MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 2, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2018.0031

Short Communication

Anti-Bovine Podoplanin Monoclonal Antibody PMab-44
Detects Goat Podoplanin in Immunohistochemistry

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Shinji Yamada,1 Mika K. Kaneko,1 Yoshikazu Furusawa,1–3 Shunsuke Itai,1,4 Masato Sano,1Takuro Nakamura,1 Miyuki Yanaka,1 Saori Handa,1 Kayo Hisamatsu,1 Yoshimi Nakamura,1Masanori Koyanagi,3 Masato Fukui,3 Hiroyuki Harada,4 and Yukinari Kato1,2

Podoplanin (PDPN) is expressed in type I alveolar cells, kidney podocytes, and lymphatic endothelial cells.
We have characterized the PDPNs of various animal species using specific anti-PDPN monoclonal antibodies (mAbs). In this study, we investigated whether these anti-PDPN mAbs cross-react with goat PDPN
(gPDPN). Flow cytometry demonstrated that the anti-bovine PDPN mAb PMab-44 (IgG1, kappa) reacts with
gPDPN, which is overexpressed in CHO-K1 cells. Using immunohistochemical analysis, type I alveolar
cells of goat lung were strongly detected by PMab-44. These results indicate that PMab-44 is useful forinvestigating gPDPN.
Keywords: goat podoplanin, PDPN, PMab-44

dothelial cells or distinguishing type I alveolar cells fromtype II alveolar cells. In addition, we have characterized
PDPNs of various animals using specific anti-PDPN mAbs,
such as anti-mouse (PMab-1),(10) anti-rat (PMab-2),(20) antirabbit (PMab-32),(21) anti-dog [PMab-38(22) and PMab-48(23)],anti-bovine (PMab-44),(24) and anti-cat (PMab-52)(25) PDPNs.
However, to date, anti-goat PDPN (gPDPN) mAbs have notbeen reported. Therefore, gPDPNs in lymphatic endothelial
cells or type I alveolar cells have not been characterized. In thisstudy, we investigated whether our anti-PDPN mAbs fromseveral animal species cross-react with gPDPN using flow
cytometry and immunohistochemical analyses.

IntroductionP

odoplanin (PDPN) is a type I transmembrane sialoglycoprotein that induces platelet aggregation throughthe C-type lectin-like receptor-2 (CLEC-2).(1,2) It comprises
three platelet aggregation-stimulating (PLAG) domains:
PLAG1–3 (EDxxVTPG).(2) Our previous work has demonstrated that PLAG3 is the most important domain for theplatelet aggregation activity of human PDPN.(2–4) The mostcritical domain for PDPN-CLEC-2 interaction remains controversial because these functional assays have been performed using their original anti-PDPN mAbs, the binding
affinity of which may vary. Cancer-specific anti-PDPN mAbs
should be used for PDPN-targeted therapy(5–9) because
PDPN is expressed in many normal tissues.
We have developed many antihuman PDPN monoclonal
antibodies (mAbs), including NZ-1.2,(10) LpMab-2,(5,6)
LpMab-3,(5) LpMab-7,(5,11) LpMab-9,(5) LpMab-10,(12)LpMab-12,(13) LpMab-13,(14) LpMab-17,(15) LpMab-19,(16)LpMab-21,(17,18) and LpMab-23.(7,8) LpMab-2 and LpMab23 were categorized as cancer-specific mAbs (CasMabs), and
LpMab-3, LpMab-9, LpMab-12, LpMab-19, and LpMab-21
were reported as antiglycopeptide mAbs. PDPN is known to
be a specific marker of lymphatic endothelial and type I alveolar cells.(2,19) Therefore, anti-PDPN mAbs are useful for
distinguishing lymphatic endothelial cells from vascular en-

Materials and Methods
Cell line and goat tissues

Chinese hamster ovary (CHO)-K1 was purchased from theAmerican Type Culture Collection (Manassas, VA). CHOK1 and transfectants were cultured in RPMI1640 medium(Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10%
heat-inactivated fetal bovine serum (Thermo Fisher Scientific,
Inc., Waltham, MA), 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. Goat tissues were collected in Nippon
Zenyaku Kogyo Co. Ltd. (Fukushima, Japan), fixed in 4%

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi, Japan.
3
Zenoaq Resource Co., Ltd., Koriyama, Fukushima, Japan.
4
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Tokyo, Japan.
296GOAT PDPN DETECTED BY PMAB-44

paraformaldehyde phosphate buffer solution (Nacalai Tesque,Inc.), and processed routinely to make paraffin-embedded tissue sections.
Overexpression of gPDPN in CHO-K1 cells

N-terminal MAP16 tag (PGTGDGMVPPGIEDKI), which isrecognized by an anti-MAP16 tag mAb [PMab-1: the samemAb against MAP tag(26)], was subcloned into a pCAG-Neo
vector (Fujifilm Wako Pure Chemical Corporation, Osaka,
Japan). Plasmids were transfected using Lipofectamine LTX
with Plus Reagent (Thermo Fisher Scientific, Inc.). Stabletransfectants were selected by limiting dilution and cultivation ina medium containing 0.5 mg/mL of G418 (Nacalai Tesque, Inc.).

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Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan)
encoding gPDPN (accession No. XM_005690821.3) plus an

97

FIG. 1. Detection of gPDPN using PMab-44. (A, B) Flow cytometry. PMab-44 at a concentration of 1 mg/mL (blue) or
10 mg/mL (red) was treated with CHO-K1 cells (A) or CHO/gPDPN cells (B) for 30 min at 4C, followed by the addition of
secondary antibodies. (C–F) Immunohistochemical analyses using goat lung tissues. Histological sections of goat lungs
were directly autoclaved in citrate buffer for 20 min. After blocking, sections were incubated with 1 mg/mL of PMab-44 (C,D) or blocking buffer (E, F) and treated with an Envision+ kit. (G, H) Hematoxylin and eosin staining. Scale bar = 100 mm.
CHO, Chinese hamster ovary; gPDPN, goat podoplanin.

98

YAMADA ET AL.

Flow cytometry

Acknowledgments

CHO/gPDPN and CHO-K1 cells were harvested after abrief exposure to 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). After washing with
0.1% bovine serum albumin in PBS, the cells were treatedwith PMab-44 (1 or 10 mg/mL) for 30 min at 4C, followed bytreatment with Alexa Fluor 488-conjugated anti-mouse IgG(1:1000; Cell Signaling Technology, Inc., Danvers, MA).
Fluorescence data were acquired using a Cell Analyzer
SA3800 (Sony Corporation, Tokyo, Japan).

This research was supported in part by AMED under grant
numbers JP18am0101078 (Y.K.), JP18am0301010 (Y.K.),
and JP18ae0101028 (Y.K.), and by JSPS KAKENHI grant
numbers 17K07299 (M.K.K.) and 16K10748 (Y.K.).
Author Disclosure Statement

No competing financial interests exist.
References

Downloaded by Yukinari Kato from www.liebertpub.com at 04/22/19. For personal use only.

Immunohistochemical analyses

Histological sections (4-mm thick) of goat tissues weredirectly autoclaved in citrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) for 20 min. After blocking with
SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher
Scientific, Inc.), sections were incubated with PMab-44(1 mg/mL) for 1 h at room temperature and treated using an
Envision+ kit (Agilent Technologies, Inc., Santa Clara, CA)
for 30 min. Color was developed using 3,3-diaminobenzidine
tetrahydrochloride (Agilent Technologies, Inc.) for 2 min,and sections were counterstained with hematoxylin (Fujifilm
Wako Pure Chemical Corporation).
Results and Discussion

We previously developed a mouse anti-bovine PDPN
mAb, PMab-44 (IgG1, kappa), which specifically detects
bovine PDPN in flow cytometry and immunohistochemistry.(24) Furthermore, a series of deletion or point mutants ofbovine PDPN were used for investigating the binding epitopes of PMab-44 using flow cytometry and Western blotting.(27) Platelet aggregation-stimulating domain 3 (PLAG3)
of bovine PDPN was identified as a critical epitope of PMab44. The comparison of amino acid sequences revealed that87% homology is observed between bovine PDPN andgPDPN (Supplementary Fig. S1). Therefore, we investigatedwhether PMab-44 cross-reacts with gPDPN in this study.
We first produced a gPDPN-stable transfectant using
CHO-K1 cells using N-terminal MAP16 tag. In flow cytometry, PMab-44 did not react with CHO-K1 cells (Fig. 1A),
but reacted with CHO/gPDPN in a dose-dependent manner (Fig. 1B). The other anti-PDPN mAbs, such as anti-rat(PMab-2),(20) anti-rabbit (PMab-32),(21) anti-dog [PMab38(22) and PMab-48(23)], and anti-cat PDPN [PMab-52(25)],
did not react with CHO/gPDPN (data not shown). These results indicate that PMab-44 is useful for detecting gPDPN.
Next, we investigated gPDPN expression using immunohistochemistry. Previous immunohistochemical studies
have reported that PMab-44 reacts with bovine tissues.(24)
Similarly, PMab-44 strongly stained type I alveolar cellsof goat lung in a membrane-staining pattern (Fig. 1C, D).
Conversely, lymphatic endothelial cells of goat colon were
not stained by PMab-44 in this study (data not shown), indicating that PMab-44 reactivity in goat exhibits tissue
specificity.
Taken together, PMab-44 is useful for detecting gPDPN inflow cytometry and immunohistochemical analyses. Further
studies are necessary to show that PMab-44 can detect
gPDPN in other normal goat tissues.

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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1, Seiryo-machi, Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: August 22, 2018
Accepted: October 14, 2018