Epitope Mapping of Monoclonal Antibody PMab-233 Against Tasmanian Devil Podoplanin

The highly O-glycosylated membrane glycoprotein podoplanin (PDPN) is frequently overexpressed in several malignant cancers, such as oral cancer, lung cancer, germinal neoplasia, mesothelioma, and brain tumor. The expression of PDPN is strongly associated with cancer progression and poor prognosis. PDPN possesses three tandem repeats of platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) and PLAG-like domain (PLD), and binds to C-type lectin-like receptor 2 (CLEC-2) on platelets, followed by PDPN-mediated platelet aggregation. We have previously established a novel anti-Tasmanian devil PDPN (tasPDPN) monoclonal antibody (mAb), PMab-233, which specifically detects tasPDPN using flow cytometry, Western blot, and immunohistochemical analyses. However, the specific binding epitope of tasPDPN for PMab-233 remains to be clarified. Herein, a series of deletion or point mutants of tasPDPN were utilized for investigating the binding epitopes of PMab-233 using flow cytometry. The findings of this study demonstrated that Asp30, Thr33, and Thr34 of tasPDPN, which are located in PLAG1, are responsible for the binding of PMab-233 to tasPDPN.