MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 37, Number 6, 2018ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2018.0036

Short Communications

Immunohistochemical Detection of Sheep Podoplanin Using
an Antibovine Podoplanin Monoclonal Antibody PMab-44

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Yukinari Kato,1,2 Shinji Yamada,1 Shunsuke Itai,1 Atsushi Kobayashi,3 Satoru Konnai,4,5 and Mika K. Kaneko1Podoplanin (PDPN) obtained from various animal species has been characterized using specific anti-PDPN
monoclonal antibodies (mAbs), namely, PMab-1, PMab-2, PMab-32, PMab-38, PMab-44, and PMab-52 againstmouse, rat, rabbit, dog, bovine, and cat PDPN, respectively. PDPN is expressed in type I alveolar cells in lungs,
lymphatic endothelial cells, and kidney podocytes. In this study, we investigated possible cross-reactions
between anti-PDPN mAbs and sheep PDPN. Type I alveolar cells from sheep lung were strongly detected byPMab-44 using immunohistochemical analyses. These results indicate that PMab-44 may be useful for thedetection of sheep PDPN.
Keywords: sheep podoplanin, sheep PDPN, PMab-44

Introduction

Podoplanin (PDPN) is a type I transmembrane sialoglycoprotein that induces platelet aggregation throughthe C-type lectin-like receptor-2 (CLEC-2).(1) It comprises
three platelet aggregation-stimulating (PLAG) domains,
namely, PLAG1–PLAG3 (EDxxVTPG sequence).(2) Previously, we have shown that PLAG3 (platelet aggregationstimulating domain 3) is the most important domain forplatelet aggregation mediated by human PDPN.(1–3) Recently, PLAG4 (EDxxT sequence), a PLAG-like domain,
was reported to be a critical sequence for the PDPN–CLEC-2
interaction.(4) The small cytoplasmic C-terminus (nine aminoacids) interacts with members of the ERM (ezrin, radixin, andmoesin) protein family. This affects cytoskeletal dynamics
and the formation or stabilization of different membrane
structures, such as filopodia, invadopodia, or ruffles.(5–7)
PDPN is a specific marker for type I alveolar cells of thelung.(2,8) Therefore, anti-PDPN monoclonal antibodies(mAbs) are useful in distinguishing type I alveolar cells of thelung from the type II cells. We characterized the PDPNs ofvarious animal species using specific anti-PDPN mAbs. We
established antimouse (PMab-1),(9) antirat (PMab-2),(10) antirabbit (PMab-32),(11) antidog (PMab-38(12) and PMab48(13)), antibovine (PMab-44),(14) and anticat (PMab-52)(15)
PDPN antibodies. PMab-38 was found to be cancer cell
specific since it did not react with lymphatic endothelial cells

and type I alveolar cells,(12) but reacted with squamous cell
carcinomas(16) and melanomas.(17) We developed several
antihuman PDPN mAbs, including NZ-1.2,(9) LpMab7,(18,19) LpMab-10,(20) LpMab-13,(21) and LpMab-17.(22) Wealso produced antiglycopeptide mAbs (GpMabs) against
human PDPN, namely, LpMab-3,(18) LpMab-9,(18) LpMab12,(23) LpMab-19,(24) and LpMab-21.(25,26) Cancer-specificmAbs (CasMabs) against human PDPN, such as LpMab2(18,27) and LpMab-23,(28,29) were also established. Because
PDPN is expressed in several normal tissues, CasMabs
against human PDPN should be utilized for clinical applications.
Antisheep PDPN mAbs have not been reported until date.
In this study, we used immunohistochemical analyses toinvestigate the potential cross-reactions between antiPDPN mAbs established for several species, particularly forsheep PDPN.
Materials and Methods
Immunohistochemical analyses

Normal sheep lungs were collected from two sheep afterautopsy at Hokkaido University, fixed in 10% neutralbuffered formalin, and processed routinely to make paraffinembedded tissue sections. Histological sections of thickness
4 mm were directly autoclaved in EnVision FLEX Target
Retrieval Solution, high pH (Agilent Technologies, Inc.,

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
3
Laboratory of Comparative Pathology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
4
Laboratory of Infectious Diseases, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University,Sapporo, Japan.
5
Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
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Santa Clara, CA) for 20 minutes. After blocking with
SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher
Scientific, Inc. Waltham, MA), sections were incubated with
PMab-44 (10 mg/mL) for 1 hour at room temperature andtreated using Envision+ Kit (Agilent Technologies, Inc.) for
30 minutes. Then color was developed using 3,3¢-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies,Inc.) for 2 minutes, and counterstaining was performed withhematoxylin (FUJIFILM Wako Pure Chemical Corporation,Osaka, Japan).
Results and Discussion

using flow cytometry and Western blotting.(30) The PLAG3of bovine PDPN was identified as the PMab-44 critical epitope. A comparison of amino acid sequences revealed an 85%
homology between bovine and sheep PDPN (SupplementaryFig. S1). Therefore, we further investigated whether PMab-44
cross-reacts with sheep PDPN.
Flow cytometry indicated that PMab-44 reacted withCHO-K1 cells that overexpressed sheep PDPN (CHO/sPDPN) (Supplementary Fig. S2). Other anti-PDPN mAbs,
such as antimouse (PMab-1),(9) antirat (PMab-2),(10) antirabbit (PMab-32),(11) antidog PMab-38(12) and PMab-48(13),and anticat (PMab-52(15)), did not react with CHO/sPDPN(data not shown). These results indicate that only PMab-44 isuseful for the detection of sheep PDPN.
Furthermore, we investigated the expression of sheep
PDPN in sheep lungs using immunohistochemical analyses.
For this purpose, we used lungs obtained from two sheep(No. 1 and No. 2). Both lungs were stained using PMab-44

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Previously, we have developed a mouse antibovine PDPNmAb, PMab-44 (IgG1, kappa), which specifically detectsbovine PDPN in immunohistochemical analyses.(14) Furthermore, a series of bovine PDPN deletion or point mutants
were used for investigating the binding epitopes of PMab-44

KATO ET AL.

FIG. 1. Immunohistochemical analyses using sheep tissues. Histological sections of the sheep tissues were directlyautoclaved in EnVision FLEX Target Retrieval Solution, high pH, for 20 minutes. After blocking, the sections wereincubated with 10 mg/mL of PMab-44 (A, B) or with blocking buffer (C, D), followed by detection using Envision+ Kit.
(E, F) Hematoxylin and eosin staining. Scale bar = 100 mm.

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SHEEP PDPN DETECTED USING PMAB-44(10 mg/mL) when EnVision FLEX Target Retrieval Solution,high pH, was used for an antigen retrieval procedure. Representative results (No. 2) are depicted in Figure 1. Type I
alveolar cells, but not the bronchial cells, were strongly
stained with PMab-44 (Fig. 1A, B). Staining was not observed without PMab-44 (Fig. 1C, D). The identical tissue(No. 2) was stained moderately using PMab-44 (1 mg/mL)
when citrate buffer (pH 6) was used for antigen retrieval(Supplementary Fig. S3), indicating that a specific staining byPMab-44 was observed at a low concentration of PMab-44(1 mg/mL) or under universal antigen retrieval conditions(i.e., when citrate buffer was used). Unfortunately, the sheep
kidney and colon were not stained by PMab-44 in this study(data not shown). This may be attributed to the varying expression levels of sheep PDPN among different tissues.
In conclusion, the antibovine PDPN mAb PMab-44 isuseful for the detection of sheep PDPN in sheep lungs using
immunohistochemical analyses. Further studies are necessary to demonstrate the capacity of PMab-44 for detection ofsheep PDPN in other tissues.
Acknowledgments

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 number 17K07299 (M.K.K.) and grant number
16K10748 (Y.K.).
Author Disclosure Statement

No competing financial interests exist.
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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery CenterTohoku University
2-1, Seiryo-machi, Aoba-ku
Sendai, Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: September 16, 2018
Accepted: November 25, 2018