FEBS Letters 581 (2007) 331–336

Functional glycosylation of human podoplanin: Glycan structureof platelet aggregation-inducing factor
Mika Kato Kanekoa,1, Yukinari Katoa,1, Akihiko Kameyamaa, Hiromi Itoa, Atsushi Kunoa,
Jun Hirabayashia, Tomomi Kubotaa, Koh Amanoa, Yasunori Chibaa, Yasushi Hasegawab,
Isoji Sasagawac, Kazuhiko Mishimad, Hisashi Narimatsua,*aResearch Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), C-2, 1-1-1 Umezono, Tsukuba,
Ibaraki 305 8568, Japanb
Department of Surgery, School of Medicine, Keio University, Tokyo, Japanc
Yamagata Tokushukai Hospital, Yamagata, Japand
Department of Neurosurgery, Saitama Medical School, Saitama, Japan
Received 4 December 2006; revised 20 December 2006; accepted 21 December 2006
Available online 5 January 2007
Edited by Judit OvádiAbstract Podoplanin (Aggrus) is a mucin-type sialoglycoprotein that plays a key role in tumor cell-induced platelet aggregation. Podoplanin possesses a platelet aggregation-stimulating(PLAG) domain, and Thr52 in the PLAG domain of human
podoplanin is important for its activity. Endogenous or recombinant human podoplanin were puriﬁed, and total glycosylation
proﬁles were surveyed by lectin microarray. Analyses of glycopeptides produced by Edman degradation and mass spectrometry
revealed that the disialyl-corel (NeuAca2-3Galbl-3(NeuAca26)GalNAcal-O-Thr) structure was primarily attached to a glycosylation site at residue Thr52. Sialic acid-deﬁcient podoplaninrecovered its activity after additional sialylation. These results
indicated that the sialylated Corel at Thr52 is critical for podoplanin-induced platelet aggregation.
Ó 2006 Federation of European Biochemical Societies. Publishedby Elsevier B.V. All rights reserved.
Keywords: Podoplanin; Platelet aggregation; Disialyl-corel,
Disialyl-T

1. Introduction
It is well known that tumor metastasis is associated withplatelet-aggregating activity possessed by human and othermammalian cancer cells [1]. A previous study has clariﬁed that
podoplanin, membranous 44 kDa and 36 kDa sialoglycoproteins in cancer cells of mice and humans, respectively, aggregated platelets with no relation to plasma components [2,3].
Podoplanin is known to be a lymphatic speciﬁc marker [4],and its expression has been reported in many tumor cells [4–11]. Podoplanin belongs to a type-I transmembrane sialomu-*
Corresponding author. Fax: +81 29 861 3191.
E-mail address: h.narimatsu@aist.go.jp (H. Narimatsu).
1

These authors contributed equally to this work.

Abbreviations: PLAG, platelet-aggregation-stimulating; CHO, Chinese
hamster ovary; MALDI-TOF-MS, matrix-assisted laser desorption/
ionization time of-ﬂight mass spectrometry; QIT, quadrupole ion trap;
HPLC, high performance liquid chromatography

cin-like glycoprotein that consisted of an extracellular domain
with abundant Ser and Thr residues as potential O-glycosylation sites, a single transmembrane portion, and a short cytoplasmic tail with putative sites for protein kinase C andcAMP phosphorylation [2,12].
We previously showed that the segment of EDxxVTPG in
the extracellular domain, designated as platelet aggregationstimulating (PLAG) domain, was critical for the activity of
podoplanin/Aggrus [2]. In particular, this motif, which ishighly conserved among species, is tandemly triplicated [13].
In the study of targeted mutagenesis to podoplanin molecules,
we obtained evidence that Thr residues of PLAG domain play
an important role for platelet aggregation [2,13]. Furthermore,unique characteristics of Chinese hamster ovary (CHO)
mutant cell lines, N-glycan-deﬁcient Lecl, CMP-sialic acidtransporter-deﬁcient Lec2, and UDP-galactose transporterdeﬁcient Lec8, revealed that sialylated O-glycan is critical forplatelet aggregation-inducing activity [14].
In this study, we puriﬁed human podoplanin from a glioblastoma cell line LN319 cells and podoplanin-transfected
CHO cells. Using a lectin microarray, Edman degradation,and matrix-assisted laser desorption/ionization time of-ﬂight
mass spectrometry (MALDI-TOF-MS), it was shown that
podoplanin possesses a disialyl-corel structure at Thr52 in
the PLAG domain, and this structure is critical for its platelet-aggregating activity.

2. Materials and methods
2.1. Production and puriﬁcation of podoplanin
We previously established FLAG-tagged human podoplanin-transfected CHO and Lec2 cell lines [14]. A glioblastoma cell line LN319,
which expresses endogenous podoplanin, was donated by Dr. Webster
K Cavenee [15]. Cells were lysed with 0.25% Triton X-100 in PBS.
Using anti-FLAG antibody-conjugated agarose gel (M2, Sigma),
podoplanin was puriﬁed from podoplanin-transfected CHO andLec2 cells, and eluted using 100 lg/ml FLAG peptide (Sigma). Endogenous podoplanin was puriﬁed from LN319 using anti-human
podoplanin antibody (NZ-1) and eluted by hpp38–51 peptide (EGGVAMPGAEDDVV) [15].
2.2. Platelet aggregation assay
The platelet aggregation assay was described previously [15]. Brieﬂy,
platelet aggregation was measured by WBA Carna (M.C. Medical)

0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2006.12.044

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M.K. Kaneko et al. / FEBS Letters 581 (2007) 331–336

with the screen ﬁltration pressure method [16]. Two hundred microliters each of heparinized mouse whole blood samples was pre-incubatedfor 2 min, followed by the addition of 12 ll each of puriﬁed protein.
Five minutes later, samples were sucked to detect aggregation pressure.
The ﬁnal platelet aggregation pressure of each reaction tube was determined at the pressure rate (%) of a pressure sensor connected to thesyringe.
2.3. Lectin microarray
The lectin microarray was basically performed as described by Kuno
et al. [17].
To generate glycan proﬁles for podoplanin, we customized a procedure using the NZ-1 antibody [15]. Interaction of podoplanin with thelectin immobilized on the glass slide was detected using biotinylated
NZ-l-Cy3-streptoavidin method to proﬁle glycans of podoplanin.
Brieﬂy, puriﬁed podoplanin was diluted to 60 ll with 1% Trition
X-100 in PBS (PBSTx) and applied to the lectin array containing triplicate spots of 43 lectin into each of eight-divided incubation baths on
the glass slide (refer to [15]). After incubation at 20 °C for 12 h, thereaction solution was discarded. The glass slide was washed three times
with PBSTx; 60 ll of biotinylated NZ-1 antibody (0.17 lg/ml) in PBS
was applied to the array and then incubated at 20 °C for 3 h. Afterwashing three times with PBSTx, Cy3-labeled streptavidin (GEHealthcare, UK) was added to the array and then incubated at
20 °C for 30 min. The glass slide was rinsed with PBSTx and scanned
using an evanescent-ﬁeld ﬂuorescence scanner (GTMASScan III; Nippon Laser & Electronics Lab, Nagoya, Japan). Lectin binding wasmeasured by ﬂuorescent intensity.
2.4. Mass spectrometryBeta-Elimination [18] and permethylation [19] were performed as
described previously. Desialylation was performed at 80 °C for 3 h in
2 M acetic acid. MS measurements were carried out using a positive
reﬂectron mode for glycans, and negative reﬂectron mode for glyco-

peptide with a MALDI-TOF mass spectrometer (Reﬂex IV; BrukerDaltonik, Germany). All collision-induced dissociation spectra wereacquired with a MALDI-QIT (quadrupole ion trap)-TOF MS spectrometer (AXIMA-QIT; Shimadzu, Kyoto, Japan). For each sample
preparation 2,5-DHB was used as the matrix. MSn ﬁngerprint matching was performed as described previously [20].
2.5. Endoproteinase digest, peptide separations, and protein sequencing
Using puriﬁed and concentrated hPod/LN319 and hPod/CHO,
endoproteinase Asp-N digestion was performed in 50 mM Tris–HCl(pH 8.0) at 37 °C for 8 h. The digests were separated by reverse phase
chromatography with a CAPCELLPAC C18 UG120 column (SHISEIDO) using high performance liquid chromatography (HPLC, Shimadzu). A linear gradient (0–50%) of acetonitrile in 0.1% triﬂuoroacetic
acid was performed at 40 °C. The glycosylation site on podoplanin
was determined by Edman microsequencing (PPSQ-23A, Shimazu),
since phenylthiohydantoin derivatives of glycosylated-Thr were notdetected.
2.6. Addition of sialic acid
Sialylation experiments were described previously [21,22]. For the
preparation of hPod/Lec2 with the sialyl Corel (NueAca2-3Galbl3GalNAcal-) residue, 25 mM HEPES buﬀer (pH 7.0) containing
30 ll of acceptor substrate (1.0 mg/ml), 10 mM MnCl2 and an appropriate concentration of CMP-NeuAc was used. Ninety microliters of
puriﬁed ST3GalI enzyme was added to the reaction mixture and incubated at 37 °C for 20 h. For the preparation of hPod/Lec2 with thedisialyl-corel (NueAca2-3Galbl-3(NeuAca2-6)GalNAcal-) residue, aliquots of supernatants of the former reaction solution were used as theacceptor substrate for the next sialylation. An appropriate concentration of CMP-NeuAc and the puriﬁed ST6GalNAcI enzyme were added
to 30 ll of supernatants and incubated at 37 °C for 20 h. Control
hPod/Lec2 sample was also treated in the same experimental condition
except for the addition of ST3GalI or ST6GalNAcI.

hPod/LN319

hPod/CHO

CBB

CBB

NZ-1

hPod/Lec2

NZ-1

45K

45K

45K

30K

30K

CBB NZ-1

30K

Pressure rate (%)

100

75

hPod/LN319
50

hPod/CHO
hPod/Lec2

25

0
0

5

10

15

Podoplanin (µg/ml)
Fig. 1. Puriﬁcation and platelet aggregation assay for endogenous and recombinant podoplanin. (A) Aﬃnity-puriﬁed human podoplanin from
LN319, podoplanin-transfected CHO, and podoplanin-transfected Lec2 cell lines were eluted using 100 lg/ml hpp38–51 peptide or FLAG peptide.
Puriﬁed proteins were electrophoresed, CBB stained, and immunoblotted with NZ-1 antibody. (B) Puriﬁed and concentrated podoplanins from
LN319, podoplanin-transfected CHO, and podoplanin-transfected Lec2 induced platelet aggregation. Platelet aggregation was measured using WBA
Carna with the screen ﬁltration pressure method.

M.K. Kaneko et al. / FEBS Letters 581 (2007) 331–336

333

3. Results and discussion
3.1. Puriﬁcation of human podoplanin and platelet aggregationassay
Puriﬁcations of endogenous or recombinant podoplaninfrom LN319, podoplanin-transfected CHO, and podoplanintransfected Lec2 were achieved by a one-step procedure using
immunoaﬃnity chromatography utilizing a monoclonal antibody (NZ-1 [15] or M2). Puriﬁed podoplanin proteins (hPod/
LN319, hPod/CHO, and hPod/Lec2) were conﬁrmed to be
more than 95% pure based on CBB staining (Fig. 1A). All
puriﬁed podoplanins induced platelet aggregation in a dosedependent manner (Fig. 1B). In our previous study, we indicated that human podoplanin possessed the ability to induce
aggregation of human and mouse platelets [2]. Furthermore,platelet aggregation induced by human podoplanin using
mouse platelet was reported to be stronger than one using human platelet. Therefore, we used mouse platelet in this wholestudy. We have investigated the platelet aggregation inducedby podoplanin using both a turbidimetric method with platelet-rich plasma (PRP) and a ﬁltration pressure method withwhole blood, and obtained the compatible results using bothmethods (data not shown). Lec2 cells are CMP-sialic acidtransporter-deﬁcient in which both glycoproteins and glycolipids lack 90% of sialic acids (SA) [23]. hPod/Lec2 possessed a
trace amount of SA on the puriﬁed protein (Fig. 2). Therefore,
hPod/Lec2 protein could induce platelet aggregation when it
was puriﬁed and concentrated.
3.2. Total glycan proﬁling of podoplanin of CHO and LN319
using lectin microarray
We investigated the total glycan proﬁle of hPod/LN319,
hPod/CHO, and hPod/Lec2 using a lectin microarray withthe detection method by biotinylated NZ-1 antibody andCy3-labeled streptavidin [15]. Lectin binding was measured
by ﬂuorescent intensity. As shown in Fig. 2, both hPod/
LN319 and hPod/CHO reacted strongly with corel ± sialic acidbinders (ABA, Jacalin, ACA, and MPA) and sialo-mucinbinders (MAH and WGA), but did not react with corel binders(BPL and PNA). These results indicated that podoplanin on
LN319 or podoplanin-transfected CHO possesses a disialyland/or monosialyl-corel structure. Predictably, hPod/Lec2
reacted intensely to corel binders (BPL and PNA). Residualsialic acid of hPod/Lec2 was detected by WGA. In particular,
Jacalin and MPA reactivity of hPod/CHO diﬀered from that of
hPod/LN319. Jacalin did not bind to disialyl-corel structure[24]. Accordingly, these results suggested that hPod/LN319
contains more disialyl-corel compared to hPod/CHO.
3.3. Glycan analysis of podoplanin of CHO and LN319 usingmass spectrometry
Next, the total glycans of hPod/LN319 and hPod/CHO wereanalyzed by mass spectrometry. Glycans were released fromhPods by reductive b-elimination [18]. The resulting alditols
were permethylated [19] and analyzed with MALDI-TOF
and MALDI-QIT-TOF mass spectrometers. MS spectra
revealed that hPod/CHO bears nearly equal amounts of(NeuAc)2-Hex-HexNAc-ol (m/z 1257) and NeuAc-Hex-HexNAc-ol (m/z 895), whereas hPod/LN319 bears mainly(NeuAc)2-Hex-HexNAc-ol (Fig. 3A). In the MS/MS spectrumof m/z 1257, the signals of m/z 620 and 659, which could be

Fig. 2. Determination of total glycan structure for endogenous andrecombinant podoplanin. Glycan proﬁles of puriﬁed podoplanins from
LN319, podoplanin-transfected CHO, and podoplanin-transfected
Lec2 cells using lectin microarray. The ﬂuorescent intensities of eachlectin on lectin array are indicated with bars. Lectin binding speciﬁcities were as follows: BPL; Galb1-3GlaNAc, TJA-II; b-GalNAc, ABA:
Galb1-3GlaNAca-Thr/Ser (T) and sialyl-T, STL; (GlcNAc)n, Jacalin;
Galb1-3GlaNAca-Thr/Ser and GlaNAca-Thr/Ser, PNA Galb1-3GalNAca-Thr/Ser, WFA; terminal GalNAc, ACA; Galb1-3GalNAcaThr/Ser, MPA; Galb1-3GlaNAca-Thr/Ser and GlaNAca-Thr/Ser,
HPA; terminal GalNAc,VVA; terminal GalNAc and GlaNAca-Thr/
Ser, SBA; terminal GalNAc, MAH; Siaa2-3Galb1–3 (Siaa2–6)GalNAca-R, WGA; (GlcNAc)n and multivalent Sia.

assigned as [permethylated NeuAc-Hex + Na]+ and [permethylated NeuAc-HexNAc-ol + Na]+, were observed (Fig. 3B). Allof these results indicated that the glycans found on podoplanin, in LN319 and podoplanin-transfected CHO, are mixtureof monosialyl- and disialyl-corel structures.
3.4. Glycosylation site of functional podoplanin
Mutation of threonine residues in the PLAG domain (52ththreonine (Thr52) of human podoplanin) abolished the platelet
aggregation-inducing abilities [2]. Furthermore, Thr52 waspredicted as a O-glycosylated site [13]. In this study, glycopeptides from hPod/LN319 and hPod/CHO digested by Asp-N
were fractionated by HPLC, followed by peptide sequencing,334

M.K. Kaneko et al. / FEBS Letters 581 (2007) 331–336

A

895.681256.81

hPod/CHO

hPod/LN319

900

1000

1100

1200

m/ z

OMe

B

OMe
MeO
AcMeN

AcMeN

MeO
OMe O
OMe OMe

MeO

OMe

O

O

OMe

O

O

MeO
MeO

m/z 398O

m/z 620m/z 398COOMe

m/z 506

m/z 881

NMeAc
OMe

OMe

m/z 659
m/z 881 881.46

COOMe

505.93619.97659.06

397.90

400

500

600

1256.67

700

800

900
m/z

1000

1100

1200

Fig. 3. The glycan structure of podoplanin analyzed by mass spectrometry. (A) MS spectra of permethylated alditols released from puriﬁed hPods/
LN319 and hPods/CHO. M/z 895.68: NeuAc-Hex-HexNAc-ol, 1256.81: (NeuAc)2-Hex-HexNAc-ol. (B) MS/MS spectrum of m/z 1257. As shown in
the inset, m/z 620 and 659 could be assigned as [permethylated NeuAc-Hex + Na]+ and [permetylated NeuAc-HexNac-ol+Na]+.

and a glycopeptide consisting of Ala23–Glu57 was obtained, inwhich four potential Asp-N digestion sites were missed. The
sequencing gap was observed at only Thr52 during Edman
degradation from Ala23 to Glu57 (Fig. 4). Other possible Oglycosylation sites, such as Thr32, Thr34, and Thr55 (notshown for others) gave the correct sequences. Therefore, only
Thr52 was O-glycosylated in this peptide. Furthermore, we
conﬁrmed using undigested hPod/LN319 and hPod/CHO proteins that Ala23 was the N-terminal amino acid of both endogenous and recombinant podoplanin (data not shown).

52T

23 ASTGQP EDDTETTG L EGGVAMPG A EDDVVTPG TSE 57
PLAG2

34T

Thr

32T

Thr

PLAG1

PLAG3

(52T)

55T

Fig. 4. Determination of glycosylation site of podoplanin. Amino acid sequence of a glycopeptide consisting of Ala23–Glu57 and Edman sequencing
chromatographs of Asp-N digested hPod/CHO protein. Glycosylated Thr52 appeared as a gap during peptide sequencing. The same results wereobtained using hPod/LN319 protein.

M.K. Kaneko et al. / FEBS Letters 581 (2007) 331–336

The MS spectrum of the glycopeptide (Ala23–Glu57)
showed a signal with m/z 4367 that was assigned as [Ala23–
GLu57 + (NeuAc)2HexHexNAc-H] . The glycan structure ofthis glycopeptide was characterized as disialyl-corel structure
by tandem mass spectrometry, using the same method
described above (data not shown). Taken together, these data
provided more convincing evidence that the sialyl-corel on the
PLAG domain (Thr52) was critical for platelet aggregationinducing activity.
3.5. Sialylation of functional podoplanin
Human podoplanin-transfected Lec2 cells could not induce
platelet aggregation [14]. We puriﬁed podoplanin (hPod/Lec2)
from podoplanin-transfected Lec2 (Fig. 1A). As shown in
Fig. 1B, puriﬁed and concentrated hPod/Lec2 induced platelet
aggregation, although podoplanin-transfected Lec2 cells did
not induce aggregation due to its low concentration on thesecell surfaces. Likewise, extremely low concentration of puriﬁed
hPod/Lec2 did not induce platelet aggregation (Fig. 5). Sialic
acid was added to hPod/Lec2 protein using recombinantsialyltransferases. Fig. 5 shows the recovery of platelet
aggregation-inducing activity of hPod/Lec2 + ST3GalT(monosialyl-corel), or hPod/Lec2 + ST3GalT + ST6GalNAcT(disialyl-corel) in spite of their extremely low concentration.
Western-blot analysis showed that sialylated hPod/Lec2
proteins were not degraded (data not shown). These results
conﬁrmed that sialic acid on podoplanin is critical for its platelet-aggregating activity.
In summary, we have clariﬁed the glycan structure of human
podoplanin, which is important for its platelet-aggregating
activity; the disialyl-corel is linked to a glycosylation site at
Thr52 in the PLAG domain of podoplanin. Further investigation is needed to identify the minimum unit of the platelet-activating glycopeptide within podoplanin. We are investigating
the target molecule for podoplanin on the platelet membrane.
However, the receptor of podoplanin has not been identiﬁed

Pressure rate (%)

8060
hPod/Lec2+ST3GalT
40

hPod/Lec2+ST3GalT+ST6GalNAcT

20

hPod/Lec2

0
0.005

0.05
Podoplanin (μg/ml)

0.5

Fig. 5. Recovery of platelet aggregation aggregation-inducing activity.
hPod/Lec2 was used as the acceptor substrate for sialylation. hPod/
Lec2 + ST3GalT (black circle) means sialyl-corel (NeuAca2–3Galb1–
3GalNAca1-) on hPod/Lec2; hPod/Lec2 + ST3GalT + ST6GalNAcT(red square) means disialyl-corel (NeuAca2–3Galb1–3(NeuAca2–
6)GalNAca1-) on hPod/Lec2. hPod/Lec2 (green triangle) was treatedin the same experimental condition without ST3GalT and ST6GalNAcT. Platelet aggregation was measured using WBA Carna with thescreen ﬁltration pressure method. (For interpretation of the referencesin colour in this ﬁgure legend, the reader is referred to the web versionof this article.)335

yet. The results may be exploited to develop new anti-coagulant therapies.
Acknowledgements: We thank Ms. Kahori Tachibana, Ms. Sanae
Furukawa, Ms. Kozue Hagiwara, Ms. Yuki Tsunoda, and Mr. Noboru Uchiyama for their technical assistance. We are very grateful toDrs. Webster K Cavenee, Naoya Fujita, and Takashi Tsuruo for theirgenerous donation of materials. This work was performed as a part of
the Medical Glycomics Project supported by NEDO (New Energy andIndustrial Technology Development Organization). This study was
supported in part by a grant for Research Fellowships from the Japanese Society for the Promotion of Science (Y. Kato), by Kanae Foundation for Life and Socio-medical Science (Y. Kato), and by the Osaka
Cancer Research Foundation (Y. Kato).

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