Molecular characterization and ligand binding specificity of the PDZ domain-containing protein GIPC3 from Schistosoma japonicum
Parasites & Vectors
Molecular characterization and ligand binding specificity of the PDZ domain-containing protein GIPC3 from Schistosoma japonicum
Yi Mu 1
Haiming Huang 1
Shuai Liu 0
Pengfei Cai 0
Youhe Gao 1
0 MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College , Beijing 100730 , P.R. China
1 National Key Laboratory of Medical Molecular Biology, Dept. of Physiology and Pathophysiology, School of Basic Medicine, Peking Union Medical College, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences , Beijing 100005 , P.R. China
Background: Schistosomiasis is a serious global health problem that afflicts more than 230 million people in 77 countries. Long-term mass treatments with the only available drug, praziquantel, have caused growing concerns about drug resistance. PSD-95/Dlg/ZO-1 (PDZ) domain-containing proteins are recognized as potential targets for the next generation of drug development. However, the PDZ domain-containing protein family in parasites has largely been unexplored. Methods: We present the molecular characteristics of a PDZ domain-containing protein, GIPC3, from Schistosoma japonicum (SjGIPC3) according to bioinformatics analysis and experimental approaches. The ligand binding specificity of the PDZ domain of SjGIPC3 was confirmed by screening an arbitrary peptide library in yeast two-hybrid (Y2H) assays. The native ligand candidates were predicted by Tailfit software based on the C-terminal binding specificity, and further validated by Y2H assays. Results: SjGIPC3 is a single PDZ domain-containing protein comprised of 328 amino acid residues. Structural prediction revealed that a conserved PDZ domain was presented in the middle region of the protein. Phylogenetic analysis revealed that SjGIPC3 and other trematode orthologues clustered into a well-defined cluster but were distinguishable from those of other phyla. Transcriptional analysis by quantitative RT-PCR revealed that the SjGIPC3 gene was relatively highly expressed in the stages within the host, especially in male adult worms. By using Y2H assays to screen an arbitrary peptide library, we confirmed the C-terminal binding specificity of the SjGIPC3-PDZ domain, which could be deduced as a consensus sequence, -[SDEC]-[STIL]-[HSNQDE]-[VIL]*. Furthermore, six proteins were predicted to be native ligand candidates of SjGIPC3 based on the C-terminal binding properties and other biological information; four of these were confirmed to be potential ligands using the Y2H system. Conclusions: In this study, we first characterized a PDZ domain-containing protein GIPC3 in S. japonicum. The SjGIPC3-PDZ domain is able to bind both type I and II ligand C-terminal motifs. The identification of native ligand will help reveal the potential biological function of SjGIPC3. These data will facilitate the identification of novel drug targets against S. japonicum infections.
Schistosoma japonicum; GIPC3; PDZ domain-containing protein; Ligand binding specificity
Schistosomiasis, caused by the parasitic blood fluke of
the genus Schistosoma, remains a major global public
health problem that affects more than 230 million
people living in the endemic areas of 77 countries
worldwide (more than 95% in Africa) (http://www.who.int/
mediacentre/factsheets/fs115/en/index.html). Three major
pathogenic schistosome species, Schistosoma mansoni,
Schistosoma haematobium, and Schistosoma japonicum,
are known to contribute to the disease loads. The
treatment of schistosomiasis is based on the long-term mass
treatment with the only available drug, praziquantel
(PZQ). However, concerns about the problem of drug
resistance have necessitated a search for alternative
treatments [1-3]. The decoding of the genomes of three
major pathogenic blood flukes has provided a valuable
entity for the systematic identification of possible drug
targets against the parasite [4-6].
Protein-protein interactions (PPIs) offer a realm of less
explored potential drug targets, and are recognized to be
the novel targets of next-generation drug development
. The PDZ domain is one of the most important PPI
modules, and is emergently regarded as a novel target of
drug discovery [8,9]. PDZ domains are built of
approximately 70 to 90 residues and form a compact globular
fold consisting of a core of six -strands (A - F) and
two -helices (A, B) . The C-terminus (4 to 5
Cterminal residues) of partners usually projects to a
hydrophobic pocket formed with the
carboxylatebinding loop (A-B loop), which contains the
conserved GLGF motif, and the ?B helix of the PDZ
domain . The PDZ binding motifs have been
conventionally placed into four classes based on the
extreme C-terminus: Class I, -[S/T]-X-*; Class II,
--X*; Class III, -[D/E/K/R]-X-*; and Class IV, -X--[D/E]
*; where X is any amino acid, is a hydrophobic
residue, is an aromatic residue, and * represents the stop
codon of the peptide . Although the PDZ domains
are conserved in a variety of organisms, no detailed
literature about the schistosome-derived PDZ domain has
been reported thus far.
As a benefit of the decoding of the genome, several
PDZ domain-containing proteins were deposited in the
public S. japonicum database. Among them, one is
homologous to the mammalian GAIP-interacting proteins, C
terminus (GIPCs, also known as TIP-2, GLUT1CBP,
RGS19IP1, SEMCAP-1, and synectin); here we have
designated the S. japonicum homologue as SjGIPC3.
GIPCs are characterized by a single, centrally located
PDZ domain for ligand binding. GIPC1 was originally
found to bind to the Gi GTPase-activating protein
RGS-GAIP and have been suggested to exert function in
a G-protein coupled complex mediating vesicular
trafficking . Accumulating data have confirmed that the
PDZ domain of GIPCs can interact with a panel of
partners through C-terminal region recognition. Most of
these partners are transmembrane receptors, such as
Tax , M-semF , Glut1 , neuropilin-1 ,
Syndecan-4 , and so on [19-28]. In addition, human
and Drosophila GIPC orthologues can also bind to the
non-receptor protein, myosin VI [29,30]. Recently, GIPC
orthologues were regarded as novel and potential targets
for therapeutic development for the treatment of human
cancers [31,32]. Several cell-permeable octapeptides
targeting the PDZ domain of GIPCs were designed based
on binding specificity and have shown their powers in
inhibition of proliferation and induction of apoptosis in
some types of cancer [31,33].
In this study, we present for the first time the
molecular characteristics of a PDZ-containing protein from S.
japonicum, GIPC3. We analyzed the expression levels of
SjGIPC3 during development of the parasite. To reveal
its ligand binding specificity, a random peptide library
constructed from human genomic DNA was used in
yeast two-hybrid (Y2H) assays to screen for the
Cterminal binding motifs of the SjGIPC3-PDZ domain.
Furthermore, the potential ligands of SjGIPC3 were
predicted by bioinformatic approaches based on the
Cterminal binding specificity, and validated in the Y2H
system. This work will facilitate the identification of
novel drug targets against S. japonicum.
Parasites and animals
The S. japonicum-infected Oncomelania hupensis were
purchased from Jiangxi Institute of Parasitic Diseases,
Nanchang, China. Cercariae were shed from the infected snails
and harvested for total RNA isolation. New Zealand White
rabbits were percutaneously infected with cercariae.
Hepatic schistosomula and adult worms were obtained from
the rabbits by hepatic-portal perfusion at 2 and 6 weeks
post-infection, respectively. Male and female adult worms
were manually separated with the aid of a light
microscope. Eggs were isolated from liver tissues of rabbits after
6 weeks of infection by the sieving and enzymatic digestion
Bioinformatics analysis of SjGIPC3
The nucleotide and deduced amino acid sequence of
SjGIPC3 were analyzed from NCBI. The PDZ domain of
SjGIPC3 protein was predicted in the Conserved
Domain Database (CDD) v.2.25 of NCBI (http://www.ncbi.
nlm.nih.gov/cdd) . The three-dimensional structure
of the SjGIPC3-PDZ domain was predicted by the
Rosetta server (http://robetta.bakerlab.org/)  with
the PDZ domain in GIPC2 (PDB code: 3GGE chain A)
as the template. The 3D structure was further refined
with PyMOL Viewer program (http://www.pymol.org/).
Multiple sequence alignment and phylogenetic analysis
The GIPC homologous sequences of other species were
retrieved from GenBank using SjGIPC3 as bait. Protein
sequences were aligned by ClustalX 2.0. The alignment
was refined with GeneDoc software. Phylogenetic
analysis was performed as previously described . The
alignment was further optimized by deletion of highly
variable N- and C-terminal regions. The phylogenetic
tree was constructed with MEGA 4 using the
neighborjoining method. Bootstrap values were expressed as
percentage of 1000 replicates.
Total RNAs of S. japonicum at different developmental
stages (cercariae, hepatic schistosomula, separated adult
male and female worms, and eggs) were extracted using
Trizol reagent (Invitrogen). The contaminating genomic
DNA was removed from RNA samples with TURBO
DNA-freeTM kit (Ambion, CA, USA). RNA
quantification and quality control was conducted by 1% agarose
gel electrophoresis and Nanodrop ND-1000
spectrophotometer (Nanodrop Technologies, Wilmington, DE). For
each sample, 1 g total RNA was reverse transcribed
into first-strand cDNA using SuperScriptTM III Reverse
Transcriptase Kit (Invitrogen), with Oligo dT (15)
primer. cDNA synthesis was performed as follows: 25C for
5 min, 50C for 1 h, 70C for 15 min. Each PCR reaction
contained 12.5 l of 2Brilliant II SYBR Green QPCR
Master Mix (Agilent, USA), 1 l diluted cDNA (20), 1
l of the forward and reverse primer pair (final
concentration: 0.2 M each primer, Additional file 1 Table S1),
and 10.5 l of sterile water. The PCR program included
40 cycles with denaturation at 95C for 30 s, followed by
annealing and extension at 60C for 1 min.
Quantification of the relative differential expression for SjGIPC3
gene was performed by normalizing against the PSMD4
transcript (26S proteasome non-ATPase regulatory
subunit 4, GenBank accession number: FN320595) and
applying the comparative 2-Ct method, according to the
SDS 1.4 software (Applied Biosystems, Foster City, USA)
Construction of bait plasmids
The DNA fragments encoding full-length SjGIPC3 and
the PDZ domain region (AA 99195) were amplified
from the adult worm cDNA templates using high fidelity
Phusion DNA polymerase (Finnzymes Oy, Finland)
(Primer sets are listed in Additional file 1 Table S1). The
PCR was performed with an initial denaturation for 1
min at 98C. Thirty PCR cycles were performed as
follows: 98C for 5 s, 50C for 30 s, and 72C for 15 s. The
final extension was 5 min at 72C. The amplicons were
digested with EcoRI and BamHI, and cloned into GAL4
BD vector, pBridge. The recombinant plasmids were
transformed into DH5 (DE3) Escherichia coli and
positive clones were selected for sequencing.
Yeast two-hybrid screening of random peptide library
Each GAL4 BD-fusion bait plasmid was transformed
into the yeast strain CG1945 using the lithium acetate
procedure. The transformants were grown on SD/-Trp
plates and lacZ assays were performed to examine
selfactivation. The transformants were further spread on
SD/-His/-Trp plates with different concentration of
3amino-1,2,4-triazole for leakage test. The random
peptide library (constructed with Tsp509I-digested human
genomic DNA fragments)  was screened following
the MATCHMAKER Two-Hybrid System protocol
(Clontech). The transformants were screened on
SD/Trp/-His/-Leu plates with different concentrations of
3amino-1,2,4-triazole (2.5 mM for full-length SjGIPC3
bait transformants, and 20 mM for SjGIPC3-PDZ bait
transformants) and further verified by the improved
LacZ assays. After rescue, the potential positive plasmids
were isolated and retransformed into the yeast strain
CG1945 containing corresponding bait plasmid. Only
the clones that were positive for all the reporter assays
and confirmed by at least two independent tests were
selected for specific interactions and sequenced .
Prediction of candidate ligands
A consensus C-terminal binding sequence of
SjGIPC3PDZ domain was deduced from sequence alignment of
positive clones from yeast two-hybrid screening. The S.
japonicum predicted protein sequences were
downloaded from SDSPB
(http://lifecenter.sgst.cn/schistosoma/cn/schdownload.do). The Tailfit software  was
used to search against the S. japonicum protein
sequences to retrieve potential ligand proteins whose
carboxyl termini matched the consensus-binding
sequence. These peptide sequences were then manually
blasted against NCBI's non-redundant protein database
to filter the truncated fragments lacking of the
Cterminus. The most promising candidate ligand proteins
were selected further based on biological information
such as subcellular localization and potential molecular
Confirmation of candidate SjGIPC3-ligand interactions
The GAL4 AD plasmids expressing the
carboxylterminus of the candidate ligands of the SjGIPC3-PDZ
domain were first constructed. The self primer
template PCR was performed to produce DNA fragments
encoding the 1011 amino acid residues of extreme
carboxyl-terminus of potential ligands (The primer sets
were shown in Additional file 1 Table S1). The PCR
procedure included thirty cycles as follows: 98C for 5
s, 50C for 25 s, and 72C for 1 s, and a final
extension at 72C for 2 min. The resulting DNA
fragments were digested with EcoRI and BamHI
endonucleases, and cloned into the EcoRI/BamHI sites of GAL4
AD vector, pGADT7 followed by sequencing. Each
constructed plasmid was co-transformed with the
SjGIPC3PDZ domain or full-length SjGIPC3 bait plasmid into
yeast strain CG1945, respectively. The positive
interactions were selected in the yeast two-hybrid system as
Molecular characteristics of S. japonicum GIPC3
By searching the S. japonicum proteomic database, we
found that at least 25 PDZ domains are distributed
among 16 proteins. S. japonicum GIPC3 is a single PDZ
domain-containing protein, which is 328 amino acids in
length, with a theoretical molecular weight of 37 kDa.
By performing a CD-search, we found that the PDZ
domain was located in the middle region of SjGIPC3
protein. The peptide sequence of SjGIPC3 was aligned with
sequences of orthologues from other taxa. The GIPC
homology 1 (GH1) and PDZ domains of GIPC proteins
were relatively well conserved, in contrast to the GIPC
homology 2 (GH2) domain and the flanking N- and
Cterminal regions (Figure 1). Homology analysis revealed
that SjGIPC3 shares a relatively high degree of sequence
homology with S. mansoni orthologue (GenBank
Accession number: XP_002569948, identity=78%, only
aa1894-2197 region was used for calculating identity)
and followed by share 46% identity with Clonorchis
sinensis orthologue (GenBank Accession number:
GAA51139). The sequence in the conserved GLGF
motif is replaced by SFGL in SjGIPC3, which is
different to that in any other GIPC orthologues (Figure 1).
Structural prediction revealed that the SjGIPC3-PDZ
domain was comprised of two -helixes and five -sheets
(lacking E, Figure 2A), with a hydrophobic pocket
formed between B and B, in which the residue F from
the unique GLGF motif (SFGL in the SjGIPC3-PDZ
domain, while ALGL or SLGL in the PDZ domains
of mammalian GIPCs) may affect the ligand binding
specificity of the PDZ domain (Figure 2B).
Phylogenetic analysis of SjGIPC3
The sequences of GIPC orthologues from other taxa
were retrieved to create a phylogenetic tree using the
Neigh-bor-Joining method. SjGIPC3 was clustered with
S. mansoni and C. sinensis orthologues to form a clade,
which was divergent from those of other phyla, even
with the parasitic nematode-derived orthologue,
AsGIPC1 (Figure 3). However, less is known about
whether this divergence of fluke GIPC orthologues is
related to adaptations specific to their parasitic life.
Transcriptional analysis of the SjGIPC3 gene at different
developmental stages of the parasite
To determine the transcriptional patterns of SjGIPC3 at
different developmental stages and between sexes,
qRTPCR was performed using an optimal reference gene,
26S proteasome non-ATPase regulatory subunit 4
(PSMD4), as an internal control . As a result, we
observed that the SjGIPC3 gene was ubiquitously
expressed at different developmental stages, but in a
stage-biased pattern. The expression level of the
SjGIPC3 gene was lower in the cercarial stage than in
any other stages in which it was detected, suggesting
that the function of SjGIPC3 is mainly exerted in the
stages within the host. In adult worms, the
transcriptional level of SjGIPC3 was significantly higher in male
than in female worms (Figure 4).
Determination of the ligand binding specificity of SjGIPC3
To investigate the binding properties of the PDZ domain
of SjGIPC3, we screened an arbitrary peptide library using
Y2H assays. When the library was screened against the
SjGIPC3-PDZ domain, 37 positive clones were sequenced,
each encoding a unique carboxy-terminus. Among them,
17 belong to Class I PDZ binding motifs, 16 belong to
Class II PDZ binding motifs, and four are unclassified
(Table 1). When the library was screened against the
full-length SjGIPC3, 42 positive clones were obtained.
Sequence analysis revealed that three pairs of clones were
identical; thus a total of 39 unique carboxy-terminal
sequences were obtained. Among them, 24 belong to
Class I PDZ binding motifs, 12 belong to Class II PDZ
binding motifs, and three are unclassified (Table 1).
In the first assay using the SjGIPC3-PDZ domain as
bait, the domain showed predominant preference for
hydrophobic amino acids at the extreme carboxyl
terminus (P0), especially Leu (37.8%), Ile (32.4%), or Val
(18.9%). The P-1 of the carboxyl terminus slightly
prioritizes polar amino acids, such as Ser (16.2%), Asn
(10.8%), or Gln (10.8%), and acidic amino acids, such as
Asp (13.5%) or Glu (13.5%). At P-2, the polar amino
acids Ser (37.8%) and Thr (10.8%) and the aliphatic
amino acids Ile (21.6%) and Leu (18.9%) were
tially selected. At P-3, Ser (13.5%), Asp (18.9%), and Glu
(24.3%) were preferred, along with the rare occurrence
of other amino acids (Figure 5A). Based on the statistical
analysis of unique carboxy-terminal sequences, a
consensus-binding sequence can be deduced as -[S/D/
In the second assay, full-length SjGIPC3 was probed to
screen the random peptide library, and a more significant
binding specificity emerged. As in the first assay,
hydrophobic amino acids, especially Ile (42.5%), Leu (21.3%), or Val
(14.2%), were predominantly preferred at P0, with a slight
bias toward the aromatic amino acid Phe (9.5%). At P-1, the
Figure 1 Sequence alignment of peptide sequences of SjGIPC3 and GIPC orthologues derived from other species with ClustalX 2.0.
Black indicates identity in all variants; grey indicates a conservative substitution in at least one variant; white indicates a non-conservative residue;
dashes indicate missing residues. GenBank Accession Numbers: SjGIPC3, Schistosoma japonicum, (AAW24514.1); SmGIPC3, Schistosoma mansoni,
(XP_002569948); CsGIPC1, Clonorchis sinensis, (GAA51139); XtGIPC3, Xenopus tropicalis, (NP_001096502); HsGIPC3, Homo sapiens, (NP_573568);
XtGIPC1, Xenopus tropicalis, (NP_001011331); HsGIPC1, Homo sapiens, (NP_005707); XtGIPC2, Xenopus tropicalis, (NP_001120269); HsGIPC2, Homo
sapiens, (NP_060125); DmKermit, Drosophila melanogaster, (NP_652028); AsGIPC1, Ascaris suum, (ADY44129); CeGIPC1, Caenorhabditis elegans,
(NP_498017). The GH1, GH2, and PDZ domains are marked with black boxes and the boundaries for these domains were defined based on the
previous study . The GLGF motif region within the PDZ domains of GIPC orthologues is highlighted by the red box.
preference for Glu (45.2%) was dramatically increased when
compared with the first assay, along with a rare selection of
other residues: His (11.9), Ser (11.9%), Asn (11.9%), or Asp
(9.5%). The P-2 position demonstrated a dramatic
preference for the polar amino acid Ser (54.8%) and the
hydrophobic amino acid Ile (21.4%); other amino acids, such as
Thr (11.9%), Val (7.1%), or Leu (4.8%), also occurred at a
low rate. At P-3, Glu (45.2%) was predominantly selected,
followed by Cys (19.0%) and Asp (14.3%) (Figure 5B).
Similar to the first assay, a consensus-binding sequence can be
inferred from the second Y2H assay: -[D/E/C]-[S/T/I]-[H/
S/N/D/E]-[V/I/L]*. Finally, based on both assays, the
Figure 2 The cartoon diagrams for the 3D structure of the SjGIPC3-PDZ domain. A. The SjGIPC3-PDZ domain is colored by
secondarystructure elements (-helices, cyan; -strands, magenta; loops, salmon). B. A hydrophobic pocket was formed by the B helix, the B strand, and
the carboxylate-binding loop. The hydrophobic amino acids within the pocket are highlighted in red. The residue F in the unique GLGF motif
(here is SFGL) may affect the selection of the C-terminal tails of its partner proteins.
consensus-binding sequence of the SjGIPC3-PDZ domain
could be reduced to -[SDEC]-[STIL]-[HSNQDE]-[VIL]*,
which fits in both class I and class II binding motifs.
Prediction and identification of native ligands of SjGIPC3
We searched for the potential native ligands of SjGIPC3 in
the S. japonicum predicted proteomic database (sjr2_protein.
fasta) with the Tailfit program using the consensus-binding
sequence -[SDEC]-[STIL]-[HSNQDE]-[VIL]* as bait; 92
proteins that physically fit the consensus binding sequence were
fished out. We also performed BLAST searching on NCBI
within taxa 6182 directly; using each unique
carboxyterminal sequence (the extreme four amino acids) obtained
from both Y2H assays as bait. A panel of proteins whose
CFigure 3 Phylogenetic tree of SjGIPC3 and its orthologues derived from other species. Bootstrap percentages are indicated at each branch.
GenBank Accession Numbers: HsGIPC2 (NP_060125), MmGIPC2 (NP_058563), XtGIPC2 (NP_001120269), XtGIPC1 (NP_001011331), HsGIPC1
(NP_005707), MmGIPC1 (NP_061241), DrGIPC3 (CAX13825), XtGIPC3 (NP_001096502), HsGIPC3 (NP_573568), MmGIPC3 (NP_683753), BmGIPC
(NP_001040127), DmKermit (NP_652028), AgGIPC (XP_308197) CsGIPC1 (GAA51139), SjGIPC3 (AAW24514.1), SmGIPC3 (XP_002569948), AsGIPC1
(ADY44129), CeGIPC1 (NP_498017).
Figure 4 Determination of relative expression levels of SjGIPC3
transcripts at different developmental stages by qRT-PCR.
Transcriptional levels were calibrated according to the comparative
2Ct method using the housekeeping gene SjPSMD4 as an
endogenous control, and were normalized relative to the cercarial
stage. Error bars represent the standard deviations of the mean from
the three replicates. C, cercariae; S, hepatic schistosomula; M, male
adult worms; F, female adult worms; E, eggs.
terminal tail matched the unique sequences was retrieved.
The protein sequences retrieved from both bioinformatics
pipelines were used to perform BLASTP searches on NCBI
to manually filter those peptides lacking the C-terminus. We
also used integrated biological information, such as
transmembrane region prediction, potential molecular functions,
and the C-terminal sequence consensus between S.
japonicum and S. mansoni to determine the most promising
candidate ligands. Finally, six proteins were selected as ligand
candidates of SjGIPC3 (Table 2). Among them, four were
further confirmed to be potential ligands of SjGIPC3 in the
Domain loss events are extensively widespread in S.
japonicum, which serves as a notable consequence of
the adoption of parasitism . It is estimated that 1,000
protein domains have been abandoned by S. japonicum
during long-term evolution, which suggests that the
remaining domains are necessary for parasitic growth,
development, and maturation. The proteomic
information revealed that at least 25 PDZ domains are
distributed among 16 S. japonicum proteins, suggesting that the
PDZ domain is one of the important modules reserved
in the parasite to mediate multiple biological processes.
In the present study, we have characterized one of the
PDZ domain-containing proteins from S. japonicum,
GIPC3, for the first time. The ligand binding properties
of the SjGIPC3-PDZ domain were determined by Y2H
assays, and its potential ligands were identified.
Screening a highly diverse peptide library provided an
opportunity to fish out the potential binding property of
SjGIPC3. Whether the SjGIPC3-PDZ domain or
fulllength SjGIPC3 were used as bait, analogous binding
specificities can be deduced from the unique C-terminal
tails obtained from both Y2H assays. However, the GH1
and GH2 domains of SjGIPC3 may still affect the binding
specificity of the PDZ domain to some degree, because
the ligand motifs were more canonical when full-length
SjGIPC3 was used as bait. For instance, at P-1, the acidic
amino acid Glu was more preferentially selected in the
second assay (45.2%) than in the first assay (13.5%).
Similarly, at P-3, 16 types of amino acids emerged in the first
assay, while only eight kinds of amino acids were
presented in the second assay. Also at this position, the
occurrence of Glu was 50.0% when screened against the
full-length SjGIPC3, twice the occurrence rate as when
the SjGIPC3-PDZ domain was used as bait. Thus, the
binding specificity screened using full-length SjGIPC3 as
bait may be more authentic to its native status.
Previously, GIPCs have been shown to interact
specifically with Class I (X-S/T-X-*), II (-X-*), and III
(X-X-C*) binding motifs . Moreover, Kermit, the
GIPC orthologue in Drosophila, and GLUT1CBP each
interact with Myosin VI through an internal sequence
within the cargo binding domain [43,44]. In this study,
we focused on the C-terminal binding specificity of
SjGIPC3 and found that the PDZ domain of SjGIPC3
preferred to bind to Class I and II motifs, consistent
with the extensive ligand-binding ability of GIPC
orthologues in other species . However, regarding the
binding of Class II motifs, the only example presented
thus far is that the PDZ domain of synectin binds to
sydecan-4 through the -EFYA* motif, in which the
aromatic amino acid Phe was selected at P-2 . In our
study, it is worth noting that this site can be occupied by
the aliphatic amino acid residues Ile and Leu, in addition
to Ser and Thr (Table 1). Moreover, in a previous report,
the binding motifs of GIPC orthologues trend toward
selecting a Val, Ala, or Cys residue at p0 ; here, the
more hydrophobic amino acids, such as Ile and Leu,
were predominantly preferred. These diverse binding
characteristics could be utilized for drug development
On average, one PDZ protein is capable of interacting with
17 partners . Although a panel of S. japonicum proteins
deposited in the public database bears a C-terminal tail
fittting in the consensus binding sequence, the bioinformatic
hints at molecular function and localization have prevented
them from being ligand candidates of SjGIPC3. Using the
Figure 5 Binding properties of the SjGIPC3-PDZ domain. The four amino acids from the extreme carboxyl terminus of positive clones
isolated from Y2H assays with the SjGIPC3-PDZ domain as bait (A), or full-length SjGIPC3 as bait (B), respectively, were aligned and the
occurrence for each amino acid at each position was statistically analyzed. The positions were numbered from the carboxyl terminus (position 0).
The tables described the percentage of each amino acid type at each given position (blank: percentage = 0%).
Table 2 Validation of the predicted ligand candidates of SjGIPC3 in the Y2H system
(S. japonicum) (S. mansoni)
glutamate receptor NMDA
-EDGNSLSA* tetraspanin (Sj25)
-DDRLCSQV* hypothetical protein
putative rhodopsin-like orphan GPCR
1: The extreme four amino acids of each unique ligand carboxyl terminus listed in Table 1 were used to BLAST on NCBI to retrieve potential ligands within taxa
6182 (S. japonicum).
2: The Tailfit software was used to search against the S. japonicum proteomic database to retrieve potential ligands with the consensus-binding sequence;
3: The validation results from Y2H assays, using the SjGIPC3-PDZ domain as bait.
4: The validation results from Y2H assays, using full-length SjGIPC3 as bait.
: positive in the Y2H system; : negative in the Y2H system.
Y2H assay, four proteins were determined to be potential
ligand candidates for SjGIPC3, which suggests that some
other potential ligands may not have been discovered yet,
likely restricted by the quality of the proteomic database, as
truncated fragments have been extensively deposited in the
S. japonicum proteomic database.
To adapt to the complexity of the life cycle,
schistosomes are equipped with a complex nervous system to
sense environmental signals . Transcriptional
analysis has revealed that the expression of SjGIPC3 was
dramatically up-regulated after invasion of the host,
suggesting that it may play an important role (e.g., signal
transduction) in the context of the parasite-host
interaction. More importantly, the preferential expression of
SjGIPC3 in male rather than female adult worms further
hinted that the potential function of the protein was
related to the processing of environmental information,
as male adult worms interface with the host and female
adult worms resided in the gynecophoral canal, more
stimulating signal would be received by male adult
worms . GIPC3 mutations have recently been
reported to cause autosomal recessive nonsyndromic
sensorineural hearing loss [48-50]. Previously, Yi and his
colleagues suggested that the GIPC orthologue in mouse
might associate with extrasynaptic NMDA receptors
through binding to the C-terminal tail ESDV, and may
be involved in the organization and trafficking of this
population of receptors . Intriguingly, among the
four potential ligands of SjGIPC3 identified in this study,
one is the glutamate receptor NMDA, which also bears
a class I motif TTTL; thus, we cannot rule out the
possibility that SjGIPC3 may also have a physiological
function in the regulation of NMDA receptor trafficking,
similar to its orthologue in mouse.
For the first time, we have presented the molecular
characterization of the PDZ domain-containing protein
GIPC3 from S. japonicum. We defined the ligand
binding specificity of the SjGIPC3-PDZ domain by screening
a random peptide library constructed with human
genomic DNA. The ligand binding ability of the
SjGIPC3PDZ domain is relatively extensive; it can bind both type
I and type II ligand motifs, which hints at its potential to
play multiple roles during the development of S.
japonicum, especially in the adult stage. Our study also
provided a practical method for predicting potential ligands
of SjGIPC3. Ongoing studies to identify more ligands
involved in signal transduction followed by improvement
of the quality of the S. japonicum proteomic dataset will
assist in the discovery of novel drug targets against the
All procedures performed on animals within this study
were conducted following animal husbandry guidelines
of the Chinese Academy of Medical Sciences and with
permission from the Experimental Animal Committee.
Additional file 1: Table S1. Primer sequences used in this study. This
file contains the information about the primers used for qRT-PCR and
YM, PC, and YG conceived and designed the experiments. YM, HH, SL, and
PC performed the experiments. YM, PC, and YG analyzed the data. YM, PC,
and YG wrote the manuscript. All authors read and approved the final
version of the manuscript.
This work was supported by the National Basic Research Program of China
(2012CB517606, 2013CB530805, 2011CB964901), the National High
Technology Research and Development Program of China (2011AA020116),
Program for Changjiang Scholars and Innovative Research Team in
University-PCSIRT (IRT0909), and 111 Project (B08007).
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