Proteomic Analysis of Tegument-Exposed Proteins ... - ACS Publications

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Proteomic Analysis of Tegument-Exposed Proteins of Female and Male Schistosoma japonicum Worms Min Zhang,† Yang Hong,† Yanhui Han,† Hongxiao Han,† Jinbiao Peng,§ Chunhui Qiu,‡ Jianmei Yang,† Ke Lu,† Zhiqiang Fu,*,† and Jiaojiao Lin*,† †

Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture of China, Shanghai 200241, P.R. China § Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, P.R. China ‡ College of Life Sciences, Fujian Agriculture and Forestry University, FuZhou, Fujian Province 350002, P.R. China S Supporting Information *

ABSTRACT: The interplay between sexes is a prerequisite for female growth, reproductive maturation, and egg production, and the basis of schistosome pathopoiesis and propagation. The tegument is in direct contact with the host environment and its surface membranes are particularly crucial for schistosome survival in the definitive host. In this study, a streptavidin−biotin affinity purification technique combined with LC−MS/MS was used to analyze putative tegumentexposed proteins in female and male adult Schistosoma japonicum worms. In total, 179 proteins were identified in females and 300 in males, including 119 proteins common to both sexes, and 60 female biased and 181 male biased proteins. Some (e.g., serpin and CD36-like class B scavenger receptor) were involved in host-schistosome interactions, while some (e.g., gynecophoral canal protein) were important in the interplay between sexes. Gene Ontology analysis revealed that proteins involved in protein glycosylation and lysosome were highly expressed in females, while proteins involved in intracellular signal transduction, regulation of actin filament polymerization, and proteasome core complex were highly expressed in males. These results might elucidate physiological differences between the sexes. Our study provides new insights into schistosome growth and sexual maturity in the final host and permits the screening of vaccine candidates or drug targets for schistosomiasis. KEYWORDS: Schistosoma japonicum, tegument-exposed proteins, proteome, female and male

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INTRODUCTION

At distinct developmental stages in the life cycle, schistosomes live in different environments, specifically fresh water, a snail invertebrate host and the mesenteric veins of the definitive vertebrate host.10 Unlike other trematode parasites, schistosomes have separate sexes. The female resides in the gynecophoric canal of male after they pair.11,12 In addition, the presence of mature male worms is a prerequisite for female worm growth, reproductive development and maintenance of maturity.13−16 Male−female interactions are likely associated with signal transduction involving intercellular or intracellular substrates that transduce signals between the surfaces of males and females.17 The mature female schistosome produces approximately 300 eggs per day, which incite a granulomatous inflammatory reaction leading to morbidity and resulting in disease propagation.18,19 Therefore, the identification of molecules that are critical for the interplay between females and males and the development of worms might contribute to an effective method for interfering with female maturation,

Schistosomiasis is one of the world’s most prevalent zoonotic diseases and remains a serious worldwide public health problem, infecting approximately 200 million people in 74 endemic countries and resulting in about 280 000 deaths annually.1,2 In China, schistosomiasis is caused by infection with Schistosoma japonicum, which is mainly distributed south of the Yangtze River.3,4 In 2011, 290 000 people had schistosomiasis, with 68.6 million at risk.5 Although chemotherapy with praziquantel is an effective treatment for schistosomiasis, resistance to praziquantel is reported in Senegal and Egypt.6−8 In addition, six vaccine candidates (glutathione-S-transferase, triose phosphate isomerase, paramyosin, 23 kDa transmembrane surface protein, myosin heavy chain, and 14 kDa fatty acid binding protein) recommended by World Health Organization (WHO) do not have ideally protective efficacy against schistosome infection.9 The screening of effective vaccine candidates and discovery of novel drug targets for schistosomiasis would help eradicate this disease, benefiting millions of people living in schistosomiasis-endemic countries. © 2013 American Chemical Society

Special Issue: Agricultural and Environmental Proteomics Received: May 22, 2013 Published: August 2, 2013 5260

dx.doi.org/10.1021/pr400476a | J. Proteome Res. 2013, 12, 5260−5270

Journal of Proteome Research

Article

biotinylation of live adult worms with OFFGEL electrophoresis and tandem mass spectrometry. However, no reports have compared the expression of female and male tegument surface membrane proteins in S. japonicum. In this study, the tegument surface membrane proteins of female and male S. japonicum worms were isolated by streptavidin−biotin affinity purification. Proteins were identified by a shotgun LC−MS/MS approach. This study might provide valuable information for screening vaccine candidates and drug targets.

preventing egg laying and pathogenesis, and interrupting disease transmission. The tegument, a syncytial layer covering the schistosome surface, is composed of the surface membrane, the basal membrane, and the matrix10,20−22 (Figure 1). The surface

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MATERIALS AND METHODS

Parasite Samples

Male New Zealand rabbits weighing 2.5−3.0 kg were used for worm collection. S. japonicum worms were maintained in rabbits and Oncomelania hupensis hupensis snails at Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (SVRI, CAAS). Animal care and all experimental procedures involving animals were conducted according to the principles for the Care and Use of Laboratory Animals of SVRI, CAAS. Cercaria of S. japonicum (Anhui isolate, Chinese mainland strain) were shed from lab-infected snails after exposure to artificial light for 5 h. Six rabbits were randomly divided into three groups and three independent challenge infections were carried out. Each time two rabbits were infected with 2000 cercariae through shaved abdominal skin, respectively. Adult worms were collected from infected rabbits at 42 days post-challenge by perfusion. The worms were incubated at 4 °C for 30 min, and the adult paired worms spontaneously separated into females and males, and then were isolated. Worms were washed five times in phosphate buffered saline (PBS, pH 7.4) at 4 °C to remove excreted and secreted proteins and other interfering substances. Worms were checked by microscope, and dead or damaged parasites were discarded. Intact worms were biotinylated immediately.

Figure 1. Diagrammatic representation of the tegument of the adult S. japonicum. Teg, tegument; SM, surface membrane; Mc, membranocalyx; Apm, apical plasma membrane; BM, basal membrane; P, pit; S, spine; MLV, multilaminate vesicle; DB, discoid body; CC, cytoplasmic connection.

membrane is a single layer with trilaminate membranes in the cercaria. In the schistosomulum and adult worms, the surface membrane is a bilayer structure, including heptalaminate membranes, interpreted as a apical plasma membrane overlaid with a secreted bilayer called the membranocalyx.23,24 During migration and growth of the schistosome in the final host, the tegument surface membrane undergoes a dynamic turnover. Its structure differentiates at parasitic developmental stages, with constant changes in biological function.25 Tegumental structures are different between female and male worms, which could lead to dissimilar biological functions in the two sexes.10 Since the tegument is in direct contact with the host environment, it is crucial for nutritional uptake, structural support, immune evasion, and modulation, excretion, osmoregulation, sensory reception, and signal transduction.26 The surface membrane proteins are responsible for these tegument functions. Therefore, the identification of these proteins is needed for screening immunological and pharmacological targets for schistosomiasis control. Recently, sequence databases for S. mansoni and S. japonicum and improvements in proteomics technology have resulted in high-throughput proteomic studies on schistosomes, particularly the tegument, and screening of molecules that are essential for the worms.27 Van Balkom et al.28 identified 43 tegument-specific proteins of adult S. mansoni using a freeze− thaw−vortex approach to isolate crude tegument preparations for LC−MS/MS. Braschi et al.21 enriched the tegument surface proteins of S. mansoni by biotinylation of live intact worms and using sequential differential extraction of whole tegument preparations followed by purification of labeled proteins using a streptavidin column. Proteins were identified by LC−MS/MS. For S. bovis, tegumental proteins of females and males were identified by trypsinization of whole adult worms and a shotgun proteomic method.29 For S. japonicum, Liu et al.30 used detergent-based extraction with 2D-nano-LC−MS to obtain 373 tegumental proteins belonging to adult females, adult males, mixed-sex adults, and hepatic schistosomula. Mulvenna et al.22 identified 54 tegument-exposed proteins using

Preparation of Biotinylated Tegument Proteins from Female and Male Worms

Tegument-exposed proteins of live worms were labeled with biotin.21 Healthy and intact flukes were washed three times in ice-cold PBS (pH 7.4) and incubated in 10 mL of ice-cold PBS containing 0.89 mM membrane-impermeable sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (EZ-Link SulfoNHS-SS-Biotin; Pierce, USA) for 30 min at 4 °C with gentle rotation mixing. Labeling was quenched using 1 mL of quenching solution (Pierce, USA). Worms were washed three times in ice-cold Tris buffered saline (TBS, pH 7.4) with protease inhibitors (protease inhibitor cocktail; Fermentas Life Sciences, USA) and snap frozen in liquid nitrogen. The effectiveness and extent of the biotin labeling of adult worms were observed by immunofluorescence microscopy before subsequent experiment and analysis. After incubation with avidin-conjugated FITC (Sigma) for 30 min at room temperature, frozen sections of biotin-labeled worms were incubated with DAPI (Beyotime, China), and then visualized using Nikon 80i fluorescence microscope (Nikon, Japan). The tegument was removed using a freeze−thaw−vortex method.31 Frozen worms were slowly thawed on ice, 1 mL of ice-cold TBS (pH 7.4) plus protease inhibitors were added, and parasites were vortexed for 10 bursts of 1 s at maximum speed. Supernatant was collected and tegument material was pelleted by centrifugation at 1000 × g for 30 min at 4 °C. The biotinylated tegument proteins were isolated by a Cell Surface 5261



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SEQUEST out file with in-house software (BuildSummary). SEQUEST search parameters were Delta CN (≥0.1) and Xcorr (one charge ≥ 1.9, two charges ≥ 2.2, three charges ≥ 3.75). Proteins with unique pep count ≥ 2 were considered accurately identified and used for bioinformatic analysis.

Protein Isolation Kit according to the manufacturer’s protocol (Pierce, USA). Totally, samples from three independent challenge infections were prepared as three biological replicates, mixed and subjected to the following SDS-PAGE and LC−MS/ MS. Protein concentration was determined by the Bradford method using Bio-Rad protein assay reagent (Bio-Rad Laboratories, USA). Samples were stored at −80 °C.

Bioinformatic Analysis

Sequences were clustered using the program CD-HIT32 with default parameter (sequence identity ≥ 95%). Molecular weight (MW) and isoelectric points (pI values) were calculated using the EMBOSS Pepstats program (http://emboss.sourceforge. net/).33 Protein annotation used BLASTP34 on the SwissProtTrEmbl database with default parameters (E value =1 × 10−6, sequence identity ≥ 30%). Associated gene ontology (GO) terms of proteins were extracted from the Gene Ontology annotation file (http://www.geneontology.org)35 by Perl scripts. A Fisher’s exact test calculated by R packages was used to assess significant differences between female and male samples (p-values ≤ 0.05) based on the number of proteins per GO category in each sample.

SDS-PAGE

Protein samples (20 μg) were mixed with an equal volume of 2× protein loading buffer (0.1 M Tris buffer, pH 6.8, 4% SDS, 0.2% β-mercaptoethanol, 40% glycerol, and 0.002% bromphenol blue) and boiled for 2 min. Samples were subjected to SDSPAGE using 5% stacking gels and 12.5% resolving gels in a mini-vertical electrophoresis system (Bio-Rad Laboratories, USA). Gels were stained with Coomassie brilliant blue (CBB) G250 (Invitrogen, USA). In-Gel Trypsin Digestion

Stained gels were cut into eight slices by protein molecular weight. Slices were transferred to a microcentrifuge tube and destained with 200 μL of 100 mM NH4HCO3 in 30% acetonitrile (ACN) at 37 °C until depigmentation. Dried gel pieces were incubated with 100 μL of 10 mM dithiotreitol (DTT) in 100 mM NH4HCO3 at 56 °C for 30 min, followed by dehydration in 100 μL of 100% ACN for 5 min. Proteins were alkylated by 60 mM iodoacetamide (IAA) in 70 mM NH4HCO3 at room temperature in the dark for 20 min. Gel pieces were washed with 100 μL of 100 mM NH4HCO3 for 15 min and dehydrated as above. After lyophilization, dried gel pieces were incubated with 5 μL of 10 ng/μL trypsin buffer at 4 °C for 60 min. After adding the same volume of trypsin buffer and 20 μL of 25 mM NH4HCO3 (pH 7.8−8.0), proteins were digested at 37 °C for 20 h. To extract digested peptides, digestion buffer was replaced with 100 μL of 0.1% trifluoroacetic acid (TFA) in 60% ACN with sonication for 15 min. Pooled extracts were lyophilized and redissolved in 20 μL of 0.1% formic acid (FA) for shotgun analysis.

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RESULTS

Global Analysis of Tegument-Exposed Proteome

The immunofluorescence microscopy revealed that the biotinylation was limited to tegument exposed proteins (Supporting Information, Figure 1). Biotinylated tegument proteins from female and male worms were separated by SDSPAGE and subjected to shotgun LC−MS/MS analysis (Figure 2). For SEQUEST, 537 proteins from female worms and 748 proteins from males were identified and assigned into 391 groups for females and 590 groups for males. To increase the identification reliability, 202 female proteins and 331 male proteins with unique pep count ≥ 2 were considered accurate. To remove redundant sequences, identified proteins were

Shotgun LC−MS/MS

Peptides were purified using reverse-phase high-performance liquid chromatography (RP-HPLC), on a surveyor LC system (Thermo Fisher, San Jose, CA) with an autosampler (Thermo Fisher, San Jose, CA). Injected samples were trapped and desalted on a column (Zorbax 300SB-C18 peptide traps, 300 μm × 65 mm, Agilent Technologies, Wilmington, DE) and separated on an analytical column (RP-C18, 150 μm × 150 mm, Column Technology, Inc., Fremont, CA). Mobile phases were 0.1% FA in Millipore water as buffer A and 0.1% FA in 84% ACN as buffer B. Peptides were eluted using a 120 min gradient of 4−50% buffer B at a flow rate of 250 nL/min. Peptides were ionized in positive ion mode and introduced into an LTQ linear ion trap mass spectrometer (Thermo Fisher, San Jose, CA) equipped with a microelectrospray source for MS/ MS. Typical experimental conditions were spray voltage, 3.0 kV; capillary temperature, 170 °C; collision energy (MS/MS), 35%. After acquisition of one full mass scan (m/z 300−1800), 10 MS/MS scans were acquired for the 10 most intense ions using dynamic exclusion settings: repeat count of 2, repeat duration of 30 s, exclusion duration of 90 s. Protein identification was via Bioworks Browser 3.1 (Thermo Fisher, San Jose, CA) against the NCBI nonredundant schistosome protein database (25135 sequences, 10/31/2012, http://www. ncbi.nlm.nih.gov/) and results were extracted from the

Figure 2. Separation of identified tegument-exposed proteins from female and male S. japonicum worms by SDS-PAGE. An amount of 20 μg of each sample was separated on a 12.5% resolving gel. Each gel lane was cut into 8 equivalent slices. 5262



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filtered to 95% sequence identity. Female and male data were clustered into 179 and 300 unique proteins, of which 153 (85.5%) and 253 (84.3%) proteins were annotated by BLASTP search in the SwissProt-TrEmbl database. A comparison of the identified proteins between female and male worms is in Figure 3. A total of 119 common identified proteins constituted 66.5%

Gene Ontology Annotation

Identified proteins from female and male samples were annotated with the GO vocabulary. Overall, 153 proteins from females were linked to 1172 GO terms and 253 proteins from males were linked to 1824 GO terms. Annotation terms were in three hierachical structures: biological processes, molecular functions, and cellular components. After redundant GO categories were removed, terms from samples were compared (Figure 5). Of 1172 GO categories in female samples, 1010 were common and 162 were unique; in male samples, 814 were unique GO categories.

Figure 3. Venn diagram of the number of identified tegument-exposed proteins from female and male worms. The overlap shows the number of common-expressed proteins between sexes. The number in parentheses denotes the proteins with annotation from the SwissProt-TrEmbl database. Figure 5. Venn diagram of the number of Gene Ontology terms from female and male samples, including all matches from Biological Process, Molecular Function, and Cellular Component. The number in parentheses denotes the total number of GO categories.

of total proteins in female worms and 39.7% in male worms (Supporting Information, Table 1). Of total proteins in different sexes, 60 (33.5%) were female biased and 181 (60.3%) were male biased (Supporting Information, Tables 2 and 3).

Statistically Significant GO Categories

Theoretical Two-Dimensional Distribution of Identified Proteins

To determine significant differences for all levels of GO, the number of proteins in each GO term from the two samples was compared using Fisher’s Exact Test. No significant differences were seen between the two samples at level 2 in three hierarchical structures (Figure 6), but 26 significant differences were detected at higher levels (Table 1). These differentially annotated categories might contain shared and unique GO terms from samples. In the biological process category, a large number of proteins were categorized into a cellular process, metabolic process, or single-organism process (Figure 6A). Less common were proteins related to biological regulation, multiorganism process, response to stimulus or developmental processes. Higher levels (above level 2) had 15 differentially annotated categories, 6 of which were enriched in females and 9 of which were enriched in males (Table 1, Supporting Information, Table 5). Among the GO terms, only autophagy was unique to females while six categories were unique to males: vegetative growth of a singlecelled organism, regulation of cellular localization, regulation of attachment of spindle microtubules to kinetochore, regulation of protein polymerization, negative regulation of autophagic vacuole size, and regulation of actin filament polymerization. In the molecular function ontology, the majority of proteins were in two major categories which were binding (more than 80% in both males and females) and catalytic activity (more than 60% in both) (Figure 6B). Compared with female samples, males had a single unique category, channel regulator

Distributions of the theoretical MW and pI of identified proteins were analyzed according to amino acid sequences (Supporting Information, Table 4). For both female and male samples, the molecular mass ranged from 6.48 kDa to 222.38 kDa, with about 90% of proteins distributed between 10 kDa and 70 kDa. The pI of identified proteins ranged from 3.98 to 12.15 with the most between 4 and 11. MW and pI distribution of sex-biased proteins were similar to the distribution of total proteins (Figure 4).

Figure 4. Theoretical 2-D (pI, MW) distribution of identified tegument-exposed proteins which are highly expressed in female or male worms. 5263



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Figure 6. Gene ontology categories for identified tegument-exposed proteins of female and male worms (GO level 2 shown): (A) Biological Process, (B) Molecular Function, (C) Cellular Component. The percent of proteins shows its proportion in total proteins of related sample with GO annotation.

activity. At higher levels (above level 2), molecular function had eight differentially annotated categories, of which threoninetype peptidase activity and threonine-type endopeptidase activity were enriched in and unique to males while the remainder were enriched in females: serine-type peptidase activity, small conjugating protein-specific isopeptidase activity,

cysteine-type peptidase activity, oligosaccharyl transferase activity, cysteine-type endopeptidase activity, dolichyl-diphosphooligosaccharide-protein glycotransferase activity (Table 1, Supporting Information, Table 5). For the cellular components category, proteins mapping to GO terms for organelle, extracellular region, cell part, 5264



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Table 1. Differential GO Categories of Identified Tegument-Exposed Proteins from Female and Male Wormsa X.level

GO_ID

female number 1

term

female number 2

male number 1

male number 2

E-value

140 140 140 140 140 140 140 140

0 4 9 0 5 9 9 0

228 228 228 228 228 228 228 228

13 0 4 9 1 4 4 7

0.003 0.022 0.039 0.029 0.034 0.039 0.039 0.049

140 140 140 140 140 140 140

1 12 9 0 0 0 4

228 228 228 228 228 228 228

11 40 4 7 7 7 19

0.037 0.044 0.039 0.049 0.049 0.049 0.048

148 148 148 148 148 148 148 148

15 9 9 0 5 9 0 5

240 240 240 240 240 240 240 240

8 2 3 10 1 2 10 1

0.014 0.008 0.014 0.016 0.035 0.008 0.016 0.035

134 134 134

0 13 9

209 209 209

10 5 2

0.016 0.007 0.009

Biological Process 4 5 5 5 6 6 6 6

GO:0072690 GO:0006914 GO:0005984 GO:0060341 GO:0045471 GO:0009101 GO:0070085 GO:0051988

6 6 7 7 7 8 8

GO:0043254 GO:0035556 GO:0006486 GO:0032271 GO:0045771 GO:0030833 GO:0019680

5 6 6 6 6 7 7 7

GO:0008236 GO:0070138 GO:0008234 GO:0070003 GO:0004576 GO:0004197 GO:0004298 GO:0004579

4 7 8

GO:0005839 GO:0000327 GO:0005764

vegetative growth of a single-celled organism autophagy disaccharide metabolic process regulation of cellular localization response to ethanol glycoprotein biosynthetic process glycosylation regulation of attachment of spindle microtubules to kinetochore regulation of protein complex assembly intracellular signal transduction protein glycosylation regulation of protein polymerization negative regulation of autophagic vacuole size regulation of actin filament polymerization L-methylmalonyl-CoA biosynthetic process Molecular Function serine-type peptidase activity small conjugating protein-specific isopeptidase activity cysteine-type peptidase activity threonine-type peptidase activity oligosaccharyl transferase activity cysteine-type endopeptidase activity threonine-type endopeptidase activity dolichyl-diphosphooligosaccharide-protein glycotransferase activity Cellular Component proteasome core complex lytic vacuole within protein storage vacuole Lysosome

a The GO categories that are statistically significantly different between the two samples are shown at all levels of GO under Biological Processes, Molecular Functions, and Cellular Components. Number 1 refers to the number of proteins that fall within each GO category at the first level while Number 2 means that the number of proteins which are classified into the differential GO subcategories. Significantly different (p ≤ 0.05) categories, which are shown, are calculated using Fisher’s Exact Test.

proteins. Liu et al.30 identified 134 and 58 tegument proteins, respectively, from adult female and male S. japonicum. Proteins were isolated from whole tegument by a detergent-based technique with no comparison of differential expression of tegument proteins between sexes. Our paper identified tegument-exposed proteins of female and male adult worms using streptavidin−biotin affinity purification, a powerful technique for isolating proteins on tegument surface membranes.22 Female- and male-identified proteins were subjected to comparative bioinformatic analysis. Our results complement those of previous studies. In our preparations, 119 proteins were commonly expressed in female and male worms, and 60 were female biased and 181 were male biased. A number of proteins have been identified as tegument-exposed in schistosome species in previous studies21,22,29,36 (Supporting Information, Tables 1−3).26,27,29,35 On the basis of functional annotations, these proteins were found to possess many molecular functions, such as binding, catalytic activity, tansporter activity, oxidoreductase activity, and so on. Among the proteins, some important tegument-exposed proteins (e.g., gynecophoral canal protein [GCP], serine protease inhibitor serpin and CD36-like class B scavenger receptor) were observed in both sexes (Table 2, Supporting Information, Table 1). Other proteins (e.g., 23 kDa integral

macromolecular complex, and membrane part were the most abundant in both female and male samples (Figure 6C). At higher levels (above level 2), three terms were significantly different between the two samples. The category of proteasome core complex occurred only in males while the categories of lytic vacuole within protein storage vacuole and lysosome were enriched in females (Table 1, Supporting Information, Table 5).

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DISCUSSION We used a streptavidin−biotin affinity purification method and a shotgun proteomic approach to identify proteins on the tegument surface membrane of female and male S. japonicum. SDS-PAGE analysis revealed clearly distinct band patterns for the two sexes (Figure 2). In total, 179 and 300 proteins were identified in female and male worms, respectively (Figure 3). In a previous study by Mulvenna et al.,22 54 proteins were identified as putatively tegument-exposed in mixed-sex adults of S. japonicum using similar techniques. The number of identified tegument-exposed proteins was different between single-sex and mixed-sex worms. We propose that the separation of an established mating pair results in the exposure of the tegument surface membrane to the female−male interface in the gynecophoric canal. This led to our identification of more 5265



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Table 2. A Part of Common Tegument-Exposed Proteins from Female and Male S. japonicum with several Annotations female database ID no.

description

male

unique pepcounta

sequence coverageb

unique pepcounta

sequence coverageb

molecular function

gi|2829279 gi|14211394

Calpain serine protease inhibitor serpin

4 3

7.50% 9.50%

11 6

19.87% 18.75%

gi|76155326 gi|76156797

similar to glucose transport protein similar to nucleotide pyrophosphatase/ phosphodiesterase 5 SNaK1 glyceraldehyde-3-phosphate dehydrogenase similar to Alkaline phosphatase

2 3

7.89% 19.07%

2 3

6.45% 19.92%

calcium ion binding; peptidase activity; peptidase activity; serine-type endopeptidase inhibitor activity transmembrane transporter activity catalytic activity

14 5

20.26% 21.89%

6 15

9.53% 56.51%

potassium-exchanging ATPase activity NAD binding; oxidoreductase activity

3

20.72%

2

14.86%

2

16.50%

2

11.17%

alkaline phosphatase activity; catalytic activity; protein binding; signal transducer activity

8

23.49%

6

12.11%

receptor activity

4 4 2 3

5.54% 7.15% 9.28% 2.08%

11 3 4 2

14.94% 5.75% 14.09% 1.61%

calcium-transporting ATPase activity;

gi|15824396 gi|56752741 gi|76155174 gi|29840985 gi|56756563 gi|3859490 gi|23664250 gi|29840937 gi|501209

transmembrane transport protein, putative similar to CD36-like class B scavenger receptor calcium ATPase 2 gynecophoral canal protein hypothetical protein with IG domain Surface protein

a The unique pepcount refer to the number of different peptides assigned to the proteins. bThe sequence coverage is defined as the ratio (%) of the protein sequence covered by the assigned peptides.

Schistosomes have a dioecious state and interplay between sexes is required for the life cycle. Mature male worms provide physical protection and transportation to females and release chemicals such as hormones, nutrients, and messengers that stimulate female growth, reproductive development, and egg production.43−45 The effects of female worms on males include influencing glutathione and lipid levels.46,47 The close interaction between the sexes requires direct contact; thus the tegument surface membrane and its proteins are critical. In our results, GCP was present in both sexes. GCP is a cell-surface glycoprotein and its sequence is similar to the developmentally regulated neural cell adhesion molecule fasciclin I. GCP is involved in female−male pairing and development of schistosomes.48,49 By immunofluorescence microscopy, GCP is detected on the entire surface of mature females and in the gynecophoric canal of mated but not nonmated males.12,50 GCP was on the tegument surface membrane of female and male worms in our results, which supports previous studies on its location at the female−male interface and suggests that it might be involved in female−male biological interaction.21,22 Over evolution, mature schistosomes have developed morphological and functional differences between the sexes.51 Therefore, identifying differential proteins between sexes may uncover distinctions between females and males.52 Proteomic comparisons of female and male schistosomes might shed light on their sexual biology. In this study, GO annotation and comparisons indicated differential expression of tegumentexposed proteins between female and male worms in protein glycosylation, lysosome, intracellular signal transduction, regulation of actin filament polymerization, and proteasome core complex. Protein glycosylation is the addition of a carbohydrate or carbohydrate derivative to a protein. For schistosomes, glycosylation might be important in host-parasite interactions, development, reproduction, protein trafficking and folding.53 Proteins in this subcategory found only in female samples include proteins similar to dolichyl-diphosphooligosaccharideprotein glycosyltransferase 48 kDa and 67 kDa subunits, both

membrane protein [Sj23] and Annexin B13a) were detected only in female samples, and the others (e.g., 20 kDa calciumbinding protein and 21.7 kDa antigen) were found only in adult male schistosomes. Adult schistosomes persist in a hostile environment of definitive hosts for decades without experiencing detectable damage. The parasite strategy is immune evasion and nutrient uptake, in addition to the exploitation of host endocrine and immune signals in which the tegument, especially its surface membranes, are important.37,38 In our study, serine protease inhibitor (serpin) and putative CD36-like class B scavenger receptor were common in females and males. Serpin belongs to a superfamily of proteins with similar structures and is involved in numerous physiological functions through inhibition of serine proteases. In parasitic helminthes, a number of serpins have been identified and deemed to be critical for worm survival by interfering with the host immune response.39 Serpin is a membrane-anchored protein on the surface of S. hematobium, identified by purification and crystallization, similar to its location on S. japonicum in our results.40 The expression of serpin, a protease inhibitor, at the host-worm interface might indicate that it counteracts host proteases in escaping immune attack. Recombinant S. japonicum serpin induces moderate protective immunity against schistosome infection in C57BL/6 mice.41 The CD36-like class B scavenger receptor is a member of a scavenger receptor family of cell surface proteins with two transmembrane regions and participates in fatty acid metabolism. Schistosomes cannot synthesize fatty acids, so the CD36-like class B scavenger receptor might help them use host lipids. A previous study indicated that this receptor binds modified low-density lipoprotein on the tegumental surface at the sporocyst stage of S. mansoni.42 Identification of this receptor on the tegument surface membranes of adult S. japonicum suggested that this protein might be important for parasite uptake of lipids from the definitive host and might support the development and survival of parasites. 5266



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SmRab was higher in males than in females.68 Rab GDP dissociation inhibitors (GDIs) constitute a family of small GTPases that regulate Rab GTPase activity.69 Depletion of a member of GDIs results in inhibition of protein transport at multiple stages of the secretory pathway, suggesting that it play an important role in membrane traffic for yeast.70 For schistosome, Rab proteins and GDIs, which were highly expressed in males in our study, might help maintain tegumental turnover in males. Regulation of actin filament polymerization modulates the frequency, rate, or extent of actin filament assembly. Proteins in this subcategory were found only in male samples and were actin-related protein 2/3 complex subunit 5, proteins similar to actin-related protein 3, 2/3 complex subunit 4, putative F-actin capping protein alpha, and putative ezrin. These three actinrelated proteins are a major constituent of the ARP 2/3 complex involved in turnover of actin filaments in schistosomes.26 Actin filaments are aligned in paracrystalline arrays in the spines located on the tegument surface and movable structures involved in host-parasite interactions.71,72 Although the schistosome tegument is vulnerable to damage by the host immune system, the motility of the spines changes the surface antigens by shifting their position and exposure.72 Spines are more abundant in males than in females, possibly because males have greater contact with the host. F-actin capping protein alpha binds actin and nucleates actin polymerization, regulating actin polymerization activated by the ARP 2/3 complex.26 As a member of the ezrin−radixin−moesin family of actin-binding proteins, ezrin attaches actin filaments to the tegument surface membranes.73 Regulation of actin polymerization is enriched in male samples, indicating a higher presence of actin filament forming proteins in the schistosome tegument. The proteasome core complex is a multisubunit barrelshaped endoprotease that is the core of the proteasome. Male samples had several biased proteins in this subcategory: 4 αsubunits and 6 β-subunits of proteasome of the 26S proteasome, a proteolytic complex responsible for degradation of mutated, unfolded, or oxygen-damaged eukaryotic proteins.74 Given its involvement in cell differentiation and replication, the proteasome are considered as a promising therapeutic target in protozoan parasites, such as Giardia, Trypanosoma, Toxoplasma spp, and so on.75 For schistosomes, in a RNA interference study conducted by Nabhan et al., nearly 80% reduction in a proteasome subunit expression caused the 78% decrease in S. mansoni viability.76 By immunization of BALB/c mice with rSjPSMA5 combined with Seppic 206 adjuvant, significant reductions of 23.29% and 35.24% are obtained in the numbers of worms and eggs in the liver, respectively.77 Therefore, proteasomal activity might be important for the development and survival of schistosomes.76−79 Antioxidants increase the expression and activity of proteasomal subunits.80 However, the quantity of antioxidants from schistosomes is related to the volume of reactive oxygen species released by host effector cell adhesion to worms.81 Since female worms live in the gynecophoric canal of males, their surface has less contact with the host immune system.29 The proteasome core complex was enriched in male samples, indicating higher expression of proteasome subunits due to a larger surface contact with the host immune system. We analyzed putative tegument-exposed proteins of female and male S. japonicum. Proteins involved in host−schistosome interaction, female−male interplay, protein glycosylation, lysosome, intracellular signal transduction, regulation of actin

of which belong to the N-oligosaccharyl transferase complex that catalyzes transfer of a high-mannose oligosaccharide from a lipid-linked oligosaccharide donor to nascent polypeptide chains. This result is similar to previous reports that dolichyldiphosphooligosaccharide-protein glycosyltransferases are female-biased genes.51,54 Sex-specific protein glycosylation in adult schistosomes has been observed using transcriptomics, proteomics, and glycomics.55 Sex-biased N-glycans are located on the tegument of females and males, indicating that tegument protein glycosylation might be distinct between sexes.53 In our study, protein glycosylation was enriched in female samples, corroborating predictions of differences in tegument protein glycosylation between the sexes. The lysosome is a lytic vacuole that contains hydrolases and has a cell cycle-independent morphology in most animal cells. Female samples had several biased proteins in this subcategory that were lacking male samples including aspartic proteinase, cathepsin B-like cysteine proteinase, preprocathepsin C, and similar to cathepsin L-like proteinase. These gene products are in the protease superfamily and are critical in schistosome invasion, migration, and nutrition through proteolytic degradation of hemoglobin and other host proteins.56,57 For example, the extracts from adult S. japonicum and S. mansoni possess aspartic proteinase activity and are able to digest hemoglobin.58 RNAi-mediated reductions in transcript levels of aspartic protease significantly retard S. mansoni growth.59 In addition, vaccination with its recombinant protein in mice lead to a significant decrease in mean total worm burdens (21−38%) and female worm burdens (22−40%) for S. japonicum.60 Although these enzymes differ in optimum pH, substrate specificity, susceptibility to host protease inhibitors, and redox potential, they might synergistically work to degrade proteins.57 Female worms degrade host hemoglobin from 330 000 red blood cells per hour, while males catabolize hemoglobin from 30 000 cells per hour.61 This finding is in agreement with our results indicating that levels and activity of proteases are higher in female worms than in males.62,63 We propose that protease activity might be more important for female than male worms. Intracellular signal transduction passes information to downstream components within a cell, propagating signals, and triggering changes in function or state. Highly expressed proteins in this subcategory found in male samples and not in females were similar to Ras-related protein Rab-1A, 2, 3, 5, 8A, 14 and putative Rab GDP-dissociation inhibitor. The Rab family is the largest branch of the Ras superfamily of small GTPases and is involved in the regulation of vesicle formation, transport, fusion, and secretion.64,65 For Plasmodium falciparum, it is reported that Ras-related proteins are cytoplasmic and plasma membrane-associated, and play an essential function in vesicular trafficking.66 The homotypic fusion between the early endosomes depends on Rab5, regulating hemoglobin endocytosis in Leishmania donovani.67 During turnover of the tegument surface membrane of schistosome, multilaminate vesicles from tegumentary cell bodies fuse with the apical membrane, releasing contents to form the membranocalyx. To prevent extension of the surface membrane, the tegument sloughs the membranocalyx into the host blood and internalizes the apical membrane as vesicles.22 Rab proteins might perform essential functions in tegument turnover. Because males have a larger surface exposed to the host defensive system than females that are in the gynecophoric canal, males might undergo more tegumental turnover in reacting with the host immune system. A previous study reported that expression of 5267



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filament polymerization, and proteasome core complex were identified. These results provide valuable information for further understanding the development and sexual maturity of schistosomes in the definitive host and for screening vaccine candidates and drug targets against schistosomiasis.

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(5) 2012 Year Book of Health in the People’s Republic of China; Peking Union Medical College Press: Peking, China, 2012. (6) Stelma, F. F.; Talla, I.; Sow, S.; Kongs, A.; Niang, M.; Polman, K.; Deelder, A. M.; Gryseels, B. Efficacy and side effects of praziquantel in an epidemic focus of Schistosoma mansoni. Am. J. Trop. Med. Hyg. 1995, 53 (2), 167−70. (7) Ismail, M.; Metwally, A.; Farghaly, A.; Bruce, J.; Tao, L. F.; Bennett, J. L. Characterization of isolates of Schistosoma mansoni from Egyptian villagers that tolerate high doses of praziquantel. Am. J. Trop. Med. Hyg. 1996, 55 (2), 214−8. (8) WHO.TDR Strategic Direction for Research: Schistosomiasis. World Health Organization: Geneva, Switzerland, 2002. (9) Pearce, E. J. Progress towards a vaccine for schistosomiasis. Acta Trop 2003, 86 (2−3), 309−13. (10) Gobert, G. N.; Stenzel, D. J.; McManus, D. P.; Jones, M. K. The ultrastructural architecture of the adult Schistosoma japonicum tegument. Int.J.Parasitol 2003, 33 (14), 1561−75. (11) Quack, T.; Beckmann, S.; Grevelding, C. G. Schistosomiasis and the molecular biology of the male-female interaction of S. mansoni. Berl. Munch. Tierarztl. Wochenschr. 2006, 119 (9−10), 365−72. (12) Aronstein, W. S.; Strand, M. A glycoprotein antigen of Schistosoma mansoni expressed on the gynecophoral canal of mature male worms. Am. J. Trop. Med. Hyg. 1985, 34 (3), 508−12. (13) Armstrong, J. C. Mating Behavior and Development of Schistosomes in the Mouse. J. Parasitol. 1965, 51, 605−16. (14) Loverde, P. T.; Chen, L. Schistosome female reproductive development. Parasitol. Today 1991, 7 (11), 303−8. (15) Vogel, H. Ueber den Einfluss des Geschlechtspartners auf Wachstum und Entwicklung bei Bilharzia mansoni und bei Kreuzpaarungen zwischen verschiedenen Bilharzia-Arten. Zentralblalt fur Bakteriologie Parasitenkunde I. Abt. Orig 1941, 148, 178−196. (16) Popiel, I.; Basch, P. F. Reproductive development of female Schistosoma mansoni (Digenea: Schistosomatidae) following bisexual pairing of worms and worm segments. J. Exp. Zool. 1984, 232 (1), 141−50. (17) LoVerde, P. T.; Niles, E. G.; Osman, A.; Wu, W. Schistosoma mansoni male−female interactions. Can. J. Zool. 2004, 82 (2), 357− 374 %@ 0008−4301.. (18) Moore, D. V.; Sandground, J. H. The relative egg producing capacity of Schistosoma mansoni and Schistosoma japonicum. Am. J. Trop. Med. Hyg. 1956, 5 (5), 831−40. (19) Pearce, E. J.; MacDonald, A. S. The immunobiology of schistosomiasis. Nat. Rev. Immunol. 2002, 2 (7), 499−511. (20) Braschi, S.; Borges, W. C.; Wilson, R. A. Proteomic analysis of the schistosome tegument and its surface membranes. Mem. Inst. Oswaldo Cruz. 2006, 101 (Suppl 1), 205−12. (21) Braschi, S.; Wilson, R. A. Proteins exposed at the adult schistosome surface revealed by biotinylation. Mol. Cell. Proteom. 2006, 5 (2), 347−56. (22) Mulvenna, J.; Moertel, L.; Jones, M. K.; Nawaratna, S.; Lovas, E. M.; Gobert, G. N.; Colgrave, M.; Jones, A.; Loukas, A.; McManus, D. P. Exposed proteins of the Schistosoma japonicum tegument. Int. J. Parasitol. 2010, 40 (5), 543−54. (23) Hockley, D. J.; McLaren, D. J. Schistosoma mansoni: changes in the outer membrane of the tegument during development from cercaria to adult worm. Int. J. Parasitol. 1973, 3 (1), 13−25. (24) Wilson, R. A.; Barnes, P. E. An in vitro investigation of dynamic processes occurring in the schistosome tegument, using compounds known to disrupt secretory processes. Parasitology 1974, 68 (2), 259− 70. (25) Zhang, M.; Han, Y.; Zhu, Z.; Li, D.; Hong, Y.; Wu, X.; Fu, Z.; Lin, J. Cloning, expression, and characterization of Schistosoma japonicum tegument protein phosphodiesterase-5. Parasitol Res. 2012, 110 (2), 775−86. (26) Jones, M. K.; Gobert, G. N.; Zhang, L.; Sunderland, P.; McManus, D. P. The cytoskeleton and motor proteins of human schistosomes and their roles in surface maintenance and host-parasite interactions. Bioessays 2004, 26 (7), 752−65.

ASSOCIATED CONTENT

S Supporting Information *

Figure 1, Immunofluorescence microscopy of an adult S. japonicum worm labeled with impermeant biotinylation reagent. Table 1, Common tegument-exposed proteins from female and male S. japonicum with annotations. Table 2, Tegumentexposed proteins which are highly expressed in female S. japonicum with annotations. Table 3, Tegument-exposed proteins which are highly expressed in male S. japonicum with annotations. Table 4, Theoretical two-dimensional (MW, pI) distributions of identified tegument-exposed proteins of female and male worms. Table 5, List of proteins in statistically significant GO categories (P-value ≤ 0.05) under biological processes, molecular functions and cellular components. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(Z.F.) E-mail: [email protected]. Tel.: +86 21 3429 3618. Fax: +86 21 3429 3619. Address: 518 Ziyue road, Minhang, Shanghai 200241, People’s Republic of China. (J.L.) E-mail: [email protected]. Tel.: +86 21 3429 3440. Fax: +86 21 5408 1818. Address: 518 Ziyue road, Minhang, Shanghai 200241, People’s Republic of China. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank Shanghai Applied Protein Technology Co. Ltd. for the technology support. Hong Yu and Shanghai Shengzi Information Tech Co., Ltd. for data analysis assistance. This work was supported by National Natural Science Foundation of China (No. 31172315, 81271871), Agro-scientific Research in the Public Interest (No. 200903036), the China Postdoctoral Science Foundation (No. 2012M510630), Science Technology and Development Foundation of Shanghai (No. 12140902700) and basic scientific research operation cost of state-level public welfare scientific research courtyard (No. 2013JB18).

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