Kanazawa T, Watanabe M, Matsushima-Hibiya Y, Kono T, Tanaka N, Koyama K, Sugimura T, Wakabayashi K
Cancer Prevention Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2226-31. doi: 10.1073/pnas.051628898.
Pierisin-1 is an 850-aa cytotoxic protein found in the cabbage butterfly, Pieris rapae, and has been suggested to consist of an N-terminal region with ADP-ribosyltransferase domain and of a C-terminal region that might have a receptor-binding domain. To elucidate the role of each region, we investigated the functions of various fragments of pierisin-1. In vitro expressed polypeptide consisting of amino acid residues 1-233 or 234-850 of pierisin-1 alone did not show cytotoxicity against human cervical carcinoma HeLa cells. However, the presence of both polypeptides in the culture medium showed some of the original cytotoxic activity. Introduction of the N-terminal polypeptide alone by electroporation also induced cell death in HeLa cells, and even in the mouse melanoma MEB4 cells insensitive to pierisin-1. Thus, the N-terminal region has a principal role in the cytotoxicity of pierisin-1 inside mammalian cells. Analyses of incorporated pierisin-1 indicated that the entire protein, regardless of whether it consisted of a single polypeptide or two separate N- and C-terminal polypeptides, was incorporated into HeLa cells. However, neither of the terminal polypeptides was incorporated when each polypeptide was present separately. These findings indicate that the C-terminal region is important for the incorporation of pierisin-1. Moreover, presence of receptor for pierisin-1 in the lipid fraction of cell membrane was suggested. The cytotoxic effects of pierisin-1 were enhanced by previous treatment with trypsin, producing "nicked" pierisin-1. Generation of the N-terminal fragment in HeLa cells was detected after application of intact entire molecule of pierisin-1. From the above observations, it is suggested that after incorporation of pierisin-1 into the cell by interaction of its C-terminal region with the receptor in the cell membrane, the entire protein is cleaved into the N- and C-terminal fragments with intracellular protease, and the N-terminal fragment then exhibits cytotoxicity.
菜粉蝶素-1是一种在菜粉蝶（Pieris rapae）中发现的由850个氨基酸组成的细胞毒性蛋白，有人认为它由一个具有ADP-核糖基转移酶结构域的N端区域和一个可能具有受体结合结构域的C端区域组成。为了阐明每个区域的作用，我们研究了菜粉蝶素-1各种片段的功能。仅由菜粉蝶素-1的氨基酸残基1-233或234-850组成的体外表达多肽对人宫颈癌HeLa细胞没有显示出细胞毒性。然而，培养基中同时存在这两种多肽时显示出了一些原始的细胞毒性活性。通过电穿孔单独导入N端多肽也会诱导HeLa细胞死亡，甚至在对菜粉蝶素-1不敏感的小鼠黑色素瘤MEB4细胞中也是如此。因此，N端区域在哺乳动物细胞内菜粉蝶素-1的细胞毒性中起主要作用。对摄入的菜粉蝶素-1的分析表明，无论它是由单个多肽组成还是由两个单独的N端和C端多肽组成，整个蛋白质都被摄入了HeLa细胞。然而，当每个多肽单独存在时，两个末端多肽都没有被摄入。这些发现表明C端区域对菜粉蝶素-1的摄入很重要。此外，还提示细胞膜脂质部分存在菜粉蝶素-1的受体。用胰蛋白酶预处理产生“缺口”菜粉蝶素-1后，菜粉蝶素-1的细胞毒性作用增强。在应用完整的菜粉蝶素-1全分子后，检测到HeLa细胞中产生了N端片段。从上述观察结果来看，有人认为菜粉蝶素-1通过其C端区域与细胞膜上的受体相互作用进入细胞后，整个蛋白质被细胞内蛋白酶切割成N端和C端片段，然后N端片段表现出细胞毒性。
