Characterization of a new apoptosis-inducing protein from the cabbage butterfly

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Pierisin-1 is a 98-kDa cytotoxic protein found in the cabbage butterfly and shows sequence similarity with ADP-ribosylating toxins such as cholera toxin. To identify a substrate protein for ADP-ribosyltransferase activity of pierisin-1, a mixture of[adenylate-^<32>P]-NAD, HeLa cell extract and pierisin-1 was incubated and analyzed by SDS/PAGE and autoradiography. The labeled material was protease resistant but DNase sensitive. This result suggested that DNA might be the substrate for ADP-ribosylation by pierisin. When a variety of oligonucleotides having different sequences were used for acceptors from the efficiency strongly correlated with the proportion of guanine base in each oligonucleotide. For structural determination of the reaction product by pierisin-1, 2'-deoxyguanosine was used as an acceptor molecule. The UV spectrum and ESI-MS suggested the presence of ADP-ribosylated dG. Several NMR analyses revealed the structure as α and β forms of N^2-(ADP-ribos-1-yl)-2'-deoxyguanosine. Finally, we independently synthesized chemically and confirmed the structural identity with the enzyme reaction product. When high-molecular-weight dsDNA was used as acceptor molecule, the same position of guanine base was ADP-ribosylated, as confirmed by structural analyses including ^1H-MNR. The ^<32>P-postlaveling method for detection of ADP-ribosylated dG indicated that ADP-ribosylation of N-2 of guanine base also occurred in HeLa cells treated with pierisin-1. From the above results, it is concluded that pierisin-1 transfers an ADP-ribosyl moiety from NAD to N-2 of guanine base of cellular DNA, and this causes apoptosis induction of the cells.