Identification and characterization of an R-Smad ortholog (SmSmad1B) from Schistosoma mansoni Joelle M Carlo1*, Ahmed Osman1,2*, Edward G Niles1, Wenjie Wu2, Marcelo R Fantappie2, Francisco M B Oliveira2 and Philip T LoVerde1,2 Department of Microbiology and Immunology, School of Medicine and Biomedical Sciences, State University of New York, NY, USA Southwest Foundation for Biomedical Research, San Antonio, TX, USA Keywords bone morphogenic protein; Schistosoma mansoni; Smad; transforming growth factor-b Correspondence P T LoVerde, South-west Foundation for Biomedical Research, PO Box 7620, NW Loop 410, San Antonio, TX 78227-5301, USA Fax: +1 210 670 3322 Tel: +1 216 258 5892 E-mail: ploverde@sfbr.org Database The nucleotide sequence described here is available in the GenBank database under the accession number AY666164 *These authors contributed equally to this work (Received 14 February 2007, revised June 2007, accepted 11 June 2007) doi:10.1111/j.1742-4658.2007.05930.x Smad proteins are the cellular mediators of the transforming growth factor-b superfamily signals Herein, we describe the isolation of a fourth Smad gene from the helminth Schistosoma mansoni, a receptor-regulated Smad (R-Smad) gene termed SmSmad1B The SmSmad1B protein is composed of 380 amino acids, and contains conserved MH1 and MH2 domains separated by a short 42 amino acid linker region The SmSmad1B gene (> 10.7 kb) is composed of five exons separated by four introns On the basis of phylogenetic analysis, SmSmad1B demonstrates homology to Smad proteins involved in the bone morphogenetic protein pathway SmSmad1B transcript is expressed in all stages of schistosome development, and exhibits the highest expression level in the cercariae stage By immunolocalization experiments, the SmSmad1B protein was detected in the cells of the parenchyma of adult schistosomes as well as in female reproductive tissues Yeast two-hybrid experiments revealed an interaction between SmSmad1B and the common Smad, SmSmad4 As determined by yeast threehybrid assays and pull-down assays, the presence of the wild-type or mutated SmTbRI receptor resulted in a decreased interaction between SmSmad1B and SmSmad4 These results suggest the presence of a nonfunctional interaction between SmSmad1B and SmTbRI that does not give rise to the phosphorylation and the release of SmSmad1B to form a heterodimer with SmSmad4 SmSmad1B, as well as the schistosome bone morphogenetic protein-related Smad SmSmad1 and the transforming growth factor-b-related SmSmad2, interacted with the schistosome coactivator proteins SmGCN5 and SmCBP1 in pull-down assays In all, these data suggest the involvement of SmSmad1B in critical biological processes such as schistosome reproductive development The multicellular, dioecious parasite Schistosoma mansoni has a complex life cycle consisting of both free-living and host-dependent stages The signaling mechanisms underlying the growth and development of S mansoni during these stages have remained largely undefined In the human host, S mansoni parasites develop from schistosomules to adults, and can survive in the host mesenteric circulation for years The implication that host molecules may be exploited by schistosomes to enhance the parasites’ development Abbreviations AP-1, activator protein-1; 3-AT, 3-amino-1,2,4-triazole; BAC, bacterial artificial chromosome; b-gal, b-galactosidase; BMP, bone morphogenetic protein; Co-Smad, common Smad; DPE, downstream promoter element; ERK, extracellular signal-regulated kinase; EST, expressed sequence tag; Gal4AD, Gal4 activation domain; Gal4BD, Gal4 DNA-binding domain; GST, glutathione S-transferase; MBP, maltose-binding protein; MH, Mad homology domain; R-Smad, receptor-regulated Smad; SmGCP, schistosome gynecophoral canal protein; TGFb, transforming growth factor-b FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS 4075 SmSmad1B, a BMP-R-Smad ortholog from S mansoni J M Carlo et al and ultimate survival within the host has prompted the need for better characterization of schistosome signaling networks [1] In recent years, several members of the transforming growth factor-b (TGFb) superfamily have been isolated from S mansoni [2–7] The involvement of the TGFb superfamily in critical cellular processes such as embryogenesis, differentiation and apoptosis makes these pathways attractive candidates for elucidating the growth and development mechanisms employed by S mansoni The TGFb superfamily comprises a large group of structurally related, secreted cytokines, including TGFb, bone morphogenetic protein (BMP), and activin [8–11] The TGFb signaling cascade is stimulated through the binding of ligand to a type II receptor, a transmembranous serine ⁄ threonine receptor kinase The ligand–type II receptor complex recruits another serine ⁄ threonine transmembrane receptor kinase, type I receptor, which is subsequently phosphorylated and activated by the type II receptor The activated type I receptor then interacts with a group of cellular mediators called receptor-regulated Smads (R-Smads) The R-Smads of the TGFb–activin pathway include Smad2 and Smad3 The R-Smads of the BMP pathway include Smads 1, and The type I receptor phosphorylates the R-Smad at its C-terminal MH2 domain, causing the R-Smad to dissociate from the receptor The phosphorylation ⁄ activation of the R-Smad allows it to interact with another component of the Smad family, called a common Smad (Co-Smad) The Smad complex translocates to the nucleus, where, in concert with other proteins, it modulates the transcription of TGFb-responsive genes In S mansoni, the first TGFb superfamily member identified was a type I receptor named SmRK1 (later referred to as SmTbRI) [3] SmTbRI was found to be expressed in the schistosome tegument, whereas RT-PCR analysis demonstrated the upregulation of SmTbRI transcript in S mansoni during stages of mammalian infection [3] These results, along with the reported binding of human TGFb to a chimeric form of SmTBRI [12], followed by the later finding of the induced association of SmTbRI with the schistosome type II receptor SmTbRII by human TGFb [7], suggested a role for TGFb signaling in host–parasite interactions Two R-Smad genes (SmSmad1 and SmSmad2) and a Co-Smad gene (SmSmad4) were also identified from S mansoni [2,5,6] It was determined that SmSmad2 acts as a substrate for receptor activation by SmTbRI, whereas the activation of SmSmad1 by SmTbRI has not been demonstrated As SmSmad1 resembles R-Smads of the BMP pathway, it was suggested that both BMP and 4076 TGFb signaling networks may be active in schistosomes, and that a second type I receptor capable of transmitting BMP-related signals may be present in the genome of S mansoni Herein, we report the isolation of a new member of the S mansoni R-Smad family, designated SmSmad1B Like SmSmad1, SmSmad1B demonstrates homology to BMP-related R-Smad genes In this study, we report the identification of SmSmad1B cDNA and present its gene structure along with the expression profiles, immunolocalization, and protein interaction properties Results Identification of SmSmad1B Through cDNA library screening with the putative SmSmad8 ⁄ expressed sequence tag (EST) as a probe, a SmSmad1B cDNA clone was isolated that contained the entire coding region and 3¢-UTR, as well as a partial 5¢-UTR sequence (68 bp) (Fig 1A) The 3¢-UTR sequence was determined to be complete by the presence of a polyA tail with the AAUAAA consensus polyadenylation signal [13] located 25 bp upstream of the polyA tail [13] The 5¢-UTR sequence was extended to its full length by employing 5¢-RACE The organization of the SmSmad1B cDNA comprises a 136 bp 5¢-UTR, a 1143 bp coding region, including a ‘TAA’ stop codon, and a 738 bp 3¢-UTR (Fig 1A) SmSmad1B protein consists of 380 amino acid residues, and contains all the typical and conserved motifs of an R-Smad As no sequence or structural properties exist to differentiate the BMP Smads from each other, a high degree of similarity among the BMP Smads is a common phenomenon SmSmad1B is one of the smallest R-Smads in terms of size, mainly due to the presence of a short linker region comprising only 42 amino acids The N-terminal MH1 domain of SmSmad1B consists of 137 amino acids, and comprises a conserved nuclear localization signal and a b-hairpin structure that serves as a DNA-binding domain (Fig 1A) The 201 amino acid MH2 domain of SmSmad1B contains a typical C-terminal SSVS phosphorylation motif, which is the site of phosphorylation by type I receptors of the TGFb superfamily (Fig 1A) The presence of the SSVS phosphorylation site identifies SmSmad1B as an R-Smad, as this motif is absent in both inhibitory and Smads and Co-Smads [9] Importantly, the L3 loop in the MH2 domain of SmSmad1B resembles that of R-Smads in being specific for transducing BMP signals (i.e amino acid residues H340 and D343) (Fig 1A) FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS J M Carlo et al SmSmad1B, a BMP-R-Smad ortholog from S mansoni Fig Structure of the SmSmad1B gene, cDNA and protein (A) Schematic representations of the SmSmad1B gene, cDNA and protein and the amino acid sequence of SmSmad1B protein Five exons interrupted by four introns constitute the SmSmad1B gene (top) A cDNA of about kb in size is transcribed from the genomic gene (middle) and translated into a 380 amino acid SmSmad1B protein (bottom) Regions encoding MH1, linker and MH2 are in light gray, gray and dark gray, respectively, and the regions representing the 5¢- and 3¢-UTRs are shown in white in the genomic gene and the cDNA Intron size in bp, domain size in bp and domain size in amino acids are indicated at the bottom of each schematic representation of the gene, cDNA, and protein, respectively The schematic representation and the amino acid sequence of SmSmad1B protein show sequence motifs (black boxed) such as nuclear localization signal (NLS), DNA-binding b-hairpin domain (DBD) and the receptor-phosphorylation motif (Pi motif) as well as the amino acid sequence of the peptide region that was used to generate SmSmad1B-specific antibody reagents (Antibody peptide) The L3 loop is also shown (gray box), with R-Smad subtype-specific amino acids in bold and underlined (B) The promoter region and the 5¢-UTR of the SmSmad1B gene The transcription start site within the Inr is designated by a broken arrow A 50 bp intron that separates exons and is shown (italics, underlined lower-case letters) The promoter region is in upper-case letters, and the exon sequences are presented in bold upper-case letters Some transcription regulatory elements are listed (boxed): Inr (initiator element); DPE; and AP-1 The underlined ATG is the codon for the translation start methionine Phylogenetic analysis Phylogenetic trees were constructed by Bayesian inference using a mixed protein substitution model with an inv-gamma distribution of rates between sites using mrbayes v3.1.1 (Fig 2) Phylogenetic analyses of both the MH1 (Fig 2A) and MH2 sequences (Fig 2B) showed that SmSmad1B clustered within the BMPrelated R-Smad group, which includes the homologous proteins Drosophila MAD and the vertebrate Smad1, FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS 4077 SmSmad1B, a BMP-R-Smad ortholog from S mansoni J M Carlo et al Fig Bayesian phylogenetic tree of SmSmad1B The dataset was analyzed using a mixed substitution model with an inv-gamma distribution of rates between sites using MRBAYES v3.1.1 The trees were started randomly; four simultaneous Markov chains were run for · 106 generations The trees were sampled every 100 generations Bayesian posterior probabilities were calculated using a Markov chain Monte Carlo sampling approach implemented in MRBAYES v3.1.1, with a burn-in value setting at 7500 generations; the values are shown at each branch point (or by arrows) The results suggested that SmSmad1B and SmSmad1 are paralogous genes; they originated from duplication of a common ancestor Smad gene after the split between platyhelminths, arthropods, and vertebrates (A) MH1 tree (B) MH2 tree The GenBank accession numbers of the analyzed sequences were as following: CeSma3 (Caenorhabditis elegans sma-3), U34902; Cem1 (Cae elegans MAD homolog 1), U10327; CeSma4 (Cae elegans SMA-4), U34596, DAD (Drosophila melanogaster DAD), AB004232; dMAD (D melanogaster MAD), U10328; dMedea (D melanogaster Medea), AF057162; dSmad2 (D melanogaster Smad2), AF101386; EmSmadB (E multilocularis SmadB), AJ548428; ckSmad8 (Gallus gallus SMAD8), AY953145; hSmad1 (Homo sapiens Smad1), U59423; hSmad2 (H sapiens Smad2), U65019; hSmad3 (H sapiens Smad3), U76622; hSmad4 (H sapiens Smad4), U44378; hSmad5 (H sapiens Smad5), U73825; hSmad6 (H sapiens Smad6), AF043640; hSmad7 (H sapiens Smad7), AF015261; mSmad2 (Mus musculus Smad2), U60530; ratSmad8 (Rattus norvegicus Smad8), AF012347; SmSmad1 (S mansoni Smad1), AF215933; SmSmad2 (S mansoni Smad2), AF232025; SmSmad4 (S mansoni Smad4), AY371484; xSmad1 (Xenopus laevis Mad1), L77888; xSmad2 (X laevis Mad2), L77885; xSmad3 (X laevis SMAD3), AJ311059; xSmad8 (X laevis Smad8), AF464927 Smad5 and Smad8 Furthermore, SmSmad1B was closely related to SmSmad1, another S mansoni Smad protein previously isolated [2], and to the tapeworm 4078 Echinococcus multilocularis Smad, EmSmadB [14] The phylogenetic data suggest that SmSmad1 and SmSmad1B are paralogous genes; they originated from FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS J M Carlo et al the duplication of a common ancestor gene after the split between the platyhelminths, arthropods, and vertebrates The same results were obtained by maximum likelihood and neighbor-joining distance analyses (supplementary Figs S1 and S2) SmSmad1B gene structure and 5¢ upstream analysis The location of the exon–intron boundaries were determined by alignment of cDNA sequence with the bacterial artificial chromosome (BAC) DNA sequence (SmBAC1 40G14) The four exon–intron junctions conform to the eukaryotic consensus GT-AG splice sites (supplementary Table S1) [15] The locations of the exon–intron junctions in SmSmad1B are shared by Smad genes from other species For example, the location of the intron within the MH1-encoding region is conserved in the human Smad4 gene, whereas the intron within the linker-encoding region of SmSmad1B is shared by human Smad3 [16] The location of the intron within the MH2-encoding region is highly conserved among human Smad9, Smad5, and Smad3, mouse Smad1, and the E multilocularis SmadB [14,16,17] PCR amplification of the SmSmad1B cDNA flanking the linker region did not produce multiple PCR products (data not shown), indicating the absence of alternative splicing in this region The beginning of exon of SmSmad1B was identified by performing 5¢-RACE Three independent rounds of 5¢-RACE produced 5¢-UTR fragments that extended no further than 136 bp upstream from the translation start site This location was determined to be the position of the putative transcription start site (Fig 1B) Analysis of the 5¢ upstream region of exon demonstrated the lack of a conserved TATA box upstream of the transcription initiation site However, an AT-rich sequence with a single nucleotide mismatch from the TATA box consensus is located at position ) 55 ⁄ ) 48 in the SmSmad1B promoter region (GATA AAAG, as compared to the consensus TATAA ⁄ TAAG ⁄ A) [18] An initiator element (Inr) is located at position ) ⁄ + that conforms to the mammalian Inr consensus rather than the Drosophila consensus Inr (TCA+1AAAC) [19,20] From position + 24 ⁄ + 29, a downstream promoter element (DPE; consensus A ⁄ G ⁄ T-C ⁄ G-A ⁄ T-C ⁄ T-A ⁄ C ⁄ G-C ⁄ T) is also located [21] The DPE is known to act in conjunction with the Inr in the initiation of transcription A potential AP-1 (activator protein-1) site is located at position ) 78 ⁄ ) 72 Interestingly, there are three core Smadbinding elements (GTCT) [22] in the upstream region (Fig 1B) SmSmad1B, a BMP-R-Smad ortholog from S mansoni Developmental expression of SmSmad1B The expression level of SmSmad1B mRNA was evaluated by performing quantitative RT-PCR on cDNA prepared from total RNA isolated from various schistosome developmental stages (Fig 3) The expression levels of SmSmad1B were compared to those of the related R-Smad gene, SmSmad1 The results demonstrate that SmSmad1B is expressed in all of the developmental stages examined The SmSmad1B expression pattern closely follows that of SmSmad1, both exhibiting the highest transcript levels in cercariae and lower levels in different developmental stages in the intermediate host, Biomphalaria glabrata snails On the other hand, expression levels show a significant drop in the stages representing different time points in the mammalian host, as early as days postinfection, and there is a gradual decrease thereafter up to 21day-old schistosomules, which represent the trough of the expression curves of both R-Smads The levels then display a slight increase, reaching maximum levels of expression in the mammalian host in paired adult worms In addition, it appears that the BMP-related Smads, SmSmad1 and SmSmad1B, exhibit relatively lower levels of expression as compared to the TGFbrelated SmSmad2 in the late stages of infection (28 day, 35 day and adult worms; SmSmad2 data not shown) Both the SmSmad1 and SmSmad2 expression Fig Quantitative RT-PCR analysis of schistosome BMP-related R-Smad genes A bar graph comparing the fold expression levels (mean ± SD) of SmSmad1 (black) and SmSmad1B (gray) normalized to the levels of Sma-tubulin throughout various stages of schistosome development The following developmental stages were tested: infected Biomphalaria glabrata snails representing daughter sporocysts (inf snail), cercariae, 3-day-old and 7-day-old cultured schistosomules, 15 day, 21 day, 28 day and 35 day parasites, adult worm pairs, separated adult female and male worms, and eggs cDNA from uninfected B glabrata snails served as a negative control FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS 4079 SmSmad1B, a BMP-R-Smad ortholog from S mansoni J M Carlo et al levels are consistent with our previously reported results that showed that SmSmad2 expression levels exceed SmSmad1 levels by approximately 45% in the later stages (35 days or older) of mammalian development [6] Identification and immunolocalization of SmSmad1B protein in adult schistosomes To detect the native SmSmad1B protein, western blots were performed using S mansoni adult worm pair protein extracts (whole and soluble) and the affinity-purified aSmSmad1B antibody A band was detected migrating at approximately 53 kDa in the S mansoni protein extracts when they were probed with the aSmSmad1B antibody (Fig 4, right panel), which is higher than the calculated molecular mass of SmSmad1B (43 kDa) The band could not be detected in the S mansoni protein extracts when they were probed with a preimmune rabbit IgG antibody (Fig 4A, middle panel) Furthermore, preincubation of aSmSmad1B antibody with varied amounts of SmSmad1B linker peptide resulted in a gradual decrease in intensity of the 53 kDa band until the native protein was no longer visualized in the presence of 10 lgỈmL)1 of the peptide (Fig 4B, right panel, lane 3), indicating that the 53 kDa band is specific The in vitro translation product of SmSmad1B runs at approximately 50 kDa (data not shown) That difference in size could be attributed to post-translational modifications that occur to the native protein, such as specific phosphorylation by type I receptor [23,24], or N-acetylation by p300, CBP, or P ⁄ CAF [25,26] Such modifications may not be seen in the in vitro translated product Immunofluorescent staining was performed to localize the expressed SmSmad1B protein in adult schistosomes Adult worm cryosections were probed with affinity-purified aSmSmad1B antibody, and the specific fluorescence was visualized at 680 nm In female adult worms, SmSmad1B was prominent in the vitellaria as well as in the reproductive ducts and subtegumental tissues (Fig 5) In male adult worms, specific fluorescence was also visualized in the subtegument but not in tissues of the reproductive system Rather, a tissue of undefined origin in the male worms demonstrated consistent, specific SmSmad1B fluorescence The signal was located in the parenchyma within the worm center, and spanned the entire length of the male worm (Fig 5B) SmSmad1B protein interactions To investigate the interaction between SmSmad1B and schistosome TGFb superfamily members, yeast two-hybrid assays were performed When Y190 yeast competent cells were cotransformed with plasmids expressing a SmSmad1B-Gal4AD fusion protein and a SmSmad4-Gal4BD fusion protein, a strong positive interaction was observed, as determined by growth on selective SD media [– Leu, – Trp, – His, + 40 mm 3-amino-1,2,4-triazole (3-AT)] (Fig 6A) and by the development of blue color in a LacZ filter lift assay (Fig 6B) The results of the yeast two-hybrid and the yeast-three hybrid experiments (described below) are summarized in greater detail in Table In comparison to the SmSmad1B–SmSmad4 interaction, a weakly positive interaction was detected with yeast cotransformed with SmSmad1B-Gal4AD and either SmTbRI0Gal4BD or SmTbRIQD-Gal4BD receptor constructs The protein interactions of the BMP-related SmSmad1 with SmSmad4, SmTbRI and SmTbRIQD were also evaluated, and found to exhibit a relatively comparable interaction pattern to that of SmSmad1B Yeast three-hybrid assays were performed to evaluate the SmSmad1B–SmSmad4 interaction in the presence of SmTbRI receptor constructs Y190 yeast competent cells were cotransformed with the SmSmad1B-Gal4AD construct and the pBridge construct that allows for the expression of both SmSmad4Gal4BD and SmTbRI (or SmTbRIQD), as described in Experimental procedures In yeast transformants with the pBridge construct, the expression of the Fig Detection of the SmSmad1B protein in S mansoni worm extract by western blot Composite photograph of SDS gel separation of soluble (S) and whole (W) adult worm extracts (left panel), and membrane strips immunoblotted with: preimmune rabbit IgG (middle panel) and affinity-purified aSmSmad1B IgG (right panel) A competition assay (right panel, lane C) was performed by preincubating the affinity-purified aSmSmad1B IgG with the SmSmad1B linker peptide (10 lgỈmL)1) The molecular size (kDa) of the band is given on the left 4080 FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS J M Carlo et al SmSmad1B, a BMP-R-Smad ortholog from S mansoni Fig Immunolocalization of SmSmad1B protein in adult schistosomes Immunofluorescent staining of SmSmad1B in adult worm cryosections Column I, phase-contrast images Column II, green autofluorescent images taken with a 522 nm filter Column III, far red immunofluorescent images taken with a 680 nm filter (200 · magnification) Worms treated with preimmune rabbit IgG (negative control) are presented in row A Rows B–E represent worms treated with affinity-purified aSmSmad1B IgG The arrows represent the area of male-specific SmSmad1B fluorescence M, male worm; F, female worm; V, vitellaria; G, gut; ST, subtegument; O, ootype FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS 4081 SmSmad1B, a BMP-R-Smad ortholog from S mansoni J M Carlo et al Fig Yeast two-hybrid analysis of SmSmad1B protein interactions (A) Growth of cotransformed Y190 yeast cells on selective SD media (– Leu, – Trp, – His, + 40 mM 3-AT) Numbers 1–8 represent the following yeast cotransformations: 1, p53–pSV40 (positive control); 2, pLamin C–pSV40 (negative control); 3, SmSmad1B-AD–SmSmad4-BD; 4, SmSmad1B-AD–SmTbRI-BD; 5, SmSmad1B-AD–SmTbRIQD-BD; 6, SmSmad1-AD–SmSmad4-BD; 7, SmSmad1-AD–SmTbRI-BD; 8, SmSmad1-AD–SmTbRI-QD-BD Cotransformation numbers and were streaked in duplicate (B) LacZ filter-lifts from transformed yeast grown on SD media lacking leucine and tryptophan Table Summary of the yeast two-hybrid and yeast-three hybrid experiments showing SmSmad1B protein interactions The following criteria were utilized in this table for designating the extent of the protein interactions: the growth rate of cotransformed Y190 yeast colonies (activation of HIS3 reporter); the duration of the development of blue color in the LacZ filter-lift assay (activation of LacZ reporter); and the b-gal units calculated from the liquid LacZ assay (activation of LacZ reporter) + ⁄ –, weak interactions (yeast growth after days of incubation, blue color development on LacZ filter-lift assay after days and ⁄ or < 0.8 b-gal units in liquid LacZ assay); +, moderate interactions (yeast growth after days of incubation, blue color development on LacZ filter-lift assay after days, and 0.9–5.5 b-gal units in liquid LacZ assay; + +, strong interactions (yeast growth after days of incubation, blue color development on LacZ filter-lift assay after day, and 1.0–7.5 b-gal units in liquid LacZ assay); + + +, stronger interactions (yeast growth after days of incubation, blue color development on LacZ filter-lift assay in less than day, and > 7.5 b-gal units in liquid LacZ assay) AD, Gal4 activation domain; BD, Gal4 binding domain Yeast two-hybrid Yeast three-hybrid SmSmad4-BD SmSmad1B-AD SmSmad1-AD SmTbRI-BD SmTbRI-QD-BD SmTbRI + SmSmad4-BD SmTbRI-QD + SmSmad4-BD ++ +++ +⁄– +⁄– +⁄– +⁄– + ++ +⁄– + receptor should be suppressed in the presence of methionine However, it was determined that the pBridge construct contains a leaky Met25 promoter that allows for the expression of the receptor constructs even in the presence of methionine-containing SD media (data not shown) Therefore, the pBridge constructs were only able to be used for three-hybrid analysis when both the SmSmad4-Gal4BD and the receptor (wildtype or active mutant) were coexpressed in yeast As compared to the SmSmad1B–SmSmad4 interaction observed in the yeast two-hybrid assay, the inclusion of SmTbRI or SmTbRIQD resulted in decreased growth of cotransformed yeast on selective SD media (– Leu, – Trp, – His, – Met, + 40 mm 3-AT) (Fig 7A) However, little change in blue color intensity was observed in the filter-lift assay (Fig 7B) Similar results 4082 were observed when SmSmad1B was replaced with SmSmad1 in the three-hybrid experiments To better examine the effect of the inclusion of TGFb receptor-containing constructs on the SmSmad1B–SmSmad4 or SmSmad1–SmSmad4 interactions, liquid LacZ assays were performed to quantify induction of b-galactosidase (b-gal) activity (Fig 7C) In the liquid LacZ assays, the SmSmad1–SmSmad4 interaction produced approximately eight b-gal units, whereas the SmSmad1B–SmSmad4 interaction produced approximately one b-gal unit The presence of the wild-type and constitutively active mutant receptor in the SmSmad1B–SmSmad4 interaction resulted in statistically significant decreases in SmSmad1B–SmSmad4 b-gal induction of 15% and 26%, respectively Decreases in b-gal units of 14% and 38% also resulted FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS J M Carlo et al SmSmad1B, a BMP-R-Smad ortholog from S mansoni Fig Yeast three-hybrid analysis of SmSmad1B protein interactions (A) Growth of cotransformed Y190 yeast cells on selective SD media (– Leu, – Trp, – His, – Met, + 40 mM 3-AT) Numbers 1–8 represent the following yeast cotransformations: 1, p53–pSV40 (positive control); 2, pLamin C–pSV40 (negative control); 3, SmSmad1BAD–SmSmad4-BD; 4, SmSmad1B-AD– SmSmad4-SmTbRI-pBridge; 5, SmSmad1BAD–SmSmad4-SmTbRI-QD-pBridge; 6, SmSmad1-AD–SmSmad4-BD; 7, SmSmad1AD–SmSmad4-SmTbRI-pBridge; 8, SmSmad1–SmSmad4-SmTbRI-QD-pBridge (B) LacZ filter-lifts from transformed yeast grown on selective SD media lacking leucine, tryptophan and methionine cotransformed with plasmids in the same order as in (A) (C) Liquid LacZ assays Induction of b-gal is reported in b-gal units, where values represent the average of three independent experiments *Represents statistically significant value (P ¼ 0.05) from the inclusion of wild-type or active receptor in the SmSmad1–SmSmad4 interaction However, only the inclusion of SmTbRIQD produced a statistically significant decrease in the SmSmad1–SmSmad4 interaction In the liquid LacZ assays, the extent of the SmSmad1– SmSmad4 interaction as compared to that of the SmSmad1B–SmSmad4 interaction is more apparent than what was observed in the filter-lift assay, due to the quantifiable nature of the liquid assays Also, the magnitude of the decrease in interaction between SmSmad1 and SmSmad4 in the presence of the receptors, specifically SmTbRIQD, is more obvious in the liquid assay than in the LacZ filter-lift assay In an attempt to confirm the SmSmad1B protein interactions in the yeast assays, maltose-binding protein (MBP) pull-down experiments were performed The resin-bound SmSmad4-MBP fusion protein was incubated with in vitro translated [35S]SmSmad1B in the presence or absence of either in vitro translated SmTbRI, unlabeled SmTbRI or SmTbRIQD MBPbound resin was used as a negative control to assess nonspecific background binding Similar to the results of the yeast two-hybrid and three-hybrid protein interaction assays, SmSmad1B was able to bind SmSmad4 in the pull-down assay (Fig 8A,B) The addition of SmTbRI resulted in a decrease in the interaction strength between SmSmad1B and SmSmad4 by 19%, and the inclusion of SmTbRIQD produced a statistically significant 52% decrease In a previous report, the inclusion of the receptor constructs in an in vitro FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS 4083 SmSmad1B, a BMP-R-Smad ortholog from S mansoni J M Carlo et al Fig In vitro interaction between SmSmad1B and schistosome TGFb superfamily members (A) Evaluation of the SmSmad1B– SmSmad4 interaction by MBP pull-down experiments; In vitro translated [35S]SmSmad1B (5 lL) was incubated with SmSmad4MBP (2 lg) in the presence or absence of unlabeled in vitro translated SmTbRI or SmTbRI-QD (10 lL) A graphical representation of the values obtained from the SmSmad1B–SmSmad4 MBP pulldowns in the presence or absence of receptor constructs is shown (bottom panel) *Represents statistically significant value (P ¼ 0.05) (B) MBP pull-down experiments demonstrating the interaction between SmSmad1B-MBP and [35S]SmTbRI or [35S]SmTbRIQD Values represent percentage binding as compared to input, and are the mean of three independent experiments Background binding, represented by (–), was accounted for in the calculation of percentage binding Lanes labeled (I) represents 10% input of 35 S-labeled in vitro translated products SmSmad1–SmSmad4 interaction assay also had a negative effect on the strength of the interaction between SmSmad1 and SmSmad4 [6] MBP pull-down assays were also employed to investigate the binding of SmSmad1B with SmTbRI or SmTbRIQD in vitro SmSmad1B was expressed as an MBP fusion protein and incubated with either in vitro translated [35S]methionine-labeled SmTbRI or SmTbRIQD, and the bound proteins were precipitated with amylose resin In 4084 the pull-down assays, SmSmad1B interacted with both SmTbRI and SmTbRIQD, with a slight binding preference for SmTbRIQD (Fig 8B) The preferential binding of SmSmad1B to SmTbRIQD in the pull-down assays, although moderate, could explain the decreased interaction between SmSmad1B and SmSmad4 in the presence of SmTbRIQD (Fig 8A), as the interaction between SmSmad1B and SmTbRIQD made SmSmad1B less available for binding to SmSmad4 Pull-down assays were performed to investigate the interaction between the schistosome coactivator proteins SmGCN5 [27] and SmCBP1 [28] and the schistosome R-Smads ) SmSmad1, SmSmad2, and SmSmad1B ) in the presence or absence of SmSmad4 For the interaction assays with SmGCN5, the schistosome R-Smads were in vitro translated as glutathione S-transferase (GST)-fusion proteins and incubated with in vitro translated 35S-labeled SmGCN5, in the presence or absence of nonlabeled SmSmad4 GSTbound glutathione Sepharose was used as a negative control to assess nonspecific background binding The results of the pull-down assays in Fig 9A show that the BMP-related R-Smads, SmSmad1 and SmSmad1B, interact at relatively higher levels with SmGCN5 as compared to the level achieved with the TGFb-related SmSmad2 In the meantime, addition of SmSmad4 resulted in decreased levels of interaction with SmGCN5 with all the tested R-Smads (Fig 9A) For SmSmad2, inclusion of SmTbRI-QD in the binding reaction not only significantly increased the interaction of SmSmad2 with SmGCN5, but also revealed the interaction of SmSmad4 with SmGCN5 and demonstrated its participation in the formation and, probably, the stabilization of the transcriptional protein complex Figure 9B shows that the presence of SmTbRI-QD significantly boosted the interaction with SmGCN5 of the 35S-labeled SmSmad4 (lane 4) and the 35 S-labeled SmSmad2 (lane 6) in the presence of either nonlabeled SmSmad2 or SmSmad4, respectively, as compared to the interaction levels attained in the presence of wild-type SmTbRI (lanes and 5) In a similar approach, a GST pull-down assay was performed to investigate the interaction between the coactivator SmCBP1 and the R-Smads SmSmad1, SmSmad1-B, and SmSmad2 The results in Fig 10A show that GST-SmCBP1 interacted with SmSmad1 and SmSmad2 but not with SmSmad1B, SmSmad4 or the receptor SmTbRI-QD, which served as a negative control Similar to the situation with SmGCN5, when SmSmad4 was included in the reactions, a reduction in interaction level with GST-SmCBP1 was observed with both SmSmad1 and SmSmad2 (Fig 10B), and again, FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS J M Carlo et al SmSmad1B, a BMP-R-Smad ortholog from S mansoni the addition of SmTbRI-QD boosted the interaction between SmSmad2 and SmCBP1, and demonstrated the presence of SmSmad4 in the protein complex (Fig 10B) Discussion Fig In vitro interaction of the coactivator SmGCN5 with different members of the schistosome TGFb signaling pathway (A) Interaction of in vitro translated 35S-labeled full-length SmGCN5 (5 lL) with glutathione Sepharose-bound GST or GST fusion proteins of SmSmad1, SmSmad1-B, or SmSmad2 (2 lg each) in the presence or absence of in vitro translated nonlabeled SmSmad4 (10 lL) Ten per cent of the radiolabeled SmGCN5 input is represented in the left lane of the gel (B) Interactions of nonlabeled, S protein-tagged fulllength SmGCN5 with 35S-labeled, non-S protein-tagged, full-length SmSmad2 or SmSmad4 (5 lL each) in the presence of non-S protein-tagged, nonlabeled in vitro translation products of SmSmad2, SmSmad4, SmTbRI-wt or SmTbRI-QD (10 lL each) Reactions were precipitated using S-protein agarose beads Precipitated products were separated by SDS ⁄ PAGE and subjected to autofluorography The top arrow points to SmSmad4, and the bottom arrow points to SmSmad2 in vitro translated, 35S-labeled proteins In lane 3, there is a distortion of the radioactive labeled protein band ([35S]Met-labeled SmSmad4) that occurred during the electrophoretic migration In this study, a new schistosome R-Smad gene was identified and designated SmSmad1B on the basis of its phylogenetic relationship with BMP-related R-Smads from other species SmSmad1B encodes a 380 amino acid protein with conserved MH1 and MH2 domains and a short, 42 amino acid linker region Protein alignment (blastp search) demonstrates that SmSmad1B exhibits a high degree of homology with BMP-related R-Smads (Smads 1, and ⁄ 9) from different species Smad5 orthologs from the domestic dog (Canis familiaris), the common chimpanzee (Pan troglodytes) and the Rhesus monkey (Macaca mulatta), as well as the closely related BMP Smad from the tapeworm (E multilocularis), EmSmadB, attained the highest homology scores with SmSmad1B However, the phylogenetic analyses suggest that both the SmSmad1 and SmSmad1B genes originated from a common ancestor gene and that gene duplication took place after the split between the platyhelminths, arthropods, and vertebrates Therefore, we considered ‘SmSmad1B’ to be a relevant designation for this gene In vertebrate homologs, the human BMP Smad9 has been reported to undergo alternate splicing within its linker region However, only one form of BMP Smad8 has been reported for both mouse and rat [17,29] SmSmad1B, which contains an intron within the Fig 10 In vitro interaction of the coactivator SmCBP1 with different members of the schistosome TGFb signaling pathway (A) GST pulldown analyses were performed to evaluate the interactions of glutathione Sepharose-bound GST or full-length GST-SmCBP1 fusion protein (5 lg each) with in vitro translated 35S-labeled SmSmad1, SmSmad1B, SmSmad2, SmSmad4, or SmTbRI-QD (5 lL each) (B) Interaction of glutathione Sepharose-bound GST or full-length GST-SmCBP1 fusion protein (5 lg each) with in vitro translated 35S-labeled SmSmad1 or SmSmad2 (5 lL each) in the presence of in vitro translated 35S-labeled SmSmad4 (5 lL per reaction), and, in the case of SmSmad2, the active mutant construct of type I receptor, SmTbRI-QD (10 lL) The top arrow points to SmSmad4, and the bottom arrow points to SmSmad2 in vitro translated, 35S-labeled proteins Binding reaction products were separated by SDS ⁄ PAGE and subjected to autofluorography FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS 4085 SmSmad1B, a BMP-R-Smad ortholog from S mansoni J M Carlo et al linker-encoding region, did not show any evidence of alternative splicing, as determined by PCR Similar to the mammalian Smad8, the remarkably short linker region of SmSmad1B lacks the consensus PPXY motif for ubiquination by Smurf1 [30] On the other hand, the SmSmad1B linker region, unlike the R-Smads from most species, lacks the PXS ⁄ TP motif for phosphorylation by extracellular signal-regulated kinase (ERK) kinase However, neither of the other two currently identified schistosome R-Smads, SmSmad1or SmSmad2, possesses ERK kinase motifs in its linker region Only schistosome Smad4 contains a functional ERK phosphorylation motif [6] The lack of ERK phosphorylation sites in all of the schistosome RSmads and its presence in the schistosome Co-Smad suggests a divergent ERK–Smad regulatory pathway in this parasite SmSmad1B demonstrates sequence motifs that are common to R-Smads, such as a nuclear localization signal and a DNA-binding domain in the MH1 domain and the L3 loop, and the C-terminal, receptor phosphorylation motif in the MH2 domain (Fig 1A) The amino acid composition in the L3 loop of R-Smads provides a clue to the types of TGFb ligand that these signaling molecules may respond to, as the L3 loop is known to mediate R-Smad–type I receptor binding specificity [31] The L3 loop of SmSmad1B groups this protein with other BMP-related R-Smads The presence of a histidine and an aspartate at positions 340 and 343 within the L3 loop of SmSmad1B (Fig 1A) is highly conserved among R-Smads that transduce BMP-like signals, and, as expected, is also conserved in SmSmad1 In contrast, schistosome SmSmad2 displays an L3 loop amino acid composition that resembles those of the R-Smads of the TGFb–activin-related pathways (i.e R613 and T616) The C-terminal SSVS phosphorylation site of SmSmad1B (Fig 1A) conforms to the reported consensus SSXS motif, which is conserved in SmSmad1 as well However, the receptor phosphorylation site of SmSmad2 and the activin-related R-Smad from the parasitic platyhelminth E multilocularis, EmSmadA, diverges slightly from the consensus with the sequence TSVS [2,5,14] As both SmSmad2 and EmSmadA were shown to be TGFb-like signal transducers, it is possible that the divergent TSVS motif may be unique to platyhelminth TGFb-related Smads Upon analysis of the SmSmad1B promoter, various regulatory elements were identified The lack of a conserved TATA element suggests that the SmSmad1B gene contains a TATA-less promoter However, the SmSmad1B promoter does contain a conserved Inr and DPE, which have been found in other TATA-less schistosome genes, such as that for glutathione peroxi4086 dase [32] In other organisms, such as Drosophila, Inr and DPE have been shown to coordinate transcription initiation in the absence of a TATA box [21] Considering that only approximately 30% of both human and Drosophila promoters contain a TATA box [33], it is not unreasonable to suggest that SmSmad1B may also be a TATA-less gene [33] The mRNA transcript levels of SmSmad1B suggest that this R-Smad may play multiple roles in the biology of S mansoni The SmSmad1B transcript was found to be expressed in all stages of development, with the highest level in cercariae As the cercariae are the free-living and infective stage of schistosomes, it is possible that the upregulation of SmSmad1B mRNA during this stage may play a role in host infection or in the transformation ⁄ development into schistosomules within the host This is the first examination of schistosome Smad transcript expression in pooled cercariae The developmental stage expressing the lowest levels of SmSmad1 and SmSmad1B transcripts comprises 21-day-old schistosomules, at a time when the majority of the parasites have migrated to the portal circulation of the liver The SmSmad1 and SmSmad1B expression levels show a moderate rise after 21 days, to reach peak expression levels in the mammalian host in paired adult worms Interestingly, expression levels in either adult male or female worms were relatively lower than those observed in paired worms These data may suggest a higher involvement of BMP-related signaling pathways associated with adult worm pairing and male–female interactions In contrast, the expression levels of SmSmad2 are at their highest in 35 day worms and adults [6] SmSmad1B was also localized in the vitellaria and reproductive ducts of the female adult worm, coinciding with the reported location of other schistosome TGFb superfamily members [5–7,34] Recently, it was reported that TGFb treatment of late-stage worms caused increased expression of the schistosome gynecophoral canal protein (SmGCP), and that the induced expression required the TGFb type II receptor [7] As the male gynecophoric canal is the structure in which the female worm resides for mating, the upregulation of SmGCP by TGFb indicates a role in worm pairing and reproduction Together with the recent report of Smad involvement in mammalian reproductive organ differentiation [35], as well as the results of the SmSmad1B RT-PCR, the immunolocalization of SmSmad1B to the female sexual organs suggests a role for SmSmad1B in schistosome reproductive development or maturation These data suggest that BMP-related SmSmad1B, in concert with other TGFb–activin family members, functions in critical processes related to schistosome sexual development FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS J M Carlo et al The protein localization of SmSmad1B in the subtegument of adult schistosomes is a common expression pattern among the schistosome Smads The presence of SmSmad1B in the subtegument further supports the hypothesis that TGFb-like signals are transmitted across the tegument via type I and type II receptors, whose Smad cellular effectors transduce the signals through the subtegument, thus enhancing the ability of the parasite to respond to changes in the surrounding environment Therefore, the expression pattern for SmSmad1B in S mansoni suggests the possibility of pleiotropic roles for SmSmad1B: a role in cercaria survival, host penetration, or migration in the early stages of infection, and a role in the development or maturation of the schistosome reproductive system and in host–parasite interactions Just as SmSmad1B exhibits motifs characteristic of BMP-related R-Smads and closely resembles schistosome SmSmad1 rather than SmSmad2, we report that the protein interaction properties of SmSmad1B resemble those of SmSmad1 as well Through protein interaction experiments, we have clearly shown that SmSmad1B interacts with the Co-Smad SmSmad4 Whereas the SmSmad1B–SmSmad4 cotransformation is considered to be a strong interaction, the SmSmad1– SmSmad4 interaction exceeds these interactions by approximately eight-fold, as determined by liquid b-gal activity Therefore, it appears that, experimentally, SmSmad1 has a greater affinity for SmSmad4 as compared to SmSmad1B The elucidation of the crystal structures of the schistosome Smads may help to explain these differences in binding preference Although a modest decrease in the interaction strength between SmSmad1B and SmSmad4 was observed in the presence of the wild-type SmTbRI construct, a significant decrease was observed in the presence of the constitutively active SmTbRI-QD construct in both in vivo and in vitro experiments The relevance of SmTbRI and SmTbRI-QD effects on the SmSmad1B–SmSmad4 interaction has yet to be determined The important point is that the constitutively active receptor did not enhance the SmSmad1B–SmSmad4 interaction, similar to what has been reported for SmSmad2 [2,6] Thus, we can infer from these studies that SmTbRI is probably not the natural receptor for SmSmad1B and SmSmad1 For this hypothesis to hold true, a second type I receptor gene must be present in the genome of S mansoni As SmTbRI was only capable of binding human TGFb ligands in concert with SmTbRII [7], the proposed second type I receptor may be activated by BMP ligands and may utilize SmSmad1B and SmSmad1 as downstream effectors As SmTbRII maintains an elevated SmSmad1B, a BMP-R-Smad ortholog from S mansoni level of expression throughout development as compared to SmTbRI, whose expression levels increase only in the later stages of schistosome development, it was also proposed that a second type I receptor must be present to work in concert with the type II receptor in the early stages of development [7] Interestingly, we have shown that SmSmad1B may play a role during an early stage of schistosome development, the cercariae, as described above Unlike most transcription factors that are sufficient to recruit the basal transcription machinery and therefore activate transcription on both naked and chromatin templates, the Smads only activate transcription from chromatin templates [36] The transcriptional coactivators p300 ⁄ CBP, P ⁄ CAF and GCN5 have been shown to interact with R-Smads [26,37–39] Therefore, we attempted to investigate the interaction profile of SmSmad1B as well as other schistosome R-Smads with the recently identified schistosome transcriptional coactivators GCN5 and p300 ⁄ CBP These assays are intended to shed some light on the nuclear phase of the SmSmad-mediated signaling pathway in schistosomes and to probe the role of SmSmad1B beyond its interaction with SmSmad4 and the formation of a Smad complex The transcription factors p300 ⁄ CBP and GCN5 possess an intrinsic histone acetyltransferase activity, which facilitates transcription by decreasing chromosome condensation through histone acetylation and by increasing the accessibility of the basal transcription machinery to transcription factors Indeed, it has been recently shown [40] that activated Smad2-containing complexes not activate transcription by directly recruiting basal transcription machinery to the promoter DNA Rather, Smads recruit the basal transcription machinery indirectly as a result of their ability to orchestrate specific histone modifications and chromatin remodeling As determined by pull-down assays, both SmSmad1 and SmSmad1B demonstrated a positive binding interaction with the schistosome coactivator protein SmGCN5, whereas SmSmad2 and SmSmad4 alone did not These results suggest a preference in binding for SmGCN5 by the BMP-related R-Smads in schistosomes, and also confirm the similarities in the protein interaction properties of SmSmad1 and SmSmad1B as described above However, in the presence of SmSmad4 and the active mutant form of TbRI, both SmSmad2 and SmSmad4 interacted with GCN5, indicating the formation of a stable Smad complex that may mimic what occurs in vivo This observation can be explained on the basis of our previous work that demonstrated that the phosphorylation of SmSmad2 by TbRI-QD FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS 4087 SmSmad1B, a BMP-R-Smad ortholog from S mansoni J M Carlo et al enhanced the interaction of SmSmad2 and SmSmad4 and resulted in the formation of a stable and functional Smad complex [6,7] The above results are consistent with the report that human GCN5 interacts with both TGFb- and BMP-related R-Smads in immunoprecipitation assays [39] Likewise, the GST pull-down assays showed that the coactivator SmCBP1 interacted with SmSmad1 and SmSmad2 but not with SmSmad1-B In the meantime, and in the absence of receptor activation, the inclusion of SmSmad4 had a negative effect on the interaction with SmCBP1 However, in the case of SmSmad2, when GST-SmCBP1 was incubated in the presence of SmSmad4 and TbRI-QD, a positive interaction was observed, in which SmSmad4 represented a part of the protein complex Thus, it seems that such complex interaction patterns with transcription coactivators are influenced by different factors, depending on the context of the developmental event and ⁄ or the response to a signal of host or parasite origin Future studies will be needed to address these interactions in in vitro and in vivo surrogate systems In this study, protein interaction experiments have demonstrated that SmSmad1B and SmSmad1 share similar binding properties This is a common finding among the BMP-related R-Smads (Smads 1, and 8), as the functions of these Smads in other organisms are highly redundant Future analysis of the target genes activated by the BMP-related Smads from schistosomes will aid in differentiating the functions of SmSmad1 and SmSmad1B in S mansoni These results also provide further evidence for the involvement of TGFb signaling in schistosome reproductive function as well as in host–schistosome interactions Experimental procedures Isolation of SmSmad1B cDNA An 1193 bp EST, generated as an overlap of six sequences (GenBank accession numbers CD081730, CD194980, CD195083, CD065319, CD182943, and CD201222) that showed homology to SmSmad8 ⁄ from different species, was obtained from the S mansoni EST genome project [41] The EST sequence was amplified from adult worm pair cDNA, the PCR product was cloned into the pCR2.1TOPO vector (Invitrogen, Carlsbad, CA, USA), and the sequence was confirmed The 1193 bp PCR product was randomly labeled with [32P]dCTP[aP] (Megaprime; GE Healthcare Biosciences, Piscataway, NJ, USA), and used to screen a kZAP II adult worm pair cDNA library to obtain the full-length cDNA, designated SmSmad1B (based on the phylogenic analysis) Positive plaques were in vivo excised and sequenced 4088 Sequence analysis and phylogenetic tree construction A phylogenetic tree was constructed using deduced sequences of MH1 and MH2 domains, respectively The sequences were aligned with clustalw (http://www.cf.ac uk/biosi/research/biosoft/Downloads/clustalw.html) Phylogenetic analysis of the dataset was carried out by Bayesian inference using a mixed protein substitution model with an inv-gamma distribution of rates between sites using mrbayes v3.1.1 [42] The trees were started randomly; four simultaneous Markov chains were run for · 106 generations and sampled every 100 generations Bayesian posterior probabilities were calculated using a Markov chain Monte Carlo sampling approach implemented in mrbayes v3.1.1, with a burn-in value setting at 7500 generations The same dataset was also tested by maximum likelihood and neighbor-joining distance [43] methods For maximum likelihood analysis, the dataset was analyzed using the Jones–Taylor–Thornton substitution model [44] with a gamma distribution of rates between sites (eight categories, parameter alpha, estimated by the program), using phyml (v2.4.4) [45] Support values for the tree were obtained by bootstrapping 100 replicates For neighbor-joining distance analysis, the dataset was analyzed using a Jones–Taylor– Thornton substitution model with a gamma distribution of rates between sites (eight categories, parameter alpha, estimated using phyml), using the phylip package v3.62 (http://evolution.genetics.washington.edu/phylip.html) Support values for the tree were obtained by bootstrapping 1000 replicates with seqboot implemented in the phylip package v3.62 Plasmid construction The cDNA coding region of SmSmad1B was PCR amplified from the original cDNA library clone, SmSmad1BpBluescript A forward primer, 5¢-CACCATGTTAGACC CAAACATTTGC-OH, was designed to allow for the directional cloning of the SmSmad1B PCR product into the Gateway entry vector pENTR ⁄ SD ⁄ D-TOPO (Invitrogen) By homologous recombination, PCR products of the entire coding region of SmSmad1B or its MH2 domain were inserted into destination vectors that were modified using the Gateway Vector Conversion System (Invitrogen) to produce the following constructs: SmSmad1B-pGADT7-DEST (yeast GAL4AD vector), SmSmad1B-pCITE-4a-DEST (in vitro transcription ⁄ translation vector), SmSmad1BpMAL-c2X-DEST (MBP-fusion prokaryotic expression vector) and SmSmad1B(MH2)-pGEX-4T1-DEST (GST-fusion prokaryotic expression vector) The following constructs utilized in this study have been described elsewhere: SmSmad4-SmTbRI-pBridge and SmSmad4-TbRIQDpBridge, SmSmad4-pBDGal4, C-terminally truncated FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS J M Carlo et al SmTbRI-pBDGal4 and SmTbRIQD-pBDGal4, SmSmad4pMAL-c2X, and SmSmad2(MH2)-pET-42a [5,6] Isolation of SmSmad1B BAC clones and gene analysis The S mansoni BAC1 library [46] was screened using the previously described [32P]dCTP[aP]-labeled SmSmad1B probe Four positive BAC clones were identified: SmBAC1 40G14, 9E14, 51H3, and 6H19 The BAC DNA was isolated, and the presence of the SmSmad1B sequence was confirmed by PCR and by sequencing the BAC DNA Exon–intron sites were located by aligning the cDNA sequence with the BAC sequence Intron size was determined by both BAC clone sequencing and by alignment of the cDNA sequence with the genomic DNA sequence obtained from the WTSI S mansoni WGS genomic database (ftp://ftp.sanger.ac.uk/ pub/databases/Trematode/S.mansoni/genome) The location of the transcription start site was identified by performing several rounds of 5¢-RACE (5¢-RACE System Kit; Invitrogen) For the 5¢-RACE, the following SmSmad1B-specific primer was used to synthesize the first-strand cDNA: 5¢-GCGATCGTGGGATAG-3.¢ Quantitative RT-PCR The expression levels of SmSmad1B mRNA in various stages of S mansoni development were evaluated by quantitative RT-PCR, using an IQ5 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) Total RNA was prepared from B glabrata snails (S mansoni intermediate host), infected or uninfected, eggs, cercariae, and 3-day-old and 7-dayold cultured schistosomules, as well as from parasites perfused from infected Syrian golden hamsters at different time points, 15 day schistosomules, 21 day worms, 28 day worms, 35 day worms, 45 day paired adult worms, and 43 day separated mature male and mature female worms cDNA was synthesized from the isolated RNA using the Superscript II Reverse Transcriptase (Invitrogen) in the presence of both random primers and oligo(dT), according to the manufacturer’s instruction Target sequences were amplified from the prepared cDNA templates using Power SYBR green PCR master mix (Applied Biosystems, Foster City, CA, USA) and specific primer pairs for each analyzed gene The expression levels of SmSmad1B in different developmental stages were compared to those of the closely related schistosome R-Smad, SmSmad1 after PCR data were normalized to the levels of the constitutively expressed gene, Sma-tubulin [47], which was used as an internal reference PCR control Quantitative PCR primers used in this study were designed using the PrimerQuest analysis tool from Integrated DNA technologies, Inc (Coralville, IA, USA; http://www.idtdna.com/Scitools/Applications/Primerquest) SmSmad1 and SmSmad1B target sequences were amplified from different cDNA samples using the following primer SmSmad1B, a BMP-R-Smad ortholog from S mansoni pairs: Smad1-fwd (5¢-ACTGTGGAAGCAGCGGAATG TCTA-3¢) and Smad1-rev (5¢-ATAGGTCCAGCAACT GTGCTGTCT-3¢) (516–539 and the reverse complement of 667–690, respectively, of the cDNA sequence, GenBank accession number AF215933); and Smad1B-fwd (5¢-TCCA GTACGCACTTCTTCACCCAA-3¢) and Smad1B-rev (5¢ACAGGCCTTAACTCATGGTGACTC-3¢) (166–190 and the reverse complement of 309–332, respectively, of the cDNA sequence, GenBank accession number AY666164), yielding 175 bp and 166 bp PCR products, respectively A forward primer, tubulin-fwd (5¢-AGCAGTTAAGCGTT GCAGAAATC-3¢), and a reverse primer, tubulin-rev (5¢GACGAGGGTCACATTTCACCAT-3¢) (851–873 and the reverse complement of 904–925, respectively, of the cDNA sequence, GenBank accession number M80214), were also used to amplify a 75 bp PCR product of the a-tubulin cDNA A melt curve protocol was run following the quantitative PCR protocol, to evaluate the efficacy of the primer pairs used and to confirm that the collected data correspond to a single amplification product for each gene Production of SmSmad1B-specific antiserum and western blot To avoid cross-reactivity with other SmSmads, a nonconserved 21 amino acid peptide within the linker region of SmSmad1B was synthesized (N¢-RHNEYPTIESTKKDSPS DETC-C¢; Proteintech, Chicago, IL, USA) The linker peptide, conjugated to KLH, was used to immunize two rabbits over the course of months (Proteintech; short protocol) The anti-SmSmad1B sera as well as preimmune rabbit sera were purified over protein G Sepharose (GE Healthcare Biosciences) to isolate the IgG fractions Specific anti-SmSmad1B IgG was isolated by affinity purification over SmSmad1B linker peptide covalently linked to CNBr-activated Sepharose resin (Amersham Biosciences) The affinitypurified anti-SmSmad1B IgG was used in western blot and immunofluorescence assays Adult worm protein extracts were prepared by homogenizing live worms in an extraction buffer containing 50 mm Tris ⁄ HCl (pH 7.5), 150 mm NaCl, 5% glycerol, 1% Triton X-100, mm phenylmethanesulfonyl fluoride, mm dithiothreitol, lm pepstatin A, and 50 lm leupeptin Approximately 30 lg of whole worm extract or soluble adult worm preparation were size separated on 10% SDS ⁄ PAGE gels and transferred to a poly(vinylidene difluoride) membrane (Immobilon P; Millipore, Billerica, MA, USA) The membranes were probed with either affinity-purified anti-SmSmad1B IgG or with preimmune rabbit IgG (0.5 lgỈmL)1), and this was followed by incubation with horseradish peroxidase-conjugated goat anti-(rabbit IgG) (Sigma, St Louis, MO, USA; : 3000 dilution) The membranes were developed using trimethylbenzidene (Zymed, San Francisco, CA, USA) according to the manufacturer’s instructions For competition experiments, 1, and 10 lgỈmL)1 of the SmSmad1B linker peptide were preincu- FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS 4089 SmSmad1B, a BMP-R-Smad ortholog from S mansoni J M Carlo et al bated with the primary antibody for h at room temperature prior to incubation with the protein blots Immunofluoresence assay Acetone-fixed adult worm cryosections were blocked in · NaCl ⁄ Pi containing 10% goat serum (Sigma) and 10 lgỈmL)1 alkaline phosphatase-conjugated streptavidin (Invitrogen) for h at room temperature The blocked sections were treated with either affinity-purified anti-SmSmad1B IgG (5 lgỈmL)1) or with preimmune rabbit IgG (5 lgỈmL)1) in · NaCl ⁄ Pi containing 3% goat serum for h at room temperature The sections were incubated with a biotin-conjugated goat anti-(rabbit IgG) (H + L) (5 lgỈmL)1: Zymed) in · NaCl ⁄ Pi containing 3% goat serum for h at room temperature Finally, the sections were treated with an AlexaFluor 647 streptavidin conjugate (5 lgỈmL)1; Molecular Probes) in · NaCl ⁄ Pi containing 3% goat serum for h at room temperature Probed slides were washed four times with · NaCl ⁄ Pi, each, following each incubation step Fluorescence was observed under a Bio-Rad MRC1024 confocal microscope with a krypton–argon laser utilizing 522 nm and 680 nm filters SmSmad1B protein interactions Yeast two-hybrid and three-hybrid assays The yeast strain Y190, a HIS3 ⁄ LacZ reporter strain, was utilized in the yeast two-hybrid assays Preparation of yeast competent cells and transformations were achieved using the Frozen-EZ Yeast Transformation II kit (Zymo Research Orange, CA, USA) To test for protein interactions, the SmSmad1B-pGADT7 plasmid that encodes for SmSmad1B fused with the Gal4 activation domain (Gal4AD) was cotransformed with the Gal4 DNA-binding domain (Gal4BD) fused with either SmSmad4, the TGFb type I receptor, SmTbRI, or the constitutively active mutant construct of the type I receptor, SmTbRI-QD SmSmad1-pGADT7 was also cotransformed with the SmSmad4 and the type I receptor constructs of Gal4BD Positive protein interactions were confirmed by the growth of transformed yeast colonies on selective synthetic dropout medium (SD) lacking leucine, tryptophan, and histidine, supplemented with 40 mm 3-AT, a histidine synthesis inhibitor added to neutralize the leaky expression of the HIS3 reporter (– Leu, – Trp, – His, + 40 mm 3-AT) Grown colonies were restreaked onto selective SD medium lacking leucine and tryptophan (– Leu, – Trp) for LacZ filter-lift assays The development of a blue color through the activation of the yeast LacZ reporter gene is another indication of a protein–protein interaction For the yeast three-hybrid experiments, SmSmad1B-pGADT7 was cotransformed with the SmSmad4-SmTbRI-pBridge or SmSmad4-SmTbRI-QD-pBridge constructs The pBridge constructs contain two multiple cloning regions and condi- 4090 tionally express two proteins: SmSmad4 in-frame with the Gal4BD, and either SmTbRI or SmTbRI-QD under the control of the Met25 promoter The Y190 cotransformants were grown on selective SD medium lacking leucine, tryptophan, histidine, and methionine, supplemented with 40 mm 3-AT (– Leu, – Trp, – His, – Met, + 40 mm 3-AT) Colonies were incubated at 30 °C, and restreaked onto selective SD medium lacking leucine, tryptophan, and methionine (– Leu, – Trp, – Met) for LacZ filter-lift assays Quantitative liquid b-gal assays were performed as described elsewhere [48] Significant differences among samples were determined by one-way anova with Tukey’s multiple comparison test P-values of 0.05 were accepted as indicating a significant difference In vitro interaction assays MBP pull-down assays were employed to evaluate the efficiency of binding of SmSmad1B with SmSmad4 The SmSmad4-MBP fusion protein was bound to amylose resin (New England BioLabs, Ipswich, MA, USA) and washed to remove contaminants SmSmad1B was in vitro translated and labeled with [35S]methionine using the Single Tube Protein System (STP3 T7 kit; EMD Bioscience, San Diego, CA, USA) according to the manufacturer’s instruction SmTbRI or SmTbRI-QD constructs were also in vitro translated by this method using unlabeled methionine The [35S]SmSmad1B protein (5 lL) was incubated in binding buffer (25 mm Tris ⁄ HCl, pH 7.5, 100 mm NaCl, 10% glycerol) with either lg of SmSmad4-MBP-bound resin or MBP resin (negative control) overnight at °C Unlabeled SmTbRI or SmTbRI-QD (10 lL) was also added to the reactions to evaluate the effect of the receptors on the SmSmad1B–SmSmad4 interaction The resin-bound proteins were washed, boiled in SDS ⁄ PAGE sample buffer, and size separated on 10% SDS ⁄ PAGE gels After electrophoresis, the gels were fixed, treated with fluorography reagent (Amplify; GE Healthcare), dried, and exposed to X-ray film at ) 80 °C MBP pull-down assays were also performed to evaluate the SmSmad1B–receptor interactions Full-length SmSmad1B was expressed in bacteria as an MBP-fusion protein and bound to amylose resin SmTbRI or SmTbRI-QD recombinant pCITE-4a vectors were in vitro translated in the presence of [35S]methionine, and lL of the labeled reactions were incubated with lg of SmSmad1B-MBP-bound resin in binding buffer overnight at °C The protein-bound resin was processed as described above Using similar approaches, pull-down assays were performed to evaluate the interaction between schistosome R-Smads and the coactivators SmGCN5 [27] and SmCBP [28] In these assays, full-length GST-fusion or in vitro translation (recombinant pCITE-2a; EMD-Bioscience) constructs of SmSmad1, SmSmad1B, SmSmad2, and SmSmad4, as well as the wild-type schistosome TGFb type I receptor, SmTbRI-wt, and the constitutively active mutant FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS J M Carlo et al construct, SmTbRI-QD cloned into the in vitro translation vector pCITE-2a, which lacks the S protein tag sequence, were used to produce recombinant proteins that were utilized along with the full-length SmGCN5 cloned into pCITE-4a (EMD-Novagen), which produces S protein-tagged in vitro translation products In vitro interaction assays were performed using [35S]methionine-labeled SmGCN5 (5 lL per reaction) and GST fusion proteins of each of the schistosome R-Smads (2 lg) bound to glutathione Sepharose beads (GE Healthcare), in the presence or absence of nonlabeled in vitro translated SmSmad4 (10 lL) GST-bound beads were used as a negative control The reactions were incubated overnight at °C, and the products were washed, separated by electrophoresis, and subjected to autofluorography as described above To evaluate the effect of the presence of type I receptor (wild type or constitutively active) on the interaction of SmSmad2 and SmSmad4 with SmGCN5, non-S proteintagged [35S]methionine-labeled SmSmad2 and SmSmad4 (5 lL each), individually and in the presence of the other SmSmad protein (nonlabeled, non S-tagged; 10 lL each), were allowed to interact with nonlabeled, S-protein tagged in vitro translated SmGCN5 (10 lL) in the presence of mm ATP and either SmTbRI-wt or SmTbRI-QD (non-S protein-tagged; 10 lL each) The reactions were incubated overnight at °C SmGCN5-S protein-tagged bound proteins were precipitated by adding 30 lL of 50% prewashed S-protein beads and incubating for h at room temperature Protein-bound beads were washed, separated by SDS ⁄ PAGE, and processed as before Full-length GST-SmCBP1 was a kind gift from R Pierce (Pasteur Institute, Lille, France) Expression of GST-SmCBP1 was done as previously described [28] GST pull-down assays were performed to evaluate the interaction of GST-SmCBP1 with schistosome R-Smads ) SmSmad1, SmSmad1B, and SmSmad ) in the presence of SmSmad4 and SmTbRI, as previously described [6] Briefly, [35S]methionine-labeled SmSmad1, SmSmad1B, SmSmad2, SmSmad4 and SmTbRI-QD (5 lL), either individually or as mixtures of more than one labeled protein, were incubated overnight in binding buffer (described above) with lg of GST-SmCBP1 bound to glutathione Sepharose beads, at °C, in the presence of mm ATP GST-bound beads were used as a negative control The beads were washed and processed as above SmSmad1B, a BMP-R-Smad ortholog from S mansoni 10 11 12 13 14 Acknowledgements This research was supported by NIH grants AI046762 and D43 TW006580 15 References 16 Dissous C, Khayath N, Vicogne J & Capron M (2006) Growth factor receptors in helminth parasites: signalling and host–parasite relationships FEBS Lett 580, 2968– 2975 Beall MJ, McGonigle S & Pearce EJ (2000) 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Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS 4093 ... 2007 FEBS 4087 SmSmad1B, a BMP -R-Smad ortholog from S mansoni J M Carlo et al enhanced the interaction of SmSmad2 and SmSmad4 and resulted in the formation of a stable and functional Smad complex... degree of homology with BMP-related R-Smads (Smads 1, and ⁄ 9) from different species Smad5 orthologs from the domestic dog (Canis familiaris), the common chimpanzee (Pan troglodytes) and the... schistosomes, and that a second type I receptor capable of transmitting BMP-related signals may be present in the genome of S mansoni Herein, we report the isolation of a new member of the S mansoni R-Smad