Epitope mapping of anti-mouse podoplanin monoclonal antibody PMab-1

Mouse podoplanin (mPDPN) is a type I transmembrane sialoglycoprotein, which is expressed on lymphatic endothelial cells, podocytes of the kidney, and type I alveolar cells of the lung. mPDPN is known as a platelet aggregation-inducing factor and possesses four platelet aggregation-stimulating (PLAG) domains: PLAG1, PLAG2, and PLAG3 in the N-terminus and PLAG4 in the middle of the mPDPN protein. mPDPN overexpression in cancers has been reportedly associated with hematogenous metastasis through interaction with the C-type lectin-like receptor 2 of platelets. We previously reported a rat anti-mPDPN monoclonal antibody clone PMab-1, which was developed by immunizing the PLAG2 and PLAG3 domains of mPDPN. PMab-1 is very useful in flow cytometry, western blot, and immunohistochemical analyses to detect both normal cells and cancers. However, the binding epitope of PMab-1 remains to be clarified. In the present study, flow cytometry, enzyme-linked immunosorbent assay, and immunohistochemical analyses were utilized to investigate the epitope of PMab-1. The results revealed that the critical epitope of PMab-1 is Asp39 and Met41 of mPDPN. These findings can be applied to the production of more functional anti-mPDPN monoclonal antibodies.


Introduction
Podoplanin (PDPN/T1alpha/gp38/Aggrus) is expressed in many normal tissues, such as renal podocytes, lymphatic endothelial cells of many tissues, and pulmonary type I alveolar cells [1][2][3][4]. Several antimouse PDPN (mPDPN) monoclonal antibodies (mAbs), such as clone 8.1.1 or clone PMab-1, have been used in many studies [5]. However, clone 8.1.1 is produced using hamsters, and clone PMab-1 is produced using rats because it is difficult to develop anti-mPDPN mAbs using mice. Recently, we developed a rat-mouse chimeric antibody, mPMab-1 of mouse IgG 2a , which was derived from a rat PMab-1 mAb [6]. Immunohistochemical analysis showed that mPMab-1 detects podocytes of the kidney, lymphatic endothelial cells of the colon, and type I alveolar cells of the lung. Importantly, mPMab-1 was shown to be more sensitive than original PMab-1.
mPDPN possesses four platelet aggregation-stimulating (PLAG) domains: PLAG1, PLAG2, and PLAG3 in the N-terminus [1] and PLAG4 in the middle of the mPDPN protein [7]. In a previous study, PMab-1 mAb was produced against the platelet aggregation-stimulating (PLAG) domain of mPDPN [5]; therefore, PMab-1 neutralizes the interaction between mPDPN and the C-type lectin-like receptor 2 [8][9][10]. The administration of PMab-1 was found to reduce lymphangiogenesis in corneal suture and ear-wound healing models [11]. PMab-1 also suppressed the infiltration of thioglycollate-induced macrophages at the site of wound healing. Furthermore, the administration of PMab-1 lead to a significant suppression of the rejection reaction in a corneal transplantation model, suggesting that mPDPN is a novel therapeutic target for suppressing lymphangiogenesis and inflammation.
In the present study, we determined the binding epitope of PMab-1 to mPDPN using flow cytometry, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical analyses.

Production of mPDPN point mutants
The cDNA of mPDPN was subcloned into a pcDNA3 vector (Thermo Fisher Scientific Inc.) [2]. Substitutions of amino acids to alanine in the mPDPN sequence were performed using a QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies Inc., Santa Clara, CA, USA).

Results and discussion
In a previous study, we developed a rat anti-mPDPN mAb PMab-1 by immunizing the PLAG domain of mPDPN [5]. We further produced a rat-mouse chimeric antibody, mPMab-1 of mouse IgG 2a , which was derived from a rat PMab-1 mAb [6]. Immunohistochemical analysis showed that both PMab-1 and mPMab-1 are capable of detecting podocytes of the kidney, lymphatic endothelial cells of the colon, and type I alveolar cells of the lung. Interestingly, mPMab-1 was shown to be more sensitive than original PMab-1 [6] probably because a high-sensitivity immunohistochemical kit can be used for mouse IgG. In the present study, we produced point mutants of mPDPN (proteins and synthesized peptides) and investigated the critical epitope of PMab-1 for mPDPN detection.
We performed a blocking assay using flow cytometry. PMab-1 reacted with the CHO/mPDPN cell line (Fig. 3). This reaction was completely neutralized by G38A. In contrast, D39A and M41A did not block the reaction of PMab-1 with CHO/mPDPN, indicating that Asp39 and Met41 of mPDPN are critical for PMab-1 detection.