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

Abstract

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 β-[adenylate-³²P]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 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-(α-ADP-ribos-1-yl)-2′-deoxyguanosine and its β form, which were determined by several spectral analyses including ¹H- and ¹³C-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 ³²P-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.