MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 34, Number 5, 2015ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2015.0018

Development of Monoclonal Antibody LpMab-10
Recognizing Non-glycosylated PLAG1/2
Domain Including Thr34 of Human Podoplanin
Satoshi Ogasawara,1* Hiroharu Oki,1,2* Mika K. Kaneko,1* Yasukazu Hozumi,3 Xing Liu,1,2
Ryusuke Honma,1,2 Yuki Fujii,1 Takuro Nakamura,1 Kaoru Goto,3 Michiaki Takagi,2 and Yukinari Kato1

Podoplanin (PDPN) is a type-I transmembrane sialoglycoprotein that possesses a platelet aggregationstimulating (PLAG) domain in the N-terminus. PLAG domain includes three tandem repeats of eight aminoacids: PLAG1, PLAG2, and PLAG3. Among the three PLAG domains, O-glycan on Thr52 of PLAG3 is criticalfor binding with C-type lectin-like receptor-2 (CLEC-2) and is essential for platelet-aggregating activity of
PDPN. In contrast, the glycosylation of Thr34 of PLAG1 of human PDPN remains to be clarified. Herein, wedeveloped and characterized a novel anti-PDPN monoclonal antibody, LpMab-10, which targets PLAG1/2
domain. LpMab-10 detects endogenous PDPN of cancer cells and normal cells independently of glycosylation.
The minimum epitope of LpMab-10 was identified as Glu33–Gly45 of PDPN using Western blot and flow
cytometry. The Thr34 of PLAG1 is critical for LpMab-10 recognition, and O-glycan is not included in LpMab10 epitope, indicating that Thr34 of PLAG1 is not O-glycosylated. In immunocytochemical and immunohistochemical analyses, LpMab-10 strongly detected PDPN-expressing tumor cells. By using monoclonal antibodies
against different Ser/Thr, including epitopes of PDPN, it becomes possible to determine whether Ser/Thr residuesof PDPN are O-glycosylated.
IntroductionP

odoplanin (PDPN/Aggrus), a platelet aggregationinducing type-I transmembrane sialoglycoprotein, is highly
expressed not only in cancer cells but also in normal tissues.(1,2)
PDPN binds to CLEC-2 and rearranges the actin cytoskeleton
in dendritic cells to promote efficient motility along stromal
surfaces.(3) The signal from CLEC-2 to PDPN controls thecontractility of fibroblastic reticular cells and lymph node
microarchitecture.(4) The physical elasticity of lymph nodes
is maintained by PDPN of stromal fibroblastic reticular
cells.(5) For embryonic blood-lymphatic vascular separation,
PDPN-CLEC-2 interaction is also important.(6) PDPN is also
very useful for detecting lymphatic endothelial cells.(7) The
development of ectopic lymphoid follicles is dependent on
Th17-expressing PDPN.(8) Expression of PDPN has beenreported in many tumors including malignant brain tumors,
mesotheliomas, oral cancer, lung cancer, esophageal cancer,
testicular tumors, and osteosarcoma.(9–22) To study thesecomplicated functions of PDPN, different epitope-possessing
anti-PDPN monoclonal antibodies (MAbs) are necessary
because site-specific glycosylation is observed in patho-

logical conditions. Almost all anti-PDPN MAbs react withplatelet aggregation-inducing (PLAG) domain of human
PDPN.(23) Rabbit polyclonal antibodies were previously
produced by Matsui and colleagues and also recognized
PLAG domain because PLAG domain was shown to be
immunodominant antigenic sites.(24) Recently, we established the platform to produce cancer-specific MAbs (CasMabs).(25) Using CasMab methods, we established different
epitope-possessing anti-PDPN MAbs such as LpMab-2,
LpMab-3, LpMab-7, and LpMab-9.(25–28) Interestingly,
LpMab-2, LpMab-3, and LpMab-9 detect site-specific Oglycosylation: LpMab-2 against Thr55/Ser56, LpMab-3
against Thr76, and LpMab-9 against Thr25. In this study,
we developed and characterized a novel anti-PDPN MAb,
LpMab-10, which possesses a novel epitope of PDPN.
Materials and Methods
Cell lines, animals, and tissues

LN229, NCI-H226, HEK-293T, Chinese hamster ovary(CHO)-K1, glycan-deficient CHO cell lines (Lec1, Lec2, andLec8), Met-5A, and P3U1 were obtained from the American

1

Department of Regional Innovation, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.
Department of Orthopaedic Surgery, 3Department of Anatomy and Cell Biology, Yamagata University Faculty of Medicine, Yamagata,Japan.
*These authors contributed equally to this work.
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LpMab-10 AGAINST NON-GLYCOSYLATED PLAG1/2 DOMAIN

Type Culture Collection (ATCC, Manassas, VA). Humanlymphatic endothelial cells (LEC) were purchased from
Cambrex (Walkersville, MD). The human glioblastoma cellline LN319 was donated by Dr. Webster K. Cavenee (Ludwig
Institute for Cancer Research, San Diego, CA). PC-10 cells
were purchased from Immuno-Biological Laboratories(Gunma, Japan). LN229, CHO-K1, Lec1, Lec2, and Lec8
were transfected with human PDPN plasmids (LN229/
hPDPN, CHO/hPDPN, Lec1/hPDPN, Lec2/hPDPN, Lec8/
hPDPN) using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA), according to the manufacturer’s instructions.(25) LN319/PDPN-knock out (KO) cells were
produced by transfecting CRISPR/Cas plasmids, which targets PDPN (Sigma-Aldrich, St. Louis, MO), using a Gene
Pulser Xcell electroporation system (Bio-Rad Laboratories,
Philadelphia, PA). CHO-K1, Lec1, Lec2, Lec8, NCI-H226,
PC-10, and P3U1 were cultured in RPMI 1640 medium(Nacalai Tesque, Kyoto, Japan), and LN229, LN319, andMet-5A were cultured in Dulbecco’s Modified Eagle’s
Medium (DMEM) medium (Nacalai Tesque), supplemented
with 10% heat-inactivated fetal bovine serum (FBS; Thermo
Fisher Scientific), 2 mM L-glutamine (Thermo Fisher Scientific), 100 U/mL of penicillin, and 100 mg/mL of streptomycin (Thermo Fisher Scientific) at 37!C in a humidified
atmosphere of 5% CO2 and 95% air. L-proline (0.04 mg/mL)
was added for Lec1, Lec2, and Lec8. LEC was cultured inendothelial cell medium EGM-2MV supplemented with 5%
FBS (Cambrex). MITO + serum extender (Thermo Fisher
Scientific) was added for Met-5A cells. Female BALB/c mice
(4 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.
This study examined one seminoma patient(11) and one osteosarcoma patient(26) who underwent surgery at Yamagata
University Hospital. The ethical committee of the Yamagata
University Faculty of Medicine approved our study. Informed
consent for obtaining samples and for subsequent data analyses was obtained from patients or the patient’s guardian.
Hybridoma production

BALB/c mice were immunized by intraperitoneal (i.p.)
injection of 1 · 108 LN229/hPDPN cells together with Imject
Alum (Thermo Fisher Scientific). After several additional
immunizations, a booster injection was given i.p. 2 days
before spleen cells were harvested. The spleen cells werefused with P3U1 cells using PEG1500 (Roche Diagnostics,
Indianapolis, IN). The hybridomas were grown in RPMI
medium with hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo Fisher Scientific). The
culture supernatants were screened using enzyme-linked
immunosorbent assay (ELISA) for binding to recombinanthuman PDPN purified from LN229/hPDPN cells.
Enzyme-linked immunosorbent assay

Purified proteins or peptides were immobilized on Nunc
Maxisorp 96-well immunoplates (Thermo Fisher Scientific)
at 1 mg/mL for 30 min. After blocking with SuperBlock T20(PBS) Blocking Buffer (Thermo Fisher Scientific), the plates
were incubated with culture supernatant or purified MAbs
(1 mg/mL) followed by 1:1000 diluted peroxidase-conjugated319

anti-mouse IgG or anti-rat IgG (Dako, Glostrup, Denmark).
The enzymatic reaction was conducted with a 1-Step Ultra
TMB-ELISA (Thermo Fisher Scientific). The optical density
was measured at 655 nm using an iMark microplate reader(Bio-Rad Laboratories). These reactions were performed
with a volume of 50 mL at 37!C.
Western blot analyses

Cell lysates (10 mg) were boiled in SDS sample buffer(Nacalai Tesque). The proteins were electrophoresed on 5–
20% polyacrylamide gels (Wako Pure Chemical Industries,
Osaka, Japan) and were transferred onto a PVDF membrane(EMD Millipore, Billerica, MA). After blocking with SuperBlock T20 (PBS) Blocking Buffer, the membrane was incubated with primary antibodies (1 mg/mL) and then withperoxidase-conjugated secondary antibodies (Dako; 1:1000
diluted), and developed with the Pierce Western Blotting
Substrate Plus (Thermo Fisher Scientific) using a SayacaImager (DRC, Tokyo, Japan).
Flow cytometry

Cell lines were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA (Nacalai Tesque).(29) After washing withphosphate buffered saline (PBS, Nacalai Tesque), the cells weretreated with primary antibodies (1 mg/mL) for 30 min at 4!C,
followed by treatment with Oregon Green 488 goat anti-mouse
IgG (Thermo Fisher Scientific). Fluorescence data were collected using a Cell Analyzer EC800 (Sony Corp., Tokyo, Japan).
Determination of binding affinity using flow cytometry

LN319 and PC-10 (2 · 105 cells) were resuspended with
100 mL of serially diluted antibody (0.02–100 mg/mL) followed by secondary anti-mouse IgG (Thermo Fisher Scientific). Fluorescence data were collected using the EC800 cell
analyzer. The dissociation constants (KD) were obtained byfitting the binding isotherms using the built-in one-sitebinding models in Prism software.
Lectin microarray

PDPN from CHO/hPDPN cells was solubilized using 1%
Triton-X100 in PBS (PBST) and was purified using a FLAGtag system (Sigma-Aldrich). Then, 100 mL of purified PDPN
(31.25–2000 ng/mL) was applied to a lectin array (LecChip,
v1.0; GlycoTechnica, Hokkaido, Japan), including triplicate
spots of 45 lectins in each of seven divided incubation bathson the glass slide. After incubation at 20!C for 17 h, 4 mL ofhuman IgG (5 mg/mL; Sigma-Aldrich) was applied to eachwell. After incubation at 20!C for 30 min, the glass slide waswashed three times with PBST; 60 mL of biotinylated LpMab10 (1 mg/mL) in PBS was applied to the array and then incubated at 20!C for 3 h. After washing three times with PBST,
Cy3-labeled streptavidin (Thermo Fisher Scientific) was added to the array and incubated at 20!C for 30 min. The glass
slide was scanned using a GlycoStation Reader 1200 (GlycoTechnica). Abbreviation of lectins: LTL, Lotus tetragolonobus lectin; PSA, Pisum sativum agglutinin; LCA, Lens
culinaris agglutinin; UEA I, Ulex europaeus agglutinin I;
AOL, Aspergillus oryzae L-fucose-specific lectin; AAL,
Aleuria aurantia lectin; MAL I, Maackia amrensis lectin I;
SNA, Sambucus nigra lectin; SSA, Sambucus sieboldiana

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agglutinin; TJA-I, Trichosanthes japonica agglutinin I; PHAL, Phaseolus vulgaris leucoagglutinin; ECA, Erythrina cristagalli agglutinin; RCA120, Ricinus communis agglutinin
120; PHA-E, Phaseolus vulgaris erythroagglutinin; DSA,
Datura stramonium agglutinin; GSL-II, Griffonia simplicifolia lectin II; NPA, Narcissus pseudonarcissus agglutinin;
ConA, Concanavalin A; GNA, Galanthus nivalis agglutinin;
HHL, Hippeastrum hybrid lectin; ACG, Agrocybe cylindricea
galectin; TxLCI, Tulipa gesneriana lectin; BPL, Bauhinia
purpurea alba lectin; TJA-II, Trichosanthes japonica agglutinin; EEL, Euonymus europaeus lectin; ABA, Agaricus bisporus agglutinin; LEL, Lycopersicon esculentum lectin; STL,
Solanum tuberosum lectin; UDA, Urtica dioica agglutinin;
PWM, pokeweed mitogen; PNA, peanut agglutinin; WFA,
Wisteria floribunda agglutinin; ACA, Amaranthus caudatus
agglutinin; MPA, Maclura pomifera agglutinin; HPA, Helix
pomatia agglutinin; VVA, Vicia villosa agglutinin; DBA,
Dolichos biflorus agglutinin; SBA, soybean agglutinin; PTL I,
Psophocarpus tetragonolobus lectin I; MAH, Maackia
amurensis hemagglutinin; WGA, wheat germ agglutinin;
GSL-I, Griffonia simplicifolia lectin I.
Production of PDPN mutants

The amplified human PDPN cDNA was subcloned into a
pcDNA3 vector (Thermo Fisher Scientific), and a FLAG
epitope tag was added at the C-terminus. Substitution ofamino acids to alanine or glycine in PDPN was performed
using a QuikChange Lightning site-directed mutagenesis kit(Agilent Technologies, Santa Clara, CA). CHO-K1 cells
were transfected with the plasmids using a Gene Pulser Xcell
electroporation system (Bio-Rad Laboratories).
Immunocytochemical analyses

Cultured cells were fixed with 4% paraformaldehyde in
0.1 M phosphate buffer (pH 7.4) for 15 min at room temperature. Cells were then treated with 10% normal goat serum
in PBS (NGS/PBS) to block nonspecific binding sites, andwere incubated with 1 mg/mL of LpMab-10 or control (PBS)
for 1 h at 4!C in a moist chamber. They were incubated withgoat anti-mouse IgG-Alexa 488 (dilution 1:200; Thermo
Fisher Scientific) for 30 min at room temperature. Cells werealso treated with DAPI (Thermo Fisher Scientific) to stain thecell nuclei. They were examined using confocal laserscanning microscopy (LSM700; Carl Zeiss, Jena, Germany).
Immunohistochemical analyses

Four-mm-thick histologic sections were deparaffinized inxylene and rehydrated. They were then autoclaved in citratebuffer (pH 6.0; Dako) for 20 min. Sections were incubatedwith 5 mg/mL of primary antibodies overnight at 4!C followed by treatment with Envision+ kit (Dako). Color wasdeveloped using 3,3-diaminobenzidine tetrahydrochloride(DAB, Dako) for 10 min; then the sections were counterstained with hematoxylin (Wako Pure Chemical Industries).
Results
Development and characterization of novelanti-PDPN MAb LpMab-10

We immunized mice with LN229/hPDPN cells to develop
novel anti-PDPN MAbs. Using ELISA, the culture superna-

OGASAWARA ET AL.

tants were screened for binding to recombinant human PDPN
purified from LN229/hPDPN cells. After the limiting dilution, LpMab-10 (mouse IgG3, kappa) was established.
LpMab-10 reacted with LN229/hPDPN, not with LN229, a
PDPN-negative cell in Western blot analysis (Fig. 1A).
LpMab-10 also recognized endogenous PDPN, which is expressed in a glioblastoma cell line LN319 and a lymphatic
endothelial cell (LEC) in Western blot analysis (Fig. 1A) andflow cytometry (Fig. 1B). Although LpMab-10 did not reactwith a malignant mesothelioma cell line NCI-H226 andHEK-293T in Western blot analysis (Fig. 1A), it strongly
recognized both cells in flow cytometry (Fig. 1B), indicatingthat LpMab-10 binds to native PDPN effectively. LN229 and
CHO-K1, which do not express PDPN, were not detected by
LpMab-10, indicating that LpMab-10 does not recognize
non-related antigens in native condition (Fig. 1B). LpMab10 also detects PDPN of a lung squamous cell carcinoma(SCC) cell line PC-10 and a normal mesothelial cell Met-5A(Fig. 1B), indicating that LpMab-10 is useful for detecting
PDPN of both cancer cells and normal cells. We also performed Western blot analysis using LpMab-10 against
several glycan-deficient PDPN transfectants, such as Lec1/
hPDPN (N-glycan deficient), Lec2/hPDPN (sialic acid deficient), and Lec8/hPDPN (O-glycan deficient) (Fig. 1A).
All transfectants of glycan-deficient PDPN were detected by
LpMab-10, indicating that the epitope of LpMab-10 is independent of glycan. To investigate whether LpMab-10 reacts with synthetic peptides, we performed ELISA using
several synthetic peptides of human PDPN (Fig. 1C). NZ-1
reacted with PLAG2/3 domain that corresponds to 38–51
amino acids of human PDPN (hpp38–51). In contrast,
LpMab-10 strongly recognized PLAG1/2 domain that corresponds to 29–47 amino acids of human PDPN (hpp29–47).
We next performed a kinetic analysis of the interaction of
LpMab-10 with LN319 and PC-10 using flow cytometry. As
shown in Figure 1D, KD was determined to be 8.755 · 10-9
M using LN319 and 9.830 · 10-9 M using PC-10, indicatingthat LpMab-10 possesses high affinity against human PDPN.
We further performed antibody-overlay lectin microarray
analysis of PDPN using LpMab-10 (Fig. 1E). PDPN strongly
reacted with a a 2–3 sialic acid binder ACG, a sialic acid –core1 binder Jacalin, and a sialo-mucin binder WGA,
moderately reacted with a core1 – sialic acid binder MPA,
and faintly reacted with a poly LacNAc binder STL, indicating that LpMab-10 is useful in antibody-overlay lectin
microarray analysis and PDPN possesses sialylated core1
structure.
Immunocytochemistry and immunohistochemistry

We next investigated whether LpMab-10 is useful for
immunocytochemical and immunohistochemical analyses.
LpMab-10 clearly stained the membrane of CHO/hPDPN,
whereas it did not react with CHO-K1 cells (Fig. 2). LpMab10 also stained endogenous PDPN expressed on LN319
cells, whereas it did not react with LN319/PDPN knock-out
cells, indicating that LpMab-10 specifically recognizes
human PDPN. As shown in Figure 3, LpMab-10 stained
seminoma cells (Fig. 3C, D) and osteosarcoma cells (Fig. 3G,H) in a membranous/cytoplasmic staining pattern, indicatingthat LpMab-10 is very useful in immunohistochemical
analysis.

FIG. 1. Characterization of a novel anti-PDPN MAb, LpMab-10. (A) Western blot analysis by LpMab-10 and NZ-1. AC15 (anti-b-actin) was used as internal control. Total cell lysate were electrophoresed on 5–20% polyacrylamide gels andtransferred onto a PVDF membrane. After blocking, the membrane was incubated with 1 mg/mL of primary antibodies andthen with peroxidase-conjugated secondary antibodies; the membrane was detected using a Sayaca-Imager. (B) Flowcytometric analysis by LpMab-10. Eight cell lines were treated with LpMab-10 (1 mg/mL; red) or control PBS (black) for
30 min at 4!C, followed by treatment with anti-mouse IgG-Oregon green. Fluorescence data were collected using a Cell
Analyzer EC800. (C) Epitope mapping of LpMab-10 and NZ-1 using ELISA. Peptides were immobilized at 1 mg/mL for
30 min. After blocking, the plates were incubated with LpMab-10 or NZ-1 (1 mg/mL) followed by 1:1000 diluted
peroxidase-conjugated anti-mouse IgG or anti-rat IgG. The enzymatic reaction was conducted with a 1-Step Ultra TMBELISA. Black bar, positive; white bar, negative. P1, PLAG1; P2, PLAG2; P3, PLAG3; TM, transmembrane. (D) Determination of binding-affinity of LpMab-10 using flow cytometry. (E) Glycan profiling of PDPN using antibody-overlay
lectin microarray. Purified PDPN was applied to a lectin array including 45 lectins. After blocking, biotinylated LpMab-10
was applied and Cy3-labeled streptavidin was added. Signal was scanned using a GlycoStation Reader 1200.
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OGASAWARA ET AL.

FIG. 2. Immunocytochemical analysis. Cells were incubated with 1 mg/mL of LpMab-10 for 1 h at 4!C in a moist
chamber. They were incubated with goat anti-mouse IgG-Alexa 488 for 30 min at room temperature. Cells were also treatedwith DAPI to stain the cell nuclei. Scale bars, 20 mm.
Epitope-mapping Western blot analysis
and flow cytometry

The epitope of LpMab-10 was shown to be PLAG1/2 in
ELISA (Fig. 1C); therefore, we next performed Western blot
analysis to determine the LpMab-10 epitope in details.
Western blot analysis showed that LpMab-10 reaction was
lost in point mutations of 33–45 amino acids (Fig. 4A). Flowcytometric analysis revealed that LpMab-10 did not reactwith L37A and E38A; weakly recognized E33A, T34A, andP44A; moderately reacted with G39A, G40A, V41A, andG45A, indicating that Leu37 and Glu38 are much more important for LpMab-10 (Fig. 4B). Taken together, the PDPN
epitope of LpMab-10 is Glu33-Gly45, and Leu37 and Glu38
are the most important amino acids.
Discussion

Until now, ours and other groups have developed manyanti-PDPN MAbs. To neutralize platelet aggregation activity
by blocking the association between PDPN and CLEC-2, we
produced NZ-1 MAb by immunizing rats with synthetic
peptides of PLAG domain.(15) NZ-1 possesses very highbinding affinity, which was clarified by several methods including ELISA, flow cytometry, Scatchard analysis, andBIAcore.(21,30) NZ-1 inhibited cancer-induced platelet aggregation and experimental metastasis by its neutralizing
activity, indicating that NZ-1 is a candidate of anti-metastatic
MAb.(15,29,31) In addition, NZ-1 was highly internalized into
glioma cells and also accumulated efficiently into tumors
in vivo.(21) NZ-1 and its rat-human chimeric anti-PDPN antibody (NZ-8) possess antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity(CDC) against PDPN-expressing glioblastoma or malignant
mesothelioma cells.(18,31) Therefore, NZ-1 is known to be an
extremely useful anti-PDPN MAb. In contrast, rat MAbs areoften avoided in immunohistochemistry, especially in pathological diagnosis, because almost all secondary antibodies
or commercially available kits for immunohistochemistry
have been developed against mouse MAbs or polyclonal
antibodies, although many rat MAbs possess high-binding
affinity and specificity. LpMab-10 shows the same reaction
pattern with NZ-1 in Western blot analysis (Fig. 1A) and flow
cytometry (Fig. 1B), demonstrating that LpMab-10 could
possess more advantages compared with NZ-1 in several

LpMab-10 AGAINST NON-GLYCOSYLATED PLAG1/2 DOMAIN

323

FIG. 3. Immunohistochemical analysis by LpMab-10 against seminoma (A–D) and osteosarcoma (E, F). Sections wereincubated with 5 mg/mL LpMab-10, followed by Envision+ kit, and color was developed using DAB and counterstainedwith hematoxylin (C, D, G, H). The serial sections were also stained by hematoxylin and eosin (A, B, E, F). Original
magnification: ·100 (A, C, E, G); ·400 (B, D, F, H). Scale bar, 100 mm.

applications. Indeed, we used Dako Envision+ kit in immunohistochemistry, which is not applied for rat MAbs, indicating LpMab-10 is more advantageous than NZ-1.
Figure 4C shows that LpMab-10 epitope is independent ofpreviously established anti-PDPN MAbs such as LpMab-2,
LpMab-3, LpMab-7, LpMab-9, and NZ-1.(25,27,28,32,33)
Furthermore, the minimum epitope of LpMab-10 is ETTGLEGGVAMPG, which consists of 13 amino acids, dem-

onstrating that LpMab-10 epitope is longer than those ofanti-PDPN MAbs. As we previously reported, Thr34 ofmouse, rat, and hamster PDPNs might be O-glycosylated
and shows the critical role for platelet aggregation.(34) Although we also demonstrated that Thr34 of human PDPN
might not be glycosylated, and is not involved in plateletaggregating activity,(35) the possibility of glycosylation of
Thr34 was suggested in platelet aggregation and in a tumor

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OGASAWARA ET AL.

FIG. 4. Epitope mapping of LpMab-10 using flow cytometry and Western blot analysis. (A) Total cell lysate of point
mutants of human PDPN were electrophoresed on 5–20% polyacrylamide gels and transferred onto a PVDF membrane.
After blocking, the membrane was incubated with 1 mg/mL of primary antibodies and then with peroxidase-conjugatedsecondary antibodies; the membrane was detected using a Sayaca-Imager. (B) Flow cytometric analysis by LpMab-10.
Point mutants of human PDPN were treated with LpMab-10 (1 mg/mL) for 30 min at 4!C, followed by treatment with antimouse IgG-Oregon green. Fluorescence data were collected using a Cell Analyzer EC800. (C) Comparison of epitopes ofanti-PDPN MAbs. Minimum epitopes of LpMab-9, LpMab-10, NZ-1, LpMab-2, LpMab-3, and LpMab-7 are TGQPED,ETTGLEGGVAMPG, MPGAEDD, TSEDRYKSGL, VTGIRIE, and RIEDL, respectively. The residues of Thr25, Thr55,
Ser56, and Thr76 were determined to be O-glycosylated using LpMab-9, LpMab-2, and LpMab-3, respectively. Oglycosylated Thr52 was detected by CLEC-2.
metastasis model in another study.(22) It was also suggested
that O-glycosylation sites are predicted by the NetOGlyc
prediction tool, and Thr32, Thr34, and Thr35 of PLAG1
might be O-glycosylated.(36) These studies did not investigate the O-glycosylation directly because there was no tool
to detect the O-glycosylation of each Thr. As shown in
Figure 4C, we have produced several site-specific MAbs

against each O-glycosylated Ser/Thr of human PDPN;
therefore, we can directly detect O-glycosylation of Thr25,
Thr34, Thr52, Thr55, Ser56, and Thr76. Because Thr34 of
PLAG1 is critical for LpMab-10 recognition (Fig. 4A, B),
and O-glycan is not included in LpMab-10 epitope (Fig.
1A), it is more likely that Thr34 of PLAG1 is not Oglycosylated.

LpMab-10 AGAINST NON-GLYCOSYLATED PLAG1/2 DOMAIN

In conclusion, LpMab-10 could be advantageous for investigating expression and function of PDPN in cancers andnormal tissues. By using MAbs against different Ser/Thrincluding epitopes of human PDPN, we can determine
whether Ser/Thr residues of PDPN are O-glycosylated. Further, different epitope-possessing anti-PDPN MAbs shouldbe established as powerful tools for investigating function of
PDPN in cancers and normal tissues in the future.
Acknowledgments

We thank Noriko Saidoh and Kanae Yoshida for their excellent technical assistance. This work was supported in partby the Platform for Drug Discovery, Informatics, and Structural Life Science (PDIS) from Japan Agency for Medical
Research and Development from AMED (Y.K.); by the Basic
Science and Platform Technology Program for Innovative
Biological Medicine from AMED (Y.K.); by the Regional
Innovation Strategy Support Program from the Ministry of
Education, Culture, Sports, Science and Technology (MEXT)
of Japan (Y.K.); and by JSPS KAKENI Grant Number
25462242 (Y.K.) and Grant Number 26440019 (M.K.K.).
Author Disclosure Statement

The authors have no financial interests to disclose.
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Address correspondence to:
Yukinari Kato
Department of Regional Innovation
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi
Aoba-ku, Sendai
Miyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp
Received: April 6, 2015
Accepted: June 12, 2015