Abstract
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.
Highlights
We have demonstrated that the critical epitope of PMab237 could include Leu82 and Thr84 of whale PDPN (wPDPN) using the deletion mutants and point mutants of wPDPN in Chinese hamster ovary (CHO)-K1 cells by flow cytometry
We developed many monoclonal antibody (mAb) against PDPNs of human,(24) mouse,(25) rat,(26) rabbit,(27) dog,(28) cat,(29) bovine,(30) horse,(31) Tasmanian devil,(32) alpaca,(33) bear,(34) tiger,(35) goat,(36) pig,(37,38) and whale.[20]. We successfully determined the binding epitope of those mAbs.[22,34,39,40,41,42,43,44,45,46,47,48] These epitope mapping results showed that almost all anti-PDPN mAbs react with platelet aggregationstimulating (PLAG) domains or PLAG-like domains (PLDs).[7,39,40,41,43,44,49,50,51] The critical epitope of PMab-237 was shown to be located in PLD (Fig. 4), suggesting that PLAG domains and PLD were advantageous to the epitope for several applications such as flow cytometry, western blotting, and immunohistochemical analyses
Using glycan-deficient CHO cell lines, such as Lec1 (N-glycan-deficient), Lec2, or Lec8,(52) we investigated whether the epitope of PMab-237 could include amino acid (AA) and glycans
Summary
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. PMab-237 detected wPDPN by flow cytometry, western blotting, and immunohistochemical analyses. 237 strongly stained pulmonary type I alveolar cells, renal podocytes, and lymphatic endothelial cells of the harbor porpoise by the immunohistochemical analysis.[22] the binding epitope of PMab-237 for wPDPN remains unknown. This study aimed to identify the epitope of PMab-237
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