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Biochemical and Biophysical Research Communications 368 (2008) 217–222
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Expression of highly sulfated keratan sulfate synthesizedin human glioblastoma cells
Norihito Hayatsu a,b, Satoshi Ogasawara a, Mika Kato Kaneko a, Yukinari Kato a,*,Hisashi Narimatsu a,baResearch Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba,
Ibaraki 305-8568, Japanb
Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
Received 9 January 2008
Available online 22 January 2008

Abstract
Keratan sulfate (KS) proteoglycan is expressed in the extracellular matrix or cell surface in numerous tissues, predominantly in thoseof the cornea, cartilage, and brain. However, its structure, function, and regulation remain poorly understood. Our investigation of KS
expression in glioblastoma cell lines using Western-blot and ﬂow cytometry with anti-KS antibody (5D4) revealed that LN229 glioblastoma cell highly expresses KS on a cell surface. Real-time PCR analysis showed that LN229 expresses a high level of keratan sulfate Gal6-sulfotransferase. Results of this study also demonstrate that recombinant 5D4-reactive aggrecan is produced in LN229. Taken
together, these results suggest that LN229 produces 5D4-reactive highly sulfated KS and is useful to investigate the KS structure andfunction in glioblastoma.
Ó 2008 Elsevier Inc. All rights reserved.
Keywords: Keratan sulfate; Proteoglycan; Glioblastoma; Glycogene

Keratan sulfate (KS) is a glycosaminoglycan that isformed through the elongation of N-glycans or O-glycans
attached covalently to scaﬀold proteins [1]. So-called KS
proteoglycans (KSPGs), which are KS-containing proteins,are found in the extracellular matrix or cell surface innumerous tissues, predominantly in those of the cornea,
cartilage, and brain. In fact, KS expression responds toembryonic development, physiological variations, andwound healing [1–3].
Actually, KS consists of a linear polymer of N-acetyllactosamine, Galb1-4GlcNAcb1-3, which is sulfated at the CAbbreviations: KS, keratan sulfate; KSPGs, KS proteoglycans;
KSGal6ST, keratan sulfate Gal-6-sulfotransferase; GlcNAc6ST, N-acetylglucosamine-6-O-sulfotransferase; b3GnT, b1,3-N-acetylglucosaminyltransferase; b4GalT, b1,4-galactosyltransferase; Gal, Galactose; GlcNAc,
N-acetylglucosamine.
*
Corresponding author. Fax: +81 29 861 3191.
E-mail addresses: yukinari-k@bea.hi-ho.ne.jp (Y. Kato), h.narimatsu@aist.go.jp (H. Narimatsu).
0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2008.01.058

6 positions of Galactose (Gal) and N-acetylglucosamine(GlcNAc) (Fig. 1A) [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 the chain is catalyzed by two carbohydrate sulfotransferases. Recent reports have described enzymes
which are involved in KS synthesis [4–10]. Both b3GnT7
and b4GalT4 are responsible for KS chain elongation.
Keratan sulfate Gal-6-sulfotransferase (KSGal6ST) is forsulfation of Gal. In addition, N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST)-1 and GlcNAc6ST-5 (alsoknown as CGn6ST) are, respectively, responsible for sulfation of GlcNAc in the brain and cornea. Results of in vitro
studies indicate that KS synthesis occurs in two stages: theproduction of GlcNAc-sulfated poly-N-acetyllactosamine
chain by b3GnT7, b4GalT4, and GlcNAc6ST-5; and the
production of highly sulfated KS through sulfation of
Gal by KSG6ST [4,5,10].

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Fig. 1. Analysis of KS expression in 10 glioblastoma cell lines. (A) Structure and synthesis of KS. (B) Cell lysates of 10 glioblastoma cells were
electrophoresed and immunoblotted using anti-KS (5D4) or anti-b-actin. (C) The LN229 lysates treated with keratanase II were analyzed using Westernblot with 5D4 or anti-b-actin. (D) Flow cytometric analyses of glioblastoma cell lines were performed using 5D4.

Most cells do not produce KS in culture because KSspeciﬁc sulfotransferases are down-regulated in culture
conditions [10,11]. Primary bovine keratocytes cultured inserum-free media continue KS synthesis in vitro at reduced
rates and with less sulfation: production of recombinant
KSPGs to analyze biological function of KS is not eﬃcient[12–14]. Furthermore, immortal cells have not beenreported to produce high levels of KS.
Results of this study show that one glioblastoma cell,
LN229, highly expresses KS using a monoclonal anti-KS
antibody (5D4). Furthermore, we investigated the relationship between expression of KS and glycogenes that are

involved in KS synthesis using quantitative real-time
PCR in 10 glioblastoma cell lines. Results also show that
the LN229 cells produce KS-containing proteoglycans carrying both N-linked and O-linked KS both endogenously
and exogenously.
Materials and methods
Cell lines and reagents. The glioblastoma cell lines [15] were donated by
Dr. Webster K. Cavenee (Ludwig Institute for Cancer Research, San
Diego, CA), and cultured at 37 °C in a humidiﬁed atmosphere of 5% CO2
and 95% air in Dulbecco’s modiﬁed Eagle’s medium (DMEM; InvitrogenN. Hayatsu et al. / Biochemical and Biophysical Research Communications 368 (2008) 217–222
Corp., Carlsbad, CA) supplemented with 10% heat-inactivated fetal
bovine serum (FBS; Sigma, St. Louis, MO) and 1% of penicillin–streptomycin solution (Invitrogen Corp., Carlsbad, CA).
Western-blot analysis. The cell lines were lysed with lysis buﬀer (50 mM
Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, and 1 mM EDTA)
including protease inhibitor solution (Sigma). Samples of the supernatantfraction were collected after centrifuging at 15,000g for 30 min. Two
micrograms of the proteins was 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), and subsequently with peroxidase-conjugated antimouse 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.).
Flow cytometry. The cell lines that were collected using EDTA treatment (10 mM EDTA in PBS) were incubated with 5D4 (1/1000 dilution)for 1 h at 4 °C. Then the cells were incubated with Oregon green-conjugated antibody (Invitrogen Corp.) for 30 min. Flow cytometry was performed using FACS Calibur (BD Biosciences, Barintree, MA).
Deglycosylation experiments. The LN229 cell lysate was treated withkeratanase II (0.25 mU/lg of protein, 10 mM sodium citrate, pH 6.2;
Seikagaku Corp.) for 12 h at 37 °C or PNGase F (0.1 mU/lg of protein;
Takara Bio Inc., Shiga, Japan) for 24 h at 37 °C according to the manufacturer’s instructions. Alkaline digestion of O-glycans on the Westernblot was performed as described [16]. Brieﬂy, transferred membrane of
LN229 cell lysate was incubated with 0.05 N NaOH for 16 h at 40 °C,
prior to probing with antibody.
Quantitative real-time PCR analysis. Total RNAs were prepared fromglioblastoma cell lines using an RNeasy mini prep kit (Qiagen Inc., Hilden, Germany). The initial cDNA strand was synthesized using SuperScript III transcriptase (Invitrogen Corp.) by priming nine randomoligomers 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 theLightCycler 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 (1 cycle), 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 wasapplied with continuous ﬂuorescence measurement. Standard curves foreach glycogene and b-actin template were generated by serial dilution of
the PCR products (1  108 to 1  102 copies/ll). The expression levels of
glycogenes were normalized by the copy number of b-actin.
RNAi experiments. Small interfering stealth RNAs (siRNAs) for
KSGal6ST were purchased from Invitrogen Corp. The target sensesequences of KSGal6ST siRNA #1, #2, and #3 were 50 -AGGGCAAGUACAUGUUGGUGCGCUA-30 , 50 -ACCUGGCUCGGAACCCUAUGAAGAA-30 , and 50 -AGCACAAAUACGGCACCGUGCGAAA-30 ,respectively. As control siRNA, stealth RNAi negative control duplex(Invitrogen Corp.) was used. The LN229 cells were transfected with 25 nM
siRNA using LipofectamineRNAiMAX (Invitrogen Corp.) in accordancewith the manufacturer’s protocol. After 3 days, total RNAs were prepared; after 4 days, cell lysates of transfected cells were prepared andanalyzed as described above.
Expression of KSGal6ST. To construct an expression vector for
KSGal6ST, we ampliﬁed cDNA encoding the KSGal6ST ORF by PCR
using BC022567 (Open Biosystems, Huntsville, AL) as the template, alongwith the following primers: 50 primer, 50 -CACCATGCAATGTT
CCTGGAAG-30 and 30 primer, 50 -TCACGAGAAGGGGCGGAAGT-30 .
The PCR product was phosphorylated with T4 polynucleotide kinase(New England Biolabs Inc., Ipswich, MA) and cloned into the EcoRV siteof pcDNA3.1 (Invitrogen Corp.). The generated plasmid was designatedas pcDNA3.1/KSGal6ST. The LN464 cells were transfected with
pcDNA3.1/KSGal6ST or empty vector using FuGENE6 (Roche) in
accordance with the manufacturer’s protocol. After 3 days, the cell lysatesof transfected cells were prepared and analyzed.

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Expression of FLAG-tagged human aggrecan G1–G2. To construct anexpression vector for FLAG-tagged human aggrecan G1–G2 (rAggrecanG1–G2) [12,17], we ampliﬁed cDNA encoding the G1–G2 domain using
PCR with BC036445 (Open Biosystems) as the template and the following
primers: 50 primer containing EcoRI site 50 -gcgaattcatcgATGACCACTTTACTCTGGGT-30 and M13 reverse primer 50 -caggaaacagctatgac-30 .
The PCR product was digested with EcoRI and BamHI, and cloned into
the EcoRI and BglII sites of pFLAG-CMV14 (Sigma). The generated
plasmid was designated as pFLAG-CMV14/rhG1–G2. The LN229 cells
were transfected with 15 lg of pFLAG-CMV14/rhG1–G2 using
FuGENE6 (Roche). After three days, the conditioned medium was
immunoprecipitated using 20 ll of anti-FLAG (M2) agarose (Sigma), andthe rAggrecanG1–G2 proteins were eluted with 100 ll of 0.2 mg/ml
3xFLAG peptide (Sigma). Subsequently, 5 ll of the eluted protein was
undigested or digested with keratanase II (0.1 mU/5 ll of the eluted
protein) for 12 h at 37 °C or PNGase F (0.25 mU/5 ll of the eluted protein) for 24 h at 37 °C, followed by Western-blot analysis using 5D4 orperoxidase-conjugated anti-FLAG antibody (M2: 1/4000 dilution; Sigma).

Results and discussion
KS expression in glioblastoma cell lines
The brain is reported to be one of tissues, which highly
expresses KS [1,3]. To investigate the expression of KS inglioblastoma, we performed Western-blot analysis against
10 glioblastoma cell lines using monoclonal anti-KS antibody, 5D4, which recognize highly sulfated KS [18]. As
shown in Fig. 1B, KS was highly expressed in one glioblastoma cell line, LN229. Keratanase II treatment of LN229
cell lysate markedly decreased the 5D4-reactivity observed
using Western-blot analysis (Fig. 1C). Next, the KS expression on cell surface was investigated using ﬂow cytometry
with 5D4. Results showed that LN229 cells expressed KS
on a cell surface at a higher level than did other glioblastoma cell lines (Fig. 1D). Although KS expression in otherhuman tumor cell lines was also examined, a high level ofKS expression was not observed (data not shown).
Comparison of expression patterns of ﬁve genes involved inKS synthesis in glioblastoma cell lines
We next performed quantitative real-time PCR analysis
to compare the respective expression levels of ﬁve genes
involved in KS synthesis in 10 glioblastoma cell lines:
KSGal6ST, GlcNAc6ST-1/-5, b3GnT7, and b4GalT4.
Fig. 2 shows that the clearest diﬀerence between LN229
and the others was that of KSGal6ST expression: LN229
cells expressed KSGal6ST at higher levels compared toother cell lines (Fig. 2A). Furthermore, LN229 cells
expressed GlcNAc6ST-1/-5 at higher levels (Fig. 2B andC); b3GnT7 is expressed in LN229 at the highest level of
all cell lines (Fig. 2D).
Eﬀect of expression of KSGal6ST on expression of 5D4reactive KS in LN229 and LN464 cells
We performed knockdown of KSGal6ST in LN229
using small interference RNAs (siRNAs) for KSGal6ST

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Fig. 3. Eﬀect of KSGal6ST on KS synthesis in LN229 and LN464 cells.
(A) siRNAs for KSGal6ST (#1, #2, and #3) were transfected into LN229
cells. Cell lysates of LN229 cells were electrophoresed and immunoblottedusing anti-KS (5D4) or anti-b-actin. (B) KSGal6ST gene was transfected
into LN464 cells. Cell lysates of LN464 cells were electrophoresed andimmunoblotted using anti-KS (5D4) or anti-b-actin.

highly sulfated KS. Results show that, KSGal6ST-transfected LN464 expresses a higher level of highly sulfated KS
than control LN464 (Fig. 3B). These results indicate that
KSGal6ST is necessary for 5D4-reactive, highly sulfated
KS in glioblastoma cells.

Production of N-linked and O-linked KS-containing
proteoglycans by LN229 cells

Fig. 2. 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 each cell line
were measured using real-time PCR. Values normalized to the level of bactin transcripts are presented.

to investigate the importance of KSGal6ST in expressionof highly sulfated KS. The siRNAs for KSGal6ST
decreased the expression level of KSGal6ST transcripts(data not shown). Furthermore, siRNAs for KSGal6ST
considerably reduced the 5D4-reactivity shown by Western-blot analysis compared to negative control siRNA(Fig. 3A). As presented in Fig. 2, another glioblastoma cell,
LN464, expresses higher level of GlcNAc6ST-1/-5 andb4GalT4 and an equal level of b3GnT7 in comparison to
LN229. However, LN464 expresses a much lower level of
KSGal6ST. We transfected KSGal6ST into LN464 cells
to clarify the importance of KSGal6ST for expression of

To determine whether LN229 cells express N-linked or
O-linked KS, the LN229 cell lysate was digested with
PNGase F, then analyzed by Western-blot using 5D4. As
depicted in Fig. 4A, the PNGase F treatment reduced most
5D4-positive signals. However, several signals were stillobserved. The PNGase F-undigested signals disappearedalmost completely by sequential alkaline hydrolysis treatment to remove O-glycans (Fig. 4A), indicating that
LN229 cells express both N-linked and O-linked KS. Next,
we examined whether N-linked and O-linked KS are
attached to recombinant proteins, such as a G1–G2 fragment of aggrecan in LN229 cells. As presented inFig. 4B, immunoprecipitated rAggrecanG1–G2 wasdetected with 5D4. The 5D4 signals were decreased by keratanase II treatment, although rAggrecanG1–G2 wasdetected using anti-FLAG antibody. Subsequent PNGase
F treatment partially decreased the molecular size of rAggrecanG1–G2. However, most 5D4-reactivity was not
aﬀected. Furthermore, PNGase F-undigested signals disappeared completely by alkaline hydrolysis (Fig. 4C). TheseN. Hayatsu et al. / Biochemical and Biophysical Research Communications 368 (2008) 217–222

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results indicating that KS expression is associated with thatmalignancy. Further study is necessary to investigate
whether KS is involved in the invasion of glioblastoma or
its malignant progression.
Acknowledgments
We thank Ms. Nana Matsuura for technical assistanceand Dr. Hiromichi Sawaki for helpful discussion. We arevery grateful to Drs. Webster K. Cavenee and KazuhikoMishima for generous donation of materials. 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).
References

Fig. 4. Analysis of KS-linkages of KSPGs produced by LN229 cells. (A)LN229 cell lysates after treatment with PNGase F were electrophoresed
and transferred onto PVDF membranes. The membranes were treatedwith PBS (left panel) or 0.05 M NaOH (right panel), and immunoblottedwith 5D4 or anti-b-actin. (B) LN229 cells were transfected with rAggrecan
G1-G2 or mock; these conditioned media were immunoprecipitated using
anti-FLAG. Immunoprecipitates were treated using keratanase II, andwere immunoblotted using 5D4 (upper panel) or anti-FLAG (lower
panel). (C) Immunoprecipitates using anti-FLAG were treated with
PNGase F, electrophoresed and transferred onto PVDF membranes.
The membranes were treated with PBS (left panel and right panel) or
0.05 M NaOH (middle panel), and immunoblotted with 5D4 (left panel
and middle panel) or anti-FLAG (right panel).

results suggest that LN229 produce rAggrecanG1–G2 withboth N-linked and O-linked KS.
In summary, we showed that a glioblastoma cell line,
LN229, strongly expresses highly sulfated KS. Experimentsof knockdown and over-expression of KSGal6ST showedthat KSGal6ST is essential for 5D4-reactive, highly sulfated KS synthesis. Deglycosylation treatments showedthat LN229 express both N-linked and O-linked KS-containing proteoglycans endogenously and exogenously.
These results suggest that LN229 might be useful to investigate the biological activities and the structure of KS
in vivo. Now, we are investigating the expression of KS inclinical glioblastoma tissues, and obtained the preliminary[1] J.L. Funderburgh, Keratan sulfate: structure, biosynthesis, andfunction, Glycobiology 10 (2000) 951–958.
[2] R. Kleene, M. Schachner, Glycans and neural cell interactions, Nat.
Rev. Neurosci. 5 (2004) 195–208.
[3] H. Zhang, K. Uchimura, K. Kadomatsu, Brain keratan sulfate andglial scar formation, Ann. N. Y. Acad. Sci. 1086 (2006) 81–90.
[4] M. Fukuta, J. Inazawa, T. Torii, K. Tsuzuki, E. Shimada, O.
Habuchi, Molecular cloning and characterization of human keratan
sulfate Gal-6-sulfotransferase, J. Biol. Chem. 272 (1997) 32321–
32328.
[5] T. Torii, M. Fukuta, O. Habuchi, Sulfation of sialyl N-acetyllactosamine oligosaccharides and fetuin oligosaccharides by keratan sulfate
Gal-6-sulfotransferase, Glycobiology 10 (2000) 203–211.
[6] T.O. Akama, A.K. Misra, O. Hindsgaul, M.N. Fukuda, Enzymaticsynthesis in vitro of the disulfated disaccharide unit of corneal
keratan sulfate, J. Biol. Chem. 277 (2002) 42505–42513.
[7] A. Seko, N. Dohmae, K. Takio, K. Yamashita, Beta 1,4-galactosyltransferase (beta 4GalT)-IV is speciﬁc for GlcNAc 6-O-sulfate. Beta
4GalT-IV acts on keratan sulfate-related glycans and a precursor
glycan of 6-sulfosialyl-Lewis X, J. Biol. Chem. 278 (2003)
9150–9158.
[8] A. Seko, K. Yamashita, beta1,3-N-Acetylglucosaminyltransferase-7(beta3Gn-T7) acts eﬃciently on keratan sulfate-related glycans, FEBS
Lett. 556 (2004) 216–220.
[9] H. Zhang, T. Muramatsu, A. Murase, S. Yuasa, K. Uchimura, K.
Kadomatsu, N-Acetylglucosamine 6-O-sulfotransferase-1 is requiredfor brain keratan sulfate biosynthesis and glial scar formation after
brain injury, Glycobiology 16 (2006) 702–710.
[10] K. Kitayama, Y. Hayashida, K. Nishida, T.O. Akama, Enzymes
responsible for synthesis of corneal keratan sulfate glycosaminoglycans, J. Biol. Chem. 282 (2007) 30085–30096.
[11] K. Nakazawa, I. Takahashi, Y. Yamamoto, Glycosyltransferase andsulfotransferase activities in chick corneal stromal cells before and afterin vitro culture, Arch. Biochem. Biophys. 359 (1998) 269–282.
[12] C.J. Poon, A.H. Plaas, D.R. Keene, D.J. McQuillan, K. Last, A.J.
Fosang, N-linked keratan sulfate in the aggrecan interglobular
domain potentiates aggrecanase activity, J. Biol. Chem. 280 (2005)
23615–23621.
[13] M.P. Beales, J.L. Funderburgh, J.V. Jester, J.R. Hassell, Proteoglycan synthesis by bovine keratocytes and corneal ﬁbroblasts: maintenance of the keratocyte phenotype in culture, Invest. Ophthalmol.
Vis. Sci. 40 (1999) 1658–1663.
[14] C.J. Long, M.R. Roth, E.S. Tasheva, M. Funderburgh, R. Smit,
G.W. Conrad, J.L. Funderburgh, Fibroblast growth factor-2 pro-

222

N. Hayatsu et al. / Biochemical and Biophysical Research Communications 368 (2008) 217–222

motes keratan sulfate proteoglycan expression by keratocytes in vitro,
J. Biol. Chem. 275 (2000) 13918–13923.
[15] Y. Kato, M.K. Kaneko, A. Kuno, N. Uchiyama, K. Amano, Y.
Chiba, Y. Hasegawa, J. Hirabayashi, H. Narimatsu, K. Mishima, M.
Osawa, Inhibition of tumor cell-induced platelet aggregation using anovel anti-podoplanin antibody reacting with its platelet-aggregationstimulating domain, Biochem. Biophys. Res. Commun. 349 (2006)
1301–1307.
[16] M. Duk, M. Ugorski, E. Lisowska, beta-Elimination of Oglycans from glycoproteins transferred to Immobilon P mem-branes: method and some applications, Anal. Biochem. 253(1997) 98–102.
[17] E. Rodriguez, S.K. Roland, A. Plaas, P.J. Roughley, The glycosaminoglycan attachment regions of human aggrecan, J. Biol. Chem. 281(2006) 18444–18450.
[18] H. Mehmet, P. Scudder, P.W. Tang, E.F. Hounsell, B. Caterson, T.
Feizi, The antigenic determinants recognized by three monoclonal
antibodies to keratan sulphate involve sulphated hepta- or larger
oligosaccharides of the poly(N-acetyllactosamine) series, Eur. J.
Biochem. 157 (1986) 385–391.