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
1 Department of Microbiology and Immunology, School of Medicine and Biomedical Sciences, State University of New York, NY, USA 2 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 5 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 fac- tor-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 com- posed 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. Sm- Smad1B transcript is expressed in all stages of schistosome development, and exhibits the highest expression level in the cercariae stage. By immuno- localization 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 Sm- Smad1B and the common Smad, SmSmad4. As determined by yeast three- hybrid 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 non- functional interaction between SmSmad1B and SmTbRI that does not give rise to the phosphorylation and the release of SmSmad1B to form a het- erodimer 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 coacti- vator 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.
largely undefined. In the human host, S. mansoni para- sites 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
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
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.
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TGFb signaling networks may be active in schisto- somes, and that a second type I receptor capable of transmitting BMP-related signals may be present in the genome of S. mansoni.
such as
and ultimate survival within the host has prompted the need for better characterization of schistosome signa- ling networks [1]. In recent years, several members of the transforming growth factor-b (TGFb) superfamily have been isolated from S. mansoni [2–7]. The involve- ment of the TGFb superfamily in critical cellular pro- cesses embryogenesis, differentiation and apoptosis makes these pathways attractive candidates for elucidating the growth and development mecha- nisms employed by S. mansoni.
Herein, we report the isolation of a new member the S. mansoni R-Smad family, designated Sm- of Smad1B. Like SmSmad1, SmSmad1B demonstrates homology to BMP-related R-Smad genes. In this the identification of SmSmad1B study, we report cDNA and present its gene structure along with the expression profiles, immunolocalization, and protein interaction properties.
related,
secreted cytokines,
Results
Identification of SmSmad1B
Through cDNA library screening with the putative SmSmad8 ⁄ 9 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 par- tial 5¢-UTR sequence (68 bp) (Fig. 1A). The 3¢-UTR sequence was determined to be complete by the pres- ence 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 organ- ization 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).
The TGFb superfamily comprises a large group of structurally including TGFb, bone morphogenetic protein (BMP), and acti- vin [8–11]. The TGFb signaling cascade is stimulated through the binding of ligand to a type II receptor, a transmembranous receptor kinase. serine ⁄ threonine 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 media- tors 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, 5 and 8. The type I receptor phos- phorylates 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.
SmSmad1B protein consists of 380 amino acid resi- dues, 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 small- est R-Smads in terms of size, mainly due to the pres- ence 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).
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 in S. mansoni during stages of SmTbRI transcript 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 the induced association of SmTbRI with the of schistosome type II receptor SmTbRII by human TGFb [7], suggested a role for TGFb signaling in host–parasite interactions. Two R-Smad genes (Sm- Smad1 and SmSmad2) and a Co-Smad gene (Sm- Smad4) 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 acti- vation of SmSmad1 by SmTbRI has not been dem- onstrated. As SmSmad1 resembles R-Smads of the BMP pathway, it was suggested that both BMP and
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Fig. 1. 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 2 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 designa- ted by a broken arrow. A 50 bp intron that separates exons 1 and 2 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 infer- ence 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 BMP- related R-Smad group, which includes the homologous proteins Drosophila MAD and the vertebrate Smad1,
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Fig. 2. Bayesian phylogenetic tree of SmSmad1B. The dataset was analyzed using a mixed substitution model with an inv-gamma distribu- tion of rates between sites using MRBAYES v3.1.1. The trees were started randomly; four simultaneous Markov chains were run for 3 · 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 duplica- tion 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; rat- Smad8 (Rattus norvegicus Smad8), AF012347; SmSmad1 (S. mansoni Smad1), AF215933; SmSmad2 (S. mansoni Smad2), AF232025; Sm- Smad4 (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 clo- sely related to SmSmad1, another S. mansoni Smad protein previously isolated [2], and to the tapeworm
Echinococcus multilocularis Smad, EmSmadB [14]. The that SmSmad1 and Sm- phylogenetic data suggest Smad1B are paralogous genes; they originated from
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Developmental expression of SmSmad1B
the duplication of a common ancestor gene after the split between the platyhelminths, arthropods, and ver- tebrates. The same results were obtained by maximum likelihood and neighbor-joining distance analyses (sup- plementary Figs S1 and S2).
SmSmad1B gene structure and 5¢ upstream analysis
stages
The expression level of SmSmad1B mRNA was evalu- ated by performing quantitative RT-PCR on cDNA prepared from total RNA isolated from various schistosome developmental stages (Fig. 3). The expres- sion levels of SmSmad1B were compared to those of the related R-Smad gene, SmSmad1. The results dem- onstrate that SmSmad1B is expressed in all of the developmental 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 3 days postinfection, and there is a gradual decrease thereafter up to 21- day-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 in paired adult expression in the mammalian host worms. In addition, it appears that the BMP-related Smads, SmSmad1 and SmSmad1B, exhibit relatively lower levels of expression as compared to the TGFb- related SmSmad2 in the late stages of infection (28 day, 35 day and adult worms; SmSmad2 data not shown). Both the SmSmad1 and SmSmad2 expression
The location of the exon–intron boundaries were deter- mined 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 loca- tion 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 con- served among human Smad9, Smad5, and Smad3, and the E. multilocularis SmadB mouse Smad1, [14,16,17]. PCR amplification of the SmSmad1B cDNA flanking the linker region did not produce mul- tiple PCR products (data not shown), indicating the absence of alternative splicing in this region.
site
is
Fig. 3. 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) normal- ized 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 para- sites, adult worm pairs, separated adult female and male worms, and eggs. cDNA from uninfected B. glabrata snails served as a neg- ative control.
The beginning of exon 1 of SmSmad1B was identi- independent fied by performing 5¢-RACE. Three 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 1 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 ) 2 ⁄ + 5 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 ⁄ is also located [21]. T-C ⁄ G-A ⁄ T-C ⁄ T-A ⁄ C ⁄ G-C ⁄ T) The DPE is known to act in conjunction with the Inr in the initiation of transcription. A potential AP-1 (activator protein-1) located at position ) 78 ⁄ ) 72. Interestingly, there are three core Smad- binding elements (GTCT) [22] in the upstream region (Fig. 1B).
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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 develop- ment [6].
tissues (Fig. 5). In male adult worms, specific fluores- cence 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 cen- ter, and spanned the entire length of the male worm (Fig. 5B).
Identification and immunolocalization of SmSmad1B protein in adult schistosomes
SmSmad1B protein interactions
indicating that
right panel,
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 1. In comparison to the SmSmad1B–SmSmad4 interaction, a weakly positive interaction was detected with yeast cotrans- formed with SmSmad1B-Gal4AD and either SmTbRI- 0Gal4BD 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.
To detect the native SmSmad1B protein, western blots were performed using S. mansoni adult worm pair pro- tein extracts (whole and soluble) and the affinity-puri- fied 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 Sm- Smad1B (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, mid- dle 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 lgÆmL)1 of the peptide the lane 3), (Fig. 4B, 53 kDa band is specific. The in vitro translation prod- uct 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 local- ize the expressed SmSmad1B protein in adult schisto- somes. 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
Yeast three-hybrid assays were performed to evalu- ate the SmSmad1B–SmSmad4 interaction in the pres- ence of SmTbRI receptor constructs. Y190 yeast competent cells were cotransformed with the Sm- Smad1B-Gal4AD construct and the pBridge construct that allows for the expression of both SmSmad4- Gal4BD and SmTbRI (or SmTbRIQD), as described transformants in Experimental procedures. In yeast the with the pBridge construct,
the expression of
Fig. 4. 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-puri- fied aSmSmad1B IgG with the SmSmad1B linker peptide (10 lgÆmL)1). The molecular size (kDa) of the band is given on the left.
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Fig. 5. Immunolocalization of SmSmad1B protein in adult schistosomes. Immunofluorescent staining of SmSmad1B in adult worm cryosec- tions. Column I, phase-contrast images. Column II, green autofluorescent images taken with a 522 nm filter. Column III, far red immunofluo- rescent 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.
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Fig. 6. 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, Sm- Smad1-AD–SmSmad4-BD; 7, SmSmad1-AD–SmTbRI-BD; 8, SmSmad1-AD–SmTbRI-QD-BD. Cotransformation numbers 4 and 5 were streaked in duplicate. (B) LacZ filter-lifts from transformed yeast grown on SD media lacking leucine and tryptophan.
Table 1. Summary of the yeast two-hybrid and yeast-three hybrid experiments showing SmSmad1B protein interactions. The following cri- teria 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 9 days of incuba- tion, blue color development on LacZ filter-lift assay after 3 days and ⁄ or < 0.8 b-gal units in liquid LacZ assay); +, moderate interactions (yeast growth after 5 days of incubation, blue color development on LacZ filter-lift assay after 2 days, and 0.9–5.5 b-gal units in liquid LacZ assay; + +, strong interactions (yeast growth after 3 days of incubation, blue color development on LacZ filter-lift assay after 1 day, and 1.0–7.5 b-gal units in liquid LacZ assay); + + +, stronger interactions (yeast growth after 3 days of incubation, blue color development on LacZ filter-lift assay in less than 1 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
SmTbRI-BD
SmTbRI-QD-BD
SmTbRI + SmSmad4-BD
SmTbRI-QD + SmSmad4-BD
SmSmad1B-AD SmSmad1-AD
+ + + + +
+ + +
+ ⁄ – +
+ ⁄ – + ⁄ –
+ ⁄ – + ⁄ –
were observed when SmSmad1B was replaced with SmSmad1 in the three-hybrid experiments.
receptor should be suppressed in the presence of methi- onine. 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 (wild- type 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
To better examine the effect of the inclusion of TGFb receptor-containing constructs on the Sm- Smad1B–SmSmad4 or SmSmad1–SmSmad4 inter- actions, 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 pro- duced 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–Sm- Smad4 b-gal induction of 15% and 26%, respectively. Decreases in b-gal units of 14% and 38% also resulted
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Fig. 7. Yeast three-hybrid analysis of SmSmad1B protein interactions. (A) Growth of cotransformed Y190 yeast cells on select- ive 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, SmSmad1B- AD–SmSmad4-BD; 4, SmSmad1B-AD– SmSmad4-SmTbRI-pBridge; 5, SmSmad1B- AD–SmSmad4-SmTbRI-QD-pBridge; 6, SmSmad1-AD–SmSmad4-BD; 7, SmSmad1- AD–SmSmad4-SmTbRI-pBridge; 8, SmS- mad1–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 signifi- cant decrease in the SmSmad1–SmSmad4 interaction. In the liquid LacZ assays, the extent of the SmSmad1– SmSmad4 interaction as compared to that of the Sm- Smad1B–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 mag- nitude of the decrease in interaction between SmSmad1 and SmSmad4 in the presence of the receptors, specific- ally SmTbRIQD, is more obvious in the liquid assay than in the LacZ filter-lift assay.
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. MBP- bound 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 inter- action 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 statisti- cally significant 52% decrease. In a previous report, the inclusion of the receptor constructs in an in vitro
In an attempt to confirm the SmSmad1B protein in the yeast assays, maltose-binding
interactions
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the pull-down assays, SmSmad1B interacted with both SmTbRI and SmTbRIQD, with a slight binding preference for SmTbRIQD (Fig. 8B). The preferen- tial 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 Sm- Smad4.
that
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 schisto- some 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. GST- bound 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 inclusion of SmTbRI-QD in the binding SmSmad2, reaction not only significantly increased the interaction of SmSmad2 with SmGCN5, but also revealed the interaction of SmSmad4 with SmGCN5 and demon- strated its participation in the formation and, prob- ably, the stabilization of the transcriptional protein complex. Figure 9B shows the presence of SmTbRI-QD significantly boosted the interaction with SmGCN5 of the 35S-labeled SmSmad4 (lane 4) and the 35S-labeled SmSmad2 (lane 6) in the presence of either nonlabeled SmSmad2 or SmSmad4, respectively, as compared to the interaction levels attained in the pres- ence of wild-type SmTbRI (lanes 3 and 5).
Fig. 8. In vitro interaction between SmSmad1B and schistosome (A) Evaluation of the SmSmad1B– TGFb superfamily members. SmSmad4 interaction by MBP pull-down experiments; In vitro translated [35S]SmSmad1B (5 lL) was incubated with SmSmad4- MBP (2 lg) in the presence or absence of unlabeled in vitro transla- ted SmTbRI or SmTbRI-QD (10 lL). A graphical representation of the values obtained from the SmSmad1B–SmSmad4 MBP pull- downs 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 interac- tion between SmSmad1B-MBP and [35S]SmTbRI or [35S]SmTbRI- QD. 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 35S-labeled in vitro translated products.
SmSmad1–SmSmad4 interaction assay also had a neg- ative effect on the strength of the interaction between SmSmad1 and SmSmad4 [6]. MBP pull-down assays were also employed to investigate the binding of Sm- Smad1B with SmTbRI or SmTbRIQD in vitro. Sm- Smad1B was expressed as an MBP fusion protein and incubated with either in vitro translated [35S]methion- ine-labeled SmTbRI or SmTbRIQD, and the bound In proteins were precipitated with amylose resin.
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,
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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
the
Fig. 9. In vitro interaction of the coactivator SmGCN5 with different members of the schistosome TGFb signaling pathway. (A) Interac- tion of in vitro translated 35S-labeled full-length SmGCN5 (5 lL) with glutathione Sepharose-bound GST or GST fusion proteins of Sm- Smad1, 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 full- length SmGCN5 with 35S-labeled, non-S protein-tagged, full-length SmSmad2 or SmSmad4 (5 lL each) in the presence of non-S pro- tein-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, 5 and 8 ⁄ 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 Sm- Smad1 and SmSmad1B genes originated from a com- mon ancestor gene and that gene duplication took split between the platyhelminths, place after 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 pull- down 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 act- ive 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.
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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]. Consid- ering 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].
in its
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 phosphoryla- tion by extracellular signal-regulated kinase (ERK) kinase. However, neither of the other two currently identified schistosome R-Smads, SmSmad1or SmS- mad2, possesses ERK kinase motifs linker region. Only schistosome Smad4 contains a functional ERK phosphorylation motif [6]. The lack of ERK phosphorylation sites in all of the schistosome R- Smads and its presence in the schistosome Co-Smad suggests a divergent ERK–Smad regulatory pathway in this parasite.
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 high- est 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 trans- formation ⁄ development into schistosomules within the host. This is the first examination of schistosome Smad transcript expression in pooled cercariae. The develop- mental stage expressing the lowest levels of SmSmad1 and SmSmad1B transcripts comprises 21-day-old schist- osomules, 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 associ- ated with adult worm pairing and male–female interac- tions. In contrast, the expression levels of SmSmad2 are at their highest in 35 day worms and adults [6].
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 phos- phorylation 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 spe- cificity [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 Sm- Smad1. 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 phosphory- lation site of SmSmad1B (Fig. 1A) conforms to the reported consensus SSXS motif, which is conserved in SmSmad1 as well. However, the receptor phosphoryla- tion 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 trans- ducers, 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 con- served 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 peroxi-
SmSmad1B was also localized in the vitellaria and reproductive ducts of the female adult worm, coinci- ding 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 gyne- cophoral 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 Sm- the immunolocalization of Sm- Smad1B RT-PCR, Smad1B 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.
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level of expression throughout development as com- pared 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.
The protein localization of SmSmad1B in the sub- tegument of adult schistosomes is a common expres- sion 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 sur- rounding environment.
the basal
coactivators
Therefore, the expression pattern for SmSmad1B in S. mansoni suggests the possibility of pleiotropic roles for SmSmad1B: a role in cercaria survival, host penet- ration, 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.
Unlike most transcription factors that are sufficient to recruit transcription machinery and therefore activate transcription on both naked and chromatin templates, the Smads only activate tran- scription from chromatin templates [36]. The tran- scriptional 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 Sm- Smad4 and the formation of a Smad complex. The transcription factors p300 ⁄ CBP and GCN5 possess an intrinsic histone acetyltransferase activity, which facili- tates transcription by decreasing chromosome conden- sation through histone acetylation and by increasing the accessibility of the basal transcription machinery to transcription factors. Indeed, it has been recently that activated Smad2-containing com- shown [40] plexes do not activate transcription by directly recruiting basal transcription machinery to the pro- moter DNA. Rather, Smads recruit the basal tran- scription machinery indirectly as a result of their ability to orchestrate specific histone modifications and chromatin remodeling.
Just as SmSmad1B exhibits motifs characteristic of BMP-related R-Smads and closely resembles schisto- some SmSmad1 rather than SmSmad2, we report that the protein interaction properties of SmSmad1B resem- ble those of SmSmad1 as well. Through protein inter- action 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 com- pared to SmSmad1B. The elucidation of the crystal the schistosome Smads may help to structures of 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 signifi- cant decrease was observed in the presence of the con- stitutively 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 recep- tor 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 sec- ond type I receptor may be activated by BMP ligands and may utilize SmSmad1B and SmSmad1 as down- stream effectors. As SmTbRII maintains an elevated
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 schisto- somes, 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
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Sequence analysis and phylogenetic tree construction
tree was
A phylogenetic 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). Phylo- genetic 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 mrb- ayes v3.1.1 [42]. The trees were started randomly; four sim- ultaneous Markov chains were run for 3 · 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.
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 immunoprecipita- tion 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 interac- tion was observed, in which SmSmad4 represented a part of the protein complex. Thus, it seems that such complex interaction patterns with transcription coacti- vators 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.
The same dataset was also tested by maximum likelihood and neighbor-joining distance [43] methods. For maximum the dataset was analyzed using the likelihood analysis, 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, esti- mated using phyml), using the phylip package v3.62 (http://evolution.genetics.washington.edu/phylip.html). Sup- port values for the tree were obtained by bootstrapping 1000 replicates with seqboot implemented in the phylip package v3.62.
Plasmid construction
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, 5 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 schisto- somes will aid in differentiating the functions of Sm- Smad1 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
constructs: following
vector), transcription ⁄ translation
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and The cDNA coding region of SmSmad1B was PCR ampli- fied from the original cDNA library clone, SmSmad1- BpBluescript. A forward primer, 5¢-CACCATGTTAGACC CAAACATTTGC-OH, was designed to allow for the direc- tional 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 inser- ted into destination vectors that were modified using the Gateway Vector Conversion System (Invitrogen) to produce the SmSmad1B-pGADT7-DEST (yeast GAL4AD vector), SmSmad1B-pCITE-4a-DEST (in vitro SmSmad1B- pMAL-c2X-DEST (MBP-fusion prokaryotic expression vec- tor) and SmSmad1B(MH2)-pGEX-4T1-DEST (GST-fusion prokaryotic expression vector). The following constructs study have been described elsewhere: utilized in this SmSmad4-TbRIQD- SmSmad4-SmTbRI-pBridge truncated pBridge, SmSmad4-pBDGal4, C-terminally 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 ⁄ 9 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 pCR2.1- TOPO 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.
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SmTbRI-pBDGal4 and SmTbRIQD-pBDGal4, SmSmad4- pMAL-c2X, and SmSmad2(MH2)-pET-42a [5,6]. and Smad1-rev
Isolation of SmSmad1B BAC clones and gene analysis
pairs: Smad1-fwd (5¢-ACTGTGGAAGCAGCGGAATG TCTA-3¢) (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 quanti- tative 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.
The S. mansoni BAC1 library [46] was screened using the pre- viously 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; Invitro- gen). For the 5¢-RACE, the following SmSmad1B-specific primer was used to synthesize the first-strand cDNA: 5¢-GCGATCGTGGGATAG-3.¢
Production of SmSmad1B-specific antiserum and western blot
Quantitative RT-PCR
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To avoid cross-reactivity with other SmSmads, a noncon- served 21 amino acid peptide within the linker region of Sm- synthesized (N¢-RHNEYPTIESTKKDSPS Smad1B was DETC-C¢; Proteintech, Chicago, IL, USA). The linker pep- tide, conjugated to KLH, was used to immunize two rabbits over the course of 2 months (Proteintech; short protocol). The anti-SmSmad1B sera as well as preimmune rabbit sera were purified over protein G Sepharose (GE Healthcare Bio- sciences) to isolate the IgG fractions. Specific anti-Sm- Smad1B IgG was isolated by affinity purification over SmSmad1B linker peptide covalently linked to CNBr-activa- ted Sepharose resin (Amersham Biosciences). The affinity- purified 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, 1 mm phenylmethanesulfonyl fluoride, 1 mm dithiothreitol, 1 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 difluo- ride) membrane (Immobilon P; Millipore, Billerica, MA, USA). The membranes were probed with either affinity-puri- fied anti-SmSmad1B IgG or with preimmune rabbit IgG (0.5 lgÆmL)1), and this was followed by incubation with horseradish peroxidase-conjugated goat anti-(rabbit IgG) (Sigma, St Louis, MO, USA; 1 : 3000 dilution). The mem- branes were developed using trimethylbenzidene (Zymed, San Francisco, CA, USA) according to the manufacturer’s instructions. For competition experiments, 1, 2 and 10 lgÆmL)1 of the SmSmad1B linker peptide were preincu- The expression levels of SmSmad1B mRNA in various sta- ges of S. mansoni development were evaluated by quantita- tive 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), infec- ted or uninfected, eggs, cercariae, and 3-day-old and 7-day- old 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 devel- opmental stages were compared to those of the closely rela- ted 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
J. M. Carlo et al.
SmSmad1B, a BMP-R-Smad ortholog from S. mansoni
bated with the primary antibody for 1 h at room temperature prior to incubation with the protein blots.
Immunofluoresence assay
tionally express two proteins: SmSmad4 in-frame with the Gal4BD, and either SmTbRI or SmTbRI-QD under the con- trol 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 (cid:2)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]. Signifi- cant 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. anti-(rabbit IgG)
In vitro interaction assays
Acetone-fixed adult worm cryosections were blocked in 1 · NaCl ⁄ Pi containing 10% goat serum (Sigma) and 10 lgÆmL)1 alkaline phosphatase-conjugated streptavidin (Invitrogen) for 1 h at room temperature. The blocked sec- tions were treated with either affinity-purified anti-SmS- mad1B IgG (5 lgÆmL)1) or with preimmune rabbit IgG (5 lgÆmL)1) in 1 · NaCl ⁄ Pi containing 3% goat serum for 1 h at room temperature. The sections were incubated with (H + L) a biotin-conjugated goat (5 lgÆmL)1: Zymed) in 1 · NaCl ⁄ Pi containing 3% goat serum for 1 h at room temperature. Finally, the sections were treated with an AlexaFluor 647 streptavidin conjugate (5 lgÆmL)1; Molecular Probes) in 1 · NaCl ⁄ Pi containing 3% goat serum for 1 h at room temperature. Probed slides were washed four times with 1 · NaCl ⁄ Pi, 5 min each, fol- lowing 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
treated with fluorography reagent 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 Pro- tein 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]SmS- mad1B protein (5 lL) was incubated in binding buffer (25 mm Tris ⁄ HCl, pH 7.5, 100 mm NaCl, 10% glycerol) with either 2 lg of SmSmad4-MBP-bound resin or MBP resin (negative control) overnight at 4 (cid:2)C. Unlabeled SmTbRI or SmTbRI-QD (10 lL) was also added to the reactions to evaluate the effect of the receptors on the SmSmad1B–SmS- mad4 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 (Amplify; GE fixed, Healthcare), dried, and exposed to X-ray film at ) 80 (cid:2)C.
the HIS3 reporter MBP pull-down assays were also performed to evaluate the SmSmad1B–receptor interactions. Full-length Sm- Smad1B 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 5 lL of the labeled reac- tions were incubated with 2 lg of SmSmad1B-MBP-bound resin in binding buffer overnight at 4 (cid:2)C. The protein-bound resin was processed as described above.
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Using similar approaches, pull-down assays were per- formed to evaluate the interaction between schistosome R-Smads and the coactivators SmGCN5 [27] and SmCBP full-length GST-fusion or in vitro [28]. In these assays, translation (recombinant pCITE-2a; EMD-Bioscience) con- structs of SmSmad1, SmSmad1B, SmSmad2, and Sm- Smad4, as well as the wild-type schistosome TGFb type I receptor, SmTbRI-wt, and the constitutively active mutant 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 SmS- mad1B-pGADT7 plasmid that encodes for SmSmad1B fused with the Gal4 activation domain (Gal4AD) was cotrans- formed 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 co- transformed with the SmSmad4 and the type I receptor con- structs 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 (– 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-
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SmSmad1B, a BMP-R-Smad ortholog from S. mansoni
and host–parasite relationships. FEBS Lett 580, 2968– 2975.
2 Beall MJ, McGonigle S & Pearce EJ (2000) Functional conservation of Schistosoma mansoni Smads in TGF- beta signaling. Mol Biochem Parasitol 111, 131–142. 3 Davies SJ, Shoemaker CB & Pearce EJ (1998) A diver- gent member of the transforming growth factor beta receptor family from Schistosoma mansoni is expressed on the parasite surface membrane. J Biol Chem 273, 11234–11240.
4 Forrester SG, Warfel PW & Pearce EJ (2004) Tegumen- tal expression of a novel type II receptor serine ⁄ threon- ine kinase (SmRK2) in Schistosoma mansoni. Mol Biochem Parasitol 136, 149–156. 5 Osman A, Niles EG & LoVerde PT (2001) Identification 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 pro- tein-tagged in vitro translation products. In vitro inter- action 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 glutathi- one 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 4 (cid:2)C, and the prod- ucts were washed, separated by electrophoresis, and subjec- ted to autofluorography as described above.
and characterization of a Smad2 homologue from Schistosoma mansoni, a transforming growth factor- beta signal transducer. J Biol Chem 276, 10072–10082. 6 Osman A, Niles EG & LoVerde PT (2004) Expression
of functional Schistosoma mansoni Smad4: role in Erk- mediated transforming growth factor beta (TGF-beta) down-regulation. J Biol Chem 279, 6474–6486.
7 Osman A, Niles EG, Verjovski-Almeida S & LoVerde PT (2006) Schistosoma mansoni TGF-beta receptor II: role in host ligand-induced regulation of a schistosome target gene. PLoS Pathog 2, 536–550. 8 Massague J (1998) TGF-beta signal transduction. Annu Rev Biochem 67, 753–791. 9 Moustakas A, Souchelnytskyi S & Heldin CH (2001) 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 protein- tagged [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 1 mm ATP and either SmTbRI-wt or SmTbRI-QD (non-S protein-tagged; 10 lL each). The reactions were incubated overnight at 4 (cid:2)C. SmGCN5-S protein-tagged bound pro- teins were precipitated by adding 30 lL of 50% prewashed S-protein beads and incubating for 1 h at room tempera- ture. Protein-bound beads were washed, separated by SDS ⁄ PAGE, and processed as before. Smad regulation in TGF-beta signal transduction. J Cell Sci 114, 4359–4369.
10 Raftery LA & Sutherland DJ (1999) TGF-beta family signal transduction in Drosophila development: from Mad to Smads. Dev Biol 210, 251–268. 11 Savage-Dunn C (2001) Targets of TGF beta-related
signaling in Caenorhabditis elegans. Cytokine Growth Factor Rev 12, 305–312. 12 Beall MJ & Pearce EJ (2001) Human transforming
growth factor-beta activates a receptor serine ⁄ threonine kinase from the intravascular parasite Schistosoma man- soni. J Biol Chem 276, 31613–31619.
Full-length GST-SmCBP1 was a kind gift from R Pierce (Pasteur Institute, Lille, France). Expression of GST-Sm- CBP1 was done as previously described [28]. GST pull-down assays were performed to evaluate the interaction of GST-SmCBP1 with schistosome R-Smads ) SmSmad1, Sm- Smad1B, and SmSmad ) in the presence of SmSmad4 and SmTbRI, as previously described [6]. Briefly, [35S]methion- ine-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 5 lg of GST-SmCBP1 bound to glutathione Sepharose beads, at 4 (cid:2)C, in the pres- ence of 1 mm ATP. GST-bound beads were used as a negative control. The beads were washed and processed as above. 13 Wahle E & Keller W (1992) The biochemistry of 3¢-end cleavage and polyadenylation of messenger RNA pre- cursors. Annu Rev Biochem 61, 419–440.
Acknowledgements
14 Zavala-Gongora R, Kroner A, Wittek B, Knaus P & Brehm K (2003) Identification and characterisation of two distinct Smad proteins from the fox-tapeworm Ech- inococcus multilocularis. Int J Parasitol 33, 1665–1677. 15 Breathnach R & Chambon P (1981) Organization and
This research was supported by NIH grants AI046762 and D43 TW006580.
expression of eucaryotic split genes coding for proteins. Annu Rev Biochem 50, 349–383.
References
FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS
4091
1 Dissous C, Khayath N, Vicogne J & Capron M (2006) 16 Huang S, Flanders KC & Roberts AB (2000) Character- ization of the mouse Smad1 gene and its expression pattern in adult mouse tissues. Gene 258, 43–53. Growth factor receptors in helminth parasites: signalling
J. M. Carlo et al.
SmSmad1B, a BMP-R-Smad ortholog from S. mansoni
transcriptional activity. Biochem Biophys Res Commun 271, 682–687. 30 Ebisawa T, Fukuchi M, Murakami G, Chiba T,
17 Watanabe TK, Suzuki M, Omori Y, Hishigaki H, Horie M, Kanemoto N, Fujiwara T, Nakamura Y & Takaha- shi E (1997) Cloning and characterization of a novel member of the human Mad gene family (MADH6). Genomics 42, 446–451.
Tanaka K, Imamura T & Miyazono K (2001) Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degrada- tion. J Biol Chem 276, 12477–12480.
18 Bucher P (1990) Weight matrix descriptions of four euk- aryotic RNA polymerase II promoter elements derived from 502 unrelated promoter sequences. J Mol Biol 212, 563–578.
31 Lo RS, Chen YG, Shi Y, Pavletich NP & Massague J (1998) The L3 loop: a structural motif determining specific interactions between SMAD proteins and TGF-beta receptors. EMBO J 17, 996–1005. 32 Mei H, Hirai H, Tanaka M, Hong Z, Rekosh D & 19 Arkhipova IR (1995) Promoter elements in Drosophila melanogaster revealed by sequence analysis. Genetics 139, 1359–1369.
LoVerde PT (1995) Schistosoma mansoni: cloning and characterization of a gene encoding cytosolic Cu ⁄ Zn superoxide dismutase. Exp Parasitol 80, 250–259. 20 Corden J, Wasylyk B, Buchwalder A, Sassone-Corsi P, Kedinger C & Chambon P (1980) Promoter sequences of eukaryotic protein-coding genes. Science 209, 1406– 1414. 33 Smale ST & Kadonaga JT (2003) The RNA polymerase 21 Kutach AK & Kadonaga JT (2000) The downstream
promoter element DPE appears to be as widely used as the TATA box in Drosophila core promoters. Mol Cell Biol 20, 4754–4764.
II core promoter. Annu Rev Biochem 72, 449–479. 34 Knobloch J, Rossi A, Osman A, LoVerde PT, Klinkert MQ & Grevelding CG (2004) Cytological and biochemi- cal evidence for a gonad-preferential interplay of SmFKBP12 and SmTbetaR-I in Schistosoma mansoni. Mol Biochem Parasitol 138, 227–236. 22 Shi Y & Massague J (2003) Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 113, 685–700.
35 Zhan Y, Fujino A, Maclaughlin DT, Manganaro TF, Szotek PP, Arango NA, Teixeira J & Donahoe PK (2006) Mullerian inhibiting substance regulates its recep- tor ⁄ SMAD signaling and causes mesenchymal transition of the coelomic epithelial cells early in Mullerian duct regression. Development 133, 2359–2369. 23 Abdollah S, Macias-Silva M, Tsukazaki T, Hayashi H, Attisano L & Wrana JL (1997) TbetaRI phosphoryla- tion of Smad2 on Ser465 and Ser467 is required for Smad2–Smad4 complex formation and signaling. J Biol Chem 272, 27678–27685. 36 Massague J, Seoane J & Wotton D (2005) Smad tran- 24 Kretzschmar M, Liu F, Hata A, Doody J & Massague J scription factors. Genes Dev 19, 2783–2810.
37 Feng XH, Zhang Y, Wu RY & Derynck R (1998) The tumor suppressor Smad4 ⁄ DPC4 and transcriptional adaptor CBP ⁄ p300 are coactivators for smad3 in TGF- beta-induced transcriptional activation. Genes Dev 12, 2153–2163. (1997) The TGF-beta family mediator Smad1 is phosphorylated directly and activated functionally by the BMP receptor kinase. Genes Dev 11, 984–995. 25 Simonsson M, Kanduri M, Gronroos E, Heldin CH & Ericsson J (2006) The DNA binding activities of Smad2 and Smad3 are regulated by coactivator-mediated acety- lation. J Biol Chem 281, 39870–39880.
38 Itoh S, Ericsson J, Nishikawa J, Heldin CH & ten Dijke P (2000) The transcriptional co-activator P ⁄ CAF potenti- ates TGF-beta ⁄ Smad signaling. Nucleic Acids Res 28, 4291–4298. 26 Tu AW & Luo K (2007) Acetylation of Smad2 by the co-activator p300 regulates activin and TGFbeta response. J Biol Chem. In press. 39 Kahata K, Hayashi M, Asaka M, Hellman U,
27 de Moraes Maciel R, de Silva Dutra DL, Rumjanek FD, Juliano L, Juliano MA & Fantappie MR (2004) Schistosoma mansoni histone acetyltransferase GCN5: linking histone acetylation to gene activation. Mol Biochem Parasitol 133, 131–135. Kitagawa H, Yanagisawa J, Kato S, Imamura T & Miyazono K (2004) Regulation of transforming growth factor-beta and bone morphogenetic protein signalling by transcriptional coactivator GCN5. Genes Cells 9, 143–151. 28 Bertin B, Oger F, Cornette J, Caby S, Noel C, Capron M,
40 Ross S, Cheung E, Petrakis TG, Howell M, Kraus WL & Hill CS (2006) Smads orchestrate specific histone modifications and chromatin remodeling to activate transcription. EMBO J 25, 4490–4502. 41 Verjovski-Almeida S, DeMarco R, Martins EA, Fantappie MR, Rumjanek FD & Pierce RJ (2006) Schistosoma mansoni CBP ⁄ p300 has a conserved domain structure and interacts functionally with the nuclear receptor SmFtz-F1. Mol Biochem Parasitol 146, 180–191.
FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS
4092
29 Kawai S, Faucheu C, Gallea S, Spinella-Jaegle S, Atfi A, Baron R & Roman SR (2000) Mouse smad8 phosphory- lation downstream of BMP receptors ALK-2, ALK-3, and ALK-6 induces its association with Smad4 and Guimaraes PE, Ojopi EP, Paquola AC, Piazza JP, Nishiyama MY Jr, Kitajima JP, Adamson RE et al. (2003) Transcriptome analysis of the acoelomate human parasite Schistosoma mansoni. Nat Genet 35, 148–157.
J. M. Carlo et al.
SmSmad1B, a BMP-R-Smad ortholog from S. mansoni
42 Huelsenbeck JP & Ronquist F (2001) MRBAYES:
48 Osman A (2004) Yeast two-hybrid assay for studying protein–protein interactions. Methods Mol Biol 270, 403–422. Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755.
Supplementary material
43 Saitou N & Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425. 44 Jones DT, Taylor WR & Thornton JM (1992) The
is available
rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci 8, 275–282.
45 Guindon S & Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52, 696–704.
The following supplementary material online: Fig. S1. Maximum likelihood tree of SmSmad1B. Fig. S2. Neighbour Joining distance tree of SmSmad1B. Table S1. Exon ⁄ Intron junctions of the SmSmad1B gene.
46 Le Paslier MC, Pierce RJ, Merlin F, Hirai H, Wu W,
This material is available as part of the online article
from http://www.blackwell-synergy.com
Williams DL, Johnston D, LoVerde PT & Le Paslier D (2000) Construction and characterization of a Schistoso- ma mansoni bacterial artificial chromosome library. Genomics 65, 87–94.
Please note: Blackwell 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 corres- ponding author for the article.
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47 Webster PJ, Seta KA, Chung SC & Mansour TE (1992) A cDNA encoding an alpha-tubulin from Schistosoma mansoni. Mol Biochem Parasitol 51, 169–170.
