Purification and properties of steroid sulfatase from human placenta.

Steroid sulfatase was purified approximately 170-fold from normal human placental microsomes and properties of the enzyme were investigated. The major steps in the purification procedure included solubilization with Triton X-100, column chromatofocusing, and hydrophobic interaction chromatography on phenylsepharose CL-4B. The purified sulfatase showed a molecular weight of 500-600 kDa on HPLC gel filtration, whereas the enzyme migrated as a molecular mass of 73 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of steroid sulfatase was estimated to be 6.7 by isoelectric focusing in polyacrylamide gel in the presence of 2% Triton X-100. The addition of phosphatidylcholine did not enhance the enzyme activity in the placental microsomes obtained from two patients with placental sulfatase deficiency (PSD) after solubilization and chromatofocusing. This result indicates that PSD is the result of a defect in the enzyme rather than a defect in the membrane-enzyme structure. Amino acid analysis revealed that the purified human placental sulfatase did not contain cysteine residue. The Km and Vmax values of the steroid sulfatase for dehydroepiandrosterone sulfate (DHA-S) were 7.8 microM and 0.56 nmol/min, while those for estrone sulfate (E1-S) were 50.6 microM and 0.33 nmol/min, respectively. The results of the kinetic study suggest the substrate specificity of the purified enzyme, but further studies should be done with different substrates and inhibitors.     

Microtiter plate cellular assay for human steroid sulfatase with fluorescence readout

Steroid sulfatase (STS; E.C. 3.1.6.2) is an enzyme involved in the local production of estrogens and androgens in target organs. Inhibitors of steroid sulfatase activity are considered novel therapeutic agents for the treatment of different pathologic conditions, including cancers of breast, endometrium, and prostate and disorders of the pilosebaceous unit. Evaluation of steroid sulfatase inhibition in cells up to now has been a cumbersome process, involving the extraction of a radioactive cleavage product into organic solvents. Here, we describe a rapid, nonradioactive cellular assay in microtiter plate format, using 4-methylumbelliferyl sulfate as a substrate. The reaction product, 4-methylumbelliferone, is read in a fluorescence microtiter plate reader. Several cell lines were assayed for sulfatase activity. To increase the sensitivity of the assay, we developed a Chinese hamster ovary (CHO) cell line stably transfected with a cDNA encoding the human steroid sulfatase. The steroid sulfatase activity in transfected cells correlated with the presence of the enzyme in these cells, as determined by immunofluorescence. For most STS inhibitors tested, including estrone-3-O-sulfamate, the results from the CHO cellular assay were in good agreement with those from a standard cell-free assay.     

The use of intrinsic protein fluorescence to quantitate enzyme-bound persulfide and to measure equilibria between intermediates in rhodanese catalysis

The intrinsic fluorescence of the enzyme rhodanese is quenched by as much as 30% when sulfur is transferred to the free enzyme form, E, giving the sulfur-substituted enzyme, ES. This fluorescence change (lambda ex = 295 nm and lambda em = 335 nm) has been used to quantitate the E and ES forms which are isolatable, obligatory intermediates in rhodanese catalysis. Fluorescence titration was performed using cyanide to irreversibly remove sulfur from ES. The results show a stoichiometry corresponding to 1 bound sulfur/molecule of the ES form of rhodanese (Mr = 33,000). The fluorescence changes were used to measure the concentrations of E and ES when these were in reversible equilibria induced by interactions with the substrates S2O3(2-) and SO3(2-). These results were compared with an equilibrium constant derived from published kinetic studies for the reaction (formula; see text) The very close agreement between the physical and kinetic methods indicate that there are no significant concentrations of intermediates other than E and ES. Overall, the results are compatible with the formation of a persulfide intermediate in rhodanese catalysis and are consistent with conclusions from x-ray crystallography and absorption spectroscopy. In addition, these procedures offer a facile method to measure equilibria between catalytic intermediates in the rhodanese reaction using functionally relevant concentrations.     

Overexpression, purification, and crystallization of the membrane-bound fumarate reductase from Escherichia coli

Quinol-fumarate reductase (QFR) from Escherichia coli is a membrane-bound four-subunit respiratory protein that shares many physical and catalytic properties with succinate-quinone oxidoreductase (EC 1.3.99.1) commonly referred to as Complex II. The E. coli QFR has been overexpressed using plasmid vectors so that more than 50% of the cytoplasmic membrane fraction is composed of the four-subunit enzyme complex. The growth characteristics required for optimal levels of expression with minimal degradation by host cell proteases and oxidation factors were determined for the strains harboring the recombinant plasmid. The enzyme is extracted from the enriched membrane fraction using the nonionic detergent Thesit (polyoxyethylene(9)dodecyl ether) in a monodisperse form and then purified by a combination of anion-exchange, perfusion, and gel filtration chromatography. The purified enzyme is highly active and contains all types of redox cofactors expected to be associated with the enzyme. Crystallization screening of the purified QFR by vapor diffusion resulted in the formation of crystals within 24 h using a sodium citrate buffer and polyethylene glycol precipitant. The crystals contain the complete four-subunit QFR complex, diffract to 3.3 A resolution, and were found to be in space group P2(1)2(1)2(1) with unit cell dimensions a = 96.6 A, b = 138.1 A, and c = 275.3 A. The purification and crystallization procedures are highly reproducible and the general procedure may prove useful for Complex IIs from other sources.     

Inhibition of steryl sulfatase activity in LNCaP human prostate cancer cells

The enzyme steryl sulfatase may help support the growth of hormone-dependent tumors, including prostate cancers, by facilitating the conversion of circulating precursor steroids to active hormones. We sought to determine the presence of steryl sulfatase activity in the androgen-dependent human prostate cancer cell line LNCaP, and to determine if this activity was inhibited by known steryl sulfatase inhibitors. Intact LNCaP cultures had steryl sulfatase activity, as determined by conversion of [3H]estrone sulfate (E(1)S) to unconjugated steroids. The level of steryl sulfatase activity was relatively low (4.6 pmol/18 h/million cells) compared to MDA-MB-231 breast cancer cells (284.0 pmol/18 h/million cells). The observed activity in both cell lines was blocked by addition of 1 microM estrone sulfamate (EMATE), an active-site-directed, steroidal inhibitor of steryl sulfatase. Steryl sulfatase activity was also inhibited by Danazol, and by (p-O-sulfamoyl)-tetradecanoyl tyramine (C2-14), a non-steroidal inhibitor. Microsomes prepared from LNCaP cultures also showed steryl sulfatase activity, as determined by hydrolysis of [3H]E(1)S and [3H]dehydroepiandrosterone sulfate (DHEAS) to unconjugated forms. LNCaP and MDA-MB-231 microsomes both hydrolyzed E(1)S about two times faster than DHEAS. Hydrolysis of E(1)S in LNCaP and MDA-MB-231 microsomes was blocked by steryl sulfatase inhibitors with the following relative potencies: EMATE>C2-14>Danazol. These data demonstrate that LNCaP prostate cancer cells contain a steryl sulfatase with properties similar to that found in human breast cancer cells, and that the activity of this enzyme can be blocked by known steryl sulfatase inhibitors. Steryl sulfatase inhibitors may be useful as an adjuvant to androgen deprivation therapy for prostate cancer.     

Complementation of multiple sulfatase deficiency in somatic cell hybrids

Multiple sulfatase deficiency (MSD) is an inherited disorder characterized by deficient activity of seven different sulfatases. Genetic complementation for steroid sulfatase (STS), arylsulfatase A, and N-acetylgalactosamine 6-SO4 sulfatase was demonstrated in somatic cell hybrids between MSD fibroblasts and mouse cells ( LA9 ) or Chinese hamster cells ( CHW ). In an electrophoretic system that separates human and rodent STS isozymes, enzyme from hybrids migrated as human enzyme. We concluded that the rodent cell complemented the MSD deficiency and allowed normal expression of the STS structural gene. Some MSD- LA9 hybrids showed significant levels of human arylsulfatase A activity, as shown by the immunoprecipitation of active enzyme by human-specific antiserum. Complementation was also suggested for N-acetylgalactosamine 6- sulfatate sulfatase (GalNAc-6S sulfatase) in several MSD- LA9 hybrids by the demonstration of a significant increase in activity (10-fold) over that of the GalNAc-6S sulfatase-deficient parental mouse and MSD cells. Thus, it was possible to demonstrate complementation for more than one sulfatase in a single MSD-rodent hybrid. Normal levels of sulfatase activity in hybrids indicate that the sulfatase structural genes are intact in MSD cells.     

Steroid sulfatase activities in human leukocytes: biochemical and clinical aspects.

Steroid sulfatase is a membrane-bound microsomal enzyme, present in various tissues. In this report, data on sulfatase activity in peripheral blood leukocytes isolated from normal women and the characterization of its enzyme are studied. In addition, sulfatase activities in placental sulfatase deficiency (PSD) and ichthyosis patients including ichthyosis vulgaris (IV) and recessive X-linked ichthyosis (RXLI) were analysed and were compared with normal subjects. Steroid sulfatase activity was measured by using tritium labeled steroid sulfate as the reaction substrate. It is demonstrated that human leukocytes contain a sulfatase activity for pregnenolone sulfate (P5-S), dehydroepiandrosterone sulfate (DHA-S) and estrone sulfate (E1-S) respectively. This enzyme has a greatest affinity for P5-S, but the activity for E1-S was the highest among the three substrates. The steroid sulfatase activity in female leukocytes is significantly stronger than that in normal males (p less than 0.001) as determined by the cleavage of DHA-S. Sulfatase in leukocytes obtained from the PSD babies and RXLI patients had lower sensitivity. In the case of the mother affected with PSD, the activity was less than half of that in normal men (p less than 0.001) and the levels did not overlap with that in normal women. In patients with IV, the activities were in the normal ranges for both males and females. The measurement of leukocyte sulfatase activity would be a clinically useful tool for the diagnosis of PSD carriers and pedigree analysis.     

Cholesteryl sulfate and sterol sulfatase in the human reproductive tract.

Cholesteryl sulfate is a component of human seminal plasma (avg. 445 mug%) and spermatozoa (15 mug/10 (9) cells) and represents more than 85% of the sterol sulfate fraction. This conjugate is avidly bound by spermatozoa when compared to other steroids or steroid sulfates. Autoradiographic localization of CS associated with the spermatozoa revealed a greater accumulation of the radioactivity in the acrosomal region in many, but not all, of those cells examined. Semen is not a site of metabolism of the sterol sulfate but the enzyme, sterol sulfatase, is present in the human female reproductive tract. This cleavage enzyme was detected in Graafian follicles and the activity in the endometrium was ten-fold that found in the Fallopian tube. These findings lead to the proposal that cholesteryl sulfate, an amphipathic molecule ideally suited for interaction with membrane components and implicated in erythrocyte membrane stabilization, may be involved in membrane modifications of the spermatozoa during the process of fertilization.     

Characterization of protein detergent complexes by NMR, light scattering, and analytical ultracentrifugation

Abstract  Bottlenecks in expression, solubilization, purification and crystallization hamper the structural study of integral membrane proteins (IMPs). Successful crystallization is critically dependent on the purity, stability and oligomeric homogeneity of an IMP sample. These characteristics are in turn strongly influenced by the type and concentration of the detergents used in IMP preparation. By utilizing the techniques and analytical tools we earlier developed for the characterization of protein-detergent complexes (PDCs) [21], we demonstrate that for successful protein extraction from E. coli membrane fractions, the solubilizing detergent associates preferentially to IMPs rather than to membrane lipids. Notably, this result is contrary to the generally accepted mechanism of detergent-mediated IMP solubilization. We find that for one particular member of the family of proteins studied (E. coli receptor kinases, which is purified in mixed multimeric states and oligomerizes through its transmembrane region), the protein oligomeric composition is largely unaffected by a 10-fold increase in protein concentration, by alteration of micelle properties through addition of other detergents to the PDC sample, or by a 20-fold variation in the detergent concentration used for solubilization of the IMP from the membrane. We observed that the conditions used for expression of the IMP, which impact protein density in the membrane, has the greatest influence on the IMP oligomeric structure. Finally, we argue that for concentrating PDCs smaller than 30 kDa, stirred concentration cells are less prone to over-concentration of detergent and are therefore more effective than centrifugal ultrafiltration devices.     

Expression, purification, and structural analysis of (HIS)UBE2G2 (human ubiquitin-conjugating enzyme)

The ubiquitin system represents a selective mechanism for intracellular proteolysis in eukaryotic cells that involves the sequential activity of three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3). The identification of these proteins and their cellular targets, as well as structural data, are essential to understanding how this system operates in the eukaryotic cell. In the present study, the open reading frame of the human ubiquitin-conjugating enzyme UBE2G2 was isolated from a human brain cDNA panel, cloned into pET28a vector and expressed in Escherichia coli. The His-tagged protein was then purified through nickel-affinity chromatography and subjected to structural and functional studies using circular dichroism (CD) and an in vitro ubiquitin-binding assay, respectively. Our results showed that the production of the HISUBE2G2 protein in bacteria, carried out with 0.1 mM of IPTG at 30 degrees C, was successfully achieved, rendering high concentrations of soluble, pure and stable enzyme after a single purification step. The recombinant protein was able to bind ubiquitin molecules when exposed to a HeLa cell extract during the ubiquitin assay. Moreover, the fact that HISUBE2G2 was expressed in its active form is supported by the typical alpha/beta secondary structure specific to other class I E2 enzymes displayed during the CD assay.     

Low-Scale expression and purification of an active putative iduronate 2-sulfate sulfatase-Like enzyme from Escherichia coli K12

The sulfatase family involves a group of enzymes with a large degree of similarity. Until now, sixteen human sulfatases have been identified, most of them found in lysosomes. Human deficiency of sulfatases generates various genetic disorders characterized by abnormal accumulation of sulfated intermediate compounds. Mucopolysaccharidosis type II is characterized by the deficiency of iduronate 2-sulfate sulfatase (IDS), causing the lysosomal accumulation of heparan and dermatan sulfates. Currently, there are several cases of genetic diseases treated with enzyme replacement therapy, which have generated a great interest in the development of systems for recombinant protein expression. In this work we expressed the human recombinant IDS-Like enzyme (hrIDS-Like) in Escherichia coli DH5α. The enzyme concentration revealed by ELISA varied from 78.13 to 94.35 ng/ml and the specific activity varied from 34.20 to 25.97 nmol/h/mg. Western blotting done after affinity chromatography purification showed a single band of approximately 40 kDa, which was recognized by an IgY polyclonal antibody that was developed against the specific peptide of the native protein. Our 100 ml-shake-flask assays allowed us to improve the enzyme activity seven fold, compared to the E. coli JM109/pUC13-hrIDS-Like system. Additionally, the results obtained in the present study were equal to those obtained with the Pichia pastoris GS1115/pPIC-9-hrIDS-Like system (3 L bioreactor scale). The system used in this work (E. coli DH5α/pGEX-3X-hrIDS-Like) emerges as a strategy for improving protein expression and purification, aimed at recombinant protein chemical characterization, future laboratory assays for enzyme replacement therapy, and as new evidence of active putative sulfatase production in E. coli.     

High-level expression, single-step immunoaffinity purification and characterization of human tetraspanin membrane protein CD81

The study of membrane protein structure and function requires their high-level expression and purification in fully functional form. We previously used a tetracycline-inducible stable mammalian cell line, HEK293S-TetR, for regulated high-level expression of G-protein coupled receptors. We here report successfully using this method for high-level expression of de novo oligo-DNA assembled human CD81 gene. CD81 is a member of the vital tetraspanin membrane protein family. It has recently been identified as the putative receptor for the Hepatitis C Virus envelope E2 glycoprotein (HCV-E2). In this study we used a single-step rho-1D4-affinity purification method to obtain >95% purity from HEK293S-TetR-inducible stable cell lines. Using ELISA assay we determined that the affinity of the purified CD81 receptor for HCV-E2 protein is 3.8+/-1.2 nM. Using fluorescent confocal microscopy we showed that the inducibly overexpressed CD81 receptor in HEK293S-TetR cells is correctly located on the plasma membrane. We demonstrated that the combination of high-level expression of CD81 with efficient single-step immunoaffinity purification is a useful method for obtaining large quantities of CD81 membrane receptor suitable for detailed structural analyses of this elusive tetraspanin protein. Furthermore, this simple single-step immunoaffinity purification to high purity of membrane protein could be useful broadly for other membrane protein purifications, thus accelerating the determination of structures for large numbers of difficult-to-obtain membrane proteins.     

In vitro and in vivo models for the evaluation of new inhibitors of human steroid sulfatase,devoid of residual estrogenic activity

The goal of our research project is to develop a new class of orally active drugs, estrone sulfatase inhibitors, for the treatment of estrogen-dependent (receptor positive) breast cancer. Several compounds were synthesized and their pharmacological potencies explored. Based on encouraging preliminary results, three of them, TX 1299, TX 1492 and TX 1506 were further studied in vitro as well as in vivo. They proved to be strong inhibitors of estrone sulfatase when measured on the whole human JEG-3 choriocarcinoma and MCF-7 breast cancer cells and their IC(50)s found to be in the range of known standard inhibitors. Their residual estrogenic activity was checked as negative in the test of induction of alkaline phosphatase (APase) activity in whole human endometrial adenocarcinoma Ishikawa cells. In addition, their effect on aromatase activity in JEG-3 cells was also examined, since the goal of inhibiting both sulfatase and aromatase activities appears very attractive. However, it has been unsuccessful so far. Then, in vivo potencies of TX 1299, the lead compound in our chemical series, were evaluated in comparison with 6,6,7-COUMATE, a non-steroidal standard, in two different rat models and by oral route. First, the absence of any residual estrogenic activity for these compounds was checked in the uterotrophic model in prepubescent female rats. Second, antiuterotrophic activity in adult ovariectomized rat supplemented with estrone sulfate (E(1)S), showed that both compounds were potent inhibitors, the power of TX 1299 relative to 6,6,7-COUMATE being around 80%. This assay was combined with uterine sulfatase level determination and confirmed the complete inhibition of this enzyme within the target organ.Preliminary studies indicated that other non-steroid compounds in the Théramex series were potent in vitro and in vivo inhibitors of estrone sulfatase in rats and further studies are in progress.     

Estrone sulfatase versus estrone sulfotransferase in human breast cancer: potential clinical applications

Estrone sulfate (E₁S) is concentrated in high levels in human breast cancer tissue. The values are particularly high in postmenopausal women and many times those circulating in the plasma. Also, the tissular concentration of this conjugate are significantly higher in tumoural tissue than in the area of the breast considered as normal. The enzyme which hydrolyzes E₁S: sulfatase, as well as the enzyme which biosynthesises this conjugate: sulfotransferase, are present in significant concentrations in breast cancer tissue. Consequently, E₁S is a balance between the activities of the two enzymes. As breast cancer tissue has all the enzymes necessary for the synthesis of estradiol (E₂), and the formation of E₂ from E₁S ‘via sulfatase’ is the main pathway, it was very attractive to explore inhibitory agents of this enzyme. It was observed that different substances including antiestrogens (4-hydroxytamoxifen, ICI 164,384) and various progestins (promegestone, nomegestrol acetate, medrogestone) as well as Org OD14 (tibolone) can block the sulfatase activity. In addition, it was demonstrated that different progestins (medrogestone, nomegestrol acetate, TX-525) and org OD14 can stimulate the sulfotransferase activity for the formation of the biologically inactive E₁S. It is concluded that the inhibition of sulfatase and the stimulation of sulfotransferase activity can open interesting possibilities to explore these effects in patients with breast cancer.     

Purification methods of mammalian catechol-O-methyltransferases

The protein purification strategies used for obtaining homogeneous rat and human soluble catechol-O-methyltransferase (S-COTM) polypeptides are reviewed. Expression and purification of recombinant rat and human S-COMT in Escherichia coli and for human S-COMT in baculovirus-infected insect cells made it possible to elucidate the S-COMT polypeptides in more detail. The application of these purification methods has allowed the crystallization of the rat S-COMT protein and the analysis of the kinetic properties of the enzyme in great detail. The availability of the pure S-COMT protein together with the structural data has also greatly enhanced the development of more potent COMT inhibitors.     

On the inhibitory action of 29 drugs having side effect gynecomastia on estrogen production

To examine the influence on aromatase and sulfatase pathways in estrogen pool by drugs reported to cause gynecomastia as the side effect, 29 ethical drugs were incubated with human placental microsomes as an enzyme source. The percent inhibition of drugs on aromatase pathway was obtained by sum of the velocity constants of two products, estrone (E1) and estradiol (E2) from testosterone (T) as the substrate, and that on sulfatase pathway was obtained as the velocity constant of production of E1 from estrone sulfate (E1S). Although several drugs including ketoconazole showed a significant inhibition effect on aromatase pathway at their non-clinical over-dose concentration (100 microM), no influence on the inhibition was observed in any drugs at their approximately therapeutic concentration (1 microM). However, several drugs including spironolactone gave the product ratio (E2/E1) having higher value than that of the control, the result means spironolactone inhibits the conversion of E2 to E1. No inhibitory effect of the drugs tested on estrogen production from E1S (sulfatase pathway) was confirmed. The results suggest the possibility that the tested drugs known to cause gynecomastia have no inhibitory effect essentially on aromatase and sulfatase pathways.     

The design,synthesis, and biochemical evaluation of derivatives of biphenyl sulfamate-based compounds as novel inhibitors of estrone sulfatase

We report the initial results of our study into the use of a potential transition state (TS) of the reaction catalyzed by the enzyme estrone sulfatase (ES) in the design of a series of simple 4'-O-sulfamoyl-4-biphenyl-based compounds as novel inhibitors of ES. The results of the study show that these compounds are: potent inhibitors, possessing greater inhibitory activity than 4-methylcoumarin-7-O-sulfamate (COUMATE); weaker inhibitors than the tricyclic derivative of COUMATE, namely 667-COUMATE and the steroidal inhibitor estrone-3-O-sulfamate (EMATE), and irreversible inhibitors of ES.     

银胶菊丙二烯氧化物合成酶的表达纯化及其活性分析

植物来源细胞色素P450的表达和结晶都十分困难,成为成功解析该类蛋白晶体结构的瓶颈。探索了使用大肠杆菌表达系统表达、纯化银胶菊丙二烯氧化物合成酶(allene oxide synthase,AOS)的方法。氨基酸序列比对分析显示,银胶菊AOS缺失N-末端的膜锚定序列和信号肽序列,推测其在大肠杆菌中应表达为水溶性蛋白质。试验中纯化得到的银胶菊AOS是一个分子量为54.5 kD的八聚体分子,均一性好,不含去垢剂,达到毫克量级,适合下一步的晶体培养研究;还原一氧化碳差示光谱显示该酶在450 nm处有特征吸收峰,酶活性测定得到该酶的米氏常数为45.3μmol/L,显示该重组AOS具有很高的酶活性。