Thr80 of Sheep Podoplanin Is a Critical Epitope of the Antisheep Podoplanin Monoclonal Antibody: PMab-256

Introduction

Sensitive and specific monoclonal antibodies (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 antisheep podoplanin (sPDPN) 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

Results

We produced 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.

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FIG. 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 minutes 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 minutes 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 minutes at 4°C, followed by staining with secondary antibodies. mAb, monoclonal antibody; sPDPN, sheep podoplanin.

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).

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FIG. 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 minutes 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 minutes 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 minutes at 4°C, followed by staining with secondary antibodies.

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FIG. 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 bar: negative reaction of PMab-256. (B) A red amino acid indicates a critical epitope of PMab-256. PLD, platelet-aggregation-stimulating-like domain; WT, wild type.

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 formalin-fixed paraffin-embedded 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 sodium dodecyl sulfate. Furthermore, PMab-256 detects sPDPN in flow cytometry.

PLAG and PLD domains bind C-type lectin-like receptor-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.