Biochemical and Biophysical Research Communications 369 (2008) 1041–1046

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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Increased expression of highly sulfated keratan sulfate synthesized
in malignant astrocytic tumors
Yukinari Kato a,*,1, Norihito Hayatsu a,b,1, Mika Kato Kaneko a, Satoshi Ogasawara a, Tetsutaro Hamano c,
Satoru Takahashi b, Ryo Nishikawa d, Masao Matsutani d, Kazuhiko Mishima d, Hisashi Narimatsu a,baResearch Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST),
Open Space Laboratory C-2, 1-1-1, Tsukuba, Ibaraki 305-8568, Japanb
Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japanc
Hamano Statistical Analysis Ofﬁce 6-7-15, Chuo-cho, Higashikurume-shi, Tokyo 203-0054, Japand
Saitama Medical University International Medical Center 1397-1 Yamane Hidaka-shi, Saitama 350-1298, Japana r t i c l e

i n f oArticle history:
Received 20 February 2008
Available online 7 March 2008
Keywords:
Keratan sulfate
Proteoglycan
Glycogene
Astrocytic tumor
Glioblastomaa b s t r a c t
Keratan sulfate (KS) proteoglycans are expressed on a subpopulation of microglia in normal adult brain.
We previously showed the up-regulated expression of KS in one of glioblastoma cell lines using anti-KS
antibody (5D4). However, it has not been clariﬁed whether KS is expressed in brain tumors and is
involved in their malignancy. In this study, 54 astrocytic tumors were investigated about KS-expression
using Western-blot with 5D4. In six of 14 anaplastic astrocytomas (43%) and 23 of 34 glioblastomas(68%), KS was detected by 5D4. KS was hardly detected by 5D4 in diffuse astrocytoma, suggesting that
KS-expression is signiﬁcantly expressed in malignant astrocytic tumors. In immunohistochemistry, KS
is highly expressed in cell surface of malignant astrocytic tumors. Taken together, KS might be associatedwith the malignancy of astrocytic tumors, and be useful for a prognostic factor of astrocytic tumors.
Ó 2008 Elsevier Inc. All rights reserved.

Keratan sulfate (KS) proteoglycans (KSPGs) consist of different
core proteoglycans that are individually glycosylated to varying
degrees with keratan sulfate chains [1]. Theses keratan sulfates
are known to be inhibitory to axonal growth. During development,
sulfated keratans are temporally and spatially located in areas inwhich restricted axonal growth occurs. In the adult, KS is also
found on a subpopulation of microglia throughout the brain; however, KS was not detected on astrocyte or oligodendrocyte [2]. KS
consists of a linear polymer of N-acetyllactosamine, Galb1-4GlcNAcb1-3, which is sulfated at the C-6 positions of galactose (Gal)and N-acetylglucosamine (GlcNAc) [1]. Elongation of the carbohydrate backbone of KS chain is catalyzed by enzymes of two glycosyltransferase families: b1,3-N-acetylglucosaminyltransferase(b3GnT) and b1,4-galactosyltransferase (b4GalT). Sulfation of thechain is catalyzed by two carbohydrate sulfotransferases. Recent
reports have described enzymes which are involved in KS synthesis[3–9]. Keratan sulfate Gal-6-sulfotransferase (KSGal6ST) is for sulfation of Gal. In addition, N-acetylglucosamine-6-O-sulfotransferAbbreviations: KS, keratan sulfate; KSPGs, KS proteoglycans; Gal, galactose; GlcNAc,
N-acetylglucosamine; KSGal6ST, keratan sulfate Gal-6-sulfotransferase; GlcNAc6ST,N-acetylglucosamine-6-O-sulfotransferase; b3GnT, b1,3-N-acetylglucosaminyltransferase; b4GalT, b1,4-galactosyltransferase.
* Corresponding author. Fax: +81 29 861 3191.
E-mail address: yukinari-k@bea.hi-ho.ne.jp (Y. Kato).
1
These authors contributed equally to this work.
0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2008.02.130

ase (GlcNAc6ST)-1 and GlcNAc6ST-5 (also known as CGn6ST) areresponsible for sulfation of N-acetylglucosamine.
Astrocytic tumors are the most common tumors of the central
nervous system (CNS) and are categorized into diffuse astrocytomas (World Health Organization (WHO) Grade II), anaplastic astrocytomas (AA; WHO Grade III) and glioblastomas (GBM; WHO
Grade IV) [10]. Despite advances in surgical techniques, radiation
therapy, and adjuvant chemotherapy, their prognoses remain poor:
the median survival time for patients with GBMs is only one year[11]. Glioblastoma may occur de novo or may result from progression of low-grade astrocytomas [12]. Molecular mechanisms oftumorigenesis and malignant progression are associated with theinactivation of tumor suppressor genes such as p53-Rb pathway
or the overexpression of oncogenes such as epidermal growth factor receptor (EGFR) [13]. However, the mechanisms of tumorigenesis and progression of astrocytic tumors have not been resolved
completely. Identiﬁcation of glycogenes or glycans that are expressed differentially in high-grade astrocytomas, low-grade tumors or normal brain tissues is important to elucidate themolecular mechanisms of tumorigenesis and to develop novel
therapeutic strategies.
In our recent study, we investigated the KS expression in glioblastoma cell lines using a monoclonal anti-KS antibody (5D4)
and revealed KS is highly expressed in one of glioblastoma celllines, LN229 [14]. Furthermore, we investigated the relationship1042

Y. Kato et al. / Biochemical and Biophysical Research Communications 369 (2008) 1041–1046

between expression of KS and glycogenes that are involved in KS
synthesis using quantitative real-time PCR, and showed clearly
that LN229 expresses a high level of KSGal6ST. However, it has
not been clariﬁed whether KS is expressed in astrocytic tumors
and is involved in their malignancy. In this study, we investigatedthe KS-expression in astrocytic tumors of various malignancies
using Western-blot and immunohistochemistry with 5D4, and discussed whether KS-expression is associated with the malignancy ofastrocytic tumors.
Materials and methods
Tissue samples. Tumor specimens were obtained during surgery from six patients with diffuse astrocytomas, 14 patients with anaplastic astrocytomas, and34 patients with glioblastomas. Informed consent had been obtained previously

from patients or their guardians. Tissue microarrays of astrocytic tumors were purchased from Cybrdi, Inc. (Frederick, MD). The histology of these tissue samples was
conﬁrmed by experienced neuropathologists.
Western-blot analysis. The tissues were lysed with lysis buffer (25 mM Tris
(pH 7.4), 50 mM NaCl, 0.5% Na deoxycholate, 2% nonidet P-40, 0.2% SDS,
1 mM phenylmethylsulfonyl ﬂuoride, and 50 mg/ml aprotinin) including
protease inhibitor solution. Samples of the supernatant fraction were collected
after centrifuging at 15,000g for 30 min. Four micrograms of the proteins were
electrophoresed under reducing conditions on 10% polyacrylamide gel (Atto
Bioscience, Tokyo, Japan). The separated proteins were transferred to a PVDF
membrane. After blocking with 3% skim milk in PBS, the membrane was incubated with 5D4 (1/5000 dilution; Seikagaku Corp., Tokyo, Japan) or anti-b-actin
antibody (1/5000 dilution; Sigma, St. Louis, MO), and subsequently withperoxidase-conjugated anti-mouse antibodies (1/5000 dilution; Bio-Rad Laboratories Inc., Hercules, CA). It was then developed for 1 min with ECL reagents(Amersham Pharmacia Biotech Inc.) using Amersham Hyperﬁlm ECL (Amersham
Pharmacia Biotech Inc.).

Fig. 1. Analysis of KS-expression in astrocytic tumors. Tissues from diffuse astrocytomas (A: lanes 1–6), anaplastic astrocytomas (B: lanes 7–20), and glioblastomas (C: lanes
21–54) were solubilized and immunoblotted using 5D4 (upper panel) or anti-b-actin antibody (lower panel). (D) The tissue lysates (patient Nos. 11, 12, 25, and 32) after
treatment with PNGase F were electrophoresed and transferred onto PVDF membranes. The membranes were treated with PBS (left panel) or 0.05 M NaOH (right panel), andimmunoblotted with 5D4 or anti-b-actin.

Y. Kato et al. / Biochemical and Biophysical Research Communications 369 (2008) 1041–1046

1043

Deglycosylation experiments. The tissue lysate was treated PNGase F (Takara Bio
Inc., Shiga, Japan; 0.1 mU/lg of protein) for 24 h at 37 °C according to the manufacturer’s instructions. Alkaline digestion of O-glycans on the Western-blot was performed as described [14]. Brieﬂy, transferred membrane of tissue lysate wasincubated with 0.05 N NaOH for 16 h at 40 °C, prior to probing with antibody.
Immunohistochemical analysis. Specimens were deparafﬁnized, rehydrated, andincubated ﬁrst with 5D4 (1/1000 dilution) at room temperature for 1 h, then withbiotin-conjugated secondary anti-mouse IgG antibody (Dako, Glostrup, Denmark)for 1 h, and ﬁnally with peroxidase-conjugated biotin–streptavidin complex (Vectastain ABC Kit; Vector Laboratories Inc., Burlingame, CA) for 1 h. Color was developed
using 3,3-diaminobenzidine tetrahydrochloride tablet sets (Dako) for 3 min. KSexpression was assessed semi-quantitatively from the percentage of tumor cellswith cytoplasmic/membrane staining: 0, no staining; +, <10%; ++, 10–50%; +++,
>50%.
Quantitative real-time PCR analysis. Total RNAs were prepared from 53 astrocytic
tumors (seven diffuse astrocytomas, 14 anaplastic astrocytomas, and 32 glioblastomas) using an RNeasy mini prep kit (Qiagen Inc., Hilden, Germany). The initial cDNA
strand was synthesized using SuperScript III transcriptase (Invitrogen Corp.,

Carlsbad, CA) by priming nine random oligomers and an oligo-dT primer according
to the manufacturer’s instructions. Real-time PCR was carried out on a LightCycler
480 Instrument with 96-well unit (Roche, Mannheim, Germany), using the LightCycler 480 SYBR Green I Master (Roche). Sets of primers were designed online with
Primer3 (http://frodo.wi.mit.edu). The PCR conditions were 95 °C for 5 min (1cycle), followed by 45 cycles of 95 °C for 10 s, 65 °C for 20 s, 72 °C for 20 s. Subsequently, a melting curve program was applied with continuous ﬂuorescencemeasurement. Standard curves for each glycogene and b-actin template were generated by serial dilution of the PCR products (1  108 copies/ll to 1  102 copies/ll).
The expression levels of glycogenes were normalized by the total RNA.
Statistical analyses. Results are expressed as the means ± standard deviation.
Student’s t-test was used to determine signiﬁcance among the groups. A value ofp < 0.05 was considered signiﬁcant.

Table 1Results of keratan sulfate immunostaining in 78 patients with astrocytic tumors

Lysates of frozen tumor specimens from 54 patients (six diffuse
astrocytomas, 14 anaplastic astrocytomas, and 34 glioblastomas)
were analyzed using Western-blot analysis with monoclonalanti-KS antibody, 5D4, which recognize highly sulfated KS [15].
As shown in Fig. 1, 5D4 detected highly sulfated KS in extracts ofanaplastic astrocytoma (Grade III) and glioblastoma (Grade IV).
One of six diffuse astrocytomas (17%), six of 14 anaplastic astrocytomas (43%), and 23 of 34 glioblastomas (68%) were detected by
5D4: two of 14 anaplastic astrocytomas (14%) and four of 34 glioblastomas (12%) were detected strongly; two of 14 anaplastic
astrocytomas (14%) and 10 of 34 glioblastomas (29%) were detected moderately. Only one of diffuse astrocytomas (17%) was de-Tumor type

Diffuse astrocytoma
Tissue microarray
Anaplastic astrocytoma
Surgical resection samples
Tissue microarray
Glioblastoma
Surgical resection samples
Tissue microarrayNo. of cases

20
20
27
3
24
31
13
18

KS immunostaining
+++

++

+

0
0
3
1
2
5
3
2

0
0
2
0
2
6
1
5

0
0
2
0
2
8
6
2

20
20
20
2
18
12
3
9

Positive rate

0.0%
0.0%
25.9%
33.3%
25.0%
61.3%
76.9%
50.0%

Results
Analysis of KS expression in astrocytic tumors

Fig. 2. Immunohistochemical detection of KS-containing proteoglycans in astrocytic tumors. No staining is apparent in a normal brain (A: 400) and in diffuse astrocytoma(B: 400). In anaplastic astrocytoma (patient No. 13), the tumor cell surface was stained positively (C: 100, D: 400). Accentuated staining is visible around an area of
microvascular proliferation in glioblastoma (patient No. 32; E: 100x, F: 400x). Bar 10 lm.

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Y. Kato et al. / Biochemical and Biophysical Research Communications 369 (2008) 1041–1046

tected weakly by 5D4. To determine whether 5D4-positive tissues
express N-linked or O-linked KS, the tissue lysate was digested
with PNGase F, then analyzed by Western-blot using 5D4. As depicted in Fig. 1D, the PNGase F treatment reduced some 5D4-positive signals. However, most signals were still observed. The PNGase
F-undigested signals disappeared almost completely by sequential
alkaline hydrolysis treatment to remove O-glycans (Fig. 1D), indicating that 5D4-positive tissues express both N-linked and Olinked KS.
Immunohistochemical staining for KS in malignant astrocytic tumors
The cellular distribution of KS in astrocytic tumors was examined immunohistochemically using 5D4. We used 62 tissues (20diffuse astrocytomas, 24 anaplastic astrocytomas, and 18 glioblas-

tomas) of tissue microarrays and 16 surgical tissues (three anaplastic astrocytomas and 13 glioblastomas) in immunohistochemistry.
KS immunoreactivity was detected in seven of 27 (26%) anaplastic
astrocytomas and in 19 of 31 (61%) glioblastomas; staining was
graded as +++ in ﬁve glioblastoma and as ++ in six glioblastoma
cases (Table 1). KS was not detected in diffuse astrocytomas. Representative staining for KS in astrocytic tumor samples is shown in
Fig. 2. Immunostaining for KS demonstrated predominantly cellsurface patterns. In anaplastic astrocytoma, the tumor cell surface
was stained using 5D4, and the regional tendency was not observed (Fig. 2C and D). In glioblastomas, KS-positive tumor cells
were prominent around microvascular proliferations (Fig. 2E and
F). Proliferating endothelial cells were negative for KS (Fig. 2F). In
non-neoplastic areas of the brain (Fig. 2A) and in diffuse astrocytoma (Fig. 2B), KS immunostaining was absent.

Fig. 3. Quantitative real-time PCR analysis of glycogenes for KS synthesis. The transcript levels for (A) KSGal6ST, (B) GlcNAc6ST-1, (C) GlcNAc6ST-5, (D) b3GnT7, and (E)b4GalT4 genes in 53 astrocytic tumors (seven diffuse astrocytomas, 14 anaplastic astrocytomas, and 32 glioblastomas) were measured using real-time PCR. Values
normalized to the level of total RNA are presented. **p < 0.01, *p < 0.05.

Y. Kato et al. / Biochemical and Biophysical Research Communications 369 (2008) 1041–1046

Comparison of expression patterns of ﬁve genes involved in KS
synthesis in astrocytic tumors
We performed quantitative real-time PCR analysis to compare
the respective expression levels of ﬁve genes (KSGal6ST, GlcNAc6ST-1/-5, b3GnT7, and b4GalT4) involved in KS synthesis in
53 astrocytic tumors (seven diffuse astrocytomas, 14 anaplastic
astrocytomas, and 32 glioblastomas). As shown in Fig. 3, theexpression of KSGal6ST, GlcNAc6ST-1/-5, and b3GnT7 gradually increased associated with tumor malignancy. Comparing anaplastic
astrocytoma with glioblastoma, the expression of KSGal6ST, GlcNAc6ST-1/-5, and b3GnT7 in glioblastoma is signiﬁcantly higher
than in anaplastic astrocytoma (p < 0.01). In comparison of diffuse
astrocytoma with anaplastic astrocytoma, the expression of GlcNAc6ST-1 in anaplastic astrocytoma is signiﬁcantly higher thanin diffuse astrocytoma (p < 0.05). The expression of b4GalT4 has
no signiﬁcant difference among different grades of astrocytic
tumors.
Discussion
Keratan sulfate (KS) is a glycosaminoglycan that is formed
through the elongation of N-glycans or O-glycans attached covalently to scaffold proteins [1,16]. KS proteoglycans (KSPGs) arefound in the extracellular matrix or cell surface in numerous tissues, predominantly in those of the cornea, cartilage, and brain.
In the normal adult brain, KS expression is restricted to a subpopulation of microglia [2]. KS expression responds to embryonicdevelopment, physiological variations, and wound healing[1,16,17]. However, it has not been investigated whether KS is expressed in brain tumors and is further involved in malignant progression of brain tumors, especially astrocytic tumors.
In this study, lysates of frozen tumor specimens were analyzed
using Western-blot analysis with monoclonal anti-KS antibody,
5D4 (Fig. 1). As a result, one of six diffuse astrocytomas (GradeII; 17%) and 29 of 48 high-grade astrocytic tumors (Grade III and
IV; 60%) were detected by 5D4, indicating that highly sulfated
KS-expression might be associated with its malignancy. Of highgrade astrocytic tumors, six of 14 anaplastic astrocytomas (43%)
and 23 of 34 glioblastomas (68%) were detected by 5D4(p < 0.05); therefore, there is signiﬁcant difference about KSexpression between anaplastic astrocytomas and glioblastomas.
We also investigated the KS expression in immunohistochemistry,
and also showed the signiﬁcant difference about KS expression between anaplastic astrocytomas (26%) and glioblastoma (61%;p < 0.05). We previously investigated the expression of podoplanin,
which is associated with malignant progression of astrocytic tumors [18,19]. Podoplanin was expressed on the cell surface in
27% of anaplastic astrocytomas and 47% of glioblastomas using
immunohistochemistry with anti-podoplanin antibody. On theother hand, podoplanin expression was not observed in diffuse
astrocytomas. Furthermore, podoplanin expression is associatedwith poor prognosis of glioblastoma patients. Interestingly, KSexpression is highly associated with podoplanin expression in diffuse astrocytomas and anaplastic astrocytomas (data not shown),
also suggesting that KS-expression might be involved in malignancy of astrocytic tumors.
In our recent study, we investigated how KS expression is regulated in glioblastoma cell lines [14]. We performed quantitative
real-time PCR analysis in 10 glioblastoma cell lines to compare therespective expression levels of ﬁve glycogenes (KSGal6ST, GlcNAc6ST-1/5, b3GnT7, and b4GalT4), which are involved in KS synthesis; LN229 cells expressed high level of these ﬁve glycogenes,
especially KSGal6ST. Knockdown of KSGal6ST in LN229 using siRNAs
for KSGal6ST considerably reduced the 5D4-reactivity shown by1045

Western-blot analysis. KSGal6ST-transfected LN464 cells express ahigher level of highly sulfated KS than control LN464. In this study,
we also performed quantitative real-time PCR analysis to compare
the respective expression levels of ﬁve glycogenes involved in KS
synthesis in 53 astrocytic tumors. Of ﬁve glycogenes, the expressionof KSGal6ST, GlcNAc6ST-1/-5, and b3GnT7 gradually increased associated with tumor malignancy (Fig. 3). Interestingly, the expressionof KSGal6ST, GlcNAc6ST-1/-5, and b3GnT7 in glioblastoma is significantly higher than in anaplastic astrocytoma. Moreover, the expression of GlcNAc6ST-1 in anaplastic astrocytoma is signiﬁcantly
higher than in diffuse astrocytoma, which is coincident with thehigh level of expression of KSGal6ST in LN229 glioblastoma cells[14]. These results suggest that glycogenes involved in KS synthesis
increased in the malignant progression of astrocytic tumors, leading
to the up-regulation of highly sulfated KS.
In summary, we revealed that KS signiﬁcantly expressed inmalignant astrocytic tumors, not in low-grade astrocytic tumors
using Western-blot. In immunohistochemistry, KS is highly expressed in cell surface of tumor cells. Taken together, KS might
be associated with the malignancy of astrocytic tumors, and be
useful for a prognostic factor of astrocytic tumors. Further study
is necessary to investigate how KS is involved in the invasion ofastrocytic tumors or their malignant progression.
Acknowledgments
We thank Ms. Nana Matsuura for technical assistance. This
study was supported in part by New Energy and Industrial Technology Development Organization (NEDO), by the Osaka Cancer
Research Foundation (MK. Kaneko), by Mitsubishi Pharma Research Foundation (Y. Kato), by the YASUDA Medical Foundation(Y. Kato), and by the Inoue Foundation for Science (Y. Kato).
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