Michael W.H. Coughtrie

A proposed nomenclature system for the cytosolic sulfotransferase (SULT) superfamily.

A nomenclature system for the cytosolic sulfotransferase (SULT) superfamily has been developed. The nomenclature guidelines were applied to 65 SULT cDNAs and 18 SULT genes that were characterized from eukaryotic organisms. SULT cDNA and gene sequences were identified by querying the GenBank databases and from published reports of their identification and characterization. These sequences were evaluated and named on the basis of encoded amino acid sequence identity and, in a few cases, a necessity to maintain historical naming convention. Family members share at least 45% amino acid sequence identity whereas subfamily members are at least 60% identical. cDNAs which encode amino acid sequences of at least 97% identity to each other were assigned identical isoform names. We also attempted to categorize orthologous enzymes between various species, where these have been identified, and the nomenclature includes a species descriptor. We present recommendations for the naming of allelic variants of SULT genes and their derived allozymes arising from single nucleotide polymorphisms and other genetic variation. The superfamily currently comprises 47 mammalian SULT isoforms, one insect isoform and eight plant enzymes, and collectively these sequences represent nine separate SULT families and 14 subfamilies. It is hoped that this nomenclature system will be widely adopted and that, as novel SULTs are identified and characterized, investigators will name their discoveries according to these guidelines.

Quantitative Evaluation of the Expression and Activity of Five Major Sulfotransferases (SULTs) in Human Tissues: The SULT Pie

Expression levels of the major human sulfotransferases (SULTs) involved in xenobiotic detoxification in a range of human tissues (i.e., SULT “pies”) are not available in a form allowing comparison between tissues and individuals. Here we have determined, by quantitative immunoblotting, expression levels for the five principal human SULTs—SULT1A1, SULT1A3/4, SULT1B1, SULT1E1, and SULT2A1—and determined the kinetic properties toward probe substrates, where available, for these enzymes in cytosol samples from a bank of adult human liver, small intestine, kidney, and lung. We produced new isoform-selective antibodies against SULT1B1 and SULT2A1, which were used alongside antibodies against SULT1A3 and SULT1A1 previously produced in our laboratory or available commercially (SULT1E1). Expression levels were derived using purified recombinant enzymes to construct standard curves for each individual isoform and immunoblot. Substantial intertissue and interindividual differences in expression were observed. SULT1A1 was the major enzyme (>50% of total, range 420-4900 ng/mg cytosol protein) in the liver, followed by SULT2A1, SULT1B1, and SULT1E1. SULT1A3 was completely absent from this tissue. In contrast, the small intestine contained the largest overall amount of SULT of any of the tissues, with SULT1B1 the major enzyme (36%), closely followed by SULT1A3 (31%), and SULT1A1, SULT1E1, and SULT2A1 more minor forms (19, 8, and 6% of total, respectively). The kidney and lung contained low levels of SULT. We provide a unique data set that will add value to the study of the role and contribution of sulfation to drug and xenobiotic metabolism in humans.

Biology and function of the reversible sulfation pathway catalysed by human sulfotransferases and sulfatases

Sulfation and sulfate conjugate hydrolysis play an important role in metabolism, and are catalysed by members of the sulfotransferase and sulfatase enzyme super-families. In general, sulfation is a deactivating, detoxication pathway, but for some chemicals the sulfate conjugates are much more reactive than the parent compound. The range of compounds which are sulfated is enormous, yet we still understand relatively little of the function of this pathway. This review summarises current knowledge of the sulfation system and the enzymes involved, and illustrates how heterologous expression of sulfotransferases (SULTs) and sulfatases is aiding our appreciation of the properties of these important proteins. The role of sulfation in the bioactivation of procarcinogens and promutagens is discussed, and new data on the inhibition of the sulfotransferase(s) involved by common dietary components such as tea and coffee are presented. The genetic and environmental factors which are known to influence the activity and expression of human SULTs and sulfatases are also reviewed.

Sulfation through the looking glass--recent advances in sulfotransferase research for the curious.

Members of the cytosolic sulfotransferase (SULT) superfamily catalyse the sulfation of a multitude of xenobiotics, hormones and neurotransmitters. Humans have at least 10 functional SULT genes, and a number of recent advances reviewed here have furthered our understanding of SULT function. Analysis of expression patterns has shown that sulfotransferases are highly expressed in the fetus, and SULTs may in fact be a major detoxification enzyme system in the developing human. The X-ray crystal structures of three SULTs have been solved and combined with mutagenesis experiments and molecular modelling, they have provided the first clues as to the factors that govern the unique substrate specificities of some of these enzymes. In the future these and other studies will facilitate prediction of the fate of chemicals metabolised by sulfation. Variation in sulfation capacity may be important in determining an individuals response to xenobiotics, and there has been an explosion in information on sulfotransferase polymorphisms and their functional consequences, including the influence of SULT1A1 genotype on susceptibility to colorectal and breast cancer. Finally, the first gene knockout experiments with SULTs have recently been described, with the generation of estrogen sulfotransferase deficient mice in which reproductive capacity is compromised. Our improved understanding of these enzymes will have significant benefits in such diverse areas as drug design and development, cancer susceptibility, reproduction and development.

Potent Inhibition of Estrogen Sulfotransferase by Hydroxylated Metabolites of Polyhalogenated Aromatic Hydrocarbons Reveals Alternative Mechanism for Estrogenic Activity of Endocrine Disrupters

Polyhalogenated aromatic hydrocarbons (PHAHs), such as polychlorinated dibenzo-p-dioxins and dibenzofurans, polybrominated diphenylethers, and bisphenol A derivatives are persistent environmental pollutants, which are capable of interfering with reproductive and endocrine function in birds, fish, reptiles, and mammals. PHAHs exert estrogenic effects that may be mediated in part by their hydroxylated metabolites (PHAH-OHs), the mechanisms of which remain to be identified. PHAH-OHs show low affinity for the ER. Alternatively, they may exert their estrogenic effects by inhibiting E2 metabolism. As sulfation of E2 by estrogen sulfotransferase (SULT1E1) is an important pathway for E2 inactivation, inhibition of SULT1E1 may lead to an increased bioavailability of estrogens in tissues expressing this enzyme. Therefore, we studied the possible inhibition of human SULT1E1 by hydroxylated PHAH metabolites and the sulfation of the different compounds by SULT1E1. We found marked inhibition of SULT1E1 by various PHAH-OHs, in particular by compounds with two adjacent halogen substituents around the hydroxyl group that were effective at (sub)nanomolar concentrations. Depending on the structure, the inhibition is primarily competitive or noncompetitive. Most PHAH-OHs are also sulfated by SULT1E1. We also investigated the inhibitory effects of the various PHAH-OHs on E2 sulfation by human liver cytosol and found that the effects were strongly correlated with their inhibitions of recombinant SULT1E1 (r = 0.922). Based on these results, we hypothesize that hydroxylated PHAHs exert their estrogenic effects at least in part by inhibiting SULT1E1-catalyzed E2 sulfation.

X-ray Crystal Structure of Human Dopamine Sulfotransferase, SULT1A3 MOLECULAR MODELING AND QUANTITATIVE STRUCTURE-ACTIVITY RELATIONSHIP ANALYSIS DEMONSTRATE A MOLECULAR BASIS FOR SULFOTRANSFERASE SUBSTRATE SPECIFICITY

Humans are one of the few species that produce large amounts of catecholamine sulfates, and they have evolved a specific sulfotransferase, SULT1A3 (M-PST), to catalyze the formation of these conjugates. An orthologous protein has yet to be found in other species. To further our understanding of the molecular basis for the unique substrate selectivity of this enzyme, we have solved the crystal structure of human SULT1A3, complexed with 3′-phosphoadenosine 5′-phosphate (PAP), at 2.5 Å resolution and carried out quantitative structure-activity relationship (QSAR) analysis with a series of phenols and catechols. SULT1A3 adopts a similar fold to mouse estrogen sulfotransferase, with a central five-stranded β-sheet surrounded by α-helices. SULT1A3 is a dimer in solution but crystallized with a monomer in the asymmetric unit of the cell, although dimer interfaces were formed by interaction across crystallographic 2-fold axes. QSAR analysis revealed that the enzyme is highly selective for catechols, and catecholamines in particular, and that hydrogen bonding groups and lipophilicity (cLogD) strongly influenced K m . We also investigated further the role of Glu146 in SULT1A3 using site-directed mutagenesis and showed that it plays a key role not only in defining selectivity for dopamine but also in preventing many phenolic xenobiotics from binding to the enzyme.

Sulfotransferases: genetics and role in toxicology

The mammalian xenobiotic-metabolizing sulfotransferases are cytosolic enzymes, which form a gene superfamily (SULT). Ten distinct human SULT forms are known. Two SULT forms represent splice variants, the other forms are encoded by separate genes. Common functional polymorphisms of the transcribed region are known for two of the forms. We have expressed 16 separate rat and human SULTs as well as some of their allelic variants, in Salmonella typhimurium TA1538 and/or V79 cells, which are target cells of commonly used mutagenicity assays. The expressed SULTs activated numerous compounds to mutagens in both assay systems. However, some promutagens were activated by only one or several of the human SULTs. Pronounced differences in promutagen activation were also detected between orthologous rat and human SULTs, and between allelic variants of human SULTs.

ESTRADIOL FORMATION BY HUMAN OSTEOBLASTS VIA MULTIPLE PATHWAYS : RELATION WITH OSTEOBLAST FUNCTION

The importance of estrogens in bone metabolism is illustrated by the accelerated bone loss and increase in osteoporotic fractures associated with postmenopausal estrogen deficiency. In this study, the expression and activity of the enzymes involved in estrogen metabolism in human osteoblastic cells were investigated in relation to differentiation of these cells. PCR reactions using mRNA from an in vitro differentiating human cell line (SV‐HFO) were performed to assess mRNA expression of the enzymes aromatase, different subtypes of 17β‐hydroxysteroid dehydrogenase (17β‐HSD), and steroid sulfatase. Aromatase, sulfatase, and 17β‐HSD type 2 and 4 were found to be expressed throughout differentiation. Expression of 17β‐HSD type 3, however, was relatively weak, except for early time points in differentiation. Type 1 17β‐HSD expression was not detected. Aromatase activity decreased during differentiation, as was demonstrated by the conversion of androstenedione (A) and testosterone (T) into estrone (E1) and estradiol (E2), respectively. The 17β‐HSD isozymes catalysing a reductive reaction convert androstenedione and estrone into testosterone and estradiol, respectively. Their activity declined with differentiation. Analysis of 17β‐HSD activity indicated both oxidative (E2 to E1; T to A) and reductive (E1 to E2; A to T) metabolism at all stages of osteoblast differentiation. Both activities declined as cells moved toward a differentiating mineralizing phenotype. However, the oxidative reaction was increasingly in favor of the reductive reaction at all times during differentiation. Sulfatase activity, as demonstrated by the conversion of estrone‐sulfate into estrone, was constant during differentiation. In conclusion, we have demonstrated that all enzymes necessary for estrogen metabolism are expressed and biologically active in differentiating human osteoblasts. The activity of aromatase and 17β‐HSD was found to be dependent on the stage of cell differentiation. In addition, human osteoblasts effectively convert estradiol into estrone. The efficacy of osteoblasts to synthesize estradiol may determine the ultimate change in rate of bone turnover after menopause, as well as the development of osteoporosis. Moreover, the enzymes involved in the metabolism of estradiol may form a target for intervention. J. Cell. Biochem. 75:528–537, 1999.

Expression profiling of human fetal cytosolic sulfotransferases involved in steroid and thyroid hormone metabolism and in detoxification

Protection against chemical insult is essential for normal development of the fetus, however many detoxification enzymes are poorly expressed during fetal development. A major exception is the sulfotransferase (SULT) family, which appears to be widely expressed in the developing human. These enzymes also play a key role in biosynthesis and homeostasis of a number of hormones, including estrogens and iodothyronines. We therefore examined the enzyme activity, protein and mRNA expression of SULT 1A, 1B, 1C, 1E and 2A families in a variety of human fetal and adult tissues. Our results show that these SULTs are expressed in the human fetus, with most present at levels equivalent to or higher than the adult. As there are no isoform-selective substrates for SULTs 1B1 and 1C2 we used immunoblot analysis to show for the first time expression of SULT1B1 at high levels in fetal small intestine, and expression of SULT1C2 in fetal liver, kidney and small intestine. SULT1C2 was not expressed in adult liver or colon. Sulfotransferase expression in the developing fetus is therefore more widespread than in the adult, and this has significant implication for our understanding of human developmental physiology.

Absolute immunoquantification of the expression of ABC transporters P-glycoprotein, breast cancer resistance protein and multidrug resistance-associated protein 2 in human liver and duodenum.

The ATP-binding cassette (ABC) transporters breast cancer resistance protein (BCRP), multidrug resistance-associated protein 2 (MRP2), and P-glycoprotein (Pgp) are important in the distribution and elimination of many drugs and endogenous metabolites. Due to their membrane location and hydrophobicity it is difficult to generate purified protein standards to quantify these transporters in human tissues. The present study generated transporter proteins fused with the S-peptide of ribonuclease for use as standards in immunoquantification in human liver and small intestine. Quantification of the S•tag™, a 15 amino acid peptide, is based on the formation of a functional ribonuclease activity upon its high affinity reconstitution with ribonuclease S-protein. S-tagged transporters were used as full-length protein standards in the immunoquantification of endogenous BCRP, MRP2, and Pgp levels in 14 duodenum and 13 liver human tissue samples. Expression levels in the duodenum were 305±248 (BCRP), 66±70 (MRP2), and 275±205 (Pgp) fmoles per cm(2). Hepatic levels were 2.6±0.9 (BCRP), 19.8±10.5 (MRP2), and 26.1±10.1 (total Pgp) pmoles per g of liver. The mean hepatic scaling factor was 35.8mg crude membrane per g of liver, and the mean duodenal scaling factor was 1.3mg crude membrane per cm(2) mucosal lining. Interindividual variability was greater in duodenal samples than liver samples. It is hoped that this innovative method of quantifying these transporters (and other membrane proteins) will improve in vivo-in vitro extrapolation and in silico prediction of drug absorption and elimination, thus supporting drug development.