Mapping the Binding between the Tetraspanin Molecule (Sjc23) of Schistosoma japonicum and Human Non-Immune IgG
et al. (2011) Mapping the Binding between the Tetraspanin Molecule (Sjc23) of Schistosoma japonicum and Human
Non-Immune IgG. PLoS ONE 6(4): e19112. doi:10.1371/journal.pone.0019112
Mapping the Binding between the Tetraspanin Molecule (Sjc23) of Schistosoma japonicum and Human Non- Immune IgG
Chuang Wu 0
Pengfei Cai 0
Qiaocheng Chang 0
Lili Hao 0
Shuai Peng 0
Xiaojing Sun 0
Huijun Lu 0
Jigang Yin 0
Ning Jiang 0
Qijun Chen 0
David Joseph Diemert, The George Washington University Medical Center, United States of America
0 1 Key Laboratory of Zoonosis, The Ministry of Education, Jilin University , Changchun , China , 2 Laboratory of Parasitology, Institute of Pathogen Biology/Institute of Basic Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College , Beijing , China , 3 College of Life Science and Technology, Southwest University of Nationalities , Chengdu , China
Background: Schistosomal parasites can establish parasitization in a human host for decades; evasion of host immunorecognition including surface masking by acquisition of host serum components is one of the strategies explored by the parasites. Parasite molecules anchored on the membrane are the main elements in the interaction. Sjc23, a member of the tetraspanin (TSP) family of Schistosoma japonicum, was previously found to be highly immunogenic and regarded as a vaccine candidate against schistosomiasis. However, studies indicated that immunization with Sjc23 generated rapid antibody responses which were less protective than that with other antigens. The biological function of this membraneanchored molecule has not been defined after decades of vaccination studies. Methodology and Principal Findings: In this study, we explored affinity pull-down and peptide competition assays to investigate the potential binding between Sjc23 molecule and human non-immune IgG. We determined that Sjc23 could bind human non-immune IgG and the binding was through the interaction of the large extra-cellular domain (LED) of Sjc23 (named Sjc23-LED) with the Fc domain of human IgG. Sjc23 had no affinity to other immunoglobulin types. Affinity precipitation (pull-down assay) in the presence of overlapping peptides further pinpointed to a 9-amino acid motif within Sjc23-LED that mediated the binding to human IgG. Conclusion and Significance: S. japonicum parasites cloak themselves through interaction with human non-immune IgG, and a member of the tetraspanin family, Sjc23, mediated the acquisition of human IgG via the interaction of a motif of 9 amino acids with the Fc domain of the IgG molecule. The consequence of this interaction will likely benefit parasitism of S. japonicum by evasion of host immune recognition or immunoresponses. This is the first report that an epitope of schistosomal ligand and its immunoglobulin receptor are defined, which provides further evidence of immune evasion strategy adopted by S. japonicum.
Funding: This study was supported by the National Natural Science Foundation of China (#30901254), the intramural grant from Institute of Pathogen Biology,
CAMS (2008IPB204), and the national science and technology specific projects (2008ZX10401), and the Grant for Young Distinguished Scientist to QC (NSFC,
30625029). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Schistosomiasis is one of the most serious parasitic diseases in
morbidity and mortality, which infects at least 207 million people
and a large number of animals in 76 countries,with an estimated
700 million people at risk (World health Organization, February
2010). Schistosomiasis japonica, which is caused by the
parasitization of adult male and female worms of Schistosoma japonicum
within mesenteric or vesicular veins of the host, is the only
zoonotic schistosomiasis that has proved to be the most difficult to
be controlled among the 5 schistosome species that infect humans
. The parasites can thrive in a human host for decades.
Vaccines based on the membrane components (or associated
membrane proteins) have been extensively studied but with little
success [4,5]. It has been well-known that schistosomal parasites
evade host immune expulsion through surface masking, molecular
mimicking, and active modulation on host immune responses .
A variety of host molecules such as immunoglobulins, major
histocompatibility complex products, complement components,
blood group antigens have been found on the surface of the
parasites inside the host [6,7]. Acquisition of host components on
the parasite surface was believed to benefit parasite by prevention
of host recognition and immune attack . So far, the
nonfilamentous paramyosin in association with parasite membrane of
both S. mansoni and S. japonicum was the only molecule characterized
as the receptor for un-specific binding of host (human and rodents)
IgG and complement components, while the other parasite ligands
that interact with host factors remain unidentified . Though it
has been hypothesized that the adherence of host serum factors on
the surface could not only block recognition of anti-parasite
antibodies, but also inhibit complement activity, it is, however, also
possible that the parasites can actively influence host immune
responses through interaction with immunoglobulins. While the
surface location of the paramyosin is still a matter of debate
[8,10,11], the tetraspanin (TSP) family proteins were also localized
to the surface of both S. mansoni and S. japonicum . The TSP
family encoded in each schistosome genome contains more than 29
members and the expression profiles of various TSP members are
quite diverse, some are with stage-specificity [15,16], while others
are universally expressed, indicating that they perform different
functions during parasite development.
We have previously found that one member of the TSP family of
S. japonicum, Sjc23, was more immunogenic than any other TSP
members . Sjc23 was expressed on the surface of de-tailed
cercariae, lung-stage schistosomula, and adult worms .
Antibodies to Sjc23 were readily detected in the sera of patients of
schistosomiasis and experimentally infected mice . Further, it
was found that antibody responses to Sjc23 were more rapid than to
other schistosomal tegument antigens in mice either infected by the
parasites or after immunization . Intriguingly, the antibodies
induced by Sjc23 were dominantly IgG2a type which has been
proved to be inefficient in complement fixation and with less
cytophilic property in ADCC (Antibody-dependent cell-mediated
cytotoxicity). Here, we further explored the function of Sjc23 and
found that it is a molecule with affinity to the Fc domain of human
non-immune IgG. The binding was mapped to a 9 amino acid
region in the loop of the large extracellular domain of Sjc23 (named
Sjc23-LED). The findings further help us to better understand the
immune evasion mechanism of S. japonicum and rational design of
vaccines based on membrane proteins such as Sjc23.
Results and Discussion
Detection of Sjc23 expression on the surface of the
In our earlier study , we showed that Sjc23 gene was actively
transcribed in cercarie, schistosomulum, adult worm and egg stages
and Sjc23 protein was detected in the parasite with Western-blot using
antibodies specific to the Sjc23-LED. Here we used the same antibody
to localise the protein on the surface of cercarie, schistosomulum and
adult stage parasites (Fig. 1 and data not shown). Thus Sjc23 is a
surface molecule as other tetraspanin family members.
Cloning and expression of recombinant proteins
In our previous investigations, we observed that the background
recognition of the recombinant Sjc23-GST protein by sera from
healthy humans were unexpectedly high. To further investigate
the possible un-specific binding between the individual protein
with human immunoglobulins, recombinant proteins of
Sjc23LED, GST and TSP-2 of S. japonicum were generated. The gene
fragment encoding Sjc23-LED was amplified by PCR (Fig. 2A)
and cloned into the pET-22b expression vector. The His-tagged
recombinant Sjc23-LED protein was expressed and purified by a
His GraviTrap column (GE Biosciences, Uppsala, Sweden). The
molecular mass of the recombinant Sjc23-LED was 12.4 kDa
(Fig. 2B). The expressed protein was confirmed by Western-blot
using a mAb specific to the His-tag (Fig. 2C). Recombinant GST
and TSP-2 were generated as described [14,16].
Sjc23-LED specifically bound human non-immune IgG
To test the possible immunoglobulin binding property of the
molecules generated above, a classical ELISA assay was performed.
The three proteins, Sjc23-LED, GST and TSP-2, were incubated
respectively with purified human IgG, IgM, IgA (Sigma, CA, USA)
and IgE (Abcam, Cambridge, UK). Only Sjc23-LED was found to
bind non-immune human IgG, while GST and TSP-2 did not show
any binding activity (Fig. 3A). Further Sjc23-LED only bound
human IgG, but not other types of immunoglobulins and albumin
(Fig. 3B, Fig. S1). This explained our earlier observation that
Sjc23LED reacted with normal human sera in ELISA assays. Thus, Sjc23
is likely another schistosomal molecule, apart from paramyosin [7
10], with immunoglobulin-binding property.
In order to confirm the binding between Sjc23-LED and
human IgG, a pull-down assay was performed. We used
Sjc23LED as a bait protein immobilized on the nickel-Sepharose beads
to capture the immunoglobulins that would interact with it. As
shown in Fig. 4, only IgG was precipitated by Sjc23-LED
immobilized Sepharose (Fig. 4A, lane 1), but not IgA, IgE or IgM.
Pull-down assays with porcine and bovine IgG were also
performed; however, very weak signal was observed with porcine
IgG (Fig. 4B, lane 1), but no signal was detected with bovine IgG
was seen (Fig. 4B, lane 3), indicating that Sjc23-LED mainly
adhered human IgG. Compared with ELISA assay, pull-down
assays require higher affinity between the ligand and receptor due
to the high stringency of particle precipitation and washing steps.
Thus the binding between Sjc23-LED and IgG was specific.
Further, S. japonicum can establish infection in domestic pigs,
though the parasitism and pathology in large stocks have not been
well established when comparing to that in human, it is likely that
the parasite is less adapted in animals than in humans. By
comparing the binding property with immunoglobulins of different
host origins, the adaptation of the parasites in different hosts may
be further investigated. Nevertheless, combining the results of
ELISA assays, we can conclude that the Sjc23 molecule of S.
japonicum is a ligand for human non-immune IgG.
Sjc23-LED bound the Fc domain of human IgG
To identify which region in human IgG that binds to
Sjc23LED, Fab and Fc fragments of human IgG (Merck, NJ, USA) were
respectively incubated with Sjc23-LED immobilized Sepharose
beads, and only the Fc fragment was precipitated (Fig. 5A lane 3),
but not the Fab fragment. Further, the binding between Fc
fragment and Sjc23-LED was also confirmed in ELISA assay
(Fig. 5B). Thus, Sjc23 of S. japonicum is another molecule in
schistosomal parasites, similar to paramyosin in binding with
human non-immune IgG. An obvious question after this finding is
the necessity of having two molecules exposed on the surface of the
parasite to bind the same Fc domain of human IgG. One
explanation could be that Sjc23 and paramyosin are differentially
exposed on the parasite surface. It was reported that paramyosin
was only exposed on the surface of schistosomula , while it was
mainly located under the tegument membrane in other stages.
Earlier studies indicated that Sjc23 was expressed in all parasite
stages in the host ; however it could not be ruled out that
the protein might be less exposed at certain stage, i.e. Sjc23 and
paramyosin were differentially expressed during parasite
development in the mammalian hosts. But the rapid immunoresponses in
the host after infection strongly indicated the Sjc23 was exposed to
the immune system from initial invasion . Thus, it is likely that
Sjc23 and paramyosin fulfilled different biological functions.
A region of 9 amino acids in Sjc23-LED mediated IgG
The relatively small molecular mass of the Sjc23-LED made it
possible to perform inhibition/competition assay with synthetic
overlapping peptides against the binding of Sjc23-LED with IgG.
Seven peptides were chemically synthesized with 910 amino acids
overlapped between adjacent peptides. In the competition assays,
peptide number 3 and 4 completely blocked the binding between
Sjc23-LED and human IgG (Fig. 6A and B), but not the other
peptides. There is a region of amino acids (-KIQTSFHCC-) that
overlapped between the two peptides, thus it is likely the structure
formed by the 9 amino acids mediated the binding of Sjc23-LED
with the Fc fragment of human IgG. Structural analysis of the
Sjc23-LED shows that the IgG-binding motif is located on the
second helix of the molecule (Fig. 7). Interestingly, the
-CCGmotif which is conserved in all TSP family proteins is located at the
C-terminal side of the identified IgG-binding motif. TSP family
proteins have been known to be promiscuous. The finding here may
explain the mechanism of Ig-binding phenomenon in other
organism . However, due to the fact that TSP-2 of S. japonicum
did not show any binding activity to Ig, it is most likely that the
binding is structure-dependent. The 6 amino acids before the
CCG- motif in Sjc23-LED are very different from the sequences of
other TSP proteins of schistosomal parasites (also see Fig. 7).
Nevertheless, this is the first report that a defined motif of a
schistosomal tegument protein with immunoglobulin-binding
property has been mapped. Earlier study on paramyosin with
fragmented recombinant proteins indicted that the binding region
to a complement element (C9) was located closed to the C-terminal
region of 122 amino acids, but the binding epitopes for both
complement and IgG in the molecule have not been defined .
The Sjc23 has a typical structure of tetraspanin family
Sequence scanning for hydrophobicity and potential
transmembrane domains in the Sjc23 sequence, we found that, like other TSP
members, Sjc23 is a molecule with 2 transmembrane domains (TM),
a small and a large extracellular domain (EC1 and EC2, here called
Sjc23-LED) and a small intracellular domain (Fig. 7A). We analyzed
the tertiary structure of Sjc23-LED by blasting the sequence against
the protein structure database (Protein Data Bank, PDB). The best
hit was the structure of human CD81 which was another member of
the TSP family  (Fig. 7). Further, Circular dichroism (CD)
spectroscopy analysis of the recombinant Sjc23 protein indicated
that the molecule was mainly composed of helical structures. The
differences between Sjc23-LED and CD81 were mainly located in
the low complex regions. In Sjc32-LED, the low complex regions
were composed of more amino acids than other TSP members of the
parasite (data not shown), this may indicate that the molecule is
under pressure from the host, given the fact that both T and B cell
epitopes were identified in the region of Sm23 .
Finally, the Sjc23 was the first tetraspanin identified in S.
japonicum 20 years ago [22,23] and it, as well as its homologous
Sm23 from S. mansoni, has been regarded as a vaccine candidate
which has been tested in several immunization and challenge trials
in animals . With the discovery of this study, it may be
necessary to reconsider the vaccine development approach based
on this antigen. To be able to generate high affinity antibodies that
can overcome the unspecific Ig-binding, i.e. to compete out the
masking immunoglobulins, might be a prerequisite for a successful
development of the vaccine based on this antigen.
In this study, we investigated the immunoglobulin binding
property of the Sjc23 molecule, a member of the TSP family,
which has been regarded as a vaccine candidate for
schistosomiasis. It was found that the large extracellular domain of Sjc23,
named Sjc23-LED, bind to human non-immune IgG via the
interaction between a motif of 9 amino acids in the molecule and
the Fc fragment of human IgG. This is the first report that the
interaction of a schistosomal ligand and its human host receptor
has been defined.
Figure 6. Defining the region of Sjc23-LED that bound human IgG. A Sequences of the synthetic peptides with overlapping regions. The
amino acid sequence of Sjc23-LED was at the top and the 7 partially overlapping peptides were listed below. The sequence region with potential
binding activity to human IgG was marked with pink color. B Inhibition of human IgG with Sjc23-LED by peptides. Sjc23-LED was incubated with
human IgG in the presence of different peptides. Only peptide 3 and 4 showed inhibitory activity. Lane 8 and 9 were no peptide and loading control
Detection of Sjc23 expression in the parasite by
Detections of Scj23 protein in cercariae, schistosomula, adult
worms were carried out with immunofluorescent assay using
antiSjc23 antibodies raised in mice according to standard protocol.
Briefly, parasites were fixed for 5 min in ice-cold acetone followed
by blocking with 10% BSA in PBS for 2 h at room temperature.
Primary antibodies to the large extracellular domain (LED) were
incubated with the parasite at 4uC overnight. After washing 4
times in PBS buffer, the parasites were further incubated with a
FITC-conjugated goat anti-mouse antibody for 2 h at room
temperature. Parasites incubated with irrelevant sera and with the
secondary antibodies alone were included as controls. The
fluorescence was visualized with a Nikon fluorescence microscope
(Japan). The expression of Sjc23 in different parasite development
stages was further confirmed by Western blot with Sjc23 specific
Cloning of the gene fragment encoding the extra-cellular
region of Sjc23 antigen (Sjc23-LED)
DNA was extracted from adult S. japonicum. PCR primers were
designed based on the gene sequence of Sjc23 (GenBank accession
number: M63706.1). The forward primer and reverse primer were
carried a BamH I and an Xho I restriction site respectively. PCR was
performed under the following conditions: 4 min at 94uC for full
denaturation,1 min at 94uC, 1 min at 58uC, 45 s at 72uC. The
amplification was run for 32 circles with a last extension at 72uC
for 10 min. The PCR product was analyzed by electrophoresis in
a 1.5% agarose gel and visualized by staining the gel with ethidium
bromide. The amplified product was gel purified using DNA Gel
Extraction Kit (AxyGen, CA, USA) and cloned into the pET-22b
expression vector. The recombinant plasmid named
pET-22bSjc23-LED was verified by restriction enzyme digestion and
Expression, purification and verification of recombinant
For protein expression,competent cells of the E. coli strain
BL21(DE3) were transformed with pET-22b-Sjc23-LED plasmid
and cultured in LB medium containing Ampicillin (100 mg/ml) at
37uC. Expression was initiated by addition of Isopropyl
b-D-1thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM
after the bacteria density reached 0.60.8 at OD600. The culture
was incubated for 7 h at 25uC. Bacteria were harvested by
centrifugation and the recombinant His-tagged Sjc23-LED
protein was purified with the His GraviTrap column (GE
Healthcare, Uppsala, Sweden) according to the manual provided
by the manufacturer. The protein was eluted using an elution
buffer containing 500 mM imidazole. The eluate was analyzed by
running on a 12% SDS-PAGE and Western-blot with a mAb to
the His-tag. The recombinant proteins of TSP-2 and GST of S.
japonicum were generated as described [14,16].
Binding of Sjc23-LED with four nonimmune human
immunoglobulins in ELISA
In an enzyme-linked immunosorbent assay (ELISA), we coated
plates with human immunoglobulins (IgG, IgA, IgE and IgM)
(250 ng/well) overnight at 4uC.The same amount of human and
bovine albumin was used as control proteins. Sjc23-LED were
added to the wells in a series of dilutions from 500 mg/ml to
62.5 mg/ml and incubated for 1 h at 37uC. The binding was
detected with an ALP-anti-His tag mAb .
human non-immune human immunoglobulins, IgG, IgA, IgE and
IgM respectively as previously described . Briefly, 100 mg of
Sjc23-LED was incubated with 50 ml Ni-NTA Sepharose resin for
30 min at room temperature. After washing to remove unbound
protein,100 mg of human IgG, IgA, IgM (Sigma, CA, USA) or IgE
(Abcam, Cambridge, UK) was mixed with the Sjc23-LED-bound
resin and incubated at 4uC for 1 h with gentle agitation. The
GST-bound-glutathione Sepharose resin was used as a control for
unspecific binding to the resin by the immunoglobulins. After
incubation, the resin was washed five times with 1 ml phosphate
buffered saline (PBS) buffer (137 mM NaCl, 2.7 mM KCl, 10 mM
sodium phosphate dibasic, and 2 mM potassium phosphate
monobasic, pH of 7.4) to remove unbound protein. Proteins were
eluted in 100 ml 1 mol/L NaCl by gentle agitation at 4uC for
30 min. Aliquots of the eluted proteins were resolved in 10%
SDSPAGE and blotted to nylon films. The binding was visualized with
a polyclonal antibody that recognized all human
Pull-Down assay of Sjc23-LED with four human
The His-tagged Sjc23-LED immobilized on Ni-NTA agarose
resin was used as a bait protein, and which was incubated with
Binding of Sjc23-LED to the Fab and Fc fragments of
Human IgG Fab and Fc fragments (Merck, NJ, USA) were
incubated with Sjc23-LED-bound Sepharose resin as described
above. The binding was detected in Western-blot with goat
antihuman IgG (Fab specific)-ALP and goat anti-human IgG (Fc
specific)-ALP (Sigma, CA, USA) respectively.
In ELISA assay, Fab and Fc fragments of human IgG were
respectively coated in ELISA plates; Sjc23-LED recombinant
protein diluted from 100 to 3.125 mg/ml was incubated with the
two antibody fragments. The binding was detected with an
Inhibition of Ig-binding with overlapping synthetic
peptides of Sjc23-LED
Seven peptides of 19 amino acids were chemically synthesized
to cover the whole sequence of Sjc23-LED  with 910 amino
acids overlapping between adjacent peptides. 100 mg of human
IgG was mixed respectively with aliquots of the peptides (from 100
to 10 mg in each reaction) and incubated with the Sjc23-LED-His
GraviTrap Sepharose beads at 4uC for 30 min. The beads were
washed for 5 times with PBS buffer. The proteins bound to the
beads were resolved in 10% SDS-PAGE and blotted to a nylon
filter film. The inhibition effect of the peptides on the binding of
Sjc23-LED and human IgG was visualized by ALP-conjugated
goat anti-human Ig antibodies as described above.
Analysis of tertiary structure of Sjc23-LED
The amino acid sequence of Sjc23 was aligned with protein
sequences in the structural database using the phyre (Protein
Homology/analogy Recognition Engine) server at Imperial
College (http://www.sbg.bio.ic.ac.uk/phyre/) . The
3-dimensional structure was further illustrated with PyMOL Viewer
Figure S1 Test of binding of Sjc23 with bovine and
human albumin. Bovine, human albumin proteins and human
IgG were coated on ELISA plate and the binding of Sjc23 to these
proteins was detected with anti-HIS mAb. The binding to IgG was
dilution dependent, while the binding of Sjc23 to both bovine and
human albumins was negative.
Conceived and designed the experiments: CW NJ QC. Performed the
experiments: CW PC QC SP XS HL. Analyzed the data: CW NJ QC.
Contributed reagents/materials/analysis tools: LH JY. Wrote the paper:
CW PC NJ QC. All authors read the manuscript and agreed upon
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