Podoplanin (PDPN) is a platelet aggregation-inducing factor, which is known as an endogenous ligand of C-type lectin-like receptor-2 (CLEC-2). PDPN is also expressed in several normal tissues, such as lung type I alveolar cells and kidney podocytes. Although many monoclonal antibodies (MAbs) against human PDPN (hPDPN) or mouse PDPN (mPDPN) have been established, anti-rat PDPN (rPDPN) MAbs, especially against platelet aggregation-stimulating (PLAG) domain (29-54 amino acids) of rPDPN, have not been developed. Therefore, functional analysis of rPDPN in normal tissues has been limited. Here, we immunized mice with rPDPN peptides (38-51 amino acids) and developed a novel mouse anti-rPDPN MAb, PMab-2 (IgG1, kappa), which possesses high affinity compared with anti-hPDPN or mPDPN MAbs. The KD of PMab-2 was determined to be 5.9 × 10−10 M. PMab-2 is useful, not only in flow cytometry and Western blot analysis against endogenous rPDPN, which is expressed in rat dermal fibroblast, but also in immunohistochemistry against normal tissues. PMab-2 showed extraordinarily high sensitivity in immunohistochemistry, indicating that PMab-2 is very advantageous for functional analysis of rPDPN.
Get full access to this article
View all access options for this article.
References
1.
Breiteneder-GeleffS, MatsuiK, SoleimanA, MeranerP, PoczewskiH, KaltR, SchaffnerG, and KerjaschkiD: Podoplanin, novel 43-kd membrane protein of glomerular epithelial cells, is down-regulated in puromycin nephrosis. Am J Pathol, 1997; 151:1141–1152.
2.
GandarillasA, SchollFG, BenitoN, GamalloC, and QuintanillaM: Induction of PA2.26, a cell-surface antigen expressed by active fibroblasts, in mouse epidermal keratinocytes during carcinogenesis. Mol Carcinog, 1997; 20:10–18.
3.
KatoY, FujitaN, KunitaA, SatoS, KanekoM, OsawaM, and TsuruoT: Molecular identification of Aggrus/T1alpha as a platelet aggregation-inducing factor expressed in colorectal tumors. J Biol Chem, 2003; 278:51599–51605.
4.
KanekoMK, KatoY, KitanoT, and OsawaM: Conservation of a platelet activating domain of Aggrus/podoplanin as a platelet aggregation-inducing factor. Gene, 2006; 378:52–57.
5.
NagaeM, Morita-MatsumotoK, KatoM, KanekoMK, KatoY, and YamaguchiY: A platform of C-type lectin-like receptor CLEC-2 for binding o-glycosylated podoplanin and nonglycosylated rhodocytin. Structure, 2014; 22:1711–1721.
Breiteneder-GeleffS, SoleimanA, KowalskiH, HorvatR, AmannG, KriehuberE, DiemK, WeningerW, TschachlerE, AlitaloK, and KerjaschkiD: Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium. Am J Pathol, 1999; 154:385–394.
PetersA, PitcherLA, SullivanJM, MitsdoerfferM, ActonSE, FranzB, WucherpfennigK, TurleyS, CarrollMC, SobelRA, BettelliE, and KuchrooVK: Th17 cells induce ectopic lymphoid follicles in central nervous system tissue inflammation. Immunity, 2011; 35:986–996.
10.
AstaritaJL, CremascoV, FuJ, DarnellMC, PeckJR, Nieves-BonillaJM, SongK, KondoY, WoodruffMC, GogineniA, OnderL, LudewigB, WeimerRM, CarrollMC, MooneyDJ, XiaL, and TurleySJ: The CLEC-2-podoplanin axis controls the contractility of fibroblastic reticular cells and lymph node microarchitecture. Nat Immunol, 2015; 16:75–84.
11.
ActonSE, FarrugiaAJ, AstaritaJL, Mourao-SaD, JenkinsRP, NyeE, HooperS, van BlijswijkJ, RogersNC, SnelgroveKJ, RosewellI, MoitaLF, StampG, TurleySJ, SahaiE, and Reis e SousaC: Dendritic cells control fibroblastic reticular network tension and lymph node expansion. Nature, 2014; 514:498–502.
12.
Ochoa-AlvarezJA, KrishnanH, PastorinoJG, NevelE, KephartD, LeeJJ, RetzbachEP, ShenY, FatahzadehM, BaredesS, KalyoussefE, HonmaM, AdelsonME, KanekoMK, KatoY, YoungMA, Deluca-RaponeL, ShienbaumAJ, YinK, JensenLD, and GoldbergGS: Antibody and lectin target podoplanin to inhibit oral squamous carcinoma cell migration and viability by distinct mechanisms. Oncotarget, 2015; 6:9045–9060.
13.
KatoY, SasagawaI, KanekoM, OsawaM, FujitaN, and TsuruoT: Aggrus: A diagnostic marker that distinguishes seminoma from embryonal carcinoma in testicular germ cell tumors. Oncogene, 2004; 23:8552–8556.
14.
Martin-VillarE, SchollFG, GamalloC, YurritaMM, Munoz-GuerraM, CrucesJ, and QuintanillaM: Characterization of human PA2.26 antigen (T1alpha-2, podoplanin), a small membrane mucin induced in oral squamous cell carcinomas. Int J Cancer, 2005; 113:899–910.
15.
KatoY, KanekoM, SataM, FujitaN, TsuruoT, and OsawaM: Enhanced expression of Aggrus (T1alpha/podoplanin), a platelet-aggregation-inducing factor in lung squamous cell carcinoma. Tumor Biol, 2005; 26:195–200.
16.
YuanP, TemamS, El-NaggarA, ZhouX, LiuD, LeeJ, and MaoL: Overexpression of podoplanin in oral cancer and its association with poor clinical outcome. Cancer, 2006; 107:563–569.
17.
KatoY, KanekoMK, KunoA, UchiyamaN, AmanoK, ChibaY, HasegawaY, HirabayashiJ, NarimatsuH, MishimaK, and OsawaM: 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.
18.
MishimaK, KatoY, KanekoMK, NakazawaY, KunitaA, FujitaN, TsuruoT, NishikawaR, HiroseT, and MatsutaniM: Podoplanin expression in primary central nervous system germ cell tumors: a useful histological marker for the diagnosis of germinoma. Acta Neuropathol, 2006; 111:563–568.
19.
MishimaK, KatoY, KanekoMK, NishikawaR, HiroseT, and MatsutaniM: Increased expression of podoplanin in malignant astrocytic tumors as a novel molecular marker of malignant progression. Acta Neuropathol, 2006; 111:483–488.
20.
AbeS, MoritaY, KanekoMK, HanibuchiM, TsujimotoY, GotoH, KakiuchiS, AonoY, HuangJ, SatoS, KishukuM, TaniguchiY, AzumaM, KawazoeK, SekidoY, YanoS, AkiyamaS, SoneS, MinakuchiK, KatoY, and NishiokaY: A novel targeting therapy of malignant mesothelioma using anti-podoplanin antibody. J Immunol, 2013; 190:6239–6249.
21.
KunitaA, KashimaTG, OhazamaA, GrigoriadisAE, and FukayamaM: Podoplanin is regulated by AP-1 and promotes platelet aggregation and cell migration in osteosarcoma. Am J Pathol, 2011; 179:1041–1049.
22.
ChandramohanV, BaoX, Kato KanekoM, KatoY, KeirST, SzafranskiSE, KuanCT, PastanIH, and BignerDD: Recombinant anti-podoplanin (NZ-1) immunotoxin for the treatment of malignant brain tumors. Int J Cancer, 2013; 132:2339–2348.
23.
KatoY, VaidyanathanG, KanekoMK, MishimaK, SrivastavaN, ChandramohanV, PegramC, KeirST, KuanCT, BignerDD, and ZalutskyMR: Evaluation of anti-podoplanin rat monoclonal antibody NZ-1 for targeting malignant gliomas. Nucl Med Biol, 2010; 37:785–794.
24.
KawaseA, IshiiG, NagaiK, ItoT, NaganoT, MurataY, HishidaT, NishimuraM, YoshidaJ, SuzukiK, and OchiaiA: Podoplanin expression by cancer associated fibroblasts predicts poor prognosis of lung adenocarcinoma. Int J Cancer, 2008; 123:1053–1059.
25.
HoshinoA, IshiiG, ItoT, AoyagiK, OhtakiY, NagaiK, SasakiH, and OchiaiA: Podoplanin-positive fibroblasts enhance lung adenocarcinoma tumor formation: podoplanin in fibroblast functions for tumor progression. Cancer Res, 2011; 71:4769–4779.
26.
SchoppmannSF, JeschB, RieglerMF, MaroskeF, SchwameisK, JomrichG, and BirnerP: Podoplanin expressing cancer associated fibroblasts are associated with unfavourable prognosis in adenocarcinoma of the esophagus. Clin Exp Metastasis, 2013; 30:441–446.
27.
ShindoK, AishimaS, OhuchidaK, FujiwaraK, FujinoM, MizuuchiY, HattoriM, MizumotoK, TanakaM, and OdaY: Podoplanin expression in cancer-associated fibroblasts enhances tumor progression of invasive ductal carcinoma of the pancreas. Mol Cancer, 2013; 12:168.
28.
InoueH, TsuchiyaH, MiyazakiY, KikuchiK, IdeF, SakashitaH, and KusamaK: Podoplanin expressing cancer-associated fibroblasts in oral cancer. Tumour Biol, 2014; 35:11345–11352.
29.
HatakeyamaK, KanekoMK, KatoY, IshikawaT, NishihiraK, TsujimotoY, ShibataY, OzakiY, and AsadaY: Podoplanin expression in advanced atherosclerotic lesions of human aortas. Thromb Res, 2012; 129:e70–76.
30.
MarksA, SutherlandDR, BaileyD, IglesiasJ, LawJ, LeiM, YegerH, BanerjeeD, and BaumalR: Characterization and distribution of an oncofetal antigen (M2A antigen) expressed on testicular germ cell tumours. Br J Cancer, 1999; 80:569–578.
31.
OgasawaraS, KanekoMK, PriceJE, and KatoY: Characterization of anti-podoplanin monoclonal antibodies: critical epitopes for neutralizing the interaction between podoplanin and CLEC-2. Hybridoma, 2008; 27:259–267.
32.
TakagiS, SatoS, Oh-haraT, TakamiM, KoikeS, MishimaY, HatakeK, and FujitaN: Platelets promote tumor growth and metastasis via direct interaction between Aggrus/podoplanin and CLEC-2. PLoS One, 2013; 8:e73609.
33.
KatoY, and KanekoMK: A cancer-specific monoclonal antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep, 2014; 4:5924.
34.
OkiH, KanekoMK, OgasawaraS, TsujimotoY, LiuX, SugawaraM, TakakuboY, TakagiM, and KatoY: Characterization of a monoclonal antibody LpMab-7 recognizing non-PLAG domain of podoplanin. Monoclon Antib Immunodiagn Immunother, 2015; 34:174–180.
OkiH, OgasawaraS, KanekoMK, TakagiM, YamauchiM, and KatoY: Characterization of monoclonal antibody LpMab-3 recognizing sialylated glycopeptide of podoplanin. Monoclon Antib Immunodiagn Immunother, 2015; 34:44–50.
37.
OgasawaraS, OkiH, KanekoMK, HozumiY, LiuX, HonmaR, FujiiY, NakamuraT, GotoK, TakagiM, and KatoY: Development of a monoclonal antibody LpMab-10 recognizing non-glycosylated PLAG1/2 domain including Thr34 of human podoplanin. Monoclon Antib Immunodiagn Immunother, 2015; 34:318–326.
38.
KanekoMK, OkiH, HozumiY, LiuX, OgasawaraS, TakagiM, GotoK, and KatoY: Monoclonal antibody LpMab-9 recognizes O-glycosylated N-terminus of human podoplanin. Monoclon Antib Immunodiagn Immunother, 2015; 34:310–317.
39.
KatoY, KunitaA, AbeS, OgasawaraS, FujiiY, OkiH, FukayamaM, NishiokaY, and KanekoMK: The chimeric antibody chLpMab-7 targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC rather than via its neutralizing activity. Oncotarget, 2015; 6:36003–36018.
40.
WatanabeM, OkochiE, SugimotoY, and TsuruoT: Identification of a platelet-aggregating factor of murine colon adenocarcinoma 26: Mr 44,000 membrane protein as determined by monoclonal antibodies. Cancer Res, 1988; 48:6411–6416.
41.
KajiC, TsujimotoY, Kato KanekoM, KatoY, and SawaY: Immunohistochemical examination of novel rat monoclonal antibodies against mouse and human podoplanin. Acta Histochem Cytochem, 2012; 45:227–237.
42.
FarrA, NelsonA, and HosierS: Characterization of an antigenic determinant preferentially expressed by type I epithelial cells in the murine thymus. J Histochem Cytochem, 1992; 40:651–664.
43.
KatoY: Specific monoclonal antibodies against IDH1/2 mutations as diagnostic tools for gliomas. Brain Tumor Pathol, 2015; 32:3–11.
44.
SteinRA, WilkinsonJC, GuyerCA, and StarosJV: An analytical approach to the measurement of equilibrium binding constants: application to EGF binding to EGF receptors in intact cells measured by flow cytometry. Biochemistry, 2001; 40:6142–6154.
