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Enzymatic Properties of Pierisin-1 and Its N- Terminal Domain, a Guanine-Specific ADP- Ribosyltransferase from the Cabbage Butterfly Masahiko Watanabe*,

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Presentation on theme: "Enzymatic Properties of Pierisin-1 and Its N- Terminal Domain, a Guanine-Specific ADP- Ribosyltransferase from the Cabbage Butterfly Masahiko Watanabe*,"— Presentation transcript:

1 Enzymatic Properties of Pierisin-1 and Its N- Terminal Domain, a Guanine-Specific ADP- Ribosyltransferase from the Cabbage Butterfly Masahiko Watanabe*, Shigeki Enomoto, Takeji Takamura-Enya, Tsuyoshi Nakano, 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 Received January 23, 2004; accepted February 5, 2004 J. Biochem, 2004, Vol. 135, No. 4, 471-477 © 2004 Oxford University Press *Oxford University Press*Oxford University Press

2 PUBMED Recent Articles 1: Shiotani B, Watanabe M, Totsuka Y, Sugimura T, Wakabayashi K. Involvement of nucleotide excision repair (NER) system in repair of mono ADP-ribosylated dG adducts produced by pierisin- 1, a cytotoxic protein from cabbage butterfly. Mutat Res. 2005 May 2;572(1-2):150-5. PMID: 15790498 [PubMed - indexed for MEDLINE] 1: Shiotani B, Watanabe M, Totsuka Y, Sugimura T, Wakabayashi K. Involvement of nucleotide excision repair (NER) system in repair of mono ADP-ribosylated dG adducts produced by pierisin- 1, a cytotoxic protein from cabbage butterfly. Mutat Res. 2005 May 2;572(1-2):150-5. PMID: 15790498 [PubMed - indexed for MEDLINE] 2: Watanabe M, Nakano T, Shiotani B, Matsushima-Hibiya Y, Kiuchi M, Yukuhiro F, Kanazawa T, Koyama K, Sugimura T, Wakabayashi K. Developmental stage-specific expression and tissue distribution of pierisin-1, a guanine-specific ADP-ribosylating toxin, in Pieris rapae. Comp Biochem Physiol A Mol Integr Physiol. 2004 Oct;139(2):125-31. PMID: 15528160 [PubMed - indexed for MEDLINE] 2: Watanabe M, Nakano T, Shiotani B, Matsushima-Hibiya Y, Kiuchi M, Yukuhiro F, Kanazawa T, Koyama K, Sugimura T, Wakabayashi K. Developmental stage-specific expression and tissue distribution of pierisin-1, a guanine-specific ADP-ribosylating toxin, in Pieris rapae. Comp Biochem Physiol A Mol Integr Physiol. 2004 Oct;139(2):125-31. PMID: 15528160 [PubMed - indexed for MEDLINE] 3: Takamura-Enya T, Watanabe M, Koyama K, Sugimura T, Wakabayashi K. Mono(ADP- ribosyl)ation of the N2 amino groups of guanine residues in DNA by pierisin-2, from the cabbage butterfly, Pieris brassicae. Biochem Biophys Res Commun. 2004 Oct 15;323(2):579-82. PMID: 15369790 [PubMed - indexed for MEDLINE] 3: Takamura-Enya T, Watanabe M, Koyama K, Sugimura T, Wakabayashi K. Mono(ADP- ribosyl)ation of the N2 amino groups of guanine residues in DNA by pierisin-2, from the cabbage butterfly, Pieris brassicae. Biochem Biophys Res Commun. 2004 Oct 15;323(2):579-82. PMID: 15369790 [PubMed - indexed for MEDLINE] 4: Watanabe M, Enomoto S, Takamura-Enya T, Nakano T, Koyama K, Sugimura T, Wakabayashi K. Enzymatic properties of pierisin-1 and its N-terminal domain, a guanine-specific ADP- ribosyltransferase from the cabbage butterfly. J Biochem (Tokyo). 2004 Apr;135(4):471-7. PMID: 15115771 [PubMed - indexed for MEDLINE] 4: Watanabe M, Enomoto S, Takamura-Enya T, Nakano T, Koyama K, Sugimura T, Wakabayashi K. Enzymatic properties of pierisin-1 and its N-terminal domain, a guanine-specific ADP- ribosyltransferase from the cabbage butterfly. J Biochem (Tokyo). 2004 Apr;135(4):471-7. PMID: 15115771 [PubMed - indexed for MEDLINE] 5: Totsuka Y, Kawanishi M, Nishigaki R, Matsukawa K, Yagi T, Takamura-Enya T, Watanabe M, Sugimura T, Wakabayashi K. Analysis of HPRT and supF mutations caused by pierisin-1, a guanine specific ADP-ribosylating toxin derived from the cabbage butterfly. Chem Res Toxicol. 2003 Aug;16(8):945-52. PMID: 12924921 [PubMed - indexed for MEDLINE] 6: Watanabe M, Takamura-Enya T, Kanazawa T, Totsuka Y, Matsushima-Hibiya Y, Koyama K, Sugimura T, Wakabayashi K. Mono(ADP-ribosyl)ation of DNA by apoptosis-inducing protein, pierisin. Nucleic Acids Res Suppl. 2002;(2):243-4. PMID: 12903196 [PubMed - indexed for MEDLINE] 5: Totsuka Y, Kawanishi M, Nishigaki R, Matsukawa K, Yagi T, Takamura-Enya T, Watanabe M, Sugimura T, Wakabayashi K. Analysis of HPRT and supF mutations caused by pierisin-1, a guanine specific ADP-ribosylating toxin derived from the cabbage butterfly. Chem Res Toxicol. 2003 Aug;16(8):945-52. PMID: 12924921 [PubMed - indexed for MEDLINE] 6: Watanabe M, Takamura-Enya T, Kanazawa T, Totsuka Y, Matsushima-Hibiya Y, Koyama K, Sugimura T, Wakabayashi K. Mono(ADP-ribosyl)ation of DNA by apoptosis-inducing protein, pierisin. Nucleic Acids Res Suppl. 2002;(2):243-4. PMID: 12903196 [PubMed - indexed for MEDLINE]

3 Introduction Pierisin-1: cytotoxic protein found in the cabbage butterfly 850 residues, MW = 98kDa Shares similarities with cholera and pertussis toxins in N-terminal region and with lectin domain of ricin superfamily in the C-terminal region Possesses a catalytic glutamate Transfers ADP-ribose to DNA (N-2 position of guanine bases) Apoptosis or gene mutations in mammalian cells are induced in a dose-dependent fashion by pierisin-1 Purpose of Study Clarify the enzymatic properties of pierisin-1, its N-terminal polypeptide and trypsin-treated full-length native pierisin-1

4 Copyright ©1999 by the National Academy of Sciences Watanabe, Masahiko et al. (1999) Proc. Natl. Acad. Sci. USA 96, 10608-10613

5 Copyright ©2001 by the National Academy of Sciences Takamura-Enya, Takeji et al. (2001) Proc. Natl. Acad. Sci. USA 98, 12414-12419

6 Copyright ©2001 by the National Academy of Sciences Kanazawa, Takashi et al. (2001) Proc. Natl. Acad. Sci. USA 98, 2226-2231

7 Fig. 1. Detection of ADP-ribosyltransferase activity of N- terminal and C-terminal polypeptides. (A) SDS- polyacrylamide gel electrophoresis of [35S]methionine-labeled polypeptides synthesized by in vitro translation. Lane 1, C- terminal Ser-234-Met-850 polypeptide; lane 2, N-terminal Met- 1-Arg-233 polypeptide; lane 3, E165Q N-terminal polypeptide; lane 4, translation without RNA. The amount of both the nonmutated and E165Q N-terminal polypeptides (lanes 2 and 3) were about 3-fold higher than that of the C-terminal fragment (lane 1). (B) ADP-ribosyltransferase activity of the polypeptides. Calf thymus DNA (1 mg/ml) and [adenylate-32P]NAD (0.1 mM, 20 mCi/mmol) were incubated in the presence of 0.25 µl of reticulocyte lysate mixture in a total volume of 25 µl for 60 min at 37°C. Incorporation of radioactivity was measured in a liquid scintillation counter

8 Fig. 2. Effects of trypsin-treatment on the ADP- ribosyltransferase activity of pierisin-1. (A) SDS- polyacrylamide gel electrophoresis. Aliquots of pierisin-1 (10 µg each) were incubated with various amounts of trypsin for 1 hour at 37°C, and 1 g each of the products were run in a 5– 20% gradient gel and silver stained. Lane M, molecular mass standard; lane 1, pierisin-1 without trypsin; lanes 2–6, pierisin-1 treated with 0.1, 0.3, 1, 3 and 10 g trypsin [trypsin:pierisin-1 = 1:100, 3:100, 10:100, 30:100, 100:100 (w/w)]. (B) ADP-ribosyltransferase activity of trypsin-treated pierisin-1. Calf thymus DNA (1 mg/ml) and [adenylate- 32P]NAD (0.1 mM, 20 mCi/mmol) were incubated in the presence of trypsin-treated pierisin-1 at 37°C. Incorporation of radioactivity was measured in a liquid scintillation counter. Activity is expressed as turnover number per second. (C) In- gel ADP-ribosyltransferase assay of N-terminal and C- terminal fragments of pierisin-1. Ten micrograms of pierisin-1 were digested with 1 µg of trypsin for 1 h at 37°C, and 1 µg of each was separated in a gel and stained with Comassie Blue R-250 (lane 1) or assessed by enzyme activity (lane 2).

9 Fig. 3. Difference in enzyme activity of pierisin-1 under various conditions. Calf thymus DNA (1 mg/ml) and [adenylate-32P]NAD (0.1 mM, 20 mCi/mmol) were incubated in the presence of the N- terminal polypeptide N1-233 (solid circles) or the trypsin-treated protein A1-850/trypsin (open circles). Incorporation of radioactivity was measured in a liquid scintillation counter. Activity is expressed as turnover number per second. (A) Effects of pH. The buffers used were: pH 4 and 5, 50 mM sodium acetate; pH 6, 50 mM BisTris-HCl, pH 7, 50 mM HEPES-NaOH; pH 8 and 9, 50 mM Tris-HCl; pH 10, 50 mM glycine- NaOH, pH 11 and 12, 50 mM sodium phosphate. Buffers other than sodium phosphate (pH 11 and 12) contained 100 mM NaCl. Due to the high ionic strength of the sodium phosphate itself, the addition of NaCl was omitted in the pH 11 and 12 buffers. All reactions were carried out at 37°C. (B) Effects of ionic strength. Reactions were carried out at 37°C in 50 mM Tris-HCl pH 9 with different concentrations of NaCl. (C) Effects of temperature. Reactions were carried out in 50 mM Tris-HCl pH 9 in the presence of 100 mM NaCl.

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11 Fig. 4. Relationship between NAD concentration and ADP-ribosyltransferase activity. Calf thymus DNA (1 mg/ml) and various concentrations of [adenylate- 32P]NAD were incubated in the presence of the N-terminal polypeptide N1-233 (solid circles) or the trypsin-treated protein A1- 850/trypsin (open circles). Incorporation of radioactivity was measured in a liquid scintillation counter. Results are demonstrated by a Lineweaver-Burk plot. Reaction velocity means turnover number per second.

12 Fig. 5. Accumulation of ADP-ribosylated DNA by pierisin-1. Calf thymus DNA (1 mg/ml) and [adenylate- 32P]NAD (0.1 mM, 20 mCi/mmol) were incubated in the presence of the N- terminal polypeptide N1-233 (solid circles) or the trypsin- treated protein A1- 850/trypsin (open circles) for up to 72 h at 37°C. Incorporation of radioactivity was measured in a liquid scintillation counter. Activity is expressed as turnover number per second.

13 Conclusions Pierisin-1 is an ADPRT enzyme with a catalytic glutamate residue N-terminal (1-233) is the catalytic domain More active than DTA or ETA (55 s -1 ) Full length enzyme is activated by nicking at Arg 233 Substrate is DNA containing guanine bases Dissociation of the N- and C-terminal domains may be physiologically relevant which would allow the 27 kDa N-terminal domain to pass through the nuclear pore to access genomic DNA of host Single enzyme molecule may be sufficient to kill a mammalian cell


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