Immunohistochemical analysis of steroid sulfatase in human tissues

Steroid sulfatase (EC 3.1.6.2) is an enzyme that removes the sulfate group from 3β-hydroxysteroid sulfates. This enzyme is best known for its role in estrogen production via the fetal adrenal–placental pathway during pregnancy; however, it also has important functions in other physiological and pathological steroid pathways. The objective of this study was to examine the distribution of steroid sulfatase in normal human tissues and in breast cancers using immunohistochemistry, employing a newly developed steroid sulfatase antibody. A rabbit polyclonal antiserum was generated against a peptide representing a conserved region of the steroid sulfatase protein. In Western blotting experiments using human placental microsomes, this antiserum crossreacted with a 65 kDa protein, the reported size of steroid sulfatase. The antiserum also crossreacted with single protein bands in Western blots of microsomes from two human breast cancer cell lines (MDA-MB-231 and MCF-7) and from rat liver; however, there were some size differences in the immunoreactive bands among tissues. The steroid sulfatase antibody was used in immunohistochemical analyses of individual human tissue slides as well as a human tissue microarray. For single tissues, human placenta and liver showed strong positive staining against the steroid sulfatase antibody. ER+/PR+ breast cancers also showed relatively strong levels of steroid sulfatase immunoreactivity. Normal human breast showed moderate levels of steroid sulfatase immunoreactivity, while ER−/PR− breast cancer showed weak immunoreactivity. This confirms previous reports that steroid sulfatase is higher in hormone-dependent breast cancers. For the tissue microarray, most tissues showed some detectable level of steroid sulfatase immunoreactivity, but there were considerable differences among tissues, with skin, liver and lymph nodes having the highest immunoreactivity and brain tissues having the lowest. These data reveal the utility of immunohistochemistry in evaluation of steroid sulfatase activity among tissues. The newly developed antibody should be useful in studies of both humans and rats.     

An estimate of unique DNA sequence heterozygosity in the human genome

Summary Patients with recessive X-linked ichthyosis Patients with recessive X-linked ichthyosis (RXLI), one hereditary form of scaly skin, lack activity of the enzyme steroid sulfatase in all tissues studied. To investigate the molecular defect underlying the lack of enzyme activity, we prepared antisera against normal enzyme by injecting normal placental microsomal suspensions or partially purified steroid sulfatase into rabbits. Antibody activity was assessed by immunoprecipitation of detergent solubilized steroid sulfatase. In addition, we prepared rabbit antisera against RXLI placental microsomal suspensions. To detect immunologically cross-reactive material in patients' placentas, extracts were studied by immunoblot techniques and by competition with normal enzyme for antibody binding. Patients' extracts did not contain immunoreactive material co-migrating on electrophoresis with purified enzyme nor did they inhibit immunoprecipitation of normal enzyme. Sera from rabbits immunized with RXLI placental microsomes contain no antibodies to normal steroid sulfatase, as judged by their failure to immunoprecipitate normal enzyme or to react with normal steroid sulfatase on immunoblot. Thus the mutation in RXLI appears to reduce steroid sulfatase enzyme protein as well as enzyme activity. Portions of this material have appeared in abstract form in Clinical Research 31:564A, 1983 and 32:138A, 1984     

Characterization of arylsulfatase C isozymes from human liver and placenta

Arylsulfatase C and steroid sulfatase were thought to be identical enzymes. However, recent evidence showed that human arylsulfatase C consists of two isozymes, s and f. In this study, the biochemical properties of the s form partially purified from human placenta were compared with those of the f form from human liver. Only the placental s form has steroid sulfatase activity and hydrolyses estrone sulfate, dehydroepiandrosterone sulfate and cholesterol sulfate. The liver f form has barely detectable activity towards these sterol sulfates. With the artificial substrate, 4-methylumbelliferyl sulfate, both forms demonstrated a similar KM but the liver enzyme has a pH optimum of 6.9 while the placental form displayed two optima at 7.3 and 5.5. The molecular weight of the native enzyme determined with gel filtration was 183,000 for the s form and 200,000 for the f form and their pI's were also similar at 6.5. However, the T50, temperature at which half of the enzyme activity was lost, was 49.5 degrees C for the f form and 56.8 degrees C for the s form. Polyclonal antibodies raised against the placental form reacted specifically against the s and not the f form. They immuno-precipitated concomitantly greater than 80% of the total placental arylsulfatase C and steroid sulfatase activities while less than 20% of the liver enzyme was immuno-precipitable. In conclusion, the two isozymes s and f of arylsulfatase C in humans purified from placenta and liver, respectively, have similar KM, pI' and native molecular weight. However, they are distinct proteins with different substrate specificity, pH optima, heat-lability and antigenic properties. Only the s form is confirmed to be steroid sulfatase.     

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.     

Synthesis and stability of steroid sulfatase in fibroblasts from multiple sulfatase deficiency

Multiple sulfatase deficiency is a lysosomal storage disorder, which can be divided into group I with severe and group II with moderate deficiencies in sulfatases. Antibodies raised against steroid sulfatase purified from human placenta were used to follow the biosynthesis and stability of this enzyme in multiple sulfatase-deficiency fibroblasts. Fibroblasts from both groups synthesized steroid sulfatase of apparently normal size and stability, while the apparent rate of enzyme synthesis and catalytic properties of steroid sulfatase were affected to a variable extent. Cell lines were observed, that synthesized normal amounts of steroid-sulfatase polypeptides, which were catalytically inactive, as well as cell lines that synthesized diminished amounts of catalytically active steroid sulfatase.     

Comparison of arylsulfatase C and steroid sulfatase from human placenta and liver

Human placental and hepatic arylsulfatase C (ASC) were purified to homogeneity and about 1,000-fold, respectively. Placental ASC hydrolyzed sterol sulfates at the same active site, whereas the major hepatic ASC did not. This major hepatic ASC isozyme was more thermolabile than placental ASC and steroid sulfatase from both placenta and liver. It was not precipitated by anti-bovine ASC IgG which quantitatively precipitated both placental ASC and steroid sulfatase activities from placenta and liver. A minor hepatic ASC isozyme with similar electrophoretic mobility to the placental enzyme copurified with the major hepatic ASC and is likely responsible for the steroid sulfatase activity in this organ. Hence, placental ASC and steroid sulfatase are biochemically and antigenically identical to hepatic steroid sulfatase. In contrast, the major hepatic ASC is a distinct protein whose catalytic and structural properties differ from all the above enzymes.     

Association of steroid sulfatase with one of the arylsulfatase C isozymes in human fibroblasts

When arylsulfatase C, a microsomal membrane-bound enzyme, is assayed with its natural substrates, the 3-beta-hydroxysteroid sulfates, it is also known as steroid sulfatase. Whether arylsulfatase C and steroid sulfatase are identical enzymes or not, however, has long been disputed. We now report that two electrophoretic variants of arylsulfatase C occur in normal human fibroblasts: one has a single anodic band of activity, "s," and the other has an additional faster migrating band, "f". The two types, s and "f + s", occur in cells from either sex. When fibroblast strains with the f + s forms of arylsulfatase C were cloned, two types of primary clones were always obtained: s and f + s. A single f band was never seen. When these primary clones were subcloned, however, the arylsulfatase C phenotype remained unchanged: primary s clones gave rise to s subclones and f + s clones to f + s subclones only. Therefore, these forms were clonal in origin and demonstrated a novel inheritance pattern in human cultured cells. The appearance of increasing amounts of the f band was correlated with up to 4-fold increase of arylsulfatase C activity, whereas the steroid sulfatase activity remained constant, thus demonstrating that arylsulfatase C was not identical with steroid sulfatase activity. Polyclonal antibodies raised against the s form immunoprecipitated activities of the s form of arylsulfatase C and steroid sulfatase but not the f form of arylsulfatase C. Therefore, we conclude that only the s form of arylsulfatase C is immunologically related to steroid sulfatase so that arylsulfatase C per se is not necessarily identical with steroid sulfatase. In addition, a novel form of genetic heterogeneity of isozymes in human fibroblasts is demonstrated.     

Localization and expression of steroid sulfatase in human fallopian tubes

Localization of steroid sulfatase, a membrane-bound microsomal enzyme, in human fallopian tubes was immunohistochemically investigated, and expression of RNA was confirmed by competitive RT-PCR. Human fallopian tubes were obtained from 10 patients in follicular and early luteal phases during gynecological laparotomy. An anti-human rabbit polyclonal antibody was prepared against sulfatase protein purified from human placenta. Total RNA was isolated from epithelium of fallopian tubes. A heterologous RNA competitor was designed, and competitive RT-PCR was carried out. Steroid sulfatase was localized to the cytoplasm of epithelial cells. With respect to the positive staining of cells, the number of positive secretory cells was higher than that of ciliated cells. A significantly higher number of positive cells was found in tissue obtained from the early luteal phase than that found in tissue from the follicular phase. An abundant expression of sulfatase mRNA in early luteal phase was also observed. This study demonstrates, for the first time, that steroid sulfatase is localized to human epithelial cells and that steroid sulfatase staining and mRNA expression changes with the menstrual cycle. These results suggest that sulfatase in the fallopian tube may be involved in controlling the local steroid environment, which appears to regulate aspects of the physiological reproductive function of the fallopian tube.     

Synthesis and structure–activity relationships of 2- and/or 4-halogenated 13β- and 13α-estrone derivatives as enzyme inhibitors of estrogen biosynthesis

Ring A halogenated 13α-, 13β-, and 17-deoxy-13α-estrone derivatives were synthesised with N-halosuccinimides as electrophile triggers. Substitutions occurred at positions C-2 and/or C-4. The potential inhibitory action of the halogenated estrones on human aromatase, steroid sulfatase, or 17β-hydroxysteroid dehydrogenase 1 activity was investigated via in vitro radiosubstrate incubation. Potent submicromolar or low micromolar inhibitors were identified with occasional dual or multiple inhibitory properties. Valuable structure–activity relationships were established from the comparison of the inhibitory data obtained. Kinetic experiments performed with selected compounds revealed competitive reversible inhibition mechanisms against 17β-hydroxysteroid dehydrogenase 1 and competitive irreversible manner in the inhibition of the steroid sulfatase enzyme.     

Steroid sulfatase. Biosynthesis and processing in normal and mutant fibroblasts

Antibodies raised against steroid sulfatase purified from human placenta were used to follow the biosynthesis of this enzyme in human skin fibroblasts. Steroid sulfatase is synthesized as a membrane-bound Mr-63 500 polypeptide with asparagine-linked oligosaccharide chains. Within 2 days, newly synthesized steroid sulfatase is processed to a mature Mr-61 000 form. The decrease in size is due to processing of the oligosaccharide chains, which are cleavable by endoglucosaminidase H in both the early and the mature form of steroid sulfatase. The processing involves mannosidase(s) sensitive to 1-deoxy-manno-nojirimycin. The half-life of the steroid sulfatase polypeptides is 4 days. Synthesis of steroid-sulfatase-related polypeptides and steroid sulfatase activity were not detectable in fibroblasts from four patients with X-linked ichthyosis.     

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.     

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.     

Human placental steroid sulfatase: purification and properties

Steroid sulfatase is recovered quantitatively from the 105,000 g h supernatant of human placental microsomes extracted with Triton X-100. The solubilized enzyme has been purified using conventional techniques. Throughout the purification procedure, steroid sulfatase appears to be heterogeneous as evidenced by certain, but not all, criteria. Following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the final preparation exhibits a major component and varying amounts of two minor ones. Antibodies raised in rabbits with the heterogeneous immunogen give rise to a single precipitation line when the native enzyme is analyzed by double immunodiffusion or by immunoelectrophoresis. In addition, using aged preparations of microsomes and immunoaffinity techniques, steroid sulfatase activity was found to be associated with the fastest migrating minor component. This finding would suggest that the apparent heterogeneity of purified steroid sulfatase is linked to degradation processes occurring within the microsomal preparations. Steroid sulfatase has a Stokes radius of 56 A, a sedimentation coefficient of 4.85 +/- 0.15S (in Triton-containing buffers) and binds 1.3 g of Triton X-100-per g of protein. The molecular weight of the Triton-protein complex was calculated to be 166,000 in which the glycoprotein portion contribution is about 43% (72,000). In contrast, the apparent molecular weight of the major polypeptide determined on calibrated SDS-gels is 62,000. The purified enzyme exhibits two pH optima with cholesterol sulfate as substrate, an acidic one at pH 5.0 and a second one at pH 7.5. The Km values for cholesterol sulfate, dehydroandrosterone sulfate and p-nitrophenylsulfate were 5.26, 14 and 1,320 microM, respectively.     

Simultaneous azo-coupling method for an estrogen sulfatase in human tissues

Summary A simultaneous azo-coupling method for the histochemical localization of d-equilenin sulfatase is described. d-Equilenin is a natural estrogenic steroid hormone, and its sulfuric acid ester was synthesized. It was found that the d-equilenin liberated during hydrolysis of d-equilenin sulfate by tissue sulfatase could be coupled with a diazonium salt to produce a purple precipitate indicating enzyme activity. d-Equilenin sulfatase was found in human tissues, but not in tissues of the rat. The optimum substrate concentration was 0.8 mM, activity was demonstrable over the wide pH range 5.0–8.0. Enzyme activity localized diffusely in the cytoplasm in optimally fixed specimens. Enzyme activity was also fairly well demonstrable in unfixed cryostat sections. Enzyme activity was completely inhibited by 0.1 M phosphate, 1 mM sodium tetraborate, 1 mM p-nitrophenyl sulfate and by 2 mM p-nitrocatechol sulfate. Estrone sulfate at concentration 0.8 mM had no effect, but at 4 mM caused marked inhibition of the reaction. At the same concentrations dehydroepiandrosterone sulfate did not inhibit the reaction. The chemical properties and tissue localizations of d-equilenin sulfatase differed from the properties of arylsulfatases A, B and C and other steroid sulfatases reported previously in the literature.     

Clinical and biochemical investigations on patients with partial deficiency of placental steroid sulfatase

Summary We report on three independent cases with a partial deficiency of placental steroid sulfatase (E.C.3.1.6.2). Upon routine pregnancy monitoring these patients were detected on the basis of low estriol excretion and failing induction of labor. In all three cases a male was delivered and subsequently the diagnosis of partial deficiency of placental steroid sulfatase was confirmed enzymatically in placenta homogenates. In one case, fibroblast cultures were established from skin explants of mother and son. In fibroblasts of the child, as in placental tissue, the activity of steroid sulfatase was only 34% of normal. Similar values were obtained for arylsulfatase C, though this enzyme is clearly separable from steroid sulfatase by electrophoresis. In cells of the mother, enzyme activities were unremarkable.     

Chemical characterization and substrate specificity of rabbit liver aryl sulfatase A

Rabbit liver aryl sulfatase A (aryl-sulfate sulfohydrolase, EC 3.1.6.1) is a glycoprotein containing 4.6% carbohydrate in the form of 25 residues of mannose, seven residues of N-acetylglucosamine, and three residues of sialic acid per enzyme monomer of molecular weight 140 000. Each monomer consists of two equivalent polypeptide chains. The protein has a relatively high content of proline, glycine and leucine, and the amino acid composition of rabbit liver aryl sulfatase A is similar to that of other known liver sulfatases. Rabbit liver aryl sulfatase A catalyzes the hydrolysis of a wide variety of sulfate esters, although it appears possible that cerebroside sulfate is a physiological substrate for the enzyme because the Km is very low (0.06 mM). The turnover rate for hydrolysis of nitrocatechol sulfate or related synthetic substrates is much higher than the rate with most naturally occurring sulfate esters such as cereroside sulfate, steroid sulfates, L-tyrosine sulfate or glucose 6-sulfate. However, the turnover rate with ascorbate 2-sulfate is comparable to the rates measured using most synthetic substrates. These results are discussed in relationship to several previously described sulfatase enzymes which were claimed to have unique specificities.     

The target sizes of the in situ and solubilized forms of human placental steroid sulfatase as measured by radiation inactivation

Whole microsomal membrane preparations and Triton X-100-solubilized human placental steroid sulfatase were subjected to radiation inactivation analysis using gamma rays from a 60Co irradiator in order to assess the size of the physiological form of the enzyme. The data indicate that the enzyme exists as a monomer of molecular weight 78600 in Triton-containing buffers and as a polymer of molecular weight 533000 within the microsomal membrane.     

Development of novel steroid sulfatase inhibitors; II. TZS-8478 potently inhibits the growth of breast tumors in postmenopausal breast cancer model rats

In postmenopausal breast cancer tissue, steroid sulfatase (STS) activity is high and much estrone sulfate also exists; these facts reveal that estrone sulfate may be involved in the growth of breast cancer as an estrogen source. Steroid sulfatase is an enzyme, which catalyzes hydrolysis from estrone sulfate to estrone, and the development of steroid sulfatase inhibitors is expected as novel therapeutic drugs for postmenopausal breast cancer. We have developed a novel compound 2',4'-dicyanobiphenyl-4-O-sulfamate (TZS-8478), which has potent steroid sulfatase-inhibitory activity and exhibits no estrogenicity in vitro and in vivo. To elucidate its usefulness as a therapeutic drug for postmenopausal breast cancer, we examined the breast cancer cell proliferation- and breast tumor growth-inhibitory activity of TZS-8478 in postmenopausal breast cancer model rats. TZS-8478 dose-dependently suppressed the estrone sulfate-stimulated proliferation of MCF-7 cells. Regarding nitrosomethylurea (NMU)-induced postmenopausal breast cancer models, furthermore, TZS-8478 (0.5 mg/kg per day) markedly inhibited the estrone sulfate-stimulated growth of breast tumors similarly to estrone sulfate-depletion. TZS-8478 completely inhibited steroid sulfatase activity in tumor, uterus and liver, and also markedly lowered plasma concentrations of estrone and estradiol. The above mentioned results suggested that TZS-8478 may be useful as a therapeutic drug for estrogen-dependent postmenopausal breast cancer.