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TiO2‑Based Phosphoproteomic Analysis of Schistosomes: Characterization of Phosphorylated Proteins in the Different Stages and Sex of Schistosoma japonicum Guofeng Cheng,*,† Rong Luo,†,∥ Chao Hu,†,∥ Jiaojiao Lin,† Zhaofang Bai,‡ Beimin Zhang,† and Hongxia Wang*,‡,§ †

Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture, 518 Ziyue Road, Shanghai, 200241, China ‡ National Center of Biomedical Analysis, 27 Taiping Road, Beijing, 100850, China S Supporting Information *

ABSTRACT: Protein phosphorylation is an important posttranslational modification in many organisms that regulates numerous cellular processes. However, it remains poorly characterized in schistosomes, the causative agent of schistosomiasis in humans and related animals. In the present study, we characterized phosphorylated proteins in different stages and sex of Schistosoma japonicum (S. japonicum) including schistosomula (14 days), adult females (35 days), and adult males (35 days) by a titanium dioxide (TiO2) based phosphoproteomic method. A total of 180 phosphopeptides were identified in 148 proteins. Our further studies revealed that heat shock protein 90 (Hsp90), one of the phosphoproteins codetected in the different stage and sex of schistosomes, may play an important role in the regulation of schistosome development by directly or indirectly interacting with other codetected signal molecules. Additionally, some phosphoproteins were shown to be detected in a gender-specific manner, and the expressions of these proteins were further validated either by immunohistochemistry or by real-time reverse transcription polymerase chain reaction (RT-PCR) at transcript levels between male and female schistosomes. In summary, these findings as well as the providing of an inventory of phosphoproteins are expected to provide new insights into schistosome development and sexual maturation and then may result in the development of novel interventions against schistosomiasis. KEYWORDS: Schistosoma japonicum, phosphoprotein, signal transduction, development, sexual maturation

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INTRODUCTION Schistosomiasis, or bilharzia, is one of the great neglected diseases caused by trematode flatworms of the genus Schistosoma, currently afflicting ∼200 million people in 76 countries worldwide.1 The most prevalent species are Schistosoma mansoni, Schistosoma japonicum (S. japonicum), and Schistosoma hematobium. Among them, schistosomiasis japonica caused by S. japonicum is one of the most serious public problems in China and south Asia.2 Currently, no successful vaccine is available for schistosomiasis.3 Praziquantel (PZQ) is now virtually the only drug being used for treating the disease. However, due to its large administration and its ineffectiveness for juvenile parasites, serious concerns about the future development of tolerance or resistance of schistosomes to PZQ have already been raised.4 Therefore, it is urgent to pursue fundamental research on schistosome biology to identify effective drug targets for schistosomiasis control. Besides that schistosomes have a complex developmental cycle associated with significant morphological alternations in different hosts,5 schistosomes have an unusual biological feature, distinct sexual dimorphism between male and female © 2012 American Chemical Society

worms. The adult male and female schistosomes are permanently paired at the mesenteric veins. Male−female pairing is a prerequisite for female development, sexual maturation, and subsequent egg production. A large number of eggs produced from adult females are primarily responsible for the pathogenesis of schistosomiasis as well as disease dissemination.6 Consequently, deeply understanding the molecular basis of these processes regarding schistosome development and sexual maturation may result in the identification of novel drug targets and vaccines to retard parasite development, to block egg production, and then to lessen the pathogenesis and disease dissemination.7 Protein phosphorylation is a key reversible post translational modification modulating crucial cellular functions ranging from signal transduction, cell differentiation, and development to cell cycle control and metabolism.8 Recent advances in proteomic methodology make it possible to identify large-scale protein phosphorylation in many organisms.9 However, due to its Received: August 20, 2012 Published: December 21, 2012 729

dx.doi.org/10.1021/pr3007864 | J. Proteome Res. 2013, 12, 729−742

Journal of Proteome Research

Article

Protein Digestion in Solution

highly dynamic nature and low stoichiometry, all large-scale phosphoprotein studies are required for phosphopeptide enrichment prior to mass spectrometric analysis.9 The most common phosphopeptide enrichment methods currently use immobilized metal affinity chromatography (IMAC) and titanium dioxide chromatography (TiO2). Although both the methods are affinity chromatographies, IMAC captures phosphopeptides based on metal ions chelated on acid-coated beads, whereas the TiO2 enriches phosphorylated peptides by using its surface.9 In a previous study, we identified the phosphoproteins in schistosomula and adult paired worms by using an IMAC-based phosphoproteomic method.10 Here, we report our improved study to identify phosphoproteins in different stages and sex of S. japonicum by using a TiO2-based phosphoproteomic method. In total, 180 phosphopeptides were identified in 148 proteins in S. japonicum. In addition, 39 proteins were shown to be cophosphorylated in the schistosomula, adult females and adult males. Then, we further characterized the functions of SjHsp90, one of the cophosphorylated proteins in these three worms, by using chemical inhibitors and RNA interference. Finally, some phosphoproteins were shown to be detected in a genderspecific manner in all of the quintuplicate MS analyses, and the expressions of these proteins were further validated either by immunohistochemistry or by real-time RT-PCR at transcript levels between male and female schistosomes.

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The parasitic lysates containing 1 mg of protein were reduced for 60 min at 55 °C by adding DTT to 10 mM and carboxyamidomethylated in 55 mM iodoacetamide (IAA) for 30 min at room temperature in the dark. The solution was diluted into 1 M urea/thiourea with 50 mM ammonium hydrogen carbonate (AmBic). Then CaCl2 was added to 5 mM, and modified trypsin (Roche) was added to a final substrate/ enzyme ratio of 50:1 (μg). The trypsin digestion was performed at 37 °C for 12 h. After digestion, the peptide mixture was acidified by the addition of 5 μL of formic acid for further enrichment analysis. Phosphopeptide Enrichment

The TiO2-based phosphopeptide enrichment was performed according to the previous publication by Bai et al.11 Briefly, approximately 5 mg TiO2 beads (GL science) were suspended with 100 μL of 100% ACN and incubated at 4 °C for 5 min. The mixture was then centrifuged at 15 000g for 2 min, and the supernatant was discarded. The TiO2 beads were equilibrated with 40 μL of loading buffer (80% ACN/5% TFA/saturated phthalic acid solution) and then centrifuged, and the supernatant was discarded again. The equilibrated TiO2 beads were mixed with an in-solution digested peptide mixture, which was dissolved in ∼100 μL of loading buffer and then agitated gently for 2 h at room temperature. The TiO2 beads were centrifuged to remove the loading buffer, washed with ∼300 μL of loading buffer, and then washed with ∼300 μL of washing buffer (2%TFA/80%ACN) three times to remove the potential remaining nonadsorbed material. The phosphopeptides were eluted with 2 × 40 μL of elution buffer (ammonia solution, pH > 10.5), followed by lyophilization and MS analysis.

MATERIALS AND METHODS

Parasite Samples

All animals' care and procedures were conducted according to the guidelines for animal use in toxicology. Study protocol was approved by the Animal Care and Use Committee of the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The life cycles of S. japonicum (Anhui isolate) were maintained using New Zealand rabbits and Oncomelania hapensis at Shanghai Veterinary Research Institute of Chinese Academy of Agricultural Sciences. New Zealand rabbits were infected with approximately 1500 cercariae via the skin of the abdomen. At 14, 22, and 35 days of post infection, schistosomula and adult schistosomes were collected from infected rabbits through the hepatic portal vein by the perfusion methods, respectively. Male and female worms were manually detached for the parasites collected at 35 days of post infection. Some parasites were snap-frozen and then stored in liquid nitrogen until use, and others were directly cultured as described below.

NanoLC−ESI−MS/MS Analysis

The enriched phosphopeptides for each worm were subjected to analysis by nanoLC−ESI−MS/MS in quintuplicate as described in our previous study.10 Briefly, a splitless nanoUltra Performance Liquid Chromatography instrument (10 kpsi nano Acquity, Waters) coupled to a Synapt high-definition mass spectrometer (HDMS) with a nanospray ion source (Waters) was employed for nanoLC−ESI−MS/MS analysis. A symmetry C18 5 μm, 180 μm × 20 mm precolumn and a BEH C18 1.7 μm, 75 μm × 250 mm, analytical reversed-phase column (Waters) were used. The mobile phases were: A, 100% H2O/0.1% formic acid, and B, 100% ACN/0.1% formic acid. The phosphopeptides were separated with a gradient of 5−40% phase B over 90 min at a flow rate of 200 nL/min, followed by a rinse with 90% phase B for 10 min. The column was re-equilibrated under the initial conditions for 20 min. The lock mass was delivered from the auxiliary pump of the Nano Acquity pump with a constant flow rate of 400 nL/min at a concentration of 100 fmol/μL of [Glu1] fibrinopeptide B to the reference sprayer of the Nano Lock Spray source of the mass spectrometer. Data-dependent acquisition was performed in positive ion mode with a resolution of 10 000 at full-width-half-maximum (fwhm). MS spectra were acquired for 1 s from m/z 350 to m/z 1990. Three of the most intense doubly or triply charged precursor ions were selected to fragment. MS/MS spectra generated with CID were acquired for 2 s from m/z 50 to m/z 1990. Two MS/MS fragmentations were performed for each precursor ion. The collision energy (CE) was calculated automatically based upon peptide charge and mass to charge ratio (m/z); a dynamic exclusion window was applied to prevent the same m/z from being selected for 45 s after its acquisition.

Protein Preparations

Frozen parasite samples (∼100 mg; schistosomula, adult female schistosomes, and adult male schistosomes) were homogenized in 2 mL of lysis buffer containing 7 M urea, 2 M thiourea, 1:100 protein phosphatase inhibitor cocktails 1 and 2 (SigmaAldrich), and 5 mM DTT using a Dounce homogenizer followed by ultrasonic disruption for 12 s. This was repeated 6 times with 2 min intervals on ice. The mixtures were gently shaken at 4 °C for 30 min and then were centrifuged at 10 000g for 10 min at 4 °C to remove tissue and cell debris. Protein concentration was determined by using the BCA protein assay kit in combination with the Compat-Able protein assay preparation set (Thermo Scientific). 730



Article

Data Analysis and Validation

sequences), Mus musculus (73 274 sequences), Drosophila melanogaster (35 349 sequences), Caenorhabditis elegans (25 324 sequences), and Saccharomyces cerevisiae (7504 sequences), respectively. BLAST alignment was classified to be significant if its corresponding E values were lower than 1 × 10−5. ClustalW was used for protein-sequence alignments, and global alignments between homologous proteins were generated by the Needle software, 17 based on the Needleman−Wunsch algorithm.17,18 Information on conserved phosphorylation sites in the five eukaryotic organisms was extracted from the annotation of the Swiss-Prot database. Stringe (http://stringdb.org/) was used to predict protein−protein interaction.

Raw data were processed using ProteinLynx Global Server 2.0.5 from Waters (smooth 2/3 Savitzky Golay and center 4 channels/80% centroid), and the resulting MS/MS data set was exported in Micromass pkl format. The pkl file was searched against the S. japonicum protein database (LSBI-Sjr, 12 657 sequences; 4 929 382 residues) downloaded from the Chinese National Genome Center at Shanghai (http:// lifecenter.sgst.cn/schistosoma/) using the in-house Mascot search engine (version 2.2) and the NCBInr protein sequence database (NCBInr 20100108: 10 291 680 sequences; 3 511 877 860 residues) using the Mascot search engine (http://www. matrixscience.com/cgi/), respectively. The search parameters used were: enzyme was trypsin; two missed cleavage sites were allowed; carbamidomethyl (C) was set as fixed modification and acetylation (protein N-terminal); oxidation (M), phosphor (ST), and phospho (Y) were set as variable modifications; MS peptide tolerance was 15 ppm, and MS/MS tolerance was 0.2 Da, respectively. Data for each nanoLC−ESI−MS/MS run were searched individually. The false discovery rate (FDR) was determined by a search of the reversed protein database and calculated from the Mascot decoy functions.12 Phosphopeptides were considered to be identified if a Mascot individual ion score was higher than 27 for searching the LSBI-Sjr database or than 54 for searching the NCBInr database (p < 0.05). Phosphopeptides assigned by Mascot were further inspected by manually checking their respective MS/MS spectra. Validation was carried out on the basis of the occurrence of at least four consecutive y or b, ions and an intense signal should be assigned to ions produced by fragmentation at the peptide bond N-terminal to proline if proline was present in the sequence. Phosphopeptides being repeatable detection in at least 3 out of 5 independent MS analyses were further annotated. For phosphorylation site determination, we report the Mascot Delta Score (MD-score) calculated by the difference between the top two Mascot ion scores of alternative phosphorylation sites in the same peptide sequence.13 To obtain MD scores for those phosphopeptides with more than two possible phosphorylation sites, we performed a second database search against both the S. japonicum protein database (LSBI-Sjr, 12 657 sequences; 4 929 382 residues) and the NCBInr protein database (NCBInr 20121003, 20 883 892 sequences; 7 158 765 844 residues) using Mascot Daemon (version 2.4) with the exact same search parameters described above. If the delta score is greater than 9, the top ranked phosphorylated site is considered confidently determined. For a delta score less than 9, the phosphorylated site assignment is considered ambiguous.14 Then, the second ranked phosphorylated site was marked out. The calculated delta scores are listed in the Supporting Information, Tables 1, 2, and 3.

Validation of Identified Phosphoproteins by Western Blot

Schistosome protein lysates (adult males and adult females) were prepared as described above and then quantified by the Bradford method. Equal amounts of protein lysates for adult males and adult females were run in 10% sodium dodecylpolyacrylamide gel electrophoresis and then electrotransferred onto polyvinylidene difluoride membranes (Sino-American Biotechnology, China). Nonspecific protein−protein interactions were blocked using 5% nonfat dry milk in PBS (pH = 7.4) containing 0.1% Tween 20 (Sigma-Aldrich). The membrane was incubated for 1 h at room temperature in primary antibodies (1:800 dilution): rabbit anti-Cdc37 (Phospho S13) (Abcam) and antialpha tubulin (Beijing Zhongshan Biotechnology, China) diluted 1:1000 in blocking buffer and washed five times for 5 min each in 0.1% Tween-20 PBS. The secondary antibodies (Kexin Bioscience, China) were horseradish peroxidase conjugated goat anti[rabbit immunoglobulin (Ig)G]. They were diluted to 1:5000 in PBS and incubated with the membrane for 1 h. The membrane was developed using Immobilon Western Kit according to the manufacturer’s instrument (Millipore). Real-Time RT-PCR Analysis of Hsp90 Transcripts in the Different Stages of Schistosomes

The different developmental stages of schistosomes were collected from the rabbits infected with S. japonicum at day 7, 14, 21, and 35 postinfection. Eggs and cercariae were obtained according to the previous publication.19 Total RNAs were isolated from different developmental stages of schistosomes using Trizol (Invitrogen) following the manufacture’s instructions, and real-time RT-PCR was performed. Briefly, total RNA was added to a reverse-transcript transcription reaction containing multi-Scribe reverse transcriptase reagent and random hexamers (Eppendorf), incubated at 25 °C for 10 min, 48 °C for 30 min, and 95 °C for 5 min. Real-time RT-PCR was performed using 1 μL of cDNA in a final volume of 25 μL containing 12.5 μL of 2× Premix Ex TaqII (TaKaRa), 10.5 μL of H2O, and 1 μL (10 μM) of each specific primer for SjHsp90 (forward primer: GTT ACG AAG AAT AAT GAC GAT GAC; reverse primer: TAG GGT AGT TGA TAA ATT GAG AGT G) in Mastercycler ep realplex (Eppendorf) using the following thermal cycling profile: 95 °C for 30 s, followed by 40 cycles of amplification (95 °C for 5 s, 62 °C for 30 s). The primers of nicotinamide adenine dinucleotide dehydrogenase (NADH) (forward primer: CGA GGA CCT AAC AGC AGA GG; reverse primer: TCC GAA CGA ACT TTG AAT CC) were used as an internal control for normalization. In the present study, quantification of the transcript of SjHSP90 relative to NADH was calculated using the following equation:20 N = 2−ΔCt, ΔCt = Cttarget − CtNADH.

Bioinformatic Analyses

Blast2GO was used to predict biological processes of S. japonicum phosphoproteins.15 ClustalW (www.ebi.ac.uk/ clustalw) was used to determine the redundant phosphoproteins for each data set, and the Scansite program (http:// scansite.mit.edu) was used for analyzing consensus sites for selected kinases and protein-docking motifs.16 The orthologs of S. japonicum phosphoproteins in five eukaryotic organisms ranging from yeast to human were identified, if available, using BLASTP (http://ca.expasy.org/tools/blast/) by imputing the full-length amino acid sequences against UniProtKB protein databases as defined the subsection of Homo sapiens (110 527 731



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RNA Interference of SjHsp90

between mock- and the Hsp90 inhibitors treated group by using a student’s t test.

Three siRNA duplexes targeting SjHsp90 (siRNA-371 sense: GUG CAG ACA UCU CGA UGA UTT, antisense: AUC AUC GAG AUG UCU GCA CTT; siRNA-1518 sense: CGG CUU UGA AGU GUU GUA UTT, antisense: AUA CAA CAC UUC AAA GCC GTT; siRNA-1841 sense: CCA GUA CUA UGG GCU AUA UTT, antisense: AUA UAG CCC AUA GUA CUG GTT) and one negative control siRNA (sense: UUC UCC GAA CGU GUC ACG UTT, antisense: ACG UGA CAC GUU CGG AGA ATT) duplex were designed, chemically synthesized, and annealed in Shanghai GenePharma (China). Then each dsRNA duplex (3 μg per shot) was electroporated (125 V, 20 ms, 1 pulse in 200 μL of RPMI 1640 media) into cultured schistosomes (22 days). Then, the parasites were transferred into a 12-well cell culture plate containing 2 mL of complete RPMI-1640 media supplemented with 2 g/L of glucose, 0.3 g/L of L-glutamine, 2.0 g/L of NaHCO3, 15% fetal bovine serum (heat inactivated), and 5% pen/strep (10 000 units of penicillin and 10 mg/streptomycin in 0.9% NaCl). At 48 or 96 h of post electroporation, the parasites were collected, and the total RNA was isolated for real-time RT-PCR analysis. Real-time RT-PCR was performed as described in the section of “Real-Time RTPCR Analysis of Hsp90 Transcripts in the Different Stages of Schistosomes”. The other primers used for real-time RT-PCR analyses were listed in the Supporting Information, Table 11.

Caspase 3/7 Assay

Caspase activities in parasites treated with 17 AAG or celastrol were carried out using a Caspase Glo 3/7 assay Kit (Promega), and the fluorescence intensity was measured by a luminometer (Berthold). The luciferase activity was normalized by the protein concentration as described above. Statistical analysis was performed to compare the difference between mock- and the Hsp90 inhibitors treated group by using a student’s t test. Schistosomiasis Animal Model

Four- to six-week-old male Balb/c mice (mean weight 25 ± 2 g) were purchased from the Shanghai Center for Experimental Animals and randomly divided into two groups: control treatment and drug treatment. Each mouse was challenged with 40 ± 5 normal S. japonicum cercariae by abdominal skin penetration. In Vivo Hsp90 Inhibitor Injection in an Animal Model

Celastrol (Sigma-Aldrich) was dissolved in the vehicle 10% DMSO, 70% Cremophor (Sigma-Aldrich)/ethanol (3:1), and 20% PBS.21 For experiments #1 and #2, starting at 7 days postinfection, each mouse in the control treatment and drug treatment group was injected by i.p. with 0.1 mL of the drug vehicle alone and the drug vehicle containing the celastrol (3 mg/kg), respectively. The mice were repeatedly injected at 10, 11, 15, 18, and 21 days postinfection, respectively. At 24 days postinfection, the parasites in each mouse were gently perfused using sterile PBS, and the number of worms in each mice was microscopically counted. The number of mice was increased in experiment #2. For experiment #3, the mice were administrated with the same dosages of celastrol in experiment #1. At 35 days postinfection, the parasites in each mouse were gently perfused, and the collected worms were counted under microscope as described above. The number of eggs in the liver of each mouse was counted as described below. Briefly, 0.5 mg of liver tissue from each infected mouse was homogenized in 10 mL of 5% NaOH. The mixture was incubated at 56 °C for 1 h. An average of three counts per 20 μL mixture was taken to estimate the number of eggs, and this was converted to eggs per gram (EPG). Statistical analysis of data was performed to compare the difference between the control-treated and drug-treated group in each experiment using a student’s t test.

Schistosome Culture and in Vitro Hsp90 Inhibitors Treatment

The schistosomes (22 days) were cultured in a 12-well flat bottom plate containing 2 mL of complete RPMI-1640 media supplemented with 2 g/L of glucose, 0.3 g/L of L-glutamine, 2.0 g/L of NaHCO3, 15% fetal bovine serum (heat inactivated), and 5% pen/strep (10 000 units of penicillin and 10 mg/ streptomycin in 0.9% NaCl) with 17-allylamino-17-demethoxygeldanamycin (17 AAG) (Merck) (5 or 10 μM) or celastrol (Sigma-Aldrich) (1.5, 3, or 6 μM) and DMSO (negative control) and incubated in a humidified 5% CO2 chamber at 37 °C. The physical activity of parasites (e.g., feeding behavior, movement, and viability) was observed and recorded at the indicated times. Fresh culture media and 17 AAG were added after 18 and 36 h. For celastrol treatment, the parasites were incubated with culture media and celastrol for 48 h. These experiments were performed in triplicate and repeated in at least three independent tests, respectively. Statistical analysis in parasitic death was performed by a student’s t test.

Real Time RT-PCR Analyses of the mRNAs Encoding the Differentially Identified Phosphoproteins being Highly Repeatable between Male and Female Schistosomes

Kinase Glo Luminescent Kinase Assay

The kinase activities of parasites were carried out by using the Kinase Glo luminescent kinase assay (Promega). Briefly, at 72 h of post treatment of 17 AAG or at 48 h of post treatment of celastrol, the parasites were collected, and the protein lysates were prepared with the lysis buffer containing 40 mM Tris-HCl (pH7.5), 20 mM MgCl2, and 0.1 mg/mL of BSA. The protein concentration of the lysates was determined using a bicinchoninic acid protein assay kit (Sangon, China). Then, the protein lysates (50 μL) were mixed with equal amounts of Glo kinase reaction substrate containing 1 μM ATP (Takara, China). The mixtures were incubated at room temperature for 10 min, and the fluorescence intensity was measured by a luminometer (Berthold). The protein concentration of each reaction solution was determined using the Pierce BCA protein assay kit and Compat-Able protein assay preparation reagent set (Thermo Scientific) for normalizing luciferase activity. Statistical analysis was performed to compare the difference

Total RNAs of adult males (35 days) and adult females (35 days) of S. japonicum were isolated using Trizol (Invitrogen), respectively. The isolated total RNA was used to perform the reverse transcription reaction as described above. Real-time RTPCR was performed using the same volume as described above by replacing with specific primers for the identified phosphoproteins (the primers used were listed in the Supporting Information, Table 11) in Mastercycler ep realplex (Eppendorf) and run under the following thermal cycling profile: 95 °C for 30 s, followed by 40 cycles of amplification (95 °C for 5 s, 60 °C for 30 s). Nicotinamide adenine dinucleotide dehydrogenase (NADH) was also used as an internal control for normalization. The relative mRNA expression was calculated using the ΔCt method as described above. Statistical analysis was performed to compare the 732



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Figure 1. Overview of schistosome phosphoproteomic analysis. (A) The stages of parasites used for phosphoproteomic analysis. Schistosomula (14 days) and adult schistosomes (35 days) were collected from the rabbits infected with cercariae at 14 and 35 days of postinfection, respectively. The adult males and adult females were manually separated. (B) Experimental workflow for phosphopeptide enrichments and nanoLC−ESI−MS/MS analyses and the results of database searching and bioinformatic analyses. *Annotated phosphopeptides means that only a phosphopeptide identified consistently in at least 3 out of 5 independent MS analyses was considered to be annotated.

difference between male and female schistosomes by using a student’s t test.

antibody with preimmune mice serum. After three 5 min washes with PBS containing 0.02% (v/v) Tween-20, the sections were treated with biotinylated goat antimice antibody for 20 min at room temperature, followed by three additional 5 min washes with PBST. Then the sections were incubated with streptavidin-horseradish peroxidase for 20 min at room temperature, followed by repeated washes, as described above. The reaction product was visualized with 3,3diaminobenzidine (DAB) at room temperature for 5 min. Sections were counterstained with hematoxylin for 30 s and rinsed with tap water, immediately dehydrated by sequential immersion in gradient ethanol and xylene, then mounted with permount on coverslips. Images were captured under a light microscope equipped with a digital camera.

Immunohistochemistry

Immunohistochemistry was performed to determine the distribution of SjWD40 protein in adult worms of S. japonicum according to standard protocol. Briefly, S. japonicum adult worms (35 days) were fixed in 4% formaldehyde solution and maintained at 4 °C. The worm sections were prepared by the paraffin section method. Then the sections were deparaffinized with xylene and rehydrated through gradient ethanol immersion. Endogenous peroxidase activity was quenched by 0.3% (v/v) hydrogen peroxide in methanol for 20 min, followed by three 5 min washes with PBS. The sections were then blocked with 10% (v/v) normal rabbit serum in PBS for 1 h, followed by overnight incubation at 4 °C with the serum against SjWD40 diluted (1:1000) in PBS containing 3% (w/v) BSA. Negative control was performed by replacing the primary 733



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Article

RESULTS

Table 1. Scansite Proteins at High Stringency (0.2%) and Low Stringency (5%) for Kinase Phosphorylation and Binding Motifs from the Data Sets of S. japonicuma

Experimental Design and Analytical Workflow

The protein lysates of schistosomula (14 days) (SS), adult females (35 days) (SF), and adult males (35 days) (SM) (Figure 1A) of S. japonicum were prepared and then were digested with trypsin. The phosphopeptides in the digested protein lysate of each worm were enriched by TiO2 bead and then were subjected to analysis by nanoLC−ESI−MS/MS in quintuplicate. Each nanoLC−ESI−MS/MS run was used to search the S. japonicum protein database (LSBI-Sjr) with inhouse Mascot server and to search the NCBInr database using Mascot via the Internet, respectively. The FDR is 0.00% and 0.34% for searching the LSBI-Sjr database and NCBInr database, respectively, in the present study. All the phosphorylated proteins and their corresponding phosphopeptides assigned by Mascot in the quintuplicate MS analyses for one sample by searching the same database were further classified into one data set to count the number of phosphopeptides (Figure 1B). To remove potential redundant phosphopeptides and corresponding phosphoproteins identified between the two databases (LSBI-Sjr and NCBInr), multiple alignments of the phosphoproteins identified among the two databases were carried out by using ClustalW. If a pair of proteins is >65% sequence identity, additionally, this sequence pair presents the same phosphopeptide and identical phosphorylation site. The phosphoprotein and its corresponding phosphopeptides associated with higher Mascot score were referred to the phosphoprotein identified, and the other one associated with the lower score was considered to be a redundant protein/isoform. Upon the removal of redundant phosphopeptides, we totally identified 232 distinct phosphopeptides in 184 proteins in SS, 138 in 113 proteins in SF, and 198 in 160 proteins in SM, respectively (Figure 1B). All phosphopeptides identified in quintuplicate MS analyses for SS, SF, and SM were provided in the Supporting Information (Table 10). Only the phosphopeptides detected three or more times within five replicates were annotated for further analyses. Finally, 127 distinct phosphopeptides in SS, 75 in SF, and 102 in SM met the criterion, and these phosphopeptides were, respectively, matching in 110 phosphoproteins in SS, 69 phosphoproteins in SF, and 91 phosphoproteins in SM (Figure 1B).

lowb

high SS

SF

SM

Phosphoserine/Threonine Binding Group 14−3−3 Mode 1 2 1 1 Basophilic Serine/Threonine Kinase Group Akt Kinase 2 2 1 Protein Kinase A 2 2 4 Calmodulin dependent Kinase 2 2 1 1 Clk2 Kinase 1 1 0 PKC alpha/beta/gamma 0 0 0 PKC delta 0 0 0 PKC zeta 0 0 0 PKC epsilon 2 1 3 DNA Damage Kinase Group DNA PK 2 1 1 ATM kinase 0 0 0 Acidophilic Serine/Threonine Kinase Group Casein Kinase 2 4 2 0 GSK3 Kinase 0 0 0 Proline-Dependent Serine/Threonine Kinase Group Cdk5 Kinase 0 0 1 Cdc2 Kinase 0 0 1 Erk1 Kinase 2 0 0

SS

SF

SM

23

14

22

15 20 10 11 4 5 5 9

10 15 9 9 5 2 4 5

13 21 12 9 4 3 3 9

14 9

11 8

7 5

25 7

18 3

14 4

25 19 17

14 12 7

22 29 10

a

Only the phosphopeptides with MD score >9 were analyzed by Scansite. bCut-off frequency value is 5 for the result of prediction at low stringency.

(casein kinase 2) as well as the potential binding sites for 14− 3−3 proteins were most predicted in the three worms (Table 1). The details of putative kinases and certain phosphorylationdependent binding motifs for all of the identified S. japonicum phosphorylated proteins are presented as Supporting Information Table 4 (SS), Table 5 (SF), and Table 6 (SM). Evolutionarily conserved phosphosites are often functionally relevant among related species.22 By alignment of the phosphoproteins identified with corresponding orthologs from yeast, worm, fly, mouse, and human, we identified 24 phosphosites conserved for the orthologs from at least two of five species investigated, which are matching in 24 proteins (Table 2). Among them, eight phosphosites were also identified in our previous study by using an IMAC-based phosphoproteomic analysis. The details of evolutionarily conserved phosphosites in S. japonicum and, if available, their corresponding phosphorylation sites of the orthologs from H. sapiens, M. musculus, D. melanogaster, C. elegans, or S. cerevisiae are listed in the Supporting Information, Table 7.

Manually Scanned Motif and Evolutionarily Conserved Phosphorylation Sites

In total, 180 phosphopeptides were annotated in the present study, which were matching in 148 proteins. The complete lists of the proteins and their corresponding phosphopeptides as well as their MS/MS spectra are presented in the Supporting Information as Tables 1, 2, and 3 and Figures 1, 2, and 3 for SS, SF, and SM, respectively. Overall, the distribution of phosphorylations among serine, threonine, and tyrosine is 90.1%:9.4%:0.5%, which is similar to our previous results from IMAC-based analysis.10 Then, we used the Scansite program to analyze the phosphosites determined confidently (MD score >9). Generally, over 70% of experimentally identified phosphosites were able to be successfully predicted by using the Scansite program when applying the default setting. As summarized in Table 1, sites phosphorylated by proline-directed kinases (Cdk5 kinase and Cdc kinase), basophilic serine/threonine kinase (Akt kinase A and Protein Kinase A), DNA damage kinase group (DNA PK), and Acidophilic serine/threonine kinase group

Validation of the Identified Cdc37 by Western Blot

Cell division cycle 37 homologue (Cdc37) was identified to be phosphorylated on serine 15 (Ser15) in S. japonicum, and the phosphosites were shown to be evolutionarily conserved with the orthologs in mammals (Figure 2A). Then, we used the antibody against human Cdc37 (Ser15) to validate the phosphorylated Cdc37 identified in S. japonicum by Western blot. As shown in Figure 2B, the band with molecular weight of expectation (approximately 45 kDa) was able to be recognized. The MS/MS spectra of Ser15 of S. japonicum Cdc37 were shown in Figure 2C. 734



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Table 2. List of Evolutionarily Conserved Phosphorylation Sites of the Proteins Identified in S. japonicum peptidesb

protein descriptions and protein IDs Signal molecules and enzymes Proliferation-associated protein 2G4 (CAX74794.1a) Heat shock protein 60 (CPRT0000007774) Cysteine string protein (AAP05964.1)a Importin 7 (CPRT0000012199)a Heat shock protein 90 (CPRT0000004202)a Glyceraldehyde 3-phosphate dehydrogenase (CPRT0000002428) Ferritin-1 heavy chain (CPRT0000000687) Beta chain spectrin (CPRT0000001619) Alkaline phosphatases (CPRT0000000345) Programmed cell death-involved protein (CPRT0000007240) Kinases cAMP-dependent protein kinase type II regulatory subunit (AAW27091.1)a Glycogen synthase kinase 3 (CPRT0000007860) cAMP-dependent protein kinase type II-alpha regulatory subunit (CPRT0000010982)a cAMP-dependent protein kinase R2 (CAX73605.1)a Cdc37 cell division cycle 37 homologue (CPRT0000002152) Protein Translation Regulation Eukaryotic translation initiation factor 4E binding protein 2 (CPRT0000002185) Eukaryotic translation initiation factor 2 alpha subunit (CPRT0000006370) Eukaryotic translation initiation factor subunit C (AAW25812.1) RNA Transcript/Protein Transport/DNA Binding Nascent polypeptide-associated complex subunit alpha (AAW26771.1) Protein DEK (CPRT0000000835)a Scaffold attachment factor B (CPRT0000001365) Heterochromatin protein 1 (CPRT0000009445) RNA binding motif protein 39 (CPRT0000003234)a Calcium-regulated heat stable protein 1 (CPRT0000006031)

worms

M.sDQEsDAEQDVLDDTVVNKYK.M R.NVIIESSWKsPK.I R.KLsTSGESLFHVLR.V K.ALGsDEDEVDEESVR.Y R.TKEVsDDEAEKEETKNESEEAEDKPK.V K.VINDNFEIVEGLMTTVHSFTAtQK.T R.VGTGLGEYTFDKETLHGEsD.R.AAtLPSGVAPESQVTSSSSASGK.K R.LTTDsGSAATAFLSGAK.G R.FIsETDSVYR.T

SS SM SS, SM, SF SS, SM, SF SS, SM, SF SF, SM SF SS, SF SF, SM SS

R.RQsVAAESFDPEKDSDDNNEEER.Q K.EMVPGTANVSyISSR.Y K.ANEsDEDEDEEPMPMPPQR.V R.RHsVAAESFNPATVNDLEPVVHPK.S M.SCLNYSKWDHIEVsDDEDDTHPNIDTPSLFR.W

SS, SM SS, SM, SF SS, SM, SF SF,SM SS, SM, SF

R.NsPAARSPPPDMIFLPEKAPNSDDK.T K.NIGGMILHsELSR.R R.IESRPLHLssDEEDIKR.I

SM SM SS

K.tVIAEESEDDEEPDASGLSEKDIELIMQQAGVSR.S K.KEFDsDEDVPLSNLTSTK.V R.sRsPPIFQSVQHRPEILEYGHR.S K.NSREsEEsGGEDEFQVEK.I R.QKsPELSPEER.D R.NRTEsQSESASTGEHGVGR.I

SS SS, SF SS,SM SS SF SM

a

Indicated that a phosphosite was also identified by an IMAC-based phosphoproteomic method in our previous study. bThe conserved phosphosites are in italics.

Phosphoproteins Coidentified in Schistosomula, Adult Males, and Adult Females

Hsp90 May Play an Important Role in Schistosome Development by Directly or Indirectly Regulating other Signaling Molecules

Key molecules coexpressed in different stages and sex of schistosomes may imply their universal functions for regulating schistosome development and likely become an ideal target for schistosomiasis control without stage bias. Comparison of the phosphoproteins identified among schistosomula, adult males, and adult females indicated that there were 39 proteins codetected in these three worms (Supporting Information, Table 8). These proteins were predicted to mainly be involved in cellular process, metabolic process, localization, biological regulation, etc. (Figure 3). It is worthy of note that these proteins included several signal molecules and kinases such as epidermal growth factor receptor pathway substrate 8 (CAX73031.1), importin 7 (CPRT0000012199), cysteine string protein (CSP, AAP05964.1), 14−3−3 protein (CPRT0000002347), heat shock protein 90 (Hsp90, CPRT0000004202), cAMP-dependent protein kinase type IIalpha regulatory subunit (PRKAR2A, CPRT0000010982), glycogen synthase kinase 3 (GSK3, CPRT0000007860), thymidylate kinase (CPRT0000005313), and cell division cycle 37 homologue (Cdc37, CPRT0000002152) (Supporting Information, Table 8). Among them, several phosphoproteins are showed to be phosphorylated in an evolutionarily conserved manner such as CSP, importin 7, Hsp90, GSK3, Cdc37, and PRKAR2A (Table 2).

Among the phosphoproteins codetected in schistosomula, adult females, and adult males, Hsp90 is now known to be a key mediator that participates in many biological processes in mammals.23 Real-time RT-PCR analyses indicated that S. japonicum Hsp90 predominantly transcribed in the stages associated with a final host such as schistosomula and adult worms (Figure 4A), suggesting that SjHsp90 may be involved in the regulation of schistosome development and parasitism in a final host. It has been demonstrated that Hsp90 plays a central role in signal transduction by interacting with its cochaperones or client proteins.23 On the basis of the human string protein− protein interaction database,24 9 out of 39 coidentified phosphoproteins in the three worms were bioinformatically predicted to directly or indirectly interact with SjHsp90 (Figure 4B). To further validate this prediction, we used small interfering RNAs (siRNAs) to silence SjHsp90 and then investigate the effect of SjHsp90 silencing on the expressions of these putative interacting proteins at the transcript levels. Briefly, three different siRNA duplexes were designed to target the different regions of SjHsp90 (starting site, respectively: 371−389, 1518−1536, and 1841−1859). Then, these three siRNA duplexes as well as one negative control duplex were electroporated into in vitro cultured S. japonicum schistosomes (22 days). At 48 h of post electroporation, the silencing effect of SjHsp90 was determined by using real-time RT-PCR. As 735



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implying that SjHsp90 and/or its mediated pathways may play an important role in the regulation of schistosome development in S. japonicum (Figure 4D). Hsp90 is Likely a Potential Target against Schistosomiasis

Hsp90 has been considered to be a potentially therapeutic target for several diseases due to its essential role in many biological processes.23 To further investigate the functions of SjHsp90 and test a potential targeting SjHsp90 against schistosomiasis, we in vitro cultured schistosomes (22 days), and then the parasites were treated with a Hsp90 inhibitor, 17(allylamino)-17-demethoxygeldanamycin (17AAG). The effect of SjHsp90 inhibition on parasitic death was observed and calculated at 24, 48, and 72 h of post treatments, respectively. As shown in Figure 5A, the significant mortality (45%) was observed at 48 h of post treatment in a higher concentration of 17 AAG (10 μM), while 99% of control remains variable. Then the mortality was further increased to 55% at 72 h of post incubation, while 80% of control remains variable under the same dosage. Both a lower dosage of treatment (5 μM) and a high dosage of treatment (10 μM) achieved similar mortality at 72 h of post incubation with 17 AAG. To corroborate this result, we further used another Hsp90 inhibitor, celastrol, which is isolated from the Chinese plant Tripterygium wilfordii Hook F,25,26 to treat in vitro cultured schistosomes. Similar results were also obtained with celastrol treatment in the cultured schistosomes (Figure 5A). To gain valuable insight into parasitic death resulting from SjHsp90 inhibition, we measured the kinase activities of the parasites treated with 17 AAG or celastrol using Kinase Glo luminescent assay. As shown in Figure 5B, the luciferase activity increased in the parasites treated with these two Hsp90 inhibitors significantly, indicating that the kinase activities significantly decreased (Figure 5B). In addition, the activities of caspase 3/7 also significantly reduced as determined by the Caspase Glo 3/7 assay (Figure 5C), suggesting that the parasitic death was independent of caspase activation. To further investigate the in vivo effect of SjHsp90 inhibition on parasite burden in a final host, starting at 7 days of post infection the mice infected with S. japonicum cercariae were systematically administrated with celastrol by intraperitoneal injection. At 24 days of post infection the mice were executed, and the worms were perfused. The number of worms in each mouse was counted under microscopy. As shown in Figure 5D, significant worm burden reductions (57%) were achieved in the mice upon the systemic administrations of celastrol (experiment #1). To further corroborate the result, we increased the number of mice to further repeat the experiment. Similarly, significant worm burden reductions were also observed in the repeated experiment, even increasing the number of mice (Figure 5D, experiment #2). Then, we further determined whether in vivo SjHsp90 inhibition can result in the reduction of liver egg burden in mice. Following administration with celastrol applied at the same dosage and under the same schedule, the mice were executed at 35 days of post infection, and the worms and liver eggs were counted. As shown in Figure 5D, significant reductions in both worm burden (58%) and liver egg (67%) were observed (experiment #3). In addition, there were no significant differences in body weights of the mice between administration of celastrol and PBS solution (data not shown).

Figure 2. Validation of the identified phosphoproptein by Western blot. (A) Alignment of partial amino acid sequences of SjCdc37 containing phosphorylated residue conserved from the species (human, Q16543; mice, Q61081; Fly, Q24276). (B) Western blot analysis of phosphorylated Cdc37 (Ser15) in the protein lysates of adult males and adult females of S. japonicum. (C) The MS/MS spectrum of the phosphopeptide of SjCdc37.

Figure 3. GO analysis of the putative biological processes of the phosphoproteins codetected between schistosomula, adult males, and adult females.

shown in Figure 4C, the best silencing effect was obtained with siRNA-371. Additionally, along with the best silencing of SjHsp90 by applying the described dosage of the siRNA, the expressions of GSK3, Cdc37, and YWHAE were shown to be concordant with the reduction of SjHsp90 at transcript levels, 736



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Figure 4. Analysis of the expressions of SjHsp90 and its putative interactors. (A) Real-time RT-PCR analysis of the expressions of SjHsp90 in the different stages of S. japoniucm. Data illustrate representative experiments and show the mean and standard error derived from triplicate experiments. (B) The putative direct or indirect interactors of SjHsp90 among the phosphoproteins codetected in schistosomula, adult females, and adult males (NUDC, Nuclear distribution gene c homologue; BCAP31, B-cell receptor-associated protein; Cdc37, cell division cycle 37 homologue; YWHAE, 14−3−3 family protein; CANX, Calnexin; PRKAR2A, cAMP-dependent protein kinase type II-alpha regulatory subunit; PFKL, Phosphofructokinase; APAK9, Sjchgc09590 protein; GSK3, Glycogen synthase kinase 3). A full line indicates direct interaction, dotted line indicates indirect interaction. (C) Effect of RNA interference on SjHsp90 at transcript levels by three different siRNA duplexes. Data illustrate representative experiments and show the mean and standard error derived from triplicate experiments. * means P ≤ 0.05 and ** means P ≤ 0.01 (student’s t test, irrelevant siRNA treatment vs SjHsp90 siRNA treatment). (D,) The expressions of some signal molecules were concordant with the reduction of SjHsp90 at transcript levels in the schistosomes electroporated with SjHsp90 siRNAs. Data illustrate representative experiments and show the mean and standard error derived from triplicate experiments. * means P ≤ 0.05 and ** means P ≤ 0.01 (student’s t test, irrelevant siRNA treatment vs SjHsp90 siRNA-371 treatment).

Different Phosphoproteins Identified Being Highly Repeatable between Adult Male and Adult Females

expressed the recombinant WD40 protein in a prokaryotic system (Supporting Information, Figure 4B). Then, the purified WD40 protein was immunized to the mice for raising polyclonal antibodies (Supporting Information, Figure 4C). By applying the polyclonal antibodies, we identified that the WD40 protein is highly expressed in male schistosomes (Supporting Information, Figure 4D) and significantly locates in the testis of schistosomes (Figure 6C). In addition, no significant staining was observed in adult female schistosomes apart from the gut where the digested host hemoglobin can react with DAB (the chromogenic substrate used in the immunohistochemical analysis) (Supporting Information, Figure 4E).

In the present study, we noted that some phosphopeptides (3 phosphopeptides for adult females and 13 phosphopeptides for adult males) were able to be detected in a specific gender in all of the quintuplicate MS/MS analyses, but they were unable to be detected once in the corresponding gender (Supporting Information, Table 9). Differential phosphopeptide identification may imply the abundance of this phosphopeptide. So, we hypothesized that these phosphopeptide are likely to be more abundant in the worms in which it was identified. Due to a lack of specific antibodies against these phosphopeptides matching proteins, alternatively, we used real-time RT-PCR to determine the mRNA transcripts of the genes encoding these proteins between adult males and females in S. japonicum. As shown in Figure 6A and 6B, the predominant mRNA transcript of the gene encoding a protein correlated with the phosphopeptide identified in the worms. It is worthy of note that WD40 protein was specifically detected in adult males of S. japonicum. Bioinformatic analysis indicated that the WD40 protein is a transmembrane protein (Supporting Information, Figure 4A). In other eukaryotic species, the WD40 protein is involved in a wide variety of functions such as signal transduction, pre-mRNA processing, and cytoskeleton assembly.27 Therefore, we cloned and

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DISCUSSION In the present study, we performed the first attempt to use a TiO2-based phosphoproteomic method to identify phosphoproteins in different stages and sex of S. japonicum. In total, 180 phosphopeptides were identified in S. japonicum, which were matching in 148 proteins. Among them, 39 phosphoproteins were shown to be cophosphorylated in schistosomula (14 days), adult females (35 days), and adult males (35 days) of S. japonicum. To increase the reliability of phosphoprotein identification, only a phosphopeptide detected in at least 3 out of 5 737



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Figure 5. Effect of SjHsp90 inhibition on kinase activity and worm death as well as worm burden reduction in animal model. (A) Effect of SjHsp90 inhibition by 17 AAG or celastrol on worm death in vitro culture studies. Data illustrate representative experiments and show the mean and standard error derived from triplicate experiments. * means P ≤ 0.05 and ** means P ≤ 0.01 (student’s t test, Hsp90 inhibitor treatment vs mock). (B) Effect of SjHsp90 inhibition by 17 AAG or celastrol on the kinase activity in the worms at 72 h of post treatment. Data illustrate representative experiments and show the mean and standard error derived from triplicate experiments. * means P ≤ 0.05 and ** means P ≤ 0.01 (student’s t test, Hsp90 inhibitor treatment vs mock). (C) Effect of SjHsp90 inhibition by 17 AAG on caspase 3/7 activities in the worms at 72 h of post treatment. Data illustrate representative experiments and show the mean and standard error derived from triplicate experiments. * means P ≤ 0.05 and ** means P ≤ 0.01 (student’s t test, Hsp90 inhibitor treatment vs mock). D. In vivo effect of SjHsp90 inhibition on the reductions of worm and egg burden in the animal model.

independent MS analyses was considered to be annotated. By applying this criterion, we found that (1) over 72% of experimentally identified phosphorylation sites were able to be predicted by the Scansite program when applying the default settings and (2) approximately 30% of the phosphopeptides identified in the present study (41 out of 127 phosphopeptides in schistosomula; 22 out of 75 phosphopeptides in adult females; 26 out of 102 phosphopeptides in adult males) were also able to be identified in the previous study by an IMACbased phosphoproteomic method. Consequently, these observations further indicated that our phosphorylation identification was highly reliable and also support the notion that the combination of TiO2 with IMAC for phosphopeptide enrichment could increase the data set of phosphoprotein identification.28 The phosphorylated status of a protein may indicate its function and/or the pathways involved for regulating specific

biological processes. In the present study, 39 proteins were identified to be cophosphorylated in different stages and sex of S. japonicum (Supporting Information, Table 8), suggesting that these proteins may play important roles in the regulation of schistosome development. Some phosphosites of these proteins were shown to be phosphorylated in an evolutionarily conserved manner such as GSK3, Csp, CDC37, Hsp90-alpha, PRKAR2A, and importin 7 (Table 2). Knowledge of the functions of the phosphosites in the orthologs of other organisms may provide perspective for understanding the functions of these phosphorylations in S. japonicum. However, most of these phosphosites remain poorly characterized so far. Among them, it has been demonstrated that phosphorylation of the tyrosine residue of GSK3 in humans leads to stimulating GSK3 activity and down-regulating beta-catenin activity.29,30 Consequently, it is likely that tyrosine 286 phosphorylation of 738



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Figure 6. Real-time RT-PCR analyses of the mRNA transcripts of the phosphorylated proteins identified being highly repeatable in a gender-specific manner and immunohistochemistry analysis of male specific WD40 protein. (A) Real-time RT-PCR analyses of the mRNA transcripts of the phosphorylated proteins specifically identified in female schistosomes. Data illustrate representative experiments and show the mean and standard error derived from triplicate experiments. * means P ≤ 0.05, and ** means P ≤ 0.01 (student’s t test, female vs male). (B) Real time RT-PCR analyses of the mRNA transcripts of the phosphorylated proteins specifically identified in male schistosomes. Data illustrate representative experiments and show the mean and standard error derived from triplicate experiments. * means P ≤ 0.05 and ** means P ≤ 0.01 (Student’s t test, male vs female). (C) Immunohistochemistry analysis of the WD40 protein in schistosomes. Vs, ventral sucker. 739



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indeed abundant in a specific gender in S. japonicum. However, it is also necessary to clarify that a phosphopeptide specifically detected in a specific gender does not mean its differential phosphorylation in this gender. The specific detection of a phosphopeptide may also be due to the potential situation that the corresponding protein is more abundant in the condition tested. In addition, it must be kept in mind that the amount of protein phosphorylation may not directly correlate with the amount of protein expression in a specific gender in schistosomes, which remains to be further determined by using the antibody against a specific phosphorylated protein or a quantitative phosphoproteomic method. It was reported that the S. japonicum worm pair usually lays at least 1000 eggs per day in a final host,37 and Fe as one of the important ions performs an important role in egg formation and embryogenesis.38 Consequently, the storage and transportation of Fe is particularly important for female schistosomes. In the present study, ferritin-1 heavy chain (FHC) was specifically detected to be phosphorylated in female schistosomes, suggesting that the phosphorylated FHC may facilitate Fe transportation and also assist in egg formation and production. It will be interesting to further determine whether targeting FHC phosphorylation may offer a potential for blocking the process of egg production for intervening schistosomiasis. WD40 proteins are implicated in a variety of functions such as signal transduction, transcription regulation, and apoptosis in other eukaryotic species.27 In the present study, we observed that the phosphorylated WD40 protein was specifically detected in male schistosomes, and our immunohistochemistry results indicated that WD40 proteins were significantly located in the testis of male schistosomes, suggesting that the WD40 protein may play an important role in schistosome sexual maturation. In addition, we noted that 7 out of 12 phosphoproteins specifically detected in male schistosomes are likely species-specific proteins, implying that male schistosomes may have specific features for regulating schistosome development and sexual maturation. Functional characterization of these proteins and their phosphorylations may broaden our vision to understand the mechanisms involved in schistosome development and sexual maturation and then may result in novel drug targets exclusively for schistosomes. Although the relatively large kinome was proposed in S. mansoni,39 we totally identified 180 phosphopeptides, which is matching to 148 proteins, in S. japonicum in the present study. We noted that a large proportion of potential phosphopeptides identified by Mascot searching were rejected when applying the stringent criteria for manual investigation. Considering that about one-third of all proteins are phosphorylated at least at some point in time,40 further improvement of phosphopeptideenrichment procedures and subsequent identification by high mass accuracy MS/MS may improve the number of protein phosphorylation identifications in S. japonicum. Although in vivo functional characterization of specific phosphorylation sites of a protein in schistosomes is challenging due to the technical difficulties of genetic manipulation,41 alternatively phosphoproteomic analyses complement gene expression profiles, and protein characterization may facilitate an improved understanding of schistosome biology and then result in the development of novel interventions against schistosomiasis.

S. japonicum GSK3 may result in down-regulation beta-catenin activity and relevant Wnt pathway. Hsp90, an evolutionarily conserved molecular chaperone, was identified to be cophosphorylated in different stages and sex of S. japonicum. Today Hsp90 is believed to function not only as a chaperone that helps proteins to fold but also in more complex roles in the maturation and/or activation of numerous “client proteins” involved in signal transduction in other organisms.31 Moreover, Hsp90 is now considered to be a potential target against protozoan infection.32 However, the functions of Hsp90 still remain undercharacterized in schistosomes. In the present study, we found that it is likely that the functions of the kinases (GSK3 and Cdc37) and signal molecule YWHAE are subject to regulate by SjHsp90 in schistosomes (Figure 4B and 4D). It has been demonstrated that Hsp90 in cooperation with the cochaperone Cdc37 plays an important role in the activation and stabilization of many client kinases in humans.31 We noted that both SjHsp90 and SjCdc37 were identified to be cophosphorylated in the different stages and sex of S. japonicum. Next, each of them had an evolutionarily conserved phosphosite with human ortholog. In addition, our real-time RT-PCR analysis indicated that there was a good correlation between the transcripts of SjHsp90 and SjCdc37 in different stages of schistosomes (data not shown). To gain insight into the mechanism of SjHsp90 involved in the activation and stabilization of its client kinases, we attempted to determine the SjHsp90−SjCdc37 interaction by using the Yeast Two-Hybrid System (Invitrogen) and CheckMate mammalian Two-Hybrid System (Promega). However, the results from both of the methods indicated very weak interaction between SjHsp90 and SjCdc37 (data not shown). Interestingly, siRNA-mediated silencing of SjHsp90 simultaneously resulted in the reduction of SjCdc37 at transcript levels (Figure 4D). Consequently, it is most likely that other chaperone(s) may be required to be involved in the interaction between SjHsp90 and SjCdc37. Schistosomes have evolved into sexual dimorphism with the separation of labor. Female schistosomes at most focus on egg production, and male schistosomes are devoted to the provisions of physical transportation and musculature to aid feeding and other maturation factors for females.33 Identification of gender-associated phosphoproteins may gain valuable insight into the molecular basis of schistosome sexual maturation and egg production. In the present study, we performed the preparations of protein lysates for schistosomula, adult males, and adult females and the following phosphopeptide enrichments in parallel. We noted that 12 phosphopeptides in males and 3 phosphopeptides in females were able to be detected in all 5 independent MS analyses in a gender-specific manner, suggesting that these phosphopeptides matching proteins are likely to be more abundant in the worms in which the phosphopeptide was detected. Expectedly, our realtime RT-PCR results (Figure 6A and 6B) and immunohistochemistry analysis (Figure 6C) further indicated that these proteins were more abundant either at transcript levels or at protein levels in the worms in which the phosphopeptides were identified. Moreover, some of such phosphopeptide matching proteins such as ferritin-1 heavy chain and translationally controlled tumor protein homologue in females and glucose transport protein in males were also reported to show genderspecific expression in schistosomes in previous studies.34−36 Collectively, these results indicated that these genderspecifically detected phosphopeptide matching proteins are 740



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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Tables and Figures. This material is available free of charge via the Internet at http://pubs.acs.org

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AUTHOR INFORMATION

Corresponding Author

*Guofeng Cheng. Tel.: 86-213-429-3659. Fax: 86-215-4081818. E-mail: [email protected]. Hongxia Wang. Tel.: 314-587-1430. Fax: 314-587-1530. E-mail: howang@ danforthcenter.org. Present Address §

Donald Danforth Plant Science Center, 975 N. Warson Road, St.Louis, MO 63132, United States. Author Contributions ∥

These authors equally contributed to this study

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The study was supported, in part or in whole, by the National Natural Science Foundation of China (Grant No. 30901068), Science and Technology Commission of Shanghai Municipality of China (Grant No.10410703400 and Grant No.10PJ1412300), and Shanghai Talent Developing Foundation of China (Grant No.2009032) for Cheng G. We thank the Chinese National Genome Center at Shanghai for making S. japonicum genome available.

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