Distribution of Cytotoxic and DNA ADP-Ribosylating Activity in Crude Extracts from Butterflies Among the Family Pieridae

Home , Anthocharis scolymus, Aporia (butterfly), Aporia hippia, Appias (butterfly), Appias lyncida, Appias nero

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

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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. analyzed data; and Y.M., T.N., M.Y., Y.M.-H., K.-I.O., O.Y., of guanine residues in DNA to yield N2-(ADP-ribos-1-yl)-2Ј- T.S., and K.W. wrote the paper. deoxyguanosine (11). The C-terminal region of pierisin-1 shares The authors declare no conflict of interest. sequence similarity with HA-33, a subcomponent of hemagglu- ‡To whom correspondence should be addressed. E-mail: [email protected]. tinin of botulinum toxin that binds to sialic acid or galactose © 2008 by The National Academy of Sciences of the USA

2516–2520 ͉ PNAS ͉ February 19, 2008 ͉ vol. 105 ͉ no. 7 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712221105 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

*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. family Pieridae. Both activities were observed in crude extracts instar larvae, pupae, and adults of Appias lyncida, whereas other from 13 species of the subtribes Pierina, Aporiina, and Appia- Appias species (Appias nero and Appias paulina) showed dina. Those extracts included a substance recognized by anti- cytotoxicity. pierisin-1 antibodies whose molecular mass was Ϸ100 kDa. Of the 13 species shown to exhibit cytotoxicity, crude extracts Moreover, the sequences constituting highly conserved catalytic from larvae and pupae of eight species were examined for their site motif among ADP-ribosyltransferases were amplified from cytotoxicity. All of the eight species exhibited toxicity much genomic DNA from the 13 species of butterflies by PCR. These stronger than that from adults. Samples from larvae and pupae findings reveal that substances that have cytotoxicity and DNA of Pieris canidia showed very potent activity, with the protein ADP-ribosylating activity are present in butterflies not only of concentration of 50% cell viability at 4.5 ng/ml and 3.9 ng/ml, the subtribe Pierina, including the genus Pieris, but also of the respectively. The value from pupae of Pieris brassicae was 1.8 subtribes Aporiina and Appiadina. The significance of presence ng/ml. Regarding the cytotoxicity of fresh adult samples, the of the substances showing pierisin-like activities in butterflies is values were 53–38,400 ng/ml, with crude extracts from Pieris discussed. napi, Pieris canidia, Pieris brassicae, and Aporia crataegi showing high activities. However, in Appias paulina, crude extracts from Results larvae, pupae, and adults exhibited low toxicity. The samples Cytotoxicity of Crude Extracts from Various Butterflies in HeLa Cells. from Talbotia naganum, Aporia gigantea, Aporia hippia, Delias When HeLa cells, being the most sensitive to pierisin-1 and -2 pasithoe, and Appias nero cases were extracts from dried adult (3, 5), were treated with crude extracts from 20 species at the butterflies and their values were 34–39,300 ng/ml. Crude extracts protein concentrations of 0.01–100 ␮g/ml, cytotoxic activity was from adults of Aporia gigantea and Aporia hippia had high detected in 13 species of six genera: genus Pieris (Pieris rapae, toxicity, with protein concentrations of 50% cell viability being Pieris canidia, Pieris napi, Pieris melete, and Pieris brassicae), 34 and 78 ng/ml, respectively. In contrast, crude extracts of adults genus Pontia (Pontia daplidice), genus Talbotia (Talbotia naga- from Talbotia naganum, Delias pasithoe, and Appias nero had low num), genus Aporia (Aporia gigantea, Aporia crataegi, and Aporia toxicity, with the values of 10,000–39,300 ng/ml. hippia), genus Delias (Delias pasithoe), and genus Appias (Appias nero and Appias paulina) (Table 1). The crude extracts from DNA ADP-Ribosylating Activity in Crude Extracts from Adult Butter- Pieris rapae and Pieris brassicae showed cytotoxicity against flies. ADP-ribosylation of DNA could be linked with cytotoxicity HeLa, as with TMK-1 cells that we reported (1). However, the in cells. Therefore, crude extracts from adult butterflies of 20 remaining seven species, Appias lyncida, Leptosia nina, Antho- species were incubated with calf thymus DNA and ␤-[adenylate- charis scolymus, Eurema hecabe, Catopsilia pomona, Catopsilia 32P]NAD ([32P]NAD), the DNA was enzymatically digested, and scylla, and Colias erate did not show cytotoxic activity, as listed the formation of ADP-ribosylated-deoxyribonucleotide adducts EVOLUTION in Table 1. Interestingly, no cytotoxicity was detected with fifth was analyzed on TLC (Table 1). Typical examples of ADP-

Matsumoto et al. PNAS ͉ February 19, 2008 ͉ vol. 105 ͉ no. 7 ͉ 2517 Fig. 1. Detection of ADP-ribosylated DNA adducts with crude extracts of various butterflies. Crude extract was incubated with calf thymus DNA and [32P]NAD and spotted on TLC sheets with (ϩ) or without (Ϫ) nuclease diges- tion, which were then developed and subjected to autoradiography. Sample 1, no protein (negative control); sample 2, pierisin-1 (40 pg, positive control); sample 3, Pieris melete; sample 4, Aporia crataegi; sample 5, Delias pasithoe; sample 6, Appias paulina; sample 7, Appias lyncida; sample 8, Anthocharis Fig. 2. Detection of substances by anti-pierisin-1 antibodies in the crude scolymus; sample 9, Colias erate. Samples 3 and 4, 1 ␮g of protein in crude extracts of various butterflies on Western blotting: Lane 1, 2.5 ng of purified extract; sample 5, 0.1 ␮g of protein in crude extract; sample 6–9, 10 ␮gof pierisin-1; lane 2, Pieris melete; lane 3, Aporia crataegi; lane 4, Delias pasithoe; protein in crude extract. The arrow indicates the Rf value of 0.05. lane 5, Appias paulina; lane 6, Appias lyncida; lane 7, Anthocharis scolymus; lane 8, Colias erate. Lane 2–4, 5 ␮g of crude extract protein from adults per lane; lane 6–8, 50 ␮g of crude extract protein from adults per lane. ribosylated DNA adducts are illustrated in Fig. 1. Adduct spots were clearly detected on TLC sheets with crude extracts from the Ϸ 13 species (Pieris rapae, Pieris canidia, Pieris napi, Pieris melete, 0.3-kb product was amplified from the genomic DNAs from 13 Pieris brassicae, Pontia daplidice, Talbotia naganum, Aporia species of butterflies that exhibited cytotoxicity and DNA gigantea, Aporia crataegi, Aporia hippia, Delias pasithoe, Appias ADP-ribosylating activity (Table 1). Typical examples are shown in Fig. 3. On the other hand, no PCR product was detected in the nero, and Appias paulina), with Rf values of 0.05, the same as those of N2-(ADP-ribos-1-yl)-2Ј-deoxyguanosine produced by genomic DNA from seven species of butterflies that had no cytotoxicity and DNA ADP-ribosylating activity. pierisin-1. The Rf values for other deoxyribonucleotides Ј (3 -phosphate form), ADP-ribose, and NAD were from 0.30 to Discussion 0.95. These 13 species showed cytotoxicity, as mentioned earlier, In the present study, we found that crude extracts from 13 species but no quantitative relation was observed between cytotoxicity of the subtribes Pierina, Aporiina, and Appiadina showed cyto- and DNA ADP-ribosylating activity (Table 1). No detectable toxicity in HeLa cells. DNA ADP-ribosylating activity and signals appeared with the crude extracts from the other seven 100-kDa substances recognized by anti-pierisin-1 antibodies species (Appias lyncida, Leptosia nina, Anthocharis scolymus, were also detected in all of the species showing cytotoxicity. The Eurema hecabe, Catopsila pomona, Catopsila scylla, and Colias 13 species showing DNA ADP-ribosylating activity with cyto- erate). The samples from these species had no cytotoxicity toxicity included Pieris rapae, Pieris napi, and Pieris brassicae, for against HeLa cells with concentrations up to 300 ␮g/ml. which cytotoxicity in TMK-1 cells was previously reported by our laboratory (1). Thus, 10 species of the subtribes Pierina (Pieris Detection of Substances Recognized by Anti-pierisin-1 Antibodies. To canidia, Pieris melete, Pontia daplidice, and Talbotia naganum), investigate whether substances recognized by anti-pierisin-1 Aporiina (Aporia gigantea, Aporia crataegi, Aporia hippia, and antibodies were present in each extract from butterflies, Western Delias pasithoe), and Appiadina (Appias nero and Appias paulina) blot analyses were performed by using the antibodies purified were newly demonstrated to be cytotoxic against mammalian from rabbit antiserum against SDS-denatured full-length pieri- cells. Cytotoxic activities of the fifth instar larvae and pupae sin-1 (6). An Ϸ100-kDa band recognized by anti-pierisin-1 were higher than those of adults in seven species (Pieris canidia, antibodies was detected in crude extracts from adult butterflies Pieris napi, Pieris melete, Pieris brassicae, Pontia daplidice, Aporia exhibiting cytotoxicity and DNA ADP-ribosylating activity crataegi, and Appias paulina), as was found in Pieris rapae (17). (Pieris rapae, Pieris canidia, Pieris napi, Pieris melete, Pieris Samples from final instar larvae, pupae, and adults of Pieris brassicae, Pontia daplidice, Talbotia naganum, Aporia gigantea, canidia were also obtained in Taipei, Taiwan, which were kindly Aporia crataegi, Aporia hippia, Delias pasithoe, Appias nero, and Appias paulina). Typical examples are shown in Fig. 2. In the other samples (Appias lyncida, Leptosia nina, Anthocharis scolymus, Eurema hecabe, Catopsila pomona, Catopsila scylla, and Colias erate) not showing cytotoxicity and ADP-ribosylating activity, the antibodies did not detect any specific fragments.

Detection of the Conserved Catalytic Site Motif of ADP-ribosyltransferase. To detect sequences containing NAD-binding sites, which are conserved in ADP-ribosyltransferases, in the genome of various butterflies, partial genomic DNA sequences of DNA ADP-ribosylating proteins were amplified by using degenerate PCR primers, deduced from pierisin-1 and -2 amino acid se- Fig. 3. PCR analysis of genomic DNA from various butterﬂies. Lane M, molecular marker, 100-bp DNA ladder (Takara Bio); lane 1, no template quences (19). The forward primer corresponded to the con- control; lane 2, Pieris rapae; lane 3, Pieris melete; lane 4, Aporia crataegi; lane served arginine residue, which maintains the reaction pocket, 5, Delias pasithoe; lane 6, Appias paulina; lane 7, Appias lyncida; lane 8, whereas the reverse primer corresponded to the conserved Anthocharis scolymus; lane 9, Colias erate. All assays were performed in glutamic acid that is a putative catalytic center domain. A triplicate and a typical result is shown.

2518 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712221105 Matsumoto et al. provided by Yu-Feng Hsu (Department of Life Science, Na- had no cytotoxic or DNA ADP-ribosylating activity, and a tional Taiwan Normal University, Taipei, Taiwan). These sam- sequence including a putative NAD-binding site was not ampli- ples showed cytotoxicity and DNA ADP-ribosylating activities fied from the genome of Appias lyncida by PCR. similar to those of Pieris canidia from Okinawa, Japan (Table 1). On the other hand, pupal forms of the other butterflies among Moreover, adults of Aporia hippia were obtained under the the family Pieridae are smoother than the tribe Pierini. Their permission by the Nagano Prefecture, Japan, and their abdo- heads are tapered apically, and the wings are strongly curved mens were homogenized and treated with saturated ammonium ventrally (type I form) (20). Leptosia nina and Anthocharis sulfate. These samples exhibited similar cytotoxicity and DNA scolymus (among the subfamily Pierinae), Eurema hecabe, Ca- ADP-ribosylating activities to those from Taibai, Shaanxi, China topsila pomona, Catopsila scylla, and Colias erate (among the (Table 1). Thus, it is suggested that the cytotoxicity and DNA subfamily Coliadinae), which form type I pupae, showed no ADP-ribosylating activity of the samples might depend on the cytotoxic and DNA ADP-ribosylating activity, and no amplifi- species, but independent of the area of sample collection. In the cation of a putative NAD-binding site sequence in their genomes present study, quantitative correlation between cytotoxicity and was observed by PCR. As shown in our previous article, Eurema DNA ADP-ribosylating activity was not observed in extracts hecabe, Colias erate, and Hebomoia glaucippe (among the Colotis from the 13 species. Because there are other factors such as the group in the subfamily Pierinae) have been reported to show no binding of pierisin-like substances on the surfaces of target cells cytotoxicity against TMK-1 cells (1). Our finding of substances and its incorporation into the cells that could affect the cytotoxic with cytotoxicity and DNA ADP-ribosylating activity in tribe activities, DNA ADP-ribosylating activity in the sample could Pierini but not in Leptosia nina (Leptosia group) and Anthocharis not be the only factor accountable for its cytotoxicity. scolymus (tribe Anthocharidini) is thus in line with speculation Recently, we reported that cytotoxicity and DNA ADP- that the branching point, at which the pierisin-like substances ribosylating activities in Pieris rapae decreased during storage were acquired, might be between Leptosia group and the tribe after killing, but persisted to some extent for at least 8 weeks. Pierini, with subtribe Appiadina butterflies on the border be- Both activities were decreased by Ϸ20% compared with fresh tween the groups with or without the pierisin-like substances. samples (18). In the present study, we found that crude extracts This branching point also corresponds to the border between two from dried adult samples of Talbotia naganum, Aporia gigantea, groups, which form type I or type II pupae. It is possible that Aporia hippia, Delias pasithoe, and Appias nero exhibited cyto- molecular analogs of pierisin had been present in all butterflies toxicity and DNA ADP-ribosylating activity. In these samples, an belonging to the family Pieridae. In this case, evolutionary Ϸ100-kDa band recognized by anti-pierisin-1 antibodies was also changes in the molecule might have resulted in the alteration of detected. In addition, cytotoxic activity of dried samples from the nucleotide sequence to undetectable levels when assayed by Aporia gigantea and Aporia hippia was higher than that of fresh PCR, leading to the loss of cytotoxic and DNA ADP-ribosylating adult samples from Pieris napi and Appias paulina (Table 1). functions in the butterfly species that had no cytotoxic and DNA Therefore, differences in cytotoxicity and DNA ADP- ADP-ribosylating activities. Another possibility is that no such ribosylating activity between species might be attributed mainly molecular analog is present in the species of butterflies without to species differences rather than sample conditions. cytotoxic and DNA ADP-ribosylating activities. Further exam- Western blot analysis showed that extracts from the 13 species inations with genomic analysis are necessary to determine with cytotoxicity and DNA ADP-ribosylating activity all contain whether the gene(s) encoding the 100-kDa substance might have Ϸ100-kDa bands similar in size to pierisin-1. Some of 100-kDa been acquired in the species of butterflies showing cytotoxic and proteins were further demonstrated to have DNA ADP- DNA ADP-ribosylating activities. ribosylation ability by in-gel assay (data not shown). Moreover, Because the 100-kDa substances with cytotoxicity and DNA PCR analysis suggested that the 13 species of butterflies with ADP-ribosylating activity are distributed in butterflies among cytotoxic and DNA ADP-ribosylating activities contain se- the subtribes Pierina, Aporiina, and Appiadina, these DNA quences including a putative NAD-binding site in their genomes. ADP-ribosylating proteins must provide some advantage to the Among these 13 species, Pieris rapae and Pieris brassicae have species. In Pieris rapae, mRNA levels of pierisin-1 reach a been reported to contain DNA ADP-ribosylating proteins, pieri- maximum in the final instar larvae and then rapidly decrease in sin-1 and -2, respectively (5, 10, 11, 16). It is also suggested that the pupal stage, and the protein is expressed during the fifth the remaining 11 species contain DNA ADP-ribosylating pro- instar larvae to early stage of pupae. Most pierisin-1 is found to teins. On the other hand, Western blot and PCR analysis be distributed in fat bodies, and a little is detected in the suggested that the seven species of butterflies without cytotoxic hemolymph and midgut (17). If the 100-kDa substances ADP- and DNA ADP-ribosylating activities do not contain 100-kDa ribosylate their own DNA, these might cause apoptosis to proteins similar in size to pierisin-1, and sequences including a remove larval tissues fated to die, for example, in the larval putative NAD-binding site. midgut and silk grand, during metamorphosis. Another possible Several molecular phylogenetic studies based on an analysis of role of DNA ADP-ribosylating proteins is in defense against nucleotide sequences of the genes have been reported, these microbes and parasites such as bacteria, viruses, or parasitic indicating that the three subtribes Pierina, Aporiina, and Ap- wasps. Understanding the roles of the 100-kDa substances with piadina are closely related (20–22). Braby’s group reported the cytotoxicity and DNA ADP-ribosylating activity and investiga- subfamily Pierinae to comprise two tribes (Anthocharidini and tion of the origin of their encoding genes should provide useful Pierini) and two groups (Colotis group and Leptosia group), and information about their biological significance. the tribe Pierini comprises three subtribes (Appiadina, Pierina, and Aporiina) and two other groups (Elodina group and Materials and Methods DixeiaϩBelenois group). Furthermore, they described that pupal Preparation of Butterfly Samples. The collection areas for the 20 species of forms of the tribe Pierini have several distinguishing character- butterﬂies examined in the present study are shown in Table 1. Live adults of istics in that the ventral surface is flat and the heads have horns Pieris rapae, Pieris napi, Pieris melete, Anthocharis scolymus, Eurema hecabe, or spike-like processes (type II form). All of the 13 species found and Colias erate were captured in Tochigi prefecture, Japan. They were fed with 10% honey solution and oviposited on fresh natural host plants: Brassica here to have cytotoxic and DNA ADP-ribosylating activity and oleracea for Pieris rapae, Arabis glabra for Pieris napi and Anthocharis a 100-kDa substance recognized by anti-pierisin-1 antibodies scolymus, Rorippa indica for Pieris melete, Albizia julibrissin for Eurema belong to these three subtribes, Pierina, Aporiina, and Appia-

hecabe, and Trifolium pratense for Colias erate in our laboratory. After EVOLUTION dina, that form type II pupae. It should be noted that samples hatching, larvae were reared on fresh natural diet at room temperature until from Appias lyncida, also belonging to the subtribe Appiadina, pupation, except for the fourth and ﬁfth larvae of Pieris rapae that reared on

Matsumoto et al. PNAS ͉ February 19, 2008 ͉ vol. 105 ͉ no. 7 ͉ 2519 artificial diet, as reported (18). Final instar larvae of Pieris canidia, Aporia Scion Image software, values being normalized to that for 1 pg of pierisin-1 in crataegi, and Appias lyncida were purchased from Eikoh Science Corp., and each test. The values from DNA adduct formation with each sample were were reared on natural host plants: Brassica oleracea for Pieris canidia, Prunus converted into picograms of pierisin-1 per microgram of protein. yedoensis for Aporia crataegi, and Crataeva religiosa for Appias lyncida at room temperature until pupation in our laboratory. Adults and final instar Western Blotting. Substances recognized by anti-pierisin-1 antibodies were larvae of Appias paulina were purchased from Butterfly Gallery Kabira, and detected as follows. In brief, crude samples obtained from various butterfly the larvae were reared on natural host plant, Drypetes matsumurae, at 25°C samples were separated by SDS-polyacrylamide gel electrophoresis and blot- until pupation in our laboratory. Live pupae of Catopsila scylla were pur- ted on PVDF transfer membranes (Millipore). Anti-pierisin-1 rabbit polyclonal chased from Mokuyo-sha, and were reared at room temperature in our antibodies were obtained as described in ref. 6. Anti-rabbit IgG, horseradish laboratory until eclosion. Live final instar larvae, pupae, and newly emerged peroxidase-linked F(abЈ)2 fragments (from donkey) (GE Healthcare) and ECL adults of Pieris rapae, Pieris canidia, Pieris napi, Pieris melete, Aporia crataegi, Western blotting detection reagents (GE Healthcare) were used to visualize Appias lyncida, Eurema hecabe, Catopsila scylla, and Colias erate or collected protein–antibody complexes. adults of Appias paulina, Leptosia nina, Anthocharis scolymus, and Catopsila pomona were used for the following experiments. PCR Amplification. Genomic DNAs were extracted from 20 species of butter- Final instar larvae, pupae, and adults of Pieris brassicae were kindly pro- flies [18 species of adults and 2 species (Pieris napi and Pontia daplidice)of vided by Masashi Ueno (Asahikawa, Hokkaido). Final instar larvae, pupae, and larvae] by using an Easy-DNA Kit (Invitrogen). Partial genomic DNA sequences adults of Pontia daplidice were purchased from Young-Woon Jung (Gyeo- corresponding to DNA ADP-ribosylating proteins were amplified by using nggi, Korea). Adults of Talbotia naganum, Appias nero, Aporia gigantea, degenerate PCR primers deduced from the pierisin-1 and -2 amino acid Aporia hippia, and Delias pasithoe were obtained at the places shown in Table sequences, including motifs of ADP-ribosyltransferase activity (19). The prim- 1, kept for some period (about a few months) after death, and were purchased ers were (5Ј-AGRCARWGRYTNGTNWGRTGGGA) and (5Ј-GCNACYTCCATYT- from Mokuyo-sha. All obtained samples were stored at Ϫ80°C until use. GRTTNGG). PCR was performed in a 20-␮l volume containing 40 pmol of each primer, 0.4 units of Phusion High-Fidelity DNA Polymerase (Finnzymes), 200 ␮ ϫ Cytotoxicity and DNA ADP-ribosylating Activity in Crude Extracts from Butter- M each dNTP and 1 accessory buffer. The PCR was carried out in iCycler flies. Whole bodies of the fifth instar larvae, pupae, or abdomens of the adults (Bio-Rad) with an initial incubation for 30 s at 98°C, followed by 35 cycles of 10 s were weighed and homogenized in nine volumes of Tris buffer (1 mM DTT, 50 at 98°C, 20 s at 56°C, and 1 min at 72°C, and a final extension for 7 min at 72°C. mM Tris⅐HCl, pH 7.5), as described in ref. 17. Protein concentrations of each crude extract were measured by the Bradford assay with a Bio-Rad Protein ACKNOWLEDGMENTS. We thank Prof. Yu-Feng Hsu (Department of Life Assay kit I (Bio-Rad). Science, National Taiwan Normal University, Taipei, Taiwan), Mr. Masashi Ueno (Asahikawa, Hokkaido), Prof. Takashi Yagi (Division of Environmental Cytotoxicity of crude extract samples against HeLa cells was examined with Genetics, Frontier Science Innovation Center, Osaka Prefecture University), Dr. WST-1 [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazo- Jae-Yoon Leem (Department of Oriental Medicine Food and Nutrition, Se- lium; Dojindo Laboratory] as described (2, 3, 18). myung University, Korea), Mr. Yasuhiro Ohshima (Biosystematics Laboratory, To estimate DNA ADP-ribosylating activity of samples, calf thymus DNA, Graduate School of Social and Cultural Studies, Kyushu University), and Prof. [32P]NAD, and 10 ␮M ␤-NAD were incubated with crude extract samples in Young-Joon Surh (National Research Laboratory of Molecular Carcinogenesis reaction buffer for 30 min at 37°C. The DNA was recovered, and digested to and Chemoprevention, College of Pharmacy Seoul National University, Korea) deoxyribonucleoside 3Ј-phosphates by treatment with micrococcal nuclease for their kind cooperation in obtaining butterfly samples. This work was supported in part by a Grant-in-Aid for the Third-Term Comprehensive 10- and phosphodiesterase II, and were spotted onto TLC sheets. After develop- Year Strategy for Cancer Control from the Ministry of Health, Labour and ment, these were exposed to Fuji Imaging Plates (Fujifilm) and DNA adducts Welfare, Japan, and by Research Resident Fellowships from the Foundation were detected with a Bio-Image Analyzer (Fujifilm) (18). Levels of adduct for Promotion of Cancer Research (Japan) for the Third-Term Comprehensive formation were calculated by densitometric analysis of DNA adducts by using 10-Year Strategy for Cancer Control (to Y.M. and M.Y.).

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2520 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712221105 Matsumoto et al.