MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 6, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0037

The Epitope of PMab-210 Is Located in Platelet
Aggregation-Stimulating Domain-3 of Pig Podoplanin

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Mika K. Kaneko,1 Yusuke Sayama,1 Masato Sano,1 and Yukinari Kato1,2

Podoplanin (PDPN)/T1alpha/Aggrus, a small mucin-type transmembrane glycoprotein, has been shown to be
expressed on lymphatic endothelial cells and epithelial cells of many organs. PDPN is also upregulated in many
cancers, and is involved in cancer metastasis and malignant progression. Human PDPN possesses three platelet
aggregation-stimulating (PLAG) domains and the PLAG-like domain, which bind to C-type lectin-like receptor2 (CLEC-2). Previously, we reported a novel antipig PDPN (pPDPN) monoclonal antibody (PMab-210) using
Cell-Based Immunization and Screening (CBIS) method. PMab-210 specifically detected pPDPN-overexpressed
Chinese hamster ovary (CHO)-K1 cells by flow cytometry and Western blot analysis. Immunohistochemical
analyses demonstrated that PMab-210 stained pulmonary type I alveolar cells strongly and renal corpuscles
weakly in pig or microminipig. However, the specific binding epitope of PMab-210 for pPDPN could not be
determined by enzyme-linked immunosorbent assay using a series of pPDPN peptides. In this study, deletionmutants or point mutants of pPDPN were produced for analyzing the PMab-210 epitope using flow cytometry.
The analysis of deletion mutants showed that N-terminus of PMab-210 epitope exists between 45th amino acid(aa) and 50th aa of pPDPN. In addition, the analysis of point mutants demonstrated that the critical epitope ofPMab-210 could include Glu47, Asp48, Tyr49, Thr50, and Val51 of pPDPN, indicating that PMab-210 epitope islocated in PLAG3 domain of pPDPN.
Keywords: pig podoplanin, monoclonal antibody, epitope, PMab-210

IntroductionP

odoplanin (PDPN)/T1alpha/Aggrus is a type I transmembrane sialoglycoprotein consisting of a heavily glycosylated extracellular domain, a single transmembrane, and ashort nine amino acid (aa) cytoplasmic tail.(1–3) PDPN has beenshown to be expressed on lymphatic endothelial cells, epithelial
cells, and fibroblasts of many organs.(2) PDPN-positive cellswith the immune cells after myocardial infarction positively
affect immune cells recruitment.(4) The PDPN-positive stromalcells play a critical role in a network of immunofibroblasts,
which are able to support the earliest phases of tertiary lymphoid structure establishment.(5) PDPN is also upregulated inmany cancers, and is involved in cancer metastasis and malignant progression,(6–9) indicating that PDPN possesses many
pathophysiological functions in both normal and malignant
tissues.
Human PDPN (hPDPN) possesses the EDxxVTPG sequences at the N-terminus of hPDPN, which were defined tobe the platelet aggregation-stimulating (PLAG) domains(PLAG1, PLAG2, and PLAG3).(10) In addition, one PLAGlike domain (PLD) of the E(D/E)xx(T/S)xx sequence has
12

been reported to be present in the middle of PDPN.(11) PLD
was also called as PLAG4,(12) which is independent of Nterminal PLAG domains. PLAG and PLD are highly conserved
among mammalian PDPNs.(10) The interaction between PDPN
on lymphatic endothelial cells and C-type lectin-like receptor-2
on platelets was shown to facilitate embryonic blood/lymphatic
vessel separation.(13)Recently, we developed a novel antipig PDPN (pPDPN)
monoclonal antibody (mAb), PMab-210, using Cell-Based
Immunization and Screening (CBIS) method.(14) PMab-210
specifically detected pPDPN by flow cytometry, Western
blotting, and immunohistochemical analysis.(14) However,the binding epitope of PMab-210 for pPDPN remains to beclarified. This study aimed to identify the epitope of PMab210 using deletion mutants and point mutants of pPDPN
using flow cytometry.
Materials and Methods
Cell lines

Chinese hamster ovary (CHO)-K1 was obtained from theAmerican Type Culture Collection (ATCC, Manassas, VA).

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

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FIG. 1. Illustration of WT and seven deletion mutants of pPDPN. Deletion mutants of pPDPN: dN30, dN40, dN45, dN50,
dN60, dN70, and dN80. Black bars: the positive reaction of PMab-210. White bars: the negative reaction of PMab-210.
PLAG domain, platelet aggregation-stimulating domain; PLD, PLAG-like domain; pPDPN, pig podoplanin; WT, wild type.

FIG. 2. Epitope mapping of PMab-210 using deletion mutants of pPDPN. (A, B) Deletion mutants of pPDPN wereanalyzed using flow cytometry. Deletion mutants were incubated with NZ-1 (anti-PA16 tag; red line, A), PMab-210 (antipPDPN mAb; red line, B), or buffer control (black line, A, B) for 30 minutes at 4C, followed by secondary antibodies.
mAb, monoclonal antibody.
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FIG. 3. Epitope mapping of PMab-210 using point mutants of pPDPN. (A, B) Transient point mutants expressing G45A,
V46A, E47A, D48A, Y49A, T50A, V51A, T52A, P53A, R54A, T55A, and R56A of pPDPN were incubated with NZ-1 (redline, A), PMab-210 (red line, B), or buffer control (black line, A, B) for 30 minutes at 4C, followed by secondary
antibodies.

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KANEKO ET AL.

The pPDPN mutation plasmids containing PA16 tag weretransfected into CHO-K1 cells using Lipofectamine LTX(Thermo Fisher Scientific, Inc., Waltham, MA). The PA16
tag comprises 16 aas (GLEGGVAMPGAEDDVV). Transiently transfected cells with deletion mutants or point mutants were cultured in Roswell Park Memorial Institute(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.

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Production of pPDPN mutants

Synthesized DNA of pPDPN was subcloned into thepCAG vector (FUJIFILM Wako Pure Chemical Corporation,
Osaka, Japan), and PA16 tag was added at the N-terminus.
Deletion mutants of pPDPN sequence were produced using a
HotStar HiFidelity Polymerase Kit (Qiagen, Inc., Hilden,
Germany) with oligonucleotides. Substitutions of aas to alanine in pPDPN sequence were conducted by QuikChangeLightning Site-Directed Mutagenesis Kits (Agilent Technologies, Inc., Santa Clara, CA). PCR fragments bearing thedesired mutations were inserted into the pCAG vector using
In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan).
Flow cytometry

Transiently transfected CHO-K1 cells were detached by
0.25% trypsin/1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.) and were collected using 10% FBS in
RPMI 1640 medium. After washing with 0.1% bovine serum
albumin/phosphate buffered saline, the cells were incubatedwith an anti-pPDPN antibody (PMab-210; 1 mg/mL) or ananti-PA16 tag antibody (NZ-1; 1 mg/mL) for 30 minutes at
4C. Alexa Fluor 488-conjugated antimouse IgG (1:1000;
Cell Signaling Technology, Inc., Danvers, MA) for detectionof PMab-210 or Oregon Green antirat IgG (1:2000; Thermo
Fisher Scientific, Inc.) for detection of NZ-1 was added toeach cell, and was incubated for 30 minutes at 4C. Fluorescence data were collected and analyzed using a Cell Analyzer EC800 (Sony Corp., Tokyo, Japan).
Results

Seven deletion mutants of pPDPN, such as dN30 (corresponding to 30–159 aas), dN40 (corresponding to 40–159aas), dN45 (corresponding to 45–159 aas), dN50 (corresponding to 50–159 aas), dN60 (corresponding to 60–159aas), dN70 (corresponding to 70–159 aas), and dN80 (corresponding to 80–159 aas), or wild type (WT) of pPDPN(corresponding to 27–159 aas), were generated using CHOK1 cells (Fig. 1). All deletion mutants and WT of pPDPN
containing N-terminal PA16 tag were detected by NZ-1
(an anti-PA16 tag mAb), indicating that the expression levelof each construct is very high (Fig. 2A). In contrast, PMab210 did not react with dN50, dN60, dN70, and dN80
(Fig. 2B), suggesting that N-terminus of PMab-210 epitope
might exist between 45th aa and 50th aa of pPDPN.
To identify the binding epitope of PMab-210, we next
produced a series of point mutants of pPDPN in CHO-K1
cells, including G45A, V46A, E47A, D48A, Y49A, T50A,

FIG. 4. Schematic illustration of the epitope recognizedby PMab-210. Underlined and red colored aa is a critical
epitope of PMab-210. Blue colored aa is a critical epitope ofPMab-213, which is the other anti-pPDPN mAb. aa, aminoacid.
V51A, T52A, P53A, R54A, T55A, and R56A. NZ-1 reactedwith all point mutants (Fig. 3A). In contrast, PMab-210 did
not react with E47A, Y49A, T50A, and V51A, and weakly
recognized D48A (Fig. 3B), indicating E47A, D48A, Y49A,
T50A, and V51A of pPDPN could be included in the critical
epitope of PMab-210 (Fig. 4).
Discussion

We have demonstrated that critical epitope of PMab-210
could include Glu47, Asp48, Tyr49, Thr50, and Val51 of
pPDPN using deletion mutants and point mutants of pPDPN
in CHO-K1 cells. In our previous study, we developed mAbagainst human,(15) mouse,(16) rat,(17) rabbit,(18) dog,(19) cat,(20)bovine,(21) horse,(22) Tasmanian devil,(23) alpaca,(24) bear,(25)tiger,(26) whale,(27) goat,(28) and pig(29) PDPNs, and determined
the binding epitope of those mAbs.(11,28,30–38) Both PMab-210
and PMab-213 for anti-pPDPN mAb could specifically detect
pPDPN by flow cytometry and Western blotting, and stained
pig tissues by immunohistochemistry. The epitope of PMab213 (the other anti-pPDPN mAb) was identified as Pro53,
Arg54, Arg56, and Tyr60 of pPDPN,(38) indicating that the
epitope of both anti-pPDPN mAbs is located in the PLAG3
domain (Fig. 4). Likewise, almost all anti-PDPN mAbs reactwith PLAG domains or PLDs,(11,12,28,30–32,35–38) suggestingthat PLAG domains and PLD were clarified to be advantageous epitopes for several applications, such as flow cytometry, Western blotting, and immunohistochemical
analyses.
Using glycan-deficient CHO cell lines, such as Lec1 (Nglycan deficient), Lec2 (sialic acid deficient), or Lec8 (galactose deficient),(39) we investigated whether the epitope ofPMab-210 could include not only aas but also O-glycans.
Flow cytometric analysis demonstrated that PMab-210 reacts
with CHO-K1/pPDPN and Lec1/pPDPN strongly and with
Lec2/pPDPN weakly, but not with Lec8/pPDPN (data notshown), indicating that sialic acid and galactose might also be
included in the PMab-210 epitope. Among Glu47, Asp48,
Tyr49, Thr50, and Val51 of pPDPN, Thr50 could be Oglycosylated (Fig. 4). Because PDPN possesses disialyl-core
1 on many Ser/Thr residues,(40) disialyl-core 1 of Thr50
might be critical for PMab-210 binding to pPDPN.

EPITOPE OF PMAB-210

In conclusion, Glu47, Asp48, Tyr49, Thr50, and Val51 in
PLAG3 domain of pPDPN are critical for PMab-210-specificbinding to pPDPN. PMab-210 did not react with glycandeficient CHO cell lines, indicating that PMab-210 was categorized as an antiglycopeptide mAb (GpMab).(41) Several
anti-hPDPN mAbs, such as LpMab-2,(42) LpMab-3,(43)LpMab-9,(44) LpMab-12,(45) LpMab-19,(46) and LpMab-21,(41)were also determined to be GpMabs. PMab-210 could be auseful tool for elucidating the pathophysiological function of
pPDPN.
Author Disclosure Statement

No competing financial interests exist.

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Funding Information

This research was supported, in part, by AMED underGrant Nos. JP19am0101078 (Y.K.), JP19am0401013 (Y.K.),
JP19ae0101028 (Y.K.), and by JSPS KAKENHI Grant No.
17K07299 (M.K.K.) and Grant No. 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: August 20, 2019
Accepted: October 1, 2019