Ogasawara Satoshi, Kaneko Mika K, Honma Ryusuke, Oki Hiroharu, Fujii Yuki, Takagi Michiaki, Suzuki Hiroyoshi, Kato Yukinari
1 Department of Regional Innovation, Tohoku University Graduate School of Medicine , Sendai, Japan .
2 Department of Orthopaedic Surgery, Yamagata University Faculty of Medicine , Yamagata, Japan .
Monoclon Antib Immunodiagn Immunother. 2016 Jun;35(3):155-62. doi: 10.1089/mab.2016.0006.
Podoplanin (PDPN)/Aggrus is a type-I transmembrane sialoglycoprotein, which possesses a platelet aggregation-stimulating (PLAG) domain. The O-glycosylation on Thr52 of human PDPN (hPDPN) is critical for the interaction of hPDPN with C-type lectin-like receptor-2 (CLEC-2), resulting in platelet aggregation. Many anti-hPDPN monoclonal antibodies (MAbs) against PLAG domains and non-PLAG domains have been established; however, mouse anti-PLAG2/3 MAb, the epitope of which is consistent with rat anti-PLAG2/3 MAb NZ-1, has not been established. NZ-1 inhibits the hPDPN-CLEC-2 interaction and is also useful for anti-PA tag MAb. We recently established CasMab technology to produce MAbs against membranous proteins. Herein, we produced a novel anti-hPDPN MAb, LpMab-13, which binds to PLAG2/3 domains. LpMab-13 recognized endogenous hPDPN of cancer cells, including glioblastoma, oral cancer, lung cancer, and malignant mesothelioma, and normal cells such as lymphatic endothelial cells and podocytes of kidney in Western blot, flow cytometry, and immunohistochemistry. LpMab-13 recognized glycan-deficient hPDPN in flow cytometry, indicating that the interaction between LpMab-13 and hPDPN is independent of its glycosylation. The minimum epitope of LpMab-13 was identified as Ala42-Asp49 of hPDPN using Western blot and flow cytometry. The combination of different epitope-possessing MAbs could be advantageous for the hPDPN-targeting diagnosis and therapy.
血小板结合蛋白（PDPN）/聚集蛋白是一种I型跨膜唾液酸糖蛋白，具有血小板聚集刺激（PLAG）结构域。人PDPN（hPDPN）第52位苏氨酸上的O-糖基化对于hPDPN与C型凝集素样受体-2（CLEC-2）的相互作用至关重要，从而导致血小板聚集。已经建立了许多针对PLAG结构域和非PLAG结构域的抗hPDPN单克隆抗体（MAb）；然而，尚未建立表位与大鼠抗PLAG2/3 MAb NZ-1一致的小鼠抗PLAG2/3 MAb。NZ-1抑制hPDPN-CLEC-2相互作用，也可用于抗PA标签MAb。我们最近建立了CasMab技术来生产针对膜蛋白的MAb。在此，我们制备了一种新型抗hPDPN MAb，LpMab-13，它与PLAG2/3结构域结合。在蛋白质免疫印迹、流式细胞术和免疫组织化学中，LpMab-13识别包括胶质母细胞瘤、口腔癌、肺癌和恶性间皮瘤在内的癌细胞以及正常细胞（如肾的淋巴管内皮细胞和足细胞）中的内源性hPDPN。LpMab-13在流式细胞术中识别糖基化缺陷的hPDPN，表明LpMab-13与hPDPN之间的相互作用与其糖基化无关。使用蛋白质免疫印迹和流式细胞术将LpMab-13的最小表位鉴定为hPDPN的Ala42-Asp49。具有不同表位的MAb组合可能有利于hPDPN靶向诊断和治疗。
