Characterization and ontogenic study of novel steroid-sulfating SULT3 sulfotransferases from zebrafish.
In vertebrates, sulfation as catalyzed by members of the cytosolic sulfotransferase (SULT) family has been suggested to be involved in the homeostasis of steroids. To establish the zebrafish as a model for investigating how sulfation functions to regulate steroid metabolism during the developmental process, we have embarked on the identification of steroid-sulfating SULTs in zebrafish. By searching the GenBank database, we identified two putative cytosolic SULT sequences from zebrafish, designated SULT3 ST1 and ST2. The recombinant proteins of these two zebrafish SULT3 STs were expressed in and purified from BL21 (DE3) cells transformed with the pGEX-2TK expression vector harboring SULT3 ST1 or ST2 cDNA. Upon enzymatic characterization, purified SULT3 ST1 displayed the strongest sulfating activity toward 17beta-estradiol among the endogenous substrates tested, while SULT3 ST2 exhibited substrate specificity toward hydroxysteroids, particularly dehydroepiandrosterone (DHEA). The pH-dependence and kinetic constants of these two enzymes with 17beta-estradiol and DHEA were determined. A developmental expression study revealed distinct patterns of the expression of SULT3 ST1 and ST2 during embryonic development and throughout the larval stage onto maturity. Collectively, these results imply that these two steroid-sulfating SULT3 STs may play differential roles in the metabolism and regulation of steroids during zebrafish development and in adulthood.
Yasuda
Tomoko
T
Department of Pharmacology, College of Pharmacy, The University of Toledo, Toledo, OH 43606, USA.
Yasuda
Shin
S
Williams
Frederick E
FE
Liu
Ming-Yih
MY
Sakakibara
Yoichi
Y
Bhuiyan
Shakhawat
S
Snow
Rhodora
R
Carter
Glendora
G
Liu
Ming-Cheh
MC
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D018507:Gene Expression Regulation, Developmental; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013256:Steroids / Q000378:metabolism*; D013379:Substrate Specificity; D013431:Sulfates / Q000378:metabolism*; D015238:Sulfotransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015262:Xenobiotics / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics*
D011994:Recombinant Proteins; D013256:Steroids; D013431:Sulfates; D015262:Xenobiotics; D015238:Sulfotransferases