TY - JOUR
T1 - Epitope Analysis of an Anti-Whale Podoplanin Monoclonal Antibody, PMab-237, Using Flow Cytometry
AU - Sayama, Yusuke
AU - Sano, Masato
AU - Kaneko, Mika K.
AU - Kato, Yukinari
N1 - Funding Information:
This research was supported in part by AMED under grant nos. JP19am0401013 (Y.K.), JP19am0101078 (Y.K.), and JP19ae0101028 (Y.K.), and by JSPS KAKENHI grant nos. 17K07299 (M.K.K.) and 19K07705 (Y.K.).
Publisher Copyright:
© Yusuke Sayama et al. 2020; Published by Mary Ann Liebert, Inc. 2020.
PY - 2020/2
Y1 - 2020/2
N2 - Podoplanin (PDPN) is a small mucin-type transmembrane glycoprotein, which was first discovered in podocytes of the kidney. PDPN is a specific lymphatic endothelial marker and is also known as T1alpha, a marker of lung type I alveolar cells, or Aggrus, a platelet aggregation-inducing factor. PDPN possesses three platelet aggregation-stimulating (PLAG) domains and PLAG-like domains (PLDs), which bind to C-type lectin-like receptor-2. Previously, we developed a novel anti-whale PDPN (wPDPN) monoclonal antibody (mAb) PMab-237 using the Cell-Based Immunization and Screening (CBIS) method and the RIEDL tag of Arg-Ile-Glu-Asp-Leu sequence. PMab-237 detected wPDPN by flow cytometry, western blot, and immunohistochemical analyses. However, the specific binding epitope of PMab-237 for wPDPN remains unknown. In this study, deletion mutants and point mutants of wPDPN with N-terminal RIEDL tag were produced to analyze the PMab-237 epitope using flow cytometry. The analysis of deletion mutants showed that the N-terminus of the PMab-237 epitope exists between the 80th amino acid (AA) and the 85th AA of wPDPN. In addition, the analysis of point mutants demonstrated that the critical epitope of PMab-237 includes Leu82 and Thr84 of wPDPN, indicating that the PMab-237 epitope is located in the PLD of wPDPN.
AB - Podoplanin (PDPN) is a small mucin-type transmembrane glycoprotein, which was first discovered in podocytes of the kidney. PDPN is a specific lymphatic endothelial marker and is also known as T1alpha, a marker of lung type I alveolar cells, or Aggrus, a platelet aggregation-inducing factor. PDPN possesses three platelet aggregation-stimulating (PLAG) domains and PLAG-like domains (PLDs), which bind to C-type lectin-like receptor-2. Previously, we developed a novel anti-whale PDPN (wPDPN) monoclonal antibody (mAb) PMab-237 using the Cell-Based Immunization and Screening (CBIS) method and the RIEDL tag of Arg-Ile-Glu-Asp-Leu sequence. PMab-237 detected wPDPN by flow cytometry, western blot, and immunohistochemical analyses. However, the specific binding epitope of PMab-237 for wPDPN remains unknown. In this study, deletion mutants and point mutants of wPDPN with N-terminal RIEDL tag were produced to analyze the PMab-237 epitope using flow cytometry. The analysis of deletion mutants showed that the N-terminus of the PMab-237 epitope exists between the 80th amino acid (AA) and the 85th AA of wPDPN. In addition, the analysis of point mutants demonstrated that the critical epitope of PMab-237 includes Leu82 and Thr84 of wPDPN, indicating that the PMab-237 epitope is located in the PLD of wPDPN.
KW - epitope
KW - monoclonal antibody
KW - PMab-237
KW - podoplanin
KW - whale
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U2 - 10.1089/mab.2019.0045
DO - 10.1089/mab.2019.0045
M3 - Article
C2 - 31934820
AN - SCOPUS:85079755365
SN - 2167-9436
VL - 39
SP - 17
EP - 22
JO - Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
JF - Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
IS - 1
ER -