Reduced SULT2B1b expression alleviates ox-LDL-induced inflammation by upregulating miR-148-3P via inhibiting the IKKß/NF-κB pathway in macrophages.

Atherosclerosis is a lipid-driven chronic inflammatory disease in which lipid-laden macrophage foam cells lead to inflamed lesions in arteries. Previous studies have proven that sulfotransferase 2B1b (SULT2B1b) has several roles in the regulation of lipid metabolism and the inflammatory response. However, little is known about the functions of SULT2B1b in ox-LDL-induced inflammation in macrophages. In this study, after treatment with either ox-LDL alone or combined with transfection of siRNAs targeting SULT2B1b, IL-6, TNF-α, NF-κB, IKKß and IκB mRNA and protein expression were determined in Raw264.7 cells by real-time PCR and Western blot, respectively. The proliferative capacity was determined by EdU staining and Cell Counting Kit-8. Our data demonstrated that SULT2B1b knockdown could reduce phosphorylated NF-κB levels and downregulate IKKß protein levels. Additionally, IκB levels were increased and the proliferation of ox-LDL stimulated cells was inhibited after SULT2B1b silencing. Downregulation of SULT2B1b expression was found to upregulate miR-148a-3p expression by microarray assay, while IKKß was a miR-148a-3p target gene. Our study suggests that SULT2B1b knockdown could promote miR148a-3p expression and inhibit activation of the IKKß/NF-κB signalling pathway, which suppressed the inflammatory response in macrophages. Therefore, targeting the SULT2B1b gene might be potentially beneficial for atherosclerosis prevention by decreasing the inflammatory response.

Finding host targets for HIV therapy.

A CRISPR screen conducted in a CD4+ T cell leukemia line has identified host factors required for HIV infection but dispensable for cellular survival. The results highlight sulfation on the HIV co-receptor CCR5 and cellular aggregation as potential targets for therapeutic intervention.

SULT2B1b sulfotransferase: induction by vitamin D receptor and reduced expression in prostate cancer.

An elevated tumor tissue androgen level, which reactivates androgen receptor in recurrent prostate cancer, arises from the intratumor synthesis of 5α-dihydrotestosterone through use of the precursor steroid dehydroepiandrosterone (DHEA) and is fueled by the steroidogenic enzymes 3ß-hydroxysteroid dehydrogenase (3ß-HSD1), aldoketoreductase (AKR1C3), and steroid 5-alpha reductase, type 1 (SRD5A1) present in cancer tissue. Sulfotransferase 2B1b (SULT2B1b) (in short, SULT2B) is a prostate-expressed hydroxysteroid SULT that converts cholesterol, oxysterols, and DHEA to 3ß-sulfates. DHEA metabolism involving sulfonation by SULT2B can potentially interfere with intraprostate androgen synthesis due to reduction of free DHEA pool and, thus, conversion of DHEA to androstenedione. Here we report that in prostatectomy specimens from treatment-naive patients, SULT2B expression is markedly reduced in malignant tissue (P < .001, Mann-Whitney U test) compared with robust expression in adjacent nonmalignant glands. SULT2B was detected in formalin-fixed specimens by immunohistochemistry on individual sections and tissue array. Immunoblotting of protein lysates of frozen cancer and matched benign tissue confirmed immunohistochemistry results. An in-house-developed rabbit polyclonal antibody against full-length human SULT2B was validated for specificity and used in the analyses. Ligand-activated vitamin D receptor induced the SULT2B1 promoter in vivo in mouse prostate and increased SULT2B mRNA and protein levels in vitro in prostate cancer cells. A vitamin D receptor/retinoid X receptor-α-bound DNA element (with a DR7 motif) mediated induction of the transfected SULT2B1 promoter in calcitriol-treated cells. SULT2B knockdown caused an increased proliferation rate of prostate cancer cells upon stimulation by DHEA. These results suggest that the tumor tissue SULT2B level may partly control prostate cancer growth, and its induction in a therapeutic setting may inhibit disease progression.

Differential use of chondroitin sulfate to regulate hyaluronan binding by receptor CD44 in Inflammatory and Interleukin 4-activated Macrophages.

CD44 is a cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan and is involved in processes ranging from leukocyte recruitment to wound healing. In the immune system, the binding of hyaluronan to CD44 is tightly regulated, and exposure of human peripheral blood monocytes to inflammatory stimuli increases CD44 expression and induces hyaluronan binding. Here we sought to understand how mouse macrophages regulate hyaluronan binding upon inflammatory and anti-inflammatory stimuli. Mouse bone marrow-derived macrophages stimulated with tumor necrosis factor α or lipopolysaccharide and interferon-γ (LPS/IFNγ) induced hyaluronan binding by up-regulating CD44 and down-regulating chondroitin sulfation on CD44. Hyaluronan binding was induced to a lesser extent in interleukin-4 (IL-4)-activated macrophages despite increased CD44 expression, and this was attributable to increased chondroitin sulfation on CD44, as treatment with ß-d-xyloside to prevent chondroitin sulfate addition significantly enhanced hyaluronan binding. These changes in the chondroitin sulfation of CD44 were associated with changes in mRNA expression of two chondroitin sulfotransferases, CHST3 and CHST7, which were decreased in LPS/IFNγ-stimulated macrophages and increased in IL-4-stimulated macrophages. Thus, inflammatory and anti-inflammatory stimuli differentially regulate the chondroitin sulfation of CD44, which is a dynamic physiological regulator of hyaluronan binding by CD44 in mouse macrophages.

Identification of MST1/STK4 and SULF1 proteins as autoantibody targets for the diagnosis of colorectal cancer by using phage microarrays.

The characterization of the humoral response in cancer patients is becoming a practical alternative to improve early detection. We prepared phage microarrays containing colorectal cancer cDNA libraries to identify phage-expressed peptides recognized by tumor-specific autoantibodies from patient sera. From a total of 1536 printed phages, 128 gave statistically significant values to discriminate cancer patients from control samples. From this, 43 peptide sequences were unique following DNA sequencing. Six phages containing homologous sequences to STK4/MST1, SULF1, NHSL1, SREBF2, GRN, and GTF2I were selected to build up a predictor panel. A previous study with high-density protein microarrays had identified STK4/MST1 as a candidate biomarker. An independent collection of 153 serum samples (50 colorectal cancer sera and 103 reference samples, including healthy donors and sera from other related pathologies) was used as a validation set to study prediction capability. A combination of four phages and two recombinant proteins, corresponding to MST1 and SULF1, achieved an area under the curve of 0.86 to correctly discriminate cancer from healthy sera. Inclusion of sera from other different neoplasias did not change significantly this value. For early stages (A+B), the corrected area under the curve was 0.786. Moreover, we have demonstrated that MST1 and SULF1 proteins, homologous to phage-peptide sequences, can replace the original phages in the predictor panel, improving their diagnostic accuracy.

Induction of high endothelial venule-like vessels expressing GlcNAc6ST-1-mediated L-selectin ligand carbohydrate and mucosal addressin cell adhesion molecule 1 (MAdCAM-1) in a mouse model of "Candidatus Helicobacter heilmannii"-induced gastritis and gastric mucosa-associated lymphoid tissue (MALT) lymphoma.

BACKGROUND: "Candidatus Helicobacter heilmannii" induce chronic gastritis, which eventually leads to gastric B-cell type mucosa-associated lymphoid tissue (MALT) lymphoma. This study was performed using an animal model of infection with "Candidatus Helicobacter heilmannii" to elucidate how this chronic inflammation is induced or maintained. MATERIALS AND METHODS: BALB/c mice were infected with the "Candidatus Helicobacter heilmannii" isolate SH4. The animals were examined at 8, 26, 54, and 83 weeks after the infection. The stomach of the animals was resected and immunostained for peripheral lymph node addressin (PNAd) and mucosal addressin cell adhesion molecule 1 (MAdCAM-1), "Candidatus Helicobacter heilmannii," and CD45R/B220. An in vitro binding assay with L- and E-selectin·IgM chimeric proteins was performed. Real-time polymerase chain reaction was used to evaluate transcripts of N-acetylglucosamine-6-O-sulfotransferases (GlcNAc6STs), which direct the expression of the PNAd and MAdCAM-1. RESULTS: Chronic gastritis developed in the infected animals, and its severity increased with the duration of the infection. B-cell type MALT lymphoma developed in some animals at 54 and 83 weeks after infection. PNAd- and MAdCAM-1-expressing high endothelial venule (HEV)-like vessels were induced in infected animals which developed chronic gastritis and MALT lymphoma. The number of HEV-like vessels increased as chronic inflammation progressed. The induced HEV-like vessels were bound by L- and E-selectin·IgM chimeric protein. mRNA expressions of GlcNAc6ST-1 and MAdCAM-1 increased in the infected animals. CONCLUSIONS: HEV-like vessels expressing GlcNAc6ST-1-mediated L-selectin ligand carbohydrate and MAdCAM-1 may play a crucial role in the pathogenesis of "Candidatus Helicobacter heilmannii"-induced chronic gastritis and MALT lymphoma.

Novel anti-carbohydrate antibodies reveal the cooperative function of sulfated N- and O-glycans in lymphocyte homing.

Cell surface glycans play pivotal roles in immune cell trafficking and immunity. Here we present an efficient method for generating anti-carbohydrate monoclonal antibodies (mAbs) using gene-targeted mice and describe critical glycans in lymphocyte homing. We immunized sulfotransferase GlcNAc6ST-1 and GlcNAc6ST-2 doubly deficient mice with sulfotransferase-overexpressing Chinese hamster ovary cells and generated two mAbs, termed S1 and S2. Both S1 and S2 bound high endothelial venules (HEVs) in the lymphoid organs of humans and wild-type mice, but not in those of doubly deficient mice. Glycan array analysis indicated that both S1 and S2 specifically bound 6-sulfo sialyl Lewis X and its defucosylated structure. Interestingly, S2 inhibited lymphocyte homing to peripheral lymph nodes by 95%, whereas S1 inhibited it by only 25%. S2 also significantly inhibited contact hypersensitivity responses and L-selectin-dependent leukocyte adhesion to HEVs. Immunohistochemical and Western blot analyses indicated that S1 preferentially bound sulfated O-glycans, whereas S2 bound both sulfated N- and O-glycans in HEVs. Furthermore, S2 strongly inhibited the N-glycan-dependent residual lymphocyte homing in mutant mice lacking sulfated O-glycans, indicating the importance of both sulfated N- and O-glycans in lymphocyte homing. Thus, the two mAbs generated by a novel method revealed the cooperative function of sulfated N- and O-glycans in lymphocyte homing and immune surveillance.

Use of a phage display antibody to measure the enzymatic activity of the Sulfs.

Sulf-1 and Sulf-2 are extracellular endoglucosamine 6-sulfatases, which selectively liberate the 6-O-sulfate groups on glucosamines present in N, 6-O, and 2-O trisulfated disaccharides of intact heparan sulfate (HS)/heparin chains. The Sulfs are known to regulate signaling of heparin/HS-binding protein ligands, such as morphogens and growth factors, presumably through their ability to decrease the association between the ligands and HS proteoglycans. These enzymes serve important roles in development and are dysregulated in many cancers. We previously described arylsulfatase and endoglucosamine 6-sulfatase assays for the Sulfs. RB4CD12 is a phage display anti-HS antibody. N-sulfation, 2-O-sulfation, and 6-O-sulfation are involved in its binding. In this chapter, we describe the application of RB4CD12 in ELISA, flow cytometry, and immunohistochemistry assays to measure the enzymatic activity of the Sulfs. These newly established methods should facilitate further investigation of the Sulfs in vitro and in vivo.

N-Acetylglucosamine 6-O-sulfotransferase-2 as a tumor marker for uterine cervical and corpus cancer.

N-Acetylglucosamine 6-O-sulfotransferase-2 (GlcNAc6ST2) is ectopically expressed in ovarian mucinous and clear cell adenocarcinoma [Kanoh et al., Glycoconj J 23:453-460, 2006]. Here we studied whether GlcNAc6ST2 protein can be detected in sera from patients with gynecological cancers and could serve as a tumor marker. First, we created a monoclonal antibody and polyclonal antiserum against GlcNAc6ST2. These antibodies were specific for GlcNAc6ST2, as shown by Western blot analysis and immunoprecipitation. Using these antibodies, we constructed a sandwich ELISA method for detecting GlcNAc6ST2 in the serum. GlcNAc6ST2 provided lower positive rates for ovarian cancer than CA125, but higher positive rates for uterine cervical and corpus cancer than SCC antigens and CA125, respectively. A significantly higher percentage of stage I uterine cervical and corpus cancers were positive for GlcNAc6ST2 than for SCC antigens and CA125, respectively. GlcNAc6ST2 could therefore be a good serological marker for detecting early-stage uterine cervical and corpus cancers.

Characterization of proline-serine-rich carboxyl terminus in human sulfotransferase 2B1b: immunogenicity, subcellular localization, kinetic properties, and phosphorylation.

The human sulfotransferase (SULT) 2B1 gene is a member of the SULT2 gene family and encodes two isoforms, SULT2B1a and SULT2B1b. Although messages for both SULT2B1a and SULT2B1b are detectable in human tissues, only SULT2B1b has been identified immunologically. Compared with other human SULTs, SULT2B1b has an extension at the proline- and serine-rich carboxyl (PSC) end of about 53 amino acids. The structure and function of this unique PSC extension were investigated. Constructs of full-length SULT2B1b as well as truncated SULT2B1b without the PSC extension were expressed in Escherichia coli. Removal of the PSC extension significantly decreased the thermostability of the expressed enzyme as well as decreasing the rate of dehydroepiandrosterone sulfation. Rabbit polyclonal antibodies were raised against both the full-length and truncated SULT2B1b proteins. Immunoblot analysis showed that antibodies raised to full-length SULT2B1b immunoreact only with full-length SULT2B1b, whereas antibodies raised to truncated SULT2B1b react with both full-length and truncated SULT2B1b. Unlike full-length SULT2B1b, truncated SULT2B1b was incapable of translocation to nuclei in transfected human BeWo choriocarcinoma cells. Phosphorylated serines were detected in the PSC extension of full-length SULT2B1b expressed in BeWo cells but not in truncated SULT2B1b. At least one phosphorylated serine was detected in expressed SULT2B1b via two-dimensional gel electrophoresis, immunoblot analysis, and mass spectroscopic analysis. Bacterially expressed full-length SULT2B1b but not truncated SULT2B1b was phosphorylated by casein kinase or Cdc2 protein kinase in vitro. This study suggests that the PSC extension of SULT2B1b is an important site in the immunogenicity, nuclear translocation, kinetic activity, and thermostability of this SULT isoform.

Estrogen sulfotransferase and steroid sulfatase in human breast carcinoma.

Estrogen sulfotransferase (EST; SULT 1E1 or STE gene) sulfonates estrogens to inactive estrogen sulfates, whereas steroid sulfatase (STS) hydrolyzes estrone sulfate to estrone. Both EST and STS have been suggested to play important roles in regulating the in situ production of estrogens in human breast carcinoma tissues. However, the expression of EST has not been examined in breast carcinoma tissues, and the biological significance of EST and STS remains unknown. Therefore, in this study, we examined the expression of EST and STS in 35 specimens of human breast carcinoma tissues using immunohistochemistry, reverse transcription-PCR (RT-PCR), and enzymatic assay. EST and STS immunoreactivity was also correlated with various clinicopathological parameters, including prognosis to examine the biological significance of these enzymes in 113 breast carcinomas. EST and STS immunoreactivity was detected in carcinoma cells and significantly associated with their mRNA levels (P = 0.0027 and 0.0158, respectively), as measured by RT/real-time PCR, and enzymatic activities (P = 0.0005 and 0.0089, respectively) in 35 breast carcinomas. In breast cancer tissues examined by laser capture microdissection/RT-PCR analyses, the mRNA for EST was localized in both carcinoma and intratumoral stromal cells, whereas that of STS was detected only in carcinoma cells. Of the 113 invasive ductal carcinomas examined in this study, EST and STS immunoreactivity was detected in 50 and 84 cases (44.2 and 74.3%), respectively. In these cases, EST immunoreactivity was inversely correlated with tumor size (P = 0.003) or lymph node status (P = 0.0027). In contrast, STS immunoreactivity was significantly correlated with tumor size (P = 0.0047). Moreover, EST immunoreactivity was significantly associated with a decreased risk of recurrence or improved prognosis by both uni (P = 0.0044, and 0.0026, respectively) and multivariate (P = 0.0429 and 0.0149, respectively) analyses. STS immunoreactivity, however, was significantly associated with an increased risk of recurrence (P = 0.0118) and worsened prognosis (P = 0.0325) by univariate analysis. Results from our present study suggest that immunoreactivities for both EST and STS are associated with their mRNA level and enzymatic activity and that EST immunoreactivity is considered to be a potent prognostic factor in human breast carcinoma.

Postnatal ontogeny and hormonal regulation of sulfotransferase SULT1B1 in male and female rats.

The ontogenic and hormonal regulation of a sulfotransferase, SULT1B1, was examined. Hepatic RNA was isolated from rats of various ages from 1 to 90 days. The mRNA for SULT1B1 is low for both sexes until a dramatic increase ( approximately 6-fold) occurs between 15 and 30 days of age in male rats. SULT1B1 expression then decreases to half of the maximal level by 90 days of age. The increase in SULT1B1 mRNA in female rats is less dramatic and occurs between 30 and 45 days of age. SULT1B1 mRNA expression plateaus from 45 to 90 days in female rats. Expression of SULT1B1 mRNA is comparable in adult male and female rats. RNA was isolated from hypophysectomized (HX) animals and HX animals treated with growth hormone [by either male (injection) or female (infusion) pattern], estradiol, progesterone, or testosterone. HX and HX plus growth hormone, or HX plus steroid replacement, did not alter SULT1B1 mRNA expression. Pituitary-intact rats were treated with steroidal compounds dexamethasone (DEX) and pregnenolone-16alpha-carbonitrile (PCN). Both DEX and PCN increased expression of SULT1B1 mRNA in male rats (4- and 3-fold, respectively). However, in female rats, only PCN induced SULT1B1 mRNA (2-fold), whereas DEX did not induce SULT1B1 in female rats. Analysis of SULT1B1 protein expression indicated that only when SULT1B1 mRNA was markedly increased, that is in DEX-treated male rats, was SULT1B1 protein increased. Thus, although adult male and female rats have similar SULT1B1 mRNA expressions, the patterns develop ontogenically differently. SULT1B1 is not regulated by pituitary hormones and DEX induces SULT1B1 protein in male rats.

Human dehydroepiandrosterone sulfotransferase pharmacogenetics: quantitative Western analysis and gene sequence polymorphisms.

Dehydroepiandrosterone sulfotransferase (DHEA ST) catalyzes the sulfation of DHEA and other hydroxysteroids. DHEA ST enzymatic activity in individual human liver biopsy samples has been shown to vary over a five-fold range, and frequency distribution histograms are bimodal, with approximately 25% of subjects included in a high activity subgroup. We set out to characterize the molecular basis for variation in human liver DHEA ST activity. The first step involved performing quantitative Western analysis of cytosol preparations from 92 human liver samples that had been phenotyped with regard to level of DHEA ST enzymatic activity. There was a highly significant correlation (r(s) = 0.635, P < 0.0001) between levels of DHEA ST activity and immunoreactive protein. We next attempted to determine whether the expression of DHEA ST might be controlled, in part, by a genetic polymorphism. DNA was isolated from three "low" and three "high" DHEA ST activity liver samples. Exons and the 5'-flanking region of the DHEA ST gene (STD) were amplified for each of these samples with the polymerase chain reaction (PCR). When compared with "wild type" STD sequence, some of the samples contained a T --> C transition at DHEA ST cDNA nucleotide 170, located within exon 2, resulting in a Met 57 --> Thr change in amino acid. Other samples contained an A --> T transversion at nucleotide 557 within STD exon 4 that resulted in a Glu 186 --> Val change. STD exons 2 and 4 were then sequenced for DNA isolated from an additional 87 liver samples that had been phenotyped with regard to level of DHEA ST enzymatic activity. The allele frequency for the exon 2 polymorphism in these samples was 0.027, whereas that for the exon 4 polymorphism was 0.038, but neither polymorphism was systematically related to the level of enzyme activity in these samples. Transient expression in COS-1 cells of cDNA that contained the nucleotide 170 and 557 polymorphisms, either separately or together, resulted in decreased expression of both DHEA ST enzymatic activity and level of immunoreactive protein, but only when the nucleotide 557 variant was present. Identification of common genetic polymorphisms within STD will now make it possible to test the hypothesis that those polymorphisms might alter in vivo expression and/or function of this important human steroid-metabolizing enzyme.

Immunolocalization of dehydroepiandrosterone sulfotransferase in normal and pathologic human adrenal gland.

Dehydroepiandrosterone sulfotransferase (DHEA-ST) catalyzes the conversion of dehydroepiandrosterone (DHEA) to dehydroepiandrosterone sulfate (DHEA-S) in the adrenals. Both DHEA and DHEA-S are quantitatively the most important corticosteroids in human. In this study, DHEA-ST was immunolocalized in normal (5 cases) and neoplastic human adrenal glands (33 cases), using a specific IgG fraction raised against the enzyme. DHEA-ST was present in almost all the zona reticularis cells and some cortical cells demonstrating lipid depletion in the zona fasciculata but not in the zona glomerulosa of the normal adrenal. This finding is consistent with adrenocorticotrophic hormone dependency of the enzyme expression. In adrenocortical adenoma, DHEA-ST immunoreactivity was observed in all the cases of Cushing's adenoma, adenoma associated with pre-Cushing's syndrome, nonfunctioning, hormonally inactive adenoma, and two of seven cases of aldosteronoma, but distribution of immunoreactivity was markedly heterogeneous among the adenoma cases. In attached non-neoplastic adrenal glands of the adenoma, intense and diffuse immunoreactivity was observed in the zona reticularis cells in all the cases of aldosteronoma and five of six of the nonfunctioning hormonally inactive adenoma, but DHEA-ST immunoreactivity was not observed or sporadic in the attached adrenal glands of Cushing's adenoma and adenoma with pre-Cushing's syndrome. These results in the attached adrenal gland may be correlated with decreased DHEA-ST expression due to autonomous neoplastic cortisol secretion and subsequent adrenocorticotrophic hormone suppression. In adrenocortical carcinoma, DHEA-ST was observed in all the cases, but the relative immunointensity of carcinoma cells was weak compared to that of the zona reticularis of the normal adrenal and adenoma.

Preparation and characterization of anti-peptide antibodies directed against human phenol and hydroxysteroid sulphotransferases.

Sulphotransferases (STs) catalyze the sulphation and, in general, detoxication of a large number of xenobiotics and endogenous compounds. A total of six synthetic peptides derived from the cDNA-derived amino acid sequences of the human phenol-sulphating form of phenosulphotransferase (P-PST) and human hydroxysteroid sulphotransferase (HST)--three from each sequence--were separately conjugated to the carrier protein keyhole limpet hemocyanin, and used to immunize rabbits. One successful antibody preparation was produced from among the P-PST peptides, and two from the HST peptides. On immunoblot analysis following SDS/PAGE, the anti-P-PST antibodies recognized two major forms of phenol ST in man, P-PST and the monoamine-sulphating form of PST, M-PST, and the two antibody preparations against HST recognized the human HST. These experiments demonstrate that it is possible to design specific antibodies against human sulphotransferases based on their amino acid sequences.

Bacterial expression and characterization of a cDNA for human liver estrogen sulfotransferase.

A distinct human estrogen sulfotransferase (hEST-1) cDNA has been isolated from a human liver lambda Zap cDNA library using a PCR procedure. The enzymatically active protein has been expressed in two bacterial expression systems and the kinetic and immunologic properties of the enzyme have been characterized. The full-length cDNA for hEST-1 is 994 base pairs in length and encodes a 294 amino acid protein with a calculated molecular mass of 35,123 Da. Purified hEST-1 migrated with an apparent molecular mass of 35,000 Da during SDS-polyacrylamide gel electrophoresis. Immunoblot analysis of hEST-1 expressed in E. coli with a rabbit anti-hEST-1 antibody yields a band of approximately 35,000 Da. The anti-hEST-1 antibody also detects a single band in human liver and jejunum cytosol which migrates with the same molecular mass as expressed hEST-1. There was also no cross-reactivity of hEST-1 with rabbit anti-hP-PST or rabbit anti-hDHEA-ST antibodies upon immunoblot analysis. hEST-1 was expressed in bacteria and purified to homogeneity. Expressed hEST-1 activity has a significantly greater affinity for estrogen sulfation than that found for the other human STs which conjugate estrogens. hEST-1 maximally sulfates beta-estradiol and estrone at concentrations of 20 nM. hEST-1 also sulfates dehydroepiandrosterone, pregnenolone, ethinylestradiol, and 1-naphthol, at significantly higher concentrations; however, cortisol, testosterone and dopamine are not sulfated. The results presented in this paper describe the expression and characterization of a human EST distinct from other human STs which sulfate estrogens. The high affinity of hEST-1 for estrogens indicates that this ST may be important in both the metabolism of estrogens and in the regulation of their activities.

APS-sulfotransferase activity is identical to higher plant APS-kinase (EC 2.7.1.25).

A cDNA from Arabidopsis thaliana L. Heynh encoding the APS-kinase (EC 2.7.1.25) was modified by deletion of a plastidic transit peptide to enable its expression in Escherichia coli. The resultant protein (MW 25,761) is enzymatically active as APS-kinase and restores prototrophic growth in an APS-kinase mutant. All transformants harbouring the modified plant DNA also acquired APS-sulfotransferase activity. In the absence of ATP but provided with DTT, a tetrameric form of recombinant APS-kinase exhibits APS-sulfotransferase activity. Monospecific polyclonal antibodies raised against the APS-kinase as immunogen also reacted against APS-sulfotransferase. We propose that APS-sulfotransferase activity is a nonphysiological side reaction of APS-kinase.

Molecular cloning and functions of rat liver hydroxysteroid sulfotransferases catalysing covalent binding of carcinogenic polycyclic arylmethanols to DNA.

Three sulfotransferases (STs) catalysing the metabolic activation of potent carcinogenic polycyclic arylmethanols were purified from female Sprague-Dawley (SD) rat liver cytosol without loss of their enzyme activities in the presence of Tween 20 used for preventing the enzymes from aggregation during purification and identified as hydroxysteroid sulfotransferases (HSTs). All the purified HSTs, STa, STb, and STc, with different electric charges had an apparently equal size of subunit (30.5 kDa) and cross-reacted with polyclonal antibody raised against STa. Our study on molecular cloning of cDNA libraries from two female SD rat livers indicated that both contained cDNA inserts coding for 5 different HST subunits, consisting of 284-285 amino acid residues (M(r), 33,084-33,535) and sharing strong amino acid sequence identity (> 83%). Of the 5 HST subunits, two had an identical amino acid sequence except for only one amino acid residue, and the other two contained only 6 amino acid substitutions in their sequences.

Purification and characterization of rat liver minoxidil sulphotransferase.

Minoxidil (Mx), a pyrimidine N-oxide, is used therapeutically as an antihypertensive agent and to induce hair growth in patients with male pattern baldness. Mx NO-sulphate has been implicated as the agent active in producing these effects. This paper describes the purification of a unique sulphotransferase (ST) from rat liver cytosol that is capable of catalysing the sulphation of Mx. By using DEAE-Sepharose CL-6B chromatography, hydroxyapatite chromatography and ATP-agarose affinity chromatography, Mx-ST activity was purified 240-fold compared with the activity in cytosol. The purified enzyme was also capable of sulphating p-nitrophenol (PNP) at low concentrations (less than 10 microM). Mx-ST was purified to homogeneity, as evaluated by SDS/PAGE and reverse-phase h.p.l.c. The active form of the enzyme had a molecular mass of 66,000-68,000 Da as estimated by gel exclusion chromatography and a subunit molecular mass of 35,000 Da. The apparent Km values for Mx, 3'-phosphoadenosine 5'-phosphosulphate and PNP were 625 microM, 5.0 microM and 0.5 microM respectively. However, PNP displayed potent substrate inhibition at concentrations above 1.2 microM. Antibodies raised in rabbits to the pure enzyme detected a single band in rat liver cytosol with a subunit molecular mass of 35,000 Da, as determined by immunoblotting. The anti-(rat Mx-ST) antibodies also reacted with the phenol-sulphating form of human liver phenol sulphotransferase, suggesting some structural similarity between these proteins.