RESUMO
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.
Assuntos
Glicosaminoglicanos/química , Proteínas Recombinantes/química , Sulfotransferases/química , Animais , Encéfalo/metabolismo , Química Encefálica , Condroitina/análise , Suplementos Nutricionais/análise , Contaminação de Medicamentos/prevenção & controle , Embrião de Mamíferos/química , Embrião de Mamíferos/metabolismo , Glucosamina/análise , Glicosaminoglicanos/metabolismo , Heparina/química , Rim/química , Rim/metabolismo , Pulmão/química , Pulmão/metabolismo , Camundongos , Músculo Liso/química , Músculo Liso/metabolismo , Miocárdio/química , Miocárdio/metabolismo , Processamento de Proteína Pós-Traducional , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/biossíntese , Coloração e Rotulagem , Sulfotransferases/biossíntese , Radioisótopos de EnxofreRESUMO
There are no reports that hypothalamus can directly affect the expression of neuron-related genes and immune-related genes in liver. We identified genes of which expression profiles showed significant modulation in rat liver after ventromedial hypothalamic (VMH) lesions. Total RNA was extracted, and differences in the gene expression profiles between rats at day 3 after VMH lesioning and sham-VMH lesioned rats were investigated using DNA microarray analysis. The result revealed that VMH lesions regulated the genes that were involved in functions related to neuronal development and immunofunction in the liver. Real-time PCR also confirmed that gene expression of SULT4A1 was upregulated, but expression of ACSL1 and CISH were downregulated at day 3 after VMH lesions. VMH lesions may change the expression of neuron-related genes and immune-related genes in rat liver.
Assuntos
Regulação da Expressão Gênica , Hipotálamo/fisiologia , Fígado/imunologia , Fígado/metabolismo , Neuroimunomodulação , Neurônios/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Feminino , Perfilação da Expressão Gênica , Hipotálamo/citologia , Fígado/inervação , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Sulfotransferases/biossíntese , Sulfotransferases/genética , Proteínas Supressoras da Sinalização de Citocina/biossíntese , Proteínas Supressoras da Sinalização de Citocina/genéticaRESUMO
Sulfotransferases are phase II drug-metabolizing enzymes. While the induction of sulfotransferases by hormones and endogenous molecules is relatively well known, induction by xenobiotics is not well studied. Isoflavones are naturally occurring phyto-oestrogens, mainly existing in soy food products. They have been described as health-promoting, disease-preventing dietary supplements and as agents with cancer-preventive activities. Recently, isoflavones have been reported to interact with nuclear receptors, including those that are known to mediate the induction of drug-metabolizing enzymes. In the present investigation, the isoflavone genistein was shown to be a xenobiotic inducer of human sulfotransferases in transformed human liver cells (HepG2) and colon carcinoma cells (Caco-2). Enzymatic activity assay, Western blot, and real-time reverse transcription-polymerase chain reaction (RT-PCR) results demonstrated that genistein significantly induced protein and mRNA expression of human simple phenol sulfotransferase (hSULT1A1) and human dehydroepiandrosterone sulfotransferase (hSULT2A1) in HepG2 and Caco-2 cells. The induction was time-dependent and dose-dependent. Western blot results agreed well with real-time RT-PCR results, suggesting that induction occurred at the gene transcription level. This isoflavone is the first nutritionally related phyto-oestrogen shown to induce human sulfotransferases in HepG2 and Caco-2 cells.
Assuntos
Arilsulfotransferase/biossíntese , Genisteína/farmacologia , Fitoestrógenos/farmacologia , Sulfotransferases/biossíntese , Arilsulfotransferase/genética , Western Blotting , Linhagem Celular Tumoral , Citosol/enzimologia , Indução Enzimática , Regulação Enzimológica da Expressão Gênica , Humanos , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfotransferases/genéticaRESUMO
Epidemiology studies have shown that consumption of fruits and vegetables is associated with the prevention of chronic diseases such as cancer and cardiovascular disease. Induction of cellular phase II detoxifying enzymes is associated with cancer preventive potential. Phenolsulfotransferases (PSTs) are traditionally known as phase II drug-metabolizing or detoxifying enzymes that facilitate the removal of drugs and other xenobiotic compounds. Phenolic acids are known to increase the activities of PSTs. In the present study, human HepG2 cells were used as model to investigate the influence of twenty vegetables on human PST activity and to evaluate the relationships to their antioxidant activity and total phenolics content. The result showed that PST-P activity was significantly (p < 0.01) induced by asparagus, broccoli, cauliflower, celery and eggplant, whereas PST-M activity was induced by asparagus, broccoli, carrot, eggplant and potato at a concentration of 100 microg/ml. The vegetable extracts that induced both forms of PSTs activities were found to have higher antioxidant capacities and total phenolic content in the oxygen radical absorbance capacity (ORAC) and Folin-Ciocalteu assay. The major polyphenols in broccoli, the most potential inducer in both forms of PSTs activities, was antioxidant phenolic acids. HPLC retention times and standard spiked indicated the presence of gallic acid, p-hydroxybenzoic acid, p-coumaric acid, gentisic acid and ferulic acid in broccoli. The overall effect of vegetables tested on the activity of PST-P was well correlated to their ORAC value and total phenolics content (r= 0.82, p < 0.05 and r = 0.78, p < 0.05). These results imply that vegetables have a capability of inducing PST activity, and the PST induction may be possibly ascribed to antioxidant phenolic acids in vegetable extracts.
Assuntos
Antioxidantes/farmacologia , Hidroxibenzoatos/análise , Sulfotransferases/metabolismo , Verduras/química , Antioxidantes/isolamento & purificação , Arilsulfotransferase , Brassica/química , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Humanos , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfotransferases/biossíntese , Sulfotransferases/genéticaRESUMO
It has been previously demonstrated in a human-derived hepatoma cell line (HepG2) that juices from cruciferous vegetables protect against the genotoxicity caused by dietary carcinogens. HepG2 cells possess different enzymes involved in the biotransformation of xenobiotics. Therefore, we investigated the effect of cruciferous juices on the activities of CYP 1A and several phase II enzymes in this cell model. For each experiment, 1 x 10(6) cells were seeded on Petri dishes. After 2 days, the juices (0.5-8 microl/ml of culture medium) were added for 48 h prior to cell harvesting. The addition of juice from water cress (Nasturtium officinalis R. Br) significantly increased the activities of ethoxyresorufin-O-deethylase at high doses only and NAD(P)H-quinone reductase in a dose-dependent manner (1.8- and 5-fold, respectively). The addition of juice from garden cress (Lepidum sativum L.) significantly increased the activities of NAD(P)H-quinone reductase and UDP-glucuronosyl-transferase with a maximal effect around the dose of 2 microl/ml juice (1.4- and 1.2-fold, respectively) while the other enzymes were not altered. Mustard (Sinapis alba L.) juice increased the activities of NAD(P)H-quinone reductase (2.6-fold at the dose of 8 microl/ml), and N-acetyl-transferase (1.4-fold at the dose of 8 microl/ml) in a dose-dependent manner while a maximal induction of UDP-glucuronosyl-transferase was obtained with a dose of 2 microl/ml (1.8-fold). These observations show that the three juices have different induction profiles: only water cress acted as a bifunctional inducer by enhancing both phase I and phase II enzymes. As a consequence, each juice may preferentially inhibit the genotoxicity of specific compounds.
Assuntos
Brassicaceae , Fígado/enzimologia , Extratos Vegetais/farmacologia , Acetiltransferases/biossíntese , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Citocromo P-450 CYP1A1/biossíntese , Indução Enzimática , Glucuronosiltransferase/biossíntese , Glutationa Transferase/biossíntese , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/biossíntese , Sulfotransferases/biossínteseRESUMO
The phytotoxin coronatine is a structural analog of octadecanoid signaling molecules, which are well known mediators of plant defense reactions. To isolate novel coronatine-regulated genes from Arabidopsis thaliana, differential mRNA display was performed. Transcript levels of CORI-7 (coronatine induced-7) were rapidly and transiently increased in coronatine-treated plants, and the corresponding cDNA was found to encode the sulfotransferase AtST5a. Likewise, upon wounding, an immediate and transient increase in AtST5a mRNA levels could be observed in both locally wounded and unwounded (systemic) leaves. Furthermore, application of octadecanoids and ethylene as compounds involved in plant wound defense reactions resulted in AtST5a gene activation, whereas pathogen defense-related signals (yeast elicitor and salicylic acid) were inactive. AtST5a and its close homologs AtST5b and AtST5c were purified as His6-tagged proteins from Escherichia coli. The three enzymes were shown to catalyze the final step in the biosynthesis of the glucosinolate (GS) core structure, the sulfation of desulfoglucosinolates (dsGSs). They accept a broad range of dsGSs as substrates. However, in a competitive situation, AtST5a clearly prefers tryptophan- and phenylalanine-derived dsGSs, whereas long chain dsGSs derived from methionine are the preferred substrates of AtST5b and AtST5c. Treatment of Arabidopsis plants with low concentrations of coronatine resulted in an increase in the amounts of specific GSs, primarily glucobrassicin and neoglucobrassicin. Hence, it is suggested that AtST5a is the sulfotransferase responsible for the biosynthesis of tryptophan-derived GSs in vivo.
Assuntos
Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , Glucosinolatos/química , Sulfotransferases/química , Aminoácidos/química , Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/química , Fenômenos Bioquímicos , Bioquímica , Northern Blotting , Catálise , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA Complementar/metabolismo , Escherichia coli/metabolismo , Etilenos/química , Perfilação da Expressão Gênica , Glucosinolatos/biossíntese , Indenos/química , Indóis/química , Modelos Químicos , Filogenia , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por Substrato , Sulfotransferases/biossíntese , Sulfotransferases/metabolismo , Fatores de Tempo , Ativação Transcricional , TriptofanoRESUMO
The response of animals in toxicity studies reflects a complex interaction of a number of variables, some intrinsic to a particular study design and others resulting from the treatment itself. The influences of strain and diet upon constitutive and benzo(a)pyrene (B(a)P) induced activities of several hepatic Phase I and II enzymes were studied in a multifactoral design. Male and female CDF and Crl:CD rats were fed a standard rodent diet ad libitum, a 75% of ad libitum restricted feeding regimen or a phytoestrogen-free diet for approximately 3 weeks. During the last five days of the study, rats were administered either corn oil (vehicle) or 15 mg/kg/day B(a)P via oral gavage. The constitutive activities of hepatic CYP1A1, CYP1A2, CYP2B1/2, and mixed isoforms of UDP-glucuronosyl transferase, sulfotransferase, and glutathione-S-transferase varied significantly by feeding regimen and strain. Responses to B(a)P administration were also observed to be influenced by diet and strain in a manner similar to that observed for constitutive activities. These findings point out the potentially significant interactions of relatively commonly encountered variables that may affect results of hazard testing, especially when employing near metabolically saturating dosages of test chemicals.
Assuntos
Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP2B1/biossíntese , Dieta , Glucuronosiltransferase/biossíntese , Glutationa Transferase/biossíntese , Sulfotransferases/biossíntese , Administração Oral , Animais , Benzo(a)pireno/farmacologia , Peso Corporal/efeitos dos fármacos , Óleo de Milho/administração & dosagem , Sistema Enzimático do Citocromo P-450/biossíntese , Indução Enzimática , Feminino , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Oxirredutases/biossíntese , Ratos , Ratos Endogâmicos , Especificidade da Espécie , Testes de Toxicidade , XenobióticosRESUMO
Precision-cut liver slices are described as a valuable tool for in vitro metabolism studies of potential drug candidates. Recently, some papers reported successful cryopreservation conditions for liver slices, facilitating a broader and more efficient use of the tissue (particularly of human origin). The aim of this study is to evaluate the effect of cryopreservation on both phase I and phase II metabolism in liver slices prepared from mouse, rat, dog, monkey and human, using rapid freezing in the presence of 18% DMSO. Glucuronidation and sulfation activities (phase II) in both freshly prepared and cryopreserved liver slices were determined by rapid LC-MS/MS analyses using 7-hydroxycoumarin as a marker substrate. Testosterone was used as a marker substrate for cytochrome P450 mediated drug metabolism (phase I). Although the metabolic patterns and rates varied among the different species, the phase I and phase II metabolic capacities of the liver slices were well maintained after cryopreservation. Despite the good biotransformation capacity of cryopreserved slices a decrease in viability, expressed as ATP content and LDH leakage, was observed. MTT reduction was well maintained after cryopreservation. The possibility to cryopreserve liver slices will allow a more efficient utilisation of tissue, in particular from human, but also from dog and monkey. Finally, cryopreserved liver slices from mouse, rat, dog, monkey and human with good phase I and II metabolism activities are a useful in vitro tool to compare metabolite profiles of new chemical entities between species.
Assuntos
Biotransformação/fisiologia , Criopreservação/métodos , Fígado/citologia , Fígado/metabolismo , Trifosfato de Adenosina/química , Animais , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Cães , Avaliação Pré-Clínica de Medicamentos , Formazans/metabolismo , Glucuronosiltransferase/biossíntese , Glucuronosiltransferase/química , Humanos , L-Lactato Desidrogenase/metabolismo , Fígado/efeitos dos fármacos , Macaca fascicularis , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Sulfotransferases/biossíntese , Sulfotransferases/química , Testosterona/metabolismo , Testosterona/farmacologia , Sais de Tetrazólio/metabolismo , Preservação de Tecido/métodos , Umbeliferonas/metabolismo , Umbeliferonas/farmacologiaRESUMO
In a previous study of nine human breast-derived cell lines, rates of metabolism of 17beta-estradiol (E(2)) were greatly enhanced when cultures were exposed to the aromatic hydrocarbon receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Elevated rates of E(2) hydroxylation at the C-2, -4, -6alpha and -15alpha positions were observed concomitant with the induction of cytochromes P450 1A1 and 1B1. In each cell line, 2- and 4-hydroxyestradiol (2- and 4-OHE(2)) were converted to 2- and 4-methoxyestradiol (2- and 4-MeOE(2)) by the action of catechol O:-methyltransferase. In this study, conjugation of these estrogen metabolites was investigated. A comparison of the levels of metabolites determined with and without prior treatment of the media with a crude beta-glucuronidase/sulfatase preparation showed that most of the 2-MeOE(2) present was in conjugated form, whereas 4-MeOE(2), 6alpha-OHE(2) and 15alpha-OHE(2) were minimally conjugated. Inhibitor studies suggested that it was the sulfatase activity of the preparation that hydrolyzed the 2-MeOE(2) conjugates in MCF-7 cell media; the presence of 2-MeOE(2)-3-sulfate in MCF-7 culture media was confirmed by electrospray ion-trap mass spectrometry. To identify the enzyme catalyzing this conjugation, the expression of mRNAs encoding five sulfotransferases (SULT1A1, SULT1A2, SULT1A3, SULT1E1 and SULT2A1) was evaluated in the nine cell lines by use of the reverse transcription-polymerase chain reaction. Only expression of SULT1A1 mRNA correlated with the observed conjugation of nanomolar levels of 2-MeOE(2) in these cell lines. Cloning and sequencing of SULT1A1 cDNA from MCF-7 cells revealed that mRNAs encoding two previously identified allelic variants, SULT1A1*1 ((213)Arg) and SULT1A1*2 ((213)His), were expressed in these cells. Heterologous cDNA-directed expression of either variant in MDA-MB-231 cells, which do not normally express SULT1A1, conferred 2-MeOE(2) sulfonation activity. The SULT1A1 allelic variants were also expressed in SF:9 insect cells, from which post-microsomal supernatants were used to determine K:(m) values of 0.90 +/- 0.12 and 0.81 +/- 0.06 microM for SULT1A1*1 and SULT1A1*2, respectively, with 2-MeOE(2) as substrate. These results show that SULT1A1 is an efficient and selective catalyst of 2-MeOE(2) sulfonation and, as such, may be important in modulating the anticarcinogenic effects of 2-MeOE(2) that have been described recently.
Assuntos
Arilsulfotransferase , Neoplasias da Mama/enzimologia , Estradiol/metabolismo , Sulfotransferases/metabolismo , 2-Metoxiestradiol , Animais , Baculoviridae/genética , Mama/enzimologia , Mama/metabolismo , Neoplasias da Mama/metabolismo , Catálise , Células Cultivadas , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Estradiol/análogos & derivados , Amplificação de Genes , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Spodoptera/enzimologia , Spodoptera/virologia , Sulfatos/metabolismo , Sulfotransferases/biossíntese , Sulfotransferases/genética , Células Tumorais CultivadasRESUMO
Using an expression cloning strategy, the cDNA encoding the human HNK-1 sulfotransferase (HNK-1ST) has been cloned. During this cloning we found that HNK-1ST and other Golgi-associated sulfotransferases cloned before share homologous sequences including the RDP motif (Ong, E., Yeh, J.-C., Ding, Y., Hindsgaul, O., and Fukuda, M. (1998) J. Biol. Chem. 223, 5190-5195). Using this conserved sequence in HNK-1ST as a probe, we identified two expressed sequence tags in EST data base which have 31.6 and 30.7% identity with HNK-1ST at the amino acid levels. Expression of these two full-length cDNAs failed to form HNK-1 glycan nor to add sulfate to CD34 or NCAM. Surprisingly, proteins expressed by these cDNAs transferred sulfate to the C-4 position of N-acetylgalactosamine in chondroitin and desulfated dermatan sulfate, thus we named these two enzymes, chondroitin 4-O-sulfotransferase 1 and -2 (C4ST-1 and C4ST-2). Both C4ST-1 and C4ST-2, however, did not form 4, 6-di-O-sulfated N-acetylgalactosamine when chondroitin sulfate C was used as an acceptor. Moreover, analysis of (35)S-labeled dermatan sulfate formed by C4ST-1 indicate that sulfation preferentially took place in GlcA-->GalNAc unit than in IdoA-->GalNAc unit, suggesting that 4-O-sulfation at N-acetylgalactosamine may precede epimerization of glucuronic acid to iduronic acid during dermatan sulfate biosynthesis. Northern analysis demonstrated that the transcript for C4ST-1 is predominantly expressed in peripheral leukocytes and hematopoietic tissues while the C4ST-2 transcript is more widely expressed in various tissues. These results indicate C4ST-1 and C4ST-2 play complementary roles in chondroitin and dermatan sulfate synthesis in different tissues.
Assuntos
Sulfotransferases/biossíntese , Sulfotransferases/genética , Sulfotransferases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Células CHO , Cromatografia Líquida de Alta Pressão , Mapeamento Cromossômico , Clonagem Molecular , Cricetinae , DNA Complementar/metabolismo , Bases de Dados Factuais , Dermatan Sulfato/metabolismo , Etiquetas de Sequências Expressas , Glicosilação , Humanos , Immunoblotting , Dados de Sequência Molecular , Filogenia , Isoformas de Proteínas , Homologia de Sequência de Aminoácidos , Sulfotransferases/química , Fatores de Tempo , Distribuição Tecidual , TransfecçãoRESUMO
Using cDNA of chick chondroitin 6-sulfotransferase (C6ST), human C6ST cDNA has been isolated. The amino acid sequence of human C6ST displayed 74% identity to chick C6ST. The major difference in amino acid sequence between chick C6ST and human C6ST was the presence of a unique hydrophilic domain in human C6ST. A 7.8-kb message of C6ST was expressed ubiquitously in various human adult tissues, indicating a rather diverse function of C6ST.
Assuntos
Sulfotransferases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Células COS , Clonagem Molecular , DNA Complementar/química , DNA Complementar/isolamento & purificação , Expressão Gênica , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , Sulfotransferases/biossíntese , Sulfotransferases/química , Transfecção , Carboidrato SulfotransferasesRESUMO
The St-20 and ST-40 cDNAs encode rat liver hydroxysteroid sulfotransferases (HS-ST) that are 90% identical in amino acid sequence but exhibit different substrate preferences for dehydroepiandrosterone (DHEA), androsterone (AD), and cortisol (CS). ST-40 is active for all three substrates, whereas ST-20 is mainly active for cortisol. To determine the domain responsible for the substrate preferences of the HS-STs, 20 chimeric HS-STs were constructed by reciprocal exchanges of DNA fragments derived from the cDNAs and were expressed in Escherichia coli. Some chimeric enzymes were enzymatically active for all three substrates, and some displayed reduced or lost CS-ST activity, with retention of DHEA- and AD-ST activities. Others lost all HS-ST activity. Analysis revealed that a central region (region III spanning amino acids 102-164 with five amino acid differences between ST-20 and ST-40) is essential for HS-ST activity, whereas regions II (amino acids 65-101) and IV (amino acids 165-219) are unimportant with regard to substrate preference. It was also shown that the parental combination of regions I (amino acids 1-64) and V (amino acids 220-284) is essential for CS-ST activity. Photoaffinity labeling with [35S]3'-phosphoadenosine 5'-phosphosulfate (PAPS) revealed that some inactive chimeras lost affinity for PAPS. These results suggested that an ordered structure formed by regions I, III, and V is required for HS-ST activity, especially for substrate preference and PAPS binding.
Assuntos
Isoenzimas/genética , Fígado/enzimologia , Proteínas Recombinantes de Fusão/genética , Sulfotransferases/genética , Androsterona/metabolismo , Animais , DNA Complementar/genética , Desidroepiandrosterona/metabolismo , Hidrocortisona/metabolismo , Isoenzimas/biossíntese , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Especificidade por Substrato , Sulfotransferases/biossínteseRESUMO
Chondroitin 6-sulfotransferase (C6ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of the N-acetylgalactosamine residue of chondroitin. The enzyme has been purified previously to apparent homogeneity from the serum-free culture medium of chick chondrocytes. The purified enzyme also catalyzed the sulfation of keratan sulfate. We have now cloned the cDNA of the enzyme. This cDNA contains a single open reading frame that predicts a protein composed of 458 amino acid residues. The protein predicts a Type II transmembrane topology similar to other glycosyltransferases and heparin/heparan sulfate N-sulfotransferase/N-deacetylases. Evidence that the predicted protein corresponds to the previously purified C6ST was the following: (a) the predicted sequence of the protein contains all of the known amino acid sequence, (b) when the cDNA was introduced in a eukaryotic expression vector and transfected in COS-7 cells, both the C6ST activity and the keratan sulfate sulfotransferase activity were overexpressed, (c) a polyclonal antibody raised against a fusion peptide, which was expressed from a cDNA containing the sequence coding for 150 amino acid residues of the predicted protein, cross-reacted to the purified C6ST, and (d) the predicted protein contained six potential sites for N-glycosylation, which corresponds to the observation that the purified C6ST is an N-linked glycoprotein. The amino-terminal amino acid sequence of the purified protein was found in the transmembrane domain, suggesting that the purified protein might be released from the chondrocytes after proteolytic cleavage in the transmembrane domain.
Assuntos
Cartilagem/enzimologia , Sulfotransferases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Cartilagem/citologia , Células Cultivadas , Embrião de Galinha , Clonagem Molecular , Sondas de DNA , DNA Complementar/genética , Biblioteca Gênica , Immunoblotting , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Testes de Precipitina , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Seleção Genética , Análise de Sequência de DNA , Sulfotransferases/biossíntese , Carboidrato SulfotransferasesRESUMO
Using two oligoprimers derived from the bovine placental estrogen sulfotransferase sequence, we amplified a probe for human placental estrogen sulfotransferase. Using this probe to screen a human placental cDNA library constructed in lambda gt11, we isolated a cDNA clone of 1.3 kb encoding human estrogen sulfotransferase. DNA analysis predicts a protein of 295 amino acids with a calculated molecular weight of 34,199. Alignment of the amino acid sequence with other sulfotransferases indicates that human placental estrogen sulfotransferase shares 68.6, 68.2 and 65.9% similarity with bovine placental, guinea pig adrenocortical, and rat liver estrogen sulfotransferase, respectively. It shows also 95.6, 57.6, 85.3, and 54.2% similarity to human phenol, human DHEA, rat phenol, and rat hydroxysteroid sulfotransferase, respectively. Transfection of expression vectors encoding human estrogen sulfotransferase and dehydroepiandrosterone (DHEA) sulfotransferase in human adrenal adenocarcinoma SW-13 cells indicates that estrogen sulfotransferase transforms estrone more specifically, whereas DHEA sulfotransferase is more specific for DHEA and pregnenolone.