TY - JOUR
T1 - Epitope mapping of the anti-california sea lion podoplanin monoclonal antibody PMab-269 using alanine-scanning mutagenesis and ELISA
AU - Tanaka, Tomohiro
AU - Asano, Teizo
AU - Sano, Masato
AU - Takei, Junko
AU - Hosono, Hideki
AU - Nanamiya, Ren
AU - Tateyama, Nami
AU - Kaneko, Mika K.
AU - Kato, Yukinari
N1 - Funding Information:
This research was supported, in part, by Japan Agency for Medical Research and Development (AMED) under Grant Nos. JP21am0401013 (Y.K.) and JP21am0101078 (Y.K.), and by the Japan Society for the Promotion of Science ( JSPS) Grants-in-Aid for Scientific Research (KAKENHI) Grant Nos. 21K15523 (to T.A.), 21K07168 (to M.K.K.), 20K16322 (to M.S.), and 19K07705 (to Y.K.).
Publisher Copyright:
© Copyright 2021, Mary Ann Liebert, Inc., publishers 2021.
PY - 2021/8/1
Y1 - 2021/8/1
N2 - Podoplanin (PDPN) plays a pivotal role in platelet aggregation, embryo development, and tumor progression. PDPN is universally expressed in many mammalian species, and is considered a typical lymphatic endothelial cell marker. We have previously developed the mouse anti-California sea lion (Zalophus californianus) PDPN (seaPDPN) monoclonal antibody (mAb), clone PMab-269, which is suitable for different experimental applications, including flow cytometry, Western blotting, and immunohistochemistry. In this study, we identified the PMab-269 epitope of the seaPDPN by enzyme-linked immunosorbent assay using deletion mutants and point mutants generated for seaPDPN. Our results demonstrated that PMab-269 recognized the peptide, corresponding to the amino acids 63-82 of seaPDPN. Furthermore, the reactions of PMab-269 to seven alanine-substituted peptides, such as P68A, D76A, F77A, H78A, L79A, E80A, and D81A, were abolished among 20 alanine-substituted peptides. We identified the seven amino acids (Pro68, Asp76, Phe77, His78, Leu79, Glu80, and Asp81) as the critical epitope targeted by PMab-269. The successful identification of the PMab-269 epitope might contribute to the pathophysiological investigations of seaPDPN.
AB - Podoplanin (PDPN) plays a pivotal role in platelet aggregation, embryo development, and tumor progression. PDPN is universally expressed in many mammalian species, and is considered a typical lymphatic endothelial cell marker. We have previously developed the mouse anti-California sea lion (Zalophus californianus) PDPN (seaPDPN) monoclonal antibody (mAb), clone PMab-269, which is suitable for different experimental applications, including flow cytometry, Western blotting, and immunohistochemistry. In this study, we identified the PMab-269 epitope of the seaPDPN by enzyme-linked immunosorbent assay using deletion mutants and point mutants generated for seaPDPN. Our results demonstrated that PMab-269 recognized the peptide, corresponding to the amino acids 63-82 of seaPDPN. Furthermore, the reactions of PMab-269 to seven alanine-substituted peptides, such as P68A, D76A, F77A, H78A, L79A, E80A, and D81A, were abolished among 20 alanine-substituted peptides. We identified the seven amino acids (Pro68, Asp76, Phe77, His78, Leu79, Glu80, and Asp81) as the critical epitope targeted by PMab-269. The successful identification of the PMab-269 epitope might contribute to the pathophysiological investigations of seaPDPN.
KW - California sea lion podoplanin
KW - PMab-269
KW - epitope
KW - monoclonal antibody
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U2 - 10.1089/mab.2021.0017
DO - 10.1089/mab.2021.0017
M3 - Article
C2 - 34283661
AN - SCOPUS:85113797461
VL - 40
SP - 196
EP - 200
JO - Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
JF - Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
SN - 2167-9436
IS - 4
ER -