A sulfotransferase dosage-dependently regulates mouthpart polyphenism in the nematode Pristionchus pacificus.

Polyphenism, the extreme form of developmental plasticity, is the ability of a genotype to produce discrete morphologies matched to alternative environments. Because polyphenism is likely to be under switch-like molecular control, a comparative genetic approach could reveal the molecular targets of plasticity evolution. Here we report that the lineage-specific sulfotransferase SEUD-1, which responds to environmental cues, dosage-dependently regulates polyphenism of mouthparts in the nematode Pristionchus pacificus. SEUD-1 is expressed in cells producing dimorphic morphologies, thereby integrating an intercellular signalling mechanism at its ultimate target. Additionally, multiple alterations of seud-1 support it as a potential target for plasticity evolution. First, a recent duplication of seud-1 in a sister species reveals a direct correlation between genomic dosage and polyphenism threshold. Second, inbreeding to produce divergent polyphenism thresholds resulted in changes in transcriptional dosage of seud-1. Our study thus offers a genetic explanation for how plastic responses evolve.

Genetic and molecular basis of drug resistance and species-specific drug action in schistosome parasites.

Oxamniquine resistance evolved in the human blood fluke (Schistosoma mansoni) in Brazil in the 1970s. We crossed parental parasites differing ~500-fold in drug response, determined drug sensitivity and marker segregation in clonally derived second-generation progeny, and identified a single quantitative trait locus (logarithm of odds = 31) on chromosome 6. A sulfotransferase was identified as the causative gene by using RNA interference knockdown and biochemical complementation assays, and we subsequently demonstrated independent origins of loss-of-function mutations in field-derived and laboratory-selected resistant parasites. These results demonstrate the utility of linkage mapping in a human helminth parasite, while crystallographic analyses of protein-drug interactions illuminate the mode of drug action and provide a framework for rational design of oxamniquine derivatives that kill both S. mansoni and S. haematobium, the two species responsible for >99% of schistosomiasis cases worldwide.

Identification and characterization of a novel kaempferol sulfotransferase from Arabidopsis thaliana.

In plants, flavonoids have been shown to be subjected to conjugation modifications such as glycosylation, methylation, and sulfation. Among these modifications, sulfation is known as an important pathway in the regulation of the levels of endogenous compounds such as steroids. Although a large variety of flavonoid sulfates also exist in plants, the detailed biochemical characterization of Arabidopsis thaliana sulfotransferases (AtSULTs) remains to be fully clarified. We report here that uncharacterized AtSULT202E1 (AGI code: At2g03770), a SULT202E subfamily member, shows the sulfating activity toward flavonoids. The general characteristics of the enzyme were studied on the optimum temperature and pH, the effect of divalent cations, and the thermal stability with kaempferol as substrate. A comparative analysis of the sulfation of flavonoids by AtSULT202E1, AtSULT202B1 and AtSULT202A1 revealed that three AtSULTs have differential substrate specificities. Surprisingly, 3-hydroxyflavone was sulfated only by AtSULT202A1 while 7-hydroxyflavone was highly sulfated by AtSULT202E1 and AtSULT202B1. These results indicate that flavonols might be sulfated in a position specific manner. In conclusion, our studies indicate that a novel AtSULT202E1 has the sulfating activity toward flavonoids together with AtSULT202B1 and AtSULT202A1. The existence of three flavonoid sulfotransferases in A. thaliana suggests that sulfation of flavonoids have an important role in regulation of their functions.

Arabidopsis Tyrosylprotein sulfotransferase acts in the auxin/PLETHORA pathway in regulating postembryonic maintenance of the root stem cell niche.

Recent identification of the Arabidopsis thaliana tyrosylprotein sulfotransferase (TPST) and a group of Tyr-sulfated peptides known as root meristem growth factors (RGFs) highlights the importance of protein Tyr sulfation in plant growth and development. Here, we report the action mechanism of TPST in maintenance of the root stem cell niche, which in the Arabidopsis root meristem is an area of four mitotically inactive quiescent cells plus the surrounding mitotically active stem cells. Mutation of TPST leads to defective maintenance of the root stem cell niche, decreased meristematic activity, and stunted root growth. We show that TPST expression is positively regulated by auxin and that mutation of this gene affects auxin distribution by reducing local expression levels of several PIN genes and auxin biosynthetic genes in the stem cell niche region. We also show that mutation of TPST impairs basal- and auxin-induced expression of the PLETHORA (PLT) stem cell transcription factor genes and that overexpression of PLT2 rescues the root meristem defects of the loss-of-function mutant of TPST. Together, these results support that TPST acts to maintain root stem cell niche by regulating basal- and auxin-induced expression of PLT1 and PLT2. TPST-dependent sulfation of RGFs provides a link between auxin and PLTs in regulating root stem cell niche maintenance.

Identification of chondroitin/dermatan sulfotransferases in the protochordate, Ciona intestinalis.

Sulfated glycosaminoglycans are important components of connective tissues. The pattern of sulfation is important for their biological functions. Ascidians, the closest relatives of vertebrates, have a simple chordate body plan. In the present study, we identified an almost complete set of genes encoding proteins homologous to chondroitin/dermatan sulfotransferases in the genome of the ascidian Ciona intestinalis. We found eight genes encoding 4-O-sulfotransferases, eight genes encoding 6-O-sulfotransferases, and three genes encoding uronyl 2-O-sulfotransferases. The number of sulfotransferase genes was unexpectedly large, considering that ascidians do not have a well-developed endoskeleton. In addition, most of the genes within each sub-family seemed to have arisen by gene duplication events that occurred in the ascidian lineage after divergence from the main chordate lineage. This suggests that a unique pattern of sulfation independently developed during ascidian evolution. Some of the genes identified in the present study showed tissue-specific expression in the epidermis, notochord, muscle, and central nervous system. Region-specific expression in the epidermis was also observed. The present study provides useful information for further comparative and functional analyses of sulfotransferases and proteoglycans in chordate embryos.

Distinct functional specificities are associated with protein isoforms encoded by the Drosophila dorsal-ventral patterning gene pipe.

Spatially regulated transcription of the pipe gene in ventral cells of the Drosophila ovary follicle cell epithelium is a key event that specifies progeny embryo dorsal-ventral (DV) polarity. pipe encodes ten putative protein isoforms, all of which exhibit similarity to vertebrate glycosaminoglycan-modifying enzymes. Expression of one of the isoforms, Pipe-ST2, in follicle cells has previously been shown to be essential for DV patterning. pipe is also expressed in the embryonic salivary gland and its expression there is required for normal viability. Here, we show that in addition to Pipe-ST2, seven of the other Pipe isoforms are expressed in the ovary, whereas all Pipe isoforms are abundantly expressed in the embryo. Of the ten isoforms, only Pipe-ST2 can restore ventral and lateral pattern elements to the progeny of otherwise pipe-null mutant females. By contrast, three Pipe isoforms, but not Pipe-ST2, support the production of a novel pipe-dependent epitope present in the embryonic salivary gland. These data indicate that differences in functional specificity, and presumably enzymatic specificity, are associated with several of the Pipe isoforms. In addition, we show that uniform expression of the Pipe-ST2 isoform in the follicle cell layer of females otherwise lacking pipe expression leads to the formation of embryos with a DV axis that is appropriately oriented with respect to the intrinsic polarity of the eggshell. This suggests the existence of a second mechanism that polarizes the Drosophila embryo, in addition to the ventrally restricted transcription of the pipe gene.

Genome-wide identification of glucosinolate synthesis genes in Brassica rapa.

Glucosinolates play important roles in plant defense against herbivores and microbes, as well as in human nutrition. Some glucosinolate-derived isothiocyanate and nitrile compounds have been clinically proven for their anticarcinogenic activity. To better understand glucosinolate biosynthesis in Brassica rapa, we conducted a comparative genomics study with Arabidopsis thaliana and identified total 56 putative biosynthetic and regulator genes. This established a high colinearity in the glucosinolate biosynthesis pathway between Arabidopsis and B. rapa. Glucosinolate genes in B. rapa share 72-94% nucleotide sequence identity with the Arabidopsis orthologs and exist in different copy numbers. The exon/intron split pattern of B. rapa is almost identical to that of Arabidopsis, although inversion, insertion, deletion and intron size variations commonly occur. Four genes appear to be nonfunctional as a result of the presence of a frame shift mutation and retrotransposon insertion. At least 12 paralogs of desulfoglucosinolate sulfotransferase were found in B. rapa, whereas only three were found in Arabidopsis. The expression of those paralogs was not tissue-specific but varied greatly depending on B. rapa tissue types. Expression was also developmentally regulated in some paralogs but not in other paralogs. Most of the regulator genes are present as triple copies. Accordingly, glucosinolate synthesis and regulation in B. rapa appears to be more complex than that of Arabidopsis. With the isolation and further characterization of the endogenous genes, health-beneficial vegetables or desirable animal feed crops could be developed by metabolically engineering the glucosinolate pathway.

Drug metabolism enzyme expression and activity in primary cultures of human proximal tubular cells.

We previously catalogued expression and activity of organic anion and cation, amino acid, and peptide transporters in primary cultures of human proximal tubular (hPT) cells to establish them as a cellular model to study drug transport in the human kidney [Lash, L.H., Putt, D.A., Cai, H., 2006. Membrane transport function in primary cultures of human proximal tubular cells. Toxicology 228, 200-218]. Here, we extend our analysis to drug metabolism enzymes. Expression of 11 cytochrome P450 (CYP) enzymes was determined with specific antibodies. CYP1B1, CYP3A4, and CYP4A11 were the only CYP enzymes readily detected in total cell extracts. These same CYP enzymes, as well as CYP3A5 and possibly CYP2D6, were detected in microsomes from confluent hPT cells, although expression levels varied among kidney samples. In agreement with Western blot data, only activity of CYP3A4/5 was detected among the enzyme activities measured. Expression of all three glutathione S-transferases (GSTs) known to be found in hPT cells, GSTA, GSTP, and GSTT, was readily detected. Variable expression of three sulfotransferases (SULTs), SULT1A3, SULT1E, and SULT2A1, and three UDP-glucuronosyltransferases (UGTs), UGT1A1, UGT1A6, and UGT2B7, was also detected. When examined over the course of cell growth to confluence, expression of all enzymes was generally maintained at readily measurable levels, although they were often lower than in fresh tissue. These results indicate that primary cultures of hPT cells possess significant capacity to metabolize many classes of drugs, and can be used as an effective model to study drug metabolism.

Sulfotransferase gene copy number variation: pharmacogenetics and function.

Pharmacogenetics is the study of the role of inheritance in variation to drug response. Drug response phenotypes can vary from adverse drug reactions at one end of the spectrum to equally serious lack of the desired effect of drug therapy at the other. Many of the current important examples of pharmacogenetics involve inherited variation in drug metabolism. Sulfate conjugation catalyzed by cytosolic sulfotransferase (SULT) enzymes, particularly SULT1A1, is a major pathway for drug metabolism in humans. Pharmacogenetic studies of SULT1A1 began over a quarter of a century ago and have advanced from biochemical genetic experiments to include cDNA and gene cloning, gene resequencing, and functional studies of the effects of single nucleotide polymorphisms (SNPs). SNP genotyping, in turn, led to the discovery of functionally important copy number variations (CNVs) in the SULT1A1 gene. This review will briefly describe the evolution of our understanding of SULT1A1 pharmacogenetics and CNV, as well as challenges involved in utilizing both SNP and CNV data in an attempt to predict SULT1A1 function. SULT1A1 represents one example of the potential importance of CNV for the evolving disciplines of pharmacogenetics and pharmacogenomics.

Inhibition of sulfotransferases by xenobiotics.

The sulfotransferase (SULT) family comprises important phase II conjugation enzymes for the detoxification of xenobiotics and modulation of the activity of physiologically important endobiotics such as thyroid hormones, steroids, and neurotransmitters. SULT enzymes catalyze the transfer of a sulfuryl group, donated by 3'-phosphoadenosine-5'-phosphosulfate (PAPS), to an acceptor substrate that may be a hydroxy group or an amine group in a process originally called sulfation, but more correctly referred to as sulfonation or sulfurylation. SULT activity may be inhibited when humans are exposed to certain xenobiotics including drugs (mefenamic acid, salicylic acid, clomiphene, danazol etc.), dietary chemicals (catechins, food colorants, flavonoids and phytoestrogens etc.), and environmental chemicals (hydroxylated polychlorinated biphenyls, hydroxylated polyhalogenated aromatic hydrocarbons, pentachlorophenol, triclosan and bisphenol A, etc.). Inhibition of individual SULT isoforms may cause adverse effects on human health. For example, hydroxylated polychlorinated biphenyls have been shown to interfere with the transport of thyroid hormones, inhibit estradiol sulfonation, and inhibit thyroid hormone sulfonation, thereby potentially disrupting the thyroid hormone system. Formation of sulfate conjugates of toxic xenobiotics usually decreases their toxicity, so inhibition of this pathway may lead to prolonged exposure to the compounds. Conversely, some sulfate conjugates are chemically reactive, inhibition of their formation may protect from toxicity. This manuscript will review the literature concerning the inhibition of SULTs by xenobiotics including isoform-selective effects, inhibition kinetics and health effects resulting from the inhibition.

The structures of the unique sulfotransferase retinol dehydratase with product and inhibitors provide insight into enzyme mechanism and inhibition.

The structure of retinol dehydratase (DHR) from Spodoptera frugiperda, a member of the sulfotransferase superfamily, in complexes with the inactive form of the cofactor PAP 3'-phosphoadenosine 5'-phosphate (PAP) and (1) the product of the reaction with retinol anhydroretinol (AR), (2) the retinoid inhibitor all-trans-4-oxoretinol (OR), and (3) the potent steroid inhibitor androsterone (AND) have been determined and compared to the enzyme complex with PAP and retinol. The structures show that the geometry of the active-site amino acids is largely preserved in the various complexes. However, the beta-ionone rings of the retinoids are oriented differently with respect to side chains that have been shown to be important for the enzymatic reaction. In addition, the DHR:PAP:AND complex reveals a novel mode for steroid binding that contrasts significantly with that for steroid binding in other sulfotransferases. The molecule is displaced and rotated approximately 180 degrees along its length so that there is no acceptor hydroxyl in close proximity to the site of sulfate transfer. This observation explains why steroids are potent inhibitors of retinol dehydratase activity, rather than substrates for sulfonation. Most of the steroid-protein contacts are provided by the alpha-helical cap that distinguishes this member of the superfamily. This observation suggests that in addition to providing a chemical environment that promotes the dehydration of a sulfonated intermediate, the cap may also serve to minimize a promiscuous sulfotransferases activity.

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.

Characterization of a zebrafish estrogen-sulfating cytosolic sulfotransferase: inhibitory effects and mechanism of action of phytoestrogens.

Cytosolic sulfotransferases (STs) are generally thought to be involved in detoxification of xenobiotics, as well as homeostasis of endogenous compounds such as thyroid/steroid hormones and catecholamine hormones/neurotransmitters. We report here the identification and characterization of a zebrafish estrogen-sulfating cytosolic ST. The zebrafish ST was bacterially expressed, purified, and examined for enzymatic activities using a variety of endogenous compounds as substrates. Results showed that the enzyme displayed much higher activities toward two endogenous estrogens, estrone (E(1)) and 17beta-estradiol (E(2)), in comparison with thyroid hormones, 3,3',5-triiodothyronine (T(3)) and thyroxine (T(4)), dopamine, dihydroxyphenylalanine (Dopa), and dehydroepiandrosterone (DHEA). The kinetic parameters, K(m), and V(max), with estrogens and thyroid hormones as substrates were determined. The calculated V(max)/K(m) for E(1), E(2), T(3), and T(4) were, respectively, 31.6, 16.7, 1.5, and 0.8 nmol min(-1) mg(-1) microM(-1), indicating clearly the estrogens being preferred physiological substrates for the enzyme. The inhibitory effects of isoflavone phytoestrogens on the sulfation of E(2) by this zebrafish ST were examined. The IC(50) determined for quercetin, genistein, and daidzein were 0.7, 2.5, and 8 microM, respectively. Kinetic analyses revealed that the mechanism underlying the inhibition by these isoflavones to be of the competitive type.

Sulphotransferases acting on mucin-type oligosaccharides.

This review summarizes the occurrence, properties and role in mucin O-glycosylation pathways of the various members of glycoprotein sulphotransferase families. Although a number of sulphotransferases have been cloned that act on mucin-type substrates in vitro, it is still difficult to determine exactly which enzymes are responsible for mucin sulphation in vivo. Sulphotransferases play a critical role in determining the chemical, physical and biological properties of mucins. Several of these enzymes have been shown to differ in expression and activity in cancer and inflammation.

Human gastrointestinal sulfotransferases: identification and distribution.

Sulfotransferases (STs) catalyze the sulfation of many structurally diverse molecules. Enzymatic assays and Western blots have been used to identify and characterize STs in the human gastrointestinal tract. Sulfation activities for 2-naphthol, dopamine, estradiol, and dehydroepiandrosterone (DHEA) from 23 donors were measured in cytosol prepared from stomach, duodenum, segments of small intestine, and colon and were compared to levels in human liver cytosol. Stomach and colon had low 2-naphthol and dopamine sulfation activities and almost no estradiol and DHEA sulfation activity. For all four substrates, small intestine has higher activities than both stomach and colon. Human small intestine 2-naphthol sulfation specific activity is approximately half that of human liver. Human small intestine dopamine sulfation activity is three times as high as that of human liver. While estrogen sulfation activity is about the same for both human intestine and human liver, human liver DHEA sulfation activity is about five times as high as that of human small intestine. The distribution of ST activities along the length of the small intestine was very different among different donors. Some donors had higher activity in the proximal segments of the small intestine, whereas other donors had higher activity in the distal segments of the small intestine. Our results also demonstrated high variation of small intestine sulfation activities compared with human liver activities among different donors. The Western blot results agreed with the enzymatic assay results. These results suggest that xenobiotics may regulate human small intestinal STs.

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.

Carbohydrate sulfotransferases in lymphocyte homing.

Sulfation is a critical modification in many instances of biological recognition. Early work in lymphocyte homing indicated that the endothelial ligands for L-selectin depended upon sulfation modifications. Subsequent studies showed that the two specific modifications, Gal-6-SO4 and GlcNAc-6-SO4, were present on actual biological ligands. Recently, a family of carbohydrate sulfotransferases capable of generating these modifications has been identified at the molecular level. Reconstitution experiments implicate members of this family as critical participants in lymphocyte homing.

Pharmacogenetics of sulfotransferase.

Cytosolic sulfotransferase catalyzes sulfoconjugation of relatively small lipophilic endobiotics and xenobiotics. At least 44 cytosolic sulfotransferases have been identified from mammals, and based on their amino acid sequences, these forms are shown to constitute five different families. In humans, 10 sulfotransferase genes have been identified and shown to localize on at least five different chromosomes. The enzymatic properties characterized in the recombinant forms indicate the association of their substrate specificity with metabolisms of such nonpeptide hormones as estrogen, corticoid, and thyroxine, although most forms are also active on the sulfation of various xenobiotics. Genetic polymorphisms are observed on such human sulfotransferases as ST1A2, ST1A3, and ST2A3.

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.