Molecular cloning of apoptosis-inducing Pierisin-like proteins, from two species of white butterfly, Pieris melete

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• 作者：Masafumi Yamamoto ; Tsuyoshi Nakano ; Yuko Matsushima-Hibiya ; Yukari Totsuka ; Azusa Takahashi-Nakaguchi ; Yasuko Matsumoto ; Takashi Sugimura ; Keiji Wakabayashi
• 关键词：DNA ADP-ribosylation ; Cytotoxicity ; Apoptosis ; Pierisin-3 ; Pierisin-4 ; DNA adducts ; Oligo-capping method ; TUNEL assay
• 刊名：Comparative Biochemistry and Physiology, Part B
• 出版年：2009
• 出版时间：November 2009
• 年：2009
• 卷：154
• 期：3
• 页码：326-333
• 全文大小：1592 K

文摘

Pierisin-1, present in cabbage butterfly, Pieris rapae, induces apoptosis against various kinds of cancer cell lines. Another cabbage butterfly, Pieris brassicae, also has an apoptosis-inducing protein, Pierisin-2. These proteins exhibit DNA ADP-ribosylating activity. Pierisin-like proteins are found to be distributed in subtribes Pierina, Aporiina and Appiadina. In this study, we performed the cDNA cloning of Pierisin-like proteins designated Pierisin-3 from gray-veined white, Pieris melete, and Pierisin-4 from black-veined white, Aporia crataegi. The nucleotide sequences of Pierisin-3 and -4 encode an 850 and an 858 amino acid protein, respectively. The partial peptide sequences of Pierisin-3 and -4 purified from pupae were identical to the deduced amino acid sequence of ORF. The deduced amino acid sequence revealed that Pierisin-3 is 93 % similar to Pierisin-1 and Pierisin-4 is 64 % . Pierisin-3 and -4 synthesized in vitro with the rabbit reticulocyte lysate exhibited apoptosis-inducing activity against human cervical carcinoma HeLa and human gastric carcinoma TMK-1 cells. Site-directed mutagenesis at a glutamic acid residue comprising the NAD-binding site resulted in a significant decrease in cytotoxicity of both proteins. Moreover, the proteins incubated with calf thymus DNA and β-NAD resulted in the formation of N²-(ADP-ribos-1-yl)-2′-deoxyguanosine, as in the case of Pierisin-1 and -2. These findings could provide useful information for understanding the importance of apoptosis-inducing ability and molecular evolution of Pierisin-like proteins in family Pieridae.