Zebrafish tyrosylprotein sulfotransferase: molecular cloning, expression, and functional characterization.
By employing the reverse transcriptase-polymerase chain reaction technique in conjunction with 3' rapid amplification of cDNA ends, a full-length cDNA encoding a zebrafish (Danio rerio) tyrosylprotein sulfotransferase (TPST) was cloned and sequenced. Sequence analysis revealed that this zebrafish TPST is, at the amino acid sequence level, 66% and 60% identical to the human and mouse TPST-1 and TPST-2, respectively. The recombinant form of the zebrafish TPST, expressed in COS-7 cells, exhibited a pH optimum at 5.75. Manganese appeared to exert a stimulatory effect on the zebrafish TPST. The activity of the enzyme determined in the presence of 20 mM MnCl2 was more than 2.5 times that determined in the absence of MnCl2. Of the other nine divalent metal cations tested at a 10 mM concentration, Co2+ also showed a considerable stimulatory effect, while Ca2+, Pb2+, and Cd2+ exerted some inhibitory effects. The other four divalent cations, Fe2+, Cu2+, Zn2+, and Hg2+, inhibited completely the sulfating activity of the zebrafish TPST. Using the wild-type and mutated P-selectin glycoprotein ligand-1 N-terminal peptides as substrates, the zebrafish TPST was shown to exhibit a high degree of substrate specificity for the tyrosine residue on the C-terminal side of the peptide. These results constitute a first study on the cloning, expression, and characterization of a zebrafish cytosolic TPST.
Mishiro
Emi
E
Biomedical Research Center, University of Texas Health Center, Tyler, 75708, USA.
Liu
Ming-Yih
MY
Sakakibara
Yoichi
Y
Suiko
Masahito
M
Liu
Ming-Cheh
MC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002413:Cations, Divalent / Q000494:pharmacology; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006863:Hydrogen-Ion Concentration; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013696:Temperature; D015027:Zebrafish / Q000235:genetics / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D002413:Cations, Divalent; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; C084882:P-selectin ligand protein; D029961:Zebrafish Proteins; D015238:Sulfotransferases; C493189:tyrosylprotein sulfotransferase, zebrafish