MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 4, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0017
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Epitope Mapping of an Anti-elephant Podoplanin Monoclonal
Antibody (PMab-295) Using Enzyme-Linked
Immunosorbent Assay

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Yuki Okada,1 Hiroyuki Suzuki,1 Mika K. Kaneko,2 and Yukinari Kato1,2

Podoplanin (PDPN) is a marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. The
overexpression of PDPN contributes to the malignant progression of tumors. Therefore, the development of antiPDPN monoclonal antibodies (mAbs) to animals is essential to evaluate the pathogenesis and cellular functions.
Using peptide immunization, we previously developed an anti-elephant PDPN (elePDPN) mAb, PMab-295, which
is useful for flow cytometry, Western blotting, and immunohistochemistry. In this study, we determined the critical
epitope of PMab-295 by enzyme-linked immunosorbent assay (ELISA). We performed ELISA with the alaninesubstituted peptides of elePDPN extracellular domain (amino acids 38–51), and found that PMab-295 did notrecognize the alanine-substituted peptides of M41A, P44A, and E47A. Furthermore, these peptides could not inhibit
the recognition of PMab-295 to elePDPN-expressing cells by flow cytometry and immunohistochemistry. The
results indicate that the binding epitope of PMab-295 includes Met41, Pro44, and Glu47 of elePDPN.
Keywords: elephant podoplanin, epitope mapping, monoclonal antibody, enzyme-linked immunosorbent assay

IntroductionP

odoplanin (PDPN) is a type I transmembrane mucin-like
glycoprotein that plays critical roles in normal lung,(1)kidney,(2) and lymphatic vessels.(3) In humans, PDPN has beenreported to be overexpressed in cancers, and plays crucial rolesin the malignant progression.(4) PDPN is also upregulated intumor microenvironment, including cancer-associated fibroblasts (CAFs)(5) and lymphocytes.(6) CAFs play a critical rolein the formation of immunosuppressive tumor microenvironment(7,8) and remodeling of the extracellular matrix. Therefore,anti-PDPN monoclonal antibodies (mAbs) are useful to identify the specific cells in tissue staining, and investigate thecellular functions in PDPN-expressing cancer cells.
The N-terminal extracellular domain of PDPN has a repeat
sequence of platelet aggregation-stimulating (PLAG)1 to
PLAG3 domains,(9) which interacts with a platelet receptor,
C-type lectin-like receptor 2, and promotes platelet aggregation.(10,11) Furthermore, several PLAG-like domains (PLD)with similar sequences were identified at the central regionof PDPN.(4,12) In the cytoplasmic tail, PDPN interacts withezrin, radixin, and moesin family proteins and regulates cellproliferation, migration, invasion, epithelial to mesenchymaltransition, and stemness.(13)We have established anti-PDPN mAbs against 20 species,(14–31) which can be used for flow cytometry and immunohistochemistry. These mAbs are expected to contribute not
only to the research of each animal but also to pathogenic
diagnosis. In a previous study, we established an anti-elephant
PDPN (elePDPN) mAb PMab-295 (IgG1, kappa) using the
peptide immunization.(15) To clarify further characteristics ofPMab-295, we performed epitope mapping using enzymelinked immunosorbent assay (ELISA).
Materials and Methods
PeptidesThe elePDPN (accession No.: XM_010593105.2) peptide(38-EGGMVIPGVEDNMV-51) and 14 alanine-substituted
peptides (Table 1) were synthesized by utilizing PEPScreen(Sigma-Aldrich Corp., St. Louis, MO).

Departments of 1Molecular Pharmacology and 2Antibody Drug Development, Tohoku University Graduate School of Medicine,
Sendai, Japan.

221

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OKADA ET AL.

Table 1. Identification of the PMab-295 Epitope
Using Alanine-Substituted elePDPN Peptides
PeptidesDownloaded by Yukinari Kato from www.liebertpub.com at 08/26/22. For personal use only.

WT (38–51 aa)
E38A
G39A
G40A
M41A
V42A
I43A
P44A
G45A
V46A
E47A
D48A
N49A
M50A
V51A

SequencesPMab-295

EGGMVIPGVEDNMVAGGMVIPGVEDNMVEAGMVIPGVEDNMVEGAMVIPGVEDNMVEGGAVIPGVEDNMVEGGMAIPGVEDNMVEGGMVAPGVEDNMVEGGMVIAGVEDNMVEGGMVIPAVEDNMVEGGMVIPGAEDNMVEGGMVIPGVADNMVEGGMVIPGVEANMVEGGMVIPGVEDAMVEGGMVIPGVEDNAVEGGMVIPGVEDNMA

+++
+++
+++
+++
+++
+
+++
+++
+
+++
+++
+++

The tissue sections were incubated with PMab-295(1 lg/mL)+each peptide for 1 hour at the room temperature,and then treated with the Envision+Kit (Agilent Technologies, Inc.) for 30 minutes. Color was developed using 3,3¢diaminobenzidine tetrahydrochloride (Agilent Technologies,Inc.) for 2 minutes, and counterstaining was performed using
hematoxylin (FUJIFILM Wako Pure Chemical Corporation,
Osaka, Japan).
Results
Epitope mapping of PMab-295 with alanine-substituted
PDPN peptide

We previously established an anti-elePDPN mAb (PMab295) by peptide immunization of elePDPN extracellular domain (38-EGGMVIPGVEDNMV-51).(14) To reveal the binding
epitope of PMab-295, we synthesized 14 alanine-substituted+++, OD655S0.5; +, 0.1&OD655 < 0.3; -, OD655 < 0.1; aa,amino acids; PDPN, podoplanin.

Enzyme-linked immunosorbent assay

Synthesized elePDPN peptides were immobilized on Nunc
Maxisorp 96-well immunoplates (Thermo Fisher Scientific,
Inc., Waltham, MA) at a concentration of 10 lg/mL for 30
minutes at 37C. After washing with phosphate-bufferedsaline (PBS) containing 0.05% Tween20 (PBST; Nacalai
Tesque, Inc., Kyoto, Japan), wells were blocked with 1%
bovine serum albumin (BSA)-containing PBST for 30 minutes at 37C. The plates were incubated with 1 lg/mL of
PMab-295, followed by peroxidase-conjugated anti-mouse
immunoglobulins (1:2000 diluted; Agilent Technologies,
Inc., Santa Clara, CA). Enzymatic reactions were performed
using the ELISA POD Substrate TMB Kit (Nacalai Tesque,Inc.). Optical density was measured at 655 nm using an iMark
microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA).
Flow cytometry

CHO/2 · RIEDL-elePDPN (CHO/elePDPN) cells(15) were
harvested after a brief exposure to 0.25% trypsin in 1 mM
ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.)and washed with 0.1% BSA (Nacalai Tesque, Inc.) in PBS(Nacalai Tesque, Inc.). PMab-295 (1 lg/mL) was incubatedwith each peptide (10 lg/mL) for 30 minutes at 4C. CHO/
elePDPN cells were treated with PMab-295+each peptide, andfurther treated with Alexa Fluor 488-conjugated anti-mouse IgG(1:2000). Fluorescence data were collected using the SA3800
Cell Analyzer (Sony Biotechnology Corp., Tokyo, Japan).
Immunohistochemical analysis

Cell blocks were produced using iPGell (Genostaff Co.,
Ltd., Tokyo, Japan)(23) and processed to make 4-lm-thickformalin-fixed paraffin-embedded (FFPE) cell sections.
Those FFPE sections were directly autoclaved in a citratebuffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) for
20 minutes, and were blocked using the SuperBlock T20(PBS) blocking buffer (Thermo Fisher Scientific, Inc.). PMab295 (1 lg/mL) was incubated with each peptide (10 lg/mL) inthe blocking buffer for 1 hour at room temperature.

FIG. 1. Determination of the PMab-295 epitope of
elePDPN by ELISA using alanine-substituted PDPN peptides. (A) The alanine-substituted elePDPN peptides were
immobilized on immunoplates. The plates were incubatedwith PMab-295 (1 mg/mL), followed by peroxidaseconjugated anti-mouse immunoglobulins. (B) Schematic illustration of elePDPN and the PMab-295 epitope. The
PMab-295 epitope involves Met41, Pro44, and Glu47 of
elePDPN. ELISA, enzyme-linked immunosorbent assay;
PDPN, podoplanin; PLAG, platelet aggregation-stimulating;
PLD, PLAG-like domain; TM, transmembrane domain.

EPITOPE MAPPING OF ANTI-ELEPHANT PDPN MAB

peptides of elePDPN (Table 1). PMab-295 exhibited reaction
with E38A, G39A, G40A, V42A, I43A, G45A, V46A, D48A,
N49A, M50A, V51A, and wild type (WT, 38-51 aa) (Fig. 1A).
In contrast, PMab-295 did not react with M41A, P44A, andE47A (Fig. 1), indicating that Met41, Pro44, and Glu47 areincluded in the critical epitope of PMab-295. The results are
summarized at Table 1. Figure 1B shows the schematic illustration of elePDPN and the critical epitope of PMab-295.
Flow cytometry using PMab-295
with alanine-substituted PDPN peptides

V46A, N49A, M50A, and V51A. Furthermore, the reaction ispartially neutralized by I43A and D48A. In contrast, M41A,
P44A, and E47A never blocked the reaction of PMab-295
with CHO/elePDPN. These results confirmed that Met41,
Pro44, and Glu47 of elePDPN are critical for PMab-295
detection by flow cytometry.
Immunohistochemistry using PMab-295
with alanine-substituted PDPN peptides

We also performed a blocking assay using immunohistochemical analysis. PMab-295 stained the CHO/elePDPN
cells-embedded section (Fig. 3B), which was completely
neutralized by WT (Fig. 3C), but not by M41A, P44A, andE47A (Fig. 3D, E), respectively. These results also confirmedthat Met41, Pro44, and Glu47 of elePDPN are critical for
PMab-295 detection by immunohistochemical analysis.

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We next performed a peptide blocking assay using flow
cytometry to confirm the importance of the PMab-295 epitope. As shown in Figure 2, PMab-295 reacted with the
CHO/elePDPN cells. This reaction was almost completely
neutralized by WT, E38A, G39A, G40A, V42A, G45A,

223

FIG. 2. Flow cytometry using PMab-295 and peptides of elePDPN. PMab-295 (1 mg/mL) plus the alanine-substituted
peptides (10 mg/mL), or control (0.1% DMSO in PBS, –peptide) was reacted with CHO/elePDPN cells for 30 minutes at
4C, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG. Filled gray represents the negative control(PBS). DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline.

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224

OKADA ET AL.

FIG. 3. Immunohistochemical analyses using PMab-295 and peptides of elePDPN. The FFPE sections of CHO/elePDPN
cells were incubated with PMab-295 (1 mg/mL) plus the alanine-substituted peptides (10 mg/mL), or control (0.1% DMSO inblocking buffer, –peptide), followed by that with the Envision+Kit. (A) no antibody (Ab), (B) PMab-295, (C) PMab-295 +WT peptide, (D) PMab-295 + M41A peptide, (E) PMab-295 + P44A peptide, (F) PMab-295 + E47A peptide. Scalebar = 100 mm. DMSO, dimethyl sulfoxide; FFPE, formalin-fixed paraffin-embedded.
Discussion

PLAG domains of PDPN possess three tandem repeats ofthe ‘‘EDxxVTPG’’ consensus sequences.(4,9) In this study,
we determined the critical epitope of PMab-295 as Met41,
Pro44, and Glu47 of elePDPN between PLAG2 and PLAG3
domains (Fig. 1B). The O-glycosylation of Thr in the PLAG3
or PLD (also named as PLAG4 domain) has been reported tobe essential for PDPN-induced platelet aggregation.(12,32)
However, the PLAG2 domain of elePDPN is the ‘‘EGGMVIPG’’ sequence, which does not possess the consensus Thr
residue, indicating that the PMab-295 recognition would not
be affected by glycosylation.
We have not examined the cross-reactivity of PMab-295 toother species. We searched the conservation of ‘‘MVIPGVE’’
sequence to other species PDPN using standard protein
BLAST (the Basic Local Alignment Search Tool, NCBI).

However, there is no species, which possess the corresponding three amino acids, suggesting that PMab-295 specificallyrecognizes elephant PDPN.
As shown in Figure 3, PMab-295 is useful for immunohistochemistry of FFPE CHO/elePDPN cells. However, we
could not obtain elephant tissues and confirm the staining
patterns using PMab-295. We showed that PMab-295 and its
blocking peptides can be used in immunohistochemistry(Fig. 3). Therefore, the use of PMab-295 with the blocking
peptides would help the identification of PDPN-expressing
cells on elephant tissues. Recently, immunohistochemical
analyses of platelet endothelial cell adhesion molecules-1
and von Willebrand factor have been reported in elephantendotheliotropic herpesvirus-hemorrhagic disease in young
Asian elephants (Elephas maximus).(33–36)
Furthermore, the reproductive tract neoplasia in adult female Asian elephant was reported in the United States.(37)EPITOPE MAPPING OF ANTI-ELEPHANT PDPN MABPDPN is overexpressed in many human tumors including
squamous cell carcinomas,(38–41) malignant gliomas,(42–52)
sarcomas,(53,54) and malignant mesotheliomas.(55–61) Therefore, the analysis of PDPN-positive cells on elephant tissues
and PDPN-expressing carcinomas by PMab-295 will help the
elucidation of the pathogenesis of the diseases.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported in part by the Japan Agency
for Medical Research and Development (AMED) underGrant Numbers to Y.K. JP22ama121008, JP22am0401013,
JP22bm1004001, JP22ck0106730, and JP21am0101078.

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EPITOPE MAPPING OF ANTI-ELEPHANT PDPN MAB

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Address correspondence to:
Yukinari Kato
Department of Molecular Pharmacology
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: April 21, 2022
Accepted: July 3, 2022