Characterization of Anti-Goat Podoplanin Monoclonal Antibody PMab-235 Using Immunohistochemistry Against Goat Tissues

Introduction

Podoplanin (PDPN)/T1alpha is a type I transmembrane glycoprotein, which is expressed in normal tissues, such as type I lung alveolar cells, renal corpuscles, and lymphatic endothelial cells.^(1,2) PDPN induces platelet aggregation through C-type lectin-like receptor-2 (CLEC-2) on platelets.^(1,3–9) The PDPN/CLEC-2 interaction facilitates the separation of embryonic blood/lymphatic vessels.⁽¹⁰⁾ The expression of human PDPN was reported in several malignant tumors, such as malignant brain tumors,^(11–14) oral squamous cell carcinomas,⁽¹⁵⁾ malignant mesotheliomas,^(16,17) and lung cancers.⁽¹⁸⁾ PDPN expression has also been associated with malignant progression and cancer metastasis.^(6,11,19)

Because pathophysiological studies of alveolar cells of the lungs, podocytes of the kidneys, or lymphatic endothelial cells of the colon using anti-goat PDPN (gPDPN) mAbs are important in many disorders, we used the Cell-Based Immunization and Screening (CBIS) method and developed specific and sensitive monoclonal antibodies (mAbs) against the gPDPN of 168 amino acids to facilitate the immunohistochemical analysis of paraffin-embedded tissue sections.⁽²⁰⁾ PMab-235 recognized CHO/gPDPN cells, but showed no reaction with CHO-K1 cells, as assessed by flow cytometry. PMab-235 cross-reacts with bovine PDPN. In contrast, PMab-235 did not react with human, mouse, rat, rabbit, dog, cat, pig, horse, Tasmanian devil, sheep, alpaca, tiger, whale, or bear PDPNs. K_D of PMab-235 for CHO/gPDPN cells was determined to be 1.5 × 10⁻⁸, indicating a moderate affinity of PMab-235 for CHO/gPDPN cells. The immunohistochemical analyses revealed that PMab-235 strongly stained type I alveolar cells of lung, podocytes of kidney, and lymphatic endothelial cells of colon. This study aimed to determine the binding epitope of PMab-235 to gPDPN using enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry.

Materials and Methods

Results

We first synthesized a series of gPDPN peptides, which are summarized in Table 1. Using ELISA, PMab-235 detected a 61–80 peptide (₆₁-TPMPTGTENVTHGHREDLPT-₈₀) of gPDPN, whereas it did not react with the other peptides: 21–40, 31–50, 41–60, 51–70, 71–90, 81–100, 91–110, 101–120, 111–130, and 121–135 peptides (Fig. 1 and Table 1).

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FIG. 1.

Epitope mapping using synthetic peptides of gPDPN. Synthesized gPDPN peptides were immobilized at 10 μg/mL, and were incubated with PMab-235, followed by secondary antibodies. Black bar: positive reaction by PMab-235. gPDPN, goat podoplanin.

We next moved on to the blocking assay to confirm the binding epitope of PMab-235. We first performed immunohistochemical analyses for goat lung tissues. PMab-235 showed very high reaction with type I alveolar cells of goat lungs in a dose-dependent manner (0.01–0.1 μg/mL; Fig. 2). Then, we performed a blocking assay using immunohistochemistry against goat lungs at a concentration of 0.02 μg/mL. PMab-235 reacted with type I alveolar cells of lung (Figs. 3A, B). These reactions were not neutralized by 21–40 peptide in lung (Figs. 3C, D). In contrast, these reactions were partially neutralized by 61–80 peptide in lung (Figs. 3E, F), indicating that 61–80 peptide is an epitope of PMab-235.

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FIG. 2.

Immunohistochemical analyses against goat lung tissues using PMab-235. Histological sections of the goat lungs were directly autoclaved in citrate buffer for 20 minutes. After blocking with SuperBlock T20 (PBS) Blocking Buffer, the sections were incubated with PMab-235 at a concentration of 0.1 μg/mL (A, B), 0.05 μg/mL (C, D), 0.02 μg/mL (E, F), and 0.01 μg/mL (G, H). Bar: 100 μm.

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FIG. 3.

Immunohistochemical analyses against goat lung tissues using PMab-235 and peptides of gPDPN. Histological sections of the goat lungs were directly autoclaved in citrate buffer for 20 minutes. After blocking with SuperBlock T20 (PBS) Blocking Buffer, the sections were incubated with (A, B) PMab-235 (0.02 μg/mL), (C, D) PMab-235 (0.02 μg/mL) + 21–40 (1 μg/mL), (E, F) PMab-235 (0.02 μg/mL) + 61–80 (1 μg/mL), or (G, H) control (blocking buffer), followed by treatment with the Envision + Kit. (I, J). Bar: 100 μm. HE, hematoxylin and eosin.

PMab-235 also showed very high reaction with lymphatic endothelial cells of goat colons in a dose-dependent manner (0.1–1 μg/mL; Fig. 4), even at a low concentration of 0.1 μg/mL (Fig. 4G, H). We performed a blocking assay using immunohistochemistry against goat colons at a concentration of 0.2 μg/mL. PMab-235 reacted with lymphatic endothelial cells of colons (Figs. 5A, B). These reactions were not neutralized by 21–40 peptide (Figs. 5C, D). In contrast, these reactions were completely neutralized by 61–80 peptide (Figs. 5E, F), also confirming that 61–80 peptide is an epitope of PMab-235.

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FIG. 4.

Immunohistochemical analyses against goat colon tissues using PMab-235. Histological sections of the goat colons were directly autoclaved in citrate buffer for 20 minutes. After blocking with SuperBlock T20 (PBS) Blocking Buffer, the sections were incubated with PMab-235 at a concentration of 1 μg/mL (A, B), 0.5 μg/mL (C, D), 0.2 μg/mL (E, F), and 0.1 μg/mL (G, H). Bar: 100 μm.

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FIG. 5.

Immunohistochemical analyses against goat colon tissues using PMab-235 and peptides of gPDPN. Histological sections of the goat colons were directly autoclaved in citrate buffer for 20 minutes. After blocking with SuperBlock T20 (PBS) Blocking Buffer, the sections were incubated with (A, B) PMab-235 (0.2 μg/mL), (C, D) PMab-235 (0.2 μg/mL) + 21–40 (1 μg/mL), (E, F) PMab-235 (0.2 μg/mL) + 61–80 (1 μg/mL), or (G, H) control (blocking buffer), followed by treatment with the Envision + Kit. (I, J). Red arrows indicate lymphatic endothelial cells. Bar: 100 μm.

We then synthesized point mutants of 61–80 peptides (Table 2). Using ELISA, PMab-235 detected T61A, P62A, M63A, P64A, T65A, G66A, T67A, E68A, N69A, V70A, T71A, H72A, G73A, H74A, E76A, D77A, and T80A. Conversely, it did not detect R75A, L78A, and P79A, indicating that R75A, L78A, and P79A are critical for PMab-235 to detect gPDPN.

Discussion

We previously developed mAbs against human,⁽²²⁾ mouse,⁽²²⁾ rat,⁽²³⁾ rabbit,⁽²⁴⁾ dog,⁽²⁵⁾ cat,⁽²⁶⁾ bovine,⁽²⁷⁾ horse,^(28–30) and pig PDPNs.^(31,32) PDPN comprises three tandem-repeat of the “EDxxVTPG” sequences, which were defined to be platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) of the N-terminus of the PDPN protein.⁽⁴⁾ There are also several PLAG-like domains (PLDs) of the “E(D/E)xx(T/S)xx” sequence. PLDs, one of which is also named as PLAG4, are reportedly important for PDPN–CLEC-2 interactions.⁽³³⁾ Almost all mAbs against PDPNs reportedly react with PLAG domains or PLDs.^(33,34) As summarized in Figure 6, PMab-235 detected PLD of gPDPN.

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FIG. 6.

Summary of PMab-235 epitope. (A–C) Immunohistochemical analyses against goat lung tissues using PMab-235 and peptides of gPDPN. Each picture is the enlargement of type I alveolar cells of Figure 3. (D–F) Immunohistochemical analyses against lymphatic endothelial cells of colon using PMab-235 and peptides of gPDPN. Each picture is the enlargement of lymphatic endothelial cells of Figure 5. Bar: 100 μm. (G) Schematic illustration of the epitope recognized by PMab-235. PLAG, platelet aggregation-stimulating; PLD, PLAG-like domain.

PMab-235 cross-reacted with bovine PDPN (bovPDPN).⁽²⁰⁾ The corresponding sequence of PMab-235 epitope “REDLPT” is also “REDLPT” about bovPDPN, which indicates 100% identity between gPDPN and bovPDPN. In the blocking assay using immunohistochemistry against goat lungs, the 61–80 peptide of goat PDPN did not completely block the PMab-235 binding to type I alveolar cells (Figs. 3E, F) because PMab-235 was developed using CBIS method, not by immunizing synthetic peptides, then the binding affinity of PMab-235 against type I alveolar cells might be higher than that for the 61–80 peptide.

Taken together, PLD of gPDPN was clarified to be advantageous epitopes for several applications, such as flow cytometry, Western blotting, and immunohistochemical analyses. These findings can be applied to the production of more functional anti-gPDPN mAbs.