Mono(ADP-ribosyl)ation of 2′-deoxyguanosine residue in DNA by an apoptosis-inducing protein, pierisin-1, from cabbage butterfly

Pierisin-1 is a potent apoptosis-inducing protein derived from the cabbage butterfly, Pieris rapae. It has been shown that pierisin-1 has an A(.)B structure-function organization like cholera or diphtheria toxin, where the A domain (N-terminal) exhibits ADP-ribosyltransferase activity. The present studies were designed to identify the target molecule for ADP-ribosylation by pierisin-1 in the presence of beta-[adenylate-P-32]NAD, and we found DNA as the acceptor, but not protein as is the case with other bacteria-derived ADP-ribosylating toxins. ADP-ribosylation of tRNAs from yeast was also catalyzed by pierisin-1, but the efficiency was around 1/10 of that for calf thymus DNA. Pierisin-1 efficiently catalyzed the ADP-ribosylation of double-stranded DNA containing dG(.)dC, but not dA(.)dT pairs. The ADP-ribose moiety of NAD was transferred to the amino group at N-2 of 2'-deoxyguanosine to yield N-2-(alpha -ADP-ribos-yl)-2'-deoxyguanosine and its beta form, which were determined by several spectral analyses including H-1- and C-13-NMR and mass spectrometry. The chemical structures were also ascertained by the independent synthesis of N-2-(D-ribos-1-yl)-2'-deoxyguanosine, which is the characteristic moiety of ADP-ribosylated dG. Using the (32)p-postlabeling method, ADP-ribosylated dG could be detected in DNA from pierisin-1-treated HeLa cells, in which apoptosis was easily induced. Thus, the targets for ADP-ribosylation by pierisin-1 were concluded to be 2'-deoxyguanosine residues in DNA. This finding may open a new field regarding the biological significance of ADP-ribosylation.