Identification and Characterization of a Smad2 Homologue from Schistosoma mansoni, a Transforming Growth Factor-beta Signal Transducer*

Ahmed Osman, Edward G. Niles, and Philip T. LoVerdeDagger

From the Department of Microbiology and Center for Microbial Pathogenesis, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, New York 14214

Received for publication, July 6, 2000, and in revised form, December 5, 2000


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Smad proteins are essential intracellular signal transducers of the transforming growth factor-beta (TGF-beta ) superfamily. The TGF-beta superfamily signals through phosphorylation and activation of R-Smad proteins, receptor-regulated Smads, by heteromeric complexes of ligand-specific type I and type II serine/threonine kinase receptors. R-Smads receive a signal from the activated receptor complex and transmit it to the nucleus. A cDNA was isolated that encodes a 649-amino acid protein found to be homologous to members of R-Smad subfamily with highest homology scored to clawed African frog and human Smad2. The Schistosoma mansoni homologue (SmSmad2) was overexpressed in bacteria as a Sj26-GST fusion protein and used to raise specific antibodies. The IgG fraction of the immunized rabbit serum identified 70- and 72-kDa protein bands in Western analysis of schistosome extracts. Treatment with alkaline phosphatase removed the 72-kDa band, which indicates that this band represents the phosphorylated form of schistosome Smad2. SmSmad2 was localized in the subtegument, parenchymal cells, and sex organs in both male and female worm cryosections. Similar results were also obtained from the analysis of the Smad2 mRNA distribution pattern revealed by in situ hybridization of adult worm pair paraffin sections. SmSmad2 mRNA levels were determined by reverse transcriptase-polymerase chain reaction in different mammalian host developmental stages and found to be constitutively expressed. SmSmad2 was also found to interact with a previously identified SmTbeta R-I, a serine/threonine type I kinase receptor. Furthermore, SmSmad2 was shown to undergo phosphorylation by constitutively active forms of SmTbeta R-I in vitro. In addition, SmSmad2 localized in the nuclei of mink lung epithelial cells upon treatment with TGF-beta 1. These data indicate that the SmSmad2 responds to the TGF-beta signals by interaction with receptor I, which phosphorylates it, whereupon it translocates into the nucleus presumably to regulate target gene transcription and consequently elicit a specific TGF-beta effect.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Schistosomiasis is a parasitic disease second only to malaria in prevalence. According to the World Health Organization records, about 300 million people in 76 countries are currently infected. More than 600 million people live in endemic areas and are at risk of contracting the disease (1). The egg is the major cause of the pathology and complications of the disease such as portal hypertension, liver fibrosis hemorrhage, and death (2, 3). Female schistosomes initiate sexual development and start laying eggs in response to a stimulus by male worms (4). Very little is known about the nature of the signal(s) involved or the mechanism(s) of this process. Furthermore, schistosomes reside in portal circulation surrounded by host molecules such as antibodies, hormones, cytokines, and growth factors. The tegument layer covering the parasite worm body represents the interface between the parasite and its host environment. Several studies reported the identification of receptors for growth factors and other host molecules on the surface of the parasite (5-7). Transforming growth factor-beta (TGF-beta )1 serine/threonine kinase receptor I (Tbeta RI) was recently identified in Schistosoma mansoni, SmTbeta R-I (5). The presence of SmTbeta RI on the surface of the parasite raises the possibility of the involvement of TGF-beta or a TGF-beta -like ligand in the host-parasite interactions or in the stimulation of female worm maturation by male schistosomes.

TGF-beta is a superfamily of secreted polypeptides including various forms of TGF-beta , bone morphogenetic proteins (BMPs), activins, inhibins, and many other structurally related factors (8). Members of this family regulate cell migration, differentiation, adhesion, multiplication, and death throughout the life span of an organism (9). These different effects result from the changes in the expression of key target genes, which depend on the cell type and developmental stage. Three classes of intracellular signal transducers are involved in the signaling cascade of the TGF-beta family as follows: a family of plasma membrane serine/threonine kinase receptors (known as type I and type II receptors); a family of cytoplasmic proteins that either transmit the signal from the activated receptor complex to the nucleus or down-regulate the signaling pathway, known as Smad family; and gene-specific nuclear DNA-binding partners that associate with Smad proteins forming transcriptional complexes (8, 10).

The signaling pathway is initiated by ligand binding to the extracellular domain of the type II receptor kinase. The cytoplasmic domain of type II receptor catalyzes the phosphorylation and concomitant activation of the specific type I receptor kinase. Once activated, the type I receptor binds to and phosphorylates a member of a subset of the Smad family, receptor-regulated Smads (R-Smads). The phosphorylation of R-Smads destabilizes the complex with type I receptor releasing the activated R-Smad (11) allowing it to associate with a related protein Smad4, known as comediator Smad or co-Smad (12, 13). The heteromeric complex (R-Smad/co-Smad) then translocates into the nucleus (14) where it associates with specific DNA-binding partner(s) that direct it to the regulatory region of target gene(s) (9).

Two structural motifs in the type I receptor and the corresponding R-Smad, the L45 loop and L3 loop, respectively, contain subtype-specific residues and determine the specificity of the interaction between these two members of the signaling cascade (15, 16). R-Smads and co-Smads consist of conserved N- and C-terminal domains and MH1 and MH2 domains. The MH1 domain possesses a DNA binding activity (17, 18), and the MH2 domain exerts a transcriptional activation function (19, 20). These conserved domains are linked by an intervening, nonconserved linker region that varies both in length and sequence (8). In the basal state, the MH1 domain of R-Smads interacts with and inhibits the transcriptional (20) and biological (19) activities of the MH2 domain. Upon phosphorylation by type I receptor kinase, this auto-inhibitory MH1-MH2 interaction of R-Smad is relieved, allowing the association of the activated R-Smad with co-Smad. The MH2 domain of R-Smads contains receptor phosphorylation sites located at the very C-terminal end of the protein (SSXS) (11, 21). Co-Smads lack this phosphorylation signature motif and consequently do not serve as substrates for receptor I kinases (11). The MH2 domain also mediates the interaction and association of R-Smads with type I receptors (11).

We report herein the identification of S. mansoni Smad2 homologue (SmSmad2). We define its expression level in different developmental stages and the localization of the protein and mRNA transcripts in adult worm sections. We also demonstrate that schistosome protein performs the biological activities exhibited by other Smad2 homologues, including its interaction with and phosphorylation by a schistosome homologue of Tbeta R-I (SmTbeta RI) and nuclear translocation in response to human TGF-beta .

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of SmSmad2 cDNA-- A fragment of about 1 kb spanning part of the MH1 domain and the linker region was coisolated during our previous work on SmRas cDNA (22). An oligonucleotide representing the 3'-end of the coding region of SmRas (5'-GACGTCGACTCATTGTATACAACATTTTGC-3') amplified a 1-kb fragment. The PCR product was cloned into TOPO-TA vector (Invitrogen) and sequenced. Sequence analysis of this fragment showed that it is related to members of R-Smad subclass of the Smad family. The DNA fragment was labeled with [alpha -32P]dCTP by Megaprime labeling kit (Amersham Pharmacia Biotech) and used as a probe to screen S. mansoni adult worm pair cDNA library. Eight positive clones were identified, and the sequence information showed that two contain the entire coding region of S. mansoni homologue of Smad2 protein (SmSmad2).

Expression of SmSmad2 and Production of Specific Antiserum-- Smad2 cDNA representing the MH1 domain was amplified from the parent phagemid with specific 5'- primer (Smad2-cod-5') containing KpnI (underlined) and SalI (italicized) sites (5'-GGTACCGTCGACCGGAATTCAAAATGAGTCTCTTTACAAGTCCAC-3') and a reverse primer representing the complementary sequence of bp 533-558. The 430-bp PCR product was subcloned in TOPO-TA vector (Invitrogen), sequenced, and then digested with KpnI at the 5'-end and HpaI at the 3'-end, a unique site present at bp 542 of the cDNA sequence. The excised DNA fragment was then recloned into the parent phagemid digested with the same enzymes yielding a cDNA lacking the 5'-untranslated region. The recombinant phagemid was cleaved with SalI and NotI, and the full-length SmSmad2 insert was subcloned in pGEX-4T-1 linearized with the same enzymes. The recombinant pGEX-SmSmad2 plasmid was overexpressed in TOP10 cells (Invitrogen) using 0.5 mM isopropyl-1-thio-beta -D-galactopyranoside at 20 °C for 5 h. The recombinant Sj26-SmSmad2 fusion protein (200 µg) was used to immunize a New Zealand rabbit with complete Freund's adjuvant for the primary injection and incomplete adjuvant for the following two booster doses with 3-week intervals between each dose. The immunized rabbit serum was then passed over protein A-Sepharose column, and the IgG fraction was eluted and used in Western blots and immunocytolocalization in adult worm pair cryosections.

Western Blot and Immunocytolocalization-- S. mansoni adult worm pairs were perfused from infected hamsters and homogenized in an extraction buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2.5 mM EDTA, 5% glycerol, 1% Triton X-100, and mammalian protease inhibitor mixture; Sigma). The homogenate was sonicated for 1 min and centrifuged at 35,000 rpm for 1 h at 4 °C, and the soluble fraction was aliquoted and stored at -80 °C until used.

Schistosome extract was treated with calf intestinal alkaline phosphatase (CIAP; Life Technologies, Inc.) at concentrations of 0.2 and 1 unit/µg extract for 30-60 min at 37 °C. The untreated extract, with and without 10× CIAP buffer, along with CIAP-treated extract were separated on 7.5% SDS-PAGE. SDS gels were transferred onto polyvinylidene difluoride membrane, and the blots were probed with IgG fractions from preimmune rabbit and SmSmad2-GST-immunized rabbit sera (0.5 µg/ml). Biotinylated goat anti-rabbit IgG (0.5 µg/ml; Zymed Laboratories Inc.) was used to probe the reacting primary antibodies, and this in turn was detected using Z-avidin horseradish peroxidase conjugate (0.5 µg/ml; Zymed Laboratories Inc.). The reactions were developed with tetramethyl benzidine reagent (Zymed Laboratories Inc.).

The IgG fractions (5 µg/ml) were also used to detect SmSmad2 native protein in adult worm cryosections. Biotinylated goat anti-rabbit IgG (5 µg/ml; Zymed Laboratories Inc.) and Z-avidin FITC conjugate (5 µg/ml; Zymed Laboratories Inc.) or streptavidin-Cy5 conjugate (10 µg/ml; Zymed Laboratories Inc.) were used to localize the primary rabbit IgG antibodies. Probed sections were evaluated using a Bio-Rad MRC100 confocal microscope equipped with a krypton laser. A 674-nm filter was used to visualize the Cy5-probed sections.

RT-PCR and in Situ Hybridization-- SmSmad2 transcripts were detected using two approaches. RT-PCR was employed to determine the total transcription level of SmSmad2 in different developmental stages. In situ hybridization was used to detect where SmSmad2 transcripts are localized and in which tissues the protein is synthesized. In RT-PCR, total RNA was purified from different life stages of S. mansoni that are related to the definitive human host, starting with 3-h schistosomules and up to paired 45-day-old adult worms using RNA-stat 60 reagent (Tel-Test, Inc.). RNA was copied by reverse transcription (22), and cDNA was used as templates for subsequent PCR amplification. A primer pair corresponding to bp 1614-1636 and the complementary sequence of bp 1902-1926 was used to amplify a fragment that spanned part of the MH2 domain. SmRXR1 (23), a clone previously identified in our laboratory and shown to be constitutively expressed in all human host-related stages of the parasite, was used as an internal control for the RT-PCRs. A primer pair representing part of the SmRXR1 AB-domain (23) was used to amplify a 460-bp DNA fragment. To remain in the linear range of amplification, 26 cycles of PCR were performed. The amplification products were size-separated in a 2.0% agarose gel, stained with ethidium bromide, and quantified using the Molecular Analyst gel documentation system (Bio-Rad). SmSmad2 PCR products were normalized to those of SmRXR to eliminate the differences in quality and/or quantity of the input RNA in the RT reaction.

For the in situ hybridization, an antisense oligonucleotide corresponding to the complementary sequence of bp 695-744 was synthesized, labeled with biotin using Bright-Star biotin labeling kit (Ambion), and used to probe paraffin-embedded sections using mRNAlocator-hyb kit (Ambion) following the manufacturer's instructions. Two primers, one represents the sense sequence of SmSmad2 (5'-GGAAGAGGTTCATCTTTTGGTTCACATCATTCTCG-3', bp 620-654) and the other represents the antisense sequence of Smp14 gene (a female-specific gene that produces an eggshell protein) (24) (5'-GCCTCCACCATATCCACTATCGCCATAACCGCTATCACAATCGCTACC-3'), were similarly processed and used as a negative and positive controls, respectively. The probed sections were developed using streptavidin-alkaline phosphatase conjugate (mRNAlocator-Biotin kit; Ambion), and the sections were lightly counterstained using nuclear fast red. The sections were examined and photographed using Nikon microphot digital microscope.

SmSmad2 Interaction with SmTbeta R-I-- To evaluate the interaction of SmSmad2 with SmTbeta R-I, DNA encoding wild type and mutant forms of SmSmad2 and SmTbeta R-I were cloned into the yeast GAL4 two-hybrid vectors (activation domain (AD) and DNA-binding domain (BD)). SmSmad2 cDNA (wild type, wt), in which the EcoRI site at position 1709 was mutated to GAGTTC to maintain the same encoded amino acid sequence was amplified. Amplification was performed using Pfu DNA polymerase (Stratagene) with Smad-5'-cod as a forward primer and a reverse primer in which NotI site (underlined) was inserted (NotI-Smad-3'; 5'-GCGGCCGCACTGACAGATGTGCAAGGATG-3'). The PCR product represents a SmSmad2 cDNA in which the stop codon was removed and DNA encoding 3 amino acids (VGG) was inserted downstream of the C-terminal motif (TSVS) of SmSmad2 (SmSmad2-3'). The PCR product was cloned in pCRII-Blunt-TOPO vector (Invitrogen). The wt SmSmad2 was digested with EcoRI/SalI and cloned in pBD-GAL4-cam and pAD-GAL4-2.1 vectors (Stratagene) downstream of GAL4 DNA-binding domain and GAL4 activation domain, respectively. SmSmad2-3' was digested with EcoRI and SalI/XhoI and cloned in pBD-GAL4-cam linearized with EcoRI and pACT2.1 (CLONTECH) and linearized with XhoI to encode fusion proteins of GAL4-BD/SmSmad2-3'and GAL4-AD/SmSmad2-3', respectively. SmTbeta R-I (wild type) (5) was mutated in its GS domain (284TCSGSGSGKPLLVQRTVARQ303) to attempt to construct both constitutively active and kinase-inactive forms (25). Three mutants were constructed to form T299D, the expected putative kinase- derivative, Q303D, the constitutively active form, and the double mutant T299D/Q303D. DNA sequence analysis was performed for the mutant forms of SmTbeta R-I to confirm the mutations. SmTbeta R-I constructs were digested with EcoRI/SalI and cloned into pBD-GAL4-cam and pAD-GAL4-2.1 vectors linearized with the same enzymes, to generate fusion proteins with GAL4-BD and GAL4-AD, respectively. The two forms of SmSmad2 and the four constructs of SmTbeta R-I in both BD and AD vectors were transformed individually, into Y190 competent cells using Fast Track Yeast Transformation Kit (Geno Technology, Inc.) as controls to evaluate the ability of these genes to activate host cell reporter gene expression. Transformed cells were plated onto synthetic dextrose medium (SD) supplemented with amino acid dropout solutions lacking either Trp or Trp, His or Leu, or Leu, His for BD vectors and AD vectors, respectively. LacZ filter assay was performed according to Stratagene Two-hybrid instruction manual. To test for an interaction, an AD vector was cotransformed with binding domain vector, and the transformed cells were plated onto SD supplemented with amino acid dropout solutions lacking either Leu, Trp, or Leu, Trp, His. LacZ filter assay was also performed on this transformation series. 3-Amino-1,2,4-triazole, a metabolic inhibitor for histidine biosynthesis, was added to histidine-lacking medium to a final concentration of 25 mM to inhibit the leaky expression of his3 reporter gene. Control plasmids, pGAL4, p53, pLaminC, pSV40, and a combination of p53 + pSV40 and pLaminC + pSV40 (Stratagene) were transformed along with each transformation experiment to provide positive and negative controls.

GST pull-down analysis was performed to attempt to confirm the results obtained from the two-hybrid interaction in yeast. In this experiment, the four constructs of SmTbeta R-I (wt and the three mutants, described above) were subcloned into pCITE-4a vector (Novagen) using appropriate restriction enzymes. Recombinant SmTbeta R-I-pCITE vectors were expressed in vitro in rabbit reticulocyte lysates with [35S]methionine (PerkinElmer Life Sciences) using STP-3 transcription/translation system (Novagen) following the supplier's instructions. The regions encoding the SmSmad2 linker and MH2 domain were PCR-amplified (primer pairs bp 488-512 and a complementary sequence of bp 1455-1480, and bp 1424-1448 and a complementary sequence of bp 2045-2068 with SalI site at the 5'-end, respectively). The PCR products were cloned into TOPO-TA vector (Invitrogen) and then subcloned into pGEX-4T-1 (Amersham Pharmacia Biotech) and pET42a (Novagen) prokaryotic expression vectors, respectively. DNA encoding SmSmad2-3' was also subcloned in pET42a vector. Recombinant prokaryotic expression vectors were overexpressed in bacteria, and the GST fusion proteins were affixed to glutathione-Sepharose beads. Binding reactions, between SmSmad2 proteins (wild type and mutant form) and SmTbeta R-I in vitro translated proteins, were performed by adding 2 µl of the translation reactions to GST fusion protein-coupled beads equivalent to about 2 µg of the fusion protein. The final volume of the reactions was adjusted to 100 µl by adding binding buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10% glycerol, 0.15% Nonidet P-40). The reactions were incubated at room temperature for 1 h and then continued overnight at 4 °C. Glutathione-Sepharose beads were added to facilitate the recovery of the reactive beads, and the samples were precipitated, washed four times, and boiled in 1× SDS loading buffer, and the bound proteins were size-separated in 12% SDS-PAGE. The gels were stained, destained, treated with Entensify (PerkinElmer Life Sciences), dried, and exposed to x-ray film. Developed x-ray films were scanned, and the specific reaction bands were quantified using Molecular Analyst Gel Documentation System (Bio-Rad). Parental pGEX-4T-1 was used to generate recombinant GST, which was affixed to glutathione beads and used to pre-treat the translation reactions to remove the nonspecific background. The equivalent of 2 µg of GST-coupled beads was used to pre-clear the amount of lysate used per reaction. GST beads were also included in the binding reactions to serve as a negative control in the pull-down experiment.

In Vitro Phosphorylation of SmSmad2 by SmTbeta R-I-- To determine whether SmSmad2 can serve as a substrate for SmTbeta R-I, we examined the ability of SmTbeta R-I to phosphorylate SmSmad2. In principle, the phosphorylation reactions were performed as outlined above for the pull-down experiment with few differences. Briefly, wild type and mutant forms of SmTbeta RI were synthesized in vitro using rabbit reticulocyte lysates and unlabeled methionine. The lysates were pre-cleared with GST-coupled beads as before and then aliquoted into four tubes (each equivalent to 4 µl of original lysate). To each was added GST or a GST fusion protein containing SmSmad2-linker, SmSmad2-MH2, or SmSmad2-3' coupled to the glutathione-Sepharose beads. Phosphorylation was performed in a 100-µl volume in the presence of 10 µCi of [32P-gamma ]ATP, for 1 h at room temperature in a buffer similar to the binding buffer (above) except it lacks Nonidet P-40. The beads were collected, washed four times with the reaction buffer, boiled in 1× SDS-loading buffer, and loaded onto 12% SDS-PAGE. The gels were processed as before and subjected to autoradiography.

To determine more precisely the phosphorylation sites in Smad2 C-terminal motif, we constructed several mutant forms of SmSmad2-MH2 domain in which the potential phosphorylation sites were mutated to alanine. Four mutant forms were constructed in which Ser649, Ser647, Ser647,649, and Thr646-Ser647,649 were mutated to alanine (TSVA, TAVS, TAVA, and AAVA, respectively). The mutant forms were generated by PCR amplification using oligonucleotides encoding the corresponding mutations. PCR products were cloned in TOPO-TA-pCR2.1 vector (Invitrogen), sequenced to confirm the presence of the specific mutations, digested with EcoRI/XhoI, and cloned in pCITE-4a vector (Novagen). Smad2-MH2-wt and SmTbeta R-I-wt and the mutant forms of both proteins were synthesized in vitro using unlabeled methionine. Kinase reactions were performed using 5 µl of each SmTbeta R-I translation reaction and 5 µl of each SmSmad2-MH2 translation reactions in a reaction buffer containing 25 mM Tris, pH 8.0, 50 mM NaCl, 5% glycerol, and 10 µCi of [gamma -32P]ATP. Reactions were allowed to proceed for 1 h at room temperature, and then 5 µg of IgG fraction of alpha -SmSmad2 rabbit antiserum was added to each reaction and incubated for 1 h at room temperature, and the antigen-antibody complexes were precipitated by adding 20 µl of protein A-Sepharose (Amersham Pharmacia Biotech). Immunoprecipitates were washed 4 times with 1× phosphate-buffered saline containing 0.1% Tween 20, boiled in 1× SDS-loading buffer, and loaded onto 12% SDS-PAGE. The gels were processed as before and subjected to autoradiography.

TGF-beta 1 Responsiveness of SmSmad2 Protein-- To determine the responsiveness of the SmSmad2 protein to TGF-beta 1 signal, SmSmad2 cDNA was cloned into the eukaryotic expression vector pCMV-GST (kindly provided by Dr. Randall Reed). The pGEX-SmSmad2 vector was digested with BamHI, and the 2.1-kb fragment was inserted in the pCMV-GST vector linearized with the same enzyme. The recombinant transformants were checked for the presence of the insert in the correct orientation by colony PCR using pGEX-fwd primer (Amersham Pharmacia Biotech) as a forward primer and a reverse primer representing the complementary sequence of bp 533-558. DNA sequence of positive samples was determined to confirm the presence of the start ATG codon of SmSmad2 cDNA in frame with the GST sequence. Double CsCl-purified pCMV-GST-SmSmad2, as well as the parent vector pCMV-GST, were used to transfect mink lung epithelial cells (MV1Lu; ATCC CCL-64) employing Effectene transfection reagent (Qiagen) following the supplier's instructions. Forty eight hours post-transfection, cells were fixed with 2% paraformaldehyde containing 0.1% Triton X-100. The fixed cells were probed with anti-GST mouse monoclonal antibody (7.5 µg/ml). Anti-mouse biotinylated IgG (7.5 µg/ml; Zymed Laboratories Inc.) was used as a secondary antibody reagent. The biotinylated antibodies were detected using Z-avidin-FITC conjugate (5 µg/ml; Zymed Laboratories Inc.). A nonrelevant plasmid (pCDNA-LacZ; Invitrogen) that lacks a GST tag was used as a negative control. Fluorescent cells were examined with Bio-Rad MRC-100 confocal microscope.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation, Identification, and Computer Analyses of SmSmad2 cDNA-- The amino acid sequence predicted by the original 1-kb DNA fragment showed strong homology to the MH1 domain of R-Smad subfamily. This DNA fragment was used to screen lambda ZAPII S. mansoni, adult worm pair cDNA library. Eight positive phages were identified of which two were shown to contain the full-length coding region of a S. mansoni homologue of Smad2 cDNA (SmSmad2). SmSmad2 cDNA encodes a 649-amino acid protein representing the largest Smad2 protein identified to date. NCBI blast search showed that SmSmad2 exhibits a high degree of homology to Smad2 proteins of Xenopus laevis (26) and humans (27-30). Homology is restricted to the MH2 domain (69% identity, 77% similarity) and, to a lesser extent, the MH1 domain (61% identity and 70% similarity, Fig. 1). SmSmad2 C-terminal phosphorylation motif is "TSVS" which is different from the standard SSXS motif, present in all R-Smads. The SmSmad2 MH2 domain also contains the L3 loop, the region that determines the specificity of interaction with the specific type I receptor (16). The sequence of the L3 loop is highly conserved and invariant among R-Smads of similar signaling specificity (e.g. Smad2 and Smad3 that are activated by Tbeta R-I and activin type I receptor, or Smad1, Smad5, and Smad8 activated by BMP or BMP-like type I receptors) (16). L3 loop in SmSmad2 has the specific subtype residues Arg613 and Thr616 (Fig. 1) which dictate the interaction of Smad2 and Smad3 with the Tbeta R-I and the activin type I receptors. Another characteristic feature maintained in the MH2 domain of SmSmad2 is the conservation of all the 5 subtype-specific residues required for the interaction of Smad2 and Smad3 with SARA (Smad Anchoring for Receptor Activation), Ile527, Phe532, Tyr552, Trp554, and Asn567 (Fig. 1) (31). SARA is an adaptor protein that interacts with both Smad2/Smad3 and type I receptor and facilitates Smad2/Smad3 MH2 domain interaction with type I receptor and consequently its phosphorylation and activation. The interaction of SARA with R-Smad subfamily is restricted to Smad2 and Smad3 (31, 32).


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Fig. 1.   Pileup analysis and peptide alignment of MH1 and MH2 domains of S. mansoni Smad2 (SmSmad2; GenBankTM accession number AF232025) with homologous domains in other R-Smad proteins. Schematic representation of the functional domains of SmSmad2 cDNA (top). Alignment of SmSmad2 MH1 and MH2 domains (bottom). DmSmad2, Drosophila melanogaster Smad2 (GenBankTM accession number AAD11458); XlSmad2, Xenopus laevis Smad2 (GenBankTM accession number L77885); HsSmad2, Smad3, and Smad1 are human Smad2, Smad3, and Smad1, respectively (GenBankTM accession numbers NP_005892, BAA22032, and S68987, respectively). %I and %S are identity and similarity scores. Black boxed sequence represents the L3 loop. White boxed sequences are R-Smad subtype-specific residues of L3 loop. White sequence represents the C-tail, and underlined sequences are the C-terminal phosphorylation motifs. "" specifies Smad2/Smad3-specific recognition residues by SARA.

Expression, Western Blot Analysis, and Immunocytolocalization-- SmSmad2 was overexpressed in bacteria as a GST fusion protein. The fusion protein was used to generate a specific rabbit polyclonal antiserum. The IgG fraction of the immunized rabbit serum detected two proteins of about 70 and 72 kDa in Western blots of schistosome worm extracts (Fig. 2, panel C, lanes 2 and 3). To determine whether the slower migrating species is a phosphorylated form of Smad2, schistosome extracts were treated with calf intestinal alkaline phosphatase prior to PAGE analyses. After CIAP treatment, only the 70-kDa protein band was detected (Fig. 2, panel C, lane 1). In this particular experiment the 70-kDa band is distorted due to the high concentration of CIAP in the sample. This result indicates that the 72-kDa protein represents a phosphorylated form of SmSmad2 in the parasite extract. Phosphorylation may result from receptor phosphorylation and activation of the SmSmad2 or mitogen-activated protein kinase phosphorylation of the linker region (33, 34). IgG fraction of preimmune rabbit serum was used as a negative control (Fig. 2, panel B).


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Fig. 2.   Identification of native SmSmad2 in adult worm extracts. Coomassie Blue-stained SDS gel (panel A) and Western blot analysis of schistosome extract using IgG fraction of preimmune (panel B) and SmSmad2-immunized (panel C) rabbit sera. Lanes 1-3 represent schistosome extract treated with CIAP (1 unit/µg protein) for 1 h at 37 °C, schistosome extract incubated with 1× CIAP buffer for 1 h at 37 °C, and untreated extract, respectively. Arrows point to 72- and 70-kDa protein.

Native SmSmad2 was localized in adult worm cryosections probed with anti-SmSmad2 antibodies. SmSmad2 was detected in the tubercles of the tegument, in parenchymal cells of male and female worms (Fig. 3, panels A and B), as well as in the vitellaria (Fig. 4, panels IC and IIIC), the developing embryo in egg (Fig. 4, panel IIC) and the ovary (Fig. 3, panel C, and Fig. 4, panel IC) of the female, and the testis of the male (data not shown). The use of Cy5 as a detection reagent enabled us to overcome the auto-fluorescence that occurs in female vitellaria and unequivocally observe SmSmad2 reactivity in the vitellaria and embryo contained within the uterine egg (Fig. 4).


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Fig. 3.   Immunocytolocalization of SmSmad2. S. mansoni adult worm cryosections were probed with anti-SmSmad2 IgG (panels A-C), and preimmune rabbit IgG (panel D) were detected using a FITC conjugate. Panel A, male (M) and female (F) cross-sections, showing fluorescence in tubercles and parenchymal cells. Panel B, male section showing fluorescence in tubercles (T) and cells of parenchyma. Panel C, female worm showing fluorescence in ovary (O). Panel D, male worm showing no fluorescence. G, gut.


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Fig. 4.   S. mansoni adult worm cryosections probed with anti-SmSmad2 IgG and detected using Cy5 conjugate. Row A shows the phase contrast fields. Row B shows background fluorescence detected when using 520-nm filter (used to visualize FITC-probed sections). Note the autofluorescence in vitellaria (groups I-III) and eggshell (group II). Row C shows the specific fluorescence of Cy5 conjugate at wavelength 674 nm. Note absence of autofluorescence in eggshell (E) (group II) and fluorescence in ovary (O) (group I), vitellaria (V) (groups I-III), developing miracidium in egg (group II) and in parenchyma and subtegumental tissues of male (group III). G, gut; F, female; and M, male. No specific fluorescence was observed in sections probed with preimmune rabbit IgG.

RT-PCR and in Situ Hybridization-- The level of SmSmad2 mRNA was determined in different schistosome developmental stages by RT-PCR. In the mammalian host stages, SmSmad2 mRNA was found to be detected as early as 4 days post-infection (Fig. 5, lane 2). Compared with the mRNA level of the internal control (SmRXR1), SmSmad2 mRNA exhibits relatively constant levels throughout development.


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Fig. 5.   SmSmad2 mRNA levels in schistosome at different stages of development. RT-PCR of SmSmad2 RNA compared with the internal control, SmRXR1. Panel A is a diagrammatic representation of the normalized mRNA levels of SmSmad2. Panel B is a photograph of 2% agarose gel showing the amplification products of SmSmad2 and SmRXR1 (arrows). Lanes 1-4 are 3-h-, 4-day-, 7-day-, and 15-day-old schistosomules, respectively. Lanes 5-8 are 20-, 28-, 32-, and 35-day worm pairs, respectively. Lanes 9-13, are single sex male worms, single sex female worms, bisex male worms, bisex female worms, and adult worm pairs, respectively.

SmSmad2 mRNA was also detected in paraffin sections of adult worms by in situ hybridization. The transcripts were localized in the subtegumental and parenchymal cells in male and female worms (Fig. 6, panels C and D) and in sexual tissues such as vitellaria (Fig. 6, panel D), uterus, and ovary (Fig. 6, panel C) in female worm. The positive control (Smp14) showed strong localization in female vitelline cells (Fig. 6, panel A) and the negative control showed minimal background reactivity in both male (Fig. 6, panel B) and female sections (data not shown).


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Fig. 6.   Localization of SmSmad2 mRNA transcripts in tissue sections of S. mansoni adult worms by in situ hybridization. Panel A represents the reaction of the positive control probe (p14 antisense primer) in the vitellaria (V) of the female worm (F). Male worms (M) were negative as expected (data not shown). Panel B shows the negative control reaction using SmSmad2 sense primer in male sections. Female sections (not shown) exhibited the same level of reactivity. Panels C and D show the specific reactions of SmSmad2 antisense probe in male and female sections. Male sections show positive reactions in subtegument and parenchymal cells. Female sections show reactivity in vitellaria (V), ovary (O), developing embryo in uterine egg (E), and subtegumental cells. G, gut; ST, subtegument; P, parenchyma.

SmSmad2 Interaction with SmTbeta R-I-- Initially, the yeast two-hybrid system was employed to evaluate the direct interaction between SmSmad2 and SmTbeta R-I. Several mutant constructs for both SmSmad2 and SmTbeta R-I were evaluated. SmSmad2-3', a SmSmad2 mutant in which the stop codon was removed to extend the reading frame beyond the TSVS phosphorylation motif, was expected to retain SmTbeta R-I binding but not to undergo receptor-mediated phosphorylation at its C-terminal end (35). Mutations in the downstream sequence of the GS region of SmTbeta R-I corresponding to Q303D and T299D were expected to yield a constitutively active and a kinase-negative version of type I receptor, respectively (21, 25, 36, 37). Both SmSmad2 (wt) and SmSmad2-3' when fused downstream to GAL4 BD strongly activated transcription of the reporter genes (lacZ and his3) in the yeast host cells Y190. This resulted in the production of blue color in the LacZ filter lift assay and the growth of the transformed cells on histidine-lacking medium (Fig. 7, panel A). Neither of these two SmSmad2 constructs activated transcription when fused to the GAL4 AD (data not shown). Based on these data, we tested the interaction of SmSmad2-AD with SmTbeta R-I fused to GAL4-BD. In vivo, SmSmad2 interacted with SmTbeta R-I under certain conditions. SmSmad2-AD (wt) strongly interacted with SmTbeta R-I-BD (wt) (Fig. 7, panel B), whereas no interaction was observed between SmSmad2-AD (wt) and the 3 mutants of SmTbeta R-I fused to GAL4-BD (not shown). On the other hand, when SmSmad2-3'-AD was used, a stronger interaction was observed with SmTbeta R-I (Q303D) and SmTbeta R-I (T299D/Q303D) (Fig. 7, panel B), whereas the wild type SmTbeta R-I and T299D mutant showed weaker interaction. In general, the results of the LacZ assay were obtained after 3-4 days, while weak interaction required incubation periods of up to 10 days.


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Fig. 7.   Interaction of SmSmad2 and SmTbeta R-I in the yeast two-hybrid system. Panel A, transactivation by SmSmad2 and SmSmad2-3' fused to GAL4-BD, positive control (pGAL4), and negative controls (p53 or pLaminC). Panel B, two-hybrid interactions between SmSmad2-AD and SmTbeta R-I-BD. Combinations of pSV40 with either p53 or pLaminC served as positive and negative controls, respectively. Diagrammatic representations of the experiments appear above the LacZ filter lift assay results.

The use of the yeast two-hybrid assay revealed that SmSmad2 interacts in vivo with SmTbeta R-I. To evaluate this interaction in vitro, we examined the ability of GST-SmSmad2 fusion constructs (linker region and MH2 domain of SmSmad2-wt and full-length SmSmad2-3'), coupled to glutathione-Sepharose beads, to bind [35S]methionine-labeled SmTbeta R-I proteins (wt and the three mutant forms). SmSmad2-MH2 domain as well as the full-length protein (SmSmad2-3') bound the four forms of SmTbeta R-I (Fig. 8, panels A-D, lanes 3 and 4). In this experiment, GST domain was used to evaluate the background interaction. Neither GST nor GST-linker fusion showed significant interaction with SmTbeta R-I proteins (Fig. 8, panels A-D, lanes 1 and 2).


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Fig. 8.   Pull-down experiment. GST or GST fusion proteins coupled to glutathione beads were incubated with in vitro translated, 35S-labeled SmTbeta R-I constructs (labeled on the sides of panels A-D). Bound proteins were separated in 12% SDS-PAGE, processed for fluorography, and exposed to x-ray films. Lanes 1-4 represent the reaction of the radiolabeled protein with resin-bound GST, GST-SmSmad2-Linker, GST-SmSmad2-MH2, and GST-SmSmad2-3' (full length), respectively. Lane 5 represents 50% of the input radiolabeled protein per binding reaction run on the same gels. Arrows point to the specific precipitated products.

Results of the yeast two-hybrid and GST pull-down analyses clearly demonstrated that certain constructs of SmSmad2 stably interact, both in vivo and in vitro, with either wt or mutant forms of SmTbeta R-I. To determine whether this interaction is functional, in vitro phosphorylation of SmSmad2 by wt and mutant constructs of SmTbeta R-I proteins was tested. In vitro synthesized SmTbeta R-I derivatives were incubated in the presence of [gamma -32P]ATP with GST or GST-SmSmad2 fusion proteins (linker, MH2 domain, and SmSmad2-3') coupled to the glutathione-Sepharose beads. After incubation for 1 h at room temperature, samples were centrifuged, the pellets washed for 4 times, resuspended in PAGE sample buffer, and denatured, and the denatured proteins were separated by electrophoresis. Neither GST nor any of the GST-SmSmad2 fusion proteins incorporated the 32P label when incubated with wild type SmTbeta R-I (Fig. 9 panel A, panel I). [32P]Phosphate was only incorporated into the MH2 domain of wt SmSmad2 by SmTbeta R-I-Q303D, T299D/Q303D, and T299D mutants (Fig. 9, panel A, lane 3, panels II-IV). The phosphorylation of the SmSmad2-MH2 domain by the mutant form SmTbeta R-I-Q303D was expected since corresponding mutations in other type I receptors resulted in constitutive phosphorylation activity in vitro (21) and ligand-independent signaling in vivo (25, 36, 37). Surprisingly, the SmTbeta R-I-T299D mutant form showed kinase activity, although less than other mutant forms. This result is in contrast to previous work, which demonstrated that a corresponding mutant to T299D in human Tbeta R-I yielded an inactive version of the receptor kinase (25). GST, GST linker, and GST-SmSmad2-3' were not phosphorylated by any of the SmTbeta R-I constructs (Fig. 9, panel A, lanes 1, 2, and 4). Indeed, the lack of phosphorylation of SmSmad2-3' by any of the SmTbeta R-I constructs was expected since this modification of the C-terminal end of SmSmad2 may result in masking the phosphorylation sites and blocking SmTbeta R-I-mediated phosphorylation of the protein.


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Fig. 9.   Panel A, in vitro phosphorylation of SmSmad2-MH2 domain by SmTbeta R-I. Different SmTbeta R-I derivatives synthesized in a rabbit reticulocyte lysate by coupled transcription and translation (shown at the top of the figure) were incubated with GST or GST fusion derivatives of wt and mutant SmSmad2, in the presence of 10 µCi of [gamma -32P]ATP. Reaction products, bound to glutathione-Sepharose beads, were pelleted by centrifugation, washed four times, denatured in SDS, separated in 12% SDS gels, and subjected to autoradiography. Lanes 1-4 represent GST, GST-SmSmad2-Linker, GST-SmSmad2-MH2, and GST-SmSmad2-3' (full length), respectively. Labeled arrows on the side of the figure point to the expected size of these products. Panel B, in vitro phosphorylation of wild type or mutant SmSmad2-MH2 by wild type or mutant forms of SmTbeta R-I. SmSmad2-MH2 and SmTbeta R-I derivatives were synthesized in vitro by coupled transcription and translation using rabbit reticulocyte lysate with unlabeled methionine. SmSmad2-MH2 proteins were incubated with the translation mix of either a negative control (no DNA transcription reaction) or one of the following SmTbeta R-I derivatives: wild type, T299D, Q303D, and T299D/Q303D, in the presence of 10 µCi [gamma -32P]ATP (panels I-V, respectively). Anti-SmSmad2 rabbit IgG was added to each reaction, and the IgG-SmSmad2-MH2 complexes were precipitated by protein A-Sepharose, washed 4 times, denatured in SDS, separated in 12% SDS gels, and subjected to autoradiography. Panel VI represents the results of immunoprecipitation of wild type and mutant [35S]Met-labeled SmSmad2-MH2 derivatives, demonstrating that the anti-SmSmad2 antibody was capable of precipitating each SmSmad2-MH2, with similar efficiency. Lanes 1-5 represent SmSmad2-MH2 with the following terminal amino acids: TSVS (wild type), TSVA, TAVS, TAVA, and AAVA, respectively. Lane 6 (panel VI) represents the immunoprecipitation background reaction using [35S]Met-labeled negative control lysate.

To confirm the phosphorylation results and to determine the specific phosphorylation sites in SmSmad2 MH2 domain C-terminal phosphorylation motif, we employed a different format of the in vitro phosphorylation reaction. In this reaction we used wt and mutant constructs of SmSmad2-MH2 domain, where the potential phosphorylation sites were mutated to alanine. Both groups of SmSmad2 and SmTbeta R-I (wt and mutant forms) were synthesized in vitro using unlabeled methionine and then the phosphorylation of SmSmad2-MH2 constructs by SmTbeta R-I forms, in the presence of [gamma -32P]-ATP, was evaluated. Fig. 9, panel B, shows that the [32P]phosphate was incorporated into the wt MH2 domain and to a lesser extent into the TSVA and TAVS mutant forms when using any of the SmTbeta R-I mutant derivatives (panels III-V, lanes 1-3). The highest incorporation level was obtained using SmTbeta R-I-Q303D followed by SmTbeta R-I-T299D/Q303D mutants. Neither the negative control (no DNA-transcription reaction) nor SmTbeta R-I-wt translation mix showed significant incorporation of the 32P label in any of the SmSmad2-MH2 constructs (panels I and II). However, minor phosphoproteins derived from the reticulocyte lysate that migrate at the same approximate size of SmSmad2-MH2, independent of SmTbeta R-I and SmSmad2, were observed in some experiments. The mutant forms of SmTbeta R-I did not show significant incorporation of the radioactive label into either TAVA or AAVA mutants of SmSmad2-MH2 domain (panels III-V, lanes 4 and 5) indicating that Thr646 is not a phosphate acceptor. Fig. 9, panel B, panel VI, shows that the anti-SmSmad2 antibody efficiently precipitated the wt as well as the mutant forms of the SmSmad2-MH2 domain.

Table I summarizes the data of SmSmad2 interaction with and in vitro phosphorylation by wt and the mutant derivatives of SmTbeta R-I.

                              
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Table I
Interaction of SmSmad2 derivatives with SmTbeta R-I
The abbreviation and symbols used are as follows: ND, not determined; ++, strong positive; +, positive; +/-, weak positive; and -, negative.

SmSmad2 Translocates to the Nucleus in Response to TGF-beta 1-- The above data indicate that SmSmad2 stably interacts with SmTbeta R-I in vitro and in yeast. The ability of SmSmad2 to interact productively with Tbeta R-I and translocate to the nucleus in a ligand-dependent way was tested in Mv1Lu cells, which are known to be responsive to induction by mammalian TGF-beta (38). Mv1Lu cells that were transfected with pCMV-GST-SmSmad2 and treated with TGF-beta 1 showed nuclear fluorescence when probed with an anti-GST monoclonal antibody (Fig. 10, panels E and F), whereas non-TGF-beta 1-treated cells showed cytoplasmic fluorescence with limited nuclear staining (Fig. 10, panel D). Cells transfected with parental pCMV-GST vector (positive control) showed cytoplasmic fluorescence only (Fig. 10, panel A). Mv1Lu cells transfected with pcDNA-LacZ, (negative control), a non-GST tag expressing, showed no fluorescence (Fig. 10, panel B).


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Fig. 10.   Nuclear translocation of SmSmad2 upon stimulation by TGF-beta 1. Transfected mink lung epithelial cells (Mv1Lu) were fixed and probed with anti-GST monoclonal antibody. Panel A shows the parental pCMV-GST vector-transfected Mv1Lu cells (positive control) expressing GST, and panel B shows typical cells from pcDNA-LacZ-transfected cells (negative control). Mv1Lu cells were transfected with pCMV-GST-Smad2 vector untreated (panel D) and stimulated with TGF-beta 1 (panels E and F). Panel C shows the phase contrast field of the examined cells in panel D. Note that there is only cytoplasmic staining in panel A and nuclear staining in panels E and F.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The life cycle of S. mansoni begins with penetration of cercariae into human skin. The parasites develop into mature worms in about 35 days when male and female worms mate and remain en copula throughout their adult life. During this period, the parasites migrate from the skin, passing through the lungs to their final destination in the portal circulation. There are numerous unanswered questions regarding the development, differentiation, and specialization of the parasite tissues, male-female worm pairing, female worm maturation, and site finding behavior during the mammalian phase of the parasite life cycle. Factors produced by the parasites as well as the host must function in these processes. Our investigation of the role of TGF-beta in parasite development may provide insight into some of these issues.

Members of TGF-beta superfamily regulate many important developmental processes, such as mesenchymal differentiation, skeletal morphogenesis, and skin formation (39). In both vertebrate and invertebrate model systems, TGF-beta family members can serve as morphogens, acting across developing tissues in a graded fashion to specify a patterned array of cell fates (40, 41). Defects in TGF-beta signaling have been implicated in multiple developmental disorders and in various human diseases, including cancer, fibrosis, and autoimmune diseases (8, 10, 42-46).

The S. mansoni Smad2 cDNA encodes a protein that is about 200 amino acids larger than other R-Smad homologues. Most of the additional amino acids are located in the linker region. The linker region contains several serine residues, which are potential phosphorylation sites for mitogen-activated protein kinase. Such phosphorylation results in inhibition of Smad2 nuclear translocation in other organisms (34, 47). The MH2 domain is highly conserved in SmSmad2. The conservation of all five residues required for interaction of MH2 domain with SARA (31) suggests the presence of SARA homologue in schistosomes. SARA would be predicted to act by presenting the SmSmad2 to the activated receptor-I to be phosphorylated and activated. L3 loop is an important and highly conserved structural element present in R-Smads-MH2 domains. SmSmad2 L3 loop possesses the specific subtype residues (Arg613 and Thr616) that specify the interaction of Smad2 and Smad3 with type I TGF-beta and activin receptors (16). Another characteristic structural feature of SmSmad2 is its unique C-terminal phosphorylation signature. Schistosome Smad2 is the only member of R-Smad subfamily with TSVS phosphorylation motif instead of the conserved SSXS motif present in all other members of the subfamily. The significance of this difference is unknown and needs further investigation. However, this difference does not appear to interfere with the biological functions of the protein. In this regard, studies on the C-terminal phosphorylation motif in other Smad proteins showed that threonine could substitute for serines (21).

RT-PCR data indicate that SmSmad2 gene expression exhibits relatively constant levels in the human host with mRNA detectable some time after 3 h but before 4 days post-infection. Native SmSmad2 was localized in female sexual tissues (vitellaria, uterus, and ovary) and in male testis, as well as cells in the subtegument and parenchyma. This pattern of distribution coincides with the pattern observed by in situ hybridization for SmSmad2 mRNA transcripts. This distribution profile of the mRNA and protein indicates that SmSmad2, or TGF-beta signaling, plays multiple roles in the development of the parasite in general, and the sexual maturation of both male and female worms. As vitelline cell development (gene expression) is regulated by a signal from the male, the finding of SmSmad2 expression in vitelline cells implicates the TGF-beta signaling cascade in female reproductive development.

To determine whether SmSmad2 could be activated by TGF-beta , the ability of SmSmad2 to translocate to the nucleus upon induction by TGF-beta was evaluated. Mink lung epithelial cells (Mv1Lu) transfected with GST-tagged SmSmad2 showed some nuclear fluorescence with most of the fluorescence localized in the cytoplasm. This observation is similar to the results of previous studies on Smad3 (48) and Xenopus Mad-like protein (XenMLP) (49). Treatment of transfected Mv1Lu cells with rhTGF-beta 1 resulted in predominant nuclear localization of SmSmad2, demonstrating that SmSmad2 responded to the signals initiated by the human ligand. These data are consistent with Nakao et al. (35) who reported that the endogenous Smad2, Smad3, and Smad4 exhibited predominant fluorescence in the cytoplasm of uninduced Mv1Lu cells, whereas the fluorescence was mainly localized in the nuclei in case of cells stimulated with TGF-beta 1. These results not only demonstrate that SmSmad2 is biologically active but also that the mechanism employed in nuclear transport in higher eukaryotes recognizes a schistosome protein.

To investigate the interaction between SmSmad2 and SmTbeta R-I, we used wild type and mutant constructs of both proteins in yeast two-hybrid and GST pull-down assays. SmSmad2-3', a mutant in which the stop codon was removed to extend the reading frame beyond the "TSVS" phosphorylation motif, was constructed. This modification of the C-terminal end of the protein was expected to block type I receptor-mediated phosphorylation of SmSmad2. Such a modification was demonstrated to abolish the ability of the C-terminal serine residues to become phosphorylated (35). For SmTbeta R-I, three mutants were constructed in which either Thr299, Gln303, or both were mutated to Asp. These constructs were expected to yield a kinase-deficient form, a constitutively active derivative (25), and a construct that contains both kinase-activating and -inactivating mutations, respectively. Wild type SmTbeta R-I interacted with wild type SmSmad2 and to a lesser extent with SmSmad2-3' in vivo in yeast two-hybrid assay, with MH2 domain of SmSmad2, and full-length SmSmad2-3' in GST pull-down experiment, but failed to phosphorylate the MH2 domain in vitro. The interaction between wild type SmSmad2 and SmTbeta R-I in yeast was unexpected since other authors have reported that the interaction between wild type human Smad2 and either wild type Tbeta R-I (11, 35) or the constitutively active form of the receptor (11) could not be detected in COS cells. Perhaps the difference in results is due to the different systems utilized in each study. Transfected COS cells followed by immunoprecipitation of the interacting proteins may not be sensitive enough to detect a weak interaction compared with transformed yeast cells, where the interaction is allowed to proceed over a relatively longer period. Consistent with our results, Zhang et al. (29) reported that the heteromeric interactions between human MAD-3 and-4 could only be detected in yeast two-hybrid assays but not by coimmunoprecipitation of transfected cultured cell lysates. The interaction of wt-SmTbeta R-I with SmSmad2-3' in vivo and in vitro was expected since the modification of the C-terminal end of SmSmad2 was likely to block the phosphorylation mediated by SmTbeta R-I resulting in stabilizing SmSmad2-SmTbeta R-I complex (11). The interaction with SmSmad2-MH2 domain in vitro was also expected as Smad2 was shown to serve as a direct substrate of the activated Tbeta R complex (Tbeta R-I activated by a ligand-bound Tbeta R-II) (11). Therefore, in the absence of Tbeta R-II, SmSmad2-MH2 would not be expected to be phosphorylated by wt-SmTbeta R-I, and thus, as shown by our results, allowed SmSmad2-MH2 to form a stable complex with the wt receptor. Our interpretation of this interaction is supported by the lack of phosphorylation of SmSmad2-MH2 by wt-SmTbeta R-I.

The activity of the SmTbeta R-I constitutive mutant Q303D showed results consistent with those obtained in other systems (25, 36, 37). This mutant was found to interact stably in vivo and in vitro with SmSmad2-3', and in vitro, although to a lesser extent, with the MH2 domain of SmSmad2. However, the in vivo interaction in yeast with wt-SmSmad2 was undetectable. SmTbeta R-I-Q303D mutant was also able to phosphorylate the MH2 domain of SmSmad2-wt in vitro. This mutant construct may exert its constitutive signaling activity due to the replacement of uncharged polar residue (Gln303) by a negatively charged residue (Asp). This would favor the initial apposition between type I receptor and SmSmad2, which should facilitate subsequent binding and phosphorylation of the substrate (31). The stable interaction between Q303D mutant and SmSmad2-3' was observed because the ability of SmTbeta R-I-Q303D to phosphorylate SmSmad2-3' was blocked by the C-terminal extension. However, in case of wt SmSmad2, which was susceptible to phosphorylation by the constitutive mutant Q303D, the interaction in vivo was only transient due to the phosphorylation of SmSmad2 and the dissociation of SmTbeta R-I-SmSmad2 complex.

The mutation T299D was expected to yield a kinase-inactive receptor I (25). Unexpectedly, this mutant did not interact with wild type SmSmad2 in vivo. However, it interacted with SmSmad2-3' in vivo, and with both full-length SmSmad2-3' and the MH2 domain of SmSmad2-wt. Surprisingly, the T299D mutant showed kinase activity, although less than Q303D mutant, in vitro. Therefore, the T299D mutant behaved similar to the Q303D construct. An explanation for the difference between SmTbeta R-I-T299D and the corresponding mutants in other type I receptors may be provided by a direct comparison of the GS domain and its downstream region in these receptors. SmTbeta R-I has one amino acid substitution (Lys292) instead of the conserved "L" at this position for all other known type I receptors. This basic amino acid located just after the GS region may participate in stabilizing the T299D mutant interaction with SmSmad2 and provide the net charge equilibrium required for phosphorylating its substrate, mimicking the effect of the Q303D mutant. Therefore, the use of "less-active kinase" term rather than "kinase-inactive" in case of T299D-SmTbeta R-I mutant would be more applicable for this system.

In most cases, an interaction was observed when SmSmad2 phosphorylation was prevented, as in absence of activation when using wild type SmTbeta R-I, or the use of a nonphosphorylatable form of SmSmad2, as in case of SmSmad2-3'. These data are in agreement with the hypothesis proposed by Macias-Silva et al. (11) who reported that this transient interaction can be stabilized by preventing phosphorylation of R-Smad either by using a kinase-deficient version of type I receptor or a mutant version of Smad2 in which the C-terminal phosphorylation is blocked. Furthermore, previous results also showed that C-terminal FLAG-tagged Smad3 stably associated with wild type Tbeta R-I (35). Our data revealed that the MH2 domain interacted in vitro with the mutant forms of SmTbeta R-I, whereas this interaction was not detected in the two-hybrid analysis using full-length SmSmad2. The interaction may have been undetectable in vivo due to its transient nature due to phosphorylation and dissociation of the complex. This would be true for Q303D and T299D in vitro in the absence of ATP.

The SmTbeta R-I-catalyzed phosphorylation of in vitro translated SmSmad2-MH2-wt and mutant forms showed results consistent with the two-hybrid and pull-down assays, as well as the in vitro phosphorylation reaction of the bacterially expressed SmSmad2-GST fusion proteins. The lack of phosphorylation of wild type or mutant SmSmad2-MH2 by wild type SmTbeta R-I confirms the inability of the wt-SmTbeta R-I to phosphorylate SmSmad2 in absence of ligand, TGF-beta . Alternatively, each mutant form of SmTbeta R-I phosphorylated the wt-MH2 domain of SmSmad2 with varied efficiencies, demonstrating that each mutant receptor I is constitutively active. SmSmad2-MH2 mutants TSVA and TAVS were phosphorylated by the mutant forms of receptor I demonstrating that neither serine is the sole phosphate acceptor. Phosphorylation of double TAVA and triple AAVA mutants was blocked, implicating both Ser647 and Ser649 as the receptor-mediated phosphorylation sites, as in the case of the human Smad2 homologue (50). In addition, the preferential phosphorylation of Ser647 suggests that Ser647 may enhance the efficient phosphorylation of Ser649. Furthermore, these results indicate that Thr646 is not a phosphate acceptor.

Both SmSmad2 and SmSmad2-3' fused to GAL4-BD activated the transcription of the reporter genes (lacZ and his3) in yeast Y190. Together with the phosphorylation data, these results indicate that SmSmad2 possesses a transactivation function, which does not depend on its ability to be phosphorylated at its C-terminal end by type I receptor kinase. Consistent with this observation, Liu et al. (20) and Hayashi et al. (51) reported that full-length Smad1 and Smad2 and Smad1 C-terminal domain could function as agonist-dependent and agonist-independent transcriptional activators, respectively, when fused to the DNA-binding domain of GAL4. Other studies also indicate that the transactivation function may not be dependent on the ability of the protein to be phosphorylated (49).

In conclusion, we showed that SmSmad2 interacts with SmTbeta R-I in vivo and in vitro. We also showed that the constitutively active mutant versions of SmTbeta R-I could phosphorylate SmSmad2 MH2 domain in vitro, although with varied efficiencies. We also showed that Ser647 and Ser649 are likely to serve as receptor-mediated phosphorylation sites of the SmSmad2. In addition, in a heterologous system, SmSmad2 was translocated to the nucleus in response to rhTGF-beta 1. Our results also showed that SmSmad2 mRNA transcript and its encoded protein localized in the subtegumental layer of the parasite. This may represent an intracellular link between the specific receptor located on the surface of the parasite (SmTbeta R-I) (5) and specific effects elicited by TGF-beta or TGF-beta -like signaling. Whether ligands are derived from the host, representing a new chapter in host-parasite interaction, or are produced by the parasite itself to elicit the development, differentiation, and specialization of its tissues, further investigations are needed. In either case, this study argues that SmSmad2 plays an important role in various stages in various tissues of the mammalian (human) phase of schistosome development. The identification of schistosome Smad2 provides a molecular tool to investigate the role of TGF-beta signaling in schistosomes. The identification of the ligand(s) as well as the cooperative partners and the responsive genes are current targets to help in understanding the role of this signaling pathway in the development of the parasite and its interaction with the human host.

    ACKNOWLEDGEMENTS

We thank Dr. Wade Sigurdson, Head of the Confocal Microscopy and Three-dimensional Imaging Laboratory, for help with microscopy and Dr. Randall Reed, Johns Hopkins University, for providing the pCMV-GST vector.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant AI46762.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF232025.

Dagger To whom correspondence should be addressed: Dept. of Microbiology, School of Medicine and Biomedical Sciences, State University of New York, 138 Farber Hall, Buffalo, NY 14214. Tel.: 716-829-2459; Fax: 716-829-2169; E-mail: loverde@buffalo.edu.

Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M005933200

    ABBREVIATIONS

The abbreviations used are: TGF-beta , transforming growth factor-beta ; SmSmad2, S. mansoni Smad2; R-Smad, receptor-regulated Smad; BMP, bone morphogenetic protein; SmTbeta R-I, S. mansoni TGF-beta receptor-I; SARA, Smad anchoring for receptor activation; MH1 and MH2, Mad homology region 1 and 2; wt, wild type; kb, kilobase pair; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; bp, base pair; CIAP, calf intestinal alkaline phosphatase; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; BD, binding domain; AD, activation domain.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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