Steroid 21-sulfatase activity in human placenta

Intravenously administered [3H]-deoxycorticosterone sulfate is not metabolized by way of deoxycorticosterone in men or non-pregnant women. Thus, it can be implied that steroid 21-sulfatase is not active in human tissues. On the other hand, evidence has accrued that deoxycorticosterone sulfate is hydrolyzed in human placenta. In the present investigation, we sought to ascertain if steroid 21-sulfatase activity were present in placenta and, if so, to characterize the enzyme activity in this tissue. Steroid 21-sulfatase activity was found to be present in microsome-enriched fractions prepared from human placental tissue; conditions of linearity of the reaction with time and protein concentration were established and the apparent KM of the enzyme for deoxycorticosterone sulfate was 100 microM. Thus, deoxycorticosterone sulfate, which is present in high concentration in plasma of the human fetus, may enter trophoblast wherein it could be hydrolyzed; the deoxycorticosterone formed could be secreted into the maternal circulation. Such a process, together with deoxycorticosterone formation from plasma progesterone in extraadrenal sites, could account for the high concentrations of deoxycorticosterone that are present in plasma of near-term pregnant women.     

New assay for steroid sulfatase (EC 3.1.6.2) and its application for studies of human placental and skin sulfatase

A new, simple, fast and highly practicable sulfatase assay and its application is described. Sterol sulfatase sulfohydrolase (EC 3.1.6.2) activity is determined by a two-phase scintillation technique separating the unreacted [4-14C]dehydroepiandrosterone sulfate from carbon-14-labeled products. The principle of the separation relies on the limited emulsifying capacity of the dioxane-based scintillation solution for water and the different partition of dehydroepiandrosterone sulfate and sulfate-free steroid products between the scintillation fluid and the aqueous phase as recently applied for determination of aromatase activity [1]. [7-3H]Dehydroepiandrosterone sulfate can also be used as a substrate for this assay. This test was applied to studies of microsomal sulfatase prepared from human term placenta and to the detection of sulfatase activity in human skin biopsies. Using placental microsomes, the Km of dehydroepiandrosterone sulfate was determined to be 5.0 X 10(7)M. Sulfatase activity in frozen scrotal skin was found to be 2-3 fold than with vaginal skin. Using an incubation time of 24h/skin sulfatase can be detected in biopsies as small as 2.5 mm2. The sulfatase assay can be applied for routine detection of human placental sulfatase deficiency and, furthermore, the application of this assay has to be demonstrated for the analysis of sulfatase activity in patients with congenital ichthyosis (X-chromosomal, recessive type).     

Human placental steryl-sulfatase. Enzyme purification, production of antisera, and immunoblotting reactions with normal and sulfatase-deficient placentas

The steryl-sulfatase of normal human placental microsomes was solubilized and enriched about 350-fold. Chromatography on Sepharose 6B of the purified enzyme preparation revealed a single protein peak which eluted according to an apparent molecular mass of 270 +/- 30 kDa; when electrophorized on sodium dodecyl sulfate polyacrylamide gel the sulfatase migrated according to a molecular mass of 64 +/- 4 kDa. Estrogensulfatase activity was co-purified with the steryl-sulfatase activity; obviously, both activities belong to the same enzyme species. The purified sulfatase was injected into three rabbits. Antisera produced by the rabbits yielded a single sharp immunoprecipitation line in Ouchterlony double diffusion experiments when tested with the isolated sulfatase or with a solubilized microsomal fraction of normal placentas. The activity of sulfatase preparations incubated with antiserum was precipitated by addition of polyethylene glycol followed by centrifugation; none of the antibodies reacting with the sulfatase therefore appeared to interfere with its enzymatic activity. Using these antisera, steryl-sulfatase protein could be detected by immunoblotting analysis in solubilized microsomal fractions of normal placentas but not in solubilized microsomal fractions of three steryl-sulfatase activity-deficient placentas. This finding argues in favour of human placental steryl-sulfatase deficiency being due to extremely diminished or absent enzyme protein in the placenta.     

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.     

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.     

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     

Human placental sterylsulfatase. Interaction of the isolated enzyme with substrates, products, transition-state analogues, amino-acid modifiers and anion transport inhibitors.

The enzymatic properties of a homogeneous sterylsulfatase preparation isolated from human term placenta were studied. The enzyme exhibited both arylsulfatase and sterylsulfatase activity: it catalysed the hydrolysis of sulfuric acid esters of (in the order of decreasing specific activity) non-steroidal phenols, of a phenolic steroid, and of neutral 3 beta-, 21- and (though at a very low rate) 17 beta-hydroxysteroids. However, among all the substrates tested only the 3-sulfates of phenolic and neutral steroids exhibited high affinity towards the sulfatase. Vitamin D3 sulfate was not hydrolysed by the sterylsulfatase but strongly inhibited its activity. The products of the catalytic reaction, free steroids or phenols as well as the sulfate anion or analogues thereof, likewise interfered with the enzyme's activity. Ki values of unconjugated steroids were ten- to hundredfold higher than Km values of the respective sulfoconjugates. Inorganic sulfate only slightly inhibited the sulfatase activity; its inhibitory potency, however, increased in a time-dependent manner when it was preincubated with the enzyme prior to assay. In contrast to sulfate, the hypothetical transition-state analogues sulfite and vanadate acted as strong inhibitors of the sulfatase activity. According to the results of an analysis of the effect of pH on sterylsulfatase kinetics, enzyme constituents with pK values of approximately 5.8 and 8.0 are involved in a general acid-base catalysed reaction. Treatment of the sulfatase with amino-acid side chain modifying reagents directed against arginine, cysteine, cystine, serine or tyrosine residues did not result in significant alteration of its activity. Diethyl-pyrocarbonate known to react primarily with histidyl groups, however, rapidly inactivated the enzyme; this inactivation reaction was markedly retarded in the presence of substrate. Histidine thus appears to be essential for the catalytic activity of the sulfatase. Taken together, the present results reveal a considerable similarity between the catalytic mechanism of human placental sterylsulfatase and the ones already proposed for the lysosomal arylsulfatases A and B. Taurocholate, salicylate, ouabain, and 4,4'-substituted stilbene-2,2'-disulfonates are well known inhibitors of carrier-mediated transport of anions across cellular membranes. With the exception of ouabain, these compounds likewise turned out to inhibit the enzymatic hydrolysis of steryl sulfates; the pattern of dose dependences of their interference with the sulfatase activity resembles the one reported for inhibition of anion transport. Since the sterylsulfatase in vivo strongly is associated with cellular membranes including the plasma membrane of the syncytiotrophoblast, this finding supports the speculation that similar molecular structures may be involved in both placental transport and hydrolysis of anionic steryl sulfates.     

Interactions of the human cytosolic sulfotransferases and steroid sulfatase in the metabolism of tibolone and raloxifene

Sulfation is important in the metabolism and inactivation of steroidal compounds and hormone replacement therapeutic (HRT) agents in human tissues. Although generally inactive, many steroid sulfates are hydrolyzed to their active forms by sulfatase activity. Therefore, the specific sulfotransferase (SULT) isoforms and the levels of steroid sulfatase (STS) activity in tissues are important in regulating the activity of steroidal and HRT compounds. Tibolone (Tib) is metabolized to three active metabolites and all four compounds are readily sulfated. Tib and the Δ4-isomer are sulfated at the 17β-OH group by SULT2A1 and the 17-sulfates are resistant to hydrolysis by human placental STS. 3-OH and 3β-OH Tib can form both 3- and 17-monosulfates as well as disulfates. Only the 3β-sulfates are susceptible to STS hydrolysis. Raloxifene monosulfation was catalyzed by at least seven SULT isoforms and SULT1E1 also synthesizes raloxifene disulfate. SULT1E1 forms both monosulfates in a ratio of approximately 8:1 with the more abundant monosulfate migrating on HPLC identical to the SULT2A1 synthesized monosulfate. The raloxifene monosulfate formed by both SULT isoforms is sensitive to STS hydrolysis whereas the low abundance monosulfate formed by SULT1E1 is resistant. The benzothiophene sulfates of raloxifene and arzoxifene were hydrolyzed by STS whereas the raloxifene 4′-phenolic sulfate was resistant. These results indicate that tissue specific expression of SULT isoforms and STS could be important in the inactivation and regeneration of the active forms of HRT agents.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Cloning and Characterization of a Novel Chondroitin Sulfate/Dermatan Sulfate 4-O-Endosulfatase from a Marine Bacterium

Sulfatases are potentially useful tools for structure-function studies of glycosaminoglycans (GAGs). To date, various GAG exosulfatases have been identified in eukaryotes and prokaryotes. However, endosulfatases that act on GAGs have rarely been reported. Recently, a novel HA and CS lyase (HCLase) was identified for the first time from a marine bacterium (Han, W., Wang, W., Zhao, M., Sugahara, K., and Li, F. (2014) J. Biol. Chem. 289, 27886–27898). In this study, a putative sulfatase gene, closely linked to the hclase gene in the genome, was recombinantly expressed and characterized in detail. The recombinant protein showed a specific N-acetylgalactosamine-4-O-sulfatase activity that removes 4-O-sulfate from both disaccharides and polysaccharides of chondroitin sulfate (CS)/dermatan sulfate (DS), suggesting that this sulfatase represents a novel endosulfatase. The novel endosulfatase exhibited maximal reaction rate in a phosphate buffer (pH 8.0) at 30 °C and effectively removed 17–65% of 4-O-sulfates from various CS and DS and thus significantly inhibited the interactions of CS and DS with a positively supercharged fluorescent protein. Moreover, this endosulfatase significantly promoted the digestion of CS by HCLase, suggesting that it enhances the digestion of CS/DS by the bacterium. Therefore, this endosulfatase is a potential tool for use in CS/DS-related studies and applications.     

A sulfatase specific for glucuronic acid 2-sulfate residues in glycosaminoglycans

Although 2-O-sulfated L-iduronic acid (IdoA) residues have been known to occur in heparin, 2-O-sulfated D-glucuronic acid (GlcA) residues have been reported only recently (Bienkowski, M. J., and Conrad, H. E. (1985) J. Biol. Chem. 250, 356-365). Disaccharides prepared by cleavage of heparin and N-deacetylated chondroitin 6-sulfate with nitrous acid were used to demonstrate a new sulfatase that catalyzed the removal of the 2-O-sulfate substituents from GlcA but not IdoA residues. The deamination products were labeled by NaB3H4 reduction to give disaccharides from heparin and chondroitin sulfate which had reducing terminal 2,5-anhydro-D-mannitol ([3H]AManR) and 2,5-anhydro-D-talitol ([3H]ATalR) residues, respectively. IdoA(2-SO4)-[3H]AManR(6-SO4) from heparin and GlcA(2-SO4)-[3H]ATalR(6-SO4) from chondroitin sulfate were purified for use as substrates. GlcA(2-SO4)-[3H]AManR(6-SO4) was prepared by epimerization of IdoA(2-SO4)-[3H]AManR(6-SO4) with hydrazine at 100 degrees C. Lysosomal enzyme preparations from chick embryo chondrocytes and from two normal human fibroblast cell lines catalyzed the removal of the 2-O-SO4 substituent from the uronic acid residues of IdoA(2-SO4)-[3H]AManR(6-SO4), GlcA(2-SO4)-[3H] AManR(6-SO4), and GlcA(2-SO4)-[3H]ATalR(6-SO4). In contrast, a lysosomal enzyme preparation from a human fibroblast cell line deficient in idurono-2-sulfatase (Hunter's-syndrome), which had no activity on the IdoA(2-SO4)-[3H]AManR(6-SO4), converted GlcA(2-SO4)-[3H]AManR(6-SO4) to a mixture of GlcA-[3H] AManR(6-SO4) and [3H]AManR(6-SO4). This enzyme also converted GlcA(2-SO4)-[3H]ATalR(6-SO4) to a mixture of GlcA-[3H]ATalR(6-SO4) and [3H]ATalR(6-SO4). Digestion of both GlcA(2-SO4)-[3H]AManR(6-SO4) and GlcA(2-SO4)-[3H]ATalR(6-SO4) was inhibited by 35SO2-4 and was arrested at the monosulfated disaccharide stage by 1,4-saccharolactone. The glucurono-2-sulfatase exhibited a pH optimum of 4. The results indicate that there exists a separate sulfatase for the removal of sulfate substituents from C-2 of GlcA residues in glycosaminoglycans.     

Exposure to common food additive carrageenan leads to reduced sulfatase activity and increase in sulfated glycosaminoglycans in human epithelial cells

The commonly used food additive carrageenan, including lambda (λ), kappa (κ) and iota (ι) forms, is composed of galactose disaccharides linked in alpha-1,3 and beta-1,4 glycosidic bonds with up to three sulfate groups per disaccharide residue. Carrageenan closely resembles the endogenous galactose or N-acetylgalactosamine-containing glycosaminoglycans (GAGs), chondroitin sulfate (CS), dermatan sulfate (DS), and keratan sulfate. However, these GAGs have beta-1,3 and beta-1,4 glycosidic bonds, in contrast to the unusual alpha-1,3 glycosidic bond in carrageenan. Since sulfatase activity is inhibited by sulfate, and carrageenan is so highly sulfated, we tested the effect of carrageenan exposure on sulfatase activity in human intestinal and mammary epithelial cell lines and found that carrageenan exposure significantly reduced the activity of sulfatases, including N-acetylgalactosamine-4-sulfatase, galactose-6-sulfatase, iduronate sulfatase, steroid sulfatase, arylsulfatase A, SULF-1,2, and heparan sulfamidase. Consistent with the inhibition of sulfatase activity, following exposure to carrageenan, GAG content increased significantly and showed marked differences in disaccharide composition. Specific changes in CS disaccharides included increases in di-sulfated disaccharide components of CSD (2S6S) and CS-E (4S6S), with declines in CS-A (4S) and CS-C (6S). Specific changes in heparin-heparan sulfate disaccharides included increases in 6S disaccharides, as well as increases in NS and 2S6S disaccharides. Study results suggest that carrageenan inhibition of sulfatase activity leads to re-distribution of the cellular GAG composition with increase in di-sulfated CS and with potential consequences for cell structure and function.     

Effects of DHA-S on placental 3 beta-hydroxysteroid dehydrogenase activity, progesterone and 20 alpha-dihydroprogesterone concentrations in placenta and serum

To study the effects of dehydroepiandrosterone sulfate (DHA-S) on placental steroid metabolism and maternal steroidal profiles at term, the following in vivo and in vitro experiments were performed. Two hundred mg of DHA-S was given to five pregnant women 30 minutes prior to delivery. After delivery, the placenta was collected and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and sulfatase activity was determined by measuring the rate of conversion of pregnenolone to progesterone and DHA-S to DHA. The amount of C21-delta 4-steroid in the placental tissue was measured by gas chromatography mass spectrometry (GC-MS) and compared with the control groups. The maternal serum concentration of several steroids was also measured by GC-MS before and after the administration of DHA-S. 3 beta-HSD activity in the placentae from the mothers who received DHA-S before delivery was significantly lower than in the controls. On the other hand, no significant change was observed in the activity of sulfatase. The serum concentration of progesterone (P) and 20 alpha-dihydro-P (20-P) before DHA-S loading decreased following the administration whereas estradiol (E), DHA, and androstenedione (A) levels increased. To study the direct effect of DHA-S and its related steroids on placental 3 beta-HSD activity, placental tissue samples were incubated with pregnenolone in vitro. Several other steroids were added simultaneously into the medium. It was observed that placental 3 beta-HSD activity was directly inhibited by DHA-S. These results indicate that DHA-S inhibits 3 beta-HSD activity in the placenta and subsequently causes a reduction in P and 20-P.