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 small middle dotB 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-(32)P]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 110 of that for calf thymus DNA. Pierisin-1 efficiently catalyzed the ADP-ribosylation of double-stranded DNA containing dG small middle dotdC, but not dA small middle dotdT 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-1-yl)-2'-deoxyguanosine and its beta form, which were determined by several spectral analyses including (1)H- and (13)C-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.
Takamura-Enya
T
T
Cancer Prevention Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-Ku, Tokyo 104-0045, Japan. tenya@gan2.ncc.go.jp
Watanabe
M
M
Totsuka
Y
Y
Kanazawa
T
T
Matsushima-Hibiya
Y
Y
Koyama
K
K
Sugimura
T
T
Wakabayashi
K
K
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D004247:DNA / Q000378:metabolism*; D003849:Deoxyguanosine / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D019476:Insect Proteins / Q000494:pharmacology*; D009243:NAD / Q000378:metabolism
D019476:Insect Proteins; C113557:pierisin protein, insect; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D004247:DNA; D036002:ADP Ribose Transferases; D003849:Deoxyguanosine