MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 37, Number 4, 2018
Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2018.0025

Elucidation of Critical Epitope of Anti-Rat Podoplanin
Monoclonal Antibody PMab-2
Yoshikazu Furusawa,1–3 Shinji Yamada,1 Shunsuke Itai,1,4 Takuro Nakamura,1Masato Fukui,3 Hiroyuki Harada,4 Mika K. Kaneko,1 and Yukinari Kato1,2

Rat podoplanin (rPDPN) is a recognized lymphatic endothelial cell marker and is expressed on the podocytes ofkidney and type I lung alveolar cells. rPDPN is a type I transmembrane sialoglycoprotein and induces platelet
aggregation via the C-type lectin-like receptor-2 of platelets. It comprises four platelet aggregation-stimulating(PLAG) domains: PLAG1–3, present in the N-terminus, and PLAG4, in the center of the PDPN protein. Previously,
we developed a mouse anti-rPDPN monoclonal antibody clone, PMab-2, by immunizing the PLAG2 and PLAG3
domains of rPDPN. PMab-2 has applications in Western blot, flow cytometry, and immunohistochemical analyses
for detection of both normal and cancer cells. However, the binding epitope of PMab-2 remains to be determined.
Herein, we investigated the epitope of PMab-2 using enzyme-linked immunosorbent assay, immunohistochemical
analysis, and flow cytometry. The results revealed that the critical epitope of PMab-2 is Leu46 and Glu47 of rPDPN.
Keywords: podoplanin, PDPN, PMab-2, epitope mapping
Introduction

Rat podoplanin (rPDPN) is a recognized lymphatic
endothelial cell marker and is expressed on the podocytesof kidney and type I lung alveolar cells.(1–4) rPDPN is a type I
transmembrane sialoglycoprotein and induces platelet aggregation via the C-type lectin-like receptor-2 (CLEC-2) ofplatelets.(5–7) It comprises four platelet aggregation-stimulating(PLAG) domains: PLAG1–3, present in the N-terminus,(1) and
PLAG4, in the center of the rPDPN protein.(8)PMab-2 monoclonal antibody (mAb) was previously produced against PLAG domain of rPDPN(9); therefore, PMab-2
could neutralize interactions between rPDPN and CLEC-2.(5–7)
PMab-2 is applicable in Western blot, flow cytometry, andimmunohistochemical analyses.
In this study, we determined the binding epitope of PMab2 to rPDPN using flow cytometry and enzyme-linked immunosorbent assay (ELISA).
Materials and Methods

CHO/rPDPN was produced in our previous study.(9) The
rPDPN mutation plasmids were transfected into CHO-K1
cells using Lipofectamine LTX (Thermo Fisher Scientific,Inc., Waltham, MA). CHO/rPDPN and transiently transfected
cells were cultured in RPMI 1640 medium (Nacalai Tesque,Inc., Kyoto, Japan), supplemented with 10% heat-inactivatedfetal bovine serum (Thermo Fisher Scientific, Inc.), 100 U/mLof penicillin, 100 mg/mL of streptomycin, and 25 mg/mL ofamphotericin B (Nacalai Tesque, Inc.) at 37C in a humidified
atmosphere of 5% carbon dioxide and 95% air.
Production of rPDPN point mutants

The complementary DNA of rPDPN was subcloned into apcDNA3 vector (Thermo Fisher Scientific, Inc.). Substitutions of amino acids to alanine in rPDPN sequence wereperformed by the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Inc., Santa Clara, CA).
Flow cytometry

Cell line

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

Cells were harvested after brief exposure to 0.25% trypsin/
1 mM ethylenediaminetetraacetic acid (Nacalai Tesque,Inc.). After washing with 0.1% bovine serum albumin in

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
3
ZENOAQ RESOURCE CO., LTD., Koriyama, Japan.
4
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,Tokyo, Japan.
2ª Yoshikazu Furusawa et al. 2018; 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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FIG. 1. Epitope mapping of PMab-2 using point mutants of rPDPN. Point mutants of rPDPN were analyzed using flow
cytometry. Point mutants were expressed on CHO-K1 cells and were then incubated with PMab-2 (2 mg/mL) or buffer
control for 30 minutes at 4C, followed by treatment with corresponding secondary antibodies.
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Table 1. Determination of PMab-2 Epitopeby Enzyme-Linked Immunosorbent Assay
Mutation

Sequence

PMab-2

G38A
D39A
D40A
M41A
V42A
N43A
P44A
G45A
L46A
E47A
D48A
R49A
I50A
E51A

ADDMVNPGLEDRIEGADMVNPGLEDRIEGDAMVNPGLEDRIEGDDAVNPGLEDRIEGDDMANPGLEDRIEGDDMVAPGLEDRIEGDDMVNAGLEDRIEGDDMVNPALEDRIEGDDMVNPGAEDRIEGDDMVNPGLADRIEGDDMVNPGLEARIEGDDMVNPGLEDAIEGDDMVNPGLEDRAEGDDMVNPGLEDRIA+++++++++++++++++++++++++++++++++++, OD655S1.0; +, 0.1&OD655<0.6; -, OD655<0.1.

phosphate-buffered saline (PBS), the cells were treated with
PMab-2 for 30 minutes at 4C, followed by treatment with
Alexa Fluor 488-conjugated anti-mouse IgG (1:1000; Cell
Signaling Technology, Inc., Danvers, MA). Fluorescencedata were acquired using the Cell Analyzer EC800 (SonyCorp., Tokyo, Japan).

Enzyme-linked immunosorbent assay

Synthesized rPDPN peptides using PEPscreen (SigmaAldrich Corp., St. Louis, MO) were immobilized on Nunc
MaxiSorp 96-well immunoplates (Thermo Fisher Scientific,Inc.) at 10 mg/mL for 30 minutes at 37C. After blocking withSuperblock T20 (PBS) blocking buffer (Thermo FisherScientific, Inc.), the plates were incubated with purified
PMab-2 (10 mg/mL), followed by a 1:2000 dilution ofperoxidase-conjugated anti-mouse IgG (Agilent Technologies, Inc.). The enzymatic reaction was conducted using 1Step Ultra TMB-ELISA (Thermo Fisher Scientific, Inc.).
Optical density was measured at 655 nm using an iMark
Microplate Reader (Bio-Rad Laboratories, Inc., Berkeley,CA). These reactions were performed at 37C with a total
sample volume of 50–100 mL.
Immunohistochemical analyses

Histological sections (4 mm thick) of rat tissues weredirectly autoclaved in citrate buffer (pH 6.0; Nichirei
Biosciences, Inc., Tokyo, Japan) for 20 minutes. After
blocking with Superblock T20 (PBS) blocking buffer,sections were incubated with PMab-2 (1 mg/mL) or PMab-2(1 mg/mL) plus peptides (5 mg/mL) for 1 hour at room
temperature, treated using an EnVision+ Kit (AgilentTechnologies, Inc.) for 30 minutes. Color was developed

FIG. 2. Immunohistochemical analyses using rat lung tissues. Histological sections of lung were directly autoclaved incitrate buffer for 20 minutes. After blocking with blocking buffer, sections were incubated with PMab-2 (1 mg/mL) or
PMab-2 (1 mg/mL) plus peptides (5 mg/mL), followed by an EnVision+ Kit. Scale bar = 100 mm. HE, hematoxylin and eosin.

FIG. 3. Immunohistochemical analyses using rat kidney tissues. Histological sections of kidney were directly autoclavedin citrate buffer for 20 minutes. After blocking with blocking buffer, sections were incubated with PMab-2 (1 mg/mL) or
PMab-2 (1 mg/mL) plus peptides (5 mg/mL), followed by an EnVision+ Kit. Scale bar = 100 mm.

EPITOPE OF PMAB-2191

FIG. 4. Immunohistochemical analyses using rat colon tissues. Histological sections of colon were directly autoclaved incitrate buffer for 20 minutes. After blocking with blocking buffer, sections were incubated with PMab-2 (1 mg/mL) or
PMab-2 (1 mg/mL) plus peptides (5 mg/mL), followed by an EnVision+ Kit. Scale bar = 100 mm.

using 3,3-diaminobenzidine tetrahydrochloride (AgilentTechnologies, Inc.) for 2 minutes and counterstained withhematoxylin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).
Results and Discussion

We previously developed a mouse anti-rPDPN mAb,PMab-2, by immunizing the PLAG domain of rPDPN.(9)In this study, we produced point mutants of rPDPN using
recombinant proteins and synthesized peptides and investigated the epitope of PMab-2 critical for rPDPN
detection.
We produced a series of point mutants of rPDPN using aQuikChange Lightning Site-Directed Mutagenesis Kit because PMab-2 was produced by immunizing mice with aminoacids 38–51 of rPDPN. Using flow cytometry, we found that
PMab-2 recognized G38A, D39A, D40A, M41A, V42A

FIG. 5. Flow cytometry using PMab-2 and point mutants of rPDPN. PMab-2 (1 mg/mL) or PMab-2 (1 mg/mL) + peptides(G38A, L46A, and E47A; 10 mg/mL) were treated with CHO/rPDPN cells for 30 minutes at 4C, followed by addition ofsecondary antibodies.

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FIG. 6. Illustration of rPDPN and epitope of PMab-2. rPDPN possesses four PLAG domains. PMab-2 was established byimmunizing PLAG2–3 domains. Leu46 and Glu47 are critical amino acids for PMab-2 recognition to rPDPN. PLAG,platelet aggregation stimulating.

N43A, P44A, G45A, D48A, R49A, I50A, and E51A (Fig. 1).
However, it did not recognize L46A and E47A, thus indicating that Leu46 and Glu47 of rPDPN are crucial for PMab2 detection.
Next, we synthesized a series of peptides: point mutants of
38–51 amino acids of rPDPN (Table 1). Using ELISA, wefound that PMab-2 detected G38A, D39A, D40A, M41A,V42A N43A, P44A, G45A, D48A, R49A, I50A, and E51A;
in contrast, it did not recognize L46A and E47A, thus confirming the result obtained via flow cytometry.
We performed a blocking assay using immunohistochemistry. PMab-2 reacted with type I alveolar cells (Fig. 2),renal podocytes (Fig. 3), and lymphatic endothelial cells ofcolon (Fig. 4). These reactions were completely or partiallyneutralized by G38A; in contrast, L46A and E47A did notblock the reactions of PMab-2 with rat tissues, thus indicating that L46A and E47A of rPDPN are critical for PMab-2detection.
In addition, we performed a blocking assay using flow cytometry and found that PMab-2 reacted with the CHO/rPDPN
cell line (Fig. 5). This reaction was completely neutralized byG38A; in contrast, L46A and E47A did not block the reactionof PMab-2 with CHO/rPDPN, which indicated that Leu46 andGlu47 of rPDPN are critical for PMab-2 detection.
Taken together, the critical epitope of PMab-2 comprises
Leu46 and Glu47 of rPDPN (Fig. 6). These findings could beapplied to the production of more functional anti-rPDPNmAbs.
Acknowledgments

The authors thank Miyuki Yanaka, Saori Handa, KayoHisamatsu, and Yoshimi Nakamura for excellent technicalassistance. This research was supported, in part, by AMED
under Grant Nos.: JP18am0101078 (Y.K.), JP18am0301010(Y.K.), and JP18ae0101028 (Y.K.); and by JSPS KAKENHI
Grant Nos.: 17K07299 (M.K.K.) and 16K10748 (Y.K.).

Author Disclosure Statement

No competing financial interests exist.
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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1, Seiryo-machi, Aoba-ku
Sendai, Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: June 27, 2018
Accepted: July 12, 2018