Sayama Yusuke, Sano Masato, Furusawa Yoshikazu, Kaneko Mika K, Kato Yukinari
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
Monoclon Antib Immunodiagn Immunother. 2019 Dec;38(6):255-260. doi: 10.1089/mab.2019.0033. Epub 2019 Oct 15.
Podoplanin (PDPN) is a mucin-type membrane glycoprotein, and possesses three platelet aggregation-stimulating (PLAG) domains: PLAG1, PLAG2, and PLAG3 at the N-terminus of PDPN, and one or two PLAG-like domains (PLDs) in the middle of PDPN. PDPN is expressed on normal tissues, such as podocytes of the kidney and type I alveolar cells of the lung, and is also overexpressed in numerous malignant cancers. Previously, we reported a novel anti-alpaca podoplanin (aPDPN) monoclonal antibody (mAb), PMab-225, using Cell-Based Immunization and Screening (CBIS) method. PMab-225 specifically detected aPDPN-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/aPDPN) cells using flow cytometry and western blotting, and strongly stained alpaca tissues such as lung type I alveolar cells by immunohistochemistry. However, the specific binding epitope of aPDPN for PMab-225 remains unclear. Thus, in this study, a series of deletion or point mutations of aPDPN were utilized for investigating the binding epitope of PMab-225 using flow cytometry. The analysis of deletion mutants showed that N-terminus of PMab-225 epitope might exist between 80 amino acid (aa) and 85 aa of aPDPN. Furthermore, the analysis of point mutants demonstrated that Thr84 of aPDPN, which exists in PLD, could be included in the critical epitope of PMab-225.
血小板结合蛋白（PDPN）是一种粘蛋白型膜糖蛋白，在PDPN的N端拥有三个血小板聚集刺激（PLAG）结构域：PLAG1、PLAG2和PLAG3，在PDPN中间有一个或两个类PLAG结构域（PLD）。PDPN在正常组织中表达，如肾脏的足细胞和肺的I型肺泡细胞，并且在许多恶性肿瘤中也过表达。此前，我们报道了一种使用基于细胞的免疫和筛选（CBIS）方法制备的新型抗羊驼血小板结合蛋白（aPDPN）单克隆抗体（mAb）PMab - 225。PMab - 225通过流式细胞术和蛋白质印迹法特异性检测过表达aPDPN的中国仓鼠卵巢（CHO）- K1（CHO/aPDPN）细胞，并通过免疫组织化学强烈染色羊驼组织，如肺I型肺泡细胞。然而，aPDPN与PMab - 225的特异性结合表位仍不清楚。因此，在本研究中，利用一系列aPDPN的缺失或点突变，通过流式细胞术研究PMab - 225的结合表位。缺失突变体分析表明，PMab - 225表位的N端可能存在于aPDPN的第80个氨基酸（aa）至第85个aa之间。此外，点突变体分析表明，存在于PLD中的aPDPN的苏氨酸84可能包含在PMab - 225的关键表位中。
