Epitope Analysis of an Antihorse Podoplanin Monoclonal Antibody PMab-219

Introduction

Podoplanin (PDPN/T1alpha/Aggrus) is a type I transmembrane glycoprotein, and is expressed in normal tissues, such as lymphatic endothelial cells, type I lung alveolar cells, and renal podocytes.^(1–3) PDPN is known as a platelet aggregation-inducing factor in highly metastatic tumor cells.^(1,4) Human PDPN (hPDPN) is frequently overexpressed in various malignant tumors, including lung cancer, mesothelioma, glioblastoma, and osteosarcoma.^(5–7) hPDPN possesses three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3), which exist at the N-terminus of hPDPN.⁽⁸⁾ In addition, one PLAG-like domain (PLD) has been reported to be present in the middle of PDPN.⁽⁹⁾ PLAG and PLD are highly conserved among mammalian PDPNs.⁽⁸⁾ The interaction between PDPN on lymphatic endothelial cells and C-type lectin-like receptor-2 (CLEC-2) on platelets was shown to facilitate embryonic blood/lymphatic vessel separation.⁽¹⁰⁾

In our previous study, we developed monoclonal antibodies (mAbs) against human,⁽¹¹⁾ mouse,⁽¹¹⁾ rat,⁽¹²⁾ rabbit,⁽¹³⁾ dog,⁽¹⁴⁾ cat,⁽¹⁵⁾ bovine,⁽¹⁶⁾ pig,⁽¹⁷⁾ Tasmanian devil,⁽¹⁸⁾ alpaca,⁽¹⁹⁾ bear,⁽²⁰⁾ tiger,⁽²¹⁾ whale,⁽²²⁾ goat,⁽²³⁾ and horse PDPNs.⁽²⁴⁾ Although PMab-202, an antihorse PDPN (horPDPN) mAb, was established by immunizing mice with synthetic peptides of horPDPN, it was not applicable for immunohistochemical analysis.⁽²⁵⁾ Highly sensitive and specific mAbs against horPDPN were needed to analyze horPDPN for pathological investigation using horse tissues.

Recently, we developed a novel anti-horPDPN mAb, PMab-219, using Cell-Based Immunization and Screening (CBIS) method. PMab-219 specifically detected horPDPN by flow cytometry, Western blotting, and immunohistochemical analysis.^(24,26) However, the binding epitope of PMab-219 for horPDPN remains to be clarified. Thus, this study aimed to identify the epitope of PMab-219 using deletion mutants and point mutants of horPDPN using flow cytometry.

Materials and Methods

Results

Seven deletion mutants of horPDPN, such as dN30 (corresponding to 30–152 aas), dN40 (corresponding to 40–152 aas), dN50 (corresponding to 50–152 aas), dN55 (corresponding to 55–152 aas), dN60 (corresponding to 60–152 aas), dN70 (corresponding to 70–152 aas), and dN80 (corresponding to 80–152 aas) or wild type (WT) of horPDPN (corresponding to 25–152 aas) were generated using CHO-K1 cells (Fig. 1A). All deletion mutants and WT of horPDPN containing N-terminus PA16 tag were detected by NZ-1 (anti-PA16 tag mAb), indicating that the expression level of each construct is very high (Fig. 1B). In contrast, PMab-219 did not react with dN60, dN70, and dN80 (Fig. 1C), suggesting that N-terminus of PMab-219 epitope might exist between 55th aa and 60th aa of horPDPN.

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FIG. 1.

Epitope mapping of PMab-219 using deletion mutants of horPDPN. (A) Illustration of WT and seven deletion mutants of horPDPN: dN30, dN40, dN50, dN55, dN60, dN70, and dN80. Black bars: the positive reaction of PMab-219. White bars: the negative reaction of PMab-219. (B, C) Deletion mutants of horPDPN were analyzed using flow cytometry. Deletion mutants were incubated with NZ-1 (anti-PA16 tag; red line, B), PMab-219 (anti-horPDPN mAb; red line, C), or buffer control (black line, B, C) for 30 min at 4°C, followed by secondary antibodies. horPDPN, horse PDPN; mAb, monoclonal antibody; PDPN, podoplanin; PLAG, platelet aggregation-stimulating; PLD, PLAG-like domain; WT, wild type.

To identify the binding epitope of PMab-219, we next produced a series of point mutants of horPDPN in CHO-K1 cells, including S55A, P56A, A57G, L58A, V59A, P60A, R61A, S62A, T63A, K64A, S65A, and T66A. NZ-1 reacted with all point mutants (Fig. 2A). In contrast, PMab-219 did not react with V59A, R61A, and S62A, and weakly recognized T63A (Fig. 2B), indicating Val59, Arg61, Ser62, and Thr63 of horPDPN could be included in the critical epitope of PMab-219 (Fig. 2C).

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FIG. 2.

Epitope mapping of PMab-219 using point mutants of horPDPN. (A, B) Transient point mutants expressing S55A, P56A, A57G, L58A, V59A, P60A, R61A, S62A, T63A, K64A, S65A, and T66A of horPDPN were incubated with NZ-1 (red line, A), PMab-219 (red line, B), or buffer control (black line, A, B) for 30 min at 4°C, followed by secondary antibodies. (C) Schematic illustration of the epitope recognized by PMab-219. Underlined amino acids are a critical epitope of PMab-219.

Discussion

We have demonstrated that critical epitope of PMab-219 could include Val59, Arg61, Ser62, and Thr63 of horPDPN using deletion mutants and point mutants of horPDPN. In our previous study, the epitope of PMab-202 (the other anti-horPDPN mAb) was identified as Lys64, Thr66, and Phe70 of horPDPN.⁽²⁷⁾ Although the binding epitope of both anti-horPDPN mAbs was shown to exist in a similar location of horPDPN, PMab-202 could not stain horse tissues by immunohistochemistry. These results suggested that CBIS method is more advantageous to obtain immunohistochemistry-applicable mAbs. Likewise, we have successfully established immunohistochemistry-applicable anti-PDPN mAbs for pig,⁽¹⁷⁾ Tasmanian devil,⁽¹⁸⁾ alpaca,⁽¹⁹⁾ bear,⁽²⁰⁾ tiger,⁽²¹⁾ whale,⁽²²⁾ and goat PDPNs⁽²³⁾ using CBIS method, but not by immunizing synthetic peptides.

Using glycan-deficient CHO cell lines, such as Lec1 (N-glycan-deficient), Lec2 (sialic acid-deficient), or Lec8 (galactose-deficient),⁽²⁸⁾ we investigated whether the epitope of PMab-219 could include not only aas but also glycans. Flow cytometric analysis demonstrated that PMab-219 strongly reacts with CHO-K1/horPDPN, Lec1/horPDPN, and Lec2/horPDPN, but weakly reacts with Lec8/horPDPN (data not shown), indicating that galactose might also be included in the PMab-219 epitope. Among Val59, Arg61, Ser62, and Thr63 of horPDPN, Ser62 and Thr63 could be O-glycosylated (Fig. 2C). In a future study, we should determine which O-glycosylation of Ser62 and/or Thr63 is essential for PMab-219 binding to horPDPN.

In conclusion, Val59, Arg61, Ser62, and Thr63 of horPDPN, which are independent of PLAG domain or PLD, are critical for PMab-219-specific binding to horPDPN. PMab-219 could be a useful tool for elucidating the pathophysiological function of horPDPN.