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.