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PubMed1982

Specificity of bile salt sulfatase activity from Clostridium sp. strains S1.

Clostridium sp. strain S1, an unnamed bile acid-desulfating strain from rat intestinal microflora (S.M. Huijghebaert, J. A. Mertens, and H. J. Eyssen, Appl. Environ. Microbiol. 43:185-192, 1982), was examined for its ability to desulfate different bile acid sulfates and steroid sulfates in growing cultures. Clostridium sp. strain S1 desulfated the 3 alpha-monosulfates of chenodeoxycholic, deoxycholic, and cholic acid, but not their 7 alpha- or 12 alpha-monosulfates. Among the 3-sulfates of the 5 alpha- and 5 beta-bile acids, only bile acid-3-sulfates with an equatorial sulfate group were desulfated. Hence, Clostridium sp. strain S1 desulfated the 3-sulfates of bile acids with a 3 alpha, 5 beta-, a 3 beta, 5 alpha- or a 3 beta, delta 5-structure. In contrast, the bile acid-3-sulfates with a 3 beta, 5 beta- or a 3 alpha, 5 alpha-structure were not desulfated. In addition, Clostridium sp. strain S1 did not hydrolyze the equatorial 3-sulfate esters of C19 and C21 steroids and cholesterol or the phenolic 3-sulfate esters of estrone and estradiol. 23-Nordeoxycholic acid with a C-23 carboxyl group was also not desulfated, in contrast to the 5 beta-bile acid 3 alpha-sulfates with a C-24 or C-26 carboxyl group. Therefore, the specificity of the sulfatase of Clostridium sp. strain S1 is related to the location of the sulfate group on the bile acid molecule, the equatorial orientation of the sulfate group, and the structure of the C-17 side chain, its carboxyl group, and chain length.

Bile Acids and Salts↗
PubMed1965

Primary alcohol sulfatase in a Pseudomonas species.

An ammonium sulfate-precipitated fraction from cell-free extracts of Pseudomonas C12B grown on a medium containing sodium dodecyl sulfate (SDS) contained alkyl sulfatase increased fourfold in specific activity over the crude. Optimal pH (7.5) and temperature (70 C) for sulfate release were determined with SDS labeled with radioactive sulfur (SDS(35)) as test substrate. Phosphate, arsenate, and certain heavy metal ions inhibited desulfation, whereas Mg(++) and Mn(++) stimulated activity of preparations which had been dialyzed against ethylenediaminetetraacetic acid. Dodecanol was recovered in semiquantitative yield from reaction mixtures containing enzyme and SDS(35). Aryl sulfates, secondary alcohol sulfates, and a phenoxyethyl sulfate failed to serve as substrate for this enzyme.

Alcohols↗
PubMed2004

Identification of iduronate-2-sulfatase in mouse pancreatic islets.

The lysosomal enzyme iduronate-2-sulfatase (IDS) is expressed in pancreatic islets and is responsible for degradation of proteoglycans, such as perlecan and dermatan sulfate. To determine the role of IDS in islets, expression and regulation of the gene and localization of the enzyme were investigated in mouse pancreatic islets and clonal cells. The Ids gene was expressed in mouse islets and beta- and alpha-clonal cells, in which it was localized intracellularly in lysosomes. The transcriptional expression of Ids in mouse islets increased with glucose in a dose-dependent manner (11.5, 40.2, 88, and 179% at 5.5, 11.1, 16.7, and 24.4 mM, respectively, P < 0.01 for 16.7 and 24.4 mM glucose vs. 3 mM glucose). This increase was not produced by glyceraldehyde (1 mM) or 6-deoxyglucose (21.4 mM) and was blocked by the addition of mannoheptulose (21.4 mM). Neither insulin content nor secretory response to glucose (16.7 mM) was altered in mouse islets infected with lentiviral constructs carrying the IDS gene in sense orientation. Furthermore, no decrease in islet cell viability was observed in mouse islets carrying lentiviral contracts compared with controls. However, insulin content was reduced (35% vs. controls, P < 0.001) in islets infected with IDS antisense construct, while the secretory response of those islets to glucose was maintained. Inhibition of IDS by antisense infection led to an increase in lysosomal size and a high rate of insulin granule degradation via the crinophagic route in pancreatic beta-cells. We conclude that IDS is localized in lysosomes in pancreatic islet cells and expression is regulated by glucose. IDS has a potential role in the normal pathway of lysosomal degradation of secretory peptides and is likely to be essential to maintain pancreatic beta-cell function.

Animals↗
PubMed2004

Steroid sulfatase gene variation and DHEA responsiveness to resistance exercise in MERET.

Genetic influences and endurance exercise have been shown to alter circulating concentrations of dehydroepiandrosterone (DHEA) and its sulfated conjugate, DHEAS. We hypothesized that acute resistance exercise (RE) and training (RET) would increase DHEA steroids, and the magnitude of the increase would be influenced by a steroid sulfatase (STS) gene variation. Fasting blood samples were collected before and after the first (S1) and last (S30) session of a 10-wk RET program in 62 men and 58 women [age: 21.0 yr (2.4)]. Acute RE increased both DHEA [+2.8 (0.4), S1; +1.6 ng/ml (0.4), S30; P < 0.001] and DHEAS [+154 (24), S1; +166 ng/ml (15), S30; P < 0.001] and decreased DHEAS:DHEA [-27 (8), S1; -15 (7), S30; P < 0.01]. RET reduced resting DHEAS (-122 ng/ml, P < 0.01) and decreased DHEA response to RE (-50%, P < 0.05). Subjects with an STS "G" allele (n = 36) had greater acute changes in DHEA [+4.4 (0.7) vs. +2.0 ng/ml (0.5), S1; +3.2 (0.6) vs. +1.0 ng/ml (0.4), S30; P < 0.01] and DHEAS:DHEA [-37 (11) vs. 5 (7), S30, P < 0.05] than those subjects with only an "A" allele (n = 84). The observed increase in DHEA and DHEAS and decrease in DHEAS:DHEA suggest RE-induced STS activation which is influenced by the STS polymorphism.

Adult↗
PubMed2000

Differential increase of steroid sulfatase activity in XX and XY trophoblast cells from human term placenta with syncytia formation in vitro.

Steroid sulfatase (STS, EC 3.1.6.2) catalyzes the hydrolysis of the sulfate ester bonds of a variety of sulfated steroids, such as cholesterol, dehydroepiandrosterone, and estrone sulfate, a reaction influencing fertility and breast cancer in mammals. The activity of the enzyme is substantially elevated in placental syncytiotrophoblasts and is lower in other somatic cells. The polypeptide sequence of the enzyme is encoded by a gene located on the distal short arm of the human X chromosome. Prior studies have shown that the STS gene escapes X-chromosome inactivation. We studied the expression of the STS gene in primary cultures of cytotrophoblasts from human term placentae and compared it with the expression of autosomally encoded placental alkaline phosphatase (PALP) and X-linked glucose-6-phosphate dehydrogenase (G6PD). During 90 h in culture, the mononucleated cytotrophoblast cells did not proliferate, but differentiated into multinucleated, syncytiotrophoblast-like cells. STS activity in freshly isolated cytotrophoblasts was low (about 17%), compared to placental tis- sue, and about 1.7-fold higher in female than in male cells. During cultivation, STS activity increased 2- to 3-fold in female, but not in male, cells. PALP activity was very low in freshly isolated cytotrophoblasts (about 5% of placental tissue), and no significant difference between female and male cells was detectable. Within 90 h in culture, PALP activity increased in all preparations about 2- to 4-fold. G6PD activity in freshly isolated cytotrophoblasts showed great variation among preparations, and no significant difference between female and male cells was detectable. In both male and female cells the activity declined to about 50% of initial activity during cultivation. We conclude that human cytotrophoblasts in primary culture show a sex-specific regulation of STS activity, perhaps as a unique feature of the STS gene. The cytotrophoblast system may offer a new possibility to study the regulation of STS gene expression.

Alkaline Phosphatase↗
PubMedDate not supplied

Association of undetectable unconjugated estriol on multiple marker screening with steroid sulfatase deficiency.

OBJECTIVE: To determine whether all patients with undetectable unconjugated estriol (uE3) on multiple marker screening (MMS) are carriers for steroid sulfatase (STS) deficiency. METHODS: This is a retrospective review of 65 pregnancies with undetectable uE3 on MMS. RESULTS: Of the 65 pregnancies, there were 21 that continued, 40 spontaneous losses, 2 lost to follow-up and 2 elective terminations. Of the 21 continuing pregnancies, 15 were determined to be carriers of the STS deletion. Twenty-seven of the 40 pregnancy losses were associated with elevated alpha-fetoprotein (AFP); about half of the losses were shown to have occurred prior to sampling. CONCLUSION: Patients with undetectable uE3 are likely to be carriers of the STS deletion, except those with associated elevated AFP. Elevated AFP with undetectable uE3 on MMS is a marker of preexisting or impending fetal demise.

Abortion, Spontaneous↗
PubMedDate not supplied

Very low maternal serum unconjugated estriol and prenatal diagnosis of steroid sulfatase deficiency.

Twenty-four women out of 7,875 pregnant women who enrolled in a prenatal screening program showed extremely low levels of unconjugated estriol (< 0.15 MOM). In 19 cases, intrauterine fetal death was reported. In 1 case anencephalus was detected. In the remaining 4 cases apparently normal healthy babies (1 female and 3 males) were born following uneventful pregnancies. Physical examination of the 3 boys at 4-6 weeks revealed mild ichthyosis compatible with the X-linked type. Two of them had a positive family history of X-linked ichthyosis. The examination of the girl did not reveal any significant findings. In both cases in which amniocentesis was performed, low levels of steroid sulfatase and arylsulfatase C were found. The prevalence of X-linked ichthyosis in this study is higher than previously reported, i.e. 1:1,300 males. Our results suggest that the prenatal screening program for neural tube defects and for Down's syndrome is useful for the prenatal detection of X-linked ichthyosis as well. These results are in accordance with two recent reports. The implications regarding genetic counseling are discussed.

Adult↗
PubMed1982

Acid mucopolysaccharide (AMPS) abnormality in multiple sulfatase deficiency: chemical compositions of AMPS in urine and liver.

Extensive chemical analyses of acid mucopolysaccharides (AMPS) were carried out in the urine and tissue (liver and brain) from a Japanese patient and two European patients with multiple sulfatase deficiency (MSD). The Japanese patient with MSD contained excessive quantities of heparan sulfate and moderately increased chondroitin sulfate A/C. Urinary excretion of AMPS in MSD heterozygotes was increased 2-fold compared to our controls. The urinary pattern of AMPS in the mother of the MSD patient showed an increase of 18% heparan sulfate and 36% dermatan sulfate whereas the urinary excretion pattern in the father was increased 21% for heparan sulfate as contrasted to controls (chondroitin sulfate A, 50-52%; chondroitin sulfate C, 38-46%; and heparan sulfate, 3-10%). Seventy-five % of the AMPS and the MSD liver was heparan sulfate rather than dermatan sulfate. The degree of accumulation of AMPS in the MSD liver was 30-50 times that of the control. Cerebral gray matter from the MSD patient contained 30-40 times that of control (relative increase of heparan and dermatan sulfate) whereas only a 5-fold increase was observed in white matter. It seems that a major site of accumulated AMPS appears to be in the gray matter. Carbohydrate analysis of the AMPS obtained from MSD urine and tissues was performed by: enzyme digestion with testicular hyaluronidase, heparitinase and chondroitinase ABC, cellulose acetate electrophoresis, Dowex-1 column chromatography and amino sugar analysis by amino acid analyzer. These findings indicate that the major accumulated AMPS in MSD urine and liver is heparan sulfate and thus, the predominant AMPS metabolic defect in MSD is heparan sulfate degradation.

Brain Chemistry↗
PubMed1997

Expression of steroid sulfatase during embryogenesis.

Neurosteroids are steroids that are synthesized de novo in the brain from cholesterol and, in general, mediate their effects through ion-gated channel receptors such as gamma-aminobutyric acidA (GABA[A]) and N-methyl-D-aspartate receptors rather than through classical nuclear steroid hormone receptors. Steroid hormones are known to exist not only as free compounds, but also as sulfated derivatives. Pharmacological studies indicate that unconjugated and sulfated steroids, such as pregnenolone and pregnenolone sulfate, may have opposite effects on GABA(A) receptors. Thus, pregnenolone acts as a potent positive allosteric modulator of gamma-aminobutyric acid action at GABA(A )receptors, whereas pregnenolone sulfate acts as a potent negative modulator. Recent experiments also suggest that dehydroepiandrosterone and dehydroepiandrosterone sulfate may have distinct effects on growth of neurites from embryonic neocortical neurons in vitro. Thus, regulation of steroid sulfation may have profound behavioral and morphological effects on the nervous system. We, therefore, studied the developmental expression of the enzyme steroid sulfatase (STS), which converts sulfated steroids to free steroids. By in situ hybridization, STS messenger RNA was expressed in the embryonic mouse cortex, hindbrain, and thalamus during the last third of gestation. The sites of expression of STS were similar to those of P450c17, suggesting that these two enzymes may have concerted actions in similar functional processes.

Animals↗
PubMed2001

Constitutive expression of the steroid sulfatase gene supports the growth of MCF-7 human breast cancer cells in vitro and in vivo.

Many human breast tumors are driven by high intratumor concentrations of 17beta-estradiol that appear to be locally synthesized. The role of aromatase is well established, but the possible contribution of the steroid sulfatase (STS), which liberates estrogens from their biologically inactive sulfates, has been inadequately assessed and remains unclear. To evaluate the role of STS further, we transduced estrogen-dependent MCF-7 human breast cancer cells with a retroviral vector directing the constitutive expression of the human STS gene. Gene integration was confirmed by Southern hybridization, production of the appropriately sized messenger RNA by Northern hybridization, and expression of functional protein by metabolism of [(3)H]estrone sulfate to [(3)H]estrone. Maximum velocity estimates of estrone formation are 64.2 pmol estrone/mg protein.h in STS-transduced cells (STS Clone 20), levels comparable to those seen in some human breast tumors. Lower levels of endogenous activity are seen in MCF-7 cells (13.0 pmol estrone/mg protein.h) and in cells transduced with vector lacking the STS gene (Vector 3 cells; 12.0 pmol estrone/mg protein.h). 17beta-Estradiol sulfate induces expression of the progesterone receptor messenger RNA only in STS Clone 20 cells, whereas estrone sulfate produces the greatest stimulation of anchorage-independent growth in these cells. STS Clone 20 cells retain responsiveness to antiestrogens, which block the ability of estrogen sulfate to increase the proportion of cells in both the S and G(2)/M phases of the cell cycle. Consistent with these in vitro observations, only STS Clone 20 cells exhibit a significant increase in the proportion of proliferating tumors in nude ovariectomized mice supplemented with 17beta-estradiol sulfate. The primary activity in vivo appears to be from intratumor STS, rather than hepatic STS. Surprisingly, 17beta-estradiol sulfate appears more effective than 17beta-estradiol when both are administered at comparable concentrations. This effect, which is seen only in STS Clone 20 cells, may reflect differences in the cellular pharmacology of exogenous estrogens compared with those released by the activity of intracellular STS. These studies directly demonstrate that intratumor STS activity can support estrogen-dependent tumorigenicity in an experimental model and may contribute to the promotion of human breast tumors.

Animals↗
PubMed2004

Steroid sulfatase: molecular biology, regulation, and inhibition.

Steroid sulfatase (STS) is responsible for the hydrolysis of aryl and alkyl steroid sulfates and therefore has a pivotal role in regulating the formation of biologically active steroids. The enzyme is widely distributed throughout the body, and its action is implicated in physiological processes and pathological conditions. The crystal structure of the enzyme has been resolved, but relatively little is known about what regulates its expression or activity. Research into the control and inhibition of this enzyme has been stimulated by its important role in supporting the growth of hormone-dependent tumors of the breast and prostate. STS is responsible for the hydrolysis of estrone sulfate and dehydroepiandrosterone sulfate to estrone and dehydroepiandrosterone, respectively, both of which can be converted to steroids with estrogenic properties (i.e., estradiol and androstenediol) that can stimulate tumor growth. STS expression is increased in breast tumors and has prognostic significance. The role of STS in supporting tumor growth prompted the development of potent STS inhibitors. Several steroidal and nonsteroidal STS inhibitors are now available, with the irreversible type of inhibitor having a phenol sulfamate ester as its active pharmacophore. One such inhibitor, 667 COUMATE, has now entered a phase I trial in postmenopausal women with breast cancer. The skin is also an important site of STS activity, and deficiency of this enzyme is associated with X-linked ichthyosis. STS may also be involved in regulating part of the immune response and some aspects of cognitive function. The development of potent STS inhibitors will allow investigation of the role of this enzyme in physiological and pathological processes.

Animals↗
PubMed2002

Systemic distribution of steroid sulfatase and estrogen sulfotransferase in human adult and fetal tissues.

Estrogens play a key role in various target tissues. Enzymes involved in the biosynthesis and metabolism of these sex steroids also regulate estrogenic actions in these tissues. Estrone sulfate (E1S) is a major circulating plasma estrogen that is converted into the biologically active estrogen, estrone (E1), by steroid sulfatase (STS). E1 is also sulfated and reverted into E1S by estrogen sulfotransferase (EST). These two enzymes have recently been shown to play important roles in the in situ estrogen actions of various sex steroid-dependent human tumors. However, the distribution of STS and EST in normal adult and fetal human tissues remains largely unknown. Therefore, in this study, in addition to examining the tissue distribution of both STS and EST mRNA in human adult and fetal tissues using RT followed by quantitative PCR, we studied the activity of these enzymes using (3)H-labeled E1/E1S as substrates in the homogenates of various human adult tissues. We also examined the localization of STS and EST protein in human adult and fetal tissues using immunohistochemistry, and that of EST mRNA in the adult kidney using laser dissection microscopy and PCR. STS mRNA, enzyme activity, and immunoreactivity were either absent or detected at very low levels in all adult and fetal tissues examined in this study. EST mRNA expression, however, was detected in all of the tissues examined, except for adult spleen and pancreas. EST enzyme activities were consistent with those of mRNA expression in the great majority of the tissues examined. Marked EST immunoreactivity was detected in hepatocytes, adrenal gland (adult, zona fasciculate to the reticularis; fetus, fetal zone), and epithelial cells of the gastrointestinal tract, smooth muscle cells of the tunica media in aorta, Leydig cells of the testis, and syncytiotrophoblast of the placenta. Patterns of EST immunolocalization were similar between adult and fetal human tissues, but EST immunoreactivity was detected in the urinary tubules of adult kidney, whereas in the fetal kidney, it was localized in the interstitial cells surrounding the urinary tubules. In the adult kidney, the presence of EST mRNA was also confirmed in the cells of urinary tubules using laser dissection microscopy and RT-PCR. Although the number of human tissues available for examination in this study was limited, our results suggest that between the enzymes involved in estrogen activation or inactivation, EST and not STS is the more widely expressed enzyme in various peripheral tissues in humans. We speculate that EST may play an important role in protecting peripheral tissues from possible excessive estrogenic effects.

Adult↗
PubMed1994

Purification and properties of bile acid sulfate sulfatase from Pseudomonas testosteroni.

The bile acid sulfate sulfatase (BSS) produced by Pseudomonas testosteroni was purified and characterized. Chromatofocusing behavior and amino acid sequence over twelve amino acid residues from N-terminus of the enzyme indicated that BSS was composed of two isoforms of which molecular weights were 125,000 and 103,000. Each isoform was a homodimer of a subunit of which molecular weight was 53,000 or 51,000, respectively. The optimum pH was 8.5 and BSS was stable at pH 5.8-8.0. The thermostability above 32 degrees C was improved by the addition of polyols, such as sorbitol, sucrose, and glycerol. BSS was a Mn(2+)-dependent enzyme and contained 1-2 atoms of manganese in its own protein molecule. All 3 alpha-sulfate esters of the bile acids routinely appearing in human serum were hydrolyzed by BSS to 3 beta-hydroxyl iso-compounds corresponding to each bile acid and sulfuric acid. We tentatively named this novel enzyme BSS (bile acid 3 alpha-sulfate sulfohydrolase).

Amino Acid Sequence↗
PubMed2006

Detection of mucopolysaccharidosis type II by measurement of iduronate-2-sulfatase in dried blood spots and plasma samples.

BACKGROUND: Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder related to a deficiency in the enzyme iduronate-2-sulfatase (IDS). Clinical trials of enzyme replacement therapy are in progress, but effective treatment will require screening assays to enable early detection and diagnosis of MPS II. Our study evaluated the diagnostic accuracy of IDS protein and enzyme activity measurements in dried blood spots and plasma. METHODS: We collected dried-blood-spot and plasma samples from unaffected control individuals and from MPS II patients. We measured IDS protein concentration with a 2-step time-delayed dissociation-enhanced lanthanide fluorescence immunoassay. To measure enzyme activity, we immobilized anti-IDS antibody on microtiter plates to capture the enzyme and measured its activity with the fluorogenic substrate 4-methylumbelliferyl sulfate. RESULTS: Dried-blood-spot samples from MPS II patients showed an almost total absence of IDS activity (0-0.075 micromol x h(-1) x L(-1)) compared with control blood spots (0.5-4.7 micromol x h(-1) x L(-1)) and control plasma (0.17-8.1 micromol x h(-1) x L(-1)). A dried-blood-spot sample from only 1 of 12 MPS II patients had detectable concentrations of IDS protein (24.8 microg/L), but no IDS protein was detected in plasma from MPS II patients. Ranges for IDS protein in control samples were 25.8-153 microg/L for blood spots and 22.8-349.4 microg/L for plasma. CONCLUSION: Measurement of the IDS protein concentration and enzyme activity (as measured by a simple fluorogenic assay with an immune capture technique) enables identification of the majority of MPS II patient samples from both dried blood spots and plasma samples.

Adolescent↗
PubMed2005

Direct comparison of measures of endurance, mobility, and joint function during enzyme-replacement therapy of mucopolysaccharidosis VI (Maroteaux-Lamy syndrome): results after 48 weeks in a phase 2 open-label clinical study of recombinant human N-acetylgalactosamine 4-sulfatase.

OBJECTIVE: Mucopolysaccharidosis VI (MPS VI; Maroteaux-Lamy syndrome) is a lysosomal storage disease caused by a deficiency of the enzyme N-acetylgalactosamine 4-sulfatase (ASB). This enzyme deficiency leads to a progressive disorder with multiple tissue and organ involvement. The disease is rare and is heterogeneous in its clinical presentation and progression. A potential treatment for this disease exists in the form of enzyme-replacement therapy (ERT) with recombinant human ASB (rhASB), and a phase 1/2 randomized, double-blind, 2-dose (0.2 and 1 mg/kg) study in 6 patients showed the treatment at 48 weeks to be well tolerated. Greater biochemical efficacy based on a urine glycosaminoglycan occurred in the high-dose (1 mg/kg) group, and functional improvement seemed greater in patients in the high-dose group with rapidly advancing disease. On the basis of the phase 1/2 results, a phase 2, open-label study in patients with rapidly advancing disease was initiated primarily to evaluate efficacy variables that measure endurance, mobility, and joint function in a larger group of patients. METHODS: This was an open-label, multinational study of 10 MPS VI patients who received 48 weekly intravenous treatments with 1.0 mg/kg rhASB and had assessments of biochemical and clinical responses at regular intervals. RESULTS: After 24 weeks of treatment, each patient on average experienced a 155-m (98%) improvement in the 12-minute walk, a 64-m (62%) improvement at the 6-minute time point of the 12-minute walk, and a 48-stair (110%) gain in the 3-minute stair climb versus the baseline mean values. Additional improvements after 48 weeks of treatment were observed, including mean values of 211 m (138%) in the 12-minute walk, 75 m (80%) at the 6-minute time point of the 12-minute walk, and 61-stair (147%) gain in the 3-minute stair climb versus the baseline mean values. Joint Pain and Stiffness Questionnaire scores improved by at least 50% by week 24 and were maintained at week 48, whereas there were only small improvements in active shoulder range of motion (<10 degrees ) and in the time taken to stand, walk, and turn starting from a seated position (Expanded Timed Get-Up and Go test). Improvement in pulmonary function based on forced vital capacity and forced expiratory volume at 1 minute in the absence of growth was observed in 3 of 6 patients, and the observed gains occurred in the 24- to 48-week treatment interval. A mean decrease of 76% in urinary excretion of glycosaminoglycans indicated that a satisfactory biochemical response was achieved and the ERT was well tolerated. CONCLUSIONS: The results suggest that a 12-minute walk extends the dynamic range of the conventional 6-minute walk and, along with the 3-minute stair climb, provide a robust approach to documenting the improvement in endurance in MPS VI patients who undergo ERT with rhASB.

Adolescent↗
PubMed2006

Both N-terminal and C-terminal regions of steroid sulfatase are important for enzyme activity.

Steroid sulfatase (STS) is localized in the endoplasmic reticulum and catalyzes desulfation of 3beta-hydroxysteroid sulfates. X-linked ichthyosis (XLI) is an inherited skin disorder caused by deficiency of STS enzyme activity. We previously reported a case in which XLI with a one-base change in the STS gene and variation in amino acid Q560P developed. In this study, we performed molecular analysis to determine the importance of terminal regions of STS and the effect of mutant STS on STS enzyme activity. To examine the effect of terminal truncated STS on the enzyme activity, N- and C-terminal truncated STS expression vectors were transfected into COS-1 cells. The activity of truncated STS lacking the N-terminal regions declined, and the activity of C-terminal-truncated STS declined with extension of the truncated C-terminal region. Although the results of pulse-chase experiments showed that a one-base mutant STS (Q560P) and C-terminal-truncated STS (deltaC2 (1-559)) had no effects on protein synthesis and degradation, the mutant STS and C-terminal-truncated STS have dominant negative effect on STS enzyme activity when the STS mutant or truncated STS protein and a wild-type STS protein coexist in cells. Results of coprecipitation of the truncated STS with an STS-FLAG fusion protein showed that STS formed a dimer conformation in cells. In this study, we have shown that both the N-terminal region and C-terminal region are important for STS enzyme activity. The C-terminal mutant has a dominant negative effect on wild-type STS.

Amino Acids↗
PubMed2006

Tissue-specific transcriptional initiation and activity of steroid sulfatase complementing dehydroepiandrosterone sulfate uptake and intracrine steroid activations in human adipose tissue.

Expression analysis by reverse transcriptase (RT)-PCR indicates that human adipose tissue is not likely to perform de novo synthesis of steroid hormones from cholesterol because the mRNAs of cytochromes P450scc and P450c17, and of the steroidogenic-related proteins, steroidogenic acute regulatory protein and steroidogenic factor 1, were not detected. Instead, our data support an intracrine role of adipose tissue, in which adrenal dehydroepiandrosterone sulfate (DHEA-S), the most abundant circulating androgen in man, is selectively uptaken, desulfated, and converted into bioactive androgens and estrogens. Three organic anion-transporting polypeptides-B, -D, and -E, presumably involved in DHEA-S transmembrane transport, were demonstrated at the mRNA level. While sulfotransferase expression was not found, the occurrence of steroid sulfatase (STS), converting DHEA-S to DHEA, was established at the mRNA, protein and catalytic activity levels. The 5'-rapid amplification of cDNA ends analysis showed that STS transcription in adipose tissue is regulated by the use of two promoters which differ from the prevalent placental one. The adipose transcripts contain a distinct untranslated first exon, 0a or 0b, followed by a common partially translated exon 1b, and nine other exons that are also shared by the main placental transcript. The presence of an upstream open reading frame in the new transcript variants could lead to an N-terminal divergence restricted to the cleavable signal peptide and thus not interfering with the catalytic activity of the mature STS protein. The adipose transcripts are also present in the placenta as minor isoforms. Western blotting revealed the characteristic approximately 64 kDa band of STS in both the placenta and adipose tissue. The specific enzymatic activity of STS in adipocytes was 118 pmol/10(6) cells per hour, about 50-100 times lower than in the placenta. A similar rate of [3H] DHEA-S uptake plus desulfation was measured in preadipocytes and adipocytes, equivalent to 40-45 pmol/10(6) cells per hour. Thus, an excessive accumulation of fat may out-compete other peripheral organs that are also dependent on intracrine DHEA-S utilization, especially when the adrenal production is low or declining with aging.

3' Untranslated Regions↗
PubMed2005

Galsulfase: arylsulfatase B, BM 102, recombinant human arylsulfatase B, recombinant human N-acetylgalactosamine-4-sulfatase, rhASB.

Galsulfase [Aryplase, arylsulfatase B, BM 102, Naglazyme, rhASB, recombinant human N-acetylgalactosamine-4-sulfatase, recombinant human arylsulfatase B] is under development with BioMarin Pharmaceutical as an enzyme replacement therapy for the treatment of mucopolysaccharidosis (MPS) VI. MPS VI (also known as Maroteaux-Lamy syndrome) is a progressive, debilitating genetic disease resulting in early death. Patients with MPS VI have a deficiency in the arylsulfatase B (ASB) enzyme that is essential for the progressive breakdown of certain complex carbohydrates. The deficiency in ASB results in the build-up of carbohydrate residues in the lysosomes in all cells of the body. Patients are usually diagnosed at 6-24 months of age, and the symptoms include deceleration of growth, enlarged liver and spleen, skeletal and joint deformities, and upper airway obstruction. Patients do not survive past 20-30 years of age in the more severe cases, but may live longer with the milder cases, but with significant medical problems. While the symptoms of MPS VI are similar to those of MPS I, mental retardation associated with the severe forms of MPS I had not been reported for patients with MPS VI. For some patients, bone marrow transplantation is a treatment, albeit risky, option. MPS VI afflicts approximately 1100 patients in the world. In November 2004, BioMarin announced that it has filed a Biologics License Application (BLA) with the the US FDA for galsulfase for the treatment of MPS VI. The company has requested a priority review as part of the BLA submission, which, if granted, is expected to be completed within 6 months of submission. The FDA accepted the filing of the BLA for galsulfase for MPS VI in February 2005, and granted it a 6-month priority review period. The FDA's decision is due on 31 May 2005. The FDA has granted galsulfase orphan drug status and fast-track designation. Orphan drug status will provide BioMarin Pharmaceutical with 7 years of marketing exclusivity for galsulfase in the US providing that galsulfase is the first agent to gain approval in the US for MPS VI. BioMarin received an orphan drug designation from the EC for galsulfase for the treatment of MPS VI. Following positive safety and efficacy results from the phase I study with galsulfase, BioMarin Pharmaceutical commenced and successfully completed a phase II trial with rhASB in ten patients with MPS VI. This 24-week, open-label, multicentre trial was conducted at two sites, in the US and Australia (at the Lysosomal Diseases Research Unit, Women's and Children's Hospital, Adelaide, Australia, by Dr John Hopwood), and evaluated the safety, efficacy and pharmacokinetics of weekly intravenous infusions of galsulfase at a dose of 1.0 mg/kg. BioMarin Pharmaceutical completed a phase I/II clinical trial of galsulfase in six patients with MPS VI in the Children's Hospital, Oakland, CA, USA, with Dr Paul Harmatz as a principal investigator. This randomised, double-blind study evaluated the safety and efficacy of two doses of galsulfase administered by weekly intravenous infusions for 24 weeks. Five patients from the phase I study had completed the 24-week, open-label extension study. Data from this study confirmed safety and good tolerability of both doses of galsulfase with the 1.0 mg/kg dose producing greater sustained effects. The patients will continue receiving therapy in the future. Seven preclinical trials with galsulfase were conducted in a naturally occurring feline model of MPS VI disease at the Lysosomal Diseases Research Unit, Women's and Children's Hospital, Adelaide, Australia, by Dr John Hopwood. The company manufactures galsulfase at a GMP facility licensed from the State of California.

Animals↗