Steroid sulfatase, arylsulfatases A and B, galactose-6-sulfatase, and iduronate sulfatase in mammary cells and effects of sulfated and non-sulfated estrogens on sulfatase activity

Sulfatase enzymes have important roles in metabolism of steroid hormones and of glycosaminoglycans (GAGs). The activity of five sulfatase enzymes, including steroid sulfatase (STS; arylsulfatase C), arylsulfatase A (ASA; cerebroside sulfatase), arylsulfatase B (ASB; N-acetylgalactosamine-4-sulfatase), galactose-6-sulfatase (GALNS), and iduronate-2-sulfatase (IDS), was compared in six different mammary cell lines, including the malignant mammary cell lines MCF7, T47D, and HCC1937, the MCF10A cell line which is associated with fibrocystic disease, and in primary epithelial and myoepithelial cell lines established from reduction mammoplasty. The effects of estrogen hormones, including estrone, estradiol, estrone 3-sulfate, and estradiol sulfate on activity of these sulfatases were determined. The malignant cell lines MCF7 and T47D had markedly less activity of STS, ASB, ASA, and GAL6S, but not IDS. The primary myoepithelial cells had highest activity of STS and ASB, and the normal epithelial cells had highest activity of GALNS and ASA. Greater declines in sulfatase activity occurred in response to estrone and estradiol than sulfated estrogens. The study findings demonstrated marked variation in sulfatase activity and in effects of exogenous estrogens on sulfatase activity among the different mammary cell types.     

Rapid degradation of steroid sulfatase in multiple sulfatase deficiency

Pulse labeling followed by SDS-PAGE electrophoresis of immunoprecipitated [35S]methionine-labeled steroid sulfatase (STS) gave a single band of molecular weight 65,000 daltons. After a chase period of 18 hours the material appeared as molecular weight approximately 64,000. No labeled STS could be detected in fibroblasts from individuals with STS deficient X-linked ichthyosis. Pulse-chase labeling of normal and multiple sulfatase deficiency (MSD) fibroblasts showed a normal rate of synthesis of STS in MSD during a 3 hour pulse but during the chase the STS of MSD cells disappeared with a half-life of 4 to 6 hours until approximately 25% of the material remained after 24 hr. STS of normal cells had a half-life of 6 days. The material produced in MSD cells had the same molecular size as normal and had the same amount of endoglycosidase sensitive carbohydrate as normal. The defect in MSD thus seems to result in degradation after the addition of N-linked oligosaccharides.     

Pathochemistry, pathogenesis and enzyme replacement in multiple-sulfatase deficiency

Multiple-sulfatase deficiency (MSD) is now considered to be heterogeneous and could be classified into three or four clinical phenotypes according to the onset of the disease: neonatal, late infantile, juvenile and possibly adult type. Neonatal-type MSD shows severe clinical involvement and practically no arylsulfatase A, B and C activities in cultured skin fibroblasts. Furthermore, arylsulfatase A activity in neonatal-type MSD was not enhanced by the addition of thiosulfate. Therefore, it is distinct from late infantile-type MSD. The degradation of 14C-sulfatide can occur in MSD-cultured skin fibroblasts and was much higher than in late infantile-type MLD. The addition of thiol protease such as leupeptin to cultured MSD skin fibroblasts enhanced arylsulfatase A activity as well as the degradation of 14C-sulfatide. This suggests that the decreased activities of arylsulfatase A is due to an acceleration of the enzyme degradation. Enzyme replacement by the addition of arylsulfatases of different sources (human liver, brain, fungus) was carried out in cultured MSD skin fibroblasts. Human enzymes of arylsulfatase A and B were mostly taken up by MSD cells rather than those of fungus origin. By the exposure to leukocytes to cultured skin fibroblasts, MSD cells corrected arylsulfatase A and B activities.     

The multiple sulfatase deficiency gene encodes an essential and limiting factor for the activity of sulfatases

In multiple sulfatase deficiency (MSD), a human inherited disorder, the activities of all sulfatases are impaired due to a defect in posttranslational modification. Here we report the identification, by functional complementation using microcell-mediated chromosome transfer, of a gene that is mutated in MSD and is able to rescue the enzymatic deficiency in patients' cell lines. Functional conservation of this gene was observed among distantly related species, suggesting a critical biological role. Coexpression of SUMF1 with sulfatases results in a strikingly synergistic increase of enzymatic activity, indicating that SUMF1 is both an essential and a limiting factor for sulfatases. These data have profound implications on the feasibility of enzyme replacement therapy for eight distinct inborn errors of metabolism.     

Multiple sulfatase deficiency: degradation of arylsulfatase A and B after endocytosis in fibroblasts

Multiple sulfatase deficiency can be classified into group I with severe and group II with moderate deficiencies in sulfatases. In fibroblasts in both groups the stability of arylsulfatase A and of the 47000-Mr form of arylsulfatase B is decreased [F. Steckel, A. Hasilik & K. von Figura (1985) Eur. J. Biochem. 151, 141-145]. After endocytosis in control fibroblasts or those from multiple sulfatase deficiency, arylsulfatase A and B derived from the latter were subjected to enhanced degradation in both types of recipient cells. The degradation was closely linked in time to endocytosis. Whereas instability of arylsulfatase A derived from different cell lines from multiple sulfatase deficiency was comparable, a marked heterogeneity was observed for the instability of the 47000-Mr polypeptide of arylsulfatase B. Each of the cell lines from multiple sulfatase deficiency synthesized arylsulfatase A and B polypeptides with normal and with decreased stability. Treatment with benzyloxycarbonyl-Phe-Ala-CHN2, an inhibitor of cysteine proteinases, stabilized arylsulfatase A polypeptides and partially restored arylsulfatase A activity in group II fibroblasts. The inhibitor had no protective effect on the 47000-Mr polypeptide or the activity of arylsulfatase B. The bearing of these findings on the yet unknown primary defect in multiple sulfatase deficiency is discussed.     

Phylogenetic conservation of arylsulfatases. cDNA cloning and expression of human arylsulfatase B

A 2.2-kilobase cDNA clone for human arylsulfatase B (ASB) and several genomic clones were isolated and sequenced. The deduced amino acid sequence of 533 amino acids contains a 41-amino acid N-terminal signal peptide and a mature polypeptide of 492 amino acid residues. Overexpression of ASB in transfected baby hamster kidney (BHK) cells resulted in up to 68-fold higher ASB activity than in untransfected BHK cells. Pulse-chase labeling showed that ASB was synthesized and secreted as a 64-kDa precursor and processed to a 47-kDa mature form in BHK cells. The 47-kDa ASB form was located in dense lysosomes. Transport of ASB to the lysosomes was accomplished in a mannose 6-phosphate receptor-dependent manner. The ASB cDNA clone hybridizes to 4.8-, 2.5-, and 1.8-kilobase species of RNA from human fibroblasts. The same pattern was observed in RNA from fibroblasts of three Maroteaux-Lamy patients who were deficient in ASB activity, as well as in RNA from fibroblasts of three patients with multiple sulfatase deficiency, in which all known sulfatases were markedly diminished. Deduced amino acid sequences of human arylsulfatase A, human ASB, human steroid sulfatase, human glucosamine-6-sulfatase, and an arylsulfatase from sea urchin showed a substantial degree of similarity suggesting that they arose from a common ancestral gene and are members of an arylsulfatase gene family.     

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.     

Genetic complementation in somatic cell hybrids of cerebroside sulfatase activator deficiency and metachromatic leukodystrophy fibroblasts

Summary Several cases of metachromatic leukodystrophy (MLD) have been described with normal or near normal activities of arylsulfatase A (cerebroside sulfatase). However, the ability of intact cultured fibroblasts to hydrolyze cerebroside sulfate was impaired. Since the impairment was corrected by cerebroside sulfatase activator, a deficiency of activator was implied. In the absence of direct demonstration of deficiency, other types of evidence were needed to support the premise that the genetic defect was not associated with the arylsulfatase A locus as in classical MLD. Therefore, somatic cell hybrids of activator deficiency and MLD fibroblasts were analyzed. Complementation was indicated by enhanced hydrolysis of cerebroside sulfate, supporting the view that cerebroside sulfatase activator deficiency and MLD are nonallelic.     

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.     

X-linked recessive ichthyosis. Enzymatic diagnosis of affected males and female carriers

Steroid sulfatase deficiency (SSD) is a sex-linked disorder characterized clinically by generalized X-linked ichthyosis. We report a study of 10 families where the clinical diagnosis of this disorder was confirmed by measuring arylsulfatase C and steroid sulfatase (STS) in cultured skin fibroblasts and/or leukocytes of patients and heterozygotes. The optimal conditions for these enzymatic determinations were determined. Our data indicate that STS measurement is a reliable test for SSD diagnosis, either in fibroblasts or in leukocytes. For the detection of heterozygotes, several enzymatic determinations in different cell types are required.     

Evidence for a dosage effect at the X-linked steroid sulfatase locus in human tissues.

Steroid sulfatase (STS) is an X-linked enzyme whose locus escapes X inactivation in human somatic cells. STS activity was determined in human fibroblasts varying in X-chromosome number from one to four. Greater STS activity was detected in 2X cell strains when compared to 1X cell strains; however, increased STS activity was not found in 3 and 4X strains as compared with the 2X strains. Greater STS activity was also observed in female hair follicles when compared with male hair follicles. These data provide evidence of a gene dosage effect at the STS locus in human tissues.     

Synthesis and stability of arylsulfatase A and B in fibroblasts from multiple sulfatase deficiency

Fibroblasts from patients with multiple sulfatase deficiency were analyzed for activities of arylsulfatase A and B, iduronate 2-sulfatase and sulfamatase. A group of patients (group I) severely deficient in all sulfatases (residual activities less than or equal to 10% of control) were differentiated from patients (group II) with residual sulfatase activities of up to 90% of control. The synthesis and stability of arylsulfatase A and B were determined in pulse-chase labelling experiments. The apparent rate of synthesis of arylsulfatase A and B varied from 30% to normal in both fibroblasts from group I and II multiple sulfatase deficiency. In group I the molecular activity of the arylsulfatase A and B was more than 10-fold lower than in control fibroblasts. In group II the molecular activity of the arylsulfatase A was twofold to threefold lower and that of arylsulfatase B half of normal. In fibroblasts of both groups the stability of arylsulfatase A polypeptides was significantly diminished. For arylsulfatase B the instability was restricted to the mature 47000-Mr polypeptide and was variable within both groups. These results demonstrate that multiple sulfatase deficiency is a heterogeneous disorder, in which the primary defects can impair both the catalytic properties and the stability of sulfatases.     

The activity of arylsulfatase A and B on tyrosine O-sulfates.

L-Tyrosine O-sulfate was hydrolyzed by pure human arylsulfatase A (arylsufate sulfohydrolase, EC 3.1.6.1). The rate of hydrolysis was 1/20 of the rate with nitrocatechol sulfate, but was comparable to the rate with cerebroside sulfate. The reaction was optimal at pH 5.3--5.5 and displayed zero order kinetics with time and enzyme concentration. The Km was about 35 mM. The enzyme showed no stereospecificity and hydrolyzed D-tyrosine O-sulfate with Km and V similar to those for the L-isomer. Arylsulfatase B was less than 5% as effective as arylsulfatase A in catalyzing the hydrolysis of the tyrosine sulfates. The daily urinary excretion of tyrosine sulfate by a patient with metachromatic leukodystrophy (arylsulfatase A deficiency) was comparable to the excretion by control subjects. The biological relevance of the tyrosine sulfatase activity of arylsulfatase A remains uncertain.     

The enzymic defect in Morquio's disease: the specificity of N-acetylhexosamine sulfatases.

Extracts of Morquio fibroblasts lack N-acetylgalactosamine 6-sulfate sulfatase activity, but exhibit normal levels of N-acetylglucosamine 6-sulfate sulfatase activity. Thus, the enzyme defective in Morquio's disease is a sulfatase specific for the 6-sulfate linked to sugars with the galactose configuration. Hydrolysis of ester sulfate by this enzyme is limited to 6-sulfate groups occurring at the non-reducing terminal.     

Expression of the neuron-specific protein, 14-3-2, and steroid sulfatase in neuroblastoma cell hybrids

Three series of neuroblastoma X fibroblast hybrid clones were isolated from crosses between mouse or human fibroblasts and mouse or human neuroblastoma cell lines by virus-mediated cell fusion. The expression of 14-3-2 protein (an acidic protein specific to neurons) and steroid sulfatase activity was studied in parental and hybrid cell lines. Steroid sulfatase was extinguished in hybrids when only one parent expressed the enzyme, but was expressed in one hybrid combination in which both parents expressed the enzyme. The neuron-specific 14-3-2 protein, on the other hand, continued to be expressed in all three series of neuroblastoma x fibroblast hybrids. In most cases where these pheno-types were expressed, they also exhibited temporal modulation; that is, specific activity is low during logarithmic growth and increases markedly during stationary phase. The glial-specific protein S-100 is absent from all parents and hybrids. The results are discussed in terms of mechanisms of regulation of differentiated phenotypes in mammalian cells.     

Regional assignment of the gene locus for steroid sulfatase

Summary The gene locus for steroid sulfatase, deficiency of which causes X-linked ichthyosis, is assigned to Xp11Xpter by analysis of 24 man-Chinese hamster somatic cell hybrids. High steroid sulfatase,activity in a hybrid clone having retained only part of Xq is explained by demonstration of an additional late-replicating human X chromosome. This observation confirms previous evidence for noninactivation of the STS locus.     

Steroid sulfatase activity and expression in mammary myoepithelial cells

PURPOSE: This investigation examined mRNA expression and enzymatic activity of steroid sulfatase (STS) in human mammary myoepithelial cells (MMECs) and MCF-7 cells and assessed the effects of 17-beta estradiol on the activity of STS. METHODS: The mRNA level of STS in MMECs was determined by RT-PCR analysis using specific primers for STS. STS enzymatic activity prior to and after treatment with 17-beta estradiol was determined by measuring 3H-metabolites formed after exposure to [3H]estrone 3-sulfate (E1S) and [3H]dehydroepiandrosterone-sulfate (DHEA-S). RESULTS: Our data demonstrate the presence of STS in the MMECs. Based on RT-PCR analysis, MMECs had slightly lower levels of STS compared to MCF-7 cells. However, sulfatase activity was about 120 times greater in the MMECs than the MCF-7 cells (E1S V(max)=2640nmol/(mg DNAh) compared to 20.9nmol/(mg DNAh)). Exposure to 17-beta estradiol was associated with 70% reduction in E1S sulfatase activity in the MCF-7 cells and 9% increase in the MMECs after 6 days. DISCUSSION: Our studies indicate for the first time the presence of STS in MMECs. This is suggestive of a previously undetermined role for MMECs in converting precursor hormones into active steroid hormones within mammary tissue. In addition, differential response of the MMECs and the MCF-7 cells to estrogen demonstrates differences in hormone metabolism between these two cell types, perhaps related to the absence of estrogen receptors in the MMECs and their presence in the MCF-7 cells. The MMECs may have an important role in hormonal regulation within mammary tissue.     

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.     

Phosphorylation of human lysosomal arylsulfatase B by cAMP-dependent protein kinase. Different sites of phosphorylation between normal and cancer tissues

We previously demonstrated that an acidic variant (B1) of lysosomal arylsulfatase B from transplanted human lung cancer is phosphorylated on its protein and carbohydrate moieties (Gasa, S., and Makita, A. (1983) J. Biol. Chem. 258, 5034-5039). The present study identifies that a cAMP-dependent protein kinase is responsible for phosphorylation of arylsulfatase B. The protein kinase activity toward the sulfatase was considerably higher in the transplanted lung cancer than in normal lung in the presence of cAMP. B enzyme purified from normal human liver was found to contain 0.6 mol/mol B enzyme, and protein kinase treatment added further 1.3 mol of Pi to give a single phosphopeptide (X). On the other hand, B1 enzyme purified from the transplanted human lung cancer which had been labeled in vivo with 32Pi revealed at least two phosphopeptides (X and Y). Assuming that the sulfatase from normal liver and lung cancer possesses the same number of available phosphorylation sites, phosphorylation of site X which was available only by deliberate phosphorylation of the native, ordinary B enzyme appears to be cancer-associated. Increasing phosphorylation of the sulfatase resulted in a maximum 50% elevation in arylsulfatase activity, followed by a decrease of the activity upon overphosphorylation, using an artificial substrate.