Biochemistry and Biophysics Reports 15 (2018) 52–56

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Biochemistry and Biophysics Reports
journal homepage: www.elsevier.com/locate/bbrep

Epitope mapping of anti-mouse podoplanin monoclonal antibody PMab-1aaab

Shinji Yamada , Shunsuke Itai , Mika K. Kaneko , Satoru Konnai , Yukinari Katoabca,c,⁎

T

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan
Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido 060-0818, JapanNew Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, JapanA R T I C LE I N FO

A B S T R A C T

Keywords:
Podoplanin
PDPN
PMab-1
Epitope mapping

Mouse podoplanin (mPDPN) is a type I transmembrane sialoglycoprotein, which is expressed on lymphatic
endothelial cells, podocytes of the kidney, and type I alveolar cells of the lung. mPDPN is known as a platelet
aggregation-inducing factor and possesses four platelet aggregation-stimulating (PLAG) domains: PLAG1,PLAG2, and PLAG3 in the N-terminus and PLAG4 in the middle of the mPDPN protein. mPDPN overexpression incancers has been reportedly associated with hematogenous metastasis through interaction with the C-type lectinlike receptor 2 of platelets. We previously reported a rat anti-mPDPN monoclonal antibody clone PMab-1, which
was developed by immunizing the PLAG2 and PLAG3 domains of mPDPN. PMab-1 is very useful in ﬂow cytometry, western blot, and immunohistochemical analyses to detect both normal cells and cancers. However, thebinding epitope of PMab-1 remains to be clariﬁed. In the present study, ﬂow cytometry, enzyme-linked immunosorbent assay, and immunohistochemical analyses were utilized to investigate the epitope of PMab-1. The
results revealed that the critical epitope of PMab-1 is Asp39 and Met41 of mPDPN. These ﬁndings can be applied
to the production of more functional anti-mPDPN monoclonal antibodies.

1. Introduction
Podoplanin (PDPN/T1alpha/gp38/Aggrus) is expressed in many
normal tissues, such as renal podocytes, lymphatic endothelial cells of
many tissues, and pulmonary type I alveolar cells [1–4]. Several antimouse PDPN (mPDPN) monoclonal antibodies (mAbs), such as clone
8.1.1 or clone PMab-1, have been used in many studies [5]. However,clone 8.1.1 is produced using hamsters, and clone PMab-1 is produced
using rats because it is diﬃcult to develop anti-mPDPN mAbs using
mice. Recently, we developed a rat–mouse chimeric antibody, mPMab1 of mouse IgG2a, which was derived from a rat PMab-1 mAb [6].
Immunohistochemical analysis showed that mPMab-1 detects podocytes of the kidney, lymphatic endothelial cells of the colon, and type I
alveolar cells of the lung. Importantly, mPMab-1 was shown to be moresensitive than original PMab-1.
mPDPN possesses four platelet aggregation-stimulating (PLAG) domains: PLAG1, PLAG2, and PLAG3 in the N-terminus [1] and PLAG4 in
the middle of the mPDPN protein [7]. In a previous study, PMab-1 mAb
was produced against the platelet aggregation-stimulating (PLAG) domain of mPDPN [5]; therefore, PMab-1 neutralizes the interaction between mPDPN and the C-type lectin-like receptor 2 [8–10]. The

administration of PMab-1 was found to reduce lymphangiogenesis incorneal suture and ear-wound healing models [11]. PMab-1 also suppressed the inﬁltration of thioglycollate-induced macrophages at thesite of wound healing. Furthermore, the administration of PMab-1 leadto a signiﬁcant suppression of the rejection reaction in a corneal
transplantation model, suggesting that mPDPN is a novel therapeutictarget for suppressing lymphangiogenesis and inﬂammation.
In the present study, we determined the binding epitope of PMab-1
to mPDPN using ﬂow cytometry, enzyme-linked immunosorbent assay(ELISA), and immunohistochemical analyses.
2. Materials and methods
2.1. Cell line
Chinese hamster ovary (CHO)-K1 cell line was purchased from theAmerican Type Culture Collection (Manassas, VA, USA). The mPDPN
mutation plasmids were transfected into CHO-K1 cells using
Lipofectamine LTX (Thermo Fisher Scientiﬁc Inc., Waltham, MA, USA).
Transiently transfected cells were cultured in RPMI 1640 medium(Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% heat-Abbreviations: PDPN, podoplanin; PLAG, platelet aggregation-stimulating; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buﬀered saline;
DAB, 3,3-diaminobenzidine tetrahydrochloride⁎
Corresponding author at: New Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan.
E-mail address: yukinarikato@med.tohoku.ac.jp (Y. Kato).
https://doi.org/10.1016/j.bbrep.2018.07.002
Received 23 June 2018; Received in revised form 1 July 2018; Accepted 2 July 2018
2405-5808/ © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

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S. Yamada et al.

Fig. 1. Epitope mapping of PMab-1 using point mutants of mPDPN. Point mutants of mPDPN were analyzed using ﬂow cytometry. Point mutants were expressedon CHO-K1 cells and then incubated with PMab-1 (2 μg/ml) or buﬀer control for 30 min at 4 °C, followed by treatment with corresponding secondary antibodies.

Inc.). Optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA). These reactions
were performed at 37 °C with a total sample volume of 50–100 μl.

inactivated fetal bovine serum (Thermo Fisher Scientiﬁc Inc.), 100
units/ml of penicillin, 100 μg/ml of streptomycin, and 25 μg/ml ofamphotericin B (Nacalai Tesque, Inc.) at 37 °C in a humidiﬁed atmosphere of 5% CO2 and 95% air.

2.5. Immunohistochemical analyses
2.2. Production of mPDPN point mutants
Histological sections (4-μm thick) of mouse tissues were directly
autoclaved in citrate buﬀer (pH 6.0; Nichirei Biosciences, Inc., Tokyo,Japan) for 20 min. After blocking with SuperBlock T20 (PBS) Blocking
Buﬀer (Thermo Fisher Scientiﬁc Inc.), sections were incubated with
mPMab-1 (1 μg/ml) or mPMab-1 (1 μg/ml) plus peptides (5 μg/ml) for
1 h at room temperature and treated using an Envision + kit (AgilentTechnologies Inc.) for 30 min. Color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.) for
2 min. Sections were counterstained with hematoxylin (FUJIFILM Wako
Pure Chemical Corporation, Osaka, Japan).

The cDNA of mPDPN was subcloned into a pcDNA3 vector (ThermoFisher Scientiﬁc Inc.) [2]. Substitutions of amino acids to alanine in themPDPN sequence were performed using a QuikChange Lightning SiteDirected Mutagenesis Kit (Agilent Technologies Inc., Santa Clara, CA,USA).
2.3. Flow cytometry
Cells were harvested after brief exposure to 0.25% trypsin/1 mM
ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). After washingwith 0.1% bovine serum albumin in PBS, the cells were treated withPMab-1 for 30 min at 4 °C, followed by treatment with Alexa Fluor 488conjugated anti-rat IgG (1:1000; Cell Signaling Technology, Inc.,Danvers, MA). Fluorescence data were acquired using the Cell Analyzer
EC800 (Sony Corp., Tokyo, Japan).

3. Results and discussion
In a previous study, we developed a rat anti-mPDPN mAb PMab-1
by immunizing the PLAG domain of mPDPN [5]. We further produced arat–mouse chimeric antibody, mPMab-1 of mouse IgG2a, which wasderived from a rat PMab-1 mAb [6]. Immunohistochemical analysis
showed that both PMab-1 and mPMab-1 are capable of detecting podocytes of the kidney, lymphatic endothelial cells of the colon, and typeI alveolar cells of the lung. Interestingly, mPMab-1 was shown to be
more sensitive than original PMab-1 [6] probably because a high-sensitivity immunohistochemical kit can be used for mouse IgG. In thepresent study, we produced point mutants of mPDPN (proteins andsynthesized peptides) and investigated the critical epitope of PMab-1for mPDPN detection.
Because PMab-1 was developed by immunizing rats with aminoacids 38–51 of mPDPN, we produced a series of point mutants of

2.4. ELISA
Synthesized mPDPN peptides using PEPScreen (Sigma-AldrichCorp., St. Louis, MO) were immobilized on Nunc Maxisorp 96-wellimmunoplates (Thermo Fisher Scientiﬁc Inc.) at 10 μg/ml for 30 min at
37 °C. After blocking with SuperBlock T20 (PBS) Blocking Buﬀer(Thermo Fisher Scientiﬁc Inc.), the plates were incubated with puriﬁed
PMab-1 (10 μg/ml), followed by a 1:2000 dilution of peroxidase-conjugated anti-rat IgG (Agilent Technologies Inc.). The enzymatic reaction
was performed using 1-Step Ultra TMB-ELISA (Thermo Fisher Scientiﬁc53

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S. Yamada et al.

Fig. 2. Illustration of mPDPN and epitope of PMab-1. mPDPN possesses four PLAG domains. PMab-1 was produced by immunizing PLAG2 and PLAG3 domains.
Asp39 and Met41 are critical amino acids for PMab-1 recognition to mPDPN.

Fig. 3. Flow cytometry using PMab-1 and point mutants of mPDPN. PMab-1 (1 μg/ml) or PMab-1 (1 μg/ml) plus peptides (G38A, D39A, and M41A; 10 μg/ml)
were reacted with CHO/mPDPN cells for 30 min at 4 °C, followed by the addition of secondary antibodies.

analysis (Fig. 2).
We performed a blocking assay using ﬂow cytometry. PMab-1 reacted with the CHO/mPDPN cell line (Fig. 3). This reaction was completely neutralized by G38A. In contrast, D39A and M41A did not block
the reaction of PMab-1 with CHO/mPDPN, indicating that Asp39 andMet41 of mPDPN are critical for PMab-1 detection.
We further performed a blocking assay using immunohistochemistry. A rat–mouse chimeric mAb mPMab-1 reactedwith type I alveolar cells (Fig. 4A), renal podocytes (Fig. 4B), andlymphatic endothelial cells of the colon (Fig. 4C and SupplementarymPDPN using a QuikChange Lightning Site-Directed Mutagenesis Kit.
As shown in Fig. 1, PMab-1 reacted with G38A, G40A, V42A, P43A,P44A, G45A, I46A, E47A, D48A, K49A, I50A, and T51A in ﬂow cytometry. In contrast, it did not react with D39A and M41A, indicating that
Asp39 and Met41 of mPDPN are critical for PMab-1 recognition.
Next, we synthesized a series of point mutants of mPDPN peptides
from the 38th to the 51st amino acid (Supplementary Table 1). Using
ELISA, PMab-1 detected G38A, G40A, V42A, P43A, P44A, G45A, I46A,E47A, D48A, K49A, I50A, and T51A. In contrast, it did not recognize
D39A and M41A, conﬁrming the result from the ﬂow cytometric
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Fig. 4. Immunohistochemical analyses. Histological sections of the lung (A), kidney (B), and colon (C) were directly autoclaved in citrate buﬀer for 20 min. After
blocking with SuperBlock T20 (PBS) Blocking Buﬀer, sections were incubated with mPMab-1 (1 μg/ml) or mPMab-1 (1 μg/ml) plus peptides (5 μg/ml), followed bytreatment with an Envision+ kit. HE, hematoxylin and eosin. Scale bar, 100 µm.

Appendix A. Transparency document

Figure 1). These reactions were completely or partially neutralized byG38A. In contrast, D39A and M41A did not block these reactions ofmPMab-1 with mouse tissues, indicating that Asp39 and Met41 ofmPDPN are critical for PMab-1 detection.
Taken together, the critical epitope of PMab-1 is Asp39 and Met41
of mPDPN. These ﬁndings can be applied to the production of morefunctional anti-mPDPN mAbs.

Supplementary data associated with this article can be found in theonline version at http://dx.doi.org/10.1016/j.bbrep.2018.07.002.
Appendix B. Supporting information
Supplementary data associated with this article can be found in theonline version at http://dx.doi.org/10.1016/j.bbrep.2018.07.002.

Acknowledgments
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We thank Takuro Nakamura, Miyuki Yanaka, Saori Handa, KayoHisamatsu, and Yoshimi Nakamura for excellent technical assistance.

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