Enzymatic Properties of Pierisin-1 and Its N-Terminal Domain, a Guanine-Specific ADP-Ribosyltransferase from the Cabbage Butterfly

Abstract

The cabbage butterfly, Pieris rapae, produces an ADP-ribosylating cytotoxic protein, pierisin-1. Unlike other ADP-ribosylating toxins, the acceptor site for ADP-ribosylation by pierisin-1 is the N-2 position of guanine bases in DNA. The present study was designed to characterize this novel guanine-specific ADP-ribosyltransferase, pierisin-1. The N-terminal polypeptide from Met-1 to Arg-233, but not the C-terminal Ser-234-Met-850 polypeptide, was found to exhibit guanine ADP-ribosyltransferase activity. Trypsin-treated pierisin-1, which is considered to be a "nicked" full-length form composed of associated N- and C-terminal fragments, also demonstrated such activity. Optimum conditions for the N-terminal polypeptide of pierisin-1 were pH 8-10, 37-40 degrees C, in the presence of 100-200 mM NaCl or KCl. Other metal ions such as Ca(2+) or Mg(2+) were not required. Kinetic studies demonstrated potent ADP-ribosyltransferase activity with a K(M) value for NAD of 0.17 mM and k(cat) of 55 per second. Under these optimum conditions, the specific activity of trypsin-treated pierisin-1 was about half (k(cat) = 25 per second). When the conditions were changed to pH 5-7 or 10-20 degrees C, some activity (6-55% or 5-20%, respectively, of that under optimal conditions) of the N-terminal polypeptide was still evident; however, almost all of the trypsin-treated enzyme activity disappeared. This implies the inhibition of the N-terminal enzyme domain by the associated C-terminal fragment. Long-term reactions indicated that a single molecule of pierisin-1 has the capacity to generate more than 10(6) ADP-ribosylated DNA adducts, which could cause the death of a mammalian cell.

The bacterium, Lysinibacillus sphaericus, as an insect pathogen

“…NAD + (K M = 45 lM; k cat = 0.04 s À1 ) (Schirmer et al, 2002a) show a somewhat greater affinity but a far lower turnover for this substrate under the experimental conditions than those reported for the related ADP-ribosyltransferase pierisin (K M = 170 lM; k cat = 55 s À1 ) (Watanabe et al, 2004).…”

How to Kill the Honey Bee Larva: Genomic Potential and Virulence Mechanisms of Paenibacillus larvae

“…KC456421; ERIC1_1c00040) is a single-chain AB toxin and belongs to an enigmatic family of toxins [31], so far comprising only few members, the larvicidal toxin MTX1 expressed by Lysinibacillus sphaericus and several pierisin-like toxins expressed by Pieridae , a large family of butterflies [32], [33]. It has been proposed that Plx1 has ADP ribosyltransferase activity [31] as already shown for MTX1 and pierisin-1 [32], [33]. Plx2 was shown to be a binary AB toxin with two separate ORFs encoding the A and B subunits [31].…”

Persistence of pierisin-1 activities in the adult cabbage white butterfly, Pieris rapae, during storage after killing

“…It was reported that the DNA ADP-ribosylating activity of purified pierisin-1 in solution at pH 9.0 decreases to around half for 42 h at 37 °C. 16) In the bodies of butterflies after killing, it is assumed that degradation of pierisin-1 might be suppressed by coexistence with other substances. We are now examining cytotoxicity and DNA ADP-ribosylating activity of crude extracts from various Pieridae butterflies obtained from many other countries.…”