Molecular cloning of apoptosis-inducing Pierisin-like proteins, from two species of white butterfly, Pieris melete and Aporia crataegi.
Pierisin-1, present in cabbage butterfly, Pieris rapae, induces apoptosis against various kinds of cancer cell lines. Another cabbage butterfly, Pieris brassicae, also has an apoptosis-inducing protein, Pierisin-2. These proteins exhibit DNA ADP-ribosylating activity. Pierisin-like proteins are found to be distributed in subtribes Pierina, Aporiina and Appiadina. In this study, we performed the cDNA cloning of Pierisin-like proteins designated Pierisin-3 from gray-veined white, Pieris melete, and Pierisin-4 from black-veined white, Aporia crataegi. The nucleotide sequences of Pierisin-3 and -4 encode an 850 and an 858 amino acid protein, respectively. The partial peptide sequences of Pierisin-3 and -4 purified from pupae were identical to the deduced amino acid sequence of ORF. The deduced amino acid sequence revealed that Pierisin-3 is 93% similar to Pierisin-1 and Pierisin-4 is 64%. Pierisin-3 and -4 synthesized in vitro with the rabbit reticulocyte lysate exhibited apoptosis-inducing activity against human cervical carcinoma HeLa and human gastric carcinoma TMK-1 cells. Site-directed mutagenesis at a glutamic acid residue comprising the NAD-binding site resulted in a significant decrease in cytotoxicity of both proteins. Moreover, the proteins incubated with calf thymus DNA and beta-NAD resulted in the formation of N(2)-(ADP-ribos-1-yl)-2'-deoxyguanosine, as in the case of Pierisin-1 and -2. These findings could provide useful information for understanding the importance of apoptosis-inducing ability and molecular evolution of Pierisin-like proteins in family Pieridae.
Yamamoto
Masafumi
M
Cancer Prevention Basic Research Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. mayamamo@ncc.go.jp
Nakano
Tsuyoshi
T
Matsushima-Hibiya
Yuko
Y
Totsuka
Yukari
Y
Takahashi-Nakaguchi
Azusa
A
Matsumoto
Yasuko
Y
Sugimura
Takashi
T
Wakabayashi
Keiji
K
D036002:ADP Ribose Transferases / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D019143:Evolution, Molecular; D006367:HeLa Cells; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity*; D007915:Lepidoptera / Q000145:classification / Q000235:genetics*; D008969:Molecular Sequence Data; D011817:Rabbits
D018076:DNA, Complementary; D019476:Insect Proteins; C488125:N(2)-(ADP-ribos-1-yl)-2'-deoxyguanosine; C113557:pierisin protein, insect; D036002:ADP Ribose Transferases; D003849:Deoxyguanosine