MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 4, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0017

Epitope Mapping of the Anti-California Sea Lion
Podoplanin Monoclonal Antibody PMab-269
Using Alanine-Scanning Mutagenesis and ELISA

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Tomohiro Tanaka,1 Teizo Asano,1 Masato Sano,1 Junko Takei,1 Hideki Hosono,1 Ren Nanamiya,1Nami Tateyama,1 Mika K. Kaneko,1 and Yukinari Kato1,2,i

Podoplanin (PDPN) plays a pivotal role in platelet aggregation, embryo development, and tumor progression.
PDPN is universally expressed in many mammalian species, and is considered a typical lymphatic endothelial
cell marker. We have previously developed the mouse anti-California sea lion (Zalophus californianus) PDPN
(seaPDPN) monoclonal antibody (mAb), clone PMab-269, which is suitable for different experimental applications, including flow cytometry, Western blotting, and immunohistochemistry. In this study, we identified the
PMab-269 epitope of the seaPDPN by enzyme-linked immunosorbent assay using deletion mutants and point
mutants generated for seaPDPN. Our results demonstrated that PMab-269 recognized the peptide, corresponding to the amino acids 63–82 of seaPDPN. Furthermore, the reactions of PMab-269 to seven alanine-substituted
peptides, such as P68A, D76A, F77A, H78A, L79A, E80A, and D81A, were abolished among 20 alaninesubstituted peptides. We identified the seven amino acids (Pro68, Asp76, Phe77, His78, Leu79, Glu80, andAsp81) as the critical epitope targeted by PMab-269. The successful identification of the PMab-269 epitope
might contribute to the pathophysiological investigations of seaPDPN.
Keywords: California sea lion podoplanin, monoclonal antibody, PMab-269, epitope
IntroductionE

pitope identification and design of immunogen are
essential for the development of monoclonal antibodies(mAbs) and vaccines.(1,2) It is important to determine whether an epitope recognized by a mAb can be used for thedevelopment of tag systems for protein extraction, purification, or detection assays.(3–8) Application of epitope tag systems facilitates the detection of membrane proteins that are
structurally difficult to recognize by antibodies alone.
Podoplanin (PDPN), also known as Aggrus or PA2.26, isa single pass transmembrane glycoprotein that is highly expressed in normal tissues, such as lymphatic endothelial cells,
type I alveolar cells, and renal glomeruli in mammals.(9–11)
The amino acids sequence of PDPN is well conserved acrossmammals.(12) PDPN is involved in platelet aggregation
by binding to its receptor, the C-type lectin-like receptor-2(CLEC-2).(13) The platelet aggregation–stimulating (PLAG)
domain, EDxxVTPG sequence, and the PLAG-like domain (PLD), E(D/E)xx(T/S)xx sequence, are critical forthe platelet-aggregating activity of PDPN.(12,14–16) PDPN–
CLEC-2 interaction is also important for blood/lymphatic

vascular separation in the embryo.(17) In the immune system,
PDPN is known to influence the maturation of natural regulatory T cells and antigen-presenting dendritic cell migration
to lymphatic vessels through interactions with the C-C Motif
Chemokine Ligand 21.(18–21) In a mouse sepsis model, binding of PDPN to the CLEC-2 on inflammatory macrophages
and platelets regulates the immune response.(22) Many studies have reported that PDPN expression in cancer cells is
associated with tumor metastasis and malignancy.(23–27)
PDPN is expressed not only by cancer cells, but also by cells
in the cancer microenvironment, such as cancer-associatedfibroblasts, inflammatory macrophages, and T helper 17
cells, and contributes to tumor progression.(28,29) PDPN has
also attracted attention as a target for cancer treatment.(28,30)
The application of anti-PDPN mAbs in chimeric antigen
receptor-T therapies and the development of cancer-specificmAbs have been proposed.(31,32)
We have established different anti-PDPN mAbs formany species by employing a cell-based immunization andscreening (CBIS) method, and characterized the targeted
epitopes.(33–47) Recently, we developed a novel anti-PDPN
mAb, clone PMab-269 (mouse IgG1, kappa), which recognizes

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan.
ORCID ID (https://orcid.org/0000-0001-5385-8201).

2
i

196

DETERMINATION OF PMAB-269 EPITOPE

197

Table 1. Identification of PMab-269 Epitope
Using Deletion Mutants
PeptideSequence

PMab-269

23–42
33–52
43–62
53–72
63–82
73–92
83–102
93–112
103–122
113–128ASTVRPEDDVTPGVEDSVVTTPGVEDSVVTLGAEDNVMTTLGAEDNVMTTGTSEEPYESGGTSEEPYESGLTALVPINTELTALVPINTESVTDFHLEDG
SVTDFHLEDGPTQESTVHARPTQESTVHAREESQSTTALNEESQSTTALNVVTHHSVEKVVVTHHSVEKVDEDTETTLEKDEDTETTLEKDGLATV+++-

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+++, OD655 S0.3; -, OD655 < 0.1.

the California sea lion (Zalophus californianus) PDPN
(seaPDPN).(48) The PMab-269 clearly detected seaPDPN protein in flow cytometry and Western blotting analysis. Furthermore, PMab-269 identified seaPDPN-positive cells informalin-fixed paraffin-embedded California sea lion specimens, including lung type I alveolar cells, kidney podocytes,
and lymphatic endothelial cells in the colon. In this study, weclarified the PMab-269 epitope of seaPDPN using enzymelinked immunosorbent assay (ELISA).

Table 2. Identification of PMab-269 Epitope
Using Point Mutants
MutantpeptideSequence

PMab-269

L63A
T64A
A65G
L66A
V67A
P68A
I69A
N70A
T71A
E72A
S73A
V74A
T75A
D76A
F77A
H78A
L79A
E80A
D81A
G82A

ATALVPINTESVTDFHLEDGLAALVPINTESVTDFHLEDGLTGLVPINTESVTDFHLEDGLTAAVPINTESVTDFHLEDGLTALAPINTESVTDFHLEDGLTALVAINTESVTDFHLEDGLTALVPANTESVTDFHLEDGLTALVPIATESVTDFHLEDGLTALVPINAESVTDFHLEDGLTALVPINTASVTDFHLEDGLTALVPINTEAVTDFHLEDGLTALVPINTESATDFHLEDGLTALVPINTESVADFHLEDGLTALVPINTESVTAFHLEDGLTALVPINTESVTDAHLEDGLTALVPINTESVTDFALEDGLTALVPINTESVTDFHAEDGLTALVPINTESVTDFHLADGLTALVPINTESVTDFHLEAGLTALVPINTESVTDFHLEDA

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

+++, OD655 S0.3; ++, 0.2& OD655 < 0.3; -, OD655 < 0.1.

FIG. 1. Determination of the PMab-269 epitope of seaPDPN by ELISA using deletion mutants. (A) Synthesized peptides of seaPDPN were immobilized on immunoplates. The plates were incubated with PMab-269 (5 mg/mL), followed
by incubation with peroxidase-conjugated anti-mouse immunoglobulins. (B) Schematic illustration of seaPDPN and the
PMab-269 epitope. ELISA, enzyme-linked immunosorbent assay; PLAG, platelet aggregation-stimulating; PLD, PLAGlike domain; seaPDPN, California sea lion (Zalophus californianus) PDPN; TMD, transmembrane domain.

198
Materials and Methods
PeptidesThe seaPDPN (Accession No.: XP_027432487) peptides,
including 10 deletion mutants (Table 1) and 20 point mutants(Table 2), were synthesized by utilizing PEPScreen (SigmaAldrich Corp., St. Louis, MO).

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Enzyme-linked immunosorbent assay

Peptides were immobilized on Nunc Maxisorp 96-wellimmunoplates (Thermo Fisher Scientific, Inc., Waltham, MA)
at a concentration of 10 lg/mL for 30 minutes at 37C. Afterwashing with phosphate-buffered saline containing 0.05%
Tween20 (PBST), wells were blocked with 1% bovine serum
albumin-containing PBST for 30 minutes at 37C. The plates
were then incubated with PMab-269 (5 lg/mL),(48) followed
by a 1:2000 dilution of peroxidase-conjugated anti-mouse
immunoglobulins (Agilent Technologies, Inc., Santa Clara,
CA). Enzymatic reactions were performed using the ELISAPOD Substrate TMB Kit (Nacalai Tesque, Inc., Kyoto,

TANAKA ET AL.

Japan). Optical density was measured at 655 nm using
an iMark microplate reader (Bio-Rad Laboratories, Inc.,
Berkeley, CA).
Results and Discussion
Epitope mapping using deletion mutants

In our previous study, we developed an anti-seaPDPN
mAb (clone PMab-269) using the CBIS method.(48) PMab269 is suitable for in vitro applications including flow cytometry, Western blotting, and immunohistochemistry. To
better characterize the epitope binding site of seaPDPN antigens recognized by PMab-269, we synthesized 10 peptides
comprising 23–42 amino acids (aa), 33–52 aa, 43–62 aa, 53–
72 aa, 63–82 aa, 73–92 aa, 83–102 aa, 93–112 aa, 103–122
aa, and 113–128 aa, respectively, of the seaPDPN protein
sequence (Table 1), and performed ELISA. The results
demonstrated that PMab-269 reacted with the 63–82 aa sequence (63-LTALVPINTESVTDFHLEDG-82) of seaPDPN(Fig. 1A). These results are summarized in Figure 1B.

FIG. 2. Determination of the PMab-269 epitope of seaPDPN by ELISA using point mutants. (A) Synthesized peptidesof seaPDPN were immobilized on immunoplates. The plates were incubated with PMab-269 (5 mg/mL), followed byperoxidase-conjugated anti-mouse immunoglobulins. (B) Schematic illustration of seaPDPN and the PMab-269 epitope.
The PMab-269 epitope of seaPDPN involves Pro68, Asp76, Phe77, His78, Leu79, Glu80, and Asp81.

DETERMINATION OF PMAB-269 EPITOPE

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Epitope mapping using point mutants

Next, we synthesized 20 different point mutants using the
63–82 aa peptide of seaPDPN (Table 2). The ELISA results
demonstrated that PMab-269 reacted with point mutants, suchas L63A, T64A, A65G, L66A, V67A, I69A, N70A, T71A,
E72A, S73A, V74A, T75A, and G82A, as well as the 63–82 aa
wild-type sequence (positive control) (Fig. 2A). In contrast,
PMab-269 did not bind to point mutants, such as the P68A,
D76A, F77A, H78A, L79A, E80A, and D81A (Fig. 2A), indicating that Pro68, Asp76, Phe77, His78, Leu79, Glu80, andAsp81 are the critical amino acids, which are included in the
PMab-269 epitope. As summarized in Figure 2B, the PMab269 binds to Glu80 and Asp81, which are residues comprising
the PLD region. Therefore, PMab-269 might possess neutralizing activity for seaPDPN-induced platelet aggregation.
In summary, this study identified the seven amino acids(Pro68, Asp76, Phe77, His78, Leu79, Glu80, and Asp81) as
the critical epitope targeted by PMab-269. In the future, thePMab-269 might contribute to pathophysiological studies
using the California sea lion model.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported, in part, by Japan Agency for
Medical Research and Development (AMED) under Grant
Nos. JP21am0401013 (Y.K.) and JP21am0101078 (Y.K.),
and by the Japan Society for the Promotion of Science ( JSPS)
Grants-in-Aid for Scientific Research (KAKENHI) Grant
Nos. 21K15523 (to T.A.), 21K07168 (to M.K.K.), 20K16322(to M.S.), and 19K07705 (to Y.K.).
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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
MiyagiJapan
E-mail: yukinarikato@med.tohoku.ac.jp
Received: April 16, 2021
Accepted: May 20, 2021