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International Journal for Parasitology

Volume 40, Issue 5, April 2010, Pages 543-554

International Journal for Parasitology journal image

Exposed proteins of the Schistosoma japonicum tegument

  1. ⁎ Corresponding author. Tel.: +61 7 3845 3726; fax: +61 7 3845 3507.
  2. a Helminth Biology Laboratory, Division of Infectious Diseases, Queensland Institute of Medical Research, Qld 4006, Australia
  3. b Molecular Parasitology Laboratory, Division of Infectious Diseases, Queensland Institute of Medical Research, Qld 4006, Australia
  4. c Parasite Cell Biology Laboratory, Division of Infectious Diseases, Queensland Institute of Medical Research, Qld 4006, Australia
  5. d The University of Queensland, School of Veterinary Sciences, Qld 4072, Australia
  6. e CSIRO Livestock Industries, Brisbane, Qld 4067, Australia
  7. f The University of Queensland, Institute for Molecular Biosciences, Qld 4072, Australia
  8. 1 These authors contributed equally to the manuscript.

Research highlights

  • Proteins exposed on the surface of parasitic worms are an important source of novel drug and vaccine targets.
  • These proteins are the most accessible to the host and likely to possess functions important for the survival of the worm.
  • Biotinylation (labelling of lysine side-chain residues with biotin) of whole worms is a useful technique for separating exposed proteins from other protein constituents of the tegument.
  • Using this technique in combination with LC-MS/MS we identified 54 proteins as putatively host-exposed in Schistosoma japonicum.
  • Using confocal and electron microscopy, the internalization of biotin-labelled proteins was observed.

Graphical abstract

Abstract

The ability of the mammalian blood fluke Schistosoma japonicum to survive in the inhospitable environment of the mammalian bloodstream can be attributed, at least in part, to its host-exposed outer surface, called the tegument. The tegument is a dynamic organ and is involved in nutrition, immune evasion and modulation, excretion, osmoregulation and signal transduction. Given its importance for parasite survival, proteins exposed to the host at the surface of the tegument are ideal targets for the development of vaccines and drugs. By biotinylating live adult worms and using a combination of OFFGEL electrophoresis and tandem mass spectrometry 54 proteins were identified as putatively host-exposed in S. japonicum. These included glucose transport proteins, an amino permease, a leucine aminopeptidase and a range of transporters, heat shock proteins and novel immune-active proteins. Members of the tetraspanin protein family and a homologue of Sm 29, a tegument membrane protein from Schistosoma mansoni, both effective vaccine antigens in S. mansoni, were also identified. The fate of labelled surface proteins was monitored over time using electron microscopy and revealed that biotinylated proteins were rapidly internalised from the surface of the tegument and trafficked into the cytoplasmic bridges that connect the distal cytoplasm of the tegument to the underlying cell bodies. The results reported herein dramatically increase the number of S. japonicum proteins known to be exposed to the host and, hence, those of interest as therapeutic targets. The ability of the parasite to rapidly internalise proteins at its surface has implications for the development of vaccines and may explain how these parasites are able to avoid the host immune system for long periods of time.

1. Introduction

Human schistosomiasis, caused mainly by infection with one of three members of the digenean flatworm genus Schistosoma, is one of the most important of the neglected tropical diseases. Approximately 207 million people worldwide are infected with schistosomiasis, primarily in sub-Saharan Africa, South America and China (Gryseels et al., 2006), with an estimated 280,000 deaths directly or indirectly attributable to the disease annually (van der Werf et al., 2003). Human infection occurs after penetration of host skin by infectious larvae, known as cercariae. Within human skin, the cercariae shed their surface layer and transform into host-adapted larvae, called schistosomula, which enter the vasculature to migrate over a period of days to the liver of the host (Jones et al., 2008). Within the liver, immature male and female worms pair and migrate to the site of patent infection, which in the case of Schistosoma mansoni and Schistosoma japonicum is the portal vasculature surrounding the human intestinal tract, and with Schistosoma haematobium is the vesicle plexus of the bladder. Adult schistosomes are long-lived and are able to avoid immune-mediated clearance from the host (Kusel et al., 2007). The ability of schistosomes to avoid host immune responses can be attributed in part to the dynamic nature of their host-exposed outer covering, or tegument, and the complex immuno-evasive strategies they employ to avoid elimination from the harsh intravascular environment (Pearce and MacDonald, 2002).

The tegument of schistosomes is a dynamic host-interactive layer involved in nutrition, immune evasion and modulation, excretion, osmoregulation, sensory reception and signal transduction (Jones et al., 2004). In adult schistosomes, as with other neodermatan parasites, the tegument is formed of a single syncytium that covers the entire body and is continuous with other epithelia, notably the foregut lining (Jones, 1998).

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This anucleate cytoplasmic layer, herein termed the surface layer of the tegument, is a highly ordered and distinct region. The layer is supported by insunken cell bodies that lie embedded in the parasite parenchyma. The surface layer maintains cytoplasmic continuity with the cell bodies by means of cytoplasmic bridges that traverse peripheral muscle bundles of the parasite body (Gobert et al., 2003). Tegumentary cell bodies contain the synthetic machinery of the syncytium including the endoplasmic reticulum and Golgi apparatus, and produce abundant vesicular products that are trafficked to the tegument along the cytoplasmic bridges (Fig. 1). The selective advantage of the syncytial tegument in schistosomes and other platyhelminth groups is poorly understood but it is undoubtedly a successful strategy that ensures survival of parasites in the harsh internal environment of their hosts. Invaginations of the surface layer and basal lamina, apparent in ultrastructural studies of schistosomes (Wilson and Barnes, 1974; Gobert et al., 2003) may provide a means for nutritional uptake and a way of avoiding the host immune response by internalising antibodies, while the dynamic turnover of the tegument may assist the parasite in avoiding immune-mediated damage by reducing antigenicity of the worm’s outer surface (Skelly and Wilson, 2006).

Figure 1

Fig. 1: Structure of the tegument and experimental plan. Worms were perfused from mice and after washing incubated with biotin for 30 min. Whole worms were removed at 5, 10, 15 and 30 min for confocal and electron microscopy. The teguments of remaining worms were removed by freeze–thawing and solubilised. The solubilised mixture was incubated with immobilised streptavidin and washed three times to remove non-biotinylated proteins. Biotinylated proteins bound to streptavidin were eluted using 50 mM DTT before proteomic characterisation.

The apical membrane of schistosomes is a complex heptalaminate structure (Hockley and McLaren, 1973). The multi-laminate appearance of this host-interactive membrane has given rise to the concept that schistosomes line their apical membrane with an accessory membrane called the membranocalyx (Wilson and Barnes, 1974), a trilaminate structure that, possibly, protects parasite molecules in the underlying tegument membrane and cytoplasm from immune assault (Skelly and Wilson, 2006). Despite the relative abundance of proteins found within the tegument (Braschi et al., 2006), very few parasite proteins appear in the apical membrane where they are likely to be exposed to the immune system (Braschi and Wilson, 2006).

Given its importance in nutrition and immune evasion, the schistosome tegument is generally accepted to present an ideal target for the development of vaccines and drugs. Indeed, a number of vaccine antigens with high efficacy against schistosomes have been reported from the tegument membrane, including the tetraspanins (TSPs) (Tran et al., 2006) and others (Da’dara et al., 2001; Cardoso et al., 2008; McManus and Loukas, 2008). A number of proteomic studies have sought to expand the range of available targets by identifying proteins that are exposed to the host at the surface of the tegument. In all studies, tandem mass-spectrometry was used together with various enrichment strategies to identify host-exposed proteins; including sequential solubilisation of whole tegument preparations (Braschi et al., 2006) and trypsinisation of whole adult parasites (Pérez-Sánchez et al., 2006). However, the most direct and discriminating method of isolating proteins exposed to the host has been biotinylation of whole adult schistosomes and the subsequent purification of labelled proteins on immobilised streptavidin. In a pioneering study, a long-chain sulfo-biotin derivative (sulfo-LC-biotin) was found to selectively label host-exposed proteins on the surface of S. mansoni without penetrating the plasma membrane (Braschi and Wilson, 2006).

In this study we have used surface biotinylation to label potential host-exposed tegumental proteins from adult S. japonicum for subsequent proteomic analysis of tegument membrane components.

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The tegumental proteome of this parasite, unlike S. mansoni, has not been well characterised although differences in morphology and biology suggest that there are likely to be subtle differences in tegumental architecture between the two schistosome species. Moreover, previous studies using this technique have relied on the fractionation of protein mixtures on polyacrylamide gels, reducing the sensitivity of the analysis due to processing required by in-gel digests. To increase the sensitivity of the analysis, and to capitalise on the recently published genomes of S. japonicum (Zhou et al., 2009) and S. mansoni (Berriman et al., 2009), we utilised the relatively new technique of OFFGEL electrophoresis (OGE) (Mulvenna et al., 2009) to fractionate tryptic peptides from biointylated tegument preparations. As biotin also enables convenient visualisation of labelled proteins in tissue sections, we have examined the distribution of biotin in the tegument of labelled worms using immuno-fluorescence and immuno-electron microscopy, and have monitored the localisation of biotin in the tegument over time.

2. Materials and methods

2.1. Harvesting of adult worms

Naturally infected Oncomelania hupensis hupensis snails were collected from an endemic focus in Anhui Province, China and imported to Australia. Adult S. japonicum worms were perfused 6 weeks after challenge from ARC Swiss mice infected percutaneously with 40 cercariae shed from the snails. Mice used in this study were housed at the Queensland Institute of Medical Research (QIMR) animal facility and all work was conducted in accordance with protocols approved by the QIMR Animal Ethics Committee. After perfusion, the parasites were washed three times with warm (37C) RPMI 1640 (Invitrogen, Melbourne, Australia) to remove excess blood products. Dead or dying flukes were removed and healthy schistosomes were used immediately for biotinylation. Some worms were removed after biotinylation for fixation followed by fluorescence or electron microscopy (see below).

2.2. Preparation of biotinylated tegument proteins by labelling live worms in culture

For proteomic studies all sample preparation steps, unless otherwise specified, were performed at 4 °C using buffers pre-chilled to the same temperature. Approximately 450 male and female parasites, representing the parasite load from 15 mice, were briefly washed five times in Hanks Balanced Salt Solution (HBSS) (Invitrogen). Washed parasites were then incubated for 30 min in 5 mL of 1 mM EZ-Link Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific)/HBSS with gentle head-over-head agitation. Biotin was then removed from the parasites and any remaining label quenched using RPMI 1640 with free amino acids. The parasites were then washed three times in RPMI 1640 (Invitrogen), resuspended in a minimal amount of RPMI 1640 with protease inhibitors and snap frozen in liquid nitrogen. Tegument removal was achieved using the freeze/thaw/vortex technique (Roberts et al., 1983b). Frozen worms were thawed slowly on ice, washed briefly with TBS (10 mM Tris/HCl, 0.84% NaCl, pH 7.4), incubated for 5 min on ice in 10 mM Tris/HCl, pH 7.4, before vortexing for 5 × 1 s bursts. The supernatant was collected and the tegumental extract pelleted at 1000g for 30 min. The tegumental extract was solubilised three times in 200 μl of solubilising solution comprising of 0.1% (w/v) SDS, 1.0% (v/v) Triton X-100 in 40 mM Tris, pH 7.4 with pelleting at 15,000g between each wash. These washes were combined and incubated with 240 μl of streptavidin–agarose beads (GE Healthcare) for 2 h at room temperature with gentle head-over-head mixing. The streptavidin beads were then pelleted, the supernatant discarded, and the beads washed three times in solubilising solution. To elute bound proteins, the beads were incubated in 50 mM DTT at room temperature for 2 h after which beads were pelleted and the supernatant removed for further analysis. For fluorescence and electron microscopy, adult worms were labelled in the same manner except for the use of a different form of biotin, sulfo-NHS-LC-biotin (Thermo Fisher Scientific).

2.3. Fluorescence and electron microscopy to observe surface biotinylation

To visualise surface biotinylation and the extent to which the biotin was incorporated into the tegument, parasites were fixed in 4% (v/v) paraformaldehyde in phosphate buffer for 30 min and embedded and frozen in OCT embedding compound. Cryostat sections (5–7 μm) were mounted onto Superfrost slides and labelled with rabbit anti-biotin antiserum (Bethyl Laboratories) diluted 1:200 in 1% (w/v) BSA (Sigma Fraction V) (BSA) in PBS, followed by goat anti-rabbit serum conjugated to CY3 (Jackson Immunodiagnostics). Sections were mounted in Vectashield mounting medium with DAPI (Vector Labs). Sections were examined using a Leica DM IRB inverted fluorescence microscope. As controls, sections of worms that had been stripped of their tegument by freeze–thawing and non-biotinylated worms were prepared and labelled as described. For immuno-electron microscopy, biotinylated parasites were fixed after exposure to biotin for 5, 15 and 30 min in 4% (v/v) paraformadehyde in 0.1 M phosphate buffer for 2 h, dehydrated in ascending concentrations of ethanol, and embedded in LR White resin (London Resin Company). Ultrathin sections were mounted onto formvar-carbon coated grids and subjected to an indirect immunocytochemistry protocol, incorporating rabbit anti-biotin antisera (as above) diluted 1:200 in BSA/PBS) and protein-A conjugated to 10 nm colloidal gold particles. After immunocytochemistry, sections were contrasted in uranyl acetate and lead citrate and examined using a JEM transmission electron microscope (JEOL) operated at 80 kV and equipped with a digital camera. For controls, non-biotinylated adult parasites were processed and labelled as described. As further controls, sections of biotinylated parasites were labelled as described above, but with omission of the anti-biotin antiserum.

2.4. OFFGEL electrophoresis

In brief, proteins were reduced and alkylated before digestion with trypsin, using established methods (Mulvenna et al., 2009). A 3100 OFFGEL Fractionator and OFFGEL Kit pH 3–10 (Agilent Technologies) with a 24-well setup were prepared according to the manufacturers’ protocols. Tryptic digests were diluted in peptide-focusing buffer, without the addition of ampholytes, to a final volume of 3.6 ml and 150 μl was loaded into each well. The samples were focused with a maximum current of 50 μA until 50 kVh was achieved. Peptide fractions were harvested and dried down using a vacuum centrifuge before mass spectrometric analysis.

2.5. LC–MS/MS analysis

OFFGEL fractions were chromatographically separated on a Dionex Ultimate 3000 HPLC using a Phenomenex C18 (2.1 mm × 25 cm) column using a linear gradient of 0–40% solvent B over 40 min with a flow rate of 250 μl/min. The mobile phase consisted of solvent A (0.1% formic acid (aq)) and solvent B (90/10 acetonitrile/0.1% formic acid (aq)). Eluates from the reverse phase (RP)-HPLC column were directly introduced into the TurboV ionisation source of a hybrid quadrupole/linear ion trap 4000 QTRAP MS/MS system (Applied Biosystems) operated in positive ion electrospray mode.

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All analyses were performed using Information Dependant Acquisition and the linear ion trap (LIT) acquisition modes. Analyst 1.4.1 software was used for data analysis. Briefly, the acquisition protocol consisted of the use of an Enhanced Mass Spectrum (EMS) scan as the survey scan. The two most abundant ions detected over the background threshold were subjected to examination using an Enhanced Resolution (ER) scan to confirm the charge state of the multiply charged ions. The ions with a charge state of +2 to +3 or with unknown charge were then subjected to collision-induced dissociation using a rolling collision energy dependent upon the m/z and the charge state of the ion. Enhanced Product Ion scans were acquired resulting in full product ion spectra for each of the selected precursors which were then used in subsequent database searches.

2.6. Protein identification

Database searching was performed using version 2.2.02 of Mascot with a 20 ppm tolerance on the precursor, 0.5 amu tolerance on the product ions, allowing for methionine oxidation and carbamidomethylation as a variable modifications, allowing for two missed cleavages, charge states of +2 and +3, trypsin as the enzyme and identifications were evaluated using MudPit scoring. A threshold of 5% probability (P < 0.05) of a false positive was used for all Mascot searches and a decoy database was used to estimate the false positive rate. Searches were conducted on the NCBI non-redundant (NCBInr) database (http://www.ncbi.nlm.nih.gov/) as of the 24th of March 2009, a custom-built database consisting of 107,410 S. japonicum expressed sequence tag (EST) sequences (effective database size 644,460 sequences) deposited in the NCBI databases as at 24th December 2008 and a custom-built database containing 267,165 protein sequences from a range of helminths, including the full protein datasets from the S. japonicum (Zhou et al., 2009) and S. mansoni (Berriman et al., 2009) genome sequencing projects, all Trematoda proteins in the NCBInr database as of the 24th of March 2009, and protein datasets from Nematode Net (http://www.nematode.net/) (Wylie et al., 2004). The criteria for accepting protein identifications was as follows: (i) the identification needed to contain at least one peptide with a score greater than the identity threshold supplied by the Mascot server; and (ii) the identification needed to contain more than one significant peptide, where significant peptides included those with scores above the identity or homology thresholds supplied by the Mascot server. On five occasions particularly high scoring single peptide identifications were verified by manual annotation of spectra. Identifications with shared proteins were retained if each contained at least one unique peptide above the significance threshold and for grouped proteins the highest scoring identification was retained.

2.7. Bioinformatic analysis

Protein descriptions were assigned to EST Mascot hits using BLASTX on the non-redundant protein database from NCBI (bit score >30) when the reading frame of the Mascot hit was the same as the blast hit. Descriptions were also assigned to proteins based on BLAST searches (bit score >30) of the S. japonicum Transcriptome and Proteome Database (http://function.chgc.sh.cn/sj-proteome/index.htm) (Liu et al., 2008), SchistoDB (http://schistodb.net/schistodb20/home.jsp) (Zerlotini et al., 2009) and SmedGD (http://smedgd.neuro.utah.edu/index.html) (Robb et al., 2008). Lutefisk v1.0.5 (Johnson and Taylor, 2002) was used to derive de novo peptide sequences from high quality unassigned spectra using the default lcq parameters. Spectra producing de novo sequence with a Pr(C) score greater than 0.60 were deemed to be high quality and used in web-based MS-BLAST searches (http://genetics.bwh.harvard.edu/msblast/) (Shevchenko et al., 2001). Trans-membrane helices were predicted using TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) (Krogh et al., 2001) and glycosylphosphatidylinositol (GPI) anchor sites were predicted using PredGPI (Pierleoni et al., 2008). Protein identifications were assigned to subcellular locations using a combination of WoLF PSORT (http://wolfpsort.org/) and literature searches.

3. Results

3.1. Fate of LC-biotin probes in tegument of live schistosomes

Adult parasites, perfused from mice and biotinylated using sulfo-NHS-LC-biotin, were subjected to immuno-fluorescence microscopy to detect the distribution of biotin probes during up to 30 min of incubation. A clear intense band of label was observed in the region of the distal cytoplasm of the tegument (Fig. 2). Biotin-immunoreactivity was not observed in the vicinity of the gastrodermis (data not shown) and thus the probe was not ingested during incubation. Fine linear and immune-positive strands were observed immediately subjacent to the distal cytoplasm of the tegument. Small immunoreactive foci were also observed surrounding some cells in peripheral regions of the parenchyma (Fig. 2). These cells were interpreted by their location, and from results of immuno-electron microscopy experiments, to be tegumentary cell bodies. Examination of ultrathin sections labelled with anti-biotin serum for immuno-electron microscopy revealed that membrane components of the distal cytoplasm, including the apical membrane–membranocalyx complex, were labelled with anti-biotin serum (Fig. 3C). This membrane-specific label was observed at all time points (5, 15 and 30 min) over the 30 min incubation, although it was also noted that the tegumentary cytoplasm contained progressively less ground substance with increasing incubation times. Signal from within the tegument matrix which appeared below the apical surface was predominantly membrane associated. This indicates that these structures correspond to infolds of the tegument and were essentially the surface of the parasite.

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Gold particles were absent from the general parenchyma of parasites and from the longitudinal and circular myofibrils that lie immediately subjacent to the distal cytoplasm of the tegument. Cytoplasmic bridges that link the distal cytoplasm with insunken cell bodies and the cell bodies themselves were labelled (Fig. 3D) at all time points, but it was not possible to ascertain whether the label was associated with membrane components on these resin sections. It is worth noting that these regions of the parasite were not retained after tegument isolation and did not contribute material to subsequent protein analyses. The negative control, in which a non-biotinylated adult worm was incubated with anti-biotin serum followed by the protein-A–gold complex, did not contain colloidal gold particles (Fig. 3A), confirming that the strong positive labelling observed in subsequent experiments was a result of the biotinylation of surface exposed proteins rather than endogenous biotin reactivity in schistosome tissues. Similarly, omission of the anti-biotin antiserum from the immunocytochemistry protocol resulted in a complete absence of labelling, indicating that protein-A–gold complexes did not label the schistosome tissues (Fig. 3B).

Figure 2

Fig. 2: Immuno-fluorescence microscopy of biotin-labelled Schistosoma japonicum after probing with anti-biotin conjugated to CY3. Anti-biotin immunoreactivity appears as red. The sections were counter-stained with DAPI (blue). Biotin was detected in the surface layer of the tegument (TEG), where it appears as a bright sinuous band of label. Positive immunoreactivity was also detected in regions subjacent to the surface layer (arrowheads), reflecting the presence of biotin in interconnecting bridges, and in discrete regions in sub-muscle cells (long arrows), likely to be tegumentary cytons.

Figure 3

Fig. 3: Ultrastructural immunocytochemistry of biotin in long-chain (LC)-biotin treated Schistosoma japonicum adults. (A) Surface layer of a non-biotinylated adult male (control) worm. (B) Region of parenchyma of an adult female exposed to biotin for 15 min. For this control section, anti-biotin antiserum was omitted from primary incubation solution in the immunocytochemistry protocol. (C) Surface layer of a biotinylated adult male. Note the clear membrane-associated label. Gold particles were frequently observed in association with internal vacuoles of the tegument (arrows). These compartments are closely associated with the apical membrane. (D) Region of parenchyma of an adult female exposed to biotin for 15 min and labelled with anti-biotin antiserum in immunocytochemistry protocol. Intercytolasmic bridges are strongly labelled by anti-biotin serum, whereas no gold is evident over myofibrils. AM, apical membrane–membranocalyx complex; BL, basal lamina of the surface layer of tegument; CM, circular musculature; LM, longitudinal musculature; N, interconnecting bridges of the tegument.

3.2. Proteomic characterisation of proteins biotinylated with thiol cleavable sulfo-NHS-SS-biotin

The use of a thiol cleavable biotin derivative enables a more efficient elution of labelled proteins from immobilised streptavidin and removes a possible source of protein degradation during the relatively harsh elution protocol required for non-cleavable sulfo-biotin.

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Accordingly, for proteomic characterisation, whole S. japonicum worms were labelled with thiol cleavable Sulfo-NHS-SS-Biotin. After purification of labelled proteins on immobilised streptavidin, mass spectrometric analysis of the 24 OGE wells from the streptavidin-bound fraction resulted in 42 non-redundant protein identifications from the NCBInr database. Identified proteins were grouped according to their predicted subcellular location and compared with findings from other proteomic, immuno-localisation and activity-based studies of the schistosome surface (Table 1). Of the 42 proteins identified, the majority (52%) were either known or predicted, by PSORT, to be membrane-associated proteins, the remainder primarily consisting of heat shock proteins (HSPs) (10%) or cytosolic proteins (21%) (Fig. 4A). Twelve parasite proteins were predicted to contain at least one trans-membrane domain, 10 of which were classified as membrane-associated. In order to fully identify all proteins in the biotinylated sample, the MS/MS data were also used to search a custom-made S. japonicum EST database and a custom-built helminth protein database. These searches produced a total of 39 and 49 non-redundant identifications, respectively, and included 11 identifications that were not made in the NCBInr protein database search (Table 2; Supplementary Tables S1 and S2). Among these extra identifications were a further seven membrane-associated proteins with homologies to the TSP protein family, myoferlin, various ATPases and a 200 kDa membrane protein. There were no identifications made in the NCBInr database search that were not also made in the EST or protein database searches.

Table 1: Protein identifications from bound fraction using the NCBI non-redundant protein databasea.

ID No. GI MS CO SC Species Description TM GPI Sm1b Sm2b Sb1b Sb2b NP
Cytosol/nuclear
23 189502922 276 37.8 7 Schistosoma japonicum 14-3-3 Protein – – – – + –
76 121697 130 35.3 6 Schistosoma japonicum Glutathione S-transferase class-mu 26 kDa isozyme – – – – + – IP (Brophy and Barrett, 1990)
68 1389744 142 31.8 4 Schistosoma japonicum Glutathione-S-transferase (28 kDa) – – – – + + IP (Brophy and Barrett, 1990)
219 38259184 83 21.7 3 Schistosoma japonicum Thioredoxin peroxidase-1 – – – – + –
26 14268828 267 21 5 Schistosoma japonicum Elongation factor 1-a – – – – + –
77 29840889 130 19.1 2 Schistosoma japonicum Cytochrome b5 reductase 1 1 – – – – –
118 149287148 101 41.7 3 Ornithodoros parkeri Ubiquitin/40S ribosomal protein S27a – – – – + –
116 76154253 104 13.8 2 Schistosoma japonicum Vacuolar proton ATPases isoform 3 – – – – – –
378 462011 55 6.9 2 Schistosoma japonicum Enolase
Cytoskeleton
5 90811719 317 23.9 7 Culex pipiens pipiens Beta-actin – – SC – + + IL (Pérez-Sánchez et al., 2006)
8 56754704 308 26.6 6 Schistosoma japonicum Actin – – SC + + + IL (Pérez-Sánchez et al., 2006)
80 17549915 127 8.3 3 Caenorhabditis elegans Tubulin, Beta family member – – – – + –
Mitochondria
168 56754309 95 24.1 4 Schistosoma japonicum ADP, ATP carrier protein 3 3 – – + + –
Heat shock proteins
66 76155249 156 28.5 3 Schistosoma japonicum Heat shock protein 70 – – SC + + –
62 56759038 197 14.9 8 Schistosoma japonicum 90 kDa Heat shock protein – – – + + –
92 194749989 118 9.2 3 Drosophila ananassae Heat shock 70 kDa protein cognate 1 – – – + + –
89 157278567 124 9.4 2 Oryzias latipes Heat shock protein 70 isoform 5 – – SC + + –
Membrane associated
2 56753019 574 39.7 12 Schistosoma japonicum Annexin A13 isoform a – – LC + – –
1 160996 635 34.3 9 Schistosoma japonicum Glyceraldehyde-3-phosphate dehydrogenase – – SC + + + IL (Goudot-Crozel et al., 1989) VP (Argiro et al., 2000)
91 56759272 121 25.2 3 Schistosoma japonicum Na, K ATPase 1 – SC + – –
67 161127 146 23.6 4 Schistosoma japonicum 22.6 kD Tegumental-associated antigen – – – + + + VP (Santiago et al., 1998)
65 76156316 175 33.5 4 Schistosoma japonicum Dysferlin – – LC + – –
3 2829279 534 20.9 12 Schistosoma japonicum Calpain – – SC + + –
75 76156797 131 12.7 2 Schistosoma japonicum Nucleotide pyrophosphatase/phosphodiesterase 5 – – LC + – – SA (Bogitsh and Krupa, 1971)
144 76152537 97 24.6 3 Schistosoma japonicum Na, K ATPase – – – + + –
61 56754313 204 15.6 4 Schistosoma japonicum Annexin - – LC + – –
229 56753245 74 8.3 2 Schistosoma japonicum Glucose transport protein 6 – – + – - IL (Jiang et al., 1996)
196 78190783 83 10.8 2 Aphrocallistes vastus ATPase alpha-subunit - – – – – –
105 56753293 115 13.2 2 Schistosoma japonicum Aquaporin-3 6 – – + – –
74 56757229 132 11.9 2 Schistosoma japonicum Annexin B13a – – LC + – –
223 56753957 81 18 1 Schistosoma japonicum Voltage–dependent anion channel 1 – – SC + – –
78 56758274 128 11.6 2 Schistosoma japonicum Myoferlin – – – – – –
334 56755685 65 6.4 3 Schistosoma japonicum Permease 1 heavy chain 1 – – – – –
71 76155174 136 14.9 1 Schistosoma japonicum Alkaline phosphatase – – LC + – – IL (Pujol et al., 1990) SA (Roberts et al., 1983a)
107 15824396 112 7.1 4 Schistosoma mansoni SNaK1 – – SC + – –
64 29840937 182 7.9 1 Schistosoma japonicum Hypothetical protein with IG domain (Basigin-like) 1 – – – – –
90 59859873 122 5.3 3 Hirudo medicinalis Na, K ATPase alpha-subunit 8 – SC + - -
128 14285348 101 4 2 Oreochromis mossambicus Plasma membrane calcium-transporting ATPase 2 7 – – + + –
73 12963455 134 2.6 2 Rana catesbeiana Plasma membrane calcium ATPase 1b 7 – – + + –
Host proteins/streptavidin/unknown
72 51653 136 4.8 1 Mus musculus Immunoglobulin gamma1 heavy chain - - - - - -
132 198917 100 3.8 2 Mus musculus Lymphocyte common antigen 2 - - - - -
69 56756665 139 22.2 3 Schistosoma japonicum SJCHGC02563 protein (No homology) - - - - - -

a ID - Assigned identification; GI - gene index number; MS - Mowse Score; CO - percentage coverage; SC - spectral count; TM - number of transmembrane domains; GPI - presence of a predicted GPI anchor point denoted by a ’+’; Sm1-2, Sb1-2 presence of the identification in a Schistosoma mansoni or Schistosoma bovis proteomics study denoted by a ’+’; NP - non-proteomic evidence of surface exposure. IL - Immuno-localisation, SA - Surface activity, VP - Vaccine protection.
b Sm1 Reference (Braschi and Wilson, 2006); SC – short chain biotin; LC – long-chain biotin; Sm2 Reference (Braschi et al., 2006) ‘+’ denotes that identification occurred in detergent soluble fractions; Sb1 Reference (Pérez-Sánchez et al., 2008); Sb2 Reference (Pérez-Sánchez et al., 2006).

Figure 4

Fig. 4: Overview of protein identifications from NCBInr Mascot database searches. (A) The proportion of total protein identifications belonging to the assigned subcellular locations for biotinylated (bound) and non-biotinylated (unbound) fractions. (B) Venn diagrams showing protein identifications for the biotinylated (bound) and non-biotinylated (unbound) fractions. Overall 23 protein identifications were made in both the bound and unbound fractions. Heat shock proteins were particularly abundant in the unbound fraction, however only four were identified in the bound fraction, one of which was unique to that fraction. Membrane-associated proteins were the most likely to be identified in both fractions with 10 protein identifications present in both fractions.

Table 2: Additional protein identifications from bound fraction in Mascot searches of custom-built expressed sequence tag (EST) and proteins databasesa.

ID No. GI DB MS CO SC BA BS Description TM GPI Sm1b Sm2b Sb1b Sb2b NP
Cytosol
587 28341294 EST 55 23.6 2 AAL30898 434 Phosphoglycerate mutase – – – – + –
39 Sjc_0053110 Prot 146 7.1 3 N/A N/A Long-chain-fatty-acid-CoA ligase 5 – – – + – –
Mitochondrial membrane
130 Sjc_0089940 Prot 65 11.3 2 N/A N/A F-type H + -transporting ATPase alpha chain – – – – – –
Cytoskeleton
157 Ovi100 Prot 57 10 2 N/A N/A Alpha tubulin protein – – – – + +
Membrane associated
473 56752696 EST 80 19.4 2 ABQ44513 286.19 Tetraspanin 3 – LC + – – IL,VP
(Tran et al., 2006)
539 56048463 EST 59 25.8 3 XP_001375250 296.98 Myoferlin – – – – – –
93 Sjc_0093040 Prot 81 7.7 2 N/A N/A Glucose transport protein 12 – – + – – IL (Jiang et al., 1996)
668 28351004 EST 51 21 1 CAX69903 209 Leucine aminopeptidase – – – – – – SA (Fripp, 1967)
185 Sjc_0214870 Prot 52 3.3 2 N/A N/A Ca2+-transporting ATPase 8 – – – – –
48 Sjc_0204190 Prot 128 8.6 2 N/A N/A Plasma membrane calcium-transporting ATPase 3 2 – – + – –
162 Sjc_0052350 Prot 55 9.1 2 N/A N/A Phospholipid-translocating ATPase 5 – – – – –
50 Sjc_0097880 Prot 123 4.7 4 N/A N/A 200-kDa GPI-anchored surface glycoprotein – + SC – + – IL (Sauma and Strand, 1990)

a ID - Assigned identification; GI - gene index number; MS - Mowse Score; CO - percentage coverage; SC - spectral count; TM - number of transmembrane domains; GPI - presence of a predicted GPI anchor point denoted by a ’+’; Sm1-2, Sb1-2 presence of the identification in a Schistosoma mansoni or Schistosoma bovis proteomics study denoted by a ’+’; NP - non-proteomic evidence of surface exposure. IL - Immuno-localisation, SA - Surface activity, VP - Vaccine protection.
b Sm1 Reference (Braschi and Wilson, 2006), SC – short chain biotin; LC – long-chain biotin; Sm2 Reference (Braschi et al., 2006) ‘+’ denotes that identification occurred in detergent soluble fractions; Sb1 Reference (Pérez-Sánchez et al., 2008); Sb2 Reference (Pérez-Sánchez et al., 2006).

In the MS/MS of the bound fraction, spectra were recorded from approximately 11,616 precursor ions of which 696 were assigned during Mascot searches. To assess the completeness of the protein identifications, unassigned spectra were filtered for quality by calculating de novo peptide sequences with the program Lutefisk. Sequences with Pr(C) greater than 0.6 were combined and used in a MS-BLAST search against the nr95_clean database. Seventeen proteins were identified using this method (Supplementary Table S3). Only three new proteins were identified in this search, a histidine kinase, an ADP-ribosylation factor and a hypothetical protein from Caenorhabditis briggsae. The latter two identifications, however, were both single peptide, low confidence identifications, suggesting that the proteins reported here account for the majority of identifiable proteins present in the tegument preparation.

3.3. Proteomic characterisation of tegument proteins that did not incorporate biotin

Proteins from the tegument fraction that did not bind to streptavidin (and therefore did not incorporate biotin during the labelling of live worms) were analysed in the same manner as the streptavidin-bound fraction. Mascot searches of MS/MS spectra generated from the 24 OGE wells provided 91 unique protein identifications (Supplementary Table S4). Unlike the bound fraction, the majority of the identifications were comprised of cytosolic, cytoskeletal or mitochondrial proteins (54%) (Fig. 4A). Cytosolic proteins provided the greatest proportion of identifications in the unbound fraction, contrasting with the bound fraction in which membrane-associated proteins alone provided over half of the identified proteins (Fig. 4A). HSPs were also abundant in the unbound fraction and comprised over 15% of the identifications. Conversely, only four HSPs were identified in the bound fraction, including one identification that was unique to that fraction. Twenty-one proteins were identified in both the bound and unbound fractions (Fig. 4B) and these were identified over the range of subcellular locations including cytosolic, mitochondrial and cytoskeletal proteins. Membrane-associated proteins were more likely to occur in both fractions with 10 of these proteins identified in both unbound and bound fractions. Only two of these proteins, one a homologue of Sm29 and the other Sj66, were from families identified solely in the unbound fraction.

3.4. Contamination and decoy database searching

All datasets were searched against the NCBInr protein database with no taxonomy restrictions. In all searches there were minimal identifications to unrelated species and none to bacterial proteins indicating that there was no contamination with non-schistosome proteins in the tegument preparations. Decoy database searches were conducted in all searches using the Mascot server. The false discovery rate (FDR) was less than 1%, as reported by the Mascot server, suggesting a true FDR of less than 2% for all searches except for one search performed against the custom-built protein database in which the FDR rate reported by the Mascot server was 3.2%.

4. Discussion

The surface layer of the schistosome tegument is covered externally by the dual membrane–membranocalyx complex. It is now generally agreed (Skelly and Wilson, 2006) that maintenance of the apical membrane and membranocalyx is mediated by the fusion of membranous vesicles, originating from the tegumentary cell bodies, with the apical membrane and the subsequent release of the membranous contents (Morris and Threadgold, 1968; Wilson and Barnes, 1977) (Fig. 5A). Fusion of the vesicles with the apical membrane replenishes the lipid bilayer while the membranous contents, after leaving the vesicle, provide material for the maintenance of the membranocalyx. To avoid unlimited expansion it is clear that the apical membrane must turn over an equal amount of lipid bilayer as is contributed during the fusion of the membranous vesicles, but a possible mechanism for this has not been established. One model of tegumental turnover proposes that the membranocalyx is turned over into the bloodstream of the host while the apical membrane is internalised and trafficked back to the cell bodies and Golgi apparatus (Skelly and Wilson, 2006). Confirmation of this has been complicated by the membranocalyx, which acts as a physical block to labelling of the apical membrane. Recently it has been shown that lipid-insoluble LC biotin is an effective reagent for the labelling of host-exposed proteins at the apical membrane (Braschi and Wilson, 2006) and in this study we have used this technique to label proteins embedded or associated with the apical membrane of adult S. Japonicum.

After incubation of freshly perfused adult schistosomes with lipid-insoluble long-chain biotin, the specificity of labelling was confirmed by using tandem mass-spectrometry to identify proteins purified on immobilised streptavidin. By using OGE on tryptic digests of these purified protein mixtures we were able to achieve greater sensitivity than traditional methods utilising polyacrylamide gels and in-gel tryptic digest. Using this technique, 46 proteins were identified as putatively biotinylated as evidenced by their purification on immobilised streptavidin. The majority of these proteins were membrane-associated proteins. Compared with the unbound fraction, which was analysed in the same manner, the bound fraction clearly contained a much greater proportion of membrane-associated proteins than did the unbound fraction, which consisted predominantly of cytosolic, mitochondrial and cytoskeletal proteins. A small number of proteins traditionally considered cytosolic were identified in the bound fraction, although an exposed location has been suggested for a number of these proteins. The 28 kDa GST provides protection when used as an anti-schistosome vaccine antigen (Balloul et al., 1987) and has been localised to the surface (Henkle et al., 1990), although its localisation and vaccine efficacy have been disputed in other studies (Holy et al., 1989; Schechtman et al., 2001).

7. J. Mulvenna et al. / International Journal for Parasitology Volume 40, Issue 5, April 2010, Pages 543-554

Figure 5

Fig. 5: Tegumental maintenance and hypothesised turnover of the apical membrane. (A) Tegumental maintenance is thought to be achieved through membranous vesicles that are abundant in the tegument. Replenishment of the tegumental complex is achieved by fusion of the membranous vesicles with the apical membrane and the incorporation of the membranous contents into the membranocalyx. (B) A concomitant reduction in the lipid bilayer of both the apical membrane and membranocalyx is necessary to avoid the unlimited growth of the tegument. One model for this process involves the sloughing of the membranocalyx into the host blood and the internalisation of apical membrane to the Golgi apparatus or endosomes. A tetraspanin (TSP) and Sj22.6, proteins that may be involved in membrane dynamics based on homologues in other organisms, are highlighted.

Both the 14-3-3 protein (Assossou et al., 2003) and enolase (Jong et al., 2003) have also been localised to the plasma membrane in other organisms. Although putatively surface exposed after identification in the bound fraction, it must be noted that these cytosolic proteins, despite the stringent detergent wash, may have been pulled down by protein–protein interactions with bona fide membrane proteins during incubation of the solubilised tegument with streptavidin. Further, although great care was taken in the perfusion and labelling of adult worms, limited tegumental damage may have led to the low-level biotinylation of internal proteins. Nonetheless, given the identification of a number of known membrane proteins and the relative lack of identifications from internal compartments, this work supports an exposed position on the tegument for the majority of the proteins identified in the bound fraction.

8. J. Mulvenna et al. / International Journal for Parasitology Volume 40, Issue 5, April 2010, Pages 543-554

HSPs were highly represented in both the bound and unbound fractions. The majority of HSPs (14 from a total of 15 proteins) were present in the unbound fraction, reflecting the traditional view of HSPs as intracellular proteins. However, a small subset of these (three proteins), together with a single unique HSP, were also identified in the bound fraction. As these proteins are known molecular chaperones, it is quite possible that their presence in the bound fraction is a result of interactions with biotinylated proteins. However, the identification of only a small subset of these proteins in the bound fraction together with a HSP unique to this fraction supports the possibility of their surface localisation. Interestingly, a number of HSPs have recently been reported to be both extracellular and associated with the plasma membrane (Tsutsumi and Neckers, 2007). Although little is known about their extracellular roles, HSPs are believed to play a role in immune responses and cellular migration (Tsutsumi and Neckers, 2007). Accordingly, the identification of biotinylated HSPs in this work may indicate similar roles for the HSPs on the surface of S. Japonicum.

Apart from cytosolic proteins and HSPs, other functional families identified in the biotinylated fraction included transport proteins, structural proteins and various enzymes, including alkaline phosphatase and SNaK1, an Na, K ATPase homologue (Skelly et al., 2001). A number of these proteins have been previously identified at the surface of various schistosomes by either proteomic or non-proteomic methods (Table 1). A role for the tegument in nutritional uptake in S. japonicum was confirmed by the identification of a protein homologous to SGTP4, a glucose transporter from S. mansoni (Skelly et al., 1994), and a S. japonicum homologue of the S. mansoni amino acid permease heavy chain (Krautz-Peterson et al., 2007). Leucine aminopeptidase (LAP) activity has been reported at the tegumental surface of S. mansoni, presumably to facilitate the breakdown of proteins into their constituent amino acids before transport across the membrane (Fripp, 1967), as well as the surface of the gut and eggs (McCarthy et al., 2004; Rinaldi et al., 2009). Two LAPs have been identified in S. japonicum (Rinaldi et al., 2009), SjLAP1 and SjLAP2, and it is noteworthy that one of these, Sj-LAP1, was identified in Mascot searches of the biotinylated fraction, although as a relatively low-scoring single peptide identification (Supplementary Fig. S1). In S. mansoni, TSPs (Tran et al., 2006) and Sm29 (Cardoso et al., 2008) have both been shown to be highly efficacious vaccine candidates and homologues to both these proteins were identified in S. japonicum. The presence of a TSP at the surface of S. japonicum makes this group of proteins potentially attractive vaccine candidates, but a recent study showed no evidence of protection when using Sj-TSP-2 as a vaccine against this schistosome (Cai et al., 2008). The homologue of Sm29 was identified in the unbound fraction, suggesting a weaker association with the tegument than that shown for Sm29 in S. mansoni (Cardoso et al., 2008). Which tegument proteins contribute to the dynamics of the apical membrane in schistosomes is currently unknown, although some proteins identified here participate in membrane dynamics in other systems. Intriguingly, mammalian TSPs are thought to participate in cargo selection and vesicle formation in the endocytic pathway (Bonifacino and Traub, 2003; Berditchevski and Odintsova, 2007), and are enriched in exosomes (van Niel et al., 2006). Likewise the identification of Sj22.6, a dynein-like tegument-associated antigen (TAA) containing two EF-hand domains, may represent part of the machinery involved in apical membrane movement. Dynein light chains (DLC) are involved in membrane dynamics and have been shown to be localised to the host-interactive distal parasite membrane where they are intimately associated with, but not embedded in, the apical membrane (Zhang et al., 2005). IgE responses to the 22.6 kDa proteins have been reported in S. japonicum (Li et al., 2000), S. mansoni (Fitzsimmons et al., 2007) and S. haematobium (Fitzsimmons et al., 2004). In S. mansoni it has been suggested that the IgE response to Sm22.6 occurs after the death of the worms in vivo (Fitzsimmons et al., 2007), but its identification here, in the absence of identifications of other components of cytoplasmic motor complexes that contain DLCs, such as dyneins or Myosin V, may indicate an exposed position for the protein at the tegument surface in adult S. Japonicum.

9. J. Mulvenna et al. / International Journal for Parasitology Volume 40, Issue 5, April 2010, Pages 543-554

The electron microscopy data presented here show that biotinylated proteins at the surface of adult schistosomes are internalised, and that this occurs within a very short period of time. Moreover, internalisation proceeded at low temperatures, in conditions designed to minimise damage to the tegument (Braschi and Wilson, 2006). The specificity of biotin labelling for membrane-associated proteins has been confirmed for S. mansoni using proteomic methods (Braschi and Wilson, 2006) and similar data presented here suggest that labelling was restricted to mostly membrane-associated proteins at the surface of the worm. The observed labelling of membrane-bound vacuolar or invaginated compartments deep within the surface layer of the tegument (Fig. 3C) is not surprising, since these compartments are essentially components of the apical membrane which invaginates extensively under normal conditions (Gobert et al., 2003). It is expected that a proportion of these basal structures is actually an extension of the apical surface that appears to be an independent structure due to the highly folded composition of the tegument and the two-dimensional limitations of transmission electron microscopy (Gobert et al., 2003). Thus, the observations of positive biotin immunoreactivity likely reflects cytoplasmic internalisation of labelled proteins, rather than artefactual penetration of a degraded apical membrane by reagents. It is known that uptake of the styryl dye FM 1-43 by the tegument of S. mansoni is inhibited by the antimalarial drug primaquine, an inhibitor of vesicular uptake in mammalian cells (Ribeiro et al., 1998), perhaps suggesting endocytotic pathways exist in the tegument, a postulate supported by studies of phospholipid dynamics in the schistosome tegument (Brouwers et al., 1999).

Utilising electron microscopy and colloidal gold markers, immunoreactivity was also observed in the intercytoplasmic bridges of the tegument. The observation of gold particles at all time points suggests a rapid transfer of membrane protein to deeper regions of the tegument that is independent of incubation temperature. Proteins in this region are not expected to contribute to the protein mix in subsequent proteomic analyses, for the freeze–thaw method of tegument isolation separates the surface layer and not underlying regions of the tegument (Roberts et al., 1983b). However, these observations are consistent with a rapid and targeted vesicular import and an organised pathway of membrane turnover in the tegumentary syncytium of schistosomes. Although indicative of surface membrane recycling, these observations require further observations of worms labelled in vivo, using fluorescent markers of tegument (Kusel et al., 2007; Krautz-Peterson et al., 2009). The use of in vivo manipulation of adult worms with cytological probes may overcome possible problems arising from tegument degradation ex vivo, and temperature effects on tegument turnover (Ribeiro et al., 1998). Nonetheless, given the results presented here, rapid turnover of the apical membrane, and the proteins therein, have a number of implications for the understanding of the biology of adult schistosomes and medical intervention in schistosomiasis. One mechanism by which the schistosome is able to avoid complement and a concerted immunological assault appears to be the adsorption of host proteins into the membranocalyx (Kemp et al., 1978; Braschi and Wilson, 2006; Skelly and Wilson, 2006) and the sloughing of the membranocalyx from the parasite into the host blood stream (Skelly and Wilson, 2006), a hypothesis supported by the identification of at least two host proteins in the biotinylated sample. In this context the rapid turnover of the apical membrane could be a by-product of the continued turnover of the membranocalyx rather than a defense mechanism aimed at the rapid internalisation of host proteins, although this may be a secondary effect. Attempts to develop effective vaccines against proteins in the tegument of adult and developing schistosomes need to contend with the presence of the membranocalyx as a physical barrier to neutralising antibodies at the surface of the parasite. The variable efficacy of vaccines targeting putative tegumental proteins, ranging from highly effective (Tran et al., 2006; Cardoso et al., 2008) to non-protective (Cai et al., 2008), indicates that further information is required on the roles and putative surface distribution of these antigens during parasite establishment and homeostasis in the host. The visibility of the apical membrane and its proteins to vaccine intervention in adult schistosomes is also poorly understood. The presence of several tegument proteins in adult excretory/secretory products (ES) (Liu et al., 2009) suggests that the apical membrane may be visible to the host, but removal and culturing of worms prior to ES characterisation may disrupt the tegument and cause the leakage of tegument proteins into the ES component. A resolution of these questions awaits a reliable method of observing tegumental dynamics in vivo, an area that is starting to be explored (Krautz-Peterson et al., 2009).

The schistosome tegument has been subjected to much investigation, due to the important role it plays in the survival of the worm in vivo, and as a source of novel drug or vaccine targets. Here we have investigated the dynamics and the exposed protein content of the tegument of S. japonicum using a combination of biotinylation, electron microscopy and proteomics. Using biotinylation of surface exposed proteins we have illustrated the dynamic nature of the schistosome tegument and have determined that, in vitro at least, surface membrane proteins, and most likely the membrane itself, are rapidly internalised. The nature of the biotinylated proteins was confirmed using proteomic analysis and it was shown that the majority of biotinylated proteins were membrane-associated. Internalisation of apical membrane may play a role in the rapid turnover of the membranocalyx and it may also contribute to immuno-evasion by the internalisation of host-derived immuno-active proteins. Given the long life span of these parasites -in vivo (Gryseels et al., 2006), this strategy may contribute to the ability of the parasite to avoid host-mounted immune response.

Acknowledgements

We would like to thank Professor Robert Parton from the University of Queensland who kindly provided colloidal gold particles. This research was supported by project and program grants from the National Health and Medical Research Council, Australia (NHMRC). J.M. is supported by a Peter Doherty Fellowship from the NHMRC and A.L. is supported by a Senior Research Fellowship from the NHMRC.

Appendix A. Supplementary data

  • Supplementary table S1:
    Protein identifications from Mascot search of custom-built Schistosoma japonicum expressed sequence tag (EST) database with MS/MS spectra from bound (biotinylated) fraction.
    View in sidebar | Download file (xls, 14k)
  • Supplementary table S2:
    Protein identifications from Mascot search of custom-built helminth parasite protein database with MS/MS spectra from bound (biotinylated) fraction.
    View in sidebar | Download file (xls, 15k)
  • Supplementary table S3:
    Protein identifications by MS-BLAST of de novo peptide sequences derived from MS/MS spectra of bound (biotinylated) fraction.
    View in sidebar | Download file (xls, 34k)
  • Supplementary table S4:
    Protein identifications from Mascot search of NCBInr (http://www.ncbi.nlm.nih.gov/) database with MS/MS spectra from unbound (non-biotinylated) fraction.
    View in sidebar | Download file (xls, 46k)
  • Supplementary figure S1:
    Spectrum matching a sequence tag from a leucine aminopeptidase (accession number CAZ30678) identified in the biotinylated fraction. The theoretical fragmentation masses are set out below the spectrum with ions observed in the spectrum highlighted in red. The sequence of the peptide is annotated on the spectrum. The x axis shows the mass to charge ratio (m/z) and the y axis, intensity in arbitrary units (a.i.).
    View in sidebar | Download file (png, 2128k)

10. J. Mulvenna et al. / International Journal for Parasitology Volume 40, Issue 5, April 2010, Pages 543-554

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