Identification of novel hydroxysteroid-sulfating cytosolic SULTs, SULT2 ST2 and SULT2 ST3, from zebrafish: cloning, expression, characterization, and developmental expression.
By searching the expressed sequence tag database, two zebrafish cDNAs encoding putative cytosolic sulfotransferases (SULTs) were identified. Sequence analysis indicated that these two zebrafish SULTs belong to the cytosolic SULT2 gene family. The recombinant form of these two novel zebrafish SULTs, designated SULT2 ST2 and SULT2 ST3, were expressed using the pGEX-2TK glutathione S-transferase (GST) gene fusion system and purified from transformed BL21 (DE3) Escherichia coli cells. Purified GST-fusion protein form of SULT2 ST2 and SULT2 ST3 exhibited strong sulfating activities toward dehydroepiandrosterone (DHEA) and corticosterone, respectively, among various endogenous compounds tested as substrates. Both enzymes displayed pH optima at approximately 6.5. Kinetic constants of the two enzymes, as well as the GST-fusion protein form of the previously identified SULT2 ST1, with DHEA and corticosterone as substrates were determined. Developmental stage-dependent expression experiments revealed distinct patterns of expression of SULT2 ST2 and SULT2 ST3, as well as the previously identified SULT2 ST1, during embryonic development and throughout the larval stage onto maturity.
Yasuda
Shin
S
Biomedical Research Center, The University of Texas Health Center, Tyler, TX 75708, USA.
Liu
Ming-Yih
MY
Yang
Yuh-Shyong
YS
Snow
Rhodora
R
Takahashi
Saki
S
Liu
Ming-Cheh
MC
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003345:Corticosterone / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D003687:Dehydroepiandrosterone / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D006914:Hydroxysteroids / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013431:Sulfates / Q000378:metabolism*; D015238:Sulfotransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000254:growth & development; D029961:Zebrafish Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D018076:DNA, Complementary; D006914:Hydroxysteroids; D007527:Isoenzymes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013431:Sulfates; D029961:Zebrafish Proteins; D003687:Dehydroepiandrosterone; D015238:Sulfotransferases; C484038:sult2st1 protein, zebrafish; D003345:Corticosterone