs, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand f Faculty of Medicine, Mahasarakham University, Maha Sarakham 44000, Thailand b c

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Of the 93 Gnathostoma spinigerum antigenic spots excised, 87 were identified by LC/MSeMS.
Twenty seven types of proteins were identified from the database search.
The set of identified antigenic molecules shows the diversity of processes.
Most protein is protein involving in metabolic process and energy generation.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 April 2015 Received in revised form 31 July 2015 Accepted 16 August 2015 Available online 25 August 2015

Gnathostoma spinigerum is the causative agent of human gnathostomiasis. The advanced third stage larva (AL3) of this nematode can migrate into the subcutaneous tissues, including vital organs, often producing severe pathological effects. This study performed immuno-proteomic analysis of antigenic spots, derived from G. spinigerum advanced third stage larva (GSAL3) and recognized by human gnathostomiasis sera, using two-dimensional (2-DE) gel electrophoresis based-liquid chromatography/tandem mass spectrometry (LC/MSeMS), and followed by the aid of a database search. The crude GSAL3 extract was fractionated using IPG strips (pH 3-11NL) and followed by SDS-PAGE in the second dimension. Each gel was stained with colloidal Coomassie blue or was electro-transferred onto a nitrocellulose membrane and probed with gnathostomiasis human sera by immunoblotting. Individual Coomassie-stained protein spots corresponding to the antigenic spots recognized by immunoblotting were excised and processed using LC/MSeMS. Of the 93 antigenic spots excised, 87 were identified by LC/MSeMS. Twenty-seven protein types were found, the most abundant being Ascaris suum37. Six spots showed good quality spectra, but could not be identified. This appears to be the first attempt to characterize antigenic proteins from GSAL3 using a proteomic approach. Immuno-proteomics shows promise to assist the search for

Keywords: Gnathostoma spinigerum Proteomic analysis Antigenic protein spots

* Corresponding author. Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. E-mail address: [email protected] (W. Maleewong). http://dx.doi.org/10.1016/j.exppara.2015.08.010 0014-4894/© 2015 Elsevier Inc. All rights reserved.

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candidate proteins for diagnosis and vaccine/drug design and may provide better understand of the hostparasite relationship in human gnathostomiasis. © 2015 Elsevier Inc. All rights reserved.

1. Introduction

2.2. Human gnathostomiasis serum

Gnathostoma spinigerum is a spirurid nematode that causes human gnathostomiasis. It is found mainly in Asian countries, especially Thailand, Japan, China and Vietnam (Daengsvang, 1981). Infection in humans usually occurs by eating undercooked freshwater fish or other meat (e.g. chicken, frog, snake, etc.) contaminated with the infective stage of G. spinigerum, the advanced third stage larva (GSAL3). The worm migrates into the subcutaneous tissues, including vital organs in which severe pathologic effects may be produced, leading to serious complications and death (Daengsvang, 1980). Human are accidental hosts. The parasite cannot mature in humans, but remains an advanced third stage larva (AL3), or may develop further into a fourth stage larva or immature adult (Daengsvang, 1981). Human gnathosomiasis is one of the serious “neglected tropical diseases” and there is an urgent need to gain insight into the mechanisms of immune evasion, hostparasite interplay and immunopathogenesis. There is additional a need to identify molecules that can assist in vaccine/drug design and to find new biomarkers for diagnostic and prognostic purposes. Previously, the amino acid sequences of some antigenic spots with molecular masses of around 23e25 kDa and isoelectric points (pI) of 8.3e8.5, were determined using liquid chromatography tandem mass spectrometry (LC/MSeMS) (Laummaunwai et al., 2008). These sequences matched portions of cyclophilin, hypothetical protein, actin, matrix metalloproteinase-like protein and intermediate filament protein B in the nr.fasta database (Laummaunwai et al., 2008). However, there are no reports regarding the biochemical properties of all the antigenic protein spots derived from GSAL3. Proteomic approaches provide one strategy for studying the protein expression patterns of organisms, using crossspecies databases to interpret mass spectrometry data (Cui et al., 2013). To date, there are no comprehensive reports characterizing the GSAL3 immuno-proteome. The aim of this study was to identify all of immunogenic proteins of GSAL3 using immunoproteomic and mass spectrometry techniques.

Pooled positive reference serum was prepared from thirteen parasitologically confirmed cases of adult human gnathostomiasis. Pooled negative reference serum was prepared from thirty healthy adult volunteers who were free from any intestinal parasitic infection, as checked by stool examination using the formalin ethyl acetate concentration technique (Elkins et al., 1986) at the time of blood collection. Informed consent was obtained from all adult participants and from parents or legal guardians of minors. The study protocol was approved by the Khon Kaen University Ethics Committee in Human Research (HE561477).

2. Materials and methods

2.3. Two-dimensional (2-DE) gel electrophoresis 2-DE was performed using the IPGphor system (GE Healthcare Bio-Sciences AB, San Francisco). For the first dimension, IEF was performed using ready-made 7 cm Immobiline DryStrip gels with a non-linear pH gradient of 3e11. Briefly, a strip was rehydrated with rehydration solution (8 M urea, 2% (w/v) CHAPS, 0.5% IPG buffer, 0.28% dithiothreitol (DTT)) and crude extract of GSAL3 (200 mg protein). Then the IPG strip was placed in the gel upside down in the holder and covered with 1 mL of IPG cover fluid. The holder was closed with a lid and the IPG strip was incubated for 13 h at 20  C followed by focusing at 8000 Vh at 20  C. After focusing, the strip was equilibrated twice for 15 min each, with the first equilibration solution (6 M urea, 30% glycerol, 2% SDS, 1% DTT, supplemented with trace amounts of bromophenol blue in 50 mM TriseHCl, pH 8.8) and with the second equilibration solution (6 M urea, 30% glycerol, 2% SDS, 2.5% iodoacetamide, supplemented with trace amounts of bromophenol blue in 50 mM TriseHCl, pH 8.8), respectively. Then, each of the focused gel strips was further subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970). After electrophoresis, each gel was stained with Coomassie blue; the interesting spots were excised for LC/MSeMS or the separated proteins were electro-transferred onto a nitrocellulose membrane for immunoblotting.

2.1. Gnathostoma spinigerum antigen The GSAL3 were collected from mice orally inoculated with early third-stage larvae recovered from copepods (Maleewong et al., 1988). Crude somatic extract of GSAL3 was prepared by homogenization and extraction as described previously (Maleewong et al., 1997). Briefly, the worms (n ¼ 40) were homogenized with a tissue grinder in a small volume of 0.1 M phosphate buffered saline (PBS) containing proteinase inhibitors (0.1 mM of phenylmethylsulfonyl fluoride, 0.1 mM of L-1-tosylamide-2phenylethylchloromethylketone and 1 mM of L-trans-3carboxyoxiran-2-carbonyl-L-leucylagmatine). The preparation was then sonicated with an ultrasonic disintegrator and centrifuged at 10,000g for 30 min at 4  C. The supernatant was dialyzed against distilled water containing the same proteinase inhibitors and kept at 20  C until further analysis. The protein content of the antigen was determined using the standard method (Lowry et al., 1951).

2.4. Western blot analysis The membrane was immersed in a blocking solution of 1% skim milk in a 100 mM PBS solution containing 0.1% Tween 20 for 30 min at room temperature and was then incubated with pooled positive or negative reference serum samples (diluted 1:100 in blocking solution) for 2 h at room temperature. Thereafter, it was washed 5 times with PBS containing 0.05% Tween 20, followed by incubation for 2 h with goat anti-human IgG (Fc) labeled with horseradish peroxidase conjugate (Zymed laboratories Inc, San Francisco, CA) (diluted 1:2500 in blocking solution). For visualization of the antibody reaction 3,30 -diaminobenzidine tetrahydrochloride (DAB) and hydrogen peroxide were used as chromogenic substrates. To ensure reproducibility of the tests, each experiment was performed in triplicate; all tests produced uniform results.

P. Janwan et al. / Experimental Parasitology 159 (2015) 53e58

2.5. 2-DE-based-liquid chromatography/tandem mass spectrometry (LC/MSeMS) and protein identification Coomassie-stained individual protein spots of interest corresponding to the antigenic spots recognized by pooled human gnathostomiasis sera by immunoblotting were excised and sent for analysis to the proteomic service at the Khon Kaen University Research Instrument Center, Thailand. Coomassie-stained spots recognized by pooled healthy human sera were excluded. Briefly, the gel plugs were destained, airdried, and subjected to tryptic digestion. One ml (10 fmol) of the BSA digest solution was used as a standard control for the experiment. The tryptic peptides were acidified and analyzed using a nano-liquid chromatography system (EASY-nLC II, Bruker Daltonik GmbH, Bremen, Germany) coupled to an ion trap mass spectrometer (Amazon Speed ETD, Bruker Daltonik GmbH) equipped with an ESI nano-sprayer. Sample volumes of 1e2 mL were loaded by the autosampler onto a EASY-Column, 10 cm, ID 75 mm, 3 mm, C18-A2 (Thermo Scientific) using a flow rate of 300 nL/min and linear gradient from solution A (0.1% formic acid) to 35% of solution B (0.1% formic acid in acetonitrile) in 35 min. The Bruker Daltonik software package, HyStar v.3.2, was used to control the ion trap device. LC-MS/MS spectra were analyzed using Compass Data Analysis v.4.0. Compound lists were exported as Mascot generic files (mgf) for further searching in MASCOT online (http://www.matrixscience.com). Protein identification was performed by searching against the protein database from NCBI (all entries) using MASCOT MS/MS Ion Search with the initial searching parameters; Enzyme: trypsin, allowed up to one missed cleavage; carbamidomethylation (C) as a fixed modification, and oxidation (HW) and oxidation (M) as variable modifications; peptide mass tolerance of 0.5 Da and fragment mass tolerance of 0.5 Da; a peptide charge state of þ1, þ2, þ3; instrument type: ESI-TRAP; and report top: auto. The molecular functions and biological roles of the identified proteins were assigned according to the gene ontology (GO) database (http://www.geneontology.org) and the Swiss-Prot/ UniProt database (http://beta.uniprot.org). 3. Results and discussion Only 93 antigenic protein spots, which were recognized by human gnathostomiasis sera but not by healthy human sera, were considered and further analyzed by mass spectrometry. Most target proteins were obtained from the acidic region of the IPG strip and their molecular masses ranged between 20 and 40 kDa (Fig. 1). Identified target proteins were excised from gels, digested with

55

trypsin and the resulting peptides analyzed by mass spectrometry (Table S1-Supplementary material). Twenty-seven types of different proteins were identified using the Mascot program (Table 1). Since there was limited information regarding Gnathostoma gene sequences, identification was based on matches to homologous proteins from related nematodes (Table 1), e.g. Caenorhabditis sp., Ascaris suum, Brugia malayi, Wuchereria bancrofti, Onchocerca volvulus, Necator americanus, Haemonchus contortus, Loa loa, Trichinella sp., Anisakis simplex, Strongyloides stercoralis and Ditylenchus destructor. The most abundant identified protein was Ascaris suum37 (As37) (17 spots). Although 6 spots show good quality spectra, they could not be identified (Table 1). In addition, Table 1 shows the biological processes and molecular functions for each immunogenic parasite protein where information was available from gene ontology (GO) term analysis. Many proteins had metabolic roles and likely exhibited catalytic activity. Some proteins had other roles: actin (locomotion), heat shock proteins & chaperone proteins (response to stimulus), cytoplasmic intermediate filament protein (binding), peroxiredoxin (antioxidant), galactoside-binding lectin (binding), Third Party Data_ inferential (TPA_inf): eukaryotic translation elongation factor 1 alpha (binding), galectin (binding), and ATPase and cell division protein 48 and vacuolar protein sorting (Vps4) oligomerisation domain containing protein (binding). We performed an immunoproteomic analysis to identify all immunogenic proteins of G. spinigerum in human gnathostomiasis. To date, there have been few proteomic studies on Gnathostoma species and human gnathostomiasis. In our previous study, 2-DE gel electrophoresis of GSAL3 crude extract, followed by immunoblotting using pooled human sera from proven gnathostomiasis cases, detected a number of prominent antigenic peptides, with at least 70 such spots having approximate molecular masses ranging from less than 21.2 to more than 108 kDa with pI between 5 and 10 (Laummaunwai et al., 2008). Peptide sequences of two of these antigenic spots, which had approximate molecular weights of 23e24 kDa and a pI of 8.1e9.3, matched with portions of cyclophilin, hypothetical protein, actin, matrix metalloproteinase (MMP)-like protein and intermediate filament protein B n et al. (2012) (Laummaunwai et al., 2008). Recently, Campista-Leo separated crude somatic antigen of Gnathostoma binucleatum AL3 using 2-DE immunoblot analysis. They found four antigenic spots, three with molecular weights of 32 kDa (pI 6.3, 6.5 and 6.9) and one with a molecular weight of 40 kDa (pI 5.6), with potential as antigens specific for serodiagnosis. Sequence analysis performed on two of the four proteins showed that they belonged to the galectin

Fig. 1. Representative images of three independent replicates are shown. Crude extract of Gnathostoma spinigerum advanced third stage larvae (GSAL3) was subjected to twodimensional gel electrophoresis. The crude GSAL3 extract (200 mg) was fractionated using IPG strip (pH 3-11NL), followed by 12% SDS-PAGE. One gel was stained with colloidal Coomassie blue (A) and the other one was electro-transferred onto a nitrocellulose membrane and probed with human gnathostomiasis sera by immunoblotting (B). Individual Coomassie-stained protein spots corresponding to the antigenic spots recognized by immunoblotting were excised and subjected to LC/MSeMS. Numbers at the left of each image indicate protein molecular weight markers. Numbers and arrows on images refer to the spot identity used in the tables.

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Table 1 Summary of the immuno-reactive protein spots of the Gnathostoma spinigerum. Type of protein

Spots

Protein [helminth]

Biological process

Molecular function

1

76, 78, 80, 82, 85, 87, 88, 89, 107, 115, 125, 127, 149, 150, 186, 199, 253 59, 60, 64 70, 255 105 254 18 25, 264 68 263

As37 [Ascaris suum]

e

e

Actin 2 [Brugia malayi] Actin 2 [Ascaris suum] Actin [Caenorhabditis elegans] Actin [Caenorhabditis elegans] Heat shock protein 90 [Ascaris suum] Heat shock protein 70 [Wuchereria bancrofti] Heat shock protein 70 [Wuchereria bancrofti] Chaperonine protein HSP60 [Onchocerca volvulus] Putative chaperone protein DnaK [Necator americanus] Phosphoenolpyruvate carboxykinase domain-containing protein [Haemonchus contortus] Phosphoenolpyruvate carboxykinase [Necator americanus] Phosphoenolpyruvate carboxykinase [Loa loa] Carboxyl transferase domain protein [Necator americanus] Carboxyl transferase domain protein, partial [Necator americanus] Enolase [Trichinella spiralis] Enolase [Anisakis simplex] Glyceraldehyde-3-phosphate dehydrogenase [Ascaris suum] Glyceraldehyde-3-phosphate dehydrogenase [Onchocerca volvulus] Glyceraldehyde 3-phosphate dehydrogenase [Brugia malayi] Peptidyl-prolylcis-trans isomerase, cyclophilin-type [Necator americanus] Cyclophilin [Gnathostoma spinigerum] Peptidyl-prolylcis-trans isomerase domain containing protein [Haemonchus contortus] Putative peptidyl-prolylcis-trans isomerase A [Necator americanus] Cytoplasmic intermediate filament protein [Caenorhabditis elegans] Peroxiredoxin [Haemonchus contortus]

Locomotion (GO:0040011)

ATP binding (GO:0005524)

Response to stress (GO:0006950)

ATP binding (GO:0005524)

Gluconeogenesis (GO:0006094)

Kinase activity (GO:0016301)

Metabolic process (GO:0008152)

Transferase activity (GO:0016740)

Glycolytic process (GO:0006096)

Phosphopyruvate hydratase activity (GO:0004634) Oxidoreductase activity (GO:0016620)

2

3

140 4

20, 23, 24, 155

21 26 5

32, 35, 42, 43 33

6 7

49, 54 51, 156, 259 74, 92, 93 79 101

8

133, 161, 170 154 159 261

9

168

10

132, 142, 262, 265

11

19, 128, 260

12

72, 73, 251

13

37, 41

14

58 256

15

Matrix metalloproteinase-like protein [Gnathostoma spinigerum] Fructose-bisphosphate aldolase [Onchocerca volvulus] 53 kDa Excretory/secretory protein [Trichinella papuae] Glu/Leu/Phe/Val dehydrogenase, dimerization domain protein [Necator americanus] Glu/Leu/Phe/Val dehydrogenase, dimerization domain protein [Necator americanus] Galectin [Brugia malayi] Galactoside-binding lectin [Necator americanus] Beta-galactoside-binding lectin [Trichinella spiralis] Putative methylmalonyl [Ascaris suum]

16

100 108 109 16

17

53

18

44

19

46

20

57

21

61

4-Hydroxybutyrate coenzyme a transferase [Ascaris suum] Hypothetical protein CAEBREN_05920 [Caenorhabditis brenneri] Independent phosphoglycerate mutase [Onchocerca volvulus] TPA_inf: eukaryotic translation elongation factor 1A [Strongyloides stercoralis] Phosphoglycerate kinase [Necator americanus]

22

104

Proteasome subunit alpha type 7-1 [Brugia malayi]

23 24

139 172

Myosin heavy chain [Brugia malayi]

Glycolytic process (GO:0006096)

Protein folding (GO:0006457)

Peptidyl-prolylcis-trans isomerase activity (GO:0003755)

e

Protein binding (GO:0005515)

Oxidation-reduction process (GO:0055114) Proteolysis (GO:0006508)

Antioxidant activity (GO:0016209)

Glycolytic process (GO:0006096) e

Metalloendopeptidase activity (GO:0004222) Fructose-bisphosphate aldolase activity (GO:0004332) e

Cellular amino acid metabolic process (GO:0006520)

Oxidoreductase activity (GO:0016491)

e

Carbohydrate binding (GO:0030246)

Metabolic process (GO:0008152)

Methylmalonyl-CoA mutase activity (GO:0004494) Transferase activity (GO:0016740)

Acetyl-CoA metabolic process (GO:0006084) Metabolic process (GO:0008152) Glucose catabolic process (GO:0006007) GTP catabolic process (GO:0006184) Glycolytic process (GO:0006096) Ubiquitin-dependent protein catabolic process (GO:0006511) Metabolic process (GO:0008152)

Ligase activity (GO:0016874) Phosphoglycerate mutase activity (GO:0004619) Translation elongation factor activity (GO:0003746) Phosphoglycerate kinase activity (GO:0004618) Threonine-type endopeptidase (GO:0004298) Motor activity (GO:0003774)

P. Janwan et al. / Experimental Parasitology 159 (2015) 53e58

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Table 1 (continued ) Type of protein

Spots

25 26

191 250

27

257

No matches

153, 192, 193, 197, 200, 258

Protein [helminth]

Biological process

Molecular function

Cytoplasmic Cu/Zn-superoxide dismutase [Ditylenchus destructor] CBN-MCE-1 protein [Caenorhabditis brenneri] ATPase and cell division protein 48 and Vps4 oligomerisation domain containing protein [Haemonchus contortus] Kinesin-2 [Ascaris suum]

Oxidation-reduction process (GO:0055114) e Metabolic process (GO:0008152)

Superoxide dismutase activity (GO:0004784) e ATP binding (GO:0005524)

Metabolic process (GO:0008152)

No matches

e

Microtubule motor activity (GO:0003777) e

family of peptides. In common with our present study, they also identified a further six proteins (cyclophilin, hypothetical proteins, actin, MMP-like protein, intermediate filament protein B and  n et al., 2012). galectin) (Laummaunwai et al., 2008; Campista-Leo These proteins are the promising antigens for the diagnosis of gnathostomiasis. After comparisons with GO databases, each protein in our study was identified as participating in at least one biological process and possessing molecular functions (except for As37, 53 kDa excretory/secretory protein and CBN-MCE-1 protein). Twenty-four proteins were assigned, according to the GO database, to seven groups; metabolic process and energy generation, cell skeleton, response to stress, oxidation-reduction, protein folding, proteolysis, and carbohydrate ligand binding protein (Table 1). The most abundant proteins were those associated with different metabolic pathways (16 proteins) (Table 1), which could be used by the parasite for energy generation and survival. Those involved in gluconeogenesis, metabolic process, glycolytic process, and catabolic process were the most represented. It is not surprising that the most immunogenic proteins were those associated with the metabolic pathways and parasite survival, since these proteins were parasitic enzyme localized to the cells (intracellular or membrane protein) in the helminth during the active larval stages and maintains redox balance in response to the accumulation of the end products (Nikolaou and Gasser, 2006) or these enzymes are present in the excretory/secretory products and have been shown previously to be immunogenic (Uparanukraw et al., 2001; Laummaunwai et al., 2010). Actin and intermediate filament are components of the cytoskeleton. Actin is one of the most abundant cytoskeleton proteins found in eukaryotic cells and is highly conserved throughout evolution. It participates in many important cellular functions, including cell motility, cell division, cytokinesis, organelle movement, cell signaling and the maintenance of cell shape (Fornelio et al., 1995). Karabinos et al. (2001) made it clear that cytoskeletal intermediate filament plays essential roles in the development of Caenorhabditis elegans. The nematode cytoskeletal proteins are a possible target for anthelmintic activity (Fornelio et al., 1990). We firstly found three proteins (peroxiredoxin, cytoplasmic Cu/ Zn-superoxide dismutase and heat shock proteins (HSP) & chaperone proteins) that are involved in response to stress and redox processes. These possibly play roles in the interactions between parasite and host. Anti-oxidant proteins appear to play specific functions in protection from the host cell's killing mechanisms, including reactive oxygen and nitrogen species generated by the host immune responses (Polla, 1991). More studies are needed in order to identify new therapeutic targets, since the host immune response includes an induced oxidative attack that parasites must neutralize and control to survive. The knocking down of the Schistosoma mansoni peroxiredoxin genes using RNA interference (RNA-i) technology dramatically increased oxidative damage to parasite proteins and lipids, which in turn reduced worm survival

(Sayed et al., 2006). In addition, the knocking down of HSP90 in adult C. elegans using RNA-i resulted in stoppage of egg production and in an embryonic lethal phenotype (Piano et al., 2000; Inoue et al., 2006). For three proteins, As37, 53 kDa excretory/secretory protein and CBN-MCE-1 protein, no function has been proposed by GO databases. As37, a highly immunoreactive 37 kDa antigen belonging to the immunoglobulin superfamily, was previously identified in the larval stage of Ascaris suum by Tsuji et al. (2002). This antigen was recently identified also in Angiostrongylus costaricensis by Rebello et al. (2011) and in Angiostrongylus cantonensis by Morassutti et al. (2012). In the latter study, the authors also described peptide molecules with high similarity to As37 in Baylisascaris schroederi and B. malayi. We found this protein among the molecules expressed by G. spinigerum and it is a possible candidate target for actin inhibition in an antihelminth vaccine (Yan et al., 2014). One spot matched with methylmalonyl-CoA epimerase (MCE) (Table 1). MCE is an enzyme involved in the propionyl-CoA metabolism that is responsible for the degradation of branched amino acids and odd-chain fatty acids. This pathway proceeds typically functions in the reversible conversion of propionyl-CoA to succinyl-CoA as similar showed the properties and functions of MCE in C. elegans (Kuhnl et al., 2005). This enzyme shows an increased resistance to oxidative stress (Kuhnl et al., 2005). Two spots matched with 53 kDa excretory/secretory protein of round worm (Table 1). This protein possibly plays an important role in immunomodulation, as reported for the recombinant Trichinella spiralis 53-kDa protein (Du et al., 2011). Finally, six spots show good quality spectra by LC/MSeMS, but could not be identified. These probably correspond to genes that have not yet to be described in the database. We have identified many key molecules of G. spinigerum using the immunoproteomic approach. The set of identified antigenic molecules showed the diversity of processes that occur in this nematode, including metabolic processes and energy generation, cell skeleton, development, redox, response to stress, protein folding, proteolysis, specific immunoreactive antigens, and virulence factors. Our study represents a novel attempt to comprehensively identify antigenic proteins of G. spinigerum, some of which may be potential vaccine candidates or valuable as protein markers for helminth infection.

Acknowledgments This research was supported by grant from the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, Thailand through the Health Cluster (SHeP-GMS) and the Faculty of Medicine, Khon Kaen University grant number TR57201. Pewpan M. Intapan and Wanchai Maleewong were supported by TRF Senior Research Scholar Grant, Thailand Research Fund grant number RTA5880001.

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