The Epitope of PMab-210 is Located in Platelet Aggregation-Stimulating Domain-3 of Pig Podoplanin
The Epitope of PMab-210 is Located in Platelet Aggregation-Stimulating Domain-3 of Pig Podoplanin
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PMab-210 的表位位于猪 Podoplanin 的血小板聚集刺激域 3
DOI：
10.1089/mab.2019.0037
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Kato Y
中科院分区：
文献类型：
--
作者：
Kaneko MK;Sayama Y;Sano M;Kato Y
Podoplanin（PDPN）/T1 alpha/Aggrus是一种小的粘蛋白型跨膜糖蛋白，已被证明在许多器官的淋巴管内皮细胞和上皮细胞上表达。PDPN在许多癌症中也上调，并且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PLAG样结构域，其结合C型凝集素样受体-2（CLEC-2）。本研究采用基于细胞的免疫筛选（Cell-Based Immunization and Screening，CBIS）方法，制备了一株抗猪PDPN（pPDPN）单克隆抗体（PMab-210）。PMab-210通过流式细胞术和Western印迹分析特异性检测pPDPN过表达的中国仓鼠卵巢（CHO）-K1细胞。免疫组化结果显示，PMab-210对猪和微型猪的I型肺泡细胞染色较强，对肾小体染色较弱。然而，PMab-210对pPDPN的特异性结合表位不能通过使用一系列pPDPN肽的酶联免疫吸附测定来确定。在这项研究中，产生pPDPN的缺失突变体或点突变体，用于使用流式细胞术分析PMab-210表位。缺失突变体分析表明，PMab-210表位的N端位于pPDPN的第45位氨基酸（aa）和第50位氨基酸（aa）之间。点突变分析表明，PMab-210的关键表位可能包括pPDPN的Glu 47、Asp 48、Tyr 49、Thr 50和Val 51，表明PMab-210表位位于pPDPN的PLAG 3结构域。
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.