Purification and cloning of pierisin-2, an apoptosis-inducing protein from the cabbage butterfly, Pieris brassicae

Abstract

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.