Epitope Analysis of an Anti-Whale Podoplanin Monoclonal Antibody, PMab-237, Using Flow Cytometry

Podoplanin (PDPN) is a small mucin-type transmembrane glycoprotein, which was first discovered in podocytes of the kidney. PDPN is a specific lymphatic endothelial marker and is also known as T1alpha, a marker of lung type I alveolar cells, or Aggrus, a platelet aggregation-inducing factor. PDPN possesses three platelet aggregation-stimulating (PLAG) domains and PLAG-like domains (PLDs), which bind to C-type lectin-like receptor-2. Previously, we developed a novel anti-whale PDPN (wPDPN) monoclonal antibody (mAb) PMab-237 using the Cell-Based Immunization and Screening (CBIS) method and the RIEDL tag of Arg-Ile-Glu-Asp-Leu sequence. PMab-237 detected wPDPN by flow cytometry, western blot, and immunohistochemical analyses. However, the specific binding epitope of PMab-237 for wPDPN remains unknown. In this study, deletion mutants and point mutants of wPDPN with N-terminal RIEDL tag were produced to analyze the PMab-237 epitope using flow cytometry. The analysis of deletion mutants showed that the N-terminus of the PMab-237 epitope exists between the 80th amino acid (AA) and the 85th AA of wPDPN. In addition, the analysis of point mutants demonstrated that the critical epitope of PMab-237 includes Leu82 and Thr84 of wPDPN, indicating that the PMab-237 epitope is located in the PLD of wPDPN.


Introduction
P odoplanin (PDPN)/T1alpha/Aggrus/PA2.26 is a type I transmembrane sialoglycoprotein consisting of a heavily glycosylated extracellular domain, a single transmembrane, and a short nine amino acid (AA) cytoplasmic tail. (1)(2)(3)(4) PDPN/Aggrus possesses the EDxxVTPG sequence at its N-terminus, which is known to be the platelet aggregationstimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3). (3,5) In addition, the PLAG-like domain (PLD) of the E(D/E)xx(T/S)xx sequence, also known as PLAG4, has been reported to be present in the middle of PDPN. (6,7) PLAG domains are highly conserved among mammalian PDPNs. (7) PDPN is expressed in lymphatic endothelial cells and is not expressed in vascular endothelial cells. (8) The interaction between PDPN (3) on lymphatic endothelial cells and C-type lectin-like receptor-2 on platelets was shown to facilitate embryonic blood/lymphatic vessel separation. (9) Because PDPN/T1alpha is expressed in type I alveolar cells but not in type II alveolar cells, it is used as a specific marker of type I alveolar cells. (10) In recent studies, other functions of PDPN were reported. The PDPN-positive cells, with the immune cells after myocardial infarction, positively affect immune cell recruitment. (11) The PDPN-positive stromal cells play a critical role in a network of immunofibroblasts, which can support the earliest phases of tertiary lymphoid structure establishment. (12) The expression of PDPN in chorionic villous stromal cells is in two important placental pathologies: preeclampsia and hydatidiform mole. (13) Moreover, PDPN is upregulated in many cancers and is involved in cancer metastasis and malignant progression. (14)(15)(16)(17) Recent reports showed that PDPN is related to a progression in oral epithelial dysplasia and oral squamous cell carcinoma through a co-expression with sex-determining region Y-related Homeo box gene 2. (18,19) Therefore, PDPN possesses many pathophysiological functions in malignant tissues.
Recently, we developed a novel anti-whale PDPN (wPDPN) monoclonal antibody (mAb), PMab-237, using the Cell-Based Immunization and Screening (CBIS) method. (20) The CBIS method was established in our previous study (21) to produce mAbs using cell lines for immunization and screening. PMab-237 specifically detected wPDPN by flow cytometry, western blotting, and immunohistochemical analyses. PMab-237 also strongly stained pulmonary type I alveolar cells, renal podocytes, and lymphatic endothelial cells of the harbor porpoise by the immunohistochemical analysis. (22) However, the binding epitope of PMab-237 for wPDPN remains unknown. This study aimed to identify the epitope of PMab-237 through flow cytometry using the deletion mutants and point mutants of wPDPN.

Production of wPDPN mutants
Synthesized DNA encoding wPDPN was subcloned into the pCAG vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and an N-terminal RIEDL tag was added. (22) The RIEDL tag was derived from the five AA sequences (Arg-Ile-Glu-Asp-Leu) of human PDPN, which was detected by clone LpMab-7. (23) Deletion mutants of the wPDPN sequence were produced using a HotStar HiFidelity Polymerase Kit (Qiagen, Inc., Hilden, Germany) with oligonucleotides. Substitutions of AAs to alanine in the wPDPN sequence were conducted by QuikChange Lightning Site-Directed Mutagenesis Kits (Agilent Technologies, Inc., Santa Clara, CA). PCR fragments bearing the desired mutations were inserted into the pCAG vector using an In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan).  SAYAMA ET AL.
All deletion mutants and WT of wPDPN containing an Nterminal RIEDL tag were detected by LpMab-7 (an anti-RIEDL tag mAb), indicating that the expression level of each construct was high ( Fig. 2A). In contrast, PMab-237 did not react with dN85, dN90, dN95, or dN100 (Fig. 2B), suggesting that the N-terminus of the PMab-237 epitope might exist between the 80th AA and 85th AA of wPDPN.
In conclusion, by using deletion mutants and point mutants of wPDPN in CHO-K1 cells, we have demonstrated that the critical epitope of PMab-237 may include Leu82 and Thr84 of wPDPN. PMab-237 could be a useful tool in elucidating the pathophysiological function of wPDPN.

Author Disclosure Statement
No competing financial interests exist.