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Aproximacion al analisis proteomico e inmunologico de extractos proteicos de cercaria y verme adulto de Schistosoma mansoni y deteccion de uno de los candidatos a vacuna, Sm28GST, de un aislado venezolano.

A combined proteomic and immunologic approach for the analysis of Schistosoma mansoni cercariae and adult worm protein extracts and the detection of one of the vaccine candidates, Sm28GST, from a Venezuelan parasite isolate.

INTRODUCTION

The trematode Schistosoma mansoni, that affects approximately 200 million persons worldwide, can survive many years in the human porto-hepatic system due to a variety of immunologic and biochemical adaptive mechanisms. Therefore, it is very important to understand the molecular organization of each parasite stage, especially those that are more vulnerable to the immune attack and chemotherapy. The identification of the major parasite proteins would give clues to the schistosome metabolism and to target molecules involved with the host immune system (1). One of the major problems we face for the development of efficient anti-schistosome vaccines is the lack of information about the degree of homology among vaccine candidate molecules from parasites of different geographic origins, since very few parasite isolates and strains have been immunochemically and biochemically characterized. Based on this, our group initially tried to circumvent that limitation evaluating if selected regions from potential protective molecules were conserved worldwide, by demonstrating if sera from mice infected with three different S. mansoni isolates, as well as four other schistosome species (S. rodhaini, S. guineensis, S. bovis, S. haematobium) (2) were able to recognize the selected peptides. The recognition of the different peptides by all of these tested sera was the guarantee that we were working with conserved regions of target molecules. One of them was the well studied Sm28GST protein, which was recognized by the sera of mice infected with three different S. mansoni isolates (2).

In the current study, the protein parasite repertoire was studied using a proteomic approach of two different parasite stages, the cercaria and adult worms from a Venezuelan isolate (JL). As far as we know, previous works were performed with the well known Puerto Rican strain (3-7) and an Egyptian strain (8), while others did not identify the parasite origin (9-11). It was important to analyze other parasite isolates in order to have a more comprehensive picture of its protein composition, studying worms from different geographic regions.

A brief review of the application of proteomics to the identification of novel schistosome candidate vaccines was recently published (12). In our study, particular emphasis was given to the identification of the gluthation S transferase protein (Sm28GST) (13) by 2D SDS-PAGE and mass spectrometry, in parallel with its immunologic detection using serum from rabbits immunized with synthetic peptides derived from the Sm28GST protein. This molecule, localized in the tegument, protonephridia and the female reproductive system (13), is one of the schistosome vaccine candidates (14) under evaluation in our laboratory as part of a multi-component synthetic peptide vaccine. Being aware that one of the strategies for survival of parasites under different conditions is the re dundancy of key molecules that could allow them to evade the host immune response (15, 16), we combined a proteomic and an immunological approach to confirm if a potential protective epitope synthesized chemically is present in different Sm28GST isoforms of isolated parasites from different geographic origin.

MATERIALS AND METHODS

Preparation of parasite material

Free swimming cercariae from the Venezuelan isolate JL, kindly provided by Dr. Italo Cesari (Instituto Venezolano de Investigaciones Cientificas, IVIC, Venezuela), were shed in water from Biomphalaria glabrata snails infected 45 days before. These larvae were frozen and afterwards lyophilized. The cercarial protein extract (CE) was achieved solubilizing the lyophilized cercariae in 7M Urea, 2M Thiourea, 4% CHAPS, 20 mM Tris pH 8, 65 mM DTT (IEF buffer). Male and female adult worms from the same isolate were collected from experimentally infected outbred golden hamsters after liver perfusion. Worms were washed and homogenized in phosphate-buffered saline (PBS) containing protease inhibitors (1mM PMSF; 1mM EDTA) in an ice bath, and centrifuged at 12,000 g for 2 h at 4[degrees]C. The supernatant was lyophilized and considered as the adult worm protein extract (AWE), which was solubilized in IEF buffer. Both extracts were processed using one precipitation with 100% acetone (v/v), overnight -20[degrees]C, followed by a wash of the pellet with 80% acetone (v/v). AWE was additionally cleaned through a Sephadex G-25 micro column for desalinization.

Two dimensional electrophoresis

After quantification using the Bradford protein determination method (17) with bovine serum albumin (Fraction V, Sigma) as a standard, 75 [micro]g of CE and 66,2 [micro]g of AWE were used for bidimensional electrophoresis in a Bio Rad IEF system (18). For first evaluations, IEF was performed with 7 cm strips with a 3-10 non-linear pH range. A 5-8 pH linear range was used afterwards for better resolution. IEF was carried out at 250 V for 15 min, 4,000 V for 2h, and then gradually increasing to a total of 20,000 V/h at 20[degrees]C, at a maximum current of 50 [micro]A/strip. After IEF, strips were reduced in equilibration buffer (6M urea, 2% SDS, 0.375M Tris HCl pH 8.8, 20% glycerol, 0.025% (w/v) bromophenol blue) containing 130 mM DTT for 15 min and were then alkylated in equilibration buffer containing 135 mM iodoacetamide for 20 min. The second dimension was performed in 12% acrylamide gels and the gels were silver stained (19). Spots were analyzed by the PDQuest Program (Bio Rad).

Mass spectrometry

Spots were excised and processed for mass spectrometry in the Plateforme Proteomique de l'Esplanade, Institut de Biologie Moleculaire et Cellulaire, Strasbourg, France.

In gel digestion. For protein identification, stained protein spots were picked out from the corresponding gels. The gel digestion procedure was carried out as described by Rabilloud et al. (20). Selected spots have been washed with 2 cycles of 100 [micro]L of 25 mM ammonium carbonate buffer (N[H.sub.4]HC[O.sub.3]) / 100 [micro]L of acetonitrile (ACN) / dehydration. Between these two cycles, reduction was achieved by 45 min treatment with 10 mM DTT in N[H.sub.4]HC[O.sub.3] buffer (100 [micro]L) at 56[degrees]C and alkylation reaction was performed by addition of 100 [micro]L of 25 mM iodoacetamide in 25 mM N[H.sub.4]HC[O.sub.3] buffer for 45 min at room temperature. All treatments were performed under shaking. The final dried spots were rehydrated with three volumes of trypsin (Promega, V5111), 12.5 ng/[micro]L, in 25 mM N[H.sub.4]HC[O.sub.3] buffer (freshly diluted) and the digestion was performed at room temperature overnight.

Afterwards, 5 [micro]L of 35% [H.sub.2]O / 60% ACN / 5% HCOOH were added and the mixture vigorously agitated for 15 min in order to extract tryptic peptides.

MALDI mass spectrometry. MALDI mass measurement was carried out on an Biflex III (Bruker-Daltonics GmbH, Bremen, Germany) matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF) used in reflector positive mode. A saturated solution of [alpha]-cyano-4-hydroxycinnamic acid in 50% water / 50% acetonitrile was used as a matrix. Mass spectra were internally calibrated with trypsin autolysis peaks (m/z = 842.510 and m/z = 2211.105). Monoisotopic peptide masses were assigned and the peak list transferred through MS BioTools[TM] program (Bruker Daltonics, Bremen, Germany) as input to search against NCBI non-redundant protein sequence database. Variable modifications (methionine oxidation and cysteine carbamidomethylation) were taken into account and the peptide mass error was limited to 50 ppm. Identifications were performed after MALDI-TOF analysis and searches on MASCOT (MatrixScience) over databanks without any restricted taxonomy.

Synthetic peptides

Peptides were manually synthesized using Merrifield's protocol (21) for the t-Boc based solid-phase peptide synthesis, modified by Houghten et al. (22) for the simultaneous multiple peptide synthesis. Glycine and cysteine amino acids were introduced at both carboxy and amino termini to allow polymerization (23, 24). Two polymerizable synthetic peptides were constructed after the sequence from the protein Sm28GST: IMT-232, (aa: 115-130) (25, 26), and IMT-234 (the carboxi-terminal region aa: 190-211) (27, 28). Both epitopes correspond to the border of the slot from the active site of the enzyme.

Rabbit immunization

Two New Zealand rabbits were immunized subcutaneously with these peptides (250 [micro]g / dose / rabbit) emulsified (1:1) in Complete Freund adyuvant (CFA) in the first dose and in Incomplete Freund adyuvant (IFA) in the second and third boosts (days 15 and 30). Rabbits were bled 10 days post third immunization, under anesthesia (ketamine chlorhydrate: 10 mg/Kg). Their maintenance and manipulations were carried out according to legislation and guidelines established by the Asociacion Venezolana de Bioterios. All sera were stored at -20[degrees]C until use.

Western blots

The immunized rabbit sera were tested against AWE and CE in 1D SDS-PAGE. AWE and CE 2D gels were transferred to PVDF membranes and exposed to the anti-IMT-232 and 234 rabbit serum at a 1:100 dilution. Anti-rabbit IgG conjugated to horseradish peroxidase was used at a 1:2,000 dilution and Western blots were developed with TMB Membrane Peroxidase Substrate (KPL; Gaithersburg MD, USA).

Schistosoma mansoni database

Search of the genes corresponding to Sm28GST was performed using SchistoDB database (www.schistodb.net) in order to investigate the gene copy number responsible for the expression of the different isoforms of this protein.

BLASTp analysis for homology

The degree of homology between cross reactive proteins was performed using BLASTp (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

RESULTS

Proteomic analysis

In the 2D SDS-PAGE of adult worm protein extract (AWE) (Fig. 1a), a total of 172 individualized spots are shown, but only the most prominent or the strongest signals (47 spots, 27.3%), based in the highest concentration of proteins in each silver stained spot, were excised, processed and analyzed by MS. Several spots were identified (Table I): probable ER-60 luminal cystein protease precursor, superoxide dismutase (Cu-Zn), thioredoxin peroxidase, actin-2, enolase, fructose biphosphate aldolase, Sm28GST, GST-Omega, 30 kD glycoprotein, among others. Among the spots analyzed, few of them were separated sequentially, suggesting the occurrence of post translational modifications in the following proteins: actin-2 molecules (19 spots), superoxide dismutases (Cu-Zn) (2 spots), enolases (2 spots), disulfide-isomerase ER-60 precursors (2 spots), Sm28GST (5 spots), fructose-biphosphate aldolases (2 spots), thioredoxin peroxidase (3 spots) and 30-kDa glycoprotein (4 spots).

In the 2D SDS-PAGE of CE (Fig. 2a), the resolution of the spots was higher than in the AWE. In fact, at least 257 individualized spots were observed, compared to the 172 observed to the mature adult worm preparation, which is expected to be composed of a more complex and rich array of functional molecules. The most prominent spots were identified as (Table I): thioredoxin peroxidase 2, actin (Sj), HSP70 (Sj), a disulfide isomerase homologue, chaperonin, ATP synthase, enolase, prohibitin, serpin, glyceraldehyde-3-phosphatedehydrogenase (GA3PDH) mayor larval surface antigen, citrate synthase, 14-3,3 protein, Sm21.7, 28K antigen, GST- Omega, and others. They correspond to 33 of the total spots detected (12.8%). The spots separated sequentially correspond to: disulfide isomerase homologue (3 spots), S. japonicum actin (4 spots), enolase (2 spots), S. japonicum citrate synthase (2 spots), serpin (2 spots), 28K antigen (2 spots) and ATP synthase (3 spots).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

The theoretical isoelectric points and molecular masses were obtained from the MASCOT software.

The able II shows the proteins found in this study and compares them with those found by other authors in previous studies. Differences will be stressed in the Discussion.

Immunochemistry

When rabbit antisera against the synthetic peptides derived from the Sm28GST molecule were tested against AWE and CE in 1D SDS-PAGE and Western blot (data not shown), it was observed a strong signal in AWE and a less intense in CE, in the range of 28 kDa. But when these rabbit sera were used against 2D gel electrophoresed AWE and CE blotted onto PVDF membranes, we observed the recognition of at least 5 sequential spots to AWE (Fig. 1b) and 2 to CE (Fig. 2b) in the range of 28 kDa and of some less intense spots, corresponding to higher molecular weights. Among them, two spots corresponding to enolase were identified in both parasite preparations.

Schistosoma mansoni database

Once it was evident that some spots were arranged in a sequential distribution on the 2D gels, we investigated if they corresponded to different isoforms of the Sm28GST and if they were originated from a single copy gene or a multi-gene family. Search on the genome of S. mansoni demonstrated that Sm28GST is a single copy gene.

BLAST analysis for homology

Based on the fact that rabbit immune anti- Sm28GST peptide sera also recognized enolase, it was carried out the analysis of the homology between these cross reactive proteins. It was found that the only region of both proteins that had a significant degree of homology (42%) contained 13 out of 22 aa of the Sm28GST peptide IMT-234.

DISCUSSION

A limited number of studies have been carried out on the proteomic analysis of the larval and adult stages of schistosomes (11, 12, 29, 30, 31) and it is noteworthy to point out that only two international S. mansoni strains, the Puerto Rican (3-7) and an Egyptian strain (8), were analyzed so far. Therefore, it is necessary to evaluate if other S. mansoni strains and isolates from different geographic origins are homogeneous from the molecular point of view, since recent studies have shown molecular diversity and polymorphism occurrence in this parasite, as reported in mucin proteins (32).

Particular emphasis was laid on the Sm28GST, since synthetic peptides derived from this protein are one of the protective peptides evaluated in outbred mice in our laboratory (unpublished results). The presence of antioxidants, such as Sm28GST are crucial in the detoxification and antioxidant mechanisms of the helminths and they are probably implicated in protection against oxidative stress, specially originated by the heme group (9) in adult worms. The importance of this identification lies in the fact that the production of neutralizing antibodies against the Sm28GST activity is related to the reduction of eggs (anti-fecundity effect) in tissues and feces (14, 33, 34), and reduction of the viability of mature eggs (27). Additionally, the previous demonstration of antigenic community among different schistosome species and different vaccine candidate molecules including Sm28GST, also at the epitopic level, argues in favor of the relevance of this conserved protein (2).

Perez-Sanchez et al. (35, 36) afforded valuable information about the protein composition of the tegument of adult S. bovis male and female worms. A range of tegumental and soluble proteins was identified using 2D immunoblots (known as immunome), including orthologs of the vaccine candidate Sm28GST. For that reason, a comparative analysis of the proteome from different studies is summarized in Table II and demonstrates the degree of homology among different parasite strains. It is noteworthy that different extracts and also different techniques for protein extraction are compared and protein representation may reflect these differences. That is the case of the studies carried out by

Curwen et al. (3), van Balkom et al. (10), and Braschi et al. (6), even when they identified some of the proteins found in this study in the S. mansoni tegument (Table II), also found others not observed in this work.

The limitations of the different proteomic studies based on the different protocols used for the preparation of the parasite material were demonstrated by Curwen et al. (4) and Knudsen et al. (5) who have shown that most cercarial proteins are secreted from the acetabular glands, but only when they artificially induced the invasive behavior in vitro. Knudsen et al. (5) found that uninduced free swimming cercaria released relatively small number of non-acetabular gland proteins including enolase, Sm28GST and actin (7), while the major secreted proteins were histolytic serine proteases that likely facilitate degradation of host skin tissue barrier, and factors that may contribute to immune evasion. In our work, we also found a schistosome serpin (serine protease inhibitor) that could possibly correspond to that previously reported in cercarial secretions (4, 7). Heat shock proteins, like HSP70 had also been identified as a major component of acetabular gland secretion (4). Sm21.7, a component of the schistosome surface or sub adjacent tegument, and Sm28GST, were released from the parasite in studies conducted by Hansell et al. (7). We also identified a group of schistosome glycolitic enzymes and kinases, known to be abundant in the cytoplasm of larval cells These are presumably released as holosecretions from the acetabular cells (4, 5) and included: GA3PDH, citrate synthase, ATP-synthase and enolase Hansell et al. (7) and Salter (37) have identified elastase (SmCE) isoforms (not identified in our study, probably because we did not work with excretory and secretory products), proteins associated with calcium function and cytosolic proteins such as heat shock protein chaperones.

Curwen et al. (3) reported the Schistosoma mansoni soluble proteome across the four different life-cycle stages. They showed a high degree of quantitative and qualitative similarities in spot patterns that were greater between adjacent stages. Their list included several of the first generation vaccine candidates such as triose phosphate isomerase, gluthation S transferase and fatty acid binding protein. They concluded that most of them were cytosolic enzymes.

In our study, the AWE lacks important excretory and secretory proteins that are poorly represented, such as the enzymes present in the vomit of this trematode, like the aspariginil endopeptidase (Sm32), cathepsin B (Sm31), cathepsin D, etc., which are only enriched when the adults worms are in vitro cultured (38). It is also possible that some of the excretory-secretory molecules, like the ones excreted by the cercarial cephalic or acetabular glands, are not represented in a detectable level by this CE preparation.

Comparing the results obtained with the JL isolate and the other two studied strains, only two proteins: SJCHGC09398 (S. japonicum) and a 30 kDa glycoprotein, were not previously reported. So far, the relevance of these two proteins remains unknown. Some spots in the 2D gel were identified as Schistosoma japonicum (Sj) proteins. The common identity of S. japonicum and S. mansoni proteins by MS is a confirmation that these parasites share conserved protein sequences.

It is noteworthy to mention the presence and abundance of 19 actin-2 spots in the AWE. Actin is a major component of the schistosome spines of the tegument (39, 40) and it seems to be one of the targets of Praziquantel (41). The abundance of spots identified as actin-2 might be the result of processing by tegument proteases and some of these fragments could play a major role in the host-parasite interaction.

A combined immunologic and proteomic approach allowed the identification of five spots corresponding to Sm28GST proteins of similar molecular weight in AWE, while in CE were observed only two Sm28K antigens Both proteins, Sm28GST and Sm28K, have identical amino acid sequences Since it has been demonstrated previously that there is only one copy of gene for the Sm28GST (42), the abundance of isoforms of the Sm28GST could be the result of post- translational modifications. The fact that the different isoforms were recognized by rabbit sera of animals immunized with synthetic peptides indicates that the selected peptides include a conserved region that is homologous to the original protein. Nevertheless, it is necessary to sequence those proteins using MS/MS techniques, since they could be different proteins sharing similar antigenic regions. On the other hand, there is a Sm28GST omega protein that is observed in the gel but is not recognized by rabbit serum. Searching in the protein databank, it seems a totally different protein from Sm28GST(43).

The less intense signals seen in the immunoblots, corresponding to higher molecular weights, could be nonspecific cross-reactions or precursors. Two of them were identified as enolase isoforms Protein sequence homology search of GST and enolase was performed by BLASTp. It was found that the only region in both protein sequences that had a significant degree of homology could explain a partial cross-reactivity of the serum observed in the figures 1b and 2b.

We conclude that the sequence contained in the polymeric synthetic peptides used for rabbit immunization was able to imitate a well known antigenic region from the original Sm28GST protein The existence of these similar proteins could only be detected by 2D SDS-PAGE It could be interesting to investigate if the same epitopes are present in Sm28GST isoforms of the Puerto Rican and the Egyptian S. mansoni strains performing a Western blot of 2D gels using protein extracts of these parasites and the antisera against the Sm28GST synthetic peptides.

The production of different isoforms for this protein may be the expression of one of the multiple evasion strategies of this very complex parasite, since it could protect itself from the immune attack using alternative isoforms when recognized by the immune system. Also, they might have different functional capabilities under the multiple environments and conditions the different parasite stages are exposed to. Therefore, a plausible explanation is that the redundancy of this protein could allow that some of the isoforms might be involved in an immune evasion mechanism.

The proteomic approach certainly allows the identification of some potential target proteins of the protective immune response that are being evaluated as members of a future anti-S mansoni vaccine. And also to demonstrate if selected peptides could induce antibodies able to recognize different isoforms of these proteins. Since there are few isolates of this species available, the results presented herein enriches the information about the composition of this rather sophisticated parasite, allowing to foresee the potential efficacy of vaccine candidates against parasites from different geographic regions.

ABBREVIATIONS

2D SDS-PAGE, two dimensional sodium dodecyl sulfate poliacrilamide gel electrophoresis; CHAPS, 3-[(3-Cholamidopropyl)dimethylammonio] propanesulfonic acid; Tris, tris(hydroxymethyl) aminomethane; DTT, Ditiotreitol; IEF, isoelectric focusing; EDTA, ethylenediaminetetraacetic acid; MALDI, Matrix-Assisted Laser Desorption/Ionization; NCBI, National Center for Biotechnology Information; IMT, Instituto de Medicina Tropical; PVDF, Polyvinylidene Fluoride

ACKNOWLEDGEMENTS

This work was funded by FONACIT-CNRS Project No 2004000012, FONACIT LANPIP Project No 2000001639 and FONACIT Project G-2005000387. We thank the anonymous referees whose comments helped to improve the manuscript.

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(39.) Cohen C, Reinhardt B, Castellani L, Norton P, Stirewalt M. Schistosome surface spines are "crystals" of actin. J Cell Biol 1982; 95(3): 987-988.

(40.) Braschi S, Wilson RA. Proteins exposed at the adult schistosome surface revealed by biotinylation. Mol Cell Proteomics 2006; 5(2):347-356.

(41.) Tallima H, El Ridi R. Re: is actin the praziquantel receptor? Int J Antimicrob Agents 2007; 30(6):566-567.

(42.) McNair AT, Dissous C, Duvaux-Miret O, Capron A. Cloning and characterisation of the gene encoding the 28-kDa Glutathione S-transferase of Schistosoma mansoni. Gene 1993; 124(2):245-249.

(43.) Girardini J, Amirante A, Zemzoumi K, Serra E. Characterization of an omega-class glutathione S-transferase from Schistosoma mansoni with glutaredoxinlike dehydroascorbate reductase and thiol transferase activities Eur J Biochem 2002; 269: 5512-5521.

Sandra Losada [1], Laurence Sabatier [2], Philippe Hammann [2], Christelle Guillier [3], Cesar Matos [4], Henry Bermudez [1], Maria Angelita Lorenzo1 and Oscar Noya [1].

[1] Seccion de Biohelmintiasis, Instituto de Medicina Tropical, Escuela de Medicina "Luis Razetti", Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela.

[2] Institut de Biologie Moleculaire et Cellulaire, CNRS, Strasbourg, France.

[3] INRA/CNRS UMR5184, Dijon, France. 4Unidad de Servicios en Trematodiasis, Centro de Microbiologia, Instituto Venezolano de Investigaciones Cientificas, IVIC, Venezuela.

Corresponding author: Oscar Noya. Seccion de Biohelmintiasis, Instituto de Medicina Tropical, Escuela de Medicina "Luis Razetti", Facultad de Medicina, Universidad Central de Venezuela. Caracas, Venezuela. Telf-Fax: 0058-212-6053563. E-mail: noyaoo@yahoo.com.
TABLE I

MS RESULTS OF AWE AND CE PROTEIN SPOTS FROM Schistosoma
mansoni JL STRAIN

Sample   Stage     Identification
Number   extract

1302     AWE       Actin-2 [Schistosoma mansoni]

4204     AWE       glutathione S-transferase omega [Schistosoma
                   mansoni]

1206     AWE       Actin-2 [Schistosoma mansoni]

2204     AWE       30 kDa glycoprotein

5203     AWE       thioredoxin peroxidase [Schistosoma mansoni]

3203     AWE       CDP-glucose 4,6-dehydratase [Vibrio fischeri
                   ES114]

6202     AWE       hypothetical protein V12B01_21691 [Vibrio
                   splendidus 12B01]

206      AWE       Actin-2 [Schistosoma mansoni]

5202     AWE       thioredoxin peroxidase [Schistosoma mansoni]

2302     AWE       Actin-2 [Schistosoma mansoni]

3204     AWE       thioredoxin peroxidase [Schistosoma mansoni]

204      AWE       Actin-2 [Schistosoma mansoni]

208      AWE       Actin-2 [Schistosoma mansoni]-C-terminal fragment

1204     AWE       Actin-2 [Schistosoma mansoni]

205      AWE       Actin-2 [Schistosoma mansoni]

4202     AWE       30 kDa glycoprotein

2205     AWE       30 kDa glycoprotein

1205     AWE       Actin-2 [Schistosoma mansoni]-C-terminal fragment

1208     AWE       Actin-2 [Schistosoma mansoni]

6201     AWE       Superoxide dismutase [Cu-Zn]

1209     AWE       Actin-2 [Schistosoma mansoni]

7202     AWE       Glutathione S-transferase 28 kDa (GST 28) (SM28
                   antigen)

2206     AWE       30 kDa glycoprotein

203      AWE       Actin-2 [Schistosoma mansoni]

301      AWE       Actin-2 [Schistosoma mansoni]

207      AWE       Actin-2 [Schistosoma mansoni]

5901     AWE       Probable protein disulfide-isomerase ER-60
                   precursor (ERP60)

6901     AWE       Probable protein disulfide-isomerase ER-60
                   precursor (ERP60)

8201     AWE       Glutathione S-transferase 28 kDa (GST 28) (SM28
                   antigen)

2301     AWE       Actin-2 [Schistosoma mansoni]

1301     AWE       Actin-2 [Schistosoma mansoni]

302      AWE       Actin-2 [Schistosoma mansoni]

1207     AWE       Actin-2 [Schistosoma mansoni]

4301     AWE       Actin-2 [Schistosoma mansoni]

8202     AWE       Glutathione S-transferase 28 kDa (GST 28) (SM28
                   antigen)

6401     AWE       Enolase

9203     AWE       SJCHGC06124 protein [Schistosoma japonicum]

7203     AWE       Superoxide dismutase [Cu-Zn]

7201     AWE       Glutathione S-transferase 28 kDa (GST 28) (SM28
                   antigen)

7401     AWE       Enolase

9301     AWE       Fructose-bisphosphate aldolase

9302     AWE       Fructose-bisphosphate aldolase

9201     AWE       Glutathione S-transferase 28 kDa (GST 28) (SM28
                   antigen)

1303     CE        actin [Xanthophyllomyces dendrorhous]

8203     CE        thioredoxin peroxidase 2 [Schistosoma mansoni]

2202     CE        similar to NM_011967 proteasome (prosome.
                   macropain) subunit. alpha type 5 in Mus musculus [
                   S. japonicum]

6302     CE        PREDICTED: similar to Actin-87E isoform 1 [Apis
                   mellifera]

9304     CE        SJCHGC05011 protein [Schistosoma
                   japonicum]=similar to T-complex protein 1 subunit
                   gamma (BLAST)

1302     CE        unknown [Schistosoma mansoni] = Serpin (BLAST)

602      CE        protein disulfide isomerase homologue [Schistosoma
                   mansoni]

603      CE        protein disulfide isomerase homologue [Schistosoma
                   mansoni]

2301     CE        unknown [Schistosoma mansoni] = Serpin (BLAST)

601      CE        protein disulfide isomerase homologue [Schistosoma
                   mansoni]

2602     CE        HSP70 [Schistosoma japonicum]

8301     CE        glutathione S-transferase omega [Schistosoma
                   mansoni]

4302     CE        actin [Schistosoma japonicum]-C terminal fragment

2601     CE        SJCHGC09424 protein [S. japonicum] = ATP synthase
                   subunit beta. mitochondrial precursor (BLAST)

305      CE        14-3-3 epsilon [Schistosoma mansoni]

302      CE        14-3-3 protein

9305     CE        Glyceraldehyde-3-phosphate dehydrogenase (Major
                   larval surface antigen) (P-37)

7301     CE        SJCHGC06488 protein [Schistosoma japonicum]=
                   prohibitin protein (BLAST)

4601     CE        SJCHGC09129 protein [Schistosoma japonicum]=GroEL
                   protein

5402     CE        actin [Schistosoma japonicum]

4401     CE        actin [Schistosoma japonicum]

1601     CE        SJCHGC09424 protein [Schistosoma japonicum]=ATP
                   synthase subunit p. mitochondrial precursor
                   (BLAST)

2401     CE        actin [Schistosoma japonicum]

9401     CE        SJCHGC00653 protein [Schistosoma japonicum] =
                   citrate synthase (BLAST)

301      CE        14-3-3 protein

9201     CE        Antigen Sm21.7

8501     CE        enolase

1602     CE        SJCHGC09424 protein [S. japonicum] = ATP synthase
                   subunit [beta]. mitochondrial precursor (BLAST)

9301     CE        28K antigen [Schistosoma mansoni]

9501     CE        enolase

9302     CE        28K antigen [Schistosoma mansoni]

5401     CE        actin [Schistosoma japonicum]

9402     CE        SJCHGC00653 protein [Schistosoma japonicum] =
                   citrate synthase (BLAST)

Sample   Theoretical   NCBI           Score    Error   %
Number   Mw/pI         Accession No   Mascot   (ppm)   cover

1302     41999/5.30    1703114        173      26      45

4204     27827/5.90    28628851       266      33      64

1206     41999/5.30    1703114        70       21      30

2204     18273/7.81    632506         65       64      54

5203     21909/6.08    5163492        156      26      63

3203     41308/5.47    59710790       83       49      23

6202     41308/5.47    59710790       60       54      22

206      41999/5.30    1703114        143      33      40

5202     21909/6.08    5163492        136      16      63

2302     41999/5.30    1703114        153      40      55

3204     21909/6.08    5163492        153      22      63

204      41999/5.30    1703114        117      41      35

208      41999/5.30    1703114        124      26      46

1204     41999/5.30    1703114        145      32      47

205      41999/5.30    1703114        111      17      33

4202     18273/7.81    632506         108      36      82

2205     18273/7.81    632506         94       26      82

1205     41999/5.30    1703114        124      32      39

1208     41999/5.30    1703114        135      29      49

6201     15883/6.09    267013         133      30      77

1209     41999/5.30    1703114        162      39      51

7202     23861/6.56    121700         183      24      69

2206     18273/7.81    632506         118      26      82

203      41999/5.30    1703114        127      18      33

301      41999/5.30    1703114        226      37      64

207      41999/5.30    1703114        156      30      46

5901     54785/6.30    729434         293      25      55

6901     54785/6.30    729434         313      22      55

8201     23861/6.56    121700         217      14      76

2301     41999/5.30    1703114        167      31      50

1301     41999/5.30    1703114        197      31      59

302      41999/5.30    1703114        216      40      65

1207     41999/5.30    1703114        163      28      47

4301     41999/5.30    1703114        189      22      63

8202     23861/6.56    121700         167      9       52

6401     47421/6.18    3023710        207      28      62

9203     36902/8.83    56758570       91       29      33

7203     15883/6.09    267013         124      33      69

7201     23861/6.56    121700         189      26      60

7401     47421/6.18    3023710        308      24      69

9301     39963/7.63    1703248        289      13      76

9302     39963/7.63    1703248        290      13      77

9201     23861/6.56    121700         209      71      83

1303     41938/5.38    1150540        82       41      28

8203     21909/6.08    10281263       165      38      76

2202     27476/5.22    29841012       84       24      38

6302     42157/5.30    66509793       68       39      25

9304     24514/9.41    76154176       70       29      32

1302     29100/5.05    2623846        108      33      45

602      54463/4.92    312018         203      37      47

603      54463/4.92    312018         113      26      27

2301     29100/5.05    2623846        89       46      33

601      54463/4.92    312018         161      38      38

2602     71613/5.12    2829289        75       28      19

8301     27827/5.90    28628851       212      24      56

4302     41999/5.30    6979994        118      24      35

2601     56074/5.85    56758584       167      25      39

305      28850/4.85    6649234        187      25      60

302      28468/4.74    790658         113      24      40

9305     36640/8.16    120709         253      28      67

7301     30259/5.54    56755505       160      31      54

4601     62614/9.51    56753359       115      47      27

5402     41999/5.30    6979994        236      50      69

4401     41999/5.30    6979994        236      27      63

1601     56074/5.85    56758584       178      19      39

2401     41999/5.30    6979994        171      20      51

9401     52487/7.98    56759284       96       24      27

301      28468/4.74    790658         149      20      50

9201     21789/6.85    417776         96       15      44

8501     47421/6.18    1002616        219      20      53

1602     56074/5.85    56758584       157      19      39

9301     23861/6.56    10164          180      31      59

9501     47421/6.18    1002616        248      39      61

9302     23861/6.56    10164          196      21      65

5401     41999/5.30    6979994        96       171     40

9402     52487/7.98    56759284       113      21      28

Note: Unidentified spots are not shown.

AWE: Adult worm protein extract.

CE: Cercarial protein extract.

Theoretical Mw/pI: Theoretical molecular weight/Isoelectric point.

% cover: proportion of the sequence that fit to the masses of the
peptides submitted for identification.

TABLE II

REVIEW TABLE COMPARING RESULTS OBTAINED IN THIS STUDY
WITH ADULT AND CERCARIAL PREPARATIONS
FROM S. mansoni JL STRAIN AND PREVIOUS WORKS

Protein                 MW/pH         AWE      CE       Curwen
                                      JL       JL       et al.,
                                      strain   strain   2004 [3]
                                                        AS
                                                        PR strain

HSP70 (S. japonicum)    71,613/5.12            X        X EGG

SJCHGC09129 protein     62,614/9.51            X
(S. japonicum) =
chaperonin

SJCHGC09424 protein     56,074/5.85            X
(S. japonicum) ATP
synthase

Probable protein        54785/6.30    X
disulfide-isomerase
ER-60 precursor
(ERP60)

Disulfide isomerase     54,463/4.92            X
homologue

SJCHGC00653 protein     52,487/7.98            X
(S.
japonicum)Citrate
synthase

Enolase                 47,421/6.18   X        X        X

Actin 2                 41,999/5.30   X        X        X

paraFructose 1,6        39,963/7.63   X                 X
biphosphate aldolase

SJCHGC06124 (S.         36,902/8.83   X
japonicum)

GA3PDH                  36,640/8.16            X        X LUNG, WORM

SJCHGC06488 protein     30,259/5.51            X
(S. japonicum)=
prohibitin

Serpin                  29,100/5.05            X        X CERCARIA

14-3-3 epsilon          28,850/4.85            X        X

14-3-3 protein          28,468/4.74            X        X

GlutationStransferase   27,827/5.90   X        X
Glutation S
transferase omega

SJCHGC05011 protein     24,514/9.41            X
(S. japonicum)=
chaperonin

28 kDa antigen          23,861/6.56   X        X        X
Glutathione
S-transferase (S.
mansoni)

Thioredoxin             21,909/6.08            X
peroxidase  2

Sm21.7 antigen          21,789/6.85            X        X LUNG, WORM

Thioredoxin             21,315/6.10   X
peroxidase  1

30 kDa glycoprotein     18,273/7.81   X

Cu/Zn superoxide        15,883/6.09   X                 X LUNG, WORM
dismutase

Protein                 Knudsen     van Balkom   Braschi     Curwen
                        et al.,     et al.,      et al.,     et al.,
                        2005 [5]    2005 [10]    2006 [6]    2006 [4]
                        CS          AWT          AWT         CS
                        PR strain   ND strain    PR strain   PR strain

HSP70 (S. japonicum)    X           X            X

SJCHGC09129 protein                 X
(S. japonicum) =
chaperonin

SJCHGC09424 protein     X           X            X
(S. japonicum) ATP
synthase

Probable protein                    X
disulfide-isomerase
ER-60 precursor
(ERP60)

Disulfide isomerase                 X            X
homologue

SJCHGC00653 protein     X           X            X
(S.
japonicum)Citrate
synthase

Enolase                 X           X            X

Actin 2                 X           X                        X

paraFructose 1,6        X           X            X           X
biphosphate aldolase

SJCHGC06124 (S.
japonicum)

GA3PDH                  X           X

SJCHGC06488 protein                 X
(S. japonicum)=
prohibitin

Serpin                              X                        X

14-3-3 epsilon                      X

14-3-3 protein          X           X            X

GlutationStransferase               X
Glutation S
transferase omega

SJCHGC05011 protein                 X
(S. japonicum)=
chaperonin

28 kDa antigen          X           X                        X
Glutathione
S-transferase (S.
mansoni)

Thioredoxin             X           X
peroxidase  2

Sm21.7 antigen          X           X            X

Thioredoxin                         X
peroxidase  1

30 kDa glycoprotein

Cu/Zn superoxide        X                        X
dismutase

Protein                 Delcroix    Hansell     El Ridi &
                        et al.,     et al.,     Tallima,
                        2007 [9]    2008 [7]    2009 [8]
                        GC          CS          SS
                        ND strain   PR strain   Egypt strain

HSP70 (S. japonicum)                X

SJCHGC09129 protein
(S. japonicum) =
chaperonin

SJCHGC09424 protein
(S. japonicum) ATP
synthase

Probable protein
disulfide-isomerase
ER-60 precursor
(ERP60)

Disulfide isomerase
homologue

SJCHGC00653 protein
(S.
japonicum)Citrate
synthase

Enolase

Actin 2                                         Similar X

paraFructose 1,6                    X           X
biphosphate aldolase

SJCHGC06124 (S.
japonicum)

GA3PDH

SJCHGC06488 protein
(S. japonicum)=
prohibitin

Serpin                              X

14-3-3 epsilon

14-3-3 protein                                  X

GlutationStransferase
Glutation S
transferase omega

SJCHGC05011 protein
(S. japonicum)=
chaperonin

28 kDa antigen                      X           X
Glutathione
S-transferase (S.
mansoni)

Thioredoxin
peroxidase  2

Sm21.7 antigen                      X

Thioredoxin                                     X
peroxidase  1

30 kDa glycoprotein

Cu/Zn superoxide        X
dismutase

AWT: adult worm tegument. CS: Cercarial secretions. SS:
Schistosomula secretions. GC: gut content.
AS: All stages. AWE: Adult worm protein extract. CE: Cercarial
protein extract. PR strain: Puerto Rican strain.
ND strain: Non described strain.
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Article Details
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Author:Losada, Sandra; Sabatier, Laurence; Hammann, Philippe; Guillier, Christelle; Matos, Cesar; Bermudez,
Publication:Investigacion Clinica
Date:Jun 1, 2011
Words:6938
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