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

Abstract

Pierisin-1, a cytotoxic protein from the cabbage butterfly ( Pieris rapae), induces apoptosis in mammalian cell lines. Binding of its C-terminal region to glycosphingolipid Gb3 and Gb4 receptors on cell membrane is necessary for incorporation into cells, while the N-terminal polypeptide catalyzes transfer of the ADP-ribose moiety of NAD at N 2 of dG in DNA. Resulting DNA adducts cause mutation if they are present at low levels. If the DNA damage is more severe, the cells undergo apoptosis. In the present study, we examined the repair system for ADP-ribosylated dG adducts using nucleotide excision repair (NER) mutants of Chinese hamster ovary (CHO) cell lines. Pierisin-1 showed cytotoxic effects in all cases: IC 50 values of them were; 650 ng/ml for AA8 (wild), 230 ng/ml for UV5, 190 ng/ml for UV20, 260 ng/ml for UV41, and 240 ng/ml for UV135. Thus, wild-type AA8 proved most resistant to pierisin-1-induced cytotoxicity. When these CHO cell lines were treated with pierisin-1, the adduct levels of ADP-ribosylated dG increased to 2.5–4.8/10 5 nucleotides time-dependently in all cell lines at 12 h. After removal of pierisin-1, the adduct levels remained constant or increased to 4–14/10 5 nucleotides in all NER mutant cells (UV5, UV20, UV41, UV135), while those rapidly decreased to 0.27/10 5 nucleotides in the repair proficient AA8 cells for 24 h. From these results, it is suggested that the NER system is involved in the repair of ADP-ribosylated dG adducts in DNA.