Original Articles

MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 42, Number 6, 2023
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2023.0026

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Epitope Mapping of an Anti-ferret Podoplanin Monoclonal
Antibody Using the PA Tag-Substituted Analysis

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Yu Isoda, Mika K. Kaneko, Tomohiro Tanaka, Hiroyuki Suzuki, and Yukinari Kato

In small animal models of severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2)
infection, ferrets (Mustela putorius furo) have been used to investigate the pathogenesis. Podoplanin (PDPN) is
an essential marker in lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells.
Monoclonal antibodies (mAbs) against ferret PDPN (ferPDPN) are useful for the pathological analyses of thosetissues. We previously established an anti-ferPDPN mAb, PMab-292 using the Cell-Based Immunization andScreening (CBIS) method. In this study, we determined the critical epitope of PMab-292 using flow cytometry.
The ferPDPN deletion mutants analysis revealed that the Val34 is located at the N-terminus of the PMab-292epitope. Furthermore, the PA tag-substituted analysis (PA scanning) showed that Asp39 is located at theC-terminus of PMab-292 epitope. The epitope sequence (VRPEDD) also exists between Val26 and Asp31 offerPDPN, indicating that PMab-292 recognizes the tandem repeat of the VRPEDD sequence of ferPDPN.
Keywords: ferret podoplanin, monoclonal antibody, epitope, PA scanning
Introduction

Ferrets (Mustela putorius furo) have been used as
an animal model for investigating the transmission andpathogenicity of human severe acute respiratory syndrome
coronaviruses (SARS-CoV1 and SARS-CoV-22). In histopathological analysis, inflammation within alveolar spaces
and perivascular mononuclear parts were observed in SARSCoV-2-infected ferrets. Microscopic observations of SARSCoV-2-infected ferrets exhibited mild bronchoalveolar oralveolar inflammation.2 However, there is a limitation of thepathological analysis owing to the lack of antibodies that canrecognize the ferret-derived antigens and distinguish thespecific cells in the lung.
Podoplanin (PDPN) is a type I transmembrane mucin-likesialoglycoprotein and an important marker in lung type I
alveolar epithelial cells,3,4 kidney podocytes,5 and lymphatic
endothelial cells.6,7 PDPN possesses a heavily glycosylated
N-terminal extracellular domain, a single transmembrane
domain, and a short intracellular domain.8,9 A repeat sequence of EDxxVTPG, named PLAG domains, is present inthe N-terminal domain. ‘‘PLAG’’ is derived from the platelet
aggregation-stimulating function of PDPN.10 Furthermore,

several PLAG-like domains (PLDs, one of which is also
named PLAG4 domain) have been identified.11
Anti-ferret PDPN (ferPDPN) monoclonal antibodies(mAbs) have been proposed as a useful tool to investigate thepathogenesis of lung type I alveolar epithelial cells, kidney
podocytes, and lymphatic endothelial cells in SARS-CoV-2
infected ferret. We previously established an anti-ferPDPN
mAb, PMab-292 using the Cell-Based Immunization andScreening (CBIS) method. PMab-292 can be used for flow
cytometry, western blotting, and immunohistochemistry.12To clarify further characteristics of PMab-292, we performed
epitope mapping using flow cytometry.
Materials and Methods
Antibodies

PMab-292 (an anti-ferPDPN mAb),12 PMab-241 (an antibear PDPN mAb),13 PMab-1 (an anti-MAP tag mAb),14 andNZ-1 (an anti-PA tag mAb)15 were described previously.
Alexa Fluor 488-conjugated anti-mouse IgG and anti-rat
IgG were purchased from Cell Signaling Technology, Inc.
(Danvers, MA).

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
This paper was previously published in preprint.org (doi: 10.20944/preprints202311.1910.v1).

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Plasmid construction and transfection

Synthesized DNA (Eurofins Genomics KK, Tokyo,Japan) encoding ferPDPN plus an N-terminal MAP tag(GDGMVPPGIEDK)16,17 were subcloned into a pCAG-Blevector (FUJIFILM Wako Pure Chemical Corporation, Osaka,Japan). The ferPDPN deletion mutants (dN33, dN34, dN35,dN36, dN37, dN38, dN39, dN40, dN41, dN42, and dN43)
were produced using a KAPA HiFi HotStart ReadyMix
polymerase chain reaction (PCR) Kit (Kapa Biosystems,Wilmington, MA), and subcloned into the pCAG-Ble vector.
For PA scanning, the substitution of PA tag (GVAMPGAEDDVV) in ferPDPN dN34 was performed with oligonucleotides containing PA tag sequence at the desired position. For example, for the substitution of the PA tag from
Val34 to Asn45 of ferPDPN, we constructed Thr33GVAMPGAEDDVV-Asn46 (34-PA-45) in ferPDPN dN34.
The PCR fragments bearing the desired mutations wereinserted into the pCAG-neo vector (FUJIFILM Wako Pure
Chemical Corporation) using an In-Fusion HD Cloning Kit(Takara Bio, Inc., Shiga, Japan). Substitutions of amino acids
to alanine (or glycine) in the ferPDPN sequence were conducted by QuikChange Lightning Site-Directed Mutagenesis
Kits (Agilent Technologies, Inc., Santa Clara, CA). PCR
fragments bearing the desired mutations were inserted intothe pCAG-neo vector using the In-Fusion HD Cloning Kit(Takara Bio, Inc.).
The ferPDPN mutant plasmids were transiently transfected
into Chinese hamster ovary (CHO)-K1 cells (the American
Type Culture Collection, Manassas, VA) using the NeonTransfection System (Thermo Fisher Scientific, Inc., Waltham, MA). CHO-K1 cells and transfectants 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 FisherScientific, Inc.), 100 units/mL of penicillin, 100 lg/mL of
streptomycin, and 0.25 lg/mL of amphotericin B (NacalaiTesque, Inc.). The cells were cultured at 37C in a humidified
atmosphere containing 5% CO2 and 95% air.
Flow cytometry

CHO-K1 cells and transfectants were harvested after abrief exposure to 0.25% trypsin in 1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.) and washedwith 0.1% bovine serum albumin in phosphate-bufferedsaline. PMab-292 (1 lg/mL), PMab-241 (1 lg/mL), PMab-1(1 lg/mL), or NZ-1 (1 lg/mL) were incubated for 30 minutes
at 4C. The cells were further treated with Alexa Fluor 488conjugated anti-mouse IgG or anti-rat IgG (1:1000). Fluorescence data were collected using the SA3800 Cell Analyzer(Sony Corp., Tokyo, Japan).
Results
Epitope mapping of PMab-292 using the N-terminal
deletion mutants of ferPDPN

We previously established an anti-ferPDPN mAb (PMab292) by the CBIS method. To determine the PMab-292 epitope, we examined the reactivity to the N-terminal ferPDPN
deletion mutants (from dN33 to dN43)-overexpressed
CHO-K1 cells by flow cytometry (Fig. 1A). As shown in
Figure 1B, PMab-292 reacted with dN33 and dN34. In

FIG. 1. Epitope determination of PMab-292 using deletion mutants of ferPDPN. (A) The ferPDPN deletionmutants were transiently expressed in CHO-K1 cells. (B)The ferPDPN mutants-expressed CHO-K1 cells were incubated with 1 mg/mL of PMab-292 (B, red line) or 1 mg/mL of
PMab-241 (C, red line), or control blocking buffer (blackline), followed by secondary antibodies treatment. The data
were analyzed using the SA3800 Cell Analyzer. CHO,
Chinese hamster ovary; ferPDPN, ferret podoplanin; PLAG,
platelet aggregation-stimulating; PLD, PLAG-like domain;ss, signal sequence; TM, transmembrane domain.
contrast, the reactivity completely disappeared in dN35, dN36,dN37, dN38, dN39, dN40, dN41, dN42, and dN43. We confirmed the cell surface expression of all deletion mutants using
PMab-241, originally developed as an antibear PDPN mAb,
which cross-react with ferPDPN (epitope amino acids: Thr86,Asp87, and Arg89).18 These results indicated that the Val34
is located at the N-terminus of PMab-292 epitope.

EPITOPE MAPPING OF ANTI-FERRET PDPN MAB

fore, we could not determine the epitope of PMab-292 using
1 · alanine or 2 · alanine scanning methods.
Flow cytometry using PMab-292 with PAtag-substituted ferPDPN

As shown in Figure 1B, PMab-292 reacted with theferPDPN dN34 mutant. We next generated PA tag (GVAMPGAEDDVV)-substituted ferPDPN dN34 mutants as shownin Figure 2A to determine the C-terminus of the PMab-292

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To identify the binding epitope of PMab-292, we generated 20 alanine (or glycine)-substituted ferPDPN(Supplementary Fig. S1A) and investigated the reactivity ofPMab-292 against CHO-K1 cells, which overexpressed
the ferPDPN mutants transiently. PMab-292 reacted with allalanine (or glycine)-substituted mutants and wild type (WT)(Supplementary Fig. S1B). We also examined the reactivityof PMab-292 against 2 · alanine (or glycine)-substituted
ferPDPN (Supplementary Fig. S2A); however, PMab-292
reacted with all mutants (Supplementary Fig. S2B). There-191

FIG. 2. Epitope determination of PMab-292 using PA tag-substituted mutants of ferPDPN. (A) The ferPDPN dN34 PA tagsubstituted mutants were transiently expressed in CHO-K1 cells. (B) The mutants-expressed CHO-K1 cells were incubated with
1 mg/mL of PMab-292 (B, red line), 1 mg/mL of PMab-241 (C, red line), 1 mg/mL of NZ-1 (D, red line), or control blocking
buffer (black line), followed by secondary antibodies treatment. The data were analyzed using the SA3800 Cell Analyzer.

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ISODA ET AL.

FIG. 3. Schematic illustration of ferPDPN and the PMab-292 epitope. The PMab-292 epitope involves tandem sequence(VRPEDD) of ferPDPN. PLAG, platelet aggregation-stimulating; PLD, PLAG-like domain; TM, transmembrane domain.
epitope. As shown in Figure 2B, the reactivity of PMab-292
almost completely disappeared in 34-PA-45, 35-PA-46,36-PA-47, 37-PA-48, 38-PA-49, and 39-PA-50 mutants offerPDPN dN34. In contrast, the reactivity of PMab-292 wasobserved in the substituted mutants from 40th to 59th aminoacids. These results indicated that the epitope of PMab-292
contains the 34-VRPEDD-39 sequence of ferPDPN.
Discussion

In this study, we performed the flow cytometry-mediatedepitope mapping of PMab-292 using ferPDPN deletionmutants (Fig. 1) and PA scanning of ferPDPN (Fig. 2). We
found that 34-VRPEDD-39 sequence of ferPDPN is an important sequence of PMab-292 epitope. In contrast, we could
not identify the critical amino acids using 1 · Ala scanning(Supplementary Fig. S1) or 2 · Ala scanning (SupplementaryFig. S2) using flow cytometry. Figure 3 shows the surrounding sequence of 34-VRPEDD-39, and found that thesame VRPEDD sequence is present between the 26th and31st amino acids of ferPDPN. This might be the reason why
we could not determine the critical amino acids of theferPDPN epitope using 1 · Ala scanning and 2 · Ala scanningin the 34-VRPEDD-39 sequence. In other words, PMab-292
probably recognizes the tandem sequences from PLAG1 toPLAG2 domains (Fig. 3). Therefore, the alanine-substitutionof two same amino acids in both 26-VRPEDD-31 and34-VRPEDD-39 sequence is essential to determine the criticalamino acids of PMab-292 epitope.

PLAG domains play a critical role in platelet aggregation.
In humans, the O-glycosylation in PLAG3 or PLD is crucial for PDPN-induced platelet aggregation.19,20 A platelet
receptor, CLEC-2 recognizes the sialylated PLAG3 or PLDsof PDPN, which is essential for PDPN-induced platelet
aggregation. We have not examined whether ferPDPN caninduce platelet aggregation and have not determined theessential O-glycosylated amino acids for platelet aggregationin PLAG domains. However, PMab-292 could contribute tothe study of platelet aggregation by ferPDPN.
We have determined the epitopes of anti-PDPN mAbs
against various species.11 It is the first example that thetandem sequence is recognized by an anti-PDPN mAb. We
performed immunohistochemistry using PMab-292 andcould detect kidney podocytes and lung alveolar epithelial
cells with high sensitivity.12 The property of PMab-292 may
contribute to the high sensitivity of immunohistochemical
analysis, which is important for the pathological analysisof lung injury by SARS-CoVs.
Authors’ Contributions

Y.I. and T.T. performed the experiments. M.K.K. and Y.K.
designed the experiments. Y.I. and M.K.K. analyzed the data.
H.S. and Y.K. wrote the manuscript. All authors have readand agreed to the published version of the manuscript.
Author Disclosure Statement

The authors have no conflict of interest.

EPITOPE MAPPING OF ANTI-FERRET PDPN MAB
Funding Information

This research was supported in part by Japan Agency for
Medical Research and Development (AMED) under Grant
Numbers: JP23ama121008 (to Y.K.), JP23am0401013(to Y.K.), 23bm1123027h0001 (to Y.K.), and JP23ck010
6730 (to Y.K.), and by the Japan Society for the Promotionof Science ( JSPS) Grants-in-Aid for Scientific Research(KAKENHI) grant numbers 21K20789 (to T.T.), 22K06995(to H.S.), 21K07168 (to M.K.K.), and 22K07224 (to Y.K.).
Supplementary Material

Supplementary Figure S1
Supplementary Figure S2

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Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinari.kato.e6@tohoku.ac.jp
Received: November 27, 2023
Accepted: December 11, 2023