MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 6, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0034

Epitope Analysis of an Antihorse Podoplanin
Monoclonal Antibody PMab-219

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

Podoplanin (PDPN), which is a mucin-type membrane glycoprotein, is expressed on lymphatic endothelial cells andepithelial cells of many organs. PDPN is also overexpressed in several malignant cancers, and its expression isassociated with cancer progression and poor prognosis. Human PDPN possesses three platelet aggregation-stimulating(PLAG) domains and the PLAG-like domain (PLD), which binds to C-type lectin-like receptor-2 (CLEC-2). Previously, we reported a novel antihorse PDPN (horPDPN) monoclonal antibody (mAb), PMab-219, using Cell-Based
Immunization and Screening (CBIS) method. PMab-219 specifically detected horPDPN-overexpressed Chinese
hamster ovary (CHO)-K1 (CHO/horPDPN) cells and FHK-TcL3.1, a horse kidney cell line, using flow cytometry. Inaddition, PMab-219 strongly stained horse tissues such as renal podocytes or lymphatic endothelial cells by immunohistochemistry. However, the specific binding epitope of PMab-219 for horPDPN remains to be clarified. In thisstudy, a series of deletion mutants or point mutants of horPDPN were produced for analyzing the PMab-219 epitope
using flow cytometry. The analysis of deletion mutants showed that N-terminus of PMab-219 epitope exists between
55th amino acid (aa) and 60th aa of horPDPN. Furthermore, the analysis of point mutants demonstrated that the critical
epitope of PMab-219, which was developed by CBIS method, could include Val59, Arg61, Ser62, and Thr63 ofhorPDPN, indicating that PMab-219 epitope is independent of PLAG domain or PLD of horPDPN.
Keywords: horse podoplanin, monoclonal antibody, epitope, PMab-219
IntroductionP

odoplanin (PDPN/T1alpha/Aggrus) is a type I
transmembrane glycoprotein, and is expressed in normal
tissues, such as lymphatic endothelial cells, type I lung alveolar cells, and renal podocytes.(1–3) PDPN is known as aplatelet aggregation-inducing factor in highly metastatic tumor cells.(1,4) Human PDPN (hPDPN) is frequently overexpressed in various malignant tumors, including lungcancer, mesothelioma, glioblastoma, and osteosarcoma.(5–7)
hPDPN possesses three platelet aggregation-stimulating(PLAG) domains (PLAG1, PLAG2, and PLAG3), which
exist at the N-terminus of hPDPN.(8) In addition, one PLAGlike domain (PLD) has been reported to be present in themiddle of PDPN.(9) PLAG and PLD are highly conserved
among mammalian PDPNs.(8) The interaction between PDPN
on lymphatic endothelial cells and C-type lectin-like receptor2 (CLEC-2) on platelets was shown to facilitate embryonicblood/lymphatic vessel separation.(10)In our previous study, we developed monoclonal antibodies(mAbs) against human,(11) mouse,(11) rat,(12) rabbit,(13) dog,(14)cat,(15) bovine,(16) pig,(17) Tasmanian devil,(18) alpaca,(19)bear,(20) tiger,(21) whale,(22) goat,(23) and horse PDPNs.(24) Although PMab-202, an antihorse PDPN (horPDPN) mAb, wasestablished by immunizing mice with synthetic peptides of12

horPDPN, it was not applicable for immunohistochemical
analysis.(25) Highly sensitive and specific mAbs against
horPDPN were needed to analyze horPDPN for pathologicalinvestigation using horse tissues.
Recently, we developed a novel anti-horPDPN mAb,
PMab-219, using Cell-Based Immunization and Screening(CBIS) method. PMab-219 specifically detected horPDPN byflow cytometry, Western blotting, and immunohistochemical
analysis.(24,26) However, the binding epitope of PMab-219
for horPDPN remains to be clarified. Thus, this study aimed
to identify the epitope of PMab-219 using deletion mutants
and point mutants of horPDPN using flow cytometry.
Materials and Methods
Cell lines

Chinese hamster ovary (CHO)-K1 was obtained from theAmerican Type Culture Collection (ATCC, Manassas, VA).
The horPDPN mutation plasmids containing PA16 tag weretransfected into CHO-K1 cells using Lipofectamine LTX(Thermo Fisher Scientific, Inc., Waltham, MA). The PA16tag comprises 16 amino acids (aas) (GLEGGVAMPGAEDDVV). Transiently transfected cells with deletionmutants or point mutants were cultured in Roswell Park
Memorial Institute (RPMI) 1640 medium (Nacalai Tesque,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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FIG. 1. Epitope mapping of PMab-219 using deletion mutants of horPDPN. (A) Illustration of WT and seven deletionmutants of horPDPN: dN30, dN40, dN50, dN55, dN60, dN70, and dN80. Black bars: the positive reaction of PMab-219.
White bars: the negative reaction of PMab-219. (B, C) Deletion mutants of horPDPN were analyzed using flow cytometry.
Deletion mutants were incubated with NZ-1 (anti-PA16 tag; red line, B), PMab-219 (anti-horPDPN mAb; red line, C),or buffer control (black line, B, C) for 30 min at 4C, followed by secondary antibodies. horPDPN, horse PDPN;
mAb, monoclonal antibody; PDPN, podoplanin; PLAG, platelet aggregation-stimulating; PLD, PLAG-like domain; WT,wild type.

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FIG. 2. Epitope mapping of PMab-219 using point mutants of horPDPN. (A, B) Transient point mutants expressing S55A,P56A, A57G, L58A, V59A, P60A, R61A, S62A, T63A, K64A, S65A, and T66A of horPDPN were incubated with NZ-1
(red line, A), PMab-219 (red line, B), or buffer control (black line, A, B) for 30 min at 4C, followed by secondary
antibodies. (C) Schematic illustration of the epitope recognized by PMab-219. Underlined amino acids are a critical epitopeof PMab-219.

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EPITOPE OF PMAB-219

Inc., Kyoto, Japan), supplemented with 10% heat-inactivatedfetal bovine serum (FBS; Thermo Fisher Scientific, Inc.),
100 U/mL of penicillin, 100 mg/mL of streptomycin, and25 mg/mL of amphotericin B (Nacalai Tesque, Inc.) at 37Cin a humidified atmosphere of 5% CO2 and 95% air.
Production of horPDPN mutants

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Synthesized DNA of horPDPN was subcloned into thepCAG vector (FUJIFILM Wako Pure Chemical Corporation,Osaka, Japan), and PA16 tag was added at the N-terminus.
Deletion mutants of horPDPN sequence were produced using aHotStar HiFidelity PCR (Qiagen, Inc., Hilden, Germany) witholigonucleotides. Substitutions of aas to alanine or glycine in
horPDPN 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 the pCAG vector using In-Fusion PCR
cloning kit (Takara Bio, Inc., Shiga, Japan).
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/phosphate-buffered saline, the cells were incubated with anti-horPDPN antibody (PMab-219; 1 mg/mL)or control anti-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
detection of PMab-219 or Oregon Green antirat IgG (1:2000;Thermo Fisher Scientific, Inc.) for detection of NZ-1 wasadded to each cell, and was incubated for 30 min at 4C.
Fluorescence data were collected and analyzed using a Cell
Analyzer EC800 (Sony Corp., Tokyo, Japan).

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Discussion

We have demonstrated that critical epitope of PMab-219
could include Val59, Arg61, Ser62, and Thr63 of horPDPN
using deletion mutants and point mutants of horPDPN. In ourprevious study, the epitope of PMab-202 (the other antihorPDPN mAb) was identified as Lys64, Thr66, and Phe70 of
horPDPN.(27) Although the binding epitope of both antihorPDPN mAbs was shown to exist in a similar location of
horPDPN, PMab-202 could not stain horse tissues by immunohistochemistry. These results suggested that CBIS method
is more advantageous to obtain immunohistochemistryapplicable mAbs. Likewise, we have successfully establishedimmunohistochemistry-applicable anti-PDPN mAbs for pig,(17)Tasmanian devil,(18) alpaca,(19) bear,(20) tiger,(21) whale,(22)and goat PDPNs(23) using CBIS method, but not by immunizing synthetic peptides.
Using glycan-deficient CHO cell lines, such as Lec1
(N-glycan-deficient), Lec2 (sialic acid-deficient), or Lec8
(galactose-deficient),(28) we investigated whether the epitope of PMab-219 could include not only aas but also glycans. Flow cytometric analysis demonstrated that PMab-219
strongly reacts with CHO-K1/horPDPN, Lec1/horPDPN, andLec2/horPDPN, but weakly reacts with Lec8/horPDPN (datanot shown), indicating that galactose might also be includedin the PMab-219 epitope. Among Val59, Arg61, Ser62, andThr63 of horPDPN, Ser62 and Thr63 could be O-glycosylated(Fig. 2C). In a future study, we should determine which
O-glycosylation of Ser62 and/or Thr63 is essential for
PMab-219 binding to horPDPN.
In conclusion, Val59, Arg61, Ser62, and Thr63 of horPDPN,
which are independent of PLAG domain or PLD, are critical for
PMab-219-specific binding to horPDPN. PMab-219 could be auseful tool for elucidating the pathophysiological function ofhorPDPN.
Author Disclosure StatementResults

Seven deletion mutants of horPDPN, such as dN30 (corresponding to 30–152 aas), dN40 (corresponding to 40–152aas), dN50 (corresponding to 50–152 aas), dN55 (corresponding to 55–152 aas), dN60 (corresponding to 60–152aas), dN70 (corresponding to 70–152 aas), and dN80 (corresponding to 80–152 aas) or wild type (WT) of horPDPN(corresponding to 25–152 aas) were generated using CHOK1 cells (Fig. 1A). All deletion mutants and WT of horPDPN
containing N-terminus PA16 tag were detected by NZ-1
(anti-PA16 tag mAb), indicating that the expression level ofeach construct is very high (Fig. 1B). In contrast, PMab-219
did not react with dN60, dN70, and dN80 (Fig. 1C), suggesting that N-terminus of PMab-219 epitope might exist
between 55th aa and 60th aa of horPDPN.
To identify the binding epitope of PMab-219, we next
produced a series of point mutants of horPDPN in CHO-K1
cells, including S55A, P56A, A57G, L58A, V59A, P60A,R61A, S62A, T63A, K64A, S65A, and T66A. NZ-1 reactedwith all point mutants (Fig. 2A). In contrast, PMab-219 did
not react with V59A, R61A, and S62A, and weakly recognized T63A (Fig. 2B), indicating Val59, Arg61, Ser62, andThr63 of horPDPN could be included in the critical epitope ofPMab-219 (Fig. 2C).

No competing financial interests exist.
Funding Information

This research was supported, in part, by AMED under Grant
Nos. JP19am0101078 (Y.K.), JP19am0401013 (Y.K.), andJP19ae0101028 (Y.K.), and by JSPS KAKENHI under 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: July 29, 2019
Accepted: September 24, 2019