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 A⅐B structure-function organization like cholera or diphtheria toxin, where the ''A'' domain (N-terminal) exhibits ADPribosyltransferase activity. The present studies were designed to identify the target molecule for ADP-ribosylation by pierisin-1 in the presence of ␤-[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 1 ⁄10 of that for calf thymus DNA. Pierisin-1 efficiently catalyzed the ADPribosylation of double-stranded DNA containing dG⅐dC, but not dA⅐dT pairs. The ADP-ribose moiety of NAD was transferred to the amino group at N 2 of 2 -deoxyguanosine to yield N 2 -(␣-ADP-ribos-1-yl)-2 -deoxyguanosine and its ␤ 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. P ierisin-1, a 98-kDa protein, is abundantly present in the fifth instar larvae and the early phase of pupae in the cabbage butterfly, Pieris rapae (1, 2). This protein has potent cytotoxic activity against various human cancer cell lines with a wide range of IC 50 values, inducing typical apoptotic cell death with characteristic morphological features, DNA fragmentation, and cleavage of poly(ADP-ribose) polymerase (2, 3). The N-terminal region of pierisin-1 has a partial regional sequence similarity with ADP-ribosylating toxins such as the A-subunit of cholera toxin, and disruption of this possible NAD-binding site by site-directed mutagenesis abolishes its apoptosis-inducing activity (4). Inhibitors of ADP-ribosyltransferase also significantly reduce its ability to induce apoptosis of cancer cells. Recently, we found that pierisin-1 is likely to have an A⅐B structure-function organization similar to that of cholera and diphtheria toxins, the ''A'' domain featuring the ADP-ribosyltransferase activity and the ''B'' domain binding to receptors in cell membranes, thereby incorporating the A domain into cells for ADP-ribosylation of intracellular target molecules. It is also suggested that the glycolipid in the membrane was a possible candidate for the receptor of pierisin-1, and the sensitivity of cancer cells to pierisin-1 depended on the presence of the receptor in cell membrane. Moreover, treatment using either the N-terminal polypeptide (amino acids...

Bioengineered silkworms with butterfly cytotoxin-modified silk glands produce sericin cocoons with a utility for a new biomaterial

“…transgenic silkworm | cytotoxin | silk proteins | hydrogel P ierisin-1 is a 98-kDa cytotoxic protein that is produced by the cabbage butterfly Pieris rapae (1)(2)(3)(4). Previous studies have shown that the addition of purified pierisin-1 to culture media can induce apoptosis of various human cancer cell lines (1)(2)(3)(4).…”

“…Previous studies have shown that the addition of purified pierisin-1 to culture media can induce apoptosis of various human cancer cell lines (1)(2)(3)(4). The N-terminal domain of pierisin-1 features an ADP ribosyltransferase that transfers the ADP ribose moiety of nicotinamideadenine dinucleotide (NAD) to the 2′-deoxyguanosine residues of DNA (2)(3)(4), whereas the C-terminal region carries a domain that mediates binding to receptors on cell membranes and uptake by target cells (4).…”

Distribution of cytotoxic and DNA ADP-ribosylating activity in crude extracts from butterflies among the family Pieridae

“…Moreover, PCR analysis suggested that the 13 species of butterflies with cytotoxic and DNA ADP-ribosylating activities contain sequences including a putative NAD-binding site in their genomes. Among these 13 species, Pieris rapae and Pieris brassicae have been reported to contain DNA ADP-ribosylating proteins, pierisin-1 and -2, respectively (5,10,11,16). It is also suggested that the remaining 11 species contain DNA ADP-ribosylating proteins.…”

“…The N-terminal region shares sequence similarity with ADPribosylating toxins from several bacteria that act on proteins as target substrates (7)(8)(9)(10). Unlike other ADP-ribosyltransferases, the N-terminal region of pierisin-1 targets the N 2 amino groups of guanine residues in DNA to yield N 2 -(ADP-ribos-1-yl)-2Ј-deoxyguanosine (11). The C-terminal region of pierisin-1 shares sequence similarity with HA-33, a subcomponent of hemagglutinin of botulinum toxin that binds to sialic acid or galactose moieties on surfaces of neuronal cells (12)(13)(14).…”

Scabin, a Novel DNA-acting ADP-ribosyltransferase from Streptomyces scabies

“…In rare cases, mART toxins may target DNA as the macromolecule substrate as in the case of Pierisin-1 (4). Those mART toxins that utilize DNA as a target macromolecule label the guanine base with an ADPribose moiety (7,8).…”