Purification, Kinetic Characterization, and Molecular Cloning of a Novel Enzyme Ecdysteroid-phosphate Phosphatase

Ryouichi Yamada,Haruyuki Sonobe

doi:10.1074/jbc.m304158200

Abstract

From eggs of the silkworm Bombyx mori, we isolated a novel enzyme that is involved in the conversion of physiologically inactive conjugated ecdysteroids, such as ecdysone 22-phosphate and 20-hydroxyecdysone 22-phosphate, to active free ecdysteroids. This enzyme, called ecdysteroid-phosphate phosphatase (EPPase), was located in the cytosol fraction and differed from nonspecific lysosomal acid phosphatases in various enzymic properties. EPPase was purified about 3,000-fold to homogeneity by seven steps of column chromatography. The cDNA clone encoding EPPase was isolated by reverse transcription polymerase chain reaction using degenerate primers on the basis of the partial amino acid sequence obtained from purified EPPase and by subsequent 3'- and 5'-rapid amplification of cDNA ends. The full-length cDNA of EPPase was found to be composed of 1620 bp with an open reading frame encoding a protein of 331 amino acid residues. A data base search showed that there was no functional protein with the amino acid sequence identical to that of EPPase. Northern blot analysis revealed that EPPase mRNA was expressed predominantly during gastrulation and organogenesis in nondiapause eggs but was not detected in diapause eggs whose development was arrested at the late gastrula stage. In nondiapause eggs, the developmental changes in the expression pattern of EPPase mRNA corresponded closely to changes in the enzyme activity and in the amounts of free ecdysteroids in eggs.

Highlights

From eggs of the silkworm Bombyx mori, we isolated a novel enzyme that is involved in the conversion of physiologically inactive conjugated ecdysteroids, such as ecdysone 22-phosphate and 20-hydroxyecdysone 22-phosphate, to active free ecdysteroids
Northern blot analysis revealed that ecdysteroid-phosphate phosphatase (EPPase) mRNA was expressed predominantly during gastrulation and organogenesis in nondiapause eggs but was not detected in diapause eggs whose development was arrested at the late gastrula stage
In B. mori, recently, it has been demonstrated that the continuous supply of 20-hydroxyecdysone (20E),1 which has been demonstrated to be an active molecule in B. mori eggs [13], is required for embryonic development and that a deficiency of 20E induces embryonic diapause [13]; that is, the cessation of embryonic development at the late gastrula stage [14, 15]

Summary

EXPERIMENTAL PROCEDURES

Animals—Moths of the silkworm B. mori that had been destined to produce diapause eggs, the commercially available bivoltine race (kinsyuϫshowa), were used as the source of eggs. Embryos ceased development at the late gastrula stage (48 –72 h after oviposition) [14, 15]. (ii) To obtain female moths destined to produce nondiapause eggs, the subesophageal ganglion, the source of diapause hormone, was removed soon after larval-pupal ecdysis [15]. In both cases, the nondiapause eggs obtained continued to develop and larvae hatched 10 days after oviposition. For RNA extraction, nondiapause eggs laid by the moths with the subesophageal ganglion removed were used, because ommochromes, which disturb first strand cDNA synthesis, are not synthesized in the nondiapause eggs

The abbreviations used are

RESULTS

DISCUSSION