MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume XX, Number XX, 2016ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2016.0016

Original Article

PMab-44 Detects Bovine Podoplaninin Immunohistochemistry
Ryusuke Honma,1,2* Satoshi Ogasawara,1* Mika K. Kaneko,1* Yuki Fujii,1 Hiroharu Oki,1,2
Takuro Nakamura,1 Michiaki Takagi,2 Satoru Konnai,3 and Yukinari Kato1

Podoplanin (PDPN)/Aggrus is a type I transmembrane O-glycoprotein, which is expressed in several normaltissues including podocytes of kidney and lymphatic endothelial cells. PDPN activates platelet aggregation bybinding to C-type lectin-like receptor-2 (CLEC-2) on platelet; however, only bovine PDPN (bovPDPN) does
not possess the platelet-aggregating activity. Although many monoclonal antibodies (mAbs) against human
PDPN, mouse PDPN, rat PDPN, and rabbit PDPN have been established, anti-bovPDPN mAbs have not beendeveloped. In this study, we immunized mice with the recombinant proteins of bovPDPN and developed
anti-bovPDPN mAbs, which are useful in immunohistochemical analysis. One of the clones, PMab-44, is usefulfor detecting podocytes and lymphatic endothelial cells in normal bovine tissues. PMab-44 also detected
bovPDPN specifically in flow cytometry. PMab-44 is expected to be useful for investigating the function ofbovPDPN.

Medium (Nacalai), supplemented with 10% heat-inactivatedfetal bovine serum (Thermo Fisher Scientific, Inc. (Thermo),
Waltham, MA), 100 U/mL of penicillin, 100 mg/mL of streptomycin, and 25 mg/mL of amphotericin B (Nacalai) at 37Cin a humidified atmosphere of 5% CO2 and 95% air. Female
BALB/c mice (four weeks old) were purchased from CLEA
Japan (Tokyo, Japan). The animals were housed underpathogen-free conditions. The Animal Care and Use Committee of Tohoku University approved the animal experiments described herein.

Introduction

Podoplanin (PDPN) is a type I transmembrane Oglycoprotein, which is expressed in several normaltissues.(1) PDPN/C-type lectin-like receptor-2 (CLEC-2)
interaction is associated with blood/lymphatic vessel separation.(2) PDPN activates platelet aggregation by binding toCLEC-2 on platelet.(3,4) The interaction with CLEC-2 wasmainly observed at Glu47 and Asp48 in the platelet aggregationstimulating (PLAG) domain and the a2,6-linked sialic acid at
Thr52 of human PDPN (hPDPN).(5) We previously reported that
only bovine PDPN (bovPDPN) lacks platelet aggregationinducing activity because bovPDPN has a sporadic deletion
mutation in amino acid sequence of the PLAG domain.(6) In thisstudy, we immunized mice with the recombinant proteins of
bovPDPN and developed novel anti-bovPDPN monoclonal
antibodies (mAbs).

Hybridoma production

Bovine PDPN with N-terminal PA tag and C-terminal
RAP tag-MAP tag (PA-bovPDPN-RAP-MAP) was inserted
into pCAG-zeo vector (Wako Pure Chemical Industries Ltd.
(Wako), Osaka, Japan). RAP tag consists of 12 amino acids(DMVNPGLEDRIE) and MAP tag consists of 12 aminoacids (GDGMVPPGIEDK). CHO-K1 was transfected withpCAG-zeo/PA-bovPDPN-RAP-MAP using Gene Pulser Xcell
electroporation system (Bio-Rad Laboratories, Inc. (Bio-Rad),
Berkeley, CA). For the purification of PA-bovPDPN-RAPMAP from cell membrane, we used the PA tag system.(7,8)
BALB/c mice were immunized by intraperitoneal (i.p.) injection of 100 mg of recombinant PA-bovPDPN-RAP-MAP together with Imject Alum (Thermo). After several additional
immunizations of 50 mg, a booster injection of 100 mg wasMaterials and Methods
Cell lines and animals

Chinese hamster ovary (CHO)-K1, Madin–Darby bovine
kidney (MDBK), and P3U1 were purchased from the American Type Culture Collection (ATCC, Manassas, VA). CHOK1, stable CHO transfectants, and P3U1 were cultured in RPMI
1640 medium (Nacalai Tesque, Inc. (Nacalai), Kyoto, Japan),and MDBK was cultured in Dulbecco’s Modified Eagle’s
1

Department of Regional Innovation, Tohoku University Graduate School of Medicine, Miyagi, Japan.
Department of Orthopaedic Surgery, Yamagata University Faculty of Medicine, Yamagata, Japan.
Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido, Japan.
*These authors contributed equally to this work.

23

1

2given i.p. 2 days before spleen cells were harvested. The spleencells were fused with P3U1 cells using PEG1500 (RocheDiagnostics, Indianapolis, IN). The hybridomas were grownin RPMI medium with hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo). Recombinant
proteins of PA-bovPDPN-RAP-MAP were immobilized onNunc Maxisorp 96-well immunoplates (Thermo) at 1 mg/mL
for 30 minutes. After blocking with 1% BSA in 0.05%
Tween20/phosphate-buffered saline (PBS), the plates wereincubated with culture supernatant followed by 1:3000 diluted
peroxidase-conjugated anti-mouse IgG (Dako; Agilent Technologies, Inc., Santa Clara, CA). The enzymatic reaction wasconducted with a 1-Step Ultra TMB-ELISA (Thermo). Theoptical density was measured at 655 nm using an iMark microplate reader (Bio-Rad).
Immunohistochemical analyses

Four micrometer-thick histologic sections were deparaffinized in xylene and rehydrated, and were autoclaved inHONMA ET AL.

citrate buffer (pH 6.0; Dako) for 20 minutes. Sections wereincubated with 1 mg/mL of primary mAbs for 1 hour at room
temperature followed by treatment with Envision+ kit
for 30 minutes (Dako). Color was developed using 3,3diaminobenzidine tetrahydrochloride (DAB; Dako) for 1
minute, and then the sections were counterstained withhematoxylin (Wako).
Flow cytometry

The pcDNA3/bovPDPN-FLAG(6) was transfected into CHOK1 cells for flow cytometry. Stable transfectant of CHO/
bovPDPN-FLAG was established using SH800 (Sony Corp.,Tokyo, Japan). The other stable transfectants (CHO/hPDPNFLAG, CHO/mPDPN-FLAG, CHO/rPDPN-His, and CHO/
PA-rabPDPN) were previously established.(9–11) Cells wereharvested by brief exposure to 0.25% Trypsin/1 mM EDTA(Nacalai). After washing with 0.1% BSA/PBS, the cells
were treated with primary mAbs (1 mg/mL) for 30 minutes at4C followed by treatment with Oregon green-conjugated

FIG. 1. Immunohistochemical analysis by anti-bovPDPN mAbs. Sections of bovine kidney and colon were autoclaved incitrate buffer (pH 6.0). After blocking, they were incubated with 1 mg/mL of PMab-43, PMab-44, PMab-45, and PMab-46,
followed by EnVision+ kit, and color was developed using DAB and counterstained with hematoxylin. PBS was used asnegative control. HE staining was performed against serial sections. Arrow heads, lymphatic vessels. Scale bar: 100 mm.
mAbs, monoclonal antibodies; PBS, phosphate-buffered saline.

FIG. 2. Flow cytometric analysis by anti-PDPN mAbs. CHO/bovPDPN-FLAG, CHO/hPDPN-FLAG, CHO/mPDPN-FLAG,CHO/rPDPN-His, CHO/PA-rabPDPN, CHO-K1, and MDBK were treated with PMab-44, LpMab-12, PMab-1, PMab-2, andPMab-32 followed by treatment with Oregon green-conjugated anti-mouse IgG against PMab-44, LpMab-12, PMab-2, and PMab32 or anti-rat IgG against PMab-1. Red line: PMab-44, LpMab-12, PMab-1, PMab-2, and PMab-32. Black line: negative control.
34anti-mouse IgG or anti-rat IgG (1:1000 diluted; Thermo).
Fluorescence data were collected using a Cell Analyzer
EC800 (Sony Corp.).
Results
Production of mAbs against bovPDPN

We first immunized mice with the recombinant proteins,which were purified from CHO/PA-bovPDPN-RAP-MAP
cells, and the ELISA screening was performed. Among
ELISA-positive wells, supernatants from four wells reactedwith bovPDPN in immunohistochemical analysis (data notshown). These hybridomas were cloned by limiting dilution.
After purification using protein G, we further investigated thesensitivity of those mAbs in immunohistochemistry. One ofthe clones, PMab-44 (IgG1, kappa), reacted with bovPDPN oflymphatic endothelial cells in kidney or colon and with podocytes and Bowman’s capsule in kidney the most sensitively
compared with PMab-43 (IgG1, kappa), PMab-45 (IgG1,kappa), and PMab-46 (IgG1, kappa) (Fig. 1). Furthermore,
PMab-44 does not need the antigen retrieval procedure todetect bovPDPN in all normal tissues; in contrast, the otheranti-bovPDPN mAbs need the antigen retrieval (data notshown). These data demonstrate that PMab-44 is useful for
immunohistochemistry using paraffin-embedded tissues.
Specificity of PMab-44 against bovine
PDPN in flow cytometryPMab-44 reacted with CHO/bovPDPN-FLAG, not with
CHO/hPDPN-FLAG, CHO/mPDPN-FLAG, CHO/rPDPNHis, CHO/PA-rabPDPN, and CHO-K1 cells in flow cytometry (Fig. 2). LpMab-12 (anti-hPDPN mAb; mouse IgG1),(12)PMab-1 (anti-mPDPN mAb; rat IgG2a),(13) PMab-2 (antirPDPN mAb; mouse IgG1),(10) and PMab-32 (anti-rabPDPN
mAb; mouse IgG1)(11) specifically reacted with CHO/hPDPNFLAG, CHO/mPDPN-FLAG, CHO/rPDPN-His, and CHO/
PA-rabPDPN, respectively. LpMab-12 reacted with CHO/
rPDPN-His and CHO/PA-rabPDPN very weakly. Furthermore, PMab-44 reacted with MDBK cells, which express
endogenous bovPDPN; in contrast, the other anti-PDPN mAbs
did not. These results indicate that PMab-44 detects not only
exogenous but also endogenous bovPDPN, and is very specificagainst bovPDPN.
Discussion

Expression of hPDPN has been reported in many malignant
tumors such as osteosarcomas,(14) malignant brain tumors,(15–18)
oral squamous cell carcinomas,(19) lung cancers,(20) esophagealcancers,(21) malignant mesotheliomas,(22) and testicular tumors.(23) Therefore, many mAbs against hPDPN have been
established.(14,18,24–29) Several mAbs against mPDPN,(13)rPDPN,(10) and rabPDPN(11,30) have also been established.
In contrast, useful mAbs against bovPDPN have not beenreported. Because we recently established specific mAbs
against rabPDPN by immunizing mice with membranous
rabPDPN, we purified membranous bovPDPN from CHO/
PA-bovPDPN-RAP-MAP cells and immunized mice withthose recombinant proteins. We used several original epitope tags such as PA tag, MAP tag, and RAP tag in thisstudy. All anti-tag mAbs detected recombinant proteins of
PA-bovPDPN-RAP-MAP (data not shown). The combina-HONMA ET AL.

tion of several tag systems is very useful not only for purification but also for establishing mAbs efficiently.
We previously constructed a phylogenetic tree of six mammalian PDPN amino acid sequences: hPDPN, mPDPN, rPDPN,bovPDPN, hamster PDPN (hamPDPN), and dog PDPN. Therodents (mPDPN, rPDPN, hamPDPN) and the bovine lineages
showed longer branches in contrast with the human and the dog
lineages.(6) It is likely that some kinds of functional changes arerelated to higher rates of amino acid substitution in the bovinelineages. In fact, bovPDPN did not induce platelet aggregation
probably because of a sporadic deletion mutation in amino acidsequence of the PLAG domain.(6) The platelet aggregation by
PDPN is known to be associated with blood/lymphatic vessel
separation in mice(2); therefore, there might be the othermechanism in bovine blood/lymphatic vessel separation. PMab44 could be useful to uncover the pathophysiological function ofbovPDPN in the near future.
Acknowledgments

We thank Noriko Saidoh and Kanae Yoshida for their excellent technical assistance. This work was supported, inpart, by the Regional Innovation Strategy Support Programfrom the Ministry of Education, Culture, Sports, Science andTechnology (MEXT) of Japan (Y.K.), by JSPS KAKENHI
Grant Number 26440019 (M.K.K.), Grant Number 25462242(Y.K.), and Grant Number 16K10748 (Y.K.) by Takeda Science Foundation (S.O.), by the Platform for Drug Discovery,Informatics, and Structural Life Science (PDIS) from Japan
Agency for Medical Research and development, AMED(Y.K.), and by the Basic Science and Platform Technology
Program for Innovative Biological Medicine from AMED
(Y.K.). This work was performed, in part, under the Cooperative Research Program of Institute for Protein Research,
Osaka University, CR-15-05 and CR-16-05.
Author Disclosure Statement

No competing financial interests exist.
References

1. Breiteneder-Geleff S, Matsui K, Soleiman A, Meraner P,Poczewski H, Kalt R, Schaffner G, and Kerjaschki D:
Podoplanin, novel 43-kd membrane protein of glomerularepithelial cells, is down-regulated in puromycin nephrosis.
Am J Pathol 1997;151:1141–1152.
2. Bertozzi CC, Schmaier AA, Mericko P, Hess PR, Zou Z,
Chen M, Chen CY, Xu B, Lu MM, Zhou D, Sebzda E,
Santore MT, Merianos DJ, Stadtfeld M, Flake AW, Graf T,
Skoda R, Maltzman JS, Koretzky GA, and Kahn ML:
Platelets regulate lymphatic vascular development through
CLEC-2-SLP-76 signaling. Blood 2010;116:661–670.
3. Kato Y, Kaneko MK, Kunita A, Ito H, Kameyama A,Ogasawara S, Matsuura N, Hasegawa Y, Suzuki-Inoue K,Inoue O, Ozaki Y, and Narimatsu H: Molecular analysis of
the pathophysiological binding of the platelet aggregationinducing factor podoplanin to the C-type lectin-like receptor CLEC-2. Cancer Sci 2008;99:54–61.
4. Kaneko MK, Kunita A, Abe S, Tsujimoto Y, Fukayama M,
Goto K, Sawa Y, Nishioka Y, and Kato Y: Chimeric antipodoplanin antibody suppresses tumor metastasis throughneutralization and antibody-dependent cellular cytotoxicity.
Cancer Sci 2012;103:1913–1919.

MABS AGAINST BOVINE PODOPLANIN

5. Nagae M, Morita-Matsumoto K, Kato M, Kaneko MK, KatoY, and Yamaguchi Y: A platform of C-Type lectin-like receptor CLEC-2 for binding O-glycosylated podoplanin andnonglycosylated rhodocytin. Structure 2014;22:1711–1721.
6. Kaneko MK, Kato Y, Kitano T, and Osawa M: Conservationof a platelet activating domain of Aggrus/podoplanin as aplatelet aggregation-inducing factor. Gene 2006;378:52–57.
7. Fujii Y, Kaneko M, Neyazaki M, Nogi T, Kato Y, and Takagi
J: PA tag: A versatile protein tagging system using a superhigh affinity antibody against a dodecapeptide derived fromhuman podoplanin. Protein Expr Purif 2014;95:240–247.
8. Fujii Y, Matsunaga Y, Arimori T, Kitago Y, Ogasawara S,Kaneko MK, Kato Y, and Takagi J: Tailored placement of aturn-forming PA tag into the structured domain of a protein toprobe its conformational state. J Cell Sci 2016;129:1512–1522.
9. Kato Y, Fujita N, Kunita A, Sato S, Kaneko M, Osawa M,and Tsuruo T: Molecular identification of Aggrus/T1alphaas a platelet aggregation-inducing factor expressed in colorectal tumors. J Biol Chem 2003;278:51599–51605.
10. Oki H, Honma R, Ogasawara S, Fujii Y, Liu X, Takagi M,
Kaneko MK, and Kato Y: Development of sensitive monoclonal antibody PMab-2 against rat podoplanin. Monoclon
Antib Immunodiagn Immunother 2015;34:396–403.
11. Honma R, Fujii Y, Ogasawara S, Oki H, Liu X, Nakamura T,
Kaneko MK, Takagi M, and Kato Y: Establishment of a novelmonoclonal antibody PMab-32 against rabbit podoplanin.
Monoclon Antib Immunodiagn Immunother 2016;35:41–47.
12. Kato Y, Ogasawara S, Oki H, Goichberg P, Honma R, Fujii
Y, and Kaneko MK: LpMab-12 Established by CasMabtechnology specifically detects sialylated O-glycan on
Thr52 of platelet aggregation-stimulating domain of human
podoplanin. PLoS One 2016;11:e0152912.
13. Kaji C, Tsujimoto Y, Kato Kaneko M, Kato Y, and Sawa Y:
Immunohistochemical Examination of novel rat monoclonal
antibodies against mouse and human podoplanin. Acta Histochem Cytochem 2012;45:227–237.
14. Kaneko MK, Oki H, Ogasawara S, Takagi M, and Kato Y:
Anti-podoplanin monoclonal antibody LpMab-7 detects
metastatic legions of osteosarcoma. Monoclon Antib Immunodiagn Immunother 2015;34:154–161.
15. Mishima K, Kato Y, Kaneko MK, Nishikawa R, Hirose T,and Matsutani M: Increased expression of podoplanin inmalignant astrocytic tumors as a novel molecular marker ofmalignant progression. Acta Neuropathol (Berl) 2006;111:483–488.
16. Mishima K, Kato Y, Kaneko MK, Nakazawa Y, Kunita A,Fujita N, Tsuruo T, Nishikawa R, Hirose T, and Matsutani M:
Podoplanin expression in primary central nervous system germ
cell tumors: A useful histological marker for the diagnosis ofgerminoma. Acta Neuropathol (Berl) 2006;111:563–568.
17. Kato Y, Vaidyanathan G, Kaneko MK, Mishima K,Srivastava N, Chandramohan V, Pegram C, Keir ST, Kuan
CT, Bigner DD, and Zalutsky MR: Evaluation of antipodoplanin rat monoclonal antibody NZ-1 for targeting
malignant gliomas. Nucl Med Biol 2010;37:785–794.
18. Kato Y, and Kaneko MK: A cancer-specific monoclonal
antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep 2014;4:5924.
19. Ogasawara S, Kaneko MK, Honma R, Oki H, Fujii Y,Takagi M, Suzuki H, and Kato Y: Establishment of mousemonoclonal antibody LpMab-13 against human podoplanin.
Monoclon Antib Immunodiagn Immunother 2016 [Epubahead of print]; DOI: 10.1089/mab.2016.0006.

520. Kato Y, Kaneko M, Sata M, Fujita N, Tsuruo T, Osawa M:
Enhanced expression of Aggrus (T1alpha/podoplanin), aplatelet-aggregation-inducing factor in lung squamous cell
carcinoma. Tumor Biol 2005;26:195–200.
21. Schoppmann SF, Jesch B, Riegler MF, Maroske F,Schwameis K, Jomrich G, and Birner P: Podoplanin expressing cancer associated fibroblasts are associated withunfavourable prognosis in adenocarcinoma of the esophagus. Clin Exp Metastasis 2013;30:441–446.
22. Kimura N, and Kimura I: Podoplanin as a marker for mesothelioma. Pathol Int 2005;55:83–86.
23. Kato Y, Sasagawa I, Kaneko M, Osawa M, Fujita N, andTsuruo T: Aggrus: A diagnostic marker that distinguishes
seminoma from embryonal carcinoma in testicular germ
cell tumors. Oncogene 2004;23:8552–8556.
24. Kato Y, Kaneko MK, Kuno A, Uchiyama N, Amano K,Chiba Y, Hasegawa Y, Hirabayashi J, Narimatsu H,
Mishima K, and Osawa M: Inhibition of tumor cell-induced
platelet aggregation using a novel anti-podoplanin antibody
reacting with its platelet-aggregation-stimulating domain.
Biochem Biophys Res Commun 2006;349:1301–1307.
25. Kaneko MK, Oki H, Hozumi Y, Liu X, Ogasawara S,Takagi M, Goto K, and Kato Y: Monoclonal antibody
LpMab-9 recognizes O-glycosylated N-terminus of humanpodoplanin. Monoclon Antib Immunodiagn Immunother2015;34:310–317.
26. Oki H, Kaneko MK, Ogasawara S, Tsujimoto Y, Liu X,Sugawara M, Takakubo Y, Takagi M, and Kato Y: Characterization of a monoclonal antibody LpMab-7 recognizing non-PLAG domain of podoplanin. Monoclon AntibImmunodiagn Immunother 2015;34:174–180.
27. Oki H, Ogasawara S, Kaneko MK, Takagi M, Yamauchi M,and Kato Y: Characterization of monoclonal antibody LpMab3 recognizing sialylated glycopeptide of podoplanin. Monoclon
Antib Immunodiagn Immunother 2015;34:44–50.
28. Ogasawara S, Kaneko MK, Price JE, and Kato Y: Characterization of anti-podoplanin monoclonal antibodies:
Critical epitopes for neutralizing the interaction between
podoplanin and CLEC-2. Hybridoma 2008;27:259–267.
29. Kaneko M, Kato Y, Kunita A, Fujita N, Tsuruo T, and Osawa
M: Functional sialylated O-glycan to platelet aggregation on
Aggrus (T1alpha/podoplanin) molecules expressed in Chinese Hamster Ovary cells. J Biol Chem 2004;279:38838–38843.
30. Honma R, Fujii Y, Ogasawara S, Oki H, Konnai S, Kagawa
Y, Takagi M, Kaneko MK, and Kato Y: Critical epitope ofanti-rabbit podoplanin monoclonal antibodies for immunohistochemical analysis. Monoclon Antib Immunodiagn
Immunother 2016;35:65–72.

Address correspondence to:
Dr. Yukinari Kato
Department of Regional Innovation
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi, Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp;
yukinarikato@med.tohoku.ac.jp
Received: March 11, 2016
Accepted: May 6, 2016