Kaneko Mika K, Sayama Yusuke, Sano Masato, Kato Yukinari
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
Monoclon Antib Immunodiagn Immunother. 2019 Dec;38(6):271-276. doi: 10.1089/mab.2019.0037. Epub 2019 Oct 30.
Podoplanin (PDPN)/T1alpha/Aggrus, a small mucin-type transmembrane glycoprotein, has been shown to be expressed on lymphatic endothelial cells and epithelial cells of many organs. PDPN is also upregulated in many cancers, and is involved in cancer metastasis and malignant progression. Human PDPN possesses three platelet aggregation-stimulating (PLAG) domains and the PLAG-like domain, which bind to C-type lectin-like receptor-2 (CLEC-2). Previously, we reported a novel antipig PDPN (pPDPN) monoclonal antibody (PMab-210) using Cell-Based Immunization and Screening (CBIS) method. PMab-210 specifically detected pPDPN-overexpressed Chinese hamster ovary (CHO)-K1 cells by flow cytometry and Western blot analysis. Immunohistochemical analyses demonstrated that PMab-210 stained pulmonary type I alveolar cells strongly and renal corpuscles weakly in pig or microminipig. However, the specific binding epitope of PMab-210 for pPDPN could not be determined by enzyme-linked immunosorbent assay using a series of pPDPN peptides. In this study, deletion mutants or point mutants of pPDPN were produced for analyzing the PMab-210 epitope using flow cytometry. The analysis of deletion mutants showed that N-terminus of PMab-210 epitope exists between 45th amino acid (aa) and 50th aa of pPDPN. In addition, the analysis of point mutants demonstrated that the critical epitope of PMab-210 could include Glu47, Asp48, Tyr49, Thr50, and Val51 of pPDPN, indicating that PMab-210 epitope is located in PLAG3 domain of pPDPN.
血小板结合蛋白（PDPN）/T1α/聚集蛋白是一种小型粘蛋白型跨膜糖蛋白，已证实在许多器官的淋巴管内皮细胞和上皮细胞中表达。PDPN在许多癌症中也上调，并参与癌症转移和恶性进展。人PDPN具有三个血小板聚集刺激（PLAG）结构域和类PLAG结构域，它们与C型凝集素样受体2（CLEC-2）结合。此前，我们报道了一种使用基于细胞的免疫和筛选（CBIS）方法制备的新型抗猪PDPN（pPDPN）单克隆抗体（PMab-210）。通过流式细胞术和蛋白质印迹分析，PMab-210能特异性检测过表达pPDPN的中国仓鼠卵巢（CHO）-K1细胞。免疫组织化学分析表明，在猪或微型猪中，PMab-210对肺I型肺泡细胞染色强烈，对肾小体染色较弱。然而，使用一系列pPDPN肽通过酶联免疫吸附测定无法确定PMab-210与pPDPN的特异性结合表位。在本研究中，制备了pPDPN的缺失突变体或点突变体，以通过流式细胞术分析PMab-210表位。缺失突变体分析表明，PMab-210表位的N端存在于pPDPN的第45个氨基酸（aa）和第50个aa之间。此外，点突变体分析表明，PMab-210的关键表位可能包括pPDPN的Glu47、Asp48、Tyr49、Thr50和Val51，这表明PMab-210表位位于pPDPN的PLAG3结构域中。
