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

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

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

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.

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

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.

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

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).

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-

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

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.

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

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-

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

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

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

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,

(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:

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-

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

+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

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 [

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