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 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.

Figure 1

HPLC analysis of reaction products from dG and β-NAD in the presence of pierisin-1. (a) HPLC profile of the reaction products on an ODS column. The UV absorbance of the eluate was monitored at 254 nm, and the peak appeared by the reaction is marked with an asterisk. (b) UV absorption spectrum with a photodiode array detector of the compound in the peak fraction marked with an asterisk.

Figure 2

Chemical structure of N²-(ADP-ribos-1-yl)-2′-deoxyguanosine (a) obtained from the reaction of dG and β-NAD catalyzed by pierisin-1, and its ¹H-NMR spectrum (b) in DMSO-d₆.

Scheme 1

Figure 3

HPLC analysis of hydrolysate of N²-(ADP-ribos-1-yl)-2′-deoxyguanosine. N²-(ADP-ribos-1-yl)-2′-deoxyguanosine was treated with phosphodiesterase I and bovine intestinal alkaline phosphatase, and separated by HPLC on an ODS column.

Figure 4

HPLC chromatogram of hydrolysate of DNA treated with β-NAD and pierisin-1. Calf thymus DNA reacted with β-NAD in the presence of pierisin-1 was recovered and digested with a nuclease mixture and alkaline phosphatase, followed by HPLC analysis.

Figure 5

Autoradiograms of DNA adducts in HeLa cells treated with pierisin-1. HeLa cells were treated with 100 ng/ml of pierisin-1 for 3 h. The exposure time of imaging plate was 1 h. DNA adduct spots derived from pierisin-1 treatment are indicated by arrowheads.