MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 39, Number 3, 2020ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2020.0010

Thr80 of Sheep Podoplanin Is a Critical Epitopeof the Antisheep Podoplanin Monoclonal Antibody:PMab-256

Downloaded by Yukinari Kato from www.liebertpub.com at 06/24/20. For personal use only.

Yukinari Kato,1,2 Masato Sano,1 Teizo Asano,1 Yusuke Sayama,1 and Mika K. Kaneko1

An antisheep podoplanin (sPDPN) monoclonal antibody (mAb), PMab-256, has recently been established.
PMab-256 shows positive immunostaining for lymphatic endothelial cells, lung type I alveolar cells, and kidney
podocytes. PDPN possesses three platelet-aggregation-stimulating (PLAG) domains, PLAG1, PLAG2, andPLAG3, and a PLAG-like domain (PLD). The binding epitope of many anti-PDPN mAbs is located in PLAG
domains or PLD. The purpose of this study is to determine the binding epitope of PMab-256. Analysis ofsPDPN deletion mutants revealed that the N-terminus of the PMab-256 epitope exists between amino acids 75
and 80 of sPDPN. Furthermore, analysis of sPDPN point mutations demonstrated that the critical epitope
includes Thr80 of sPDPN, indicating that the PMab-256 epitope is in the PLD of sPDPN.
Keywords: sheep podoplanin, monoclonal antibody, epitope, PMab-256

sPDPN mutation plasmids containing a RAP14 tag(15) weretransfected into CHO-K1 cells using Lipofectamine LTX(Thermo Fisher Scientific, Inc., Waltham, MA). The RAP14
tag comprises 14 amino acids (aa), (DMVNPGLEDRIEDL),
and is recognized by PMab-2 mAb(16) and LpMab-7.(1) Cells
transiently transfected with deletion or point mutations 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 lg/mL of
streptomycin, and 0.25 lg/mL of amphotericin B (NacalaiTesque, Inc.) at 37C in a humidified atmosphere of 5% CO2and 95% air.

Introduction

S

ensitive and specific monoclonal antibodies (mAbs)
against podoplanin (PDPN) of various species including
human,(1) mouse,(2) and whale(3) are necessary for the analysis of expression and pathophysiological functions of PDPN.
PDPN is used for discriminating lymphatic endothelial cells(LECs) from vascular endothelial cells(4) or type I alveolarcells from type II alveolar cells.(5) An antisheep podoplanin(sPDPN) mAb, PMab-256, was obtained using the Cell-Based
Immunization and Screening (CBIS) method.(6–8) Like othertypical anti-PDPN mAbs, PMab-256 demonstrated positive
immunoreaction for LECs,(4) type I alveolar cells,(5) and kidney podocytes.(9)The binding epitope of many anti-PDPN mAbs is locatedin three platelet aggregation-stimulating (PLAG) domains,
PLAG1, PLAG2, and PLAG3,(10) at the N-terminus or a
PLAG-like domain (PLDs)(3,11–14) in the middle of the PDPN
protein. The purpose of this study was to determine thebinding epitope of PMab-256.

Production of sPDPN mutants

Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan)
encoding sPDPN (accession no.: XM_004013802.4) wassubcloned into the pCAG vector (FUJIFILM Wako Pure
Chemical Corporation, Osaka, Japan), and an RAP14 tag wasadded at the N-terminus. Deletion mutants of the sPDPN
sequence were produced using a HotStar HiFidelity Polymerase Kit (Qiagen, Inc., Hilden, Germany) with oligonucleotides. Substitutions of aa with alanine in the sPDPN
sequence were produced by QuikChange Lightning SiteDirected Mutagenesis Kits (Agilent Technologies, Inc., Santa
Clara, CA). Polymerase chain reaction fragments bearing

Materials and Methods
Cell lines

Chinese hamster ovary (CHO)-K1 cells were obtained
from the American Type Culture Collection (Manassas, VA).
12Department of Antibody Drug Development, Graduate School of Medicine, Tohoku University, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.

95

Downloaded by Yukinari Kato from www.liebertpub.com at 06/24/20. For personal use only.

FIG. 1. Epitope mapping of PMab-256 using deletion mutations. (A) Deletion mutants of sPDPN were analyzed usingflow cytometry. Mutants were incubated with PMab-2 (anti-RAP14 tag mAb; red line) or buffer control (black line) for 30
minutes at 4C, followed by staining with secondary antibodies. (B) Deletion mutants of sPDPN were analyzed using flowcytometry. Mutants were incubated with LpMab-7 (anti-RAP14 tag mAb; red line) or buffer control (black line) for 30
minutes at 4C, followed by staining with secondary antibodies. (C) Deletion mutants of sPDPN were analyzed using flow
cytometry. Mutants were incubated with PMab-256 (anti-sPDPN mAb; red line) or buffer control (black line) for 30 minutes
at 4C, followed by staining with secondary antibodies. mAb, monoclonal antibody; sPDPN, sheep podoplanin.
96

Downloaded by Yukinari Kato from www.liebertpub.com at 06/24/20. For personal use only.

FIG. 2. Epitope mapping of PMab-256 using point mutations. (A) Transient point mutants expressing G75A, E76A,D77A, L78A, P79A, T80A, A81G, E82A, S83A, T84A, T85A, and A86G of sPDPN were incubated with PMab-2 (antiRAP14 tag; red line), or buffer control (black line) for 30 minutes at 4C, followed by staining with secondary antibodies.
(B) Transient point mutants expressing G75A, E76A, D77A, L78A, P79A, T80A, A81G, E82A, S83A, T84A, T85A, andA86G of sPDPN were incubated with LpMab-7 (anti-RAP14 tag; red line) or buffer control (black line) for 30 minutes at4C, followed by staining with secondary antibodies. (C) Transient point mutants expressing G75A, E76A, D77A, L78A,
P79A, T80A, A81G, E82A, S83A, T84A, T85A, and A86G of sPDPN were incubated with PMab-256 (anti-sPDPN mAb;red line) or buffer control (black line) for 30 minutes at 4C, followed by staining with secondary antibodies.
97

Downloaded by Yukinari Kato from www.liebertpub.com at 06/24/20. For personal use only.

98

KATO ET AL.

FIG. 3. Schematic illustration of the epitope recognized by PMab-256. (A) Illustration of WT sPDPN and its sevendeletion mutants: dN30, dN40, dN50, dN60, dN70, dN75, and dN80. Black bars: positive reactions of PMab-256. White
bar: negative reaction of PMab-256. (B) A red amino acid indicates a critical epitope of PMab-256. PLD, plateletaggregation-stimulating-like domain; WT, wild type.
desired mutations were inserted into the pCAG vector using
the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan).

acted with all point mutants (Fig. 2A). By contrast, PMab256 did not react with T80A (Fig. 2B). Thr80 of sPDPN isessential for PMab-256 binding (Fig. 3).

Flow cytometry

Transiently transfected CHO-K1 cells were detached by
0.25% trypsin/1 mM ethylenediaminetetraacetic acid
(EDTA; Nacalai Tesque, Inc.) and collected using 10% FBS
in RPMI 1640 medium. After washing with 0.1% bovine
serum albumin in phosphate-buffered saline, cells were incubated with anti-sPDPN antibody (PMab-256; 1 lg/mL) andan anti-RAP14 tag antibody (PMab-2; 1 lg/mL or LpMab-7;1 lg/mL) for 30 minutes at 4C. Alexa Fluor 488-conjugatedantimouse IgG (1:2000; Cell Signaling Technology, Inc.,Danvers, MA) was added to each cell suspension and incubation continued for 30 minutes at 4C. Fluorescence data
were collected and analyzed using a Cell Analyzer EC800(Sony Corp., Tokyo, Japan).
Results

We produced seven deletion mutants of sPDPN in CHOK1 cells, namely, dN30, with aa 30–168 deletion; dN40, aa
40–168; dN50, aa 50–168; dN60, aa 60–168; dN70, aa 70–168; dN75, aa 75–168; dN80, aa 80–168; or wild type (WT)sPDPN, aa 27–168. All deletion mutants and WT containing
the N-terminal RAP14 tag were recognized by PMab-2 orLpMab-7 (anti-RAP14 tag mAbs), indicating that the expression level of each construct is high (Fig. 1A). PMab-256
recognized dN30, dN40, dN50, dN60, dN70, and dN75, but
not dN80 (Fig. 1B), suggesting that the N-terminus of thePMab-256 epitope exists between sPDPN aa 75 and 80.
Next, a series of point mutants of sPDPN, G75A, E76A,
D77A, L78A, P79A, T80A, A81G, E82A, S83A, T84A,
T85A, and A86G, were obtained. PMab-2 and LpMab-7 re-Discussion

A variety of mAbs against pig,(17,18) horse,(19,20) Tasmaniandevil,(21) alpaca,(22) tiger,(23) whale,(24) goat,(25,26) bear,(27,28)
and sheep(29) PDPNs using the CBIS method are available.(6–8)
An anti-sPDPN mAb PMab-256 is useful for immunohistochemical analyses using formalin-fixed paraffin-embedded
tissues of sheep for detecting LECs of many organs, type Ialveolar cells, and renal epithelial cells.(29) PMab-256 is also
useful for Western blot analysis, indicating that PMab-256
recognizes sPDPN that is fixed in formalin and denatured bysodium dodecyl sulfate. Furthermore, PMab-256 detects
sPDPN in flow cytometry.
PLAG and PLD domains bind C-type lectin-like receptor2 and induce platelet aggregation and cancer metastasis.(30)
Anti-PDPN mAbs, epitopes of which are located in PLAG
domains or PLD, could neutralize platelet aggregation.(30)
The epitope of PMab-256 is also located in PLD of sPDPN.
PMab-256 is thus not only a useful mAb for research but
also could be a functional mAb with the ability to neutralize
induction of platelet aggregation.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported, in part, by AMED underGrant Nos. JP20am0401013 (Y.K.), JP20am0101078 (Y.K.),
and JP20ae0101028 (Y.K.), and by JSPS KAKENHI Grant
No. 17K07299 (M.K.K.) and Grant No. 19K07705 (Y.K.).

EPITOPE OF PMAB-256

Downloaded by Yukinari Kato from www.liebertpub.com at 06/24/20. For personal use only.

References

1. Kato Y, and Kaneko MK: A cancer-specific monoclonal
antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep 2014;4:5924.
2. Yamada S, Itai S, Kaneko MK, Konnai S, and Kato Y:Epitope mapping of anti-mouse podoplanin monoclonal
antibody PMab-1. Biochem Biophys Rep 2018;15:52–56.
3. Sayama Y, Sano M, Kaneko MK, and Kato Y: Epitope
analysis of an anti-whale podoplanin monoclonal antibody,PMab-237, using flow cytometry. Monoclon Antib Immunodiagn Immunother 2020;39:17–22.
4. Breiteneder-Geleff S, Soleiman A, Kowalski H, Horvat R,Amann G, Kriehuber E, Diem K, Weninger W, Tschachler
E, Alitalo K, and Kerjaschki D: Angiosarcomas express
mixed endothelial phenotypes of blood and lymphatic
capillaries: Podoplanin as a specific marker for lymphatic
endothelium. Am J Pathol 1999;154:385–394.
5. Rishi AK, Joyce-Brady M, Fisher J, Dobbs LG, Floros J,VanderSpek J, Brody JS, and Williams MC: Cloning,
characterization, and development expression of a rat lungalveolar type I cell gene in embryonic endodermal andneural derivatives. Dev Biol 1995;167:294–306.
6. Itai S, Fujii Y, Nakamura T, Chang YW, Yanaka M, Saidoh
N, Handa S, Suzuki H, Harada H, Yamada S, Kaneko MK,and Kato Y: Establishment of CMab-43, a sensitive andspecific anti-CD133 monoclonal antibody, for immunohistochemistry. Monoclon Antib Immunodiagn Immunother2017;36:231–235.
7. Yamada S, Itai S, Nakamura T, Yanaka M, Chang YW,Suzuki H, Kaneko MK, and Kato Y: Monoclonal antibody L1Mab-13 detected human PD-L1 in lung cancers.
Monoclon Antib Immunodiagn Immunother 2018;37:110–115.
8. Yamada S, Itai S, Nakamura T, Yanaka M, Kaneko MK,and Kato Y: Detection of high CD44 expression in oral
cancers using the novel monoclonal antibody, C44Mab-5.
Biochem Biophys Rep 2018;14:64–68.
9. Breiteneder-Geleff S, Matsui K, Soleiman A, Meraner P,
Poczewski H, Kalt R, Schaffner G, and Kerjaschki D: Podoplanin, novel 43-kd membrane protein of glomerularepithelial cells, is down-regulated in puromycin nephrosis.
Am J Pathol 1997;151:1141–1152.
10. Kaneko MK, Kato Y, Kitano T, and Osawa M: Conservation of a platelet activating domain of Aggrus/podoplanin
as a platelet aggregation-inducing factor. Gene 2006;378:52–57.
11. Kato Y, Sayama Y, Sano M, and Kaneko MK: Epitope
analysis of an antihorse podoplanin monoclonal antibody
PMab-219. Monoclon Antib Immunodiagn Immunother2019;38:266–270.
12. Kato Y, Takei J, Furusawa Y, Sayama Y, Sano M, Konnai
S, Kobayashi A, Harada H, Takahashi M, Suzuki H, Yamada S, and Kaneko MK: Epitope mapping of anti-bearpodoplanin monoclonal antibody PMab-247. Monoclon
Antib Immunodiagn Immunother 2019;38:230–233.
13. Sano M, Kaneko MK, and Kato Y: Epitope mapping ofmonoclonal antibody PMab-233 against tasmanian devil
podoplanin. Monoclon Antib Immunodiagn Immunother2019;38:261–265.
14. Sayama Y, Sano M, Furusawa Y, Kaneko MK, and Kato Y:
Epitope mapping of PMab-225 an anti-alpaca podoplanin
monoclonal antibody using flow cytometry. Monoclon
Antib Immunodiagn Immunother 2019;38:255–260.

99

15. Kato Y, Yamada S, Itai S, Kobayashi A, Konnai S, andKaneko MK: Immunohistochemical detection of sheep
podoplanin using an antibovine podoplanin monoclonal
antibody PMab-44. Monoclon. Antib. Immunodiagn. Immunother 2018;37:265–268.
16. Oki H, Honma R, Ogasawara S, Fujii Y, Liu X, Takagi M,
Kaneko MK, and Kato Y: Development of sensitive
monoclonal antibody PMab-2 against rat podoplanin.
Monoclon Antib Immunodiagn Immunother 2015;34:396–403.
17. Kato Y, Yamada S, Furusawa Y, Itai S, Nakamura T, Yanaka M, Sano M, Harada H, Fukui M, and Kaneko MK:
PMab-213: A monoclonal antibody for immunohistochemical analysis against pig podoplanin. Monoclon AntibImmunodiagn Immunother 2019;38:18–24.
18. Furusawa Y, Yamada S, Itai S, Sano M, Nakamura T,
Yanaka M, Fukui M, Harada H, Mizuno T, Sakai Y, Takasu
M, Kaneko MK, and Kato Y: PMab-210: A monoclonal
antibody against pig podoplanin monoclon. Antib Immunodiagn Immunother 2019;38:30–36.
19. Furusawa Y, Yamada S, Itai S, Sano M, Nakamura T,Yanaka M, Handa S, Mizuno T, Maeda K, Fukui M, Harada H, Kaneko MK, and Kato Y: Establishment of monoclonal antibody PMab-202 against horse podoplanin.
Monoclon Antib Immunodiagn Immunother 2018;37:233–237.
20. Kato Y, Yamada S, Itai S, Kobayashi A, Konnai S, andKaneko MK: Anti-horse podoplanin monoclonal antibody
PMab-219 is useful for detecting lymphatic endothelial
cells by immunohistochemical analysis. Monoclon AntibImmunodiagn Immunother 2018;37:272–274.
21. Furusawa Y, Yamada S, Itai S, Nakamura T, Takei J, SanoM, Harada H, Fukui M, Kaneko MK, and Kato Y: Establishment of a monoclonal antibody PMab-233 for immunohistochemical analysis against Tasmanian devil podoplanin.
Biochem Biophys Rep 2019;18:100631.
22. Kato Y, Furusawa Y, Yamada S, Itai S, Takei J, Sano M,and Kaneko MK: Establishment of a monoclonal antibody
PMab-225 against alpaca podoplanin for immunohistochemical analyses. Biochem Biophys Rep 2019;18:100633.
23. Furusawa Y, Kaneko MK, Nakamura T, Itai S, Fukui M,Harada H, Yamada S, and Kato Y: Establishment of amonoclonal antibody PMab-231 for tiger podoplanin.
Monoclon Antib Immunodiagn Immunother 2019;38:89–95.
24. Kato Y, Furusawa Y, Itai S, Takei J, Nakamura T, Sano M,Harada H, Yamada S, and Kaneko MK: Establishment ofan anticetacean podoplanin monoclonal antibody PMab237 for immunohistochemical analysis. Monoclon AntibImmunodiagn Immunother 2019;38:108–113.
25. Furusawa Y, Yamada S, Nakamura T, Sano M, Sayama Y,
Itai S, Takei J, Harada H, Fukui M, Kaneko MK, and Kato
Y: PMab-235: A monoclonal antibody for immunohistochemical analysis against goat podoplanin. Heliyon 2019;5:e02063.
26. Takei J, Itai S, Harada H, Furusawa Y, Miwa T, Fukui M,Nakamura T, Sano M, Sayama Y, Yanaka M, Handa S,
Hisamatsu K, Nakamura Y, Yamada S, Kaneko K, M., andKato Y: Characterization of anti-goat podoplanin monoclonal antibody PMab-235 using immunohistochemistry
against goat tissues. Monoclon Antib Immunodiagn Immunother 2019; DOI: 10.1089/mab.2019.0022.
27. Takei J, Furusawa Y, Yamada S, Nakamura T, Sayama Y,Sano M, Konnai S, Kobayashi A, Harada H, Kaneko MK,

100

Downloaded by Yukinari Kato from www.liebertpub.com at 06/24/20. For personal use only.

and Kato Y: PMab-247 detects bear podoplanin in immunohistochemical analysis. Monoclon Antib Immunodiagn
Immunother 2019;38:171–174.
28. Takei J, Yamada S, Konnai S, Ishinazaka T, Shimozuru M,
Kaneko MK, and Kato Y: PMab-241 specifically detects
bear podoplanin of lymphatic endothelial cells in the lungof brown bear. Monoclon Antib Immunodiagn Immunother2019;38:282–284.
29. Kato Y, Furusawa Y, Sano M, Takei J, Nakamura T,
Yanaka M, Okamoto S, Handa S, Komatsu Y, Asano T,
Sayama Y, and Kaneko MK: Development of an antisheep podoplanin monoclonal antibody PMab-256 forimmunohistochemical analysis of lymphatic endothelial
cells. Monoclon Antib Immunodiagn Immunother 2020;
39:82–90.
30. Kato Y, Kaneko MK, Kunita A, Ito H, Kameyama A,
Ogasawara S, Matsuura N, Hasegawa Y, Suzuki-Inoue K,
Inoue O, Ozaki Y, and Narimatsu H: Molecular analysis ofKATO ET AL.

the pathophysiological binding of the platelet aggregationinducing factor podoplanin to the C-type lectin-like receptor CLEC-2. Cancer Sci 2008;99:54–61.

Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery CenterTohoku University
2-1 Seiryo-machi
Aoba-ku
Sendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: March 13, 2020
Accepted: April 19, 2020