Elucidation of Critical Epitope of Anti-Rat Podoplanin Monoclonal Antibody PMab-2

Rat podoplanin (rPDPN) is a recognized lymphatic endothelial cell marker and is expressed on the podocytes of kidney and type I lung alveolar cells. rPDPN is a type I transmembrane sialoglycoprotein and induces platelet aggregation via the C-type lectin-like receptor-2 of platelets. It comprises four platelet aggregation-stimulating (PLAG) domains: PLAG1–3, present in the N-terminus, and PLAG4, in the center of the PDPN protein. Previously, we developed a mouse anti-rPDPN monoclonal antibody clone, PMab-2, by immunizing the PLAG2 and PLAG3 domains of rPDPN. PMab-2 has applications in Western blot, flow cytometry, and immunohistochemical analyses for detection of both normal and cancer cells. However, the binding epitope of PMab-2 remains to be determined. Herein, we investigated the epitope of PMab-2 using enzyme-linked immunosorbent assay, immunohistochemical analysis, and flow cytometry. The results revealed that the critical epitope of PMab-2 is Leu46 and Glu47 of rPDPN.

In this study, we determined the binding epitope of PMab-2 to rPDPN using flow cytometry and enzyme-linked immunosorbent assay (ELISA).

Cell line
Chinese hamster ovary (CHO)-K1 was purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Production of rPDPN point mutants
The complementary DNA of rPDPN was subcloned into a pcDNA3 vector (Thermo Fisher Scientific, Inc.). Substitutions of amino acids to alanine in rPDPN sequence were performed by the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Inc., Santa Clara, CA).

FIG. 1.
Epitope mapping of PMab-2 using point mutants of rPDPN. Point mutants of rPDPN were analyzed using flow cytometry. Point mutants were expressed on CHO-K1 cells and were then incubated with PMab-2 (2 mg/mL) or buffer control for 30 minutes at 4°C, followed by treatment with corresponding secondary antibodies.

Results and Discussion
We previously developed a mouse anti-rPDPN mAb, PMab-2, by immunizing the PLAG domain of rPDPN. (9) In this study, we produced point mutants of rPDPN using recombinant proteins and synthesized peptides and investigated the epitope of PMab-2 critical for rPDPN detection.
We performed a blocking assay using immunohistochemistry. PMab-2 reacted with type I alveolar cells (Fig. 2), renal podocytes (Fig. 3), and lymphatic endothelial cells of colon (Fig. 4). These reactions were completely or partially neutralized by G38A; in contrast, L46A and E47A did not block the reactions of PMab-2 with rat tissues, thus indicating that L46A and E47A of rPDPN are critical for PMab-2 detection.
In addition, we performed a blocking assay using flow cytometry and found that PMab-2 reacted with the CHO/rPDPN cell line (Fig. 5). This reaction was completely neutralized by G38A; in contrast, L46A and E47A did not block the reaction of PMab-2 with CHO/rPDPN, which indicated that Leu46 and Glu47 of rPDPN are critical for PMab-2 detection.
Taken together, the critical epitope of PMab-2 comprises Leu46 and Glu47 of rPDPN (Fig. 6). These findings could be applied to the production of more functional anti-rPDPN mAbs.