MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 1, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0055
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Immunohistochemical Analysis Using Monoclonal Antibody
PMab-269 Against Steller Sea Lion Podoplanin

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Guanjie Li,1 Hiroyuki Suzuki,1 Junko Takei,2,3 Masaki Saito,1 Nohara Goto,1,4 Kazuyuki Uchida,5Takayuki Nakagawa,6 Hiroyuki Harada,3 Tomohiro Tanaka,2 Teizo Asano,2Mika K. Kaneko,2 and Yukinari Kato1,2

Monoclonal antibodies (mAbs) that specifically target podoplanin (PDPN), a marker for type I alveolar cells,
are required for immunohistochemical analyses. Anti-PDPN mAbs are available for many species, including
human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, bear, sheep,
and California sea lion PDPNs. However, no anti-Steller sea lion PDPN (stePDPN) antibody has been developed. Immunohistochemical analysis showed that an anti-California sea lion PDPN mAb (PMab-269) reactedwith type I alveolar cells from the Steller sea lion lung, renal glomeruli and Bowman’s capsules from kidney,
and lymphatic endothelial cells from the colon, indicating that PMab-269 is useful for detecting stePDPN.
Keywords: Steller sea lion, podoplanin, PDPN, PMab-269

short intracellular domain. PDPN is identical to T1a, which
encodes an antigen expressed at the apical membrane of lungtype I alveolar epithelium.(4,5) The name ‘‘podoplanin’’ isderived from its expression in kidney podocytes.(6) In a rat
model of human minimal change nephrotic syndrome, thereduced expression of PDPN was observed at the proteinuric
stage. The amount of urinary PDPN markedly increased on
day 1 of PAN nephropathy.(7)PDPN expression in lymphatic endothelium was also reported and named as ‘‘E11 antigen.’’(8) PDPN is one of the mosthighly expressed lymphatic-specific genes but is not expressedin blood vascular endothelial cells.(9,10) Therefore, PDPN is oneof the important markers of lymphatic endothelial cells.
PDPN binds to its receptor, the C-type lectin-like receptor2, and is involved in platelet aggregation.(11) The platelet
aggregation-stimulating (PLAG) domain(12–14) and the
PLAG-like domain(15) in PDPN ectodomain play a vital rolein platelet aggregation.

Introduction

S

teller sea lions (Eumetopias jubatus) inhabit thecoastal waters in the northern Pacific Rim, stretchingfrom central California to northern Japan.(1) The western
stock of the Steller sea lion in the northern Pacific Ocean has
reduced by >80% in four decades. This resulted in its listingas an endangered species in 1997. Information on the distributions and cause of the reduction is required for management.(2) The closest relative to the Steller sea lion with asequenced genome is the California sea lion (Zalophus californianus). Both species have a diploid karyotype with 18
chromosomes. Alignment of the Steller sea lion assembly to
the California sea lion assembly shows a 99.5% similarity
between the two genomes.(3)Podoplanin (PDPN) is a type I transmembrane mucin-likesialoglycoprotein having a heavily glycosylated N-terminal
extracellular domain, a single transmembrane domain, and a

Departments of 1Molecular Pharmacology and 2Antibody Drug Development, Tohoku University Graduate School of Medicine,
Sendai, Japan.
3
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Bunkyo-ku, Japan.
4
Department of Experimental Pathology, Graduate School of Comprehensive Human Sciences, Faculty of Medicine, University ofTsukuba, Tsukuba, Japan.
5
Laboratory of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Japan.
6
Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Japan.

39

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LI ET AL.

Anti-PDPN monoclonal antibodies (mAbs) have been
generated in various species, including human,(16) mouse,(17)rat,(18) rabbit,(19) dog,(20) cat,(21) cow,(22) pig,(23) Tasmaniandevil,(24) alpaca,(25) tiger,(26) whale,(27) goat,(28) horse,(29)bear,(30) sheep,(31,32) and California sea lion.(33) All mAbs
are available for Western blotting, flow cytometry, andimmunohistochemistry.
In this study, immunohistochemistry was performed using
Steller sea lion tissues and the reactivity of PMab-269 (developed as an anti-California sea lion PDPN mAb) to Steller
sea lion PDPN (stePDPN) was investigated.

Target Retrieval Solution High pH (Agilent Technologies,
Inc., Santa Clara, CA, USA) for 20 minutes. After blocking
with SuperBlock T20 (phosphate-buffered saline) Blocking
Buffer (Thermo Fisher Scientific, Inc., Waltham, MA, USA),sections were incubated using PMab-269 (5 lg/mL) for
1 hour at room temperature and treated with EnVision+ Kit(Agilent Technologies, Inc.) for 30 minutes. The color wasdeveloped using 3,3¢-diaminobenzidine tetrahydrochloride(DAB; Agilent Technologies, Inc.) for 2 minutes and counterstained using hematoxylin and eosin (FUJIFILM Wako
Pure Chemical Corporation, Osaka, Japan).

Materials and Methods

Results

Immunohistochemical analysis

Immunohistochemical analyses

Normal Steller sea lion tissues obtained from routine
necropsies performed at the Laboratory of Veterinary Pathology, the University of Tokyo, fixed in 10% neutralbuffered formalin, were processed to make formalin-fixedparaffin-embedded (FFPE) tissue sections. Histological sections (4 lm thickness) were autoclaved in EnVision FLEX

We investigated whether PMab-269 can be used for immunohistochemical analyses using FFPE Steller sea lion
tissue sections. The normal lung, kidney, and colon tissue
from Steller sea lions were examined, all of which express
PDPN in different species, including humans,(16) mouse,(17)rat,(18) rabbit,(19) dog,(20) cat,(21) cow,(22) pig,(23) Tasmanian

FIG. 1. Immunohistochemical analysis of Steller sea lion lung tissue. Histological sections of Steller sea lion lung weredirectly autoclaved in EnVision FLEX Target Retrieval Solution High pH and incubated with 5 mg/mL PMab-269 (A, B) or
blocking buffer (C, D), followed by the use of EnVision+ Kit. (E, F) Hematoxylin and eosin staining. Arrows indicate thelymphatic endothelial cells. Scale bar = 100 mm.

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ANTI-STELLER SEA LION PDPN MAB

41

FIG. 2. Immunohistochemical analysis of Steller sea lion kidney tissue. Histological sections of Steller sea lion kidney
were directly autoclaved in EnVision FLEX Target Retrieval Solution High pH and incubated with 5 mg/mL PMab-269(A, B) or blocking buffer (C, D), followed by the use of EnVision+ Kit. (E, F) Hematoxylin and eosin staining. Scalebar = 100 mm.

devil,(24) alpaca,(25) tiger,(26) whale,(27) goat,(28) horse,(29)bear,(30) sheep,(31,32) and California sea lion.(33) As indicatedin Figure 1, PMab-269 strongly and specifically identified
type I alveolar cells within the lung. PMab-269 also identified
renal glomeruli and Bowman’s capsules (Fig. 2). In addition,
lymphatic endothelial cells in the lung and submucosa of thecolon were detected by PMab-269, respectively (Figs. 1 and
3, arrows). These results show that PMab-269 is useful indetecting stePDPN-positive cells in FFPE tissue.
Discussion

The usefulness of PMab-269 was demonstrated for thepathophysiological analysis of lung, kidney, and lymphatic
tissues of the Steller sea lion. PMab-269 was originally developed as an anti-California sea lion PDPN mAb, which hasstrong cross-reactivity to cat and tiger, but not to the dog
PDPN.(33) The binding epitope of PMab-269 as Pro68,
Asp76, Phe77, His78, Leu79, Glu80, and Asp81 of California
sea lion PDPN was also determined.(34) The sequence iscompletely identical to that of stePDPN. Alternatively, cat
and tiger PDPN match the six of seven amino acids, and dog
PDPN matches the five of seven. The Pinnipedia (including
sea lions) is assigned to Califormia, which is a suborder of
dog-like carnivorans. Califormia stands in contrast to theother suborder of Carnivora, the Feliformia (cat-like carnivorans). At least in the binding epitope of PMab-269, sea
lions are similar to the Feliformia.
The number of Steller sea lions of the western stock has
reduced. Among several suspected causes for the decline areadverse effects of chemical pollutants, including polychlorinated biphenyls (PCBs). PCBs and their effects on animals have been widely studied.(35–37) PCBs show a broad rangeof toxic effects, including reproduction failure(38) and immune
disorders.(39) Wang et al. investigated 145 individual PCBs in
the tissues of sea lions. They determined the mean toxic
equivalents in the kidney, liver, and blubber samples, proposing the possibility of adverse effects on animals.(40) PMab269 might contribute the pathological studies to investigate thetissue damage, including podocyte injury in the kidney.

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LI ET AL.

FIG. 3. Immunohistochemical analyses of Steller sea lion colon tissue. Histological sections of Steller sea lion colon weredirectly autoclaved in EnVision FLEX Target Retrieval Solution High pH and incubated with 5 mg/mL PMab-269 (A, B) or
blocking buffer (C, D), followed by the use of EnVision + Kit. (E, F) Hematoxylin and eosin staining. Arrows show the
PDPN expressed lymphatic endothelial cells. Scale bar = 100 mm. PDPN, podoplanin.
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 (to Y.K.) and JP21am0101078(to Y.K.), and by the Japan Society for the Promotion of
Science ( JSPS) Grants-in-Aid for Scientific Research (KAKENHI) Grant Nos. 21K06059 (to M.S.), 21K07168(to M.K.K.), 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
Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: November 17, 2021
Accepted: January 11, 2021