Matsushima-Hibiya Yuko, Watanabe Masahiko, Hidari Kazuya I-P, Miyamoto Daisei, Suzuki Yasuo, Kasama Takeshi, Kasama Takashi, Koyama Kotaro, Sugimura Takashi, Wakabayashi Keiji
Cancer Prevention Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
J Biol Chem. 2003 Mar 14;278(11):9972-8. doi: 10.1074/jbc.m212114200.
Pierisin-1, a cytotoxic protein found naturally in the cabbage butterfly, induces apoptosis of mammalian cells. Our recent studies suggest that pierisin-1 consists of an N-terminal ADP-ribosyltransferase domain, and a C-terminal region that binds to receptors on the surfaces of target cells and incorporates the protein into cells. The present study was undertaken to identify receptors for pierisin-1. The cross-linking and cloning experiments suggested that the proteins on cell membrane had no binding ability to pierisin-1. Inhibitory assays of fractionated lipids from human cervical carcinoma HeLa cells, which are highly sensitive to pierisin-1, indicated neutral glycosphingolipids on the cell surface to show receptor activity. Inhibitory assays and TLC immunostaining using anti-pierisin-1 antibodies demonstrated two neutral glycosphingolipids as active components. Analysis of their structures with glycosphingolipid-specific antibodies and negative secondary ion mass spectrometry identified them as globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4). The receptor activities of Gb3 and Gb4 for pierisin-1 were also confirmed with these authentic compounds. Pierisin-1-insensitive mouse melanoma MEB4 cells were found to lack pierisin-1 receptors, including Gb3 and Gb4, but pretreatment of the cells with glycosphingolipid Gb3 or Gb4 enhanced their sensitivity to pierisin-1. Thus, Gb3 and Gb4 were proven to serve as pierisin-1 receptors. The C-terminal region of pierisin-1 consists of possible lectin domains of a ricin B-chain, containing QXW sequences, which are essential for its structural organization. Alteration of QXW by site-directed mutagenesis caused marked reduction of pierisin-1 cytotoxicity. Thus, our results suggest that pierisin-1 binds to Gb3 and Gb4 receptors at the C-terminal region, in a manner similar to ricin, and then exhibits cytotoxicity after incorporation into the cell.
菜粉蝶素-1是一种在菜粉蝶中天然存在的细胞毒性蛋白，可诱导哺乳动物细胞凋亡。我们最近的研究表明，菜粉蝶素-1由一个N端ADP核糖基转移酶结构域和一个C端区域组成，该C端区域与靶细胞表面的受体结合并将该蛋白摄入细胞内。本研究旨在鉴定菜粉蝶素-1的受体。交联和克隆实验表明细胞膜上的蛋白质对菜粉蝶素-1没有结合能力。对人宫颈癌HeLa细胞（对菜粉蝶素-1高度敏感）的分级脂质进行的抑制试验表明，细胞表面的中性糖鞘脂具有受体活性。使用抗菜粉蝶素-1抗体进行的抑制试验和薄层层析免疫染色显示两种中性糖鞘脂为活性成分。用糖鞘脂特异性抗体和负离子二次离子质谱对其结构进行分析，确定它们为Globotriaosylceramide（Gb3）和Globotetraosylceramide（Gb4）。这些纯化物也证实了Gb3和Gb4对菜粉蝶素-1的受体活性。发现对菜粉蝶素-1不敏感的小鼠黑色素瘤MEB4细胞缺乏包括Gb3和Gb4在内的菜粉蝶素-1受体，但用糖鞘脂Gb3或Gb4对细胞进行预处理可增强它们对菜粉蝶素-1的敏感性。因此，已证明Gb3和Gb4可作为菜粉蝶素-1的受体。菜粉蝶素-1的C端区域由蓖麻毒素B链可能的凝集素结构域组成，包含QXW序列，这对其结构组织至关重要。通过定点诱变改变QXW会导致菜粉蝶素-1细胞毒性显著降低。因此，我们的结果表明，菜粉蝶素-1在C端区域以类似于蓖麻毒素的方式与Gb3和Gb4受体结合，然后在摄入细胞后表现出细胞毒性。
