Epitope mapping of the anti-california sea lion podoplanin monoclonal antibody PMab-269 using alanine-scanning mutagenesis and ELISA

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.