Article pubs.acs.org/jpr
A Proteomic Approach To Investigate the Distribution and Abundance of Surface and Internal Fasciola hepatica Proteins during the Chronic Stage of Natural Liver Fluke Infection in Cattle Orçun Haçarız,*,† Gearóid Sayers,‡ and Ahmet Tarık Baykal† ̇ AK Marmara Research Center, Genetic Engineering and Biotechnology Institute, P.O. Box 21, 41470, Gebze, Kocaeli, TÜ BIT Turkey ‡ Veterinary Sciences Centre, School of Agriculture, Food Science and Veterinary Medicine, College of Life Sciences, University College Dublin, Belfield, Dublin 4, Ireland †
S Supporting Information *
ABSTRACT: Fasciola hepatica, a trematode helminth, causes an economically important disease (fasciolosis) in ruminants worldwide. Proteomic analysis of the parasite provides valuable information to understand the relationship between the parasite and its host. Previous studies have identified various parasite proteins, some of which are considered as vaccine candidates or important drug targets. However, the approximate distribution and abundance of the proteins on the surface and within internal parts of the liver fluke are unknown. In this study, two fractions including surface protein fraction (representing surface part of the parasite, near subplasma membrane of the tegument and above the basal membrane of the tegument) and internal protein fraction (representing internal part of the parasite, mainly deeper sides of the tegument including subbasal membrane and other further internal elements of the parasite) were obtained. Components of these two fractions were investigated by an advanced proteomics approach using a high-definition mass spectrometer with nano electrospray ionization source coupled to a high-performance liquid chromatography system (nanoUPLC−ESI−qTOF−MS). FABP1 was found highly abundant in the SPF fraction. Potentially novel F. hepatica proteins showing homology with AKT interacting protein (Xenopus tropicalis), sterol O-acyltransferase 2 (Homo sapiens), and integrin beta 7 (Mus musculus) were identified with high quantities in only the surface fraction of the parasite and may be possible candidates for future control strategies. KEYWORDS: Fasciola hepatica, surface protein fraction, label-free proteomics, sterol O-acyltransferase 2, integrin beta 7, AKT interacting protein parasite and host−parasite interaction.6−11 The integration of proteomics and transcriptomic data (such as expressed sequence tags) using bioinformatics based protein homology tools is useful to identify potentially novel liver fluke proteins, as a complete proteome or whole genome sequence of the liver fluke has not yet been published.6,7,9,11−13 In recent proteomics studies, the EST database available at the Wellcome Trust Sanger Institute has been used to increase the number of identified proteins.6,7,9 More recently, larger EST databases, reported for F. hepatica (consist of approximately 45 000 sequences)8 and for Fasciola gigantica (consist of more than 30 000 sequences),14 are very important to facilitate the proteomics based studies in terms of identification of previously unknown proteins. Besides, other F. hepatica databases created by Cancela et al.10 and Wilson et al.11 are also useful for the current proteomic studies for the same purpose. Since 2000, most previous studies have focused on the proteomic analysis of the excretory-secretory products of F.
1. INTRODUCTION The liver fluke, Fasciola hepatica, is the causative agent of fasciolosis, leading to significant economic losses in the livestock industry on a global scale, in addition to also becoming an emerging pathogen of humans in many countries (reviewed in refs 1, 2). Development of immunoprophylactic control strategies for liver fluke infection are favored over anthelmintic treatment, because of increasing parasite resistance to such drugs (triclabendazole and albendazole).3,4 Proteomic studies identifying essential parasite proteins are advantageous in generating more effective vaccines and discovery of useful targets for drug development or RNA interference (RNAi) studies.5−7 To date, 23 reviewed F. hepatica proteins are in the public current protein knowledgebase (UniProtKB; www.uniprot.org, 15 March 2012). Although these parasite proteins have been previously identified, their relative abundance and importance toward fluke survival within the chronic stage of the fluke infection in vivo remain undefined. The ‘omics’ based strategies is now applied to the liver fluke giving valuable information in understanding the biology of the © 2012 American Chemical Society
Received: January 7, 2012 Published: May 29, 2012 3592
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hepatica.6,15−18 Findings were mainly the identification of proteases (such as cathepsin L proteases and leucyl aminopeptidase) and antioxidants (such as glutathione transferases, fatty acid binding proteins and thioredoxin peroxidase), important for the digestion of the host tissue and the parasite’s defense mechanism (detoxification of reactive oxygen elements), respectively. The proteolytic enzymes and antioxidants have been proposed as vaccine candidates (reviewed in refs 19, 20), and others including β-tubulins in the tegument are important as drug (such as for benzimidazole drugs) targets.12,21−23 Recent proteomic studies have characterized other F. hepatica proteins including a developmentally regulated heat shock protein9 and 14-3-3z protein.7 More recently, the tegumental fraction of the liver fluke has been isolated using a method derived from a protocol for the isolation of the tegument of a similar helminth parasite (Schistosoma mansoni) and various related tegumental proteins of the liver fluke with distinct biological mechanisms have been identified.11 The anatomic structure of the tegument of the liver fluke has been previously demonstrated (reviewed in ref 24). The outer part of the tegument is surrounded with thick glycocalyx structure and the tegumental plasma membrane (apical part of the tegument) lies just below this structure. Below the plasma membrane there is an interstitial area rich in mitochondria and the bottom of this area is covered with the basement (basal) membrane of the tegument. Below the basal membrane there are circular and longitudinal muscle layers which remain just above the tegumental cells. However, these tegumental cells are biologically connected to the upside of the basal membrane, and cytosolic components including proteins are passed through the protoplasmic tubes and accumulated within the surface area (between the plasma and basal membranes of the tegument). For research purposes, isolation of the tegumental layer is important to identify and quantify both previously known and novel F. hepatica proteins expressed at this site.11 However, the tegumental structure of F. hepatica is highly complex; therefore, applying different approaches including different extraction methods and protein identification techniques may reveal novel proteins and comparison of the tegumental and nontegumental fractions based on the identified proteins may further enlighten the roles of these parts of the parasite in its survival and evasion. As a first step of a program to identify alternative protein targets for F. hepatica control, this study investigates the abundance and distribution of previously identified and potentially new F. hepatica proteins expressed by the adult fluke parasite during the infectious stage, using a modified version of previously published extraction method for the F. hepatica tegument25 and an advance proteomic approach, highdefinition mass spectrometer with nano electrospray ionization source coupled to a high-performance liquid chromatography system (nanoUPLC−ESI−qTOF−MS). Efforts were aimed at obtaining two protein fractions for analysis, the first approximating to the surface component of the liver fluke (surface, near subplasma membrane of the tegument, above the basal membrane of the tegument) and second, a fraction approximating to the internal components of the parasite (subbasal membrane of the tegument and further deeper sides of the parasite body).
2. MATERIALS AND METHODS 2.1. Materials
Acetonitrile (LC−MS grade), water (LC−MS grade), dithiothreitol (DTT), trifluoroacetic acid (TFA), formic acid (FA), iodoacetamide (IAA), and sequencing grade modified trypsin (proteomic grade) were purchased from Sigma-Aldrich. Ammonium bicarbonate (NH4HCO3) was from Fluka. RapiGest, an MS compatible detergent and the internal standard MassPREP yeast enolase digest (Uniprot accession no. P00924) were from Waters Corp., Milford, MA. 2.2. Parasites
Livers from naturally infected cattle were collected at an abattoir (near to the location of the Institute) in Istanbul, Turkey. Adult flukes were removed from the livers and immediately placed in warm sterile (autoclaved) phosphate buffered saline [PBS, pH 7.4, including 140 mM NaCl (Sigma), 2.7 mM KCl (Sigma), 8.1 mM Na2HPO4 (Sigma), and 2 mM KH2PO4 (Sigma)]. Observation of the liver flukes in the bile ducts confirmed the chronic phase of the infection.26−29 The drug resistance status of the parasites was not determined. 2.3. Preparation of F. hepatica Fractions
The flukes were washed with warm sterile PBS and two fractions of F. hepatica were prepared as described below. (a) Surface protein fraction (SPF): This fraction was isolated using a previously published method of tegumental protein isolation with some modifications.25 First, instead of washing the parasites at ice-cold PBS repeatedly as described in the published protocol, the parasites were subjected to a snap −80 °C freeze, from in vivo status, to keep the parasite proteins at their exact locations and also to totally inactivate the expression of excretory/secretory products and their release, parasite enzymes, and muscle constrictions of the oral and ventral suckers in order to avoid contamination of the excretory/secretory products. The most outer part of the tegument, a thick glycocalix (carbohydrate, glycoprotein rich zone) layer, was to be undamaged or minimally damaged as freeze based methods (such as freeze-substitution) are utilized to preserve glycocalix structures (as used for the glycocalix layer of Cryptosporidium parvum oocytes).30 Later, in laboratory setting, the frozen flukes were then quickly thawed to 4 °C and washed with cold PBS gently to further remove possible attached excretory/secretory proteins. The flukes were then incubated with cold PBS containing 1% Nonidet P40 (NP40) (Sigma) substitute detergent solution (1 parasite/2 mL of PBS−1% NP 40) at 4 °C for 30 min with gentle shaking for enrichment of the proteins around the surface (upper side of the basal membrane of the tegument) of the parasite. The supernatant, containing proteins that are obtained from the surface of the parasite, was collected and labeled as surface protein fraction (SPF). (b) Internal protein fraction (IPF): The remaining flukes were washed with cold PBS and homogenized with 0.5 mm metal beads using a homogenizer for 15 min.31 Both fractions were vortexed before being aliquoted into several tubes and stored at −80 °C. 2.4. Trypsin Digestion of Proteins in the Fractions
The fractions (100 μL) were mixed with equal volumes of 0.1% RapiGest (Waters Corp., Milford, MA), an MS compatible 3593
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detergent [prepared in 50 mM ammonium bicarbonate (NH4HCO3) (Fluka)] and subjected to ultra sonication (10 s on, 10 s off, three cycles). The samples were desalted with 5 kDa cutoff spin columns (Vivaspin, Sartorius) using a centrifuge (21 000g, 4 °C for 5−15 min). After centrifugation (21 000g, 4 °C for 15 min) to eliminate contaminating particles, the protein concentration for each sample was measured using the Bradford method.32 Calculated amounts of protein mixture (50 μg) for each fraction were pipetted and made up to 50 μL total volume with 0.1% Rapigest solution. Disulphite bonds were broken by DTT (Sigma-Aldrich) addition (5 mM, 15 min incubation at 60 °C). Free thiols were alkylated by the addition of IAA (Sigma-Aldrich) (10 mM, 30 min incubation in the dark at room temperature). Proteolytical digestion of proteins was done by the addition of 50 μL of proteomics grade trypsin (sequencing grade modified trypsin) (Sigma-Aldrich) (20 ng/ μL) in 50 mM NH4HCO3 and incubated at 37 °C overnight. Samples were stored at −80 °C until analysis. 2.5. LC−MS−MS Analysis
Each sample containing the digested peptides was suspended with 2 μL of acetonitrile (ACN) (LC−MS grade, SigmaAldrich), 2 μL of TFA (Sigma-Aldrich), 5 μL of internal standard (50 fmol) (MassPREP Enolase Digestion Standard) (Waters Corp., Milford, MA) and made up to 200 μL total volume with 50 mM NH4HCO3. This solution was mixed at 400 rpm and at 60 °C for 2 h and then centrifuged at 21 000g and 4 °C for 15 min. Each sample was analyzed in triplicate (3 technical replicates per sample). A 2 μL volume of sample (containing 500 ng of tryptic peptide mixture) was loaded on the LC−ESI−qTOF system [nanoACQUITY ultra presssure liquid chromatography (UPLC) and SYNAPT high definition mass spectrometer with nanolockspray ion source, Waters]. Prior to the injection, the columns are equilibrated with 97% mobile phase A [water (LC−MS grade, Sigma) with 0.1% FA] and 3% mobile phase B (ACN containing 0.1% FA). The column temperature was set to 35 °C. First, peptides were trapped on a nanoACQUITY UPLC Symmetry C18 Trap column (5 μm particle size, 180 μm i.d. × 20 mm length) at 5 μL/min flow rate for 5 min. Peptides were eluted from the trap column by gradient elution onto an analytical column (nanoACQUITY UPLC BEH C18 Column, 1.7 μm particle size, 75 μm i.d. × 250 mm length, Waters), at 300 nL/min flow rate with a linear gradient from 5 to 40% ACN over 90 min. Data independent acquisition mode (MSE) was carried out by operating the instrument at positive ion V mode, applying the MS and MS/MS functions over 1,5 s intervals with 6 V low energy and 15−40 V high energy collusions to collect the peptide mass to charge ratio (m/z) and the product ion information to deduce the amino acid sequence. To correct for the mass drift, the internal mass calibrant glu-fibrinopeptide (500 pmol/μL) (Sigma-Aldrich) was infused every 45 s through the nanolockspray ion source at 300 nL/min flow rate. Peptide signal data between 50 and 1600 m/z values were collected.
Figure 1. Experimental flowchart for the qualitative and quantitative LC−MSE protein analysis, and integration of the ESTs data using tblastn. Prior to in-solution digestion, the proteins are extracted and modified by DTT reduction following IAA methylation. Through alternate scanning mode (MSE), two sets of raw data are collected which compile the precusor information and the fragment ion information. It is via these two sets of data that PLGS software deduces the amino acid sequences and protein identifications.
higher collusion energies. Tandem mass spectra extraction and charge state deconvolution and deisotoping steps were processed with ProteinLynx Global Server v2.3 software (PLGS) (Waters Corp., Milford, MA). Protein databanks from the online resources (UniProtKB and NCBI, over 5 million protein sequences, December 2010−March 2011) were used to generate the in-silico reference sequences, as this method facilitates both the identification and quantification is a simultaneous process. First, the raw peptide sequences (processed by the software PLGS) were searched with IDENTITYE algorithm against the reviewed protein database of F. hepatica. IdentityE was set up to search null assuming the digestion enzyme trypsin and search with a fragment ion mass tolerance of 0.028 Da and a parent ion tolerance of 0.011 Da. Then, to expand the analysis, the processed raw data were searched with the same algorithm against the databases of S. mansoni, Schistosoma japonicum, metazoan, and all reviewed protein sequences (obtained from the UniProtKB and NCBI Web sites), subsequently. Proteins of species that were taxonomically close to the liver fluke were considered when the same protein from different species was identified. The Conserved Domain Search (CD Search) was carried out on the NCBI Web site (http://www.ncbi.nlm.nih. gov) with the default settings.33 The accession numbers of the top-ranking protein identified by the PLGS program for the F. hepatica and homologue proteins (orthologous) were reported. The identified peptide sequences and full-length protein sequences (obtained from the Uniprot or NCBI databases) of the homologue proteins were compared with the F. hepatica expressed sequence tags (ESTs) databases including the database (contains around 14 000 entries) available at the Wellcome Trust Sanger Institute ftp site (ftp://ftp.sanger.ac.
2.6. Protein Identification and Quantification
Figure 1 shows the proteomics analysis workflow and integration of the ESTs search applied for the identification. Proteins from the two fractions of F. hepatica were identified and quantified through a data independent proteomics analysis method with a nanoUPLC−ESI−qTOF setup. Within the mass spectrometer, peptide mass measurement was achieved at low collusion energy and peptide sequence data was obtained at 3594
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Table 1. Proteins from the Fractions of the Parasite Identified with the nLC−ESI−MS/MS Methoda fraction SPF functional group
accesion
IPF
description
mW
pI
PLGS score
cov. (%)
NP
PLGS score
cov. (%)
NP
16680 20498 21646 24561 24518
7.9 10.4 5.0 7.7 9. 0
1954 196 542 * 1398
47 59 58 * 46
11 10 15 * 18
462 * * 807 863
19 * * 40 40
4 * * 10 7
25298 55187 14702
5.9 4.9 5.8
4198 236 17274
71 37 85
37 17 37
2754 103 7088
59 30 77
16 11 22
5934
5.9
5934
88
28
3016
62
15
2390
9.5
6160
80
23
2390
48
11
76981 60410
7.8 5.7
47 23
3 8
2 4
109 *
3 *
2 *
59856
8.6
41
8
4
*
*
*
Chaperone
P0CG48 P11983
Cholestrol absorption Chromosome related
O75908
Hemoglobin F2 (F. hepatica) Ferritin like protein (F. hepatica) Thioredoxin peroxidase (F. hepatica) Thioredoxin peroxidase (F. hepatica) Glutathione transferase sigma class(F. hepatica) Glutathione transferase (F. hepatica) Protein disulfide isomerase (F. hepatica) Fatty acid binding protein Fh15 (FABP1) (F. hepatica) Fatty acid binding protein type 2 (F. hepatica) Fatty acid binding protein type 3 (F. hepatica) Polyubiquitin C (H. sapiens) T complex protein 1 subunit alpha (M. musculus) Sterol O-acyltransferase 2 (H. sapiens)
P02299
Histone H3 (D. melanogaster)
15378
11.7
*
*
*
2132
27
4
P35059 B0B5G3
Histone H4 (A. formosa) Alpha tubulin (F. hepatica)
11374 50030
11.8 4.8
* *
* *
* *
269 115
42 13
4 4
B0B5G4 B0B5G8 B0B5H1 AAW25537.1 P53471 Q05870 Q26519 P48667
Alpha tubulin (F. hepatica) Beta tubulin (F. hepatica) Beta tubulin (F. hepatica) SJCHGC06318 protein (S. japonicum) Actin 2 (S. mansoni) Paramyosin (S. mansoni) Tropomyosin (S. japonicum) Keratin, type II cytoskeletal 6 (H. sapiens) Protein bicaudal D homologue 1 (H. sapiens) Stefin 1 (F. gigantica)
49978 49829 49788 41808 41713 100480 33004 42442
4.8 4.6 4.7 5.3 5.2 5.1 4.4 5.1
159 * 192 4296 2303 * * 50
19 * 34 21 59 * * 15
6 * 16 9 31 * * 5
* 679 * * 1956 65 125 *
* 27 * * 42 25 14 *
* 10 * * 20 18 4 *
110681
5.5
26
7
5
*
*
*
11042
7.9
*
*
*
564
56
10
Glyceraldehyde 3 phosphate dehydrogenase fragment (F. hepatica) Enolase (F. hepatica) Propionyl CoA carboxylase alpha chain mitochondrial (C. elegans) Glutamate dehydrogenase mitochondrial (C. aceratus) Glutamate dehydrogenase 1 (S. japonicum) Triosephosphate isomerase (S. japonicum) Succinate dehydrogenase ubiquinone flavoprotein subunit mitochondrial (H. sapiens) Fructose bisphosphate aldolase B (S. aurata) NADH dehydrogenase subunit I (F. gigantica) Mitochondrial acetate succinate CoA transferase (F. hepatica) Succinate semialdehyde dehydrogenase NADP (D. radiodurans) Oxoglutarate dehydrogenase mitochondrial (B. taurus) Probable phospholipid transporting ATPase ID (M. musculus)
23344
7.1
3457
40
14
1990
36
10
46245 79712
6.3 7.6
853 *
49 *
21 *
658 56
32 12
11 9
55359
7.3
32
4
2
*
*
*
58730
8.7
*
*
*
1140
29
10
27644
6.7
72
41
11
30
31
3
72645
7.0
89
13
70
80
10
5
39620
8.1
86
9
7
*
*
*
20001
9.2
*
*
*
945
49
14
52400
8.5
607
41
15
355
29
9
51208
4.9
48
12
4
*
*
*
115734
6.3
*
*
*
40
6
5
136879
6.6
23
5
5
*
*
*
Antioxidant
B5KY09 Q2L7A9 P91883 B6DT35 Q06A71 B5AK46 O76945 Q7M4G0 Q7M4G1 Q9U1G6
Cytoskeleton/ motility
Q96G01 Cysteine type endopeptidase inhibitor Energy
ACS35603.1 Q95V10 Q27655 Q19842 P82264 CAX70245.1 Q27775 P31040 P53447 BAJ41359.1 C6EUD4 O32507 Q148N0 P98199
3595
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Table 1. continued fraction SPF functional group
accesion P21360 P06801
Fibronectin binding Neuronal inhibition Protein biosynthesis
Q45UT3 P26011 P19019 P02993 O43781 Q5DAA3
Proteolysis
Regulation of apoptosis Signaling
Q9GRW5 Q9NB30 Q7JNQ9 Q17TZ3 CAM57967.1 CAD32937.1 Q28IA3 O46121 Q9NIG5 Q6IX08 P59644 AAZ20312.1
Stress
B1NI97 ACE00520.1 O57521 Q930Y0
Transferase
P49724
description
pI
63214
6.4
63958
PLGS score
IPF
cov. (%)
NP
cov. (%)
NP
44
21
8
*
*
*
7.2
80
9
6
*
*
*
46969 87354
9.2 5.2
155 23
33 5
14 4
* *
* *
* *
Gamma aminobutyric acid receptor beta 3 subunit pr (G. gallus) Elongation factor 1 alpha (A. salina)
54397
9.2
*
*
*
49
7
4
50535
9.4
185
18
6
81
9
4
Dual specificity tyrosine phosphorylation regulated kinase 3 (H. sapiens) 40S ribosomal protein S3a (S. japonicum) Cathepsin L1 Fragment (F. hepatica) Cathepsin L (F. hepatica) Secreted cathepsin L 1(F. hepatica) Leucyl aminopeptidase (F. hepatica) Unnamed protein product (F. hepatica) Pro cathepsin B2 (F. hepatica) AKT interacting protein (X. tropicalis)
65771
9.6
*
*
*
33
2
5
29270
10.3
188
42
16
*
*
*
35173 37124 36749 56358 35988 38045 33231
5.2 5.5 6.3 7.0 5.3 6.8 9.5
1515 414 * * * * 96
45 47 * * * * 10
18 13 * * * * 2
302 * 788 336 1688 1451 *
30 * 28 23 26 24 *
10 * 10 10 6 10 *
Calcium binding protein (F. hepatica) Putative calcium binding protein (F. hepatica) 14−3−3 epsilon2 isoform (F. hepatica) Phosphatidylinositol 4,5-bisphosphate 5phosphatase A (M. musculus) Tegumental calcium binding EF hand protein (F. gigantica) Heat shock protein 70 (F. gigantica) Alpha Crystallin containing small heat shock protein variant NtermFhHSP35a (F. hepatica) Heat shock protein HSP 90 beta (D. rerio) 60 kDa chaperonin 3 Protein Cpn60 3 groEL prote (R. meliloti) Ornithine aminotransferase mitochondrial (D. ananassae)
22013 7661
5.1 5.5
403 1492
34 57
10 6
197 453
12 22
3 2
28173 107537
5.2 9.7
101 29
26 8
8 7
185 *
17 *
5 *
22077
5.1
1177
76
24
*
*
*
70723 48329
5.4 6.1
397 751
28 35
19 20
344 *
25 *
17 *
83305
4.7
*
*
*
51
6
5
57486
4.9
43
12
5
*
*
*
47345
7.8
148
3
1
*
*
*
Malate synthase glyoxysomal (P. angusta) NADP dependent malic enzyme (M. musculus) Phosphoglycerate kinase (F. hepatica) Integrin beta 7 (M. musculus)
mW
PLGS score
a
Accession number, protein description, molecular weight (mW), isoelectric point (pI), PLGS score, sequence coverage [Cov. (%)], number of identified peptide (NP) and functional group for each identified molecules in the fractions are listed. The PLGS Score, calculated by the Protein Lynx Global Server (PLGS) using a Monte Carlo algorithm, is a statistical data for the accuracy of peptide assignation and a higher score indicates greater confidence of protein identity.66,67 The cutoff value for the PLGS score was 10068 for the F. hepatica proteins in the Uniprot or NCBI databases; however, lower cutoff values, but not less than 20, were considered for the homolog proteins.69 The functional groups are various including antioxidant, chaperone, cholesterol absorption, chromosome related, cytoskeleton/motility, energy, fibronectin binding, neuronal inhibiton, protein biosynthesis, proteolysis, regulation of apoptosis, signaling, stress, and transferase. The accession numbers and corresponding scores belong to the PLGS-derived identifications on the UniProtKB or NCBI databases. (*): unidentified.
organism parameter was set to Fasciola sp. or Schistosoma sp.) using tBlastx procedure with default settings34 (E value
