Portal de Periódicos da CAPES

Fasciola hepatica Surface Tegument: Glycoproteins at the Interface of Parasite and Host

2016; Elsevier BV; Volume: 15; Issue: 10 Linguagem: Inglês

10.1074/mcp.m116.059774

1535-9484

Alessandra Ravidà, Krystyna Cwiklinski, Allison Aldridge, Paul Clarke, Róisı́n Thompson, Jared Q. Gerlach, Michelle Kilcoyne, Cornelis H. Hokke, John P. Dalton, Sandra M. O’Neill,

Parasites and Host Interactions

Fasciola hepatica, commonly known as liver fluke, is a trematode that causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterization of FhTeg glycosylation using lectin microarrays to characterize carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. Although some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica. Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components that could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine. Fasciola hepatica, commonly known as liver fluke, is a trematode that causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterization of FhTeg glycosylation using lectin microarrays to characterize carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. Although some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica. Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components that could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine. Fasciola hepatica is a parasitic flatworm of livestock and the causative agent of Fasciolosis, a disease that results in major economic losses to the agricultural industry globally, estimated at $3 billion annually (1.Boray J.C. Chemotherapy of infections with fasciolidae.in: Boray JC. Immunology, Pathobiology and Control of Fasciolosis. 1994: 83-97Google Scholar). Fasciolosis has recently been acknowledged by the World Health Organization (WHO) as a neglected tropical zoonotic disease, with as many as 2 to 17 million people infected worldwide and 180 million at risk of infection (2.Mas-Coma S. Bargues M.D. Valero M.A. Fascioliasis and other plant-borne Trematode zoonoses.Int. J. Parasitol. 2005; 35: 1255-1278Crossref PubMed Scopus (659) Google Scholar). Infection in animals is currently treated chemically with drugs such as triclabendazole, albendazole, and oxyclosanide, although drug-resistance is now widespread across Europe and globally (3.Fairweather I. Reducing the future threat from (liver) fluke: realistic prospect or quixotic fantasy?.Vet. Parasitol. 2011; 180: 133-143Crossref PubMed Scopus (110) Google Scholar). Although vaccines are considered a safe, economically viable and environmentally friendly solution none have been commercially developed to date and there is a dearth of promising candidates with potent immunoprotective efficacy in the pipeline (4.Wilson R.A. Wright J.M. de Castro-Borges W. Parker-Manuel S.J. Dowle A.A. Ashton P.D. Young N.D. Gasser R.B. Spithill T.W. Exploring the Fasciola hepatica tegument proteome.Int. J. Parasitol. 2011; 41: 1347-1359Crossref PubMed Scopus (99) Google Scholar). It is only recently that the fluke surface tegument has been the subject of close investigations by molecular techniques (4.Wilson R.A. Wright J.M. de Castro-Borges W. Parker-Manuel S.J. Dowle A.A. Ashton P.D. Young N.D. Gasser R.B. Spithill T.W. Exploring the Fasciola hepatica tegument proteome.Int. J. Parasitol. 2011; 41: 1347-1359Crossref PubMed Scopus (99) Google Scholar, 5.Haçarız O. Sayers G. Baykal A.T. 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.J. Proteome Res. 2012; 11: 3592-3604Crossref PubMed Scopus (45) Google Scholar, 6.Morphew R.M. Hamilton C.M. Wright H.A. Dowling D.J. O'Neill S.M. Brophy P.M. Identification of the major proteins of an immune modulating fraction from adult Fasciola hepatica released by Nonidet P40.Vet. Parasitol. 2013; 191: 379-385Crossref PubMed Scopus (24) Google Scholar, 7.Haçarız O. Baykal A.T. Akgün M. Kavak P. Sağıroğlu M. Ș Sayers G.P. Generating a detailed protein profile of Fasciola hepatica during the chronic stage of infection in cattle.Proteomics. 2014; 14: 1519-1530Crossref PubMed Scopus (21) Google Scholar) with the aim of discovering novel potential targets for both drug and vaccine development. The F. hepatica tegument is a metabolically active layer that is continuously sloughed off and replaced during infection. The tegument is intimately associated with host tissues and performs a number of important functions including the synthesis and secretion of substances, absorption of nutrients, osmoregulation, and protection against host enzymes (8.Fairweather I. Threadgold L.T. Hanna R.E.B. Development of Fasciola hepatica in the mammalian host./Fasciolosis.in: Dalton J.P. CAB International, 1999: 47-111Google Scholar). Closely packed spines that point posteriorly are distributed throughout the tegument and help maintain the position of the fluke within the tissues and bile duct. The spines also facilitate feeding of the obligate blood-feeding adult through the erosion of the epithelium and puncturing of small bloods vessels (8.Fairweather I. Threadgold L.T. Hanna R.E.B. Development of Fasciola hepatica in the mammalian host./Fasciolosis.in: Dalton J.P. CAB International, 1999: 47-111Google Scholar). It is highly likely that the shed tegumental coat is released into the blood stream and studies have shown the presence of anti-tegumental antibodies in serum form infected animals (9.Haçarız O. Sayers G. Mulcahy G. A preliminary study to understand the effect of Fasciola hepatica tegument on naïve macrophages and humoral responses in an ovine model.Vet. Immunol. Immunopathol. 2011; 139: 245-249Crossref PubMed Scopus (23) Google Scholar). The outer-most surface of the tegument is shielded by a glycocalyx that is comprised predominantly of glycoconjugates (10.Threadgold L.T. Fasciola hepatica: ultrastructure and histochemistry of the glycocalyx of the tegument.Exp Parasitol. Feb. 1976; 39: 119-134Crossref PubMed Scopus (43) Google Scholar). Glycoproteins and glycolipids of parasitic helminths often contain a mixture of oligosaccharide motifs similar or identical to those present in their hosts, as well as structurally-unusual, pathogen-specific motifs (11.Prasanphanich N.S. Mickum M.L. Heimburg-Molinaro J. Cummings R.D. Glyco-conjugates in host-heminth interactions.Front. Immunol. 2013; 4: 240Crossref PubMed Scopus (62) Google Scholar). These glycoconjugates can play key roles in the immunoregulatory activity of the parasite by interacting with C-type lectin receptors (CLRs) 1The abbreviations used are: CTRsC-type lectin receptorsDCsDendritic CellsFhTegFasciola hepatica Tegumental AntigensMAPKMitogen-activated protein kinasesNK-kBNuclear factor kappa BSOCS3Suppressor of cytokine signalling (SOCS) 3STATsignal transducers and activators of transcription3TLRToll-like receptorTRILTLR4 interactor with leucine-rich repeatsVTCN-1V-set domain-containing T cell activation inhibitor-1WHOWorld Health Organisation. (12.Meevissen M.H. Yazdanbakhsh M. Hokke C.H. Schistosoma mansoni egga glycoproteins and C-type lectins of host immune cells: molecular partners that shape immune responses.Exp. Parasitol. 2012; 132: 14-21Crossref PubMed Scopus (41) Google Scholar). We have previously shown that a preparation of tegumental antigen (FhTeg) exhibits potent Th1 immune suppressive properties in vivo by reducing serum levels of IFNγ and IL-12p70 in the mouse model of septic shock (13.Hamilton C.M. Dowling D.J. Loscher C.E. Morphew R.M. Brophy P.M. O'Neill S.M. The Fasciola hepatica tegumental antigen suppresses dendritic cell maturation and function.Infect. Immun. 2006; 77: 2488-2498Crossref Scopus (103) Google Scholar). FhTeg-activated dendritic cells and mast cells are hypo-responsive to Toll-like Receptors (TLR) activation and thereby suppress the production of inflammatory cytokines and co-stimulatory molecules important in driving adaptive immune responses (14.Vukman K.V. Adams P.A. Metz M. Maurer M. O'Neill S.M. Fasciola hepatica Tegumental Coat Impairs Mast Cells' Ability to Drive Th1 Immune Responses.J. Immunol. 2013; 43: 531-539Google Scholar). The FhTeg mechanism of action is independent of TLRs and has been linked to the suppression of NF-κB and MAPK pathways and enhanced expression levels of suppressor of cytokine signaling (SOCS) 3, a negative regulator of the TLR and STAT3 pathways that is observed in vitro and following F. hepatica infection (15.Vukman K.V. Adams P.A. O'Neill S.M. Fasciola hepatica tegumental coat antigen suppresses MAPK signalling in dendritic cells and up-regulates the expression of SOCS3.Parasite Immunol. 2013; 35: 234-238Crossref PubMed Scopus (32) Google Scholar, 16.Rojas-Caraballo J. López-Abán J. Fernández-Soto P. Vicente B. Collía F. Muro A. Gene expression profile in the liver of BALB/c mice infected with Fasciola hepatica.PLoS ONE. 2015; 10: e0134910Crossref PubMed Scopus (14) Google Scholar). C-type lectin receptors Dendritic Cells Fasciola hepatica Tegumental Antigens Mitogen-activated protein kinases Nuclear factor kappa B Suppressor of cytokine signalling (SOCS) 3 signal transducers and activators of transcription3 Toll-like receptor TLR4 interactor with leucine-rich repeats V-set domain-containing T cell activation inhibitor-1 World Health Organisation. The contribution of glycoconjugates to the development of protective immunity to Fasciola infection has yet to be established and, even if these oligosaccharides are not antigenic per se, they can influence the immunogenicity of glycoprotein-based vaccines by contributing to correct protein folding and biophysical properties (17.Geldhof P. De Maere V. Vercruysse J. Claerebout E. Recombinant expression systems: the obstacle to helminth vaccines?.Trends Parasitol. 2007; 23: 527-532Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), as well as to the modulation of antigen processing and activation of antigen presenting cells. One of the first steps of understanding the role of Fasciola glycoconjugates is to identify the glycoproteins on its tegumental coat. Here we employed orthogonal lectin assays to probe the F. hepatica tegumental components and profile the variety of glycoprotein and carbohydrate moieties at or close to the parasite surface. Selective enrichment of these tegumental glycoproteins was performed using lectin chromatography and these were subjected to proteomic analysis. This study provides a comprehensive and novel insight regarding glycoprotein composition of the FhTeg and has identified those that likely play critical functions at the interface between parasite and host. F. hepatica tegumental extract (FhTeg) was prepared as previously reported (13.Hamilton C.M. Dowling D.J. Loscher C.E. Morphew R.M. Brophy P.M. O'Neill S.M. The Fasciola hepatica tegumental antigen suppresses dendritic cell maturation and function.Infect. Immun. 2006; 77: 2488-2498Crossref Scopus (103) Google Scholar). In brief, F. hepatica adult worms following collection from sheep at an abattoir in Ireland were washed in sterile phosphate-buffered saline (PBS) and incubated in 1% Nonidet P-40 (Nonidet P-40 (Sigma Aldrich, Wicklow, Ireland) in PBS) for 30 min. Supernatant was collected and Nonidet P-40 removed using detergent-removing biobeads (Bio-Rad laboratories, Fannin Ltd, Dublin, Ireland), and the remaining supernatant was centrifuged at 14,000 × g for 30 min at 4 °C prior to being filtered/concentrated using compressed air, and then stored at −20 °C. All protein concentrations were determined using a bicinchoninic acid protein assay kit (Promeaga, Madison, WI). FhTeg was fluorescently labeled with Alexa Fluor 555 via carboxylic acid succinimidyl ester conjugation strategy according to manufacturer instructions (ThermoFischer Scientific. MSc Co ltd, Dublin, Ireland). Excess dye was removed from FhTeg-555 using a desalting column (7 kDa molecular weight cut off (MWCO) (Invitrogen, Paisley, UK)). Final protein concentration and labeling efficiency was calculated according to the manufacturer's instructions by measuring sample absorbance at 280 and 550 nm and using the arbitrary values for molecular mass (100,000) and extinction coefficient (E of 10). A panel of 48 lectins (supplemental Table S1) was printed on Nexterion® Slide H microarray slides in a 62% (±2%) humidity environment using a SciFLEXARRAYER S3 (Scienion AG, Berlin, Germany) equipped with a 90 μm uncoated glass dispenser capillary to construct the lectin microarray as previously described (18.Gerlach J.Q. Kilcoyne M. Joshi L. Microarray evaluation of the effects of lectin and glycoprotein orientation and data filtering on glycoform discrimination.Analytical Methods. 2014; 6: 440-449Crossref Google Scholar). Microarray incubations were carried out essentially as previously described (18.Gerlach J.Q. Kilcoyne M. Joshi L. Microarray evaluation of the effects of lectin and glycoprotein orientation and data filtering on glycoform discrimination.Analytical Methods. 2014; 6: 440-449Crossref Google Scholar) and all procedures were carried out in the dark. In brief, FhTeg-555 was diluted in Tris-buffered saline supplemented with Ca2+ and Mg2+ ions (TBS; 20 mm Tris-HCl, 100 mm NaCl, 1 mm CaCl2, 1 mm MgCl2, pH 7.2) and with 0.05% Tween 20 (TBS-T) for incubation on the microarrays using an eight-well gasket (Agilent Technologies, Cork, Ireland) at room temperature for 1 h with gentle rotation (4 rpm) in the dark. Initially several concentrations (2.9–14.4 μg/ml) were titrated to determine the best concentration for optimal signal to noise ratio. Based on the binding interactions observed from the titration experiments, samples were also coincubated with 100 mm Gal and Man in parallel on the lectin microarrays to verify carbohydrate-mediated binding (19.Gerlach J.Q. Kilcoyne M. Eaton S. Bhavanandan V. Joshi L. Non-Carbohydrate Mediated Interactions of Lectins with Plant Proteins, Molecular Immunology of Complex Carbohydrates-3.in: Wu A.M. Adv. Exp. Med. Biol. Springer, 2011: 257-269Google Scholar). Incubations were carried out in triplicate using the optimal concentration of 8.6 μg/ml FhTeg in TBS-T. After incubation, the microarray slides were disassembled under TBS-T, washed three times in TBS-T for 2 min each with gentle agitation in a Coplin jar, with a final 2 min wash in TBS. The slides were centrifuged until dry and scanned immediately with the 532 nm laser (90% laser power, 5 μm resolution) of an Agilent G2505B microarray scanner. Three replicate experiments were performed. Microarray data extraction was performed as previously described (18.Gerlach J.Q. Kilcoyne M. Joshi L. Microarray evaluation of the effects of lectin and glycoprotein orientation and data filtering on glycoform discrimination.Analytical Methods. 2014; 6: 440-449Crossref Google Scholar). In brief, raw intensity values were extracted from the image files using GenePix Pro v6.1.0.4 (Molecular Devices, Berkshire, UK) and a proprietary address file (*.gal) using adaptive diameter (70–130%) circular alignment based on 230 μm features and exported as text to Excel (version 2007, Microsoft). Local background-corrected median feature intensity data (F543median-B543) was selected and the median of six replicate spots per subarray was handled as a single data point for graphical and statistical analysis. Data were normalized to the mean of three replicate microarray slides (subarray by subarray using subarray total intensity), and binding data was presented in histogram form of mean intensity with one standard deviation of three experimental replicates (n = 3, 18 data points in total). Unsupervised clustering of normalized lectin binding data was performed with Hierarchical Clustering Explorer v3.5 (HCE 3.5; Human-Computer Interaction Lab, University of Maryland, http://www.cs.umd.edu/hcil/hce/hce3.html). Mean normalized data was initially clustered with the following parameters: no prefiltering, complete linkage and Euclidean distance. Precast 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels (Thermo Fisher Scientific) were run under standard reducing conditions. Samples were loaded at a concentration of 10 μg/ml and gels were subjected to Coomassie or silver staining (20.Candiano G. Bruschi M. Musante L. Santucci L. Ghiggeri G.M. et al.Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis.Electrophoresis. 2004; 25: 1327-1333Crossref PubMed Scopus (1594) Google Scholar), or transferred onto nitrocellulose membranes by iBlot® Dry blotting system (Thermo Fisher Scientific) for Western analysis. After standard Western blotting procedures with primary (goat anti-biotin antibody and biotinylated lectins as per manufacturer's instructions (Vector Laboratories Ltd, Peterborough, UK) and secondary antibody (IRDye 800 Streptavidin and IRDye 680 anti-goat; Li-COR Biosciences, Lincoln, NE), the membranes were scanned using Odyssey Infrared Imaging System (Li-COR Biosciences). Data analysis was performed with Odyssey V 3.0 software (Li-COR Biosciences). Adult liver flukes were flat-fixed in 4% paraformaldehyde and incubated with fluorescein-labeled lectins or with biotin-MAL II followed by incubation with fluorescein-labeled anti-biotin (Vector Laboratories). Parasites were mounted on glass microscope slides with Vectashield® anti-fading solution with or without DAPI (Vector Laboratories). Specimens were viewed using a Leica DM IL LED microscope using 10×, 20×, and 40× HI PLAN I objectives (Leica Microsystems, Ashbourne, Ireland) equipped with epifluorescent source and a filter system for FITC and TRITC fluorescence. Images were processed with Adobe Photoshop CS4 software (Adobe System Inc. San Jose, CA). FhTeg glycoproteins (Glyco-FhTeg) were isolated by biotinylating the carbohydrates with carbohydrate-specific EZ-link hydrazide-biotin (Thermo Fisher Scientific) according to manufacturer guidelines (see supplemental Fig. S1A). Western blot analysis with IR-labeled streptavidin complex confirmed the efficient biotinylation of the FhTeg glycoproteins (FhTeg-B; supplemental Fig. S1B). FhTeg-B was purified using the biotin/avidin system using a monomeric avidin-agarose column (Thermo Scientific). Nonbiotinylated (nonglycosylated) proteins flowed through the column constituting the Avidin FT fraction, whereas biotinylated (glycosylated) proteins (Avidin B) were displaced from avidin-immobilized beads by elution with concentrated biotin in reducing conditions. This purified biotinylated glycoprotein fraction is referred to as Glyco-FhTeg. Western blot analysis (supplemental Fig. S1C) confirmed the presence of biotinylated molecules in the starting material (FhTeg-B), bound fraction (FhTeg-B Avidin B) but not in the flow through material (FhTeg-B Avidin FT). Mannose-rich glycoprotein components of the FhTeg were obtained by lectin affinity chromatography using plant lectins ConA, LCA, and GNL. FhTeg was buffer exchanged in lectin binding buffer (20 mm Tris-HCl, 0.15 m NaCl, 1 mm CaCl2, 1 mm MnCl2, pH 7.2) using 7 kDa MWCO Zeba spin columns (Thermo Fisher Scientific) and then incubated with ConA-, LCA-, or GNL-agarose slurry (Vector Laboratories ltd) with end-over-end agitation overnight at 4 °C. The beads were centrifuged and the unbound fractions decanted. Following extensive washing, the beads were incubated for 3 h at room temperature with two column volumes of inhibiting sugars to elute bound oligosaccharides (0.5 m alpha-methylmannoside and 0.5 m alpha-methylglucoside for ConA; 0.5 m methylmannoside for GNL and LCA). High concentrations of salts and monosaccharides were removed from both unbound and bound fractions by diafiltration in Amicon Ultra centrifugal filters with 3 kDa MWCO (Millipore Ireland B.V., Cork, Ireland). Isolation yields and efficiency were monitored by measuring total protein content of fractions with BCA assay, SDS-PAGE and lectin blot probed with ConA, LCA, and GNL (supplemental Fig. S1), as described above, before subjecting these to proteomic analysis. The combined proteome of the three lectin affinity purified fractions (ConA, LCA, and GNL) is referred to Man-FhTeg. Microarray, lectin blots, and micrographs were performed with at least two biological replicates. For the proteomics study, whole Fasciola tegument was initially analyzed by LC-MS/MS. Because the aim of this experiment was to profile the molecules associated with fluke tegument, no biological replicates were used and therefore no quantitation was performed. Selected F. hepatica fractions were subjected to proteomics analysis by mass spectrometry (MS by Proteomique Platform of the Quebec Genomics Center, CHU de Quebec Research Centre, Quebec, Canada). Liquid chromatography/tandem mass spectrometry (LC-MS/MS) was carried out according to a previously reported protocol (21.Gallaud E. Caous R. Pascal A. Bazile F. Gagné J.P. Huet S. Poirier G.G. Chrétien D. Richard-Parpaillon L. Giet R. Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells.J. Cell Biol. 2014; 204: 1111-1121Crossref PubMed Scopus (37) Google Scholar). Briefly, proteins were resolved by SDS-PAGE, the gel was stained with SYPRO Ruby gel stain according to the manufacturer's instructions (Bio-Rad Laboratories). The protein bands were cut into gel slices and deposited in 96-well plates. A liquid handling station (MassPrep; Waters, Dublin, Ireland) was used, with sequencing-grade modified trypsin (Promega, Madison, WI) for in-gel protein digestion according to the manufacturer's instructions. Peptide extracts were then dried by evaporation in a SpeedVac (Thermo Fisher Scientific). LC-MS/MS experiments were performed with an TripleTOF 5600 mass spectrometer connected to a Thermo Surveyor MS pump and equipped with a nanoelectrospray ion source (all from Thermo Fisher Scientific). The peptides were separated in a PicoFrit BioBasic C18 column (0.075 mm I.D. x 10 cm; New Objective), with a linear gradient from 2 to 50% solvent B (acetonitrile, 0.1% formic acid) over 30 min at a flow rate of 200 nl/min. Mass spectra were acquired in the data-dependent acquisition mode (Xcalibur software, version 2.0). Each full-scan mass spectrum (m/z 400–2000) was followed by the collision-induced dissociation of the seven ions giving the most intense signals. The dynamic exclusion function was enabled (30 s of exclusion), and the relative collisional fragmentation energy was set at 35%. Peak list files were generated by Protein Pilot v5.0 (SCIEX, co AB Sciex UK Limited, Cheshire, UK) using default parameters and exported to Mascot v2.4.1 (Matrix Science, Zürich, Switzerland) for database searching. All MS/MS spectra were analyzed with Mascot (version 2.4.1; Matrix Science), set up to search against a database comprised of gene models identified from the recently sequenced F. hepatica draft genome (version 1.0, 101,780 entries; 22) and the Uniprot sheep protein data bank (27,174 entries), assuming digestion with trypsin with two missed cleavages permitted. A subset of this database was used for the tandem spectra also assuming trypsin. The F. hepatica gene model sequences can be accessed through WormBase ParaSite (http://parasite.wormbase.org/) under accession PRJEB6687 (genomic read data and gene model transcripts). Fragment and parent ion mass tolerance were set at 0.100 Da. Carbamidomethylation of cysteine was as a fixed modification and the dehydration of the N terminus, Glu->pyro-Glu of the N terminus, ammonia-loss of the N terminus, Gln->pyro-Glu of the N terminus, deamidation of asparagine and glutamine, oxidation of methionine, hex of arginine and threonine and biotin of lysine were specified as variable modifications. Scaffold (version 4.3.4, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95% probability to achieve an FDR less than 1.0% by the Peptide Prophet algorithm (24.Nesvizhskii A.I. Keller A. Kolker E. Aebersold R.A. Statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3633) Google Scholar) with Scaffold delta-mass correction. Protein identifications were accepted if they could be established at greater than 95.0% probability to achieve an FDR less than 1.0% and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (24.Nesvizhskii A.I. Keller A. Kolker E. Aebersold R.A. Statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3633) Google Scholar). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Putative annotation of the F. hepatica gene models was assigned using in silico tools, Uniprot, Gene Ontology (GO), and InterproScan (20.Candiano G. Bruschi M. Musante L. Santucci L. Ghiggeri G.M. et al.Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis.Electrophoresis. 2004; 25: 1327-1333Crossref PubMed Scopus (1594) Google Scholar). The identified proteins in this study were categorised according to their GO classification. Glycosylation predictive analysis was carried out using NetNGlyc 1.0 and NetOGlyc 4.0 Servers (25..http://www.cbs.dtu.dk/services/NetNGlyc/,Google Scholar). The glycosylation of FhTeg was characterized on a lectin microarray featuring a comprehensive panel of plant, bacterial and fungal lectins (Fig. 1A and supplemental Table S1). FhTeg exhibited a particularly high affinity for the Man-binding lectins (Calsepa, NPA, GNA, HHA and Con A; supplemental Table S1), which suggested a predominance of glycoproteins modified with oligomannose type N-linked oligosaccharide structures at the parasite surface. Binding to LEL and DSA indicated the presence of N-acetylglucosamine (GlcNAc) residues, but these residues were likely predominantly part of the chitobiose core (GlcNAc-β-(1,4)-GlcNAc) of N-linked structures and/or N-acetyllactosamine (Gal-β-(1,4)-GlcNAc) in the antennae rather than terminal GlcNAc residues as other typical GlcNAc-binding lectins such as GSL-II displayed only moderate binding. FhTeg also bound to the fucose-binding lectins AAL, LTA and UEA-I and to the terminal α-linked galactose-(Gal-) binding lectins GSL-I-B4, MPA, VRA, and MOA. The presence of complex-type N-linked oligosaccharides was also confirmed by binding to PHA-L and -E and CPA (Fig. 1A). Structures with terminal β-linked Gal and residues were inferred to be relatively abundant because of moderate binding intensity with the lectins RCA-I, AIA, PNA, and PHA-E with N-acetylgalactosamine (GalNAc) residues further indicated by more intense binding with SNA-II (supplemental Table S1). Additionally, the binding of AIA and PNA may further indicate the presence of O-linked oligosaccharides in FhTeg protein extract, more specifically the T antigen (Gal-β-(1,3)-GalNAc-O-Ser/Thr), and PNA binding suggested that these structures were not sialylated (supplemental Table S1). Interestingly, only low binding was observed for lectins with binding affinities for sialic acid-containing moieties, SNA-I, MAA, and WGA, except for CCA, which recognizes 9-O-acetyl- and 4-O-acetylneuraminic acids (26.Ravindranath M.H. Herman H.H. Copper E.L. Paulson J.C. Purification and characterisation of an O-acetylsialic aci