Sulfation of hydroxychlorobiphenyls. Molecular cloning, expression, and functional characterization of zebrafish SULT1 sulfotransferases.

As a first step toward developing a zebrafish model for investigating the role of sulfation in counteracting environmental estrogenic chemicals, we have embarked on the identification and characterization of cytosolic sulfotransferases (STs) in zebrafish. By searching the zebrafish expressed sequence tag database, we have identified two cDNA clones encoding putative cytosolic STs. These two zebrafish ST cDNAs were isolated and subjected to nucleotide sequencing. Sequence data revealed that the two zebrafish STs are highly homologous, being approximately 82% identical in their amino acid sequences. Both of them display approximately 50% amino acid sequence identity to human SULT1A1, rat SULT1A1, and mouse SULT1C1 ST. These two zebrafish STs therefore appear to belong to the SULT1 cytosolic ST gene family. Recombinant zebrafish STs (designated SULT1 STs 1 and 2), expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as approximately 35 kDa proteins on SDS/PAGE. Purified zebrafish SULT1 STs 1 and 2 displayed differential sulfating activities toward a number of endogenous compounds and xenobiotics including hydroxychlorobiphenyls. Kinetic constants of the two enzymes toward two representative hydroxychlorobiphenyls, 3-chloro-4-biphenylol and 3,3',5,5'-tetrachloro-4,4'-biphenyldiol, and 3,3',5-triiodo-l-thyronine were determined. A thermostability experiment revealed the two enzymes to be relatively stable over the range 20-43 degrees C. Among 10 different divalent metal cations tested, Co2+, Zn2+, Cd2+, and Pb2+ exhibited considerable inhibitory effects, while Hg2+ and Cu2+ rendered both enzymes virtually inactive.     

Identification of a novel estrogen-sulfating cytosolic SULT from zebrafish: molecular cloning, expression, characterization, and ontogeny study

By searching the expressed sequence tag database, a zebrafish cDNA encoding a putative cytosolic sulfotransferase (SULT) was identified. Sequence analysis indicated that this zebrafish SULT belongs to the SULT1 cytosolic SULT gene family. The recombinant form of this novel zebrafish SULT, expressed using the pGEX-2TK expression system and purified from transformed BL21 (DE3) Escherichia coli cells, displayed sulfating activities specifically for estrone and 17beta-estradiol among various endogenous compounds tested as substrates. The enzyme also exhibited sulfating activities toward some xenobiotic phenolic compounds. This new zebrafish SULT showed dual pH optima, at 6.5 and 10-10.5, with estrone or n-propyl gallate as substrate. Kinetic constants of the sulfation of estrone, 17beta-estradiol, and n-propyl gallate were determined. Developmental stage-dependent expression experiments revealed a significant level of expression of this novel zebrafish estrogen-sulfating SULT at the beginning of the hatching period during embryogenesis, which continued throughout the larval stage onto maturity.     

Identification of a novel thyroid hormone-sulfating cytosolic sulfotransferase, SULT1 ST5, from zebrafish

By employing RT-PCR in conjunction with 3'-RACE, a full-length cDNA encoding a novel zebrafish cytosolic sulfotransferase (SULT) was cloned and sequenced. Sequence analysis revealed that this zebrafish SULT (designated SULT1 ST5) is, at the amino acid sequence level, close to 50% identical to human and dog SULT1B1 (thyroid hormone SULT). A recombinant form of zebrafish SULT1 ST5 was expressed using the pGEX-2TK bacterial expression system and purified from transformed BL21 (DE3) cells. Purified zebrafish SULT1 ST5 migrated as a 34 kDa protein and displayed substrate specificity for thyroid hormones and their metabolites among various endogenous compounds tested. The enzyme also exhibited sulfating activities toward some xenobiotic phenolic compounds. Its pH optima were 6.0 and 9.0 with 3,3',5-triiodo-l-thyronine (l-T3) as substrate and 6.0 with beta-naphthol as substrate. Kinetic constants of the enzyme with thyroid hormones and their metabolites as substrates were determined. Quantitative evaluation of the regulatory effects of divalent metal cations on the l-T3-sulfating activity of SULT1 ST5 revealed that Fe2+, Hg2+, Co2+, Zn2+, Cu2+, Cd2+ and Pb2+ exhibited dramatic inhibitory effects, whereas Mn2+ showed a significant stimulation. Developmental stage-dependent expression experiments revealed a significant level of expression of this novel zebrafish thyroid hormone-sulfating SULT at the beginning of the hatching period during embryogenesis, which gradually increased to a high level of expression throughout the larval stage into maturity.     

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.     

Molecular cloning,expression, and functional characterization of a novel zebrafish cytosolic sulfotransferase

By searching the zebrafish expressed sequence tag (EST) database, we have identified a cDNA clone encoding a putative zebrafish cytosolic sulfotransferase (ST). This cDNA was isolated and subjected to nucleotide sequencing. Analysis of the sequence data revealed that this novel zebrafish ST displays 32-35% amino acid sequence identity to members of all major cytosolic ST gene families. Therefore, this zebrafish ST, while belonging to the cytosolic ST gene superfamily, appears to be independent from all known constituent ST gene families. Recombinant zebrafish ST, expressed using the pET23c prokaryotic expression vector and purified from transformed Escherichia coli cells, migrated as a 34-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified zebrafish ST displayed sulfating activities toward dopamine and thyroid hormones (T(3) and T(4)), with a pH optimum spanning 7-9. The enzyme also exhibited activities toward a number of xenobiotics including some flavonoids, isoflavonoids, and other phenolic compounds. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 and 48 degrees C. Among 10 divalent metal cations tested, Fe(++), Hg(++), Co(++), Zn(++), Cu(++), and Cd(++) exhibited dramatic inhibitory effects on the activity of the enzyme. These results constitute a first study on the cloning, expression, and characterization of a zebrafish cytosolic ST.     

Enzymatic properties, tissue-specific expression, and lysosomal location of two highly homologous rat SULT1C2 sulfotransferases

We have isolated two highly homologous but distinct rat sulfotransferase cDNAs termed ratSULT1C2 and ratSULT1C2A encoding polypeptides of 297 amino acids each. The amino acid sequence of ratSULT1C2 is 84% identical to the human SULT1C2 and 81% identical to a rabbit SULT1C2 sulfotransferase. ratSULT1C2 and ratSULT1C2A are 92% identical but differ in 22 amino acids. The majority of these amino acid substitutions in ratSULT1C2A is not found in the human and rabbit SULT1C2, which identifies ratSULT1C2 as the orthologue of these sulfotransferases, whereas SULT1C2A is a closely related but distinct enzyme. ratSULT1C2 and 2A sulfotransferases do not sulfonate steroids, dopamine, acetaminophen, or alpha-naphthol, but only p-nitrophenol. Prokaryotically expressed ratSULT1C2A is less active than ratSULT1C2. ratSULT1C2/2A mRNAs are abundant in kidney and less abundant in stomach and liver. The enzymes are expressed as 34-kDa polypeptides in rat kidney, liver, and stomach. In addition, a 28-kDa cross-reacting polypeptide is found in kidney only. Immunohistochemistry revealed expression of ratSULT1C2/2A in the epithelial cells of the proximal tubules of the kidney, bile duct epithelia, hepatocytes, and the epithelium of the gastric mucosal glands. Although the cDNA predicted amino acid sequence identifies both sulfotransferases as cytosolic enzymes, in tissue sections, in the kidney cell line NRK 52, and in transiently transfected BHK cells a considerable fraction of the enzyme was found in a granular perinuclear compartment. Costaining with a lysosomal marker in gastric mucosa tissue sections and cultured cells identifies these structures as lysosomes.     

Identification and expression of a novel class of glutathione-S-transferase from amphioxus Branchiostoma belcheri with implications to the origin of vertebrate liver

Glutathione-S-transferases have been identified in all the living species examined so far, yet little is known to date about them in amphioxus, a model organism for insights into the origin and evolution of vertebrates. We have isolated a cDNA encoding an amphioxus (Branchiostoma belcheri) glutathione-S-transferase with a predicted molecular mass of approximately 26 kDa, from the gut cDNA library. The glutathione-S-transferase had 43.7-51.8% identity to most glutathione-S-transferases identified from aquatic organisms including fish and green alga, but it was much less identical (<27%) to other cytosolic glutathione-S-transferase classes. The phylogenetic analysis revealed that the glutathione-S-transferase was grouped together with most piscine and algal glutathione-S-transferases, separating from other cytosolic glutathione-S-transferase classes. Moreover, the glutathione-S-transferase had an exon-intron organization typical of zebrafish putative GST, red sea bream GSTR1 and plaice GSTA1 genes. The recombinant glutathione-S-transferase has been successfully expressed and purified, which showed a relatively high catalytic activity (3.37+/-0.1 unit/mg) toward 1-chloro-2, 4-dinitrobenzene and a moderate activity toward ethacrynic acid (0.41+/-0.01 unit/mg), although it had no detectable activity toward 1, 2-dichloro-4-nitrobenzene, 4-hydroxynonenal, 4-nitrobenzyl chloride and cumene hydroperoxide. In addition, we have revealed a tissue-specific expression pattern of the glutathione-S-transferase gene in B. belcheri, with the most abundant expression in the hepatic caecum. All these indicate that the amphioxus glutathione-S-transferase belongs to a novel rho-class of glutathione-S-transferases with a tissue-specific expression pattern. The relation between the glutathione-S-transferase expression in amphioxus hepatic caecum and the origin of vertebrate liver is also discussed.     

Purification and cDNA cloning of chloroplastic monodehydroascorbate reductase from spinach

The chloroplastic isoform of monodehydroascorbate (MDA) radical reductase was purified from spinach chloroplasts and leaves. The cDNA of chloroplastic MDA reductase was cloned, and its deduced amino acid sequence, consisting of 497 residues, showed high homology with those of putative organellar MDA reductases deduced from cDNAs of several plants. The amino acid sequence of the amino terminal of the purified enzyme suggested that the chloroplastic enzyme has a transit peptide consisting of 53 residues. A southern blot analysis suggested the occurrence of a gene encoding another isoform homologous to the chloroplastic isoform in spinach. The recombinant enzyme was highly expressed in Eschericia coli using the cDNA, and purified to a homogeneous state with high specific activity. The enzyme properties of the chloroplastic isoform are presented in comparison with those of the cytosolic form.     

Differential xenoestrogen-sulfating activities of the human cytosolic sulfotransferases: molecular cloning,expression, and purification of human SULT2B1a and SULT2B1b sulfotransferases

Environmental xenoestrogens have been implicated in human reproductive disorders and an increased incidence of breast cancer. Sulfation, a Phase II detoxification mechanism involving the cytosolic sulfotransferases (STs), may be an important mechanism in vivo for fending off these compounds. In this study, we report on the molecular cloning, expression, and purification of two human cytosolic STs, SULT2B1a and SULT2b1b. The activities of these two enzymes, as well as the other eight known human cytosolic STs previously prepared, toward representative environmental xenoestrogens were examined. Activity data showed that P-form (SULT1A1) PST displayed the highest activity toward these compounds, while SULT1C ST #2 also showed considerable activity, indicating that these enzymes may play a more important role in detoxification of environmental xenoestrogens. SULT1C ST #1, SULT2B1a ST, SULT2B1b ST and NST showed negligible or undetectable activity toward these compounds. The other four enzymes, M-form (SULT1A3) PST, SULT1B2 ST, SULT2A1 ST and SULT1E ST showed intermediate levels of activity toward some of these compounds. Kinetic studies on the sulfation of xenoestrogens by P-form (SULT1A1) PST were performed. The results are interpreted in the context of the endocrine-disrupting nature of these xenoestrogens.     

Differential xenoestrogen-sulfating activities of the human cytosolic sulfotransferases: molecular cloning,expression, and purification of human SULT2B1a and SULT2B1b sulfotransferases

Environmental xenoestrogens have been implicated in human reproductive disorders and an increased incidence of breast cancer. Sulfation, a Phase II detoxification mechanism involving the cytosolic sulfotransferases (STs), may be an important mechanism in vivo for fending off these compounds. In this study, we report on the molecular cloning, expression, and purification of two human cytosolic STs, SULT2B1a and SULT2b1b. The activities of these two enzymes, as well as the other eight known human cytosolic STs previously prepared, toward representative environmental xenoestrogens were examined. Activity data showed that P-form (SULT1A1) PST displayed the highest activity toward these compounds, while SULT1C ST #2 also showed considerable activity, indicating that these enzymes may play a more important role in detoxification of environmental xenoestrogens. SULT1C ST #1, SULT2B1a ST, SULT2B1b ST and NST showed negligible or undetectable activity toward these compounds. The other four enzymes, M-form (SULT1A3) PST, SULT1B2 ST, SULT2A1 ST and SULT1E ST showed intermediate levels of activity toward some of these compounds. Kinetic studies on the sulfation of xenoestrogens by P-form (SULT1A1) PST were performed. The results are interpreted in the context of the endocrine-disrupting nature of these xenoestrogens.     

Isolation and characterisation of a novel rabbit sulfotransferase isoform belonging to the SULT1A subfamily

Sulfotransferases (SULTs) catalyse the sulfonation of both endogenous and exogenous compounds including hormones, catecholamines, drugs and xenobiotics. While in most occasions, sulfonation is a detoxication pathway, in the case of certain drugs and carcinogens, it leads to metabolic activation. Since, the rabbit has been extensively used for both pharmacological and toxicological studies, the purpose of this study was to further characterise the sulfotransferase system of this animal. In the present study, a novel sulfotransferase isoform (GenBank Accession no. AF360872) was isolated from a rabbit liver cDNA lambdaZAP II library. The full-length sequence of the clone was 1138 bp long and contained a coding region of 888 bp encoding a cytosolic protein of 295 amino acids (deduced molecular weight 34,193 Da). The amino acid sequence of this novel SULT isoform showed >70% identity with members of the SULT1A subfamily of sulfotransferases from other species. Upon expression of the encoded rabbit sulfotransferase in Escherchia coli (E. coli), it was shown that the enzyme was capable of sulfonating both p-nitrophenol (K(m) and Vmax values of 0.15 microM and 897.5 nmol/min/mg protein, respectively) and dopamine (K(m) and V(max) values of 175.3 microM and 151.1 nmol/min/mg protein, respectively). Based on the sequence data obtained and substrate specificity, this new rabbit sulfotransferase was named rabSULT1A1. Immunoblotting was used to demonstrate that rabSULT1A1 protein is expressed in liver, duodenum, jejunum, ileum, colon and rectum.     

cDNA cloning and expression of a bovine phenol UDP-glucuronosyltransferase, BovUGT1A6

A full-length cDNA encoding a phenol UDP-glucuronosyltransferase was isolated by plaque hybridization, RT-PCR and 5'-RACE from a cDNA library prepared from the bovine liver. The deduced amino acid sequence (529 amino acid residues) has A signal sequence (23 amino acid residues) at the amino terminus and a transmembrane-anchoring domain (17 amino acid residues) at the carboxyl terminus. The encoded protein has a potential asparagine-linked glycosylation site (Asn291). The cloned cDNA was named bovUGT1A6 on the basis of the amino acid similarity. BovUGT1A6 cloned in the pAAH5 expression vector was transformed into Saccharomyces cerevisiea AH22 cells to obtain an active 54-kDa bovUGT1A6 enzyme. The expressed enzyme represented UDP-glucuronosyltransferase activities toward 1-naphthol and 4-methylumbelliferone, confirming that the isolated cDNA is an isoform of bovine phenol UDP-glucuronosyltransferase. Microsomal UDP-glucuronosyltransferase activity toward 1-naphthol in the bovine kidney cortex was found to be higher than that in the liver and other organs, and mRNA of bovUGT1A6 was more strongly detected in the kidney on Northern blotting analysis. These results suggest that the bovine kidney, which strongly expresses bovUGT1A6, is a significant organ for xenobiotics glucuronidation.     

Identification and characterization of the zebrafish glutathione S‐transferase Pi‐1

Zebrafish has in recent years emerged as a popular vertebrate model for use in pharmacological and toxicological studies. While there have been sporadic studies on the zebrafish glutathione S‐transferases (GSTs), the zebrafish GST gene superfamily still awaits to be fully elucidated. We report here the identification of 15 zebrafish cytosolic GST genes in NCBI GenBank database and the expression, purification, and enzymatic characterization of the zebrafish cytosolic GST Pi‐1 (GSTP1). The cDNA encoding the zebrafish GSTP1 was cloned from a 3‐month‐old female zebrafish, expressed in Eschelichia coli host cells, and purified. Purified GSTP1 displayed glutathione‐conjugating activity toward 1‐chloro‐2,4‐dinitrobenzene as a representative substrate. The enzymatic characteristics of the zebrafish GSTP1, including pH‐dependency, effects of metal cations, and kinetic parameters, were studied. Moreover, the expression of zebrafish GSTP1 at different developmental stages during embryogenesis, throughout larval development, onto maturity was examined.     

Sequence analysis and functional study of thymidylate synthase from zebrafish, Danio rerio

The thymidylate synthase (TS), an important target for many anticancer drugs, has been cloned from different species. But the cDNA property and function of TS in zebrafish are not well documented. In order to use zebrafish as an animal model for screening novel anticancer agents, we isolated TS cDNA from zebrafish and compared its sequence with those from other species. The open reading frame (ORF) of zebrafish TS cDNA sequence was 954 nucleotides, encoding a 318-amino acid protein with a calculated molecular mass of 36.15 kDa. The deduced amino acid sequence of zebrafish TS was similar to those from other organisms, including rat, mouse and humans. The zebrafish TS protein was expressed in Escherichia coli and purified to homogeneity. The purified zebrafish TS showed maximal activity at 28 degrees C with similar K(m) value to human TS. Western immunoblot assay confirmed that TS was expressed in all the developmental stages of zebrafish with a high level of expression at the 1-4 cell stages. To study the function of TS in zebrafish embryo development, a short hairpin RNA (shRNA) expression vector, pSilencer 4.1-CMV/TS, was constructed which targeted the protein-coding region of zebrafish TS mRNA. Significant change in the development of tail and epiboly was found in zebrafish embryos microinjected pSilencer4.1-CMV/TS siRNA expression vector.