RESEARCH ARTICLE

LpMab-12 Established by CasMab
Technology Specifically Detects Sialylated OGlycan on Thr52 of Platelet AggregationStimulating Domain of Human Podoplanin
Yukinari Kato1*, Satoshi Ogasawara1, Hiroharu Oki1, Polina Goichberg2,
Ryusuke Honma1, Yuki Fujii1, Mika K. Kaneko1

a11111

1 Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2–1 Seiryo-machi,
Aoba-ku, Sendai, Miyagi 980–8575, Japan, 2 Department of Anesthesia, Brigham and Women’s Hospital,
Harvard Medical School, 75 Francis Street, Boston, MA 02115, United States of America* yukinari-k@bea.hi-ho.ne.jp

OPEN ACCESS
Citation: Kato Y, Ogasawara S, Oki H, Goichberg P,
Honma R, Fujii Y, et al. (2016) LpMab-12 Establishedby CasMab Technology Specifically Detects
Sialylated O-Glycan on Thr52 of Platelet
Aggregation-Stimulating Domain of Human
Podoplanin. PLoS ONE 11(3): e0152912.
doi:10.1371/journal.pone.0152912
Editor: Yves St-Pierre, INRS, CANADA
Received: January 7, 2016
Accepted: March 21, 2016
Published: March 31, 2016
Copyright: This is an open access article, free of all
copyright, and may be freely reproduced, distributed,
transmitted, modified, built upon, or otherwise usedby anyone for any lawful purpose. The work is madeavailable under the Creative Commons CC0 publicdomain dedication.
Data Availability Statement: All relevant data arewithin the paper and its Supporting Information files.
Funding: This work was supported in part by the
Regional Innovation Strategy Support Program from
the Ministry of Education, Culture, Sports, Scienceand Technology (MEXT) of Japan (Y.K.); by the
Platform for Drug Discovery, Informatics, andStructural Life Science (PDIS) from Japan Agency for
Medical Research and development, AMED (Y.K.); by
the Basic Science and Platform Technology Programfor Innovative Biological Medicine from AMED (Y.K.);
by Japan Society for the Promotion of Science

Abstract
Podoplanin (PDPN), also known as Aggrus, possesses three tandem repeat of platelet
aggregation-stimulating (PLAG) domains in its N-terminus. Among the PLAG domains, sialylated O-glycan on Thr52 of PLAG3 is essential for the binding to C-type lectin-like receptor-2 (CLEC-2) and the platelet-aggregating activity of human PDPN (hPDPN). Althoughvarious anti-hPDPN monoclonal antibodies (mAbs) have been generated, no specific mAb
has been reported to target the epitope containing glycosylated Thr52. We recently established CasMab technology to develop mAbs against glycosylated membrane proteins.
Herein, we report the development of a novel anti-glycopeptide mAb (GpMab), LpMab-12.
LpMab-12 detected endogenous hPDPN by flow cytometry. Immunohistochemical analyses also showed that hPDPN-expressing lymphatic endothelial and cancer cells wereclearly labeled by LpMab-12. The minimal epitope of LpMab-12 was identified as Asp49–
Pro53 of hPDPN. Furthermore, LpMab-12 reacted with the synthetic glycopeptide ofhPDPN, corresponding to 38–54 amino acids (hpp3854: 38-EGGVAMPGAEDDVVTPG-54),
which carries α2–6 sialylated N-acetyl-D-galactosamine (GalNAc) on Thr52. LpMab-12 did
not recognize non-sialylated GalNAc-attached glycopeptide, indicating that sialylated GalNAc on Thr52 is necessary for the binding of LpMab-12 to hPDPN. Thus, LpMab-12 could
serve as a new diagnostic tool for determining whether hPDPN possesses the sialylation on
Thr52, a site-specific post-translational modification critical for the hPDPN association with
CLEC-2.

Introduction
Podoplanin (PDPN), the endogenous ligand of C-type lectin-like receptor-2 (CLEC-2) [1,2], ishighly expressed not only in various tumors including oral cancer, lung cancer, esophageal

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Monoclonal Antibody against Thr52 of Human Podoplanin(JSPS) KAKENHI Grant Number Grant Number
25462242 (Y.K.) and 26440019 (M.K.K.); by Takeda
Science Foundation (S.O.); and by the Grant-in-Aid
from the American Heart Association (P.G.).
Competing Interests: The authors have declaredthat no competing interests exist.
Abbreviations: mAb, monoclonal antibody; PLAG,
platelet aggregation-stimulating; GalNAc, N-acetyl-Dgalactosamine; GpMab, anti-glycopeptide mAb;
CLEC-2, C-type lectin-like receptor-2; LEC, lymphatic
endothelial cell.

cancer, malignant brain tumors, mesotheliomas, testicular tumors, and osteosarcoma [3–13],
but also in normal cells such as lymphatic endothelial cells and podocytes [14,15]. PDPN isalso abundant in lung type I alveolar cells where it is called "T1α" [16]. We previously named
PDPN as "Aggrus" because PDPN possesses a platelet aggregation-inducing activity, which is
associated with cancer metastasis [3]. Further, PDPN is known as a specific lymphatic endothelial marker [17], and PDPN-CLEC-2 signaling leads to platelet aggregation, which is critical forthe embryonic blood-lymphatic vascular separation [18].
Interaction of human PDPN (hPDPN) with CLEC-2 mainly involves Glu47 and Asp48 inthe platelet aggregation-stimulating domain-3 (PLAG3) and the α2–6 linked sialic acid residue[19]. The sequence motif is conserved among PDPNs of various species [20]. CLEC-2 has a recognition motif in the form ‘‘EDXXXT/S,” where X is any amino acid, and T (or S) contains disialyl-core 1 [21]. In our previous studies, we established that the glycosylation on Thr52 is
critical for the binding of hPDPN to CLEC-2, and the sialylated O-glycan on Thr52 is required
for the platelet aggregating activity of hPDPN [3,21]. Therefore, the detection of site-specificglycosylation on Thr52 is important for determining whether in given pathophysiological conditions, hPDPN is prone to the CLEC-2 binding and has the potential to cause platelet
aggregation.
Here, we describe the development and characterization of a new anti-hPDPN mAb,
LpMab-12, which specifically binds to glycosylated Thr52, and might serve as a novel modality
to study hPDPN-CLEC-2 interaction.

Materials and Methods
Cell lines, animals, and tissues
Chinese hamster ovary (CHO)-K1, LN229, HEK-293T, COS-7, and P3U1 were obtained fromthe American Type Culture Collection (ATCC, Manassas, VA). Human lymphatic endothelial
cell (LEC) was purchased from Cambrex (Walkersville, MD). The human glioblastoma cellline, LN319, was donated by Dr. Kazuhiko Mishima (Saitama Medical University, Saitama,
Japan). LN229 was transfected with human PDPN plasmids (LN229/hPDPN) using Lipofectamine 2000 (Thermo Fisher Scientific Inc., Waltham, MA) according to the manufacturer’s
instructions [22]. LN319/hPDPN-knock out (KO) cells (PDIS-6), HEK-293T/hPDPN-KO cells(PDIS-2), and COS-7/hPDPN-KO cells (PDIS-4) were produced by transfecting CRISPR/Cas
plasmids, which targets hPDPN (Sigma-Aldrich Corp., St. Louis, MO), using a Gene Pulser
Xcell electroporation system (Bio-Rad Laboratories Inc., Philadelphia, PA). The amplified
hPDPN cDNA was subcloned into a pcDNA3 vector (Thermo Fisher Scientific Inc.) and a
FLAG epitope tag was added at the C-terminus. Substitution of amino acids to alanine in
hPDPN was performed using a QuikChange Lightning site-directed mutagenesis kit (Agilent
Technologies Inc., Santa Clara, CA). CHO-K1 cells were transfected with the plasmids using a
Gene Pulser Xcell electroporation system (Bio-Rad Laboratories Inc.). P3U1 and CHO-K1 celllines, and their counterparts transfected with hPDPN were cultured in L-glutamine-containing
RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), and LN229, LN319, HEK-293T,
COS-7 cell lines and their transfected counterparts were cultured in L-glutamine-containing
Dulbecco’s Modified Eagle’s Medium (DMEM) medium (Nacalai Tesque, Inc.), supplemented
with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific Inc.) at 37°C in ahumidified atmosphere of 5% CO2 and 95% air. LEC was cultured in endothelial cell medium
EGM-2MV supplemented with 5% FBS (Cambrex Corp.). Antibiotics including 100 units/mlof penicillin, 100 μg/ml of streptomycin, and 25 μg/ml of amphotericin B (Nacalai Tesque,Inc.) were added to all media.

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Monoclonal Antibody against Thr52 of Human Podoplanin

Three female BALB/c mice (four-weeks old) were purchased from CLEA Japan (Tokyo,
Japan). Animals were housed under pathogen-free conditions. "The Animal Care and Use
Committee of Tohoku University" approved the animal experiments described herein. The
use of one oral cancer tissue was reviewed and approved by "Tokyo Medical and Dental University Institutional Review Board" [23]. Written informed consent was obtained for thehuman cancer tissue samples used in this study. The use of human heart tissue sections for
immunohistochemical analysis was reviewed and approved by the "Partners Institutional
Review Board".

Hybridoma production
Three BALB/c mice were immunized by intraperitoneal (i.p.) injection of 1 × 108 LN229/
hPDPN cells together with Imject Alum (Thermo Fisher Scientific Inc.), as previously
described [22]. After several additional immunizations, a booster injection was given i.p. twodays before mice were euthanized by cervical dislocation and spleen cells were harvested. The
spleen cells were fused with P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN).
The fused cells were grown in RPMI medium with hypoxanthine, aminopterin, and thymidine
selection medium supplement (Thermo Fisher Scientific Inc.). The culture supernatants werescreened using enzyme-linked immunosorbent assay (ELISA) for binding to recombinant
hPDPN purified from LN229/hPDPN cells.

Enzyme-linked immunosorbent assay (ELISA)
Recombinant hPDPN or glycopeptides were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific Inc.) at a concentration of 1 μg/ml for 30 min. After blocking
with 1% BSA in 0.05% Tween20/phosphate buffered saline (PBS, Nacalai Tesque, Inc.), theplates were incubated with culture supernatant followed by 1:1000 diluted peroxidase-conjugated anti-mouse IgG or anti-rat IgG (Dako; Agilent Technologies, Inc., Glostrup, Denmark).
The enzymatic reaction was conducted with a 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific Inc.). The optical density was measured at 655 nm using an iMark microplate reader (BioRad Laboratories Inc.).

Western blot analyses
Cell lysates (10 μg) were boiled in sodium dodecyl sulfate (SDS) sample buffer (Nacalai Tesque, Inc.). The proteins were electrophoresed on 5–20% polyacrylamide gels (Wako Pure
Chemical Industries Ltd.) and were transferred onto a PVDF membrane (EMD Millipore
Corp., Billerica, MA). After blocking with 4% skim milk (Nacalai Tesque, Inc.) in 0.05%
Tween20/PBS, the membrane was incubated with 1 μg/ml of LpMab-12, LpMab-7, 1E6(anti-FLAG; Wako Pure Chemical Industries Ltd.), RcMab-3 (anti-IDH1) [24], or AC-15(anti-β-actin; Sigma-Aldrich Corp.) and then with peroxidase-conjugated anti-mouse IgG(1:1000 diluted; Dako), and developed with the ImmunoStar LD Chemiluminescence
Reagent (Wako Pure Chemical Industries Ltd.) using a Sayaca-Imager (DRC Co. Ltd.,Tokyo, Japan).

Flow cytometry
Cell lines were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA (Nacalai Tesque,Inc.). After washing with PBS, the cells were incubated with LpMab-12 (1 μg/ml) for 30 min at
4°C, followed by the incubation with Oregon Green 488 goat anti-mouse IgG (Thermo Fisher

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Monoclonal Antibody against Thr52 of Human Podoplanin

Scientific Inc.). Fluorescence data were collected using a Cell Analyzer EC800 (Sony Corp.,
Tokyo, Japan).

Determination of the apparent binding affinity using flow cytometry
LN319 (2 × 105 cells) and LEC (1 × 105 cells) were resuspended in 100 μl of serially diluted
LpMab-12 (0.061–100 μg/ml) followed by Oregon Green 488 goat anti-mouse IgG (Thermo
Fisher Scientific Inc.). Fluorescence data were collected using a cell analyzer (EC800; Sony
Corp.). The apparent dissociation constants (KD) were obtained by fitting the binding isotherms using the built-in one-site binding models in GraphPad PRISM 6 (GraphPad software,Inc., La Jolla, CA).

Immunohistochemical analysis of human heart tissues
Four-μm-thick histologic sections of the myocardium were deparaffinized in xylene, rehydrated, and subjected to 10 min heat-induced antigen retrieval in citric buffer (pH 6.0). Samples were blocked in 10% normal donkey serum (Jackson ImmunoResearch Inc., West Grove,PA) for 30 min at room temperature, incubated with 10 μg/ml of LpMab-12 overnight at 4°C,
and then with Alexa Fluor 568-conjugated donkey anti-mouse IgG (Thermo Fisher ScientificInc.) for 1 h at 37°C. Subsequently, the sections were incubated with goat anti-human LYVE-1(10 μg/ml; R&D Systems, Inc., Minneapolis, MN) and mouse anti-α-sarcomeric actin (α-SA)(1:200 diluted; Sigma-Aldrich Corp.) for 2 h at 37°C, followed by fluorescein isothiocyanate(FITC)-conjugated donkey anti-goat IgG and Alexa Fluor 647-conjugated donkey anti-mouse
IgM (15 μg/ml each; Jackson ImmunoResearch Inc.) and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (1 μg/ml; Sigma-Aldrich Corp.) for 1 h at 37°C. The sections were thentreated with 1% solution of Sudan Black B (Sigma-Aldrich Corp.) for 30 min at room temperature, and mounted in Vectashield medium (Vector Laboratories, Inc., Road Burlingame, CA).
Images were acquired with Olympus FluoView FV100 laser scanning confocal microscope
equipped with CCD camera (Bio-Rad Laboratories Inc.).

Immunohistochemical analyses of oral cancer
Four-μm-thick histologic sections were deparaffinized in xylene and rehydrated. Without antigen retrieval procedure, sections were incubated with 1 μg/ml of LpMab-12 or LpMab-7 for 1 h
at room temperature followed by treatment with Envision+ kit (Dako) for 30 min. Color wasdeveloped using 3, 3-diaminobenzidine tetrahydrochloride (DAB; Dako), and then the sections
were counterstained with hematoxylin (Wako Pure Chemical Industries Ltd.).

Production of hPDPN glycopeptide
The hPDPN glycopeptide (hpp3854) with a GalNAc residue was purchased from Peptide Institute (Osaka, Japan), and used as an acceptor substrate. For synthesis of the sialylated GalNAc
on hpp3854, 25 mM HEPES (pH 7.0) containing 30 μM of acceptor substrate, 10 mM MnCl2(Nacalai Tesque, Inc.), and 250 μM CMP-Neu5Ac (Sigma-Aldrich Corp.) was used. A half volume of purified ST6GalNAcT-I enzyme was added to the reaction mixture and incubated at
37°C for 24 h. The recombinant ST6GalNAcT-I enzyme was bound to anti-FLAG M2 affinitygel (Sigma-Aldrich Corp.). After enzymatic reaction, the resin was removed by filtration using
an Ultrafree-MC column (EMD-Millipore). Then, the glycopeptides were purified using areversed-phase SPE cartridge (ZipTip C18; EMD-Millipore). The other glycopeptides were
produced sequentially as previously described [2].

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Monoclonal Antibody against Thr52 of Human Podoplanin

Assessment of antibody-mediated inhibition of hPDPN binding to
hCLEC-2
Inhibition assays were performed by ELISA. The recombinant proteins of hPDPN-Fc [2] andhCLEC-2-Fc [19] were produced in our previous studies. The hPDPN-Fc was immobilized on
Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific Inc.) at 1 μg/ml for 30 min.
After blocking with SuperBlock T20 (PBS) Blocking Buffer, LpMab-2 [22], LpMab-3 [25],
LpMab-9 [26], LpMab-12, LpMab-3 + LpMab-12, or isotype control (PMab-32) [27,28] wereadded at 10 μg/ml for 30 min. The plates were incubated with biotinylated hCLEC-2-Fc (1 μg/ml) followed by 1/1000 diluted peroxidase-conjugated streptavidin (GE Healthcare, Piscataway, NJ). The enzymatic reaction was conducted with a 1-Step Ultra TMB-ELISA (Thermo
Fisher Scientific Inc.). The optical density was measured at 655 nm using an iMark microplate
reader (Bio-Rad Laboratories Inc.). All data were shown as means ± SD. Statistical analysis by
one-way ANOVA was performed using GraphPad Prism 6 (GraphPad Software Inc., La Jolla,CA).

Results
Establishment and characterization of a novel anti-hPDPN mAb LpMab12
We immunized mice with hPDPN-expressing LN229 glioma cells (LN229/hPDPN), which
possess cancer-type glycan patterns including highly sulfated polylactosamine and aberrant
sialylation [22]. Spleen cells were harvested and fused with P3U1 cells. Selection of hybridoma
was performed using ELISA and flow cytometry, and a novel anti-hPDPN LpMab-12 mAb(mouse IgG1, kappa) was developed. Fig 1A shows that LpMab-12 reacted with LN229/hPDPN
and endogenous PDPN (HEK-293T, LN319, COS-7), and did not react with LN229 and
PDPN-KO cells (HEK-293T/hPDPN-KO, LN319/hPDPN-KO, COS-7/hPDPN-KO), indicating that LpMab-12 reliably detects hPDPN by immunohistochemistry.
As shown in Fig 1B and 1C, lymphatic endothelial cells, identified by a lymphatic marker
lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), were clearly stained by LpMab12 in myocardial samples from human hearts, indicating that LpMab-12 is applicable for reliably detecting hPDPN by immunohistochemistry.
Using flow cytometry analysis, apparent dissociation constant of LpMab-12 was determined
to be 1.2 × 10−8 M for LN319 and 1.8 × 10−8 M for LEC, suggesting that the binding affinity of
LpMab-12 is comparable with previously established anti-hPDPN mAbs [22] for hPDPNexpressing cancer cells and normal cells (Fig 1D).
To confirm the utility of LpMab-12 for immunolabeling of tissues, we compared the reactivity of LpMab-12 with LpMab-7, the most sensitive anti-hPDPN mAb for this type of analysis[29]. Both LpMab-12 (Fig 2A and 2B) and LpMab-7 (Fig 2E and 2F) strongly stained tumor
cells in a membranous/cytoplasmic-staining pattern. Lymphatic vessels were immunolabeledclearly without background by LpMab-12 (Fig 2C and 2D) and LpMab-7 (Fig 2G and 2H).
Blood vessels were not stained by both mAbs (Fig 2C, 2D, 2G and 2H).

Epitope mapping
To determine the critical epitope for the LpMab-12 interaction with hPDPN, we compared themAb binding to the hPDPN carrying different point mutations. Using Western blot, we foundthat LpMab-12 did not detect protein sequences with the following amino acid substitutions:
D49A, V51A, T52A, and P53A (Fig 3A).

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Monoclonal Antibody against Thr52 of Human Podoplanin

Fig 1. Characterization of an anti-hPDPN mAb LpMab-12. (A) Flow cytometry assessment of LpMab-12
binding to LN229/hPDPN and LN229 cells, endogenous hPDPN-expressing (HEK-293T, LN319, COS-7),
and hPDPN-KO cells (HEK-293T/hPDPN-KO, LN319/hPDPN-KO, COS-7/hPDPN-KO). (B, C) Human
myocardial samples were indirectly immunolabeled with LpMab-12 (red), the lymphatic endothelial epitope,
LYVE-1 (green), and the myocytes were stained with anti-α-sarcomeric actin (α-SA) (grey). Nuclei werecounterstained with DAPI (blue). Arrows, lymphatic endothelial cells; arrowheads, vascular endothelial cellsof an artery. Scale bars: 100 μm (B) and 20 μm (C). Note the preferential labeling of lymphatic endothelium by
LpMab-12. (D) Determination of apparent binding affinity against LN319 and LEC by flow cytometry. The
apparent dissociation constants (KD) were obtained by fitting the binding isotherms using the built-in one-sitebinding models.
doi:10.1371/journal.pone.0152912.g001

In agreement, flow cytometry analysis demonstrated that LpMab-12 did not react with
D49A, T52A, and P53A mutant proteins (Fig 3B) Thus, our results indicate that the epitope of
LpMab-12 is Asp49-Pro53. In our previous study we established that the sialylated O-glycan
on Thr52 is critical for platelet aggregating activity of hPDPN [21]. Therefore, the data pointthat the epitope of LpMab-12 contains the sialylated O-glycan on Thr52 in the Asp49-Pro53
sequence of hPDPN.
To further clarify the essential epitope of LpMab-12, especially the essential glycan structure
detected by LpMab-12, we synthesized several glycopeptides of hPDPN, which include thePLAG2 and PLAG3 domains (Fig 4). Specifically, we generated SAα2-6GalNAc + hpp3854;
Gal + GalNAc + hpp3854; SAα2-3Gal + GalNAc + hpp3854; Gal + SAα2-6GalNAc +hpp3854; and SAα2-3Gal + SAα2-6GalNAc + hpp3854. LpMab-12 detected SAα2-6GalNAc +hpp3854, Gal + SAα2-6GalNAc + hpp3854, and SAα2-3Gal + SAα2-6GalNAc + hpp3854(Table 1). LpMab-9, the epitope of which was identified as residues 25–30 of hPDPN [26], did

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Monoclonal Antibody against Thr52 of Human Podoplanin

Fig 2. Immunohistochemical analysis of the oral cancer and heart tissue samples using LpMab-12
and LpMab-7. Serial sections of the tissues with oral cancer were incubated with LpMab-12 (A-D) or LpMab7 (E-H), followed by the development with the EnVision+ kit and counterstaining with hematoxylin, or the HE
staining (I-L). Arrows, lymphatic endothelial cells; arrowheads, vascular endothelial cells. Scale bars:
100 μm. LpMab-12 stains lymphatic vessels with high efficiency, similarly to LpMab-7.
doi:10.1371/journal.pone.0152912.g002

not react with any glycopeptides of hpp3854. In contrast, LpMab-13 and LpMab-20, whichwere recently established using CasMab technology [30], recognized all the glycopepties of
hpp3854. Collectively, these data indicate that the essential epitope of LpMab-12 is 49-DVVT(SAα2-6GalNAc)P-53 (Fig 5).

Neutralization assays of hPDPN and CLEC-2
Finally, we determined whether LpMab-12 inhibits hPDPN-CLEC-2 interaction using ELISA.
Additional anti-hPDPN mAbs such as LpMab-2, LpMab-3, and LpMab-9 were employed ascontrols. Similarly to LpMab-12, LpMab-2 [22], LpMab-3 [25] and LpMab-9 [26] include both
peptide and glycan as their epitopes; accordingly, we collectively named these mAbs as antiglycopeptide mAbs (GpMabs). Anti-rabbit PDPN antibody PMab-32 was used as an additional
negative control, since it is of the same isotype (mouse IgG1, kappa) with LpMab-12 [27]. As
shown in Fig 6, LpMab-12 impaired the binding of hCLEC-2-Fc to hPDPN-Fc (10.3% inhibition), whereas LpMab-2 did not affect the hPDPN/hCLEC-2 interaction. Interestingly,
LpMab-3, the epitope of which includes Thr76 of hPDPN [25] also moderately reduced thehPDPN/hCLEC-2 binding (7.1% inhibition); and LpMab-9, the epitope of which includes
Thr25 of hPDPN, impaired the hPDPN/hCLEC-2 interaction to a lesser extent (3.6% inhibition). Importantly, the combination of LpMab-12 and LpMab-3 reduced the hPDPN/hCLEC-2
interaction more effectively (14.7% inhibition) than either LpMab-12 or LpMab-3 alone, indicating that a combination of several sialic acids in the hPDPN protein might be important for
its optimal interaction with hCLEC-2 in this in vitro assay.

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Monoclonal Antibody against Thr52 of Human Podoplanin

Fig 3. Epitope mapping of LpMab-12 by Western blot analysis and flow cytometry. (A) CHO-K1 cells
were transfected with a plasmid expressing wild-type hPDPN with the FLAG-tag added to the C-terminus(WT), or the FLAG-tag hPDPN containing a point mutation in the sequence E47A-E57A, as indicated in the
figure. Total cell lysates from the transfected cell lines were analyzed by Western blot with LpMab-12 or
LpMab-7, as a positive control for hPDPN expression. Immunoblot with anti-FLAG antibody was also used aswell to establish the expression of exogenous hPDPN. Anti-IDH1 and anti-β-actin mAbs were used asinternal controls to show that total proteins are equal protein load. Red arrow, 40-kDa; blue arrow, 30-kDa. (B)
CHO-K1 cells transfected as in (A) were analyzed by flow cytometry using indirect immunolabeling with
LpMab-12. Cells exposed to the secondary anti-mouse IgG only were used as a negative control (Control).
doi:10.1371/journal.pone.0152912.g003

Discussion
As of today, almost all anti-hPDPN mAbs produced using conventional methods react with
the non-glycosylated peptide spanning the PLAG1-3 domains [7,12,31] or PLAG4 domain[32]. Our group also produced numerous mAbs against mouse, rat and rabbit PDPN proteins[27,33,34]. Rabbit polyclonal antibodies, which were reported by Matsui et al., also recognize
PLAG1-3 domains, which are shown to be immunodominant antigenic sites of PDPN [35].
Recently, we established the CasMab technology for the production of cancer-specific mAbs
and anti-glycopeptide mAbs (GpMabs). Using CasMab platform, we generated multiple mAbs,
including LpMab-2, LpMab-3, LpMab-7, LpMab-9, LpMab-10, and LpMab-17, which target
different epitopes of hPDPN [22,23,25,26,29,36–38]. Furthermore, using CasMab approach,

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Monoclonal Antibody against Thr52 of Human Podoplanin

Fig 4. Binding assay of LpMab-12 against sialylated glycopeptide of hPDPN using ELISA. Strategy forthe sialylated glycopeptide synthesis. SA, sialic acid; Gal, galacose; GalNAc, N-acetyl-D-galactosamine.
doi:10.1371/journal.pone.0152912.g004

we produced mAbs that detect residue-specific O-glycosylation in hPDPN: LpMab-2 on
Thr55/Ser56, LpMab-3 on Thr76, and LpMab-9 on Thr25. Although the glycosylation on
Thr52 is the most critical for the binding of hPDPN to CLEC-2 and platelet aggregating-activity of hPDPN [2,19], no GpMab against Thr52-containing epitope has been developed. The
direct detection of glycosylation on Thr52 using specific mAb might be implemented for investigating the function of hPDPN or clinical diagnosis.
In this study, we successfully developed LpMab-12 (mouse IgG1, kappa), which specificallydetects the glycosylation on Thr52 of hPDPN by flow cytometry (Figs 1 and 3), Western blot
(Fig 3), and immunohistochemical analysis (Figs 1 and 2). Because this modification was previously shown to be of critical importance for hPDPN-CLEC-2 interaction [2,19], we hypothesized that LpMab-12 might interfere with the hPDPN-binding to CLEC-2. We found that
LpMab-12 only partially and weakly reduced the hPDPN binding to hCLEC-2, yet with ahigher efficiency than the other anti-hPDPN glycopeptide mAbs (GpMabs), such as LpMab-3
and LpMab-9 (Fig 6). These results indicate that hCLEC-2 might interact with several sialic
acids attached to Ser/Thr of hPDPN. Indeed, a novel platelet aggregation-stimulating domain4 (PLAG4) of hPDPN (Fig 5) was recently suggested [32], further supporting the notion thatcomplex interactions might be required for an optimal association of hPDPN with hCLEC-2.
Our data show that LpMab-12 is advantageous for the use for hPDPN detection in fixedparaffin-embedded tissue sections, since, unlike other anti-hPDPN antibodies, including
Table 1. The reaction of LpMab-12 against glycopeptides of hPDPN.
Anti-hPDPN mAbs
Glycopeptides
GalNAc + hpp3854

LpMab-9

LpMab-12

LpMab-13

LpMab-20

-

-

+++

+++

Gal + GalNAc + hpp3854

-

-

+++

+++

SAα2-6GalNAc + hpp3854

-

+++

+++

+++

SAα2-3Gal + GalNAc + hpp3854

-

-

+++

+++

Gal + SAα2-6GalNAc + hpp3854

-

+++

+++

+++

SAα2-3Gal + SAα2-6GalNAc + hpp3854

-

+++

+++

+++

+++, 0.5≦OD655; -, negative
doi:10.1371/journal.pone.0152912.t001

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Monoclonal Antibody against Thr52 of Human Podoplanin

Fig 5. Schematic summary of the epitopes for several anti-hPDPN mAbs. Glycosylation sites are shown(O-glycan). Numbers indicate amino acid position. GpMab, anti-glycopeptide mAb; PLAG, platelet aggregationstimulating.
doi:10.1371/journal.pone.0152912.g005

LpMab-2 and LpMab-3 [22,25], or D2-40 and 18H5 [31], LpMab-12 does not necessitate antigen retrieval (Fig 2). Further, in most PDPN immunolabeling protocols, the antibodies have to
be used at a concentration of 1 μg/ml or higher [22,25,31], whereas relatively low concentrations of LpMab-12 (less than 0.1 μg/ml) are sufficient to detected the lymphatic endothelial
cells in fixed samples (data not shown).
Lec2 mutant of CHO cells lacks a CMP-sialic acid transporter, and is not able to add sialic
acid to glycans. In contrast, Lec8 mutant of CHO cells lacks a UDP-Gal transporter and is notable to add Gal to glycans [39]. Our results show that LpMab-12 detects hPDPN with sialylated
O-GalNAc (Fig 4 and Table 1); therefore, LpMab-12 did not react with Lec2/hPDPN (S1B Fig).
Surprisingly, we observed that LpMab-12 did not react with Lec8/hPDPN cells even at

Fig 6. The hPDPN-hCLEC-2 interaction was reduced by LpMab-12. Inhibition assay was performed using
ELISA. Recombinant immobilized hPDPN-Fc was incubated with PMab-32 (Control), LpMab-2, LpMab-3,
LpMab-9, LpMab-12, or LpMab-3 + LpMab-12, followed by sequential exposure to biotinylated hCLEC-2-Fc
and peroxidase-conjugated streptavidin. The enzymatic reaction was conducted with a 1-Step Ultra
TMB-ELISA. The optical density was measured at 655 nm. Each data bar represents the average of fiveindependent wells. Error bars show standard deviation (SD). N.S., not significant, * P < 0.05, ** P < 0.01,****P < 0.0001, as determined by one-way ANOVA.
doi:10.1371/journal.pone.0152912.g006

PLOS ONE | DOI:10.1371/journal.pone.0152912 March 31, 201610 / 13

Monoclonal Antibody against Thr52 of Human Podoplanin

relatively high concentrations of 10 μg/ml or 100 μg/ml (S1C Fig). Future studies are warranted
to determine the reason for the deficiency in O-GalNAc sialylation in Lec8/hPDPN.

Conclusion
Our study suggests that LpMab-12 is useful for determining whether hPDPN possesses thesite-specific sialylation on Thr52, an important post-translational modification for the association of hPDPN with CLEC-2 and activation of platelet aggregation. Furthermore, the combination of different epitope-specific mAbs, especially GpMabs, might be advantageous for the
PDPN-targeting therapies or disease diagnosis.

Supporting Information
S1 Fig.
(TIFF)
S1 File.
(DOCX)

Acknowledgments
We thank Takuro Nakamura, Noriko Saidoh, Hazuki Kanno, and Kanae Yoshida for their
excellent technical assistance. We also thank Prof. Hiroyuki Harada for providing us with thetissue samples.

Author Contributions
Conceived and designed the experiments: YK. Performed the experiments: SO HO PG RH YF.
Analyzed the data: MKK. Contributed reagents/materials/analysis tools: SO HO PG RH YF.
Wrote the paper: YK PG MKK.

References
1.

Suzuki-Inoue K, Kato Y, Inoue O, Kaneko MK, Mishima K, Yatomi Y, et al. (2007) Involvement of thesnake toxin receptor CLEC-2, in podoplanin-mediated platelet activation, by cancer cells. J Biol Chem
282: 25993–26001. PMID: 17616532

2.

Kato Y, Kaneko MK, Kunita A, Ito H, Kameyama A, Ogasawara S, et al. (2008) Molecular analysis of
the pathophysiological binding of the platelet aggregation-inducing factor podoplanin to the C-type lectin-like receptor CLEC-2. Cancer Sci 99: 54–61. PMID: 17944973

3.

Kato Y, Fujita N, Kunita A, Sato S, Kaneko M, Osawa M, et al. (2003) Molecular identification of Aggrus/
T1alpha as a platelet aggregation-inducing factor expressed in colorectal tumors. J Biol Chem 278:
51599–51605. PMID: 14522983

4.

Kato Y, Sasagawa I, Kaneko M, Osawa M, Fujita N, Tsuruo T (2004) Aggrus: A diagnostic marker that
distinguishes seminoma from embryonal carcinoma in testicular germ cell tumors. Oncogene 23:8552–8556. PMID: 15361850

5.

Martin-Villar E, Scholl FG, Gamallo C, Yurrita MM, Munoz-Guerra M, Cruces J, et al. (2005) Characterization of human PA2.26 antigen (T1alpha-2, podoplanin), a small membrane mucin induced in oral
squamous cell carcinomas. Int J Cancer 113: 899–910. PMID: 15515019

6.

Kato Y, Kaneko M, Sata M, Fujita N, Tsuruo T, Osawa M (2005) Enhanced expression of Aggrus(T1alpha/podoplanin), a platelet-aggregation-inducing factor in lung squamous cell carcinoma. Tumor
Biol 26: 195–200.

7.

Kato Y, Kaneko MK, Kuno A, Uchiyama N, Amano K, Chiba Y, et al. (2006) Inhibition of tumor cellinduced platelet aggregation using a novel anti-podoplanin antibody reacting with its platelet-aggregation-stimulating domain. Biochem Biophys Res Commun 349: 1301–1307. PMID: 16979138

8.

Mishima K, Kato Y, Kaneko MK, Nakazawa Y, Kunita A, Fujita N, et al. (2006) Podoplanin expression inprimary central nervous system germ cell tumors: a useful histological marker for the diagnosis of germinoma. Acta Neuropathol (Berl) 111: 563–568.

PLOS ONE | DOI:10.1371/journal.pone.0152912 March 31, 201611 / 13

Monoclonal Antibody against Thr52 of Human Podoplanin

9.

Mishima K, Kato Y, Kaneko MK, Nishikawa R, Hirose T, Matsutani M (2006) Increased expression of
podoplanin in malignant astrocytic tumors as a novel molecular marker of malignant progression. Acta
Neuropathol (Berl) 111: 483–488.

10.

Abe S, Morita Y, Kaneko MK, Hanibuchi M, Tsujimoto Y, Goto H, et al. (2013) A novel targeting therapy
of malignant mesothelioma using anti-podoplanin antibody. J Immunol 190: 6239–6249. doi: 10.4049/
jimmunol.1300448 PMID: 23690472

11.

Kunita A, Kashima TG, Ohazama A, Grigoriadis AE, Fukayama M (2011) Podoplanin is regulated byAP-1 and promotes platelet aggregation and cell migration in osteosarcoma. Am J Pathol 179: 1041–
1049. doi: 10.1016/j.ajpath.2011.04.027 PMID: 21801875

12.

Kato Y, Vaidyanathan G, Kaneko MK, Mishima K, Srivastava N, Chandramohan V, et al. (2010) Evaluation of anti-podoplanin rat monoclonal antibody NZ-1 for targeting malignant gliomas. Nucl Med Biol
37: 785–794. doi: 10.1016/j.nucmedbio.2010.03.010 PMID: 20870153

13.

Kunita A, Kashima TG, Morishita Y, Fukayama M, Kato Y, Tsuruo T, et al. (2007) The platelet aggregation-inducing factor aggrus/podoplanin promotes pulmonary metastasis. Am J Pathol 170: 1337–1347.
PMID: 17392172

14.

Breiteneder-Geleff S, Matsui K, Soleiman A, Meraner P, Poczewski H, Kalt R, et al. (1997) Podoplanin,novel 43-kd membrane protein of glomerular epithelial cells, is down-regulated in puromycin nephrosis.
Am J Pathol 151: 1141–1152. PMID: 9327748

15.

Schacht V, Dadras SS, Johnson LA, Jackson DG, Hong YK, Detmar M (2005) Up-regulation of the lymphatic marker podoplanin, a mucin-type transmembrane glycoprotein, in human squamous cell carcinomas and germ cell tumors. Am J Pathol 166: 913–921. PMID: 15743802

16.

Rishi AK, Joyce-Brady M, Fisher J, Dobbs LG, Floros J, VanderSpek J, et al. (1995) Cloning, characterization, and development expression of a rat lung alveolar type I cell gene in embryonic endodermal
and neural derivatives. Dev Biol 167: 294–306. PMID: 7851650

17.

Breiteneder-Geleff S, Soleiman A, Kowalski H, Horvat R, Amann G, Kriehuber E, et al. (1999) Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium. Am J Pathol 154: 385–394. PMID: 10027397

18.

Bertozzi CC, Schmaier AA, Mericko P, Hess PR, Zou Z, Chen M, et al. (2010) Platelets regulate lymphatic vascular development through CLEC-2-SLP-76 signaling. Blood 116: 661–670. doi: 10.1182/
blood-2010-02-270876 PMID: 20363774

19.

Nagae M, Morita-Matsumoto K, Kato M, Kaneko MK, Kato Y, Yamaguchi Y (2014) A Platform of CType lectin-like receptor CLEC-2 for binding O-glycosylated podoplanin and nonglycosylated rhodocytin. Structure 22: 1711–1721. doi: 10.1016/j.str.2014.09.009 PMID: 25458834

20.

Kaneko MK, Kato Y, Kitano T, Osawa M (2006) Conservation of a platelet activating domain of Aggrus/
podoplanin as a platelet aggregation-inducing factor. Gene 378: 52–57. PMID: 16766141

21.

Kaneko MK, Kato Y, Kameyama A, Ito H, Kuno A, Hirabayashi J, et al. (2007) Functional glycosylationof human podoplanin: glycan structure of platelet aggregation-inducing factor. FEBS Lett 581: 331–
336. PMID: 17222411

22.

Kato Y, Kaneko MK (2014) A cancer-specific monoclonal antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep 4: 5924. doi: 10.1038/srep05924 PMID: 25080943

23.

Kato Y, Ogasawara S, Oki H, Honma R, Takagi M, Fujii Y, et al. (2016) Novel monoclonal antibody
LpMab-17 developed by CasMab technology distinguishes human podoplanin from monkey podoplanin. Monoclon Antib Immunodiagn Immunother.

24.

Kato Y (2014) Specific monoclonal antibodies against IDH1/2 mutations as diagnostic tools for gliomas.
Brain Tumor Pathol 32: 3–11. doi: 10.1007/s10014-014-0202-4 PMID: 25324168

25.

Oki H, Ogasawara S, Kaneko MK, Takagi M, Yamauchi M, Kato Y (2015) Characterization of monoclonal antibody LpMab-3 recognizing sialylated glycopeptide of podoplanin. Monoclon Antib Immunodiagn
Immunother 34: 44–50. doi: 10.1089/mab.2014.0087 PMID: 25723283

26.

Kaneko MK, Oki H, Hozumi Y, Liu X, Ogasawara S, Takagi M, et al. (2015) Monoclonal antibody
LpMab-9 recognizes O-glycosylated N-terminus of human podoplanin. Monoclon Antib Immunodiagn
Immunother 34: 310–317. doi: 10.1089/mab.2015.0022 PMID: 26492618

27.

Honma R, Fujii Y, Ogasawara S, Oki H, Liu X, Nakamura T, et al. (2016) Establishment of a novel
monoclonal antibody PMab-32 against rabbit podoplanin. Monoclon Antib Immunodiagn Immunother
35: 41–47. doi: 10.1089/mab.2015.0066 PMID: 26788987

28.

Honma R, Fujii Y, Ogasawara S, Oki H, Konnai S, Kagawa Y, et al. (2016) Critical Epitope of Anti-Rabbit Podoplanin Monoclonal Antibodies for Immunohistochemical Analysis Monoclon Antib Immunodiagn Immunother.

PLOS ONE | DOI:10.1371/journal.pone.0152912 March 31, 201612 / 13

Monoclonal Antibody against Thr52 of Human Podoplanin29.

Kaneko MK, Oki H, Ogasawara S, Takagi M, Kato Y (2015) Anti-podoplanin monoclonal antibody
LpMab-7 detects metastatic legions of osteosarcoma. Monoclon Antib Immunodiagn Immunother 34:
154–161. doi: 10.1089/mab.2014.0091 PMID: 26090592

30.

Ogasawara S, Kaneko MK, Honma R, Oki H, Fujii Y, Takagi M, et al. (2016) Establishment of Mouse
Monoclonal Antibody LpMab-13 against Human Podoplanin Monoclon Antib Immunodiagn Immunother.

31.

Ogasawara S, Kaneko MK, Price JE, Kato Y (2008) Characterization of anti-podoplanin monoclonal
antibodies: critical epitopes for neutralizing the interaction between podoplanin and CLEC-2. Hybridoma 27: 259–267. doi: 10.1089/hyb.2008.0017 PMID: 18707544

32.

Sekiguchi T, Takemoto A, Takagi S, Takatori K, Sato S, Takami M, et al. (2015) Targeting a novel
domain in podoplanin for inhibiting plateletmediated tumor metastasis. Oncotarget.

33.

Kaji C, Tsujimoto Y, Kato Kaneko M, Kato Y, Sawa Y (2012) Immunohistochemical Examination ofNovel Rat Monoclonal Antibodies against Mouse and Human Podoplanin. Acta Histochem Cytochem
45: 227–237. PMID: 23012488

34.

Oki H, Honma R, Ogasawara S, Fujii Y, Liu X, Kaneko MK, et al. (2015) Development of a sensitive
monoclonal antibody PMab-2 against rat podoplanin. Monoclon Antib Immunodiagn Immunother 34:
396–403. doi: 10.1089/mab.2015.0041 PMID: 26683179

35.

Matsui K, Breiteneder-Geleff S, Kerjaschki D (1998) Epitope-specific antibodies to the 43-kD glomerular membrane protein podoplanin cause proteinuria and rapid flattening of podocytes. J Am Soc
Nephrol 9: 2013–2026. PMID: 9808087

36.

Oki H, Kaneko MK, Ogasawara S, Tsujimoto Y, Liu X, Sugawara M, et al. (2015) Characterization of a
monoclonal antibody LpMab-7 recognizing non-PLAG domain of podoplanin. Monoclon Antib Immunodiagn Immunother 34: 174–180. doi: 10.1089/mab.2014.0090 PMID: 26090595

37.

Ogasawara S, Oki H, Kaneko MK, Hozumi Y, Liu X, Honma R, et al. (2015) Development of monoclonal
antibody LpMab-10 recognizing non-glycosylated PLAG1/2 domain including Thr34 of human podoplanin. Monoclon Antib Immunodiagn Immunother 34: 318–326. doi: 10.1089/mab.2015.0018 PMID:
26492619

38.

Kato Y, Kunita A, Abe S, Ogasawara S, Fujii Y, Oki H, et al. (2015) The chimeric antibody chLpMab-7
targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC rather than via its
neutralizing activity. Oncotarget 6: 36003–36018. doi: 10.18632/oncotarget.5339 PMID: 26416352

39.

Kaneko M, Kato Y, Kunita A, Fujita N, Tsuruo T, Osawa M (2004) Functional sialylated O-glycan toplatelet aggregation on Aggrus (T1alpha/podoplanin) molecules expressed in Chinese Hamster Ovarycells. J Biol Chem 279: 38838–38843. PMID: 15231832

PLOS ONE | DOI:10.1371/journal.pone.0152912 March 31, 201613 / 13