Molecular cloning of an apoptosis-inducing protein, pierisin, from cabbage butterfly: Possible involvement of ADP-ribosylation in its activity

Self Cite

Significance Specific gene functions have been successfully suppressed by gene silencing or editing in many organisms. However, genetic manipulation to suppress the function of a target tissue has not been achieved using cytotoxin genes. We established transgenic silkworms with posterior silk glands (PSGs) that express the enzymatic domain of the cytotoxin pierisin-1A (P1A). The larvae with the modified PSGs produced the sericin cocoons with potential utilities in tissue engineering. The targeted P1A expression was found to cause site-specific repression of certain protein synthesis that appeared to have no impact on the developmental stages of individuals. Thus, the new approach through targeted P1A expression could be applicable to the development of biologically useful model organisms with tissue-specific dysfunction.

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Otsuki

Yamamoto

Matsumoto

et al. 2017

Self Cite

“…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.…”

mentioning

confidence: 99%

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

Takamura-Enya¹,

Watanabe²,

Totsuka³

et al. 2001

Self Cite

108

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...

“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

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

Matsumoto¹,

Nakano²,

Yamamoto³

et al. 2008

Self Cite

Cabbage butterflies, Pieris rapae and Pieris brassicae , contain strong cytotoxic proteins, designated as pierisin-1 and -2, against cancer cell lines. These proteins exhibit DNA ADP-ribosylating activity. To determine the distribution of substances with cytotoxicity and DNA ADP-ribosylating activity among other species, crude extracts from 20 species of the family Pieridae were examined for cytotoxicity in HeLa cells and DNA ADP-ribosylating activity. Both activities were detected in extracts from 13 species: subtribes Pierina ( Pieris rapae , Pieris canidia , Pieris napi , Pieris melete , Pieris brassicae , Pontia daplidice , and Talbotia naganum ), Aporiina ( Aporia gigantea , Aporia crataegi , Aporia hippia , and Delias pasithoe ), and Appiadina ( Appias nero and Appias paulina ). All of these extracts contained substances recognized by anti-pierisin-1 antibodies, with a molecular mass of ≈100 kDa established earlier for pierisin-1. Moreover, sequences containing NAD-binding sites, conserved in ADP-ribosyltransferases, were amplified from genomic DNA from 13 species of butterflies with cytotoxicity and DNA ADP-ribosylating activity by PCR. Extracts from seven species, Appias lyncida , Leptosia nina , Anthocharis scolymus , Eurema hecabe , Catopsilia pomona , Catopsilia scylla , and Colias erate , showed neither cytotoxicity nor DNA ADP-ribosylating activity, and did not contain substances recognized by anti-pierisin-1 antibodies. Sequences containing NAD-binding sites were not amplified from genomic DNA from these seven species. Thus, pierisin-like proteins, showing cytotoxicity and DNA ADP-ribosylating activity, are suggested to be present in the extracts from butterflies not only among the subtribe Pierina, but also among the subtribes Aporiina and Appiadina. These findings offer insight to understanding the nature of DNA ADP-ribosylating activity in the butterfly.