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

• Published in: Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 2009-11, Vol.154 (3), p.326-333
• Main Authors: Yamamoto, Masafumi, Nakano, Tsuyoshi, Matsushima-Hibiya, Yuko, Totsuka, Yukari, Takahashi-Nakaguchi, Azusa, Matsumoto, Yasuko, Sugimura, Takashi, Wakabayashi, Keiji
• Format: Article
• Language: English
• Subjects: ADP Ribose Transferases - chemistry; ADP Ribose Transferases - metabolism; ADP Ribose Transferases - toxicity; Amino Acid Sequence; Animals; Apoptosis; Apoptosis - drug effects; Cloning, Molecular; Conserved Sequence; Cytotoxicity; Deoxyguanosine - analogs & derivatives; Deoxyguanosine - metabolism; DNA adducts; DNA ADP-ribosylation; DNA, Complementary - genetics; Evolution, Molecular; HeLa Cells; Humans; Insect Proteins - chemistry; Insect Proteins - genetics; Insect Proteins - metabolism; Insect Proteins - toxicity; Lepidoptera - classification; Lepidoptera - genetics; Molecular Sequence Data; Oligo-capping method; Pierisin-3; Pierisin-4; Rabbits; TUNEL assay
• ISSN: 1096-4959; 1879-1107
• DOI: 10.1016/j.cbpb.2009.07.007

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 N 2-(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.