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, 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.     

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.     

Hunter syndrome: presence of material cross-reacting with antibodies against iduronate sulfatase

Summary Polyclonal antibodies were obtained from rabbits by injection of iduronate sulfatase purified 35,000-fold from human placenta, after elution of the enzyme from sodium dodecyl sulfate (SDS) polyacrylamide gels. The specificity of these antibodies towards iduronate sulfatase was demonstrated by immunoprecipitation of enzyme activity; the level of other lysosomal hydrolases and sulfatases remained constant. Immunoblot of iduronate sulfatase from various human sources showed that the antibody recognises a polypeptide of mol.wt. 72,000 daltons in placenta and serum, and a form of mol.wt. 60,000 daltons in fibroblasts. No immunoprecipitable peptide was found in urine or in the culture medium of fibroblasts. Polypeptides of the same molecular weight were recognised in serum and in fibroblasts of Hunter patients. The presence of altered proteins in these patients was also shown by competition experiments. The addition of Hunter proteins alters the binding of normal enzyme to the antibody.     

Steroid sulfatase activity in leukocytes: a comparative study in 45,X; 46,Xi(Xq) and carriers of steroid sulfatase deficiency

The enzyme steroid sulfatase (STS) hydrolyses 3-beta-hydroxysteroid sulfates. The female-male STS activity ratio is 1.04-1.7:1 in several cell lines in adults and reaches 2:1 in prepubertal subjects. In fibroblasts, STS values in X-chromosome abnormalities show a partial positive correlation according to the number of X-chromosomes. X-linked ichthyosis (XLI) carriers, with only one copy of the STS gene, present lower STS levels than normal controls. This study analyzes the STS activity in leukocytes of 46,Xi(Xq); 45,X; XLI carriers and normal controls using 7-[3H]-dehydroepiandrosterone sulfate as substrate. X-monosomy (1.07 +/- 0.18 pmol/mg protein/h), Xq isochromosome (1.02 +/- 0.12 pmol/mg protein/h) and normal females (1.03 +/- 0.11 pmol/mg protein/h) had similar STS values (p > 0.05). XLI-carriers and males showed the lowest STS levels (0.34 +/- 0.04 pmol/mg protein/h, p < 0.001 and 0.82 +/- 0.14 pmol/mg protein/h, p < 0.05, respectively). Female-male STS activity ratio in leukocytes was 1.3:1. These data indicate that a complex mechanism regulates the STS expression depending on each type of cell line.     

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.     

N-acetylgalactosamine-6-sulfate sulfatase in human placenta: purification and characteristics.

N-Acetylgalactosamine-6-sulfate sulfatase from human placenta was purified 33,600-fold using beta-N-acetyl-D-galactosamine 6-sulfate-(1----4)-beta-D-glucuronic acid-(1----3)-N-acetyl-D-[3H]galactosaminitol 6-sulfate as the substrate. This enzyme is an oligomer with a molecular mass of 120 kDa and consists of polypeptides of 40 and 15 kDa. The 15 kDa polypeptide is a glycoprotein. This purified protein has activities of N-acetylgalactosamine-6-sulfate sulfatase and galactose-6-sulfate sulfatase. Rabbit antiserum was raised against the purified protein. The antibody titrated N-acetylgalactosamine-6-sulfate sulfatase and galactose-6-sulfate sulfatase. The size of the precursor of the enzyme is 60 kDa, as determined by cell-free translation. The optimal pH values of the N-acetylgalactosamine-6-sulfate sulfatase and galactose-6-sulfate sulfatase activities are pH 3.8-4.0, and the Kms are 8 and 13 microM, respectively. Sulfate and phosphate ions are potent competitive inhibitors for the enzyme and their inhibition constants are 35 and 200 microM, respectively. Cross-reactive materials of 40 and 15 kDa were detected by immunoblot analysis, in the placenta, liver, and normal fibroblasts, but not in fibroblasts from a patient with Morquio disease.     

Tissue-specific expression of human arylsulfatase-C isozymes and steroid sulfatase.

Steroid sulfatase (STS; E.C.3.1.6.2), which acts on 3-hydroxysteroid sulfates, and arylsulfatase-C (ARC; E.C.3.1.6.1), assayed with aromatic artificial substrates, are both membrane-bound, microsomal enzymes with alkaline pH optima. Although they copurify during preparation and their gene loci are mapped to the short arm of the human X chromosome where they appear to have escaped from X inactivation, it has not been settled whether STS and ARC are the same enzyme or not. Recent work from our laboratory has shown that ARC exists in two electrophoretically distinct forms in human fibroblasts. We now report that these two forms--the faster migrating (F) and more slowly migrating (S)--occur in human tissues. Each of 11 human tissue types from 10 subjects showed a consistent pattern of ARC isozymes. Thyroid, heart, spleen, skeletal muscle, and adrenal tissue mainly had the S form. In contrast, kidney, liver, and pancreas tissue had mainly the F form, while gonadal, lung, and intestinal tissue had both the S and the F forms. The question of escape of their gene locus from X-chromosome inactivation was examined by comparing the specific activities of ARC and STS in male-derived vis-à-vis female-derived tissues. The majority of the tissues did not show any significant difference in these activities between the sexes, the exceptions being heart muscle, gonadal, and kidney tissue. None showed the 1:2 ratio between male- and female-derived tissues expected of a locus that had escaped X inactivation. The question of identity between ARC and STS was examined by comparing the ratios of their activities in these tissue types: if the enzymes were identical, the ratios of their activities should have remained constant across the different tissue types. It was thus shown that ARC activity varied by as much as 100-fold, depending on the ARC isozymic pattern of the tissue. STS, measured as estrone sulfatase and dehydroepiandrosterone sulfatase, did not show similar variations. This provides further evidence that ARC activity is not necessarily identical to that of STS.     

Steroid sulfatase gene in XX males.

The human X and Y chromosomes pair and recombine at their distal short arms during male meiosis. Recent studies indicate that the majority of XX males arise as a result of an aberrant exchange between X and Y chromosomes such that the testis-determining factor gene (TDF) is transferred from a Y chromatid to an X chromatid. It has been shown that X-specific loci such as that coding for the red cell surface antigen, Xg, are sometimes lost from the X chromosome in this aberrant exchange. The steroid sulfatase functional gene (STS) maps to the distal short arm of the X chromosome proximal to XG. We have asked whether STS is affected in the aberrant X-Y interchange leading to XX males. DNA extracted from fibroblasts of seven XX males known to contain Y-specific sequences in their genomic DNA was tested for dosage of the STS gene by using a specific genomic probe. Densitometry of the autoradiograms showed that these XX males have two copies of the STS gene, suggesting that the breakpoint on the X chromosome in the aberrant X-Y interchange is distal to STS. To obtain more definitive evidence, cell hybrids were derived from the fusion of mouse cells, deficient in hypoxanthine phosphoribosyltransferase, and fibroblasts of the seven XX males. The X chromosomes in these patients could be distinguished from each other when one of three X-linked restriction-fragment-length polymorphisms was used. Hybrid clones retaining a human X chromosome containing Y-specific sequences in the absence of the normal X chromosome could be identified in six of the seven cases of XX males.(ABSTRACT TRUNCATED AT 250 WORDS)     

SUMF1 enhances sulfatase activities in vivo in five sulfatase deficiencies

Sulfatases are enzymes that hydrolyse a diverse range of sulfate esters. Deficiency of lysosomal sulfatases leads to human diseases characterized by the accumulation of either GAGs (glycosaminoglycans) or sulfolipids. The catalytic activity of sulfatases resides in a unique formylglycine residue in their active site generated by the post-translational modification of a highly conserved cysteine residue. This modification is performed by SUMF1 (sulfatase-modifying factor 1), which is an essential factor for sulfatase activities. Mutations in the SUMF1 gene cause MSD (multiple sulfatase deficiency), an autosomal recessive disease in which the activities of all sulfatases are profoundly reduced. In previous studies, we have shown that SUMF1 has an enhancing effect on sulfatase activity when co-expressed with sulfatase genes in COS-7 cells. In the present study, we demonstrate that SUMF1 displays an enhancing effect on sulfatases activity when co-delivered with a sulfatase cDNA via AAV (adeno-associated virus) and LV (lentivirus) vectors in cells from individuals affected by five different diseases owing to sulfatase deficiencies or from murine models of the same diseases [i.e. MLD (metachromatic leukodystrophy), CDPX (X-linked dominant chondrodysplasia punctata) and MPS (mucopolysaccharidosis) II, IIIA and VI]. The SUMF1-enhancing effect on sulfatase activity resulted in an improved clearance of the intracellular GAG or sulfolipid accumulation. Moreover, we demonstrate that the SUMF1-enhancing effect is also present in vivo after AAV-mediated delivery of the sulfamidase gene to the muscle of MPSIIIA mice, resulting in a more efficient rescue of the phenotype. These results indicate that co-delivery of SUMF1 may enhance the efficacy of gene therapy in several sulfatase deficiencies.     

Correction of Steroid Sulfatase Deficiency by Gene Transfer into Basal Cells of Tissue-Cultured Epidermis from Patients with Recessive X-Linked Ichthyosis

To develop an experimental model for somatic gene therapy we have tried to correct the steroid sulfatase (STS) deficiency in tissue-cultured primary epidermal keratinocytes from patients suffering from recessive X-linked ichthyosis. An efficient Epstein-Barr virus-based vector was constructed, in which full-length steroid sulfatase cDNA is located between an SV40 early promotor and processing signals. After STS gene transfer into cultured basal cells from ichthyotic skin, the cells produce large amounts of enzymatically active steroid sulfatase protein. The subpopulation of transfected cells can be made to produce approximately 100 times more STS activity than normal keratinocytes. Keratinocytes from patients suffering from recessive X-linked ichthyosis display an abnormal phenotype when developing a multilayered tissue in culture: Initially an extensive burst of keratinization is observed, followed by rapid, premature shedding and degradation of most suprabasal cell layers, leaving a culture with hyperproliferative relatively immature keratinocytes. Transfection of these immature ichthyotic cells with the functional STS construct led to an increase in the amount of retained cell material in the culture medium, indicating an increased cell maturation. It is possible to genetically label individual transfected epidermal cells with a reporter gene. Cotransfection experiments with STS and reporter gene vectors show that the cohort of transfected cells had a tendency to develop less rapidly since they became overrepresented in the smaller size classes at the same time the total population was somewhat shifted toward higher cell sizes. We interpret these results as an indication that restoration of the enzymatic activity induces a more normal maturation of the transfected keratinocytes.     

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 activity in homogenates, microsomes and purified Leydig cells from adult rat testis

Steroid sulfatase (STS) activity was studied in the Long-Evans rat testis. The rate of dehydroepiandrosterone sulfate (DHA-S) hydrolysis determined in whole testis homogenates was low compared to that of the corresponding microsomal fractions, which was, in contrast, as high as that expressed in homogenates from purified Leydig cells. Such an increment in STS activity between total homogenates and the corresponding microsomes was not observed for the seminiferous tubules. The STS affinity reported for total testicular microsomes (Km = 3.47 +/- 0.54 microM; mean +/- SEM) was of the same magnitude as that previously reported for Leydig cells, but was about 3 times higher than that measured for whole testis homogenate (Km = 10.11 +/- 0.92 microM). In vivo hCG treatment decreased the STS affinity in total testicular microsomes without affecting this kinetic parameter in whole testis homogenate. These data suggest that the steroid sulfatase expressed in total testicular microsomes (activity and regulation by hCG) could be considered as a good index of Leydig cell STS activity.     

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.     

6-(2-adamantan-2-ylidene-hydroxybenzoxazole)-O-sulfamate: a potent non-steroidal irreversible inhibitor of human steroid sulfatase

We report the synthesis and results from the in vitro evaluation of 6-(adamantan-2-ylidene-hydroxybenzoxazole)-O-sulfamate 1 as an irreversible inhibitor of human steroid sulfatase (STS). Highly straightforward, condensation of 2-methyl-6-hydroxybenzoxazole with 2-adamantanone, subsequent elimination of water and sulfamoylation provide the title compound in 45% overall yield from the inexpensive 2,4-dihydroxyacetophenone. 1 was found to be a potent irreversible inhibitor of purified human steroid sulfatase (STS) and specific for this enzyme relative to human arylsulfatases A and B. In cellular assays with human keratinocytes, sebocytes and fibroblasts, 1 blocked STS activity with IC₅₀ values in the range of 0.15–0.8 nM, and in MCF-7 breast cancer cells with IC₅₀=2.3 nM, while it did not bind to estrogen receptors and β. Thus, 1 is a candidate for further investigation of its potential as a drug to be used in androgen- and estrogen-dependent diseases.     

Activity of steroid sulfatase in fibroblasts with numerical and structural X chromosome aberrations

Summary In cultured fibroblasts of patients with numerical and structural X chromosome aberrations the activity of steroid sulfatase (STS) is correlated with the number of functional STS gene copies. While normally, this X-linked gene is not inactivated, our data suggest that it may be subject to inactivation when carried on a structurally altered X-chromosome. Similar inactivation patterns have been reported earlier for the Xg locus which, like STS, is located on the distal protion of Xp.     

Long-range physical mapping around the human steroid sulfatase locus

The region of the human X chromosome containing the steroid sulfatase locus was analyzed by pulsed-field gel electrophoresis. Restriction site maps were generated for the X chromosome in the blood of a normal male individual and that in the mouse-human hybrid cell line ThyB-X; these maps extend over approximately 4.3 Mb of DNA of the former, and 3.2 Mb of the latter. Physical linkage was defined between the STS locus and sequences detected by the probes GMGX9 (DXS237), GMGXY19 (DYS74), CRI-S232 (DXS278), and dic56 (DXS143), and the order telomere--(STS, DYS74)--DXS237--DXS278--DXS143--centromere was deduced. The pulsed-field maps were used to demonstrate a deletion of 180 kb of DNA from the X chromosome of an individual with X-linked ichthyosis. Also, possible locations for the Kallmann syndrome gene were revealed, and the distance between the steroid sulfatase locus and the pseudoautosomal region was estimated to be at least 4 Mb.     

N-Acyl arylsulfonamides as novel, reversible inhibitors of human steroid sulfatase

Steroid sulfatase (STS) is an attractive target for a range of oestrogen- and androgen-dependent diseases. In search of novel chemotypes of STS inhibitors, we had previously identified nortropinyl-arylsulfonylureas 1; however, while these compounds were good inhibitors of purified STS (lowest K(i)=76 nM), they showed only weak inhibition of STS activity in cells (lowest IC(50) around 2 microM). Extended structure-activity relationship studies involving modification of the phenylacetyl side chain and replacement of the nortropine element by simpler scaffolds led to the discovery of N-acyl arylsulfonamides, more specifically N-(Boc-piperidine-4-carbonyl)-benzenesulfonamides, as STS inhibitors, some of which exhibit improved cellular potency (best IC(50)=270 nM).     

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.