SRPX2 Is a Novel Chondroitin Sulfate Proteoglycan That
Is Overexpressed in Gastrointestinal Cancer
Kaoru Tanaka1,2, Tokuzo Arao1, Daisuke Tamura1, Keiichi Aomatsu1, Kazuyuki Furuta1, Kazuko
Matsumoto1, Hiroyasu Kaneda1,2, Kanae Kudo1, Yoshihiko Fujita1, Hideharu Kimura1, Kazuyoshi
Yanagihara3, Yasuhide Yamada4, Isamu Okamoto2, Kazuhiko Nakagawa2, Kazuto Nishio1*
1Department of Genome Biology, Kinki University School of Medicine, Osaka-Sayama, Osaka, Japan, 2Department of Medical Oncology, Kinki University School of
Medicine, Osaka-Sayama, Osaka, Japan, 3Department of Life Science, Faculty of Pharmacy, Yasuda Women’s University, Asaminami-ku, Hiroshima, Japan, 4Department of
Medical Oncology, National Cancer Center Hospital, Chuo-ku, Tokyo, Japan
SRPX2 (Sushi repeat-containing protein, X-linked 2) has recently emerged as a multifunctional protein that is involved in
seizure disorders, angiogenesis and cellular adhesion. Here, we analyzed this protein biochemically. SRPX2 protein was
secreted with a highly posttranslational modification. Chondroitinase ABC treatment completely decreased the molecular
mass of purified SRPX2 protein to its predicted size, whereas heparitinase, keratanase and hyaluroinidase did not. Secreted
SRPX2 protein was also detected using an anti-chondroitin sulfate antibody. These results indicate that SRPX2 is a novel
chondroitin sulfate proteoglycan (CSPG). Furthermore, a binding assay revealed that hepatocyte growth factor dose-
dependently binds to SRPX2 protein, and a ligand-glycosaminoglycans interaction was speculated to be likely in
proteoglycans. Regarding its molecular architecture, SRPX2 has sushi repeat modules similar to four other CSPGs/lecticans;
however, the molecular architecture of SRPX2 seems to be quite different from that of the lecticans. Taken together, we
found that SRPX2 is a novel CSPG that is overexpressed in gastrointestinal cancer cells. Our findings provide key
glycobiological insight into SRPX2 in cancer cells and demonstrate that SRPX2 is a new member of the cancer-related
Citation: Tanaka K, Arao T, Tamura D, Aomatsu K, Furuta K, et al. (2012) SRPX2 Is a Novel Chondroitin Sulfate Proteoglycan That Is Overexpressed in
Gastrointestinal Cancer. PLoS ONE 7(1): e27922. doi:10.1371/journal.pone.0027922
Editor: Hana Algu ¨l, Technische Universita ¨t Mu ¨nchen, Germany
Received June 28, 2011; Accepted October 27, 2011; Published January 5, 2012
Copyright: ? 2012 Tanaka et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by funds for the Comprehensive 3rd Term of the 10-Year Strategy for Cancer Control, a Grant-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and Technology of Japan (19209018), and a fund from the Health and Labor Scientific Research Grants.
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.
* E-mail: firstname.lastname@example.org
Sushi repeat protein X-linked 2 (SRPX2) was first identified as a
gene up-regulated in pro-B leukemia cells and was described as
sushi-repeat protein up-regulated in leukemia (SPRUL, ).
Several years later, SRPX2 was found to be responsible for
rolandic seizures associated with oral and speech dyspraxia and
mental retardation . The disease-causing mutation (N327S) and
a second mutation (Y72S) of SRPX2 were identified, and these
mutations resulted in the gain-of-N-glycosylated form of the
mutant protein . Although the molecular and biological
functions of SRPX2 have been unknown for a long time, a recent
study clearly demonstrated that SRPX2 binds to urokinase
plasminogen activator receptor (uPAR) in a ligand/receptor
interaction and that SRPX2 mutations led to an increase in the
SRPX2/uPAR binding affinity . In the vascular endothelial
cells, Srpx2 regulates endothelial cell migration and tube
formation, and the interaction of SRPX2 and uPAR is also
involved in the early phases of endothelial remodeling during
Recently, we demonstrated that SRPX2 is overexpressed in
gastric cancer tissue and that expression was associated with a poor
clinical outcome . SRPX2 enhances cellular migration and
adhesion in gastric cancer cells and, interestingly, the conditioned-
medium obtained from SRPX2-producing cells increased the
cellular migration activity and cellular adhesion . We further
examined SRPX2, focusing on a biochemical analysis in this
Materials and Methods
HEK293 was maintained in DMEM medium and SNU-16 and
MKN7 were maintained in RPMI1640 medium supplemented
with 10% FBS. HUVEC (human umbilical vein endothelial cells)
was maintained in Humedia-EG2 (KURABO, Tokyo, Japan)
medium with 1% FBS under the addition of EGF and FGF-2. The
cells were maintained in a 5% CO2-humidified atmosphere at
37uC. These cell lines were obtained from the Japanese Collection
of Research Bioresources Collection (Sennan-shi, Osaka).
Western blotting analysis
The western blotting analysis has been previously described .
In belief, cell pellets were lysed in RIPA buffer (Tris-HCl: 50 mM,
pH 7.4; NP-40: 1%; Na-deoxycholate: 0.25%; NaCl: 150 mM;
EDTA: 1 mM; phenylmethyl-sulfonyl fluoride: 1 mM; aprotinin,
PLoS ONE | www.plosone.org1 January 2012 | Volume 7 | Issue 1 | e27922
leupeptin, pepstatin: 1 mg/ml each; Na3VO4: 1 mM; NaF:
1 mM). Cell extracts were electrophoresed on 7.5% (w/v)
polyacrylamide gels and transferred to a polyvinylidene di-fluoride
membrane (Nihon Millipore, Tokyo, Japan). The membrane was
incubated in Tris-buffered saline containing 0.5% Tween 20 with
3% BSA and then reacted with the primary antibodies and the
HRP-conjugated secondary antibody for 90 min each. Visualiza-
tion was achieved with an enhanced chemiluminescent detection
reagent (Amersham Biosciences, Buckinghamshire, UK). The
following antibodies were used: anti-HA high affinity (Roche
Applied Science, Mannheim, Germany), anti-SRPX2  and
anti-chondroitin sulfate (CS-56; Seikagaku Kogyo, Tokyo, Japan).
Detection of endogenous SRPX2 protein
The culture medium was dialyzed against 50 mM of ammoni-
um bicarbonate and lyophilized. The residue was dissolved in
50 mM of Tris-HCl (pH 7.4) and centrifuged at 20,000 rpm for
30 min. The supernatant was filtered through a 0.22-mm filter.
The filtrate was subjected to fast protein liquid chromatography
(FPLC; GE Healthcare UK Ltd. Buckinghamshire, England)
separation on HiTrap Q HP columns (5 mL; GE Healthcare).
The columns were equilibrated with 50 mM of Tris-HCl (pH 7.4).
The samples were then injected onto the columns, which were
washed with the same buffer and eluted at a flow rate of 4 mL/
min using a linear gradient consisting of 0–2 M NaCl in 50 mM
Tris-HCl (pH 7.4) over 45 min. The SRPX2 protein-containing
fractions were then performed using gel-filtration chromatography
(Superdex200 column, 16 mm660 mm; GE Healthcare).
Expression constructs and purification of SRPX2-HA/His
The method for producing the expression constructs was
previously described . Empty and SRPX2-HA/His vectors
were then transfected into HEK293 cells using FuGENE6
transfection reagent (Roche Diagnostics, Basel, Switzerland), and
the cells were then selected with hygromycin. The stable
transfectant HEK293 cells were designated as HEK293-Mock
and HEK293-SRPX2-HA/His. The conditioned medium of the
HEK293-Mock and HEK293-SRPX2-HA/His cells was subject-
ed to FPLC loading at 3 mL/min on a 5-mL HisTrap HP column
(GE Healthcare). The bound protein was washed with 15 mL of
wash buffer (WB: 50 mM Na2HPO4, 10 mM Tris-HCl, 20 mM
imidazole [pH 8.0] and 600 mM NaCl,) and eluted in elution
buffer (EB: WB+230 mM imidazole). The SRPX2-HA/His
protein-containing fractions were applied to an FPLC Super-
dex200 column (16 mm660 mm; GE Healthcare) equilibrated
with 0.15 M of ammonium bicarbonate. Elution was carried out
using the same buffer at a flow rate of 1 mL/min. The SRPX2-
HA/His-containing fractions were verified using western blotting
Digestion of SRPX2 by specific GAG-degrading enzymes
Purified SRPX2-HA/His protein was digested with several
specific enzymes including chondroitinase ABC and chondroiti-
nase AC II (0.1 units in 40 mM Tris-HCl, 40 mM sodium acetate
[pH 8.0] at 37uC for 2 h), chondroitinase B (0.02 units in 20 mM
Tris-HCl, 0.25 mM calcium acetate [pH 7.5] at 37uC for 2 h),
heparinase I and heparinase II (0.05 units in 5 mM calcium
acetate, 50 mM sodium acetate [pH 7.0] 37uC for h), keratanase
(0.1 units in 7.5 mM Tris-HCl [pH 7.4] at 37uC for 2 h), and
hyaluroinidase (0.02 M acetate buffer, 0.15 M NaCl [pH 6.0] at
60uC for 2 h). Enzymes were purchased from Seikagaku Kogyo.
The samples were then analyzed using western blotting.
An IAsys resonant mirror biosensor (Affinity Sensors, Cam-
bridge,UK) with a carboxymethyl dextran-sensing cuvette wasused
to determine the kinetic constants of hepatocyte growth factor
(HGF) binding to immobilized SRPX2-HA/His. SRPX2-HA/His
was dissolved in 10 mM sodium formate (pH 4.0) and immobilized
on the carboxymethyl dextran surface of the cuvette, according to
the manufacturer’s instructions. Binding experiments were per-
formed in PBS. Changes in the resonant angle were monitored at 1-
s intervals for approximately 600 s. Experiments were performed at
25uC with a stirrer speed of 80 rpm. The binding parameters were
calculated from the association and dissociation phases of the
binding reactions using the non-linear curve fitting FastFit (Affinity
Sensors). Bovine serum albumin (BSA) was used as a control.
The clinical samples of the paired colorectal cancers (CRCs),
microarray procedure and analysis method have been previously
described . This study was approved by the institutional review
board, and written informed consent was obtained from all the
patients. All microarray data has been deposited to Center for
Information Biology gene Expression database (CIBEX, http://
cibex.nig.ac.jp/index.jsp) as accession number #CBX205. All
data is MIAME compliant and that the raw data has been
deposited in a MIAME compliant database (CIBEX), as detailed
on the MGED Society website http://www.mged.org/Work-
Patients and samples
The 30 CRC and 10 paired non-cancerous colonic mucosa
samples were analyzed using real-time RT-PCR. The RNA
extraction method and the quality check protocol have been
previously described . This study was approved by the
institutional review board of the National Cancer Center Hospital,
and written informed consent was obtained from all the patients.
Real-time reverse transcription PCR and western blot
The methods used in this section have been previously
Overexpression of SRPX2 in CRC tissues
We evaluated the mRNA expression of SRPX2 in clinical samples
of CRCs in addition to its homologue SRPX (SRPX1) using
microarray data. SRPX2 expression was markedly up-regulated
(20.5 fold, p=0.00014) in cancer tissues, compared with paired
noncancerous mucosa samples, whereas the putative tumor
suppressor gene SRPX was down-regulated (0.7 fold, p=0.029) in
cancer (Fig. 1). The result indicates that SRPX2 is overexpressed in
CRC during carcinogenesis and tumor progression, unlike SRPX.
Real-time RT-PCR for the 30 CRC and 10 paired non-cancerous
colonic mucosa samples confirmed that SRPX2 mRNA was
markedlyoverexpressed inthe CRCsamplesbutwasonlyexpressed
at a very low level in non-cancerous colonic mucosa (Figure 1B).
Secreted SRPX2 protein is suspected to be modified
The predicted molecular mass of SRPX2 protein was 53 kDa;
however, western blotting revealed that the molecular mass of the
secreted SRPX2 protein was highly increased, with smeared bands
at an apparent molecular mass of 100–150 kDa in SNU-16 and
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MKN7 cell lines (Fig. 2A). Next, we evaluated the exogenously
expressed SRPX2 protein derived from HEK293-Mock and
HEK293-SRPX2-HA/His cells. The molecular mass of intracel-
lular SRPX2 protein was similar to the predicted size, while the
molecular mass of the secreted-SRPX2 protein was highly
increased (100–150 kDa). Smeared bands were also detected
using both anti-HA and anti-SRPX2 antibodies (Fig. 2B). The
non-smeared bands at 120 kDa in cell lysate are endogenous
SRPX2. These results suggested that secreted SRPX2 protein may
undergo posttranslational modifications.
SRPX2 is a novel chondroitin sulfate proteoglycan
Based on the appearance of the smeared bands at a highly
increased molecular mass, we hypothesized that SRPX2 is a
proteoglycan with glycosaminoglycan (GAG) chains. Accordingly,
we treated purified-SRPX2 protein obtained from the cultured
medium of HEK293-Mock (empty control) or HEK293-SRPX2-
HA/His cells with chondroitinase ABC, heparitinase 1, hepar-
itinase 2, keratanase, chondroitinase AcII, chondroitinase B, and
hyaluroinidase. Western blotting revealed that the molecular mass
of the secreted SRPX2 protein was clearly decreased by
chondroitinase ABC digestion, but not by heparitinase or
keratanase or hyaluroinidase (Fig. 3A, 3B). Further chondroitinase
treatment showed that chondroitinase ABC and chondroitinase
AcII completely digested GAGs on SRPX2, but that chondroi-
tinase B partially digested these chains (Fig. 3B). A small digested
SRPX2 protein was also detected using anti-SRPX2 antibody
(Fig. 3C, 3D). These results indicate that SRPX2 contains
chondroitin sulfate GAG chains and is a novel chondroitin sulfate
proteoglycan (CSPG). In addition, the partial digestion by
chondroitinase B suggests that a dermatan sulfate component
may be included in the chondroitin sulfate GAG chains. Next, we
Figure 1. SRPX2 is overexpressed in colorectal cancer (CRC). (A) The mRNA expression of SRPX2 and its homologue SRPX in 15 CRC and paired
normal colonic mucosa specimens. The values indicate the normalized signal intensity obtained from the microarray data. (B) mRNA expression levels
of SRPX2 determined using real-time RT-PCR. CRC: colorectal cancer, Rel mRNA: normalized mRNA expression levels (SRPX2/GAPD6106).
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confirmed the results of enzymatic digestion against endogenous
SRPX2 from HUVEC using western blotting with anti-SRPX2
antibody and a similar result was obtained (Fig. 4A). Anti-
chondroitin sulfate antibody (CS-56) also detected the chondroitin
sulfate GAG on SRPX2 (Fig. 4B). The non-smeared bands at
120 kDa in cell lysate are endogenous SRPX2.
HGF binds to SRPX2
It is well known that several ligands including HGF, heparin-
binding EGF-like growth factor, fibroblast growth factor 2 and
vascular endothelial growth factor are capable of binding to the
GAG chain and that such interactions are considered to be a
unique characteristic of GAGs and proteoglycans . According
to a report on CSPG endocan and HGF binding , we examined
the interaction between HGF and GAGs using an IAsys resonant
mirror biosensor. HGF dose-dependently bound to the GAGs of
SRPX2, while control BSA did not (Fig. 5A). The Kdvalue of this
interaction, calculated from the ratio of Kdiss/Kass, was 5.6 nM;
these data were similar to those for previously reported data on
HGF and endocan . Next, we examined the biological function
of SRPX2 on HGF. HGF increased the proliferation of HUVECs,
and the addition of purified SRPX2 protein into the medium
significantly increased HGF-induced proliferation (Figure 5B).
These results suggest that the interaction of HGF with SRPX2 has
a positive effect on angiogenesis.
SRPX2 has unique molecular architectures compared
with other sushi repeat module-containing CSPG
Data from publicly available databases (http://smart.embl-
heidelberg.de/) and a previous report  showed that SRPX2
has three sushi repeat modules (also known as complement control
protein modules or short consensus repeats) and one hyaline
domain (Fig. 6). Interestingly, four CSPG (agrrecan, versican,
neurocan and brevican; also known as lecticans) are present
among the sushi repeat module-containing family, and their
common molecular architectures consist of one immunoglobulin-
like domain, 2,4 LINK domains, one EGF-like domain, one C-
type lectin, and one sushi repeat module (Fig. 6). The presence of a
sushi repeat module and classification as a CSPG are the same for
SRPX2 and lecticans, but the other molecular architectures of
SRPX2 are quite different.
Taken together, these findings indicate that SRPX2 is a novel
CSPG that is overexpressed in gastrointestinal cancer cells.
The extensive use and structural diversity of sushi repeat
modules presumably reflects the versatility of a structural scaffold
that has been adapted by evolution to suit many purposes, both
architectural and functional, such as the mediation of specific
protein-protein and protein-carbohydrate interactions [10–12].
Meanwhile, SRPX2 has one hyaline domain, which appears to be
involved in cellular adhesion. Hyaline domains have been
identified in several eukaryotic proteins and are often associated
with sushi repeat modules or arranged in multiple copies .
These characteristics of the molecular architectures of SRPX2,
based on knowledge of protein-protein interactions, may contrib-
ute to ligand/receptor interactions between SRPX2 and uPAR,
with implications for disorders of the language cortex, cognition,
and angiogenesis [3,4].
We have demonstrated that SRPX2 is a novel CSPG,
suggesting that SRPX2 may have additional as yet unknown
biological functions as a proteoglycan, including interactions with
Figure 2. Secreted SRPX2 protein is suspected to be modified posttranslationally. (A) Secreted form of endogenous SRPX2 protein
obtained from culture medium (CM) in SNU-16 and MKN7 cells. CM was subjected to ion exchange chromatography and used for western blotting
analysis using anti-SRPX2 antibody. (B) Western blotting for exogenous SRPX2 protein obtained from cell lysate and CM using anti-SRPX2 and anti-HA
antibody. Stable transfectant HEK293 cells, introducing the full-length cDNA fragment encoding human SRPX2 with HA and the His-tag vector or
empty vector, were used for analysis. The non-smeared bands at 120 kDa in cell lysate are endogenous SRPX2. Mock: HEK293-Mock cells, SRPX2:
HEK293-SRPX2-HA/His cells. IB: immunoblotting, Lysate: cell lysate, CM: culture medium.
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various extracellular signaling molecules such as growth factors,
morphogens, enzymes and chemokines and/or may act at the cell-
extracellular-matrix interface to modulate cell signaling. The
conditioned-medium of SRPX2-producing cells markedly en-
hanced cellular adhesion in various cancer cell lines ; this result
can be explained by the biological function of SRPX2 as a
proteoglycan.Inaddition,althoughwe haveonly demonstratedthat
HGF can bind to SRPX2, our results suggest that other known
GAG-interacting ligands may be capable of binding to the GAG
chain of SRPX2. Therefore, the function of ligand-SRPX2 binding
may widely affect the activities of signaling pathway critical to
cancer cells, including cellular proliferation, apoptosis, migration
and survival . In addition, SRPX2 was found to be secreted and
may act as an extracellular matrix protein similar to other
proteoglycans; indeed coating the culture dish with SRPX2 protein
markedly enhanced cellular adhesion , supporting this idea.
Vascular endothelial cells HUVEC markedly express SRPX2 to
the same extent as high-expressing cancer cell lines . A recent
report demonstrated that Srpx2 is a novel mediator of angiogen-
esis and a key molecule involved in the invasive migration of
angiogenic endothelium through its role as a ligand for vascular
uPAR . Our findings also support the involvement of SRPX2 in
angiogenesis from another aspect of proteoglycans. Since endocan
is well-known as a vascular endothelial cells-specific CSPG ,
SRPX2 may be categorized as a vascular-related CSPG similar to
In conclusion, we found that SRPX2 is a novel chondroitin
sulfate proteoglycan that is overexpressed in gastrointestinal
Figure 3. Effects of chondroitinases on SRPX2. (A, B) Purified SRPX2 protein obtained from cultured medium of HEK293-Mock or HEK293-
SRPX2-HA/His cells were digested with chondroitinase ABC, heparitinase 1, heparitinase 2, keratanase, chondroitinase AcII, chondroitinase B and
hyaluroinidase. The effect of digestion of the glycosaminoglycan chains was detected using western blotting using anti-HA (A, B) and anti-SRPX2 (C,
D) antibody. IB: immunoblotting, CM: culture medium. Mock: HEK293-Mock cells, SRPX2: HEK293-SRPX2-HA/His cells.
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Figure 4. Detection of chondroitin sulfate glycosaminoglycan and binding of HGF to SRPX2. (A) Chondroitinase ABC digestion for
endogenous SRPX2 protein derived from HUVEC (human umbilical vein endothelial cells). The SRPX2 protein was detected using anti-SRPX2
antibody. (B) Western blotting for SRPX2 protein using anti-chondroitin sulfate antibody (CS-56). The non-smeared bands at 120 kDa in cell lysate are
endogenous SRPX2. IB: immunoblotting, Lysate: cell lysate, CM: culture medium. Mock: HEK293-Mock cells, SRPX2: HEK293-SRPX2-HA/His cells.
Figure 5. Binding of HGF to SRPX2 at the indicated concentrations. (A) IAsys resonant mirror biosensor was used for analysis. Bovine serum
albumin (BSA) was used as a negative control. (B) Cell proliferation of HUVECs evaluated using an MTT assay. The HUVECs were stimulated with or
without 10 ng/mL of HGF and 5 mg/mL of purified SRPX2 protein for 72 hours. *, SRPX2 (2) vs. (+), p,0.05.
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PLoS ONE | www.plosone.org6 January 2012 | Volume 7 | Issue 1 | e27922
cancer. Our findings provide key glycobiological knowledge of this Download full-text
protein in cancer cells.
We thank Mrs. Eiko Honda (Life Science Center, Kinki University School
of Medicine) for her technical assistance and Mr. Kiyotaka Okada
(Department of Physiology, Kinki University School of Medicine) for
Conceived and designed the experiments: TA K. Nakagawa K. Nishio.
Performed the experiments: KT DT KA KF KM H. Kaneda KK KY YF.
Analyzed the data: TA H. Kimura KY IO. Contributed reagents/
materials/analysis tools: YY. Wrote the paper: KT TA K. Nishio.
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Figure 6. Molecular architectures of SRPX2. The data was obtained from the public database SMART (http://smart.embl-heidelberg.de/). SRPX2
has three sushi repeat modules and one hyaline domain. Four sushi repeat module-containing CSPG (agrrecan, versican, neurocan and brevican; also
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