Tài liệu Báo cáo khoa học: Identification and characterization of an R-Smad ortholog (SmSmad1B) from Schistosoma mansoni pdf

Identification and characterization of an R-Smad ortholog
(SmSmad1B) from Schistosoma mansoni
Joelle M. Carlo
1
*, Ahmed Osman
1,2
*, Edward G. Niles
1
, Wenjie Wu
2
, Marcelo R. Fantappie
2
,
Francisco M. B. Oliveira
2
and Philip T. LoVerde
1,2
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
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 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
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:
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.
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
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 such as embryogenesis, differentiation and
apoptosis makes these pathways attractive candidates

for elucidating the growth and development mecha-
nisms employed by S. mansoni.
The TGFb superfamily comprises a large group of
structurally related, secreted cytokines, 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 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 media-
tors called receptor-regulated Smads (R-Smads). The
R-Smads of the TGF b–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.
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 (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
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.
Herein, we report the isolation of a new member
of the S. mansoni
R-Smad family, designated Sm-
Smad1B. 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 ⁄ 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).
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).
SmSmad1B, a BMP-R-Smad ortholog from S. mansoni J. M. Carlo et al.
4076 FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS
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,
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.

J. M. Carlo et al. SmSmad1B, a BMP-R-Smad ortholog from S. mansoni
FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS 4077
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
phylogenetic data suggest that SmSmad1 and Sm-
Smad1B are paralogous genes; they originated from
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 · 10
6
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.
SmSmad1B, a BMP-R-Smad ortholog from S. mansoni J. M. Carlo et al.
4078 FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS
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
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,
mouse Smad1, and the E. multilocularis SmadB
[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.
The beginning of exon 1 of SmSmad1B was identi-
fied 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 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
+1
AAAC) [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 Smad-
binding elements (GTCT) [22] in the upstream region
(Fig. 1B).
Developmental expression of SmSmad1B
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 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 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
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 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
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.

J. M. Carlo et al. SmSmad1B, a BMP-R-Smad ortholog from S. mansoni
FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS 4079
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].
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 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
(Fig. 4B, right panel, lane 3), indicating that the
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
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).
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 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.
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
in Experimental procedures. In yeast transformants
with the pBridge construct, the expression of the
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.
SmSmad1B, a BMP-R-Smad ortholog from S. mansoni J. M. Carlo et al.
4080 FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS

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.
J. M. Carlo et al. SmSmad1B, a BMP-R-Smad ortholog from S. mansoni
FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS 4081
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
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 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
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 m
M 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 + + + + ⁄ –+⁄ –++ +
SmSmad1B, a BMP-R-Smad ortholog from S. mansoni J. M. Carlo et al.
4082 FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS

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.
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 [
35
S]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
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 m
M 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).
J. M. Carlo et al. SmSmad1B, a BMP-R-Smad ortholog from S. mansoni
FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS 4083
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 SmTb RI or SmTbRIQD in vitro. Sm-
Smad1B was expressed as an MBP fusion protein and
incubated with either in vitro translated [
35
S]methion-
ine-labeled SmTbRI or SmTbRIQD, and the bound
proteins were precipitated with amylose resin. In
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.
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
35
S-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
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 demon-
strated its participation in the formation and, prob-
ably, the stabilization of the transcriptional protein
complex. Figure 9B shows that the presence of
SmTbRI-QD significantly boosted the interaction with
SmGCN5 of the
35
S-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 pres-
ence of wild-type SmTbRI (lanes 3 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 SmT bRI-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,
Fig. 8. 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 [
35
S]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 [
35
S]SmTbRI or [
35
S]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
35
S-labeled in vitro translated products.

SmSmad1B, a BMP-R-Smad ortholog from S. mansoni J. M. Carlo et al.
4084 FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS
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
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
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 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
35
S-labeled SmSmad1, SmSmad1B, SmSmad2, SmSmad4, or SmTbRI-QD (5 l L each). (B) Interaction of
glutathione Sepharose-bound GST or full-length GST-SmCBP1 fusion protein (5 lg each) with in vitro translated
35
S-labeled SmSmad1 or
SmSmad2 (5 lL each) in the presence of in vitro translated
35
S-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,
35
S-labeled proteins. Binding reaction products were separated by SDS ⁄ PAGE and subjected to autofluorography.
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
35
S-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
35

S-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,
35
S-labeled proteins. In lane 3, there is
a distortion of the radioactive labeled protein band ([
35
S]Met-labeled
SmSmad4) that occurred during the electrophoretic migration.
J. M. Carlo et al. SmSmad1B, a BMP-R-Smad ortholog from S. mansoni
FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS 4085
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 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 R-

Smads 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 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-
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].
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 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 TGF b 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-
Smad1B RT-PCR, the immunolocalization of Sm-
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.
SmSmad1B, a BMP-R-Smad ortholog from S. mansoni J. M. Carlo et al.
4086 FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS
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.
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.
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
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 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
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.
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 tran-
scription from chromatin templates [36]. The tran-
scriptional 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 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
shown [40] that activated Smad2-containing com-
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.
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
J. M. Carlo et al. SmSmad1B, a BMP-R-Smad ortholog from S. mansoni
FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS 4087
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.
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
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 [
32
P]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.
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 (.
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 · 10
6
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, esti-
mated using phyml), using the phylip package v3.62
( Sup-
port 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 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 following constructs: SmSmad1B-pGADT7-DEST
(yeast GAL4AD vector), SmSmad1B-pCITE-4a-DEST

(in vitro transcription ⁄ translation vector), SmSmad1B-
pMAL-c2X-DEST (MBP-fusion prokaryotic expression vec-
tor) 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-TbRIQD-
pBridge, SmSmad4-pBDGal4, C-terminally truncated
SmSmad1B, a BMP-R-Smad ortholog from S. mansoni J. M. Carlo et al.
4088 FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS
SmTbRI-pBDGal4 and SmTbRIQD-pBDGal4, SmSmad4-
pMAL-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 pre-
viously described [
32
P]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 ( />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.¢
Quantitative RT-PCR
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;
/>SmSmad1 and SmSmad1B target sequences were amplified
from different cDNA samples using the following primer

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 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.
Production of SmSmad1B-specific antiserum and
western blot
To avoid cross-reactivity with other SmSmads, a noncon-
served 21 amino acid peptide within the linker region of Sm-
Smad1B was synthesized (N¢-RHNEYPTIESTKKDSPS
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-
J. M. Carlo et al. SmSmad1B, a BMP-R-Smad ortholog from S. mansoni
FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS 4089
bated with the primary antibody for 1 h at room temperature

prior to incubation with the protein blots.
Immunofluoresence assay
Acetone-fixed adult worm cryosections were blocked in
1 · NaCl ⁄ P
i
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
)in1· NaCl ⁄ P
i
containing 3% goat serum for
1 h at room temperature. The sections were incubated with
a biotin-conjugated goat anti-(rabbit IgG) (H + L)
(5 lgÆmL
)1
: Zymed) in 1 · NaCl ⁄ P
i
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 ⁄ P
i
containing
3% goat serum for 1 h at room temperature. Probed slides
were washed four times with 1 · NaCl ⁄ P
i
, 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
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 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-
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 °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.
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 [
35

S]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 [
35
S]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 °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
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 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 [
35
S]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 °C. The protein-bound
resin was processed as described above.
Using similar approaches, pull-down assays were per-

formed 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) 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
SmSmad1B, a BMP-R-Smad ortholog from S. mansoni J. M. Carlo et al.
4090 FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS
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 [
35
S]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 °C, and the prod-
ucts were washed, separated by electrophoresis, and subjec-
ted 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 protein-
tagged [

35
S]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
1mm ATP and either SmTbRI-wt or SmTbRI-QD (non-S
protein-tagged; 10 lL each). The reactions were incubated
overnight at 4 °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.
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, [
35
S]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 °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.
Acknowledgements

This research was supported by NIH grants AI046762
and D43 TW006580.
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Supplementary material
The following supplementary material is available
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.
This material is available as part of the online article
from
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.
J. M. Carlo et al. SmSmad1B, a BMP-R-Smad ortholog from S. mansoni
FEBS Journal 274 (2007) 4075–4093 ª 2007 The Authors Journal compilation ª 2007 FEBS 4093