Recombinant Sulfotransferases Research Articles

Increased 3-O-sulfated heparan sulfate in Alzheimer's disease brain is associated with genetic risk gene HS3ST1.

HS3ST1 is a genetic risk gene associated with Alzheimer's disease (AD) and overexpressed in patients, but how it contributes to the disease progression is unknown. We report the analysis of brain heparan sulfate (HS) from AD and other tauopathies using a LC-MS/MS method. A specific 3-O-sulfated HS displayed sevenfold increase in the AD group (n = 14, P < 0.0005). Analysis of the HS modified by recombinant sulfotransferases and HS from genetic knockout mice revealed that the specific 3-O-sulfated HS is made by 3-O-sulfotransferase isoform 1 (3-OST-1), which is encoded by the HS3ST1 gene. A synthetic tetradecasaccharide (14-mer) carrying the specific 3-O-sulfated domain displayed stronger inhibition for tau internalization than a 14-mer without the domain, suggesting that the 3-O-sulfated HS is used in tau cellular uptake. Our findings suggest that the overexpression of HS3ST1 gene may enhance the spread of tau pathology, uncovering a previously unidentified therapeutic target for AD.

Coelenterazine sulfotransferase from Renilla muelleri.

The luciferin sulfokinase (coelenterazine sulfotransferase) of Renilla was previously reported to activate the storage form, luciferyl sulfate (coelenterazine sulfate) to luciferin (coelenterazine), the substrate for the luciferase bioluminescence reaction. The gene coding for the coelenterazine sulfotransferase has not been identified. Here we used a combined proteomic/transcriptomic approach to identify and clone the sulfotransferase cDNA. Multiple isoforms of coelenterazine sulfotransferase were identified from the anthozoan Renilla muelleri by intersecting its transcriptome with the LC-MS/MS derived peptide sequences of coelenterazine sulfotransferase purified from Renilla. Two of the isoforms were expressed in E. coli, purified, and partially characterized. The encoded enzymes display sulfotransferase activity that is comparable to that of the native sulfotransferase isolated from Renilla reniformis that was reported in 1970. The bioluminescent assay for sensitive detection of 3’-phosphoadenosine 5’-phosphate (PAP) using the recombinant sulfotransferase is demonstrated.

Sulfation and Its Effect on the Bioactivity of Magnolol, the Main Active Ingredient of Magnolia Officinalis.

Magnolol, the main active ingredient of Magnolia officinalis, has been reported to display anti-inflammatory activity. Sulfation plays an important role in the metabolism of magnolol. The magnolol sulfated metabolite was identified by the ultra-performance liquid chromatography to quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and a proton nuclear magnetic resonance (1H-NMR). The magnolol sulfation activity of seven major recombinant sulfotransferases (SULTs) isoforms (SULT1A1*1, SULT1A1*2, SULT1A2, SULT1A3, SULT1B1, SULT1E1, and SULT2A1) was analyzed. The metabolic profile of magnolol was investigated in liver S9 fractions from human (HLS9), rat (RLS9), and mouse (MLS9). The anti-inflammatory effects of magnolol and its sulfated metabolite were evaluated in RAW264.7 cells stimulated by lipopolysaccharide (LPS). Magnolol was metabolized into a mono-sulfated metabolite by SULTs. Of the seven recombinant SULT isoforms examined, SULT1B1 exhibited the highest magnolol sulfation activity. In liver S9 fractions from different species, the CLint value of magnolol sulfation in HLS9 (0.96 µL/min/mg) was similar to that in RLS9 (0.99 µL/min/mg) but significantly higher than that in MLS9 (0.30 µL/min/mg). Magnolol and its sulfated metabolite both significantly downregulated the production of inflammatory mediators (IL-1β, IL-6 and TNF-α) stimulated by LPS (p < 0.001). These results indicated that SULT1B1 was the major enzyme responsible for the sulfation of magnolol and that the magnolol sulfated metabolite exhibited potential anti-inflammatory effects.

Identification of Galeterone and Abiraterone as Inhibitors of Dehydroepiandrosterone Sulfonation Catalyzed by Human Hepatic Cytosol, SULT2A1, SULT2B1b, and SULT1E1

Galeterone and abiraterone acetate are antiandrogens developed for the treatment of metastatic castration-resistant prostate cancer. In the present study, we investigated the effect of these drugs on dehydroepiandrosterone (DHEA) sulfonation catalyzed by human liver and intestinal cytosols and human recombinant sulfotransferase enzymes (SULT2A1, SULT2B1b, and SULT2E1) and compared their effects to those of other antiandrogens (cyproterone acetate, spironolactone, and danazol). Each of these chemicals (10 μM) inhibited DHEA sulfonation catalyzed by human liver and intestinal cytosols. Enzyme kinetic analysis showed that galeterone and abiraterone acetate inhibited human liver cytosolic DHEA sulfonation with apparent Ki values at submicromolar concentrations, whereas cyproterone acetate, spironolactone, and danazol inhibited it with apparent Ki values at low micromolar concentrations. The temporal pattern of abiraterone formation and abiraterone acetate depletion suggested that the metabolite abiraterone, not the parent drug abiraterone acetate, was responsible for the inhibition of DHEA sulfonation in incubations containing human liver cytosol and abiraterone acetate. Consistent with this proposal, similar apparent Ki values were obtained, regardless of whether abiraterone or abiraterone acetate was added to the enzymatic incubation. Abiraterone was more effective than abiraterone acetate in inhibiting DHEA sulfonation when catalyzed by human recombinant SULT2A1 or SULT2B1b. In conclusion, galeterone and abiraterone are novel inhibitors of DHEA sulfonation, as determined in enzymatic incubations containing human tissue cytosol (liver or intestinal) or human recombinant SULT enzyme (SULT2A1, SULT2B1b, or SULT1E1). Our findings on galeterone and abiraterone may have implications in drug-drug interactions and biosynthesis of steroid hormones.

Sulfation and amidinohydrolysis in the biosynthesis of giant linear polyenes.

Clethramycin from Streptomyces malaysiensis DSM4137, and mediomycins (produced together with clethramycin from Streptomyces mediocidicus), are near-identical giant linear polyenes apparently constructed from, respectively, a 4-guanidinobutanoate or 4-aminobutanoate starter unit and 27 polyketide extender units, and bearing a specific O-sulfonate modification at the C-29 hydroxy group. We show here that mediomycins are actually biosynthesised not by use of a different starter unit but by direct late-stage deamidination of (desulfo)clethramycin. A gene (slf) encoding a candidate sulfotransferase has been located in both gene clusters. Deletion of this gene in DSM4137 led to accumulation of desulfoclethramycin only, instead of a mixture of desulfoclethramycin and clethramycin. The mediomycin gene cluster does not encode an amidinohydrolase, but when three candidate amidinohydrolase genes from elsewhere in the S. mediocidicus genome were individually expressed in Escherichia coli and assayed, only one of them (medi4948), located 670 kbp away from the mediomycin gene cluster on the chromosome, catalysed the removal of the amidino group from desulfoclethramycin. Subsequent cloning of medi4948 into DSM4137 caused mediomycins A and B to accumulate at the expense of clethramycin and desulfoclethramycin, respectively, a rare case where an essential biosynthetic gene is not co-located with other pathway genes. Clearly, both desulfoclethramycin and clethramycin are substrates for this amidinohydrolase. Also, purified recombinant sulfotransferase from DSM4137, in the presence of 3'-phosphoadenosine-5'-phosphosulfate as donor, efficiently converted mediomycin B to mediomycin A in vitro. Thus, in the final steps of mediomycin A biosynthesis deamidination and sulfotransfer can take place in either order.

In vitro synthesis and characterisation of three fenoterol sulfoconjugates detected in fenoterol post-administration urine samples

Fenoterol, a fast-acting β2-adrenergic agonist, is used in the therapy of obstructive pulmonary diseases and for the inhibition of premature labour obstetrics. Doping control for β2-agonists, which are prohibited in sports by the World Anti-Doping Agency, is commonly performed by liquid chromatography/mass spectrometry after hydrolysis of phase II metabolites. The continuing development of analytical procedures has led to direct injection of urine samples without sample preparation becoming a viable tool. For the detection of substances without sample preparation, including hydrolysis, detailed information of the phase II metabolism of the substances is essential. In this study, human S9 fractions of different tissues and two recombinant sulfotransferases were investigated for their potential to form fenoterol sulfoconjugates, which were characterised in detail. Two mono-sulfoconjugates and one bis-sulfoconjugate were synthesised and their structures confirmed by liquid chromatography–high-resolution/high-accuracy mass spectrometry. All of the metabolites were identified as esterified phenolic compounds. Excretion studies with orally and inhalatively administered fenoterol proved the occurrence of the sulfoconjugates in vivo. Inhalatively administered fenoterol resulted in the detection of the two monosulfoconjugates in low amounts in urine due to the lower inhalation dose of fenoterol compared to the oral dose. After oral uptake of fenoterol, the two mono-sulfoconjugates and a fenoterol bis-sulfoconjugate were detected in urine. This is the first report of the bis-sulfoconjugate.

LC-MS and LC-MS/MS studies of incorporation of 34SO3 into glycosaminoglycan chains by sulfotransferases

The specificities of glycosaminoglycan (GAG) modification enzymes, particularly sulfotransferases, and the locations and concentrations of these enzymes in the Golgi apparatus give rise to the mature GAG polysaccharides that bind protein ligands. We studied the substrate specificities of sulfotransferases with a stable isotopically labeled donor substrate, 3'-phosphoadenosine-5'-phosphosulfate. The sulfate incorporated by in vitro sulfation using recombinant sulfotransferases was easily distinguished from those previously present on the GAG chains using mass spectrometry. The enrichment of the [M + 2] isotopic peak caused by (34)S incorporation, and the [M + 2]/[M + 1] ratio, provided reliable and sensitive measures of the degree of in vitro sulfation. It was found that both CHST3 and CHST15 have higher activities at the non-reducing end (NRE) units of chondroitin sulfate, particularly those terminating with a GalNAc monosaccharide. In contrast, both NDST1 and HS6ST1 showed lower activities at the NRE of heparan sulfate (HS) chains than at the interior of the chain. Contrary to the traditional view of HS biosynthesis processes, NDST1 also showed activity on O-sulfated GlcNAc residues.

Enzymatic Production of Heparin Anticoagulants for Human Use

Currently, the pharmaceutical heparin being used as an anticoagulant is isolated from bovine lungs or porcine intestines. The primary problem resulting from heparin isolation from animal sources is that this process leaves the heparin open to contamination from animal pathogens that may also be infectious to humans. Enzymatic production of heparin anticoagulant is an attractive alternative to animal derived heparin as it removes the potential for contamination with animal pathogens and also potentially reduces molecular heterogeneity. Heparosan, produced in bacteria, has a structure similar to the backbone structure of heparin and therefore is used as a precursor in the enzymatic production of heparin anticoagulant using various recombinant sulfotransferases. After production, the synthetic heparin was assessed for anticoagulant activity. This approach offers a great promise for human use in the coming years.Supported by NIHLBI grant HL107152.

Detoxication of Benzo[a]pyrene-7,8-dione by Sulfotransferases (SULTs) in Human Lung Cells

Polycyclic aromatic hydrocarbons (PAH) are environmental and tobacco carcinogens. Human aldo-keto reductases catalyze the metabolic activation of proximate carcinogenic PAH trans-dihydrodiols to yield electrophilic and redox-active o-quinones. Benzo[a]pyrene-7,8-dione a representative PAH o-quinone is reduced back to the corresponding catechol to generate a futile redox-cycle. We investigated whether sulfonation of PAH catechols by human sulfotransferases (SULT) could intercept the catechol in human lung cells. RT-PCR identified SULT1A1, -1A3, and -1E1 as the isozymes expressed in four human lung cell lines. The corresponding recombinant SULTs were examined for their substrate specificity. Benzo[a]pyrene-7,8-dione was reduced to benzo[a]pyrene-7,8-catechol by dithiothreitol under anaerobic conditions and then further sulfonated by the SULTs in the presence of 3'-[(35)S]phosphoadenosine 5'-phosphosulfate as the sulfonate group donor. The human SULTs catalyzed the sulfonation of benzo[a]pyrene-7,8-catechol and generated two isomeric benzo[a]pyrene-7,8-catechol O-monosulfate products that were identified by reversed phase HPLC and by LC-MS/MS. The various SULT isoforms produced the two isomers in different proportions. Two-dimensional (1)H and (13)C NMR assigned the two regioisomers of benzo[a]pyrene-7,8-catechol monosulfate as 8-hydroxy-benzo[a]pyrene-7-O-sulfate (M1) and 7-hydroxy-benzo[a]pyrene-8-O-sulfate (M2), respectively. The kinetic profiles of three SULTs were different. SULT1A1 gave the highest catalytic efficiency (k(cat)/K(m)) and yielded a single isomeric product corresponding to M1. By contrast, SULT1E1 showed distinct substrate inhibition and formed both M1 and M2. Based on expression levels, catalytic efficiency, and the fact that the lung cells only produce M1, it is concluded that the major isoform that can intercept benzo[a]pyrene-7,8-catechol is SULT1A1.

Sulfation of 4-Hydroxy Toremifene: Individual Variability, Isoform Specificity, and Contribution to Toremifene Pharmacogenomics

Toremifene (TOR) is a selective estrogen receptor modulator used in adjuvant therapy for breast cancer and in clinical trials for prostate cancer prevention. The chemical structure of TOR differs from that of tamoxifen (TAM) by the presence of a chlorine atom in the ethyl side chain, resulting in a more favorable toxicity spectrum with TOR. In addition, some patients who fail on TAM therapy benefit from high-dose TOR therapy. Several studies have indicated that functional genetic variants in the TAM metabolic pathway influence response to therapy, but pharmacogenomic studies of patients treated with TOR are lacking. In this study, we examined individual variability in sulfation of 4-hydroxy TOR (4-OH TOR) (the active metabolite of TOR) in human liver cytosols from 104 subjects and found approximately 30-fold variation in activity. 4-OH TOR sulfation was significantly correlated (r = 0.98, P < 0.0001) with β-naphthol sulfation (diagnostic for SULT1A1) but not with 17β estradiol sulfation, a diagnostic substrate for SULT1E1(r = 0.09, P = 0.34). Examination of recombinant sulfotransferases (SULTs) revealed that SULT1A1 and SULT1E1 catalyzed 4-OH TOR sulfation, with apparent Km values of 2.6 and 6.4 μM and Vmax values of 8.5 and 5.5 nmol x min(-1) x mg protein(-1), respectively. 4-OH TOR sulfation was inhibited by 2,6-dichloro-4-nitrophenol (IC50 = 2.34 ± 0.19 μM), a specific inhibitor of SULT1A1. There was also a significant association between SULT1A1 genotypes and copy number and 4-OH TOR sulfation in human liver cytosols. These results indicate that variability in sulfation could contribute to response to TOR in the treatment of breast and prostate cancer.

Sulfation of fulvestrant by human liver cytosols and recombinant SULT1A1 and SULT1E1

Fulvestrant (Faslodex™) is a pure antiestrogen that is approved to treat hormone receptor-positive metastatic breast cancer in postmenopausal women. Previous studies have demonstrated that fulvestrant metabolism in humans involves cytochromes P450 and UDP-glucuronosyltransferases (UGTs). To date, fulvestrant sulfation has not been characterized. This study examined fulvestrant sulfation with nine recombinant sulfotransferases and found that only SULT1A1 and SULT1E1 displayed catalytic activity toward this substrate, with Km of 4.2 ± 0.99 and 0.2 ± 0.16 μM, respectively. In vitro assays of 104 human liver cytosols revealed marked individual variability that was highly correlated with β-naphthol sulfation (SULT1A1 diagnostic substrate; r = 0.98, P < 0.0001), but not with 17β-estradiol sulfation (SULT1E1 diagnostic substrate; r = 0.16, P = 0.10). Fulvestrant sulfation was correlated with both SULT1A1*1/2 genotype (P value = 0.023) and copy number (P < 0.0001). These studies suggest that factors influencing SULT1A1/1E1 tissue expression and/or enzymatic activity could influence the efficacy of fulvestrant therapy.

Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases

Sulfated glycans play critical roles during the development, differentiation and growth of various organisms. The most well-studied sulfated molecules are sulfated glycosaminoglycans (GAGs). Recent incidents of heparin drug contamination convey the importance of having a convenient and sensitive method for detecting different GAGs. Here, we describe a molecular method to detect GAGs in biological and biomedical samples. Because the sulfation of GAGs is generally not saturated in vivo, it is possible to introduce the radioisotope (35)S in vitro using recombinant sulfotransferases, thereby allowing detection of minute quantities of these molecules. This strategy was also successfully applied in the detection of other glycans. As examples, we detected contaminant GAGs in commercial heparin, heparan sulfate and chondroitin samples. The identities of the contaminant GAGs were further confirmed by lyase digestion. Oversulfated chondroitin sulfate was detectable only following a simple desulfation step. Additionally, in vitro sulfation by sulfotransferases allowed us to map glycan epitopes in biological samples. This was illustrated using mouse embryo and rat organ tissue sections labeled with the following carbohydrate sulfotransferases: CHST3, CHST15, HS3ST1, CHST4 and CHST10.

Abstract 2619: Sulfation of toremifene: Individual variability, isoform specificity, and contribution to toremifene pharmacogenomics

Abstract Toremifene (TOR) is a selective estrogen receptor modulator (SERM) used in adjuvant therapy for breast cancer, and more recently is in clinical trials for prostate cancer prevention in patients with high grade PIN. The chemical structure of TOR differs from tamoxifen (TAM) only by the presence of a chlorine atom in the ethyl side chain. That difference results in distinct breakdown products of the two drugs, which may provide for a more favorable toxicity spectrum with TOR compared to TAM. TOR has a similar mechanism of action to that of TAM, in that its 4-hydroxy metabolites bind specifically to estrogen receptors and, as an estrogen receptor-TOR complex, to chromatin. Interestingly, studies have indicated that a subset of patients who fail on TAM therapy benefit from high-dose TOR therapy, indicating that pharmacogenomics could be an important determinant of response to treatment with TOR. Several studies have indicated that functional genetic variants in the TAM metabolic pathway influence response to therapy, but to date, pharmacogenomic studies of patients treated with TOR are lacking. In order to perform candidate gene association studies, it is necessary to identify the enzymes involved in TOR metabolism. While the Phase I metabolism of TOR has been well-characterized, Phase II metabolism of the active 4-OH-TOR is not well-defined. In this study, we examined individual variability in sulfation of TOR, and found 20-fold variation in human liver cytosols from 100 subjects. Examination of recombinant sulfotransferases revealed that SULT1A1, SULT1E1, and to a lesser extent, SULT2A1 catalyzed the sulfation of TOR. SULT1A1 is the most highly expressed hepatic sulfotransferase, and there was a significant association between SULT1A1*1/*2 genotype and TOR sulfation in human liver cytosols (p=0.001). Since target tissue metabolism is also a critical determinant of clinical efficacy, we also examined the presence of sulfotransferase isoforms in normal prostate (n= 8), BPH tissue (n= 12), and prostate cancer (n=4). Quantitative RT-PCR analysis showed the presence of SULT1A1 and SULT2B1b in BPH and Prostate cancer tissues. These results indicate that variability in sulfation, in terms of both first-pass metabolism and target tissue metabolism, contributes to response to TOR for prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2619.

In-vitro sulfation of piceatannol by human liver cytosol and recombinant sulfotransferases

Abstract Objectives The aim of this study was to investigate the concentration-dependent sulfation of piceatannol, a dietary polyphenol present in grapes and wine and known for its promising anticancer and anti-inflammatory activity. Methods Sulfation of piceatannol was investigated in human liver cytosol as well as using a panel of recombinant sulfotransferase isoforms. Furthermore, the chemical structures of novel sulfates were identified by liquid chromatography/mass spectrometry (LC/MS). Key findings In the presence of 3′-phosphoadenosine-5′-phosphosulfate, three metabolites could be detected whose structures were identified by LC/MS/MS as piceatannol disulfate (M1) and two monosulfates (M2, M3). The kinetics of M1 formation exhibited a pattern of substrate inhibition with a Ki of 21.8 ± 11.3 μM and a Vmax/Km of 7.63 ± 1.80 μl/mg protein per min. Formation of M2 and M3 showed sigmoidal kinetics with apparent Km and Vmax values of 27.1 ± 2.90 μM and 118.4 ± 4.38 pmol/mg protein per min, respectively, for M2; and 35.7 ± 2.70 μM and 81.8 ± 2.77 pmol/mg protein per min, respectively, for M3. Incubation in the presence of human recombinant sulfotransferases (SULTs) demonstrated that M1 was formed equally by SULT1A1*1 and SULT1B1 and to a lesser extent by SULT1A1*2. M2 was preferentially catalysed by SULT1A1*2, 1A3 and 1E1. The formation of M3, however, was mainly catalysed by SULT1A2*1 and SULT1A3. Conclusions Our results elucidate the importance of piceatannol sulfation in human liver, which must be taken into account in humans after dietary intake of piceatannol.

Inhibitory effects of kynurenic acid, a tryptophan metabolite, and its derivatives on cytosolic sulfotransferases

KYNA (kynurenic acid) is an endogenous metabolite of tryptophan in the kynurenine pathway and has been characterized as an antagonist of ionotropic glutamate receptors. In addition, we have reported this endogenous compound as a potent inhibitor of SULTs (cytosolic sulfotransferases). In the present study we characterized the inhibitory effects of KYNA on several human (h) and mouse (m) recombinant SULTs. No sulfate metabolite of KYNA was detected with mouse and human SULTs examined under the conditions used, suggesting that it is a bona fide inhibitor of SULTs. Among the mouse enzymes examined, KYNA exhibited selective inhibitory effects on Sult1b1-mediated sulfation of various compounds with IC50 values in the low micromolar range (2.9-4.9 microM). KYNA also exerted an inhibitory activity towards hSULT1A1 and hSULT1B1. The inhibitory potency of KYNA for mSult1b1 was stronger than that of 2,6-dichloro-4-nitrophenol, a known non-specific SULT inhibitor, whereas the potencies of these two inhibitors for hSULT1B1 were comparable. The inhibitory characteristics of KYNA were clearly distinct from those of mefenamic acid, a selective inhibitor of SULT1A enzymes. The KYNA derivatives 5,7-dichlorokynurenic acid and L689,560 exhibited preferential inhibitory effects on hSULT1A1 and hSULT1B1 respectively. Interestingly, gavestinel, another KYNA derivative, was found to be an extremely potent inhibitor of hSULT1B1. Finally, we have demonstrated that the mechanism underlying the KYNA inhibition varied depending on the enzyme and substrate involved. Taken together, the present results unveil another distinct aspect of KYNA and its derivatives as an inhibitor of SULTs.

In-vitro sulfation of piceatannol by human liver cytosol and recombinant sulfotransferases

The aim of this study was to investigate the concentration-dependent sulfation of piceatannol, a dietary polyphenol present in grapes and wine and known for its promising anticancer and anti-inflammatory activity. Sulfation of piceatannol was investigated in human liver cytosol as well as using a panel of recombinant sulfotransferase isoforms. Furthermore, the chemical structures of novel sulfates were identified by liquid chromatography/mass spectrometry (LC/MS). In the presence of 3'-phosphoadenosine-5'-phosphosulfate, three metabolites could be detected whose structures were identified by LC/MS/MS as piceatannol disulfate (M1) and two monosulfates (M2, M3). The kinetics of M1 formation exhibited a pattern of substrate inhibition with a Ki of 21.8 +/- 11.3 microm and a Vmax/Km of 7.63 +/- 1.80 microl/mg protein per min. Formation of M2 and M3 showed sigmoidal kinetics with apparent Km and Vmax values of 27.1 +/- 2.90 microm and 118.4 +/- 4.38 pmol/mg protein per min, respectively, for M2; and 35.7 +/- 2.70 microm and 81.8 +/- 2.77 pmol/mg protein per min, respectively, for M3. Incubation in the presence of human recombinant sulfotransferases (SULTs) demonstrated that M1 was formed equally by SULT1A1*1 and SULT1B1 and to a lesser extent by SULT1A1*2. M2 was preferentially catalysed by SULT1A1*2, 1A3 and 1E1. The formation of M3, however, was mainly catalysed by SULT1A2*1 and SULT1A3. Our results elucidate the importance of piceatannol sulfation in human liver, which must be taken into account in humans after dietary intake of piceatannol.

The Suggested Physiologic Aryl Hydrocarbon Receptor Activator and Cytochrome P4501 Substrate 6-Formylindolo[3,2-b]carbazole Is Present in Humans

Dioxins and other polycyclic aromatic compounds formed during the combustion of waste and fossil fuels represent a risk to human health, as well as to the well being of our environment. Compounds of this nature exert carcinogenic and endocrine-disrupting effects in experimental animals by binding to the orphan aryl hydrocarbon receptor (AhR). Understanding the mechanism of action of these pollutants, as well as the physiological role(s) of the AhR, requires identification of the endogenous ligand(s) of this receptor. We reported earlier that activation of AhR by ultraviolet radiation is mediated by the chromophoric amino acid tryptophan (Trp), and we suggested that a new class of compounds derived from Trp, in particular 6-formylindolo[3,2-b]carbazole (FICZ), acts as natural high affinity ligands for this receptor. Here we describe seven new FICZ-derived indolo[3,2-b]carbazole-6-carboxylic acid metabolites and two sulfoconjugates, and we demonstrate the following. (i) FICZ is formed efficiently by photolysis of Trp upon exposure to visible light. (ii) FICZ is an exceptionally good substrate for cytochromes P450 (CYP) 1A1, 1A2, and 1B1, and its hydroxylated metabolites are remarkably good substrates for the sulfotransferases (SULTs) 1A1, 1A2, 1B1, and 1E1. Finally, (iii) sulfoconjugates of phenolic metabolites of FICZ are present in human urine. Our findings indicate that formylindolo[3,2-b]carbazols are the most potent naturally occurring activators of the AhR signaling pathway and may be the key substrates of the CYP1 and SULT1 families of enzymes. These conclusions contradict the widespread view that xenobiotic compounds are the major AhR ligands and CYP1 substrates.

Inhibitory effects of various beverages on human recombinant sulfotransferase isoforms SULT1A1 and SULT1A3

Sulfotransferase (SULT) 1A1 and SULT1A3 play important roles in the presystemic inactivation of beta(2) agonists in the liver and intestine, respectively. The study aimed to investigate the inhibitory effects of grapefruit juice, orange juice, green tea, black tea and oolong tea and their constituents on the activities of SULT1A1 and SULT1A3. The activities of both SULT1A1 and SULT1A3 were significantly inhibited by all the beverages investigated at a concentration of 10%. The beverage constituents were tested in concentration ranges considered to be physiologically relevant. The grapefruit constituent, quercetin, completely inhibited SULT1A1, while quercetin and naringin both partially inhibited SULT1A3. The orange constituents, tangeretin and nobiletin, also completely inhibited SULT1A1. The tea constituents, (-)-epicatechin gallate and (-)-epigallocatechin gallate, both almost completely inhibited SULT1A1 and SULT1A3. Moreover, the theaflavin and thearubigin fractions of black tea both completely inhibited SULT1A1 and strongly inhibited SULT1A3. The inhibitory action of green tea on SULT1A3 was competitive, while that of black tea and oolong tea was mixed competitive/non-competitive. Mechanism-based inhibition was not observed with any beverage. In conclusion, various beverages, especially teas, inhibit the function of SULT1A3, and therefore may have the potential to increase the bioavailability of orally administered substrates of SULT1A3, such as beta(2) agonists.

Sulfonation of 17β-estradiol and inhibition of sulfotransferase activity by polychlorobiphenylols and celecoxib in channel catfish, Ictalurus punctatus

The sulfonation of 17β-estradiol (E2) by human liver and recombinant sulfotransferases is influenced by environmental contaminants such as hydroxylated metabolites of polychlorinated biphenyls (OH-PCBs), which are potent inhibitors, and the therapeutic drug, celecoxib, which affects positional sulfonation of E2. In some locations, the aquatic environment is contaminated by PCBs, OH-PCBs and widely used therapeutic drugs. The objectives of this study were to investigate the sulfonation kinetics of E2 in liver cytosol from channel catfish ( Ictalurus punctatus); to examine the effect of OH-PCBs on E2 sulfonation; and to determine if celecoxib altered the position of E2 sulfonation, as it does with human liver cytosol. E2 was converted to both 3- and 17-sulfates by catfish liver cytosol. At E2 concentrations below 1 μM, formation of E2-3-sulfate (E2-3-S) predominated, but substrate inhibition was observed at higher concentrations. Rates of E2-3-S formation at different E2 concentrations were fit to a substrate inhibition model, with K ′ m and V ′ max values of 0.40 ± 0.10 μM and 91.0 ± 4.7 pmol/min/mg protein, respectively and K i of 1.08 ± 0.09 μM. The formation of E2-17-S fit Michaelis-Menten kinetics over the concentration range 25 nM to 2.5 μM, with K m and V max values of 1.07 ± 0.23 μM and 25.7 ± 4.43 pmol/min/mg protein, respectively. The efficiency ( V max/ K m) of formation of E2-3-S was 9.8-fold higher than that of E2-17-S. Several OH-PCBs inhibited E2 3-sulfonation, measured at an E2 concentration of 1 nM. Of those tested, the most potent inhibitor was 4′-OH-CB79, with two chlorine atoms flanking the OH group (IC 50: 94 nM). The inhibition of estrogen sulfonation by OH-PCBs may disrupt the endocrine system and thus contribute to the known toxic effects of these compounds. Celecoxib did not stimulate E2-17-S formation, as is the case with human liver cytosol, but did inhibit the formation of E2-3-S (IC 50: 44 μM) and to a lesser extent, E2-17-S (IC 50: >160 μM), suggesting the previously found effect of celecoxib on E2-17-S formation may be specific to human SULT2A1.

Enzymatic Redesigning of Biologically Active Heparan Sulfate

Heparan sulfate carries a wide range of biological activities, regulating blood coagulation, cell differentiation, and inflammatory responses. The sulfation patterns of the polysaccharide are essential for the biological activities. In this study, we report an enzymatic method for the sulfation of multimilligram amounts of heparan sulfate with specific functions using immobilized sulfotransferases combined with a 3'-phosphoadenosine 5'-phosphosulfate regeneration system. By selecting appropriate enzymatic modification steps, an inactive precursor has been converted to the heparan sulfate having three distinct biological activities, associated with binding to antithrombin, fibroblast growth factor-2, and herpes simplex virus envelope glycoprotein D. Because the recombinant sulfotransferases are expressed in bacteria, and the method uses a low cost sulfo donor, it can be readily utilized to synthesize large quantities of anticoagulant heparin drug or other biologically active heparan sulfates.