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

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.In insects, ecdysteroids, which regulate development and reproduction, are synthesized in prothoracic glands. However, in many insect species, ecdysteroids have been demonstrated to be synthesized in ovaries as well, to accumulate in mature ovaries, and to be transferred to eggs (1-3). Most of these ecdysteroids are physiologically inactive phosphoric esters and synthesized by the enzyme, ATP:ecdysteroid-phosphotransferase (ecdysteroid kinase), which catalyzes phosphorylation of ecdysteroids ( Fig. 1) (4,5). It has been suggested that free ecdysteroids are liberated from maternal ecdysteroid-phosphates during embryonic development and participate in the secretion of serosal cuticle and/or the induction of embryonic cuticulogenesis (embryonic molt) in various insects, such as Locusta migratoria (6), Schistocerca greagaria (7), Blaberus craniifer (8), and Manduca sexta (9). Furthermore, the involvement of ecdysteroids in embryonic diapause has also been suggested in Lepidosphes ulmi (10), Bombyx mori (11), and L. migratoria (12).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). Furthermore, 20E was demonstrated to be formed by de novo synthesis (16 -18) and dephosphorylation of 20-hydroxyecdysone 22-phosphate (20E22P) and ecdysone 22-phosphate (E22P) (in the c...

Multidomain STS/TULA proteins are novel cellular regulators

“…Their phosphatase activity is directed at phosphosteroids and, thus, appears to be crucial for mobilizing active ecdysteroids from an inactive phosphorylated pool (19,20). The ecdysteroid-phosphate phosphatase of Bombyx mori contains little but the TULA-type phosphatase domain.…”

Purification, Kinetic Characterization, and Molecular Cloning of a Novel Enzyme, Ecdysteroid 22-Kinase

“…In B. mori (21,49), Lepidosaphes ulmi (50), and Locusta migratoria (51), 20-hydroxyecdysone plays an indispensable role in the developmental difference between diapause eggs and nondiapause eggs. In B. mori eggs, 20-hydroxyecdysone has been demonstrated to originate from both de novo biosynthesis (23,38,52,53) and dephosphorylation of ovary-derived ecdysteroid phosphates (27)(28)(29)53). Furthermore, in L. migratoria (54), S. gregaria (55), Blaberus craniifer (56), and Manduca sexta (14,15), free ecdysteroids liberated from ovary-derived ecdysteroid phosphates are suggested to participate in controlling the early events of embryonic development, such as embryonic cuticulogenesis and germ band elongation, which occur before the prothoracic glands differentiate during embryonic development.…”

“…The former specifically catalyzes the dephosphorylation of ecdysteroid 22-phosphate, whereas the latter contains nonspecific phosphatase activity. The procedures of each enzymatic hydrolysis have been described previously (27,28,37).…”

“…Enzymatic Hydrolysis of Ecdysteroid Phosphates-Ecdysteroid phosphates were hydrolyzed to free ecdysteroids by recombinant EPPase (27) or the H. pomatia digestive enzyme preparation (Nakarai Tesque). The former specifically catalyzes the dephosphorylation of ecdysteroid 22-phosphate, whereas the latter contains nonspecific phosphatase activity.…”

“…Recently, in B. mori eggs, a novel enzyme ecdysteroid-phosphate phosphatase (EPPase) 3 ( Fig. 1), which is specifically involved in dephosphorylation of ecdysteroid phos-phates, was isolated, characterized, and revealed to be a vital enzyme that may control the "on/off switch" of embryonic development (27)(28)(29).…”