MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 37, Number 2, 2018ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2017.0067

Epitope Mapping of Monoclonal Antibody PMab-52
Against Cat Podoplanin

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Yao-Wen Chang,1 Mika K. Kaneko,1 Shinji Yamada,1 and Yukinari Kato1,2

The mucin-type membrane glycoprotein podoplanin (PDPN) is frequently overexpressed in numerous malignant cancers, including squamous cell carcinoma, germinal neoplasia, mesothelioma, lung cancer, oral cancer,
and brain tumor. PDPN expression is strongly associated with cancer progression and poor prognosis. Furthermore, PDPN binds to C-type lectin-like receptor 2 (CLEC-2) on platelets, followed by PDPN-mediatedplatelet aggregation to facilitate tumor metastasis. We have previously reported a novel anti-cat PDPN (cPDPN)
monoclonal antibody (mAb), PMab-52, which specifically detects cPDPN using flow cytometry analysis andsuccessfully identifies cPDPN in feline squamous cell carcinomas. However, the specific binding epitope ofcPDPN for PMab-52 remains unelucidated. In this study, a series of deletion or point mutants of cPDPN were
utilized for investigating the binding epitopes of PMab-52 using flow cytometry and Western blotting. The
findings of this study revealed that the critical epitopes of platelet aggregation-stimulating domain 4 (PLAG4)
of cPDPN are responsible for the binding of PMab-52 to cPDPN.
Keywords: cat podoplanin, monoclonal antibody, epitope
cytometry analysis and successfully recognizes cPDPN in
feline squamous cell carcinomas.(32) However, the specificbinding region of cPDPN for PMab-52 remains to be elucidated. In this study, we investigated the binding epitopes ofPMab-52 by analyzing a series of deletion or point mutants ofcPDPN using flow cytometry and Western blotting.

IntroductionP

odoplanin (PDPN/T1a/Aggrus), a highly Oglycosylated transmembrane glycoprotein,(1–3) is extensively distributed in normal tissues and cells, including
kidney podocytes, lung alveolar cells, lymphatic endothelial
cells, myofibroblasts, mesothelial cells, heart, and central
nervous system.(4–8) Versatile physiological functions of
PDPN have been reported to play crucial roles in embryoniccardiac development,(8,9) blood/lymphatic vessel separation,(10,11) and high endothelial venule integrity.(12) However,a high level of PDPN expression has been observed in several
malignant tumors, including brain tumors,(13,14) oral cancers,(15) lung cancers,(16) melanomas,(17) mesotheliomas,(5)breast cancers,(18,19) central nervous system tumors,(20) andosteosarcomas.(21–23) Clinical data also indicated the expression of PDPN to be correlated with poor prognosis and tumor
malignancy in lung carcinomas, oral squamous cell carcinomas, and breast cancers.(8,24–27) Furthermore, PDPN facilitates
hematogenous metastasis by eliciting tumor cell-induced
platelet aggregation response through its interaction withplatelet C-type lectin-like receptor 2 (CLEC-2).(28–31) These
pieces of evidence imply the importance of developing antiPDPN monoclonal antibodies (mAbs) for cancer therapeutic
treatment.
We have recently established a novel PMab-52 mAb,
which specifically detects cat PDPN (cPDPN) using flow
1
2

Materials and Methods
Cell lines

Chinese hamster ovary (CHO)-K1 was purchased from theAmerican Type Culture Collection (ATCC, Manassas, VA).
The cPDPN mutation plasmids containing MAP tag weretransfected into CHO-K1 cells using Lipofectamine LTX(Thermo Fisher Scientific, Inc., Waltham, MA). Transiently
transfected cells with delete/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.
Production of cPDPN mutants

The cDNA of cPDPN was subcloned into a pCAG vector(Wako Pure Chemical Industries Ltd., Osaka, Japan), and an

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

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MAP tag was added at the N-terminus. Deletion mutants ofcPDPN sequence were performed using a HotStar HiFidelity
PCR (Qiagen, Inc., Hilden, Germany) with oligonucleotides
containing the desired mutations. Substitutions of aminoacids to alanine in cPDPN sequence were conducted by
QuikChange lightning site-directed mutagenesis kit (Agilent
Technologies, Inc., Santa Clara, CA). PCR fragments bearing
the desired mutations were inserted into pCAG vector using
In-Fusion PCR cloning kit (Clontech, Palo Alto, CA).
Flow cytometry

tibody (PMab-52; 1 mg/mL) or control anti-MAP tag antibody(PMab-1; 1 mg/mL) for 30 minutes at 4C followed by treatment with Alexa Fluor 488-conjugated antimouse IgG (1:1000;
Cell Signaling Technology, Inc., Danvers, MA) and Oregon
Green-conjugated antirat IgG (1:1000; Thermo Fisher Scientific, Inc.), respectively. Fluorescence data were collected using
a Cell Analyzer EC800 (Sony Corp., Tokyo, Japan).
Western blot analysis

Whole cell lysates of the deletion and point mutants werecollected, lysed, and then boiled in sodium dodecyl sulfate(SDS) sample buffer (Nacalai Tesque, Inc.). The extracted
proteins were electrophoresed using 5%–20% SDSpolyacrylamide gel electrophoresis (SDS-PAGE) (Wako Pure
Chemical Industries Ltd.) and transferred to polyvinylidene
difluoride (PVDF) membranes (Merck KGaA, Darmstadt,

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Transiently transfected CHO-K1 cells were detached by
0.25% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA)(Nacalai Tesque, Inc.) and collected in RPMI 1640 medium.
After washing with 0.1% bovine serum albumin /phosphatebuffered saline, the cells were incubated with anti-cPDPN an-

CHANG ET AL.

FIG. 1. Epitope mapping of PMab-52 using deletion mutants of cPDPN. (A) Illustration of nine cPDPN deletion mutantsof dN23, dN37, dN46, dN55, dN65, dN75, dN85, dN95, and dN105. (B) Deletion mutants of cPDPN were analyzed using
flow cytometry. Deletion mutants were expressed on CHO-K1 cells and were then incubated with anti-MAP tag PMab-1(left panel, red line), PMab-52 (right panel, blue line), or buffer control (black line) for 30 minutes at 4C, followed bytreatment with corresponding secondary antibodies. CHO, Chinese hamster ovary; cPDPN, cat PDPN; PLAG4, platelet
aggregation-stimulating domain 4.

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FIG. 2. Epitope mapping of PMab-52 using point mutants of cPDPN. (A, B) Flow cytometry. Transient point mutants
expressing H80A, I81A, E82A, D83A, G84A, P85A, T86A, Q87A, E88A, S89A, and T90A of cPDPN reacted with PMab-1(A) or PMab-52 (B) for 30 minutes at 4C, followed by treatment with corresponding secondary antibodies. (C) The celllysates from point mutants were collected for Western blotting with anti-cPDPN (PMab-52) antibody. The anti-MAP tag(PMab-1) antibody was used as a control. (D) Schematic illustration of the epitope recognized by PMab-52.
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Germany). The membranes were blocked with 4% skim milk
(Nacalai Tesque, Inc.) for 1 hour and then were incubated with
1 mg/mL of PMab-52 and 1 mg/mL of anti-MAP tag (PMab-1),
then with peroxidase-conjugated antimouse and antirat IgG(1:1000; Agilent Technologies, Inc.), respectively. The proteins were finally detected using ImmunoStar LD (Wako Pure
Chemical Industries Ltd.) with a Sayaca-Imager (DRC Co.
Ltd., Tokyo, Japan).

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Results and Discussion

We have previously established a novel PMab-52 mAb,
which can be efficiently utilized for the immunohistochemical
detection of cPDPN in normal feline tissues, including kidney,lung, and rectum.(32) Furthermore, PMab-52 specifically recognized cPDPN expression in feline squamous cell carcinomas, including carcinomas of the mouth floor, skin, ear, andtongue.(33) Flow cytometry and Western blotting analyses also
revealed that PMab-52 specifically detects only cPDPN, not
PDPNs of other species such as humans, dogs, cattle, rabbits,
and mice.(32) On the basis of these results, the epitope mappingof PMab-52 could be beneficial in uncovering the pathophysiological function of cPDPN in feline squamous cell carcinomas and cPDPN-related antibody-based therapy.
First, we constructed nine deletion mutants of cPDPN(Fig. 1A). Transient transfections of cPDPN-mutant clones
were produced using CHO-K1 cells, including dN23 (corresponding to 23–163 amino acids [aa]); dN37 (corresponding to 37–163 aa); dN46 (corresponding to 46–163 aa); dN55
(corresponding to 55–163 aa); dN65 (corresponding to 65–
163 aa); dN75 (corresponding to 75–163 aa); dN85 (corresponding to 85–163 aa); dN95 (corresponding to 95–163 aa);and dN105 (corresponding to 105–163 aa). All deletionmutants of cPDPN contain N-terminal MAP tags and wereanalyzed using flow cytometry for epitope mapping of PMab52. PMab-1 (anti-MAP tag mAb) detected all deletion mutants of cPDPN, including dN23, dN37, dN46, dN55, dN65,
dN75, dN85, dN95, and dN105 (Fig. 1B, left). On the contrary, PMab-52 lost the reaction with dN85, dN95, and dN105(Fig. 1B, right). Further experiments revealed that PMab-52
could detect deletion mutants of dN80 of cPDPN (data notshown). These results imply that the epitope-binding regionof PMab-52 is located between the 80th and 90th amino acids, which contain new identified platelet aggregationstimulating domain 4 (PLAG4), which plays a crucial role in
PDPN-mediated cancer metastasis.(34)
Next, we investigated the epitope-binding region of PMab52 by producing 11 point mutants of cPDPN, including H80A,
I81A, E82A, D83A, G84A, P85A, T86A, Q87A, E88A, S89A,
and T90A. All these mutants can be recognized by PMab-1(Fig. 2A). Remarkably, we observed that PMab-52 reacted withall these mutants, except with Q87A and E88A using flow
cytometry (Fig. 2B).
To identify the epitope of cPDPN for PMab-52 binding, we
also performed Western blotting using these point mutants.
The results revealed that PMab-52 could not recognize thepoint mutants of D83A-P85A and Q87A-E88A (Fig. 2C),
which are located in the PLAG4 domain (82-EDGPTQE-88).
Both these results further confirmed that PMab-52 interacts
with certain crucial amino acid residues (Asp83, Gly84,
Pro85, Gln87, and Glu88) of the PLAG4 domain (Fig. 2D).

CHANG ET AL.

In conclusion, through this study, we characterized the
crucial binding region of PLAG4 domain of cPDPN for PMab52-specific binding. PMab-52 can be a useful tool in elucidating the pathophysiological function of cPDPN.
Acknowledgments

This research was supported in part by AMED under Grant
Numbers: JP17am0301010 (Y.K.), JP17am0101078 (Y.K.), andJP17ae0101028 (Y.K.), and by JSPS KAKENHI Grant Number
17K07299 (M.K.K.) and Grant Number 16K10748 (Y.K.). This
work was performed in part under the Cooperative Research
Program of Institute for Protein Research, Osaka University, CR17-05 and by the Grant for Joint Research Project of the Instituteof Medical Science, the University of Tokyo.
Author Disclosure Statement

Y.K. received research funding from Ono PharmaceuticalCo., Ltd. All other authors have nothing to disclose.
References

1. Kato Y, Fujita N, Kunita A, Sato S, Kaneko M, Osawa M,
and Tsuruo T: Molecular identification of Aggrus/T1alphaas a platelet aggregation-inducing factor expressed in colorectal tumors. J Biol Chem 2003;278:51599–51605.
2. Martin-Villar E, Scholl FG, Gamallo C, Yurrita MM,
Munoz-Guerra M, Cruces J, and Quintanilla M: Characterization of human PA2.26 antigen (T1alpha-2, podoplanin), a small membrane mucin induced in oral squamouscell carcinomas. Int J Cancer 2005;113:899–910.
3. Kaneko MK, Kato Y, Kitano T, and Osawa M: Conservationof a platelet activating domain of Aggrus/podoplanin as aplatelet aggregation-inducing factor. Gene 2006;378:52–57.
4. Matsui K, Breitender-Geleff S, Soleiman A, Kowalski H,
and Kerjaschki D: Podoplanin, a novel 43-kDa membraneprotein, controls the shape of podocytes. Nephrol Dial
Transplant 1999;14 Suppl 1:9–11.
5. Kimura N, and Kimura I: Podoplanin as a marker for mesothelioma. Pathol Int 2005;55:83–86.
6. 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.
7. Ugorski M, Dziegiel P, and Suchanski J: Podoplanin—Asmall glycoprotein with many faces. Am J Cancer Res 2016;
6:370–386.
8. Astarita JL, Acton SE, and Turley SJ: Podoplanin: Emerging functions in development, the immune system, andcancer. Front Immunol 2012;3:283.
9. Mahtab EA, Wijffels MC, Van Den Akker NM, Hahurij
ND, Lie-Venema H, Wisse LJ, Deruiter MC, Uhrin P,
Zaujec J, Binder BR, Schalij MJ, Poelmann RE, andGittenberger-De Groot AC: Cardiac malformations andmyocardial abnormalities in podoplanin knockout mouseembryos: Correlation with abnormal epicardial development. Dev Dyn 2008;237:847–857.
10. Bertozzi CC, Schmaier AA, Mericko P, Hess PR, Zou Z,
Chen M, Chen CY, Xu B, Lu MM, Zhou D, Sebzda E,
Santore MT, Merianos DJ, Stadtfeld M, Flake AW, Graf T,
Skoda R, Maltzman JS, Koretzky GA, and Kahn ML:
Platelets regulate lymphatic vascular development through
CLEC-2-SLP-76 signaling. Blood 2010;116:661–670.

Downloaded by Yukinari Kato from www.liebertpub.com at 04/30/18. For personal use only.

EPITOPE OF PMAB-52

11. Hess PR, Rawnsley DR, Jakus Z, Yang Y, Sweet DT, Fu J,
Herzog B, Lu M, Nieswandt B, Oliver G, Makinen T, Xia
L, and Kahn ML: Platelets mediate lymphovenous hemostasis to maintain blood-lymphatic separation throughout
life. J Clin Invest 2014;124:273–284.
12. Herzog BH, Fu J, Wilson SJ, Hess PR, Sen A, McDaniel
JM, Pan Y, Sheng M, Yago T, Silasi-Mansat R, McGee S,
May F, Nieswandt B, Morris AJ, Lupu F, Coughlin SR,
McEver RP, Chen H, Kahn ML, and Xia L: Podoplaninmaintains high endothelial venule integrity by interactingwith platelet CLEC-2. Nature 2013;502:105–109.
13. Kato Y, Vaidyanathan G, Kaneko MK, Mishima K, Srivastava N, Chandramohan V, Pegram C, Keir ST, Kuan
CT, Bigner DD, and Zalutsky MR: Evaluation of antipodoplanin rat monoclonal antibody NZ-1 for targeting
malignant gliomas. Nucl Med Biol 2010;37:785–794.
14. Kato Y, and Kaneko MK: A cancer-specific monoclonal
antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep 2014;4:5924.
15. Ochoa-Alvarez JA, Krishnan H, Pastorino JG, Nevel E,
Kephart D, Lee JJ, Retzbach EP, Shen Y, Fatahzadeh M,
Baredes S, Kalyoussef E, Honma M, Adelson ME, Kaneko
MK, Kato Y, Young MA, Deluca-Rapone L, Shienbaum
AJ, Yin K, Jensen LD, and Goldberg GS: Antibody andlectin target podoplanin to inhibit oral squamous carcinoma
cell migration and viability by distinct mechanisms. Oncotarget 2015;6:9045–9060.
16. Kato Y, Kaneko M, Sata M, Fujita N, Tsuruo T, and Osawa
M: Enhanced expression of Aggrus (T1alpha/podoplanin),a platelet-aggregation-inducing factor in lung squamouscell carcinoma. Tumour Biol 2005;26:195–200.
17. Abe S, Kaneko MK, Tsuchihashi Y, Izumi T, Ogasawara S,
Okada N, Sato C, Tobiume M, Otsuka K, Miyamoto L,
Tsuchiya K, Kawazoe K, Kato Y, and Nishioka Y: Antitumor effect of novel anti-podoplanin antibody NZ-12
against malignant pleural mesothelioma in an orthotopic
xenograft model. Cancer Sci 2016;107:1198–1205.
18. Wahal SP, Goel MM, and Mehrotra R: Lymphatic vessel
assessment by podoplanin (D2-40) immunohistochemistryin breast cancer. J Cancer Res Ther 2015;11:798–804.
19. Schoppmann SF, Berghoff A, Dinhof C, Jakesz R, Gnant
M, Dubsky P, Jesch B, Heinzl H, and Birner P: Podoplaninexpressing cancer-associated fibroblasts are associated withpoor prognosis in invasive breast cancer. Breast Cancer Res
Treat 2012;134:237–244.
20. Mishima K, Kato Y, Kaneko MK, Nakazawa Y, Kunita A,
Fujita N, Tsuruo T, Nishikawa R, Hirose T, and Matsutani M:
Podoplanin expression in primary central nervous systemgerm cell tumors: A useful histological marker for the diagnosis of germinoma. Acta Neuropathol 2006;111:563–568.
21. Kunita A, Kashima TG, Ohazama A, Grigoriadis AE, andFukayama M: Podoplanin is regulated by AP-1 and promotes platelet aggregation and cell migration in osteosarcoma. Am J Pathol 2011;179:1041–1049.
22. Ariizumi T, Ogose A, Kawashima H, Hotta T, Li G, Xu Y,
Umezu H, Sugai M, and Endo N: Expression of podoplanin inhuman bone and bone tumors: New marker of osteogenic andchondrogenic bone tumors. Pathol Int 2010;60:193–202.
23. Kaneko MK, Oki H, Ogasawara S, Takagi M, and Kato Y:
Anti-podoplanin monoclonal antibody LpMab-7 detects
metastatic lesions of osteosarcoma. Monoclon Antib Immunodiagn Immunother 2015;34:154–161.
24. Xu Y, Ogose A, Kawashima H, Hotta T, Ariizumi T, Li G,
Umezu H, and Endo N: High-level expression of podo-

99

planin in benign and malignant soft tissue tumors: Immunohistochemical and quantitative real-time RT-PCR
analysis. Oncol Rep 2011;25:599–607.
25. Yuan P, Temam S, El-Naggar A, Zhou X, Liu DD, Lee JJ,
and Mao L: Overexpression of podoplanin in oral cancer
and its association with poor clinical outcome. Cancer
2006;107:563–569.
26. Yurugi Y, Wakahara M, Matsuoka Y, Sakabe T, Kubouchi
Y, Haruki T, Nosaka K, Miwa K, Araki K, Taniguchi Y,
Shiomi T, Nakamura H, and Umekita Y: Podoplanin expression in cancer-associated fibroblasts predicts poor
prognosis in patients with squamous cell carcinoma of thelung. Anticancer Res 2017;37:207–213.
27. Pula B, Jethon A, Piotrowska A, Gomulkiewicz A, Owczarek T,
Calik J, Wojnar A, Witkiewicz W, Rys J, Ugorski M, DziegielP, and Podhorska-Okolow M: Podoplanin expression bycancer-associated fibroblasts predicts poor outcome in invasive
ductal breast carcinoma. Histopathology 2011;59:1249–1260.
28. 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 of
the pathophysiological binding of the platelet aggregationinducing factor podoplanin to the C-type lectin-like receptor CLEC-2. Cancer Sci 2008;99:54–61.
29. Chang YW, Hsieh PW, Chang YT, Lu MH, Huang TF,
Chong KY, Liao HR, Cheng JC, and Tseng CP: Identification of a novel platelet antagonist that binds to CLEC-2
and suppresses podoplanin-induced platelet aggregationand cancer metastasis. Oncotarget 2015;6:42733–42748.
30. Kunita A, Kashima TG, Morishita Y, Fukayama M, Kato
Y, Tsuruo T, and Fujita N: The platelet aggregationinducing factor aggrus/podoplanin promotes pulmonary
metastasis. Am J Pathol 2007;170:1337–1347.
31. Kaneko MK, Kunita A, Abe S, Tsujimoto Y, Fukayama M,
Goto K, Sawa Y, Nishioka Y, and Kato Y: Chimeric antipodoplanin antibody suppresses tumor metastasis throughneutralization and antibody-dependent cellular cytotoxicity.
Cancer Sci 2012;103:1913–1919.
32. Yamada S, Itai S, Nakamura T, Yanaka M, Saidoh N, Chang
YW, Handa S, Harada H, Kagawa Y, Ichii O, Konnai S, Kaneko
MK, and Kato Y: PMab-52: Specific and sensitive monoclonal
antibody against cat podoplanin for immunohistochemistry.
Monoclon Antib Immunodiagn Immunother 2017;36:224–230.
33. Itai S, Yamada S, Kaneko MK, Harada H, Kagawa Y,
Konnai S, and Kato Y: Expression of cat podoplanin infeline squamous cell carcinomas. Monoclon Antib Immunodiagn Immunother 2017;36:243–250.
34. Sekiguchi T, Takemoto A, Takagi S, Takatori K, Sato S,
Takami M, and Fujita N: Targeting a novel domain in
podoplanin for inhibiting platelet-mediated tumor metastasis. Oncotarget 2016;7:3934–3946.

Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1 Seiryo-machi
Aoba-ku
Sendai 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp
Received: December 7, 2017
Accepted: January 4, 2018