Corrections EVOLUTION. For the article ‘‘Distribution of cytotoxic and DNA that in Table 1, columns 6 and 7 carried incorrect footnote ADP-ribosylating activity in crude extracts from butterflies symbols. These columns should carry the † footnote symbol, among the family Pieridae,’’ by Yasuko Matsumoto, Tsuyoshi corresponding to: ‘‘–, No detection of cytotoxicity, DNA ADP- Nakano, Masafumi Yamamoto, Yuko Matsushima-Hibiya, Ken- ribosylating activity, specific bands recognized by anti-pierisin-1 Ichi Odagiri, Osamu Yata, Kotaro Koyama, Takashi Sugimura, antibodies and amplification of NAD-binding site sequence by and Keiji Wakabayashi, which appeared in issue 7, February 19, PCR. ϩ, Detection of specific bands recognized by the antibod- 2008, of Proc Natl Acad Sci USA (105:2516–2520; first published ies and amplification of NAD-binding site sequence by PCR.’’ February 6, 2008; 10.1073͞pnas.0712221105), the authors note The corrected table appears below. Table 1. Pierisin-like activity in crude extracts from various butterflies, and areas where the samples were collected DNA ADP- ribosylating activity, Concentration of 50% cell viability, ng/ml*† pg/␮g†‡ Western Species Fifth larvae Pupae Adults Adults blot† PCR† Collection area Pieris rapae 24.3 (3.6–38.9) 23.9 (10.8–43.0) 691 (428–812) 22.88 ϩϩTochigi, Japan Pieris canidia 4.5 (3.8–5.1) 3.9 (2.3–4.1) 53 (36–165) 263.71 ϩϩOkinawa, Japan Pieris napi 9.0 (5.0–10.7) 11.5 (10.7–77.4) 109 (29–134) 14.47 ϩϩTochigi, Japan Pieris melete 22.4 (3.6–39.4) 272 (77–308) 649 (129–4,380) 37.01 ϩϩTochigi, Japan Pieris brassicae 16.6 (3.1–40.1) 1.8 (1.7–2.3) 123 (43–147) 113.26 ϩϩHokkaido, Japan Pontia daplidice 53.8 (35–75) 86 (59–107) 450 (409–908) 44.92 ϩϩGyeonggi, Korea Talbotia naganum§ N.E. N.E. 10,000 (5,100–10,100) 19.21 ϩϩNan hin, Laos Aporia gigantea§ N.E. N.E. 34 (31–340) 419.1 ϩϩHagian, Vietnam Aporia crataegi 4.5 (2.9–6.5) 18.1 (11.5–22.3) 77 (38–146) 29.35 ϩϩHokkaido, Japan Aporia hippia§ N.E. N.E. 78 (11–159) 31.46 ϩϩShaanxi, China Delias pasithoe§ N.E. N.E. 19,300 (14,600–23,200) 95.36 ϩϩHong Kong, China Appias nero§ N.E. N.E. 39,300 (7,000–45,200) 2.55 ϩϩNan hin, Laos Appias paulina 1,255 (1,210–2,040) 10,560 (10,400–18,500) 38,400 (30,000–40,600) 3.45 ϩϩOkinawa, Japan Appias lyncida –– Ϫ ––Ϫ Okinawa, Japan Leptosia nina N.E. N.E. Ϫ ––Ϫ Okinawa, Japan Anthocharis –– Ϫ ––Ϫ Tochigi, Japan scolymus Eurema hecabe –– Ϫ ––Ϫ Tochigi, Japan Catopsilia pomona N.E. N.E. Ϫ ––Ϫ Okinawa, Japan Catopsilia scylla N.E. – Ϫ ––Ϫ Malaysia Colias erate –– Ϫ ––Ϫ Tochigi, Japan *Median values of 50% cell viability (minimum-maximum) are shown. The data were obtained from three independent assays by using two wells for each assay. N.E., not examined. †Ϫ, No detection of cytotoxicity, DNA ADP-ribosylating activity, specific bands recognized by anti-pierisin-1 antibodies and amplification of NAD-binding site sequence by PCR. ϩ, Detection of specific bands recognized by the antibodies and amplification of NAD-binding site sequence by PCR. ‡DNA ADP-ribosylating activities expressed as picograms of pierisin-1 per microgram of protein. §Samples extracted from dried butterflies. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801904105 CORRECTIONS www.pnas.org PNAS ͉ April 8, 2008 ͉ vol. 105 ͉ no. 14 ͉ 5649 Downloaded by guest on September 30, 2021 Distribution of cytotoxic and DNA ADP-ribosylating activity in crude extracts from butterflies among the family Pieridae Yasuko Matsumoto*, Tsuyoshi Nakano*, Masafumi Yamamoto*, Yuko Matsushima-Hibiya*, Ken-Ichi Odagiri†, Osamu Yata†, Kotaro Koyama*, Takashi Sugimura*, and Keiji Wakabayashi*‡ *Cancer Prevention Basic Research Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan; and †Biosystematics Laboratory, Graduate School of Social and Cultural Studies, Kyushu University, 4-2-1 Ropponmatsu, Fukuoka 810-8560, Japan Contributed by Takashi Sugimura, December 26, 2007 (sent for review November 30, 2007) Cabbage butterflies, Pieris rapae and Pieris brassicae, contain moieties on surfaces of neuronal cells (12–14). Receptors for strong cytotoxic proteins, designated as pierisin-1 and -2, against pierisin-1 on mammalian cells have been found to be the neutral cancer cell lines. These proteins exhibit DNA ADP-ribosylating glycosphingolipids, including globotriaosylceramide and globo- activity. To determine the distribution of substances with cytotox- tetraosylceramide, and their expression levels largely determine icity and DNA ADP-ribosylating activity among other species, crude sensitivity to the toxic protein (15). extracts from 20 species of the family Pieridae were examined for The other cabbage white butterfly, Pieris brassicae, also con- cytotoxicity in HeLa cells and DNA ADP-ribosylating activity. Both tains the cytotoxic protein and the protein is isolated and named activities were detected in extracts from 13 species: subtribes pierisin-2. Its amino acid sequence is 91% identical to that of Pierina (Pieris rapae, Pieris canidia, Pieris napi, Pieris melete, Pieris pierisin-1 (5). Pierisin-2 targets DNA, and the structure of the brassicae, Pontia daplidice, and Talbotia naganum), Aporiina (Apo- DNA adduct produced by pierisin-2 is the same as that produced ria gigantea, Aporia crataegi, Aporia hippia, and Delias pasithoe), by pierisin-1 (16). and Appiadina (Appias nero and Appias paulina). All of these Pierisin-1 is mainly distributed in fat bodies during the final extracts contained substances recognized by anti-pierisin-1 anti- larval instar and is highly expressed in fifth instar larvae and bodies, with a molecular mass of Ϸ100 kDa established earlier for early pupae (17). Thus, pierisin-1 may play important roles in pierisin-1. Moreover, sequences containing NAD-binding sites, con- induction of apoptosis to remove larval cells in the pupation of served in ADP-ribosyltransferases, were amplified from genomic Pieris rapae. Another possibility is that the strong cytotoxicity of DNA from 13 species of butterflies with cytotoxicity and DNA pierisin-1 might be effective as a protective agent against mi- ADP-ribosylating activity by PCR. Extracts from seven species, crobes and/or parasitoids. In any case, it would be expected that Appias lyncida, Leptosia nina, Anthocharis scolymus, Eurema he- proteins, such as pierisin-1 and -2, having cytotoxicity and DNA cabe, Catopsilia pomona, Catopsilia scylla, and Colias erate, ADP-ribosylating activity might be distributed in various but- showed neither cytotoxicity nor DNA ADP-ribosylating activity, terflies, not only in the genus Pieris, but also in other genera. and did not contain substances recognized by anti-pierisin-1 Previously, we reported analysis of cytotoxicity of 18 kinds of antibodies. Sequences containing NAD-binding sites were not butterflies against TMK-1 cells, positive results being obtained amplified from genomic DNA from these seven species. Thus, with extracts from Pieris rapae, Pieris brassicae, and Pieris napi pierisin-like proteins, showing cytotoxicity and DNA ADP-ribosy- among the genus Pieris (1). However, no cytotoxicity was lating activity, are suggested to be present in the extracts from observed in the other extracts from examined butterflies: Eu- butterflies not only among the subtribe Pierina, but also among the rema hecabe, Colias erate, and Hebomoia glaucippe of the family subtribes Aporiina and Appiadina. These findings offer insight to Pieridae; Papilio bianor, Papilio helenus, Papilio maackii, Papilio understanding the nature of DNA ADP-ribosylating activity in the machaon, Papilio protenor, and Papilio xuthus of the family butterfly. Papilionidae; Dichorragia nesimachus, Vanessa indica, Sasakia charonda, and Hestina japonica of the family Nymphalidae; pierisin ͉ NAD-binding site ͉ Pierina ͉ Aporiina ͉ Appiadina Celastrina argiolus and Lycaena phlaeas of the family Lycaenidae (1). We need to examine many species to obtain accurate data ierisin-1 was initially identified as a cytotoxic protein from regarding the distribution of substances showing pierisin-like Ppupae of the cabbage white butterfly, Pieris rapae, against activities in butterflies. Recently, we reported that cytotoxic and TMK-1 human gastric cancer cell line (1, 2). Subsequent studies DNA ADP-ribosylating activities of pierisin-1 in adults of Pieris showed pierisin-1 to exhibit potent cytotoxic effects against rapae persisted to some extent in the body after killing. Both various human cancer cell lines, with measured pierisin-1 con- activities decreased to Ϸ20% for 8 weeks (18). This is valuable centration of 50% cell viability ranging from 0.043 to 270 ng/ml information in that adult specimens after death can still be used (3), apoptosis being induced by a mitochondrial pathway involv- for examination of the presence of substance having pierisin-like ing Bcl-2 and caspases (4). Among 13 mammalian cell lines so far activity, at least for a few months, thus increasing the availability tested, human cervical carcinoma HeLa cells were the most of species in this study. sensitive to the cytotoxic effects of pierisin-1 (3, 5, 6). We here examined cytotoxic activity against HeLa cells and Pierisin-1 is a 98-kDa protein comprising 850 aa consisting of DNA ADP-ribosylating activity in butterflies of 20 species of the the N-terminal region (27 kDa) and C-terminal region (71 kDa). The N-terminal region shares sequence similarity with ADP- ribosylating toxins from several bacteria that act on proteins as Author contributions: Y.M. and K.W. designed research; Y.M., T.N., M.Y., and Y.M.-H. target substrates (7–10). Unlike other ADP-ribosyltransferases, performed research; K.-I.O., O.Y., and K.K. contributed new reagents/analytic tools; Y.M., the N-terminal region of pierisin-1 targets the N2 amino groups T.N., M.Y., Y.M.-H., T.S., and K.W.