MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 37, Number 6, 2018ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2018.0037

Detection of Alpaca Podoplanin by Immunohistochemistry
Using the Antibovine Podoplanin Monoclonal
Antibody PMab-44

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Yukinari Kato,1,2 Shinji Yamada,1 Shunsuke Itai,1 Satoru Konnai,3,4 Atsushi Kobayashi,5 and Mika K. Kaneko1

Podoplanin (PDPN) is expressed in type I alveolar cells of the lungs, lymphatic endothelial cells, and podocytesof the kidneys, and induces platelet aggregation through the C-type lectin-like receptor-2. PDPNs of various
animal species have been characterized using specific anti-PDPN monoclonal antibodies (mAbs). However,
alpaca PDPN has not previously been characterized because antialpaca PDPN mAbs have not yet been developed. In this study, we investigated the potential cross-reaction between established antibovine PDPN mAbs
and alpaca PDPN. Using immunohistochemical analysis, type I alveolar cells of the alpaca lungs were detectedby the antibovine PDPN mAb, PMab-44. These results indicate that PMab-44 may be useful for the detection ofalpaca PDPN.
Keywords: alpaca podoplanin, alpaca PDPN, PMab-44

Introduction

Podoplanin (PDPN) studied in a variety of animal
species is a type I transmembrane sialoglycoprotein.(1)Although PDPN lacks any recognizable catalytic activity, its
N-terminus extracellular domain and C-terminus intracellular domain play important roles in cellular physiology
through protein–protein interactions. The small cytoplasmic
C-terminus (nine amino acids) interacts with the members of
the ERM (ezrin, radixin, and moesin) protein family,(2–4)
impacting the cytoskeleton dynamics and formation or stabilization of membrane structures, such as filopodia, invadopodia, or ruffles. The extracellular domain of PDPN isextensively O-glycosylated, enabling interactions with a variety of proteins, including the C-type lectin-like receptor-2(CLEC-2),(5) CD44,(3) CD9,(6) galectin8,(7) CCL21,(8) orHspA9.(9) Importantly, three platelet aggregation-stimulating(PLAG) domains termed PLAG1, PLAG2, and PLAG3(EDxxVTPG sequence) of the N-terminus induce platelet
aggregation through CLEC-2 of the platelets.(10) We havepreviously shown that PLAG3 is the most important domain
for platelet aggregation by human PDPN.(10–12)
Anti-PDPN monoclonal antibodies (mAbs) are useful indistinguishing type I from type II alveolar cells of the lungs,
since PDPN serves as a specific marker for type I alveolar

cells.(11,13) We characterized the PDPNs from a variety ofanimal species using specific anti-PDPN mAbs and established antimouse (PMab-1),(14) antirat (PMab-2),(15) antirabbit (PMab-32),(16) antidog (PMab-38(17) and PMab-48(18)),
antibovine (PMab-44),(19) and anticat (PMab-52) PDPN
mAbs.(20)Antialpaca PDPN mAbs have not previously been reported. In this study, we investigated the potential crossreaction between the alpaca PDPN and our set of anti-PDPN
mAbs established against diverse species using immunohistochemical analyses.
Materials and Methods
Immunohistochemical analyses

Normal alpaca lungs were collected after autopsy atHokkaido University, fixed in 10% neutral-buffered formalin, and processed routinely to make paraffin-embedded tissue sections. Histological sections of thickness 4 mm weredirectly autoclaved in EnVision FLEX Target Retrieval Solution, high pH (Agilent Technologies, Inc., 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 and treated using

1Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
3
Laboratory of Infectious Diseases, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University,Sapporo, Japan.
4
Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
5
Laboratory of Comparative Pathology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
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Envision+ Kit (Agilent Technologies, Inc.) for 30 minutes.
Then color was developed using 3,3¢-diaminobenzidine tetrahydrochloride (Agilent Technologies, Inc.) for 2 minutes,and counterstaining was performed with hematoxylin(FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).

Results and Discussion

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In our previous work, we developed a mouse antibovine
PDPN mAb, PMab-44 (IgG1, kappa), which specifically detects bovine PDPN in immunohistochemistry.(19) The
PLAG3 of bovine PDPN was identified as a critical epitope ofPMab-44(21) (Supplementary Fig. S1). The comparison ofamino acid sequences revealed 85% homology between thebovine PDPN and alpaca PDPN. Furthermore, the alpaca
PDPN possesses PMab-44 epitope (VEDYTT). Therefore, inthis study, we investigated the cross-reaction between PMab44 and alpaca PDPN.

KATO ET AL.

In flow cytometry studies, PMab-44 reacted with alpaca
PDPN-overexpressed CHO-K1 (CHO/aPDPN) cells (Supplementary Fig. S2). No reaction with CHO/aPDPN wasobserved for the other anti-PDPN mAbs, such as PMab-1,(14)PMab-2,(15) PMab-32,(16) PMab-38(17) and PMab-48,(18) andPMab-52(20) (data not shown). These results indicate that
only PMab-44, among the anti-PDPN mAbs, is useful forexperimental detection of alpaca PDPN.
We investigated the expression of alpaca PDPN in several
alpaca tissues. Previously, it has been demonstrated that
PMab-44 reacts with podocytes of bovine kidney(19) (Supplementary Fig. S3). As depicted in Figure 1, a membranestaining pattern was observed, with strong staining of type I
alveolar cells of the alpaca lungs by PMab-44. In contrast,
alpaca kidney and colon cells were not stained by PMab-44 inthis study (data not shown). This may be attributed to thevarying expression levels of alpaca PDPN in different tissues.
In conclusion, antibovine PDPN (PMab-44) is useful forthe detection of alpaca PDPN using immunohistochemicalFIG. 1. Immunohistochemical analyses using alpaca tissues. Histological sections of the alpaca tissues were directly
autoclaved 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.

ALPACA PDPN DETECTED USING PMAB-44

analyses. Further studies are necessary to explore whether
PMab-44 is able to detect alpaca PDPN in tissues other thantype 1 alveolar cells of the lungs.
Acknowledgments

This research was supported, in part, by AMED underGrant Nos. JP18am0101078 (Y.K.), JP18am0301010 (Y.K.),
and JP18ae0101028 (Y.K.), and by JSPS KAKENHI Grant
No. 17K07299 (M.K.K.) and Grant No. 16K10748 (Y.K.).
Author Disclosure Statement

No competing financial interests exist.

Downloaded by Yukinari Kato from www.liebertpub.com at 12/28/18. For personal use only.

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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1, Seiryo-machi, Aoba-ku
MiyagiSendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: September 18, 2018
Accepted: November 25, 2018