Purification and cloning of pierisin-2, an apoptosis-inducing protein from the cabbage butterfly, Pieris brassicae.
The cabbage butterfly Pieris rapae contains a strong apoptosis-inducing substance, pierisin, against human cancer cell lines, which is thought to act via ADP-ribosylation. Here we report the purification and cloning of an apoptosis-inducing substance, designated as pierisin-2, from another cabbage butterfly, Pieris brassicae. Pierisin-2 was purified from pupae by sequential chromatography and its cytotoxic and apoptosis-inducing activities to various cancer cells were similar to those of pierisin, designated as pierisin-1, from P. rapae. cDNA cloning of pierisin-2 was performed on the basis of the partial amino-acid sequence. The nucleotide sequence indicated that the cDNA encodes an 850-amino-acid protein with a calculated molecular mass of 97 986. The deduced amino-acid sequence of pierisin-2 was 91% identical with that of pierisin-1. In vitro expressed protein in the reticulocyte lysate exhibited apoptosis-inducing activities against human gastric carcinoma TMK-1 and cervical carcinoma HeLa cells, similar to the purified native pierisin-2 from the pupae. Pierisin-2 shows regional sequence similarities with certain ADP-ribosylating toxins such as the A-subunit of cholera toxin. The results from site-directed mutagenesis at Glu165, a conserved residue among ADP-ribosylating enzymes necessary for NAD binding, and from experiments with ADP-ribosylating enzyme inhibitors suggested that pierisin-2 could be considered as an ADP-ribosylating toxin like pierisin-1.
Matsushima-Hibiya
Y
Y
Cancer Prevention Division, National Cancer Center Research Institute, Tokyo, Japan. yhibiya@gan2.ncc.go.jp
Watanabe
M
M
Kono
T
T
Kanazawa
T
T
Koyama
K
K
Sugimura
T
T
Wakabayashi
K
K
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D001549:Benzamides / Q000494:pharmacology; D002080:Butterflies / Q000737:chemistry; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014407:Tumor Cells, Cultured; D020298:U937 Cells
D001549:Benzamides; D018076:DNA, Complementary; D019476:Insect Proteins; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; C113557:pierisin protein, insect; C037689:benzamide; C025160:3-aminobenzamide; D036002:ADP Ribose Transferases