MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 39, Number 1, 2020
Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0045

Epitope Analysis of an Anti-Whale Podoplanin Monoclonal
Antibody, PMab-237, Using Flow Cytometry
Yusuke Sayama,1 Masato Sano,1 Mika K. Kaneko,1 and Yukinari Kato1,2

Podoplanin (PDPN) is a small mucin-type transmembrane glycoprotein, which was first discovered in podocytes of
the kidney. PDPN is a specific lymphatic endothelial marker and is also known as T1alpha, a marker of lung type I
alveolar cells, or Aggrus, a platelet aggregation-inducing factor. PDPN possesses three platelet aggregationstimulating (PLAG) domains and PLAG-like domains (PLDs), which bind to C-type lectin-like receptor-2.
Previously, we developed a novel anti-whale PDPN (wPDPN) monoclonal antibody (mAb) PMab-237 using the CellBased Immunization and Screening (CBIS) method and the RIEDL tag of Arg-Ile-Glu-Asp-Leu sequence. PMab-237
detected wPDPN by flow cytometry, western blot, and immunohistochemical analyses. However, the specific binding
epitope of PMab-237 for wPDPN remains unknown. In this study, deletion mutants and point mutants of wPDPN
with N-terminal RIEDL tag were produced to analyze the PMab-237 epitope using flow cytometry. The analysis ofdeletion mutants showed that the N-terminus of the PMab-237 epitope exists between the 80th amino acid (AA) andthe 85th AA of wPDPN. In addition, the analysis of point mutants demonstrated that the critical epitope of PMab-237
includes Leu82 and Thr84 of wPDPN, indicating that the PMab-237 epitope is located in the PLD of wPDPN.
Keywords: whale, podoplanin, monoclonal antibody, epitope, PMab-237
critical role in a network of immunofibroblasts, which cansupport the earliest phases of tertiary lymphoid structure
establishment.(12) The expression of PDPN in chorionic villous stromal cells is in two important placental pathologies:
preeclampsia and hydatidiform mole.(13) Moreover, PDPN
is upregulated in many cancers and is involved in cancer
metastasis and malignant progression.(14–17) Recent reports
showed that PDPN is related to a progression in oral epithelial dysplasia and oral squamous cell carcinoma through a
co-expression with sex-determining region Y-related Homeobox gene 2.(18,19) Therefore, PDPN possesses many pathophysiological functions in malignant tissues.
Recently, we developed a novel anti-whale PDPN (wPDPN)
monoclonal antibody (mAb), PMab-237, using the Cell-Based
Immunization and Screening (CBIS) method.(20) The CBIS
method was established in our previous study(21) to produce
mAbs using cell lines for immunization and screening.
PMab-237 specifically detected wPDPN by flow cytometry,western blotting, and immunohistochemical analyses. PMab237 also strongly stained pulmonary type I alveolar cells,
renal podocytes, and lymphatic endothelial cells of the harbor
porpoise by the immunohistochemical analysis.(22) However,the binding epitope of PMab-237 for wPDPN remains unknown. This study aimed to identify the epitope of PMab-237

IntroductionP

odoplanin (PDPN)/T1alpha/Aggrus/PA2.26 is a
type I transmembrane sialoglycoprotein consisting of aheavily glycosylated extracellular domain, a single transmembrane, and a short nine amino acid (AA) cytoplasmic tail.(1–4)
PDPN/Aggrus possesses the EDxxVTPG sequence at its
N-terminus, which is known to be the platelet aggregationstimulating (PLAG) domains (PLAG1, PLAG2, and
PLAG3).(3,5) In addition, the PLAG-like domain (PLD) of theE(D/E)xx(T/S)xx sequence, also known as PLAG4, has beenreported to be present in the middle of PDPN.(6,7) PLAG
domains are highly conserved among mammalian PDPNs.(7)PDPN is expressed in lymphatic endothelial cells and is
not expressed in vascular endothelial cells.(8) The interaction
between PDPN(3) on lymphatic endothelial cells and C-type
lectin-like receptor-2 on platelets was shown to facilitate
embryonic blood/lymphatic vessel separation.(9) Because
PDPN/T1alpha is expressed in type I alveolar cells but not intype II alveolar cells, it is used as a specific marker of type I
alveolar cells.(10) In recent studies, other functions of PDPN
were reported. The PDPN-positive cells, with the immune
cells after myocardial infarction, positively affect immune
cell recruitment.(11) The PDPN-positive stromal cells play a1
2

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.

ª Yusuke Sayama et al. 2020; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of theCreative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.

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SAYAMA ET AL.

FIG. 1. Schematic illustration of deletion mutants of anti-wPDPN. Illustration of WT and 10 deletion mutants of wPDPN:
dN30, dN40, dN50, dN60, dN70, dN80, dN85, dN90, dN95, and dN100. Black bars: the positive reaction of PMab-237.
White bars: the negative reaction of PMab-237. PLAG, platelet aggregation-stimulating; PLD, PLAG-like domain; wPDPN,
whale podoplanin; WT, wild type.
through flow cytometry using the deletion mutants and point
mutants of wPDPN.
Materials and Methods
Production of wPDPN mutants

Synthesized DNA encoding wPDPN was subcloned into
the pCAG vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and an N-terminal RIEDL tag wasadded.(22) The RIEDL tag was derived from the five AA

sequences (Arg-Ile-Glu-Asp-Leu) of human PDPN, which
was detected by clone LpMab-7.(23) Deletion mutants of the
wPDPN sequence were produced using a HotStar HiFidelity
Polymerase Kit (Qiagen, Inc., Hilden, Germany) with oligonucleotides. Substitutions of AAs to alanine in the wPDPN
sequence were conducted by QuikChange Lightning SiteDirected Mutagenesis Kits (Agilent Technologies, Inc., Santa
Clara, CA). PCR fragments bearing the desired mutations
were inserted into the pCAG vector using an In-Fusion HD
Cloning Kit (Takara Bio, Inc., Shiga, Japan).

FIG. 2. Epitope mapping of PMab-237 using deletion mutants of wPDPN. Deletion mutants of wPDPN were analyzed
using flow cytometry. Deletion mutants were incubated with LpMab-7 (anti-RIEDL tag; red line, A), PMab-237 (antiwPDPN mAb; red line, B), or buffer control (black line, A, B) for 30 minutes at 4C, followed by secondary antibodies.
mAb, monoclonal antibody.

EPITOPE OF PMAB-237
Cell lines and culture condition

Chinese hamster ovary (CHO)-K1 was obtained fromthe American Type Culture Collection (Manassas, VA). The
wPDPN mutation plasmids containing the RIEDL tag weretransfected into CHO-K1 cells using Lipofectamine LTX(Thermo Fisher Scientific, Inc., Waltham, MA). Transiently
transfected cells with deletion mutants or point mutants were
cultured in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto,
Japan), supplemented with 10% heat-inactivated fetal bovine
serum (FBS) (Thermo Fisher Scientific, Inc.), 100 U/mL of
penicillin, 100 mg/mL of streptomycin, and 25 mg/mL of amphotericin B (Nacalai Tesque, Inc.) at 37C in a humidified
atmosphere of 5% CO2 and 95% air.
Flow cytometry

Transiently transfected CHO-K1 cells were detached by
0.25% trypsin/1 mM of ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.), and were collected using 10% FBS in
RPMI 1640 medium. After washing with 0.1% bovine serum
albumin and phosphate-buffered saline, the cells were incubated with an anti-wPDPN antibody (PMab-237(22); 1 mg/mL)

19

or an anti-RIEDL tag antibody (LpMab-7(23); 1 mg/mL) for 30
minutes at 4C. Alexa Fluor 488-conjugated anti-mouse IgG(1:1000; Cell Signaling Technology, Inc., Danvers, MA) for
detection of PMab-237 and LpMab-7 were added to each celland incubated for 30 minutes at 4C. Fluorescence data werecollected and analyzed using a Cell Analyzer EC800 (SonyCorp., Tokyo, Japan).

Results
Epitope analysis using deletion mutants of wPDPN

Ten deletion mutants of wPDPN, such as dN30 (corresponding to 30–161 AA), dN40 (corresponding to 40–161
AA), dN50 (50–161 AA), dN60 (60–161 AA), dN70 (70–161 AA), dN80 (80–161 AA), dN85 (85–161 AA), dN90
(90–161 AA), dN95 (95–161 AA), and dN100 (100–161
AA), or wild type (WT) of wPDPN (corresponding to 23–161
AA) were generated using CHO-K1 cells (Fig. 1).
All deletion mutants and WT of wPDPN containing an Nterminal RIEDL tag were detected by LpMab-7 (an antiRIEDL tag mAb), indicating that the expression level of each

FIG. 3. Epitope mapping of PMab-237 using point mutants of wPDPN. Transient point mutants expressing E80A, D81A,
L82A, P83A, T84A, A85G, E86A, S87A, T88A, I89A, H90A, S91A, Q92A, G93A, Q94A, S95A, Q96A, S97A, T98A,
T99A, T100A, and L101A of wPDPN were incubated with LpMab-7 (red line, A), PMab-237 (red line, B), or buffer control(black line, A, B) for 30 minutes at 4C, followed by secondary antibodies.

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SAYAMA ET AL.

FIG. 4. Schematic illustration of the epitope recognized by PMab-237. The critical epitope of PMab-237 includes Leu82
and Thr84 of wPDPN. Both Leu82 and Thr84 are included in the PLD.
construct was high (Fig. 2A). In contrast, PMab-237 did notreact with dN85, dN90, dN95, or dN100 (Fig. 2B), suggestingthat the N-terminus of the PMab-237 epitope might exist
between the 80th AA and 85th AA of wPDPN.
Epitope analysis using point mutants of wPDPN

To identify the binding epitope of PMab-237, we then produced a series of point mutants of wPDPN using CHO-K1 cells,
including E80A, D81A, L82A, P83A, T84A, A85G, E86A,
S87A, T88A, I89A, H90A, S91A, Q92A, G93A, Q94A, S95A,
Q96A, S97A, T98A, T99A, T100A, and L101A.
LpMab-7 reacted with all point mutants, indicating that alltransfectants express wPDPN (Fig. 3A). In contrast, PMab237 weakly recognized L82A and did not react with T84A,
indicating that the L82A and T84A of wPDPN could be included in the critical epitope of PMab-237 (Fig. 3B). Taken
together, the epitope of PMab-237 is located in the PLD ofwPDPN (Fig. 4).
Discussion

We have demonstrated that the critical epitope of PMab237 could include Leu82 and Thr84 of wPDPN using thedeletion mutants and point mutants of wPDPN in CHO-K1
cells by flow cytometry. In our previous studies, we developed many mAbs against PDPNs of human,(24) mouse,(25)rat,(26) rabbit,(27) dog,(28) cat,(29) bovine,(30) horse,(31) Tasmanian devil,(32) alpaca,(33) bear,(34) tiger,(35) goat,(36) pig,(37,38)
and whale.(20) We successfully determined the binding epitope of those mAbs.(22,34,39–48) These epitope mapping results
showed that almost all anti-PDPN mAbs react with PLAG
domains or PLDs.(7,39–41,43,44,49–51) The critical epitope ofPMab-237 was also shown to be located in PLD (Fig. 4),
suggesting that PLAG domains and PLD were advantageous to
the epitope for several applications such as flow cytometry,western blotting, and immunohistochemical analyses.
Furthermore, using glycan-deficient CHO cell lines, such as
Lec1 (N-glycan-deficient), Lec2 (sialic acid-deficient), or Lec8
(galactose-deficient),(52) we investigated whether the epitopeof PMab-237 could include not only AAs but also glycans.
A flow cytometric analysis demonstrated that PMab-237 reacts

not only with CHO-K1/wPDPN but also with Lec1/wPDPN,
Lec2/wPDPN, and Lec8/wPDPN (data not shown), indicatingthat glycans of wPDPN might not be included in the PMab-237
epitope. This analysis further suggests that no glycan is
attached to Thr84 in the PLD of wPDPN (Fig. 4).
In conclusion, by using deletion mutants and point mutantsof wPDPN in CHO-K1 cells, we have demonstrated that thecritical epitope of PMab-237 may include Leu82 and Thr84
of wPDPN. PMab-237 could be a useful tool in elucidating
the pathophysiological function of wPDPN.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported in part by AMED under grantnos. JP19am0401013 (Y.K.), JP19am0101078 (Y.K.), andJP19ae0101028 (Y.K.), and by JSPS KAKENHI grant nos.
17K07299 (M.K.K.) and 19K07705 (Y.K.).
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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: November 12, 2019
Accepted: December 17, 2019