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.     

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

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

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.     

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.     

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

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

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

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

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.     

Solubilization and partial purification of steroid sulfatase from rat liver: characterization of estrone sulfatase.

Steroid sulfatase, a membrane-bound enzyme present in many mammalian tissues, was extracted from rat liver microsomes by treatment with Miranol H2M, a zwitterion detergent, and sonication. It has been purified approximately 33-fold. All steps of the purification, which included salt and solvent fractionation, hydroxylapatite treatment, ion-exchange chromatography, and gel filtration were performed in the presence of Miranol H2M, most of which was removed from the final preparation by gel filtration. The final preparation did not contain any detectable NADPH-cytochrome c reductase or glucose-6-phosphate phophatase activities. According to the elution volume on a Sephadex G-200 column, steroid sulfatase has a molecular weight of approximately 130,000. Polyacrylamide-gel electrophoresis in the presence of Miranol H2M revealed one major protein band which was enzymatically active. Purified steroid sulfatase hydrolyzes all the sulfate esters of estrone, dehydroepiandrosterone, pregnenolone, testosterone, and cholesterol as well as p-nitrophenyl sulfate, the substrate for arylsulfatase C, during the purification. However, estrone sulfatase and arylsulfatase C activities were enriched more than the others. Analysis of kinetic data and the effects of different buffers and of Miranol H2M also suggested that estrone sulfatase and arylsulfatase C are identical but that they are distinct from the other sulfatases. Competitive inhibition studies suggest that estrone sulfatase also catalyzes the hydrolysis of the sulfate esters of other estrogens.     

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.     

Cloning and expression of human arylsulfatase A

A full length cDNA for human arylsulfatase A was cloned and sequenced. The predicted amino acid sequence comprises 507 residues. A putative signal peptide of 18 residues is followed by the NH2-terminal sequence of placental arylsulfatase A. One of the arylsulfatase A peptides ends 3 residues ahead of the predicted COOH terminus. This indicates that proteolytic processing of arylsulfatase A is confined to the cleavage of the signal peptide. The predicted sequence contains three potential N-glycosylation sites, two of which are likely to be utilized. The sequence shows no homology to any of the known sequences of lysosomal enzymes but a 35% identity to human steroid sulfatase. Transfection of monkey and baby hamster kidney cells resulted in an up to 200-fold increase of the arylsulfatase A activity. The arylsulfatase A was located in lysosome-like structures and transported to dense lysosomes in a mannose 6-phosphate receptor-dependent manner. The arylsulfatase A cDNA hybridizes to 2.0- and 3.9-kilobase species in RNA from human fibroblasts and human liver. RNA species of similar size were detected in metachromatic leukodystrophy fibroblasts of two patients, in which synthesis of arylsulfatase A polypeptides was either detectable or absent.     

STEROID SULFATASE IN BRAIN: COMPARISON OF SULFOHYDROLASE ACTIVITIES FOR VARIOUS STEROID SULFATES IN NORMAL AND PATHOLOGICAL BRAINS, INCLUDING THE VARIOUS FORMS OF METACHROMATIC LEUKODYSTROPHY

Abstract– The enzymatic hydrolysis by brain homogenate of the sulfate esters of estrone, pregnenolone, dehydroepiandrosterone, testosterone, cholesterol and p-nitrophenol was studied. With homogenate of young rat brain, the pH optima of estrone sulfatase ⁴ 4 The term steroid sulfatase is used as a general name for the enzyme(s) which hydrolyzes the sulfate ester of a steroid. Simplified terms, such as estrone sulfatase, instead of the more formal terms, such as estrone sulfate sulfohydrolase, have been used throughout.
and arysulfatase C (p-nitrophenyl sulfate as substrate) were 8.2 and all other steroid sulfatases had pH optima at 6.6. Apparent K_ms for these steroid sulfates were widely different. The highest K_m value was 32.2 μm for estrone sulfate and the lowest was 0.66 μm for testosterone sulfate; the K_m for p-nitrophenyl sulfate was 30 fold higher than for estrone sulfate. Specific activity was also highest with estrone sulfatase and lowest with testosterone sulfatase; specific activity with aryl sulfatase C was over 3 fold higher than with estrone sulfatase. Estrone sulfatase activity was inhibited noncompetitively by sulfate esters of dehydroepiandrosterone, pregnenolone, and cholesterol; on the other hand, other steroid sulfatases were inhibited by these latter three sulfates competitively. Developmental changes of these sulfohydrolase activities in rat brain were almost identical with the exception of testosterone sulfatase activity; the latter sulfatase had a peak activity at 30 days old, while all other sulfatase had a peak at 20 days old. Thermal stability of all these activities was identical. Testosterone sulfatase activity in neurological mouse mutants, jimpy, msd, and quaking mice, was less than one half of littermate controls, while other steroid sulfatase levels in these mutants' brain were normal. All sulfatase activities were diminished in the brain of a metachromatic leukodystrophy patient with multiple sulfatase deficiency. The brains of classical metachromatic leukodystrophy patients contained normal levels of all steroid sulfatases and arylsulfatase C, with the single exception of testosterone sulfatase which level was less than 50% of control.     

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

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

Biochemical characterization of arylsulfatase-C isozymes in human fibroblasts

Arylsulfatase-C and sterol sulfatase were thought to be identical enzymes whose X-linked locus escapes inactivation. However, recent evidence shows that they are not identical but that arylsulfatase-C in human fibroblasts exists in two isozymic forms, designated as slow and fast. We now report that the two forms are enzymatically different. When assayed with an artificial fluorogenic substrate, the slow form showed a pH optimum of 8.00 and a Km of 228 microM. In contrast, the fast form showed a pH optimum of 7.67 and a Km of 86.7 microM with substrate inhibition occurring above 0.33 mM. The heat stability of the fast form was slightly below that of the slow form. Polyclonal antibodies raised against the slow form did not cross-react with the fast form. Hence, the two isozymic forms of arylsulfatase-C are enzymatically and structurally different and the slow form is associated with sterol sulfatase activity.     

Structure and tissue-specific developmental expression of a sea urchin arylsulfatase gene

Arylsulfatases are a group of enzymes that remove sulfate moieties from a diverse set of substrates including glycoproteins, steroids, and cerebrosides. We have isolated recombinant cDNA clones corresponding to an arylsulfatase (SpARS) message that encodes an abundant protein of pluteus larvae of the sea urchin Strongylocentrotus purpuratus. Although vertebrate arylsulfatases have broad tissue distributions, in situ hybridization with a probe for SpARS shows that the sea urchin message accumulates in the embryo only in the single cell type of aboral ectoderm and its precursors. The message is first detectable by RNase protection assays around hatching blastula stage and accumulates through pluteus larva stage. The open reading frame of cDNA clones is 1701 nt long and encodes a deduced protein with a predicted molecular mass of 61 kDa. Analysis of corresponding genomic DNA clones reveals that the pre-mRNA contains six exons. Consistent with the fact that arylsulfatase enzyme activity is extracellular, this polypeptide has a hydrophobic leader sequence and three potential glycosylation sites. Furthermore, hybridization in situ shows that in blastulae arylsulfatase message is preferentially concentrated around nuclei at the basal sides of cells. The S. purpuratus sequence is very similar to that recently reported for the same enzyme from Hemicentrotus pulcherrimus and 30% of the amino acid residues are also identical to those of both human arylsulfatase C (steroid sulfatase) and arylsulfatase A. Sequence relationships among these four mRNAs suggest that, assuming equal rates of evolution, the duplication separating the human genes occurred at about the time of separation of the echinoderm and vertebrate lineages.     

Morquio disease: isolation, characterization and expression of full-length cDNA for human N-acetylgalactosamine-6-sulfate sulfatase.

We cloned and sequenced a full-length cDNA of human placental N-acetylgalactosamine-6-sulfate sulfatase, the enzyme deficient in Morquio disease. The 2339-nucleotide sequence contained 1566 nucleotides which encoded a polypeptide of 522 amino acid residues. The deduced amino acid sequence was composed of a 26-amino acid N-terminal signal peptide and a mature polypeptide of 496 amino acid residues including two potential asparagine-linked glycosylation sites. Expression of the cDNA in transfected deficient fibroblasts resulted in higher production of this sulfatase activity than in untransfected deficient fibroblasts. The cDNA clone was hybridized to only a 2.3-kilobase species of RNA in human fibroblasts. The amino acid sequence of N-acetylgalactosamine-6-sulfate sulfatase showed a high degree of homology with those of other sulfatases such as human arylsulfatases A, B or C, glucosamine-6-sulfatase, iduronate-2-sulfatase and sea urchin arylsulfatase.     

The sulfatase gene family: cross-species PCR cloning using the MOPAC technique.

Several human sulfatase cDNAs have recently been cloned, revealing highly conserved domains of protein similarity. We have used this information for the isolation of sulfatase genes in different species using the polymerase chain reaction (PCR). Degenerate oligonucleotide primers corresponding to these regions of identity among human arylsulfatases A, B, and steroid sulfatase (ARSA, ARSB, and STS) were designed. The primers were used in the PCR amplification of reverse transcribed RNA (RT-PCR) from multiple tissues in human and mouse. Amplification products were obtained from mouse liver and from human liver, lymphoblasts, kidney, intestine, heart, muscle, and brain cDNA samples. Each of the PCR products was subcloned into a plasmid vector, and several subclones were characterized by colony hybridization and DNA sequencing. All the previously identified human ARSA, ARSB, and STS were found among our clones, indicating the power of the technique. Sequence analysis of two mouse clones showed high degrees of homology with the human ARSA and ARSB sequences, respectively, and likely represent the murine homologues of these enzymes. These are the first sulfatase genes isolated in the mouse. A murine equivalent for STS could not be identified, suggesting its strong diversity from the human homologue.     

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

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