Thr80 of sheep podoplanin is a critical epitope of the anti-sheep podoplanin monoclonal antibody, PMab-256

Degi July 30, 2023

Thr80 of sheep podoplanin is a critical epitope of the anti-sheep podoplanin monoclonal antibody, PMab-256

Yukinari Kato1, 2, Masato Sano1, Teizo Asano1, Yusuke Sayama1, Mika K. Kaneko1

1Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan
2New Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan

Address correspondence to: Yukinari Kato; New Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan; E-mail: yukinarikato@med.tohoku.ac.jp

Abstract
An anti-sheep podoplanin (sPDPN) monoclonal antibody (mAb), PMab-256, has recently been established. PMab-256 shows positive immunostaining for lymphatic endothelial cells (LECs), lung type I alveolar cells, and kidney podocytes. PDPN possesses three platelet-aggregation-stimulating (PLAG) domains, PLAG1, PLAG2, and PLAG3, and a PLAG-like domain (PLD). The binding epitope of many anti-PDPN mAbs is located in PLAG domains or PLD. The purpose of the present study is to determine the binding epitope of PMab-256. Analysis of sPDPN deletion mutants revealed that the N-terminus of the PMab-256 epitope exists between amino acids (aa) 75 and 80 of sPDPN. Further, analysis of sPDPN point mutations demonstrated that the critical epitope includes Thr80 of sPDPN, indicating that the PMab-256 epitope is in the PLD of sPDPN.

Key words: sheep podoplanin; monoclonal antibody; epitope, PMab-256

Introduction
              Sensitive and specific mAbs against podoplanin (PDPN) of various species including human (1), mouse (2), and whale (3) are necessary for the analysis of expression and pathophysiological functions of PDPN. PDPN is used for discriminating lymphatic endothelial cells (LECs) from vascular endothelial cells (4) or type I alveolar cells from type II alveolar cells (5). An anti-sheep podoplanin (sPDPN) monoclonal antibody (mAb), PMab-256, was obtained using the Cell-Based Immunization and Screening (CBIS) method (6-8). Like other typical anti-PDPN mAbs, PMab-256 demonstrated positive immunoreaction for LECs (4), type I alveolar cells (5), and kidney podocytes (9).
              The binding epitope of many anti-PDPN mAbs is located in three platelet aggregation-stimulating (PLAG) domains, PLAG1, PLAG2, and PLAG3, (10) at the N-terminus or a PLAG-like domain (PLDs) (3, 11-14) in the middle of the PDPN protein. The purpose of this study was to determine the binding epitope of PMab-256.

Materials and Methods
              Cell lines
              Chinese hamster ovary (CHO)-K1 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). sPDPN mutation plasmids containing a RAP14 tag (15) were transfected into CHO-K1 cells using Lipofectamine LTX (Thermo Fisher Scientific Inc., Waltham, MA, USA). The RAP14 tag is comprised of 14 amino acids (aa), (DMVNPGLEDRIEDL), and is recognized by PMab-2 mAb (16) and LpMab-7 (1). Cells transiently transfected with deletion or point mutations were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific Inc.), 100 units/ml of penicillin, 100 μg/ml of streptomycin, and 25 μg/ml of amphotericin B (Nacalai Tesque, Inc.) at 37°C in a humidified atmosphere of 5% CO2 and 95% air.

              Production of sPDPN mutants
              Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan) encoding sPDPN (Accession No.: XM_004013802.4) was subcloned into the pCAG vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and a RAP14 tag was added at the N-terminus. Deletion mutants of the sPDPN sequence were produced using a HotStar HiFidelity Polymerase Kit (Qiagen Inc., Hilden, Germany) with oligonucleotides. Substitutions of amino acids with alanine in the sPDPN sequence were produced by QuikChange Lightning Site-Directed Mutagenesis Kits (Agilent Technologies Inc., Santa Clara, CA, USA). PCR fragments bearing desired mutations were inserted into the pCAG vector using the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan).

              Flow cytometry
              Transiently transfected CHO-K1 cells were detached by 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.) and collected using 10% FBS in RPMI 1640 medium. After washing with 0.1% bovine serum albumin in phosphate-buffered saline, cells were incubated with anti-sPDPN antibody (PMab-256; 1 μg/ml) and an anti-RAP14 tag antibody (PMab-2; 1 μg/ml or LpMab-7; 1 μg/ml) for 30 min at 4°C. Alexa Fluor 488-conjugated anti-mouse IgG (1:2000; Cell Signaling Technology, Inc., Danvers, MA, USA) was added to each cell suspension and incubation continued for 30 min at 4°C. Fluorescence data were collected and analyzed using a Cell Analyzer EC800 (Sony Corp., Tokyo, Japan).

Results
              Seven deletion mutants of sPDPN in CHO-K1 cells, namely: dN30, with aa 30–168 deletion; dN40, aa 40–168; dN50, aa 50–168; dN60, aa 60–168; dN70, aa 70–168; dN75, aa 75–168; dN80, aa 80–168; or wild type (WT) sPDPN, aa 27–168. All deletion mutants and WT containing the N-terminal RAP14 tag were recognized by PMab-2 or LpMab-7 (anti-RAP14 tag mAbs), indicating that the expression level of each construct is high (Fig. 1A). PMab-256 recognized dN30, dN40, dN50, dN60, dN70, and dN75, but not dN80 (Fig. 1B), suggesting that the N-terminus of the PMab-256 epitope exists between sPDPN aa 75 and 80.
              Next, a series of point mutants of sPDPN: G75A, E76A, D77A, L78A, P79A, T80A, A81G, E82A, S83A, T84A, T85A, and A86G were obtained. PMab-2 and LpMab-7 reacted with all point mutants (Fig. 2A). By contrast, PMab-256 did not react with T80A (Fig. 2B). Thr80 of sPDPN is essential for PMab-256 binding (Fig. 3).

Discussion
              A variety of mAbs against pig (17, 18), horse (19, 20), Tasmanian devil (21), alpaca (22), tiger (23), whale (24), goat (25, 26), bear (27, 28), and sheep (29) PDPNs using the CBIS method are available (6-8). An anti-sPDPN mAb PMab-256 is useful for immunohistochemical analyses using FFPE tissues of sheep for detecting LECs of many organs, type I alveolar cells, and renal epithelial cells (29). PMab-256 is also useful for western blot analysis, indicating that PMab-256 recognizes sPDPN that is fixed in formalin and denatured by SDS. Further, PMab-256 detects sPDPN in flow cytometry.
              PLAG and PLD domains bind C-type lectin-like receptor-2 (CLEC-2), and induce platelet aggregation and cancer metastasis (30). Anti-PDPN mAbs, epitopes of which are located in PLAG domains or PLD, could neutralize platelet aggregation (30). The epitope of PMab-256 is also located in PLD of sPDPN. PMab-256 is thus not only a useful mAb for research but also could be a functional mAb with the ability to neutralize induction of platelet aggregation.

Conflict of Interest
The authors have no conflict of interest.

Acknowledgments
This research was supported in part by AMED under Grant Numbers: JP20am0401013 (Y.K.), JP20am0101078 (Y.K.), and JP20ae0101028 (Y.K.), and by JSPS KAKENHI Grant Number 17K07299 (M.K.K.) and Grant Number 19K07705 (Y.K.).

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Figure Legends
Figure 1. Epitope mapping of PMab-256 using deletion mutations. (A) Deletion mutants of sPDPN were analyzed using flow cytometry. Mutants were incubated with PMab-2 (anti-RAP14 tag mAb; red line) or buffer control (black line) for 30 min at 4°C, followed by staining with secondary antibodies. (B) Deletion mutants of sPDPN were analyzed using flow cytometry. Mutants were incubated with LpMab-7 (anti-RAP14 tag mAb; red line) or buffer control (black line) for 30 min at 4°C, followed by staining with secondary antibodies. (C) Deletion mutants of sPDPN were analyzed using flow cytometry. Mutants were incubated with PMab-256 (anti-sPDPN mAb; red line) or buffer control (black line) for 30 min at 4°C, followed by staining with secondary antibodies. sPDPN, sheep podoplanin.

Figure 2. Epitope mapping of PMab-256 using point mutations. (A) Transient point mutants expressing G75A, E76A, D77A, L78A, P79A, T80A, A81G, E82A, S83A, T84A, T85A, and A86G of sPDPN were incubated with PMab-2 (anti-RAP14 tag; red line), or buffer control (black line) for 30 min at 4°C, followed by staining with secondary antibodies. (B) Transient point mutants expressing G75A, E76A, D77A, L78A, P79A, T80A, A81G, E82A, S83A, T84A, T85A, and A86G of sPDPN were incubated with LpMab-7 (anti-RAP14 tag; red line) or buffer control (black line) for 30 min at 4°C, followed by staining with secondary antibodies. (C) Transient point mutants expressing G75A, E76A, D77A, L78A, P79A, T80A, A81G, E82A, S83A, T84A, T85A, and A86G of sPDPN were incubated with PMab-256 (anti-sPDPN mAb; red line) or buffer control (black line) for 30 min at 4°C, followed by staining with secondary antibodies. sPDPN, sheep podoplanin.

Figure 3. Schematic illustration of the epitope recognized by PMab-256. (A) Illustration of WT sPDPN and its seven deletion mutants: dN30, dN40, dN50, dN60, dN70, dN75, and dN80. Black bars: positive reactions of PMab-256. White bars: negative reactions of PMab-256. WT, wild type. (B) A red amino acid indicates a critical epitope of PMab-256. PLD, PLAG-like domain.







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