Identification of glycosphingolipid receptors for pierisin-1, a guanine-specific ADP-ribosylating toxin from the cabbage butterfly
Identification of glycosphingolipid receptors for pierisin-1, a guanine-specific ADP-ribosylating toxin from the cabbage butterfly
复制标题
DOI：
10.1074/jbc.m212114200
复制
发表时间：
2003-03-14
影响因子：
4.8
通讯作者：
Wakabayashi, K
中科院分区：
文献类型：
--
作者：
Matsushima-Hibiya, Y;Watanabe, M;Wakabayashi, K
Pierisin-1是一种在卷心菜蝴蝶中天然发现的细胞毒性蛋白，可诱导哺乳动物细胞凋亡。我们最近的研究表明，pierisin-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，该区域与靶细胞表面的受体结合并将蛋白质整合到细胞中。本研究旨在鉴定pierisin-1的受体。交联和克隆实验表明，细胞膜上的蛋白与pierisin-1无结合能力。对pierisin-1高度敏感的人宫颈癌HeLa细胞分离脂质进行抑制实验，发现细胞表面中性鞘糖脂具有受体活性。抑制实验和使用抗pierisin-1抗体的TLC免疫染色表明两种中性鞘糖脂是有效成分。经糖鞘脂特异性抗体和负二次离子质谱分析，鉴定为globotriaosylneuroide （Gb3）和globotetraosylneuroide （Gb4）。Gb3和Gb4对pierisin-1的受体活性也得到了证实。小鼠黑色素瘤MEB4细胞缺乏pierisin-1受体，包括Gb3和Gb4，但鞘糖脂Gb3或Gh4预处理细胞可增强其对pierisin-1的敏感性。因此，Gb3和Gb4被证明是pierisin-1受体。pierisin-1的c端区域由蓖麻毒素b链的可能的凝集素结构域组成，包含对其结构组织至关重要的QXW序列。位点诱变对QXW的改变导致pierisin-1细胞毒性显著降低。因此，我们的研究结果表明，pierisin-1以类似蓖麻毒素的方式在c端区域与Gb3和Gb4受体结合，然后在并入细胞后表现出细胞毒性。
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-l-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 Gh4 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.