Biochemical characterization of neonatal multiple sulfatase deficient (MSD) disorder cultured skin fibroblasts

Cultured skin fibroblasts of a patient with a neonatal onset of MSD can be distinguished from usual type of MSD cells by enzyme assays of arylsulfatases A, B and C activities, effect of thiosulfate on arylsulfatase A activity and ³⁵SO₄-acid mucopolysaccharide accumulation and degradation. These data suggest that neonatal onset of MSD is a distinct disorder from usual type of MSD in term of genetic occurence.     

Elevated levels of arylsulfatase C activity in cultured skin fibroblasts of patients with autosomal dominant ichthyosis vulgaris

Summary Assays of arylsulfatase C and steroidsulfatase activities in cultured skin fibroblasts from 20 persons with autosomal dominant ichthyosis and 8 controls showed significantly elecated arylsulfatase C activities in cells from patients with autosomal dominant ichthyosis in comparison with those in control cells. This difference was not detected for steroidsulfatase activity. In leukocytes, activities of arylsulfatase C and of steroidsulfatase were similar for patients and controls.     

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.     

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.     

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

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

The effect of (+) --cyanidanol on lysosomal enzymes of I-cell fibroblasts.

(+)--Cyanidanol, a water-soluble flavonoid, when added to cultured skin fibroblasts of a patient with I-cell disease raised the intracellular concentration of beta-galactosidase but did not affect the distribution of arylsulfatase. A, alpha-mannosidase or beta-glucuronidase. The elevated accumulation of 35SO4 by I-cell, Hunter and Maroteaux-Lamy fibroblasts was decreased by the addition of (+)--cyanidanol to the culture medium, but the degradation of previously labeled, intracellular glycosaminoglycans was not. It is concluded that (+)--cyanidanol does not produce a biochemical correction of the enzymic abnormalities existing in I-cell fibroblasts.     

Correction of sulfatide metabolism after transfer of prosaposin cDNA to cultured cells from a patient with SAP-1 deficiency.

The lysosomal removal of the sulfate moiety from sulfatide requires the action of two proteins, arylsulfatase A and sphingolipid activator protein-1 (SAP-1). Recently, patients have been identified who have a variant form of metachromatic leukodystrophy which is characterized by mutations in the gene coding for SAP-1, which is also called "prosaposin." All of the mutations characterized in these patients result in (a) deficient mature SAP-1, as determined by immunoblotting after SDS-PAGE of tissue and cell extracts, and (b) decreased ability of cultured skin fibroblasts to metabolize endocytosed [14C]-sulfatide. We now report the insertion of the full-length prosaposin cDNA into the Moloney murine leukemia virus-derived retroviral vector, pLJ, and the infection of cultured skin fibroblasts from a newly diagnosed and molecularly characterized patient with SAP-1 deficiency. The cultured cells infected with the prosaposin cDNA construct now show both production of normal levels of mature SAP-1 and completely normal metabolism of endocytosed [14C]-sulfatide. These studies demonstrate that the virally transferred prosaposin cDNA is processed normally and is localized within lysosomes, where it is needed for interaction between sulfatide and arylsulfatase A. In addition, normal as well as mutant sequences can now be found by allele-specific oligonucleotide hybridization of PCR-amplified genomic DNA by using exonic sequences as primers.     

Association of steroid sulfatase with one of the arylsulfatase C isozymes in human fibroblasts

When arylsulfatase C, a microsomal membrane-bound enzyme, is assayed with its natural substrates, the 3-beta-hydroxysteroid sulfates, it is also known as steroid sulfatase. Whether arylsulfatase C and steroid sulfatase are identical enzymes or not, however, has long been disputed. We now report that two electrophoretic variants of arylsulfatase C occur in normal human fibroblasts: one has a single anodic band of activity, "s," and the other has an additional faster migrating band, "f". The two types, s and "f + s", occur in cells from either sex. When fibroblast strains with the f + s forms of arylsulfatase C were cloned, two types of primary clones were always obtained: s and f + s. A single f band was never seen. When these primary clones were subcloned, however, the arylsulfatase C phenotype remained unchanged: primary s clones gave rise to s subclones and f + s clones to f + s subclones only. Therefore, these forms were clonal in origin and demonstrated a novel inheritance pattern in human cultured cells. The appearance of increasing amounts of the f band was correlated with up to 4-fold increase of arylsulfatase C activity, whereas the steroid sulfatase activity remained constant, thus demonstrating that arylsulfatase C was not identical with steroid sulfatase activity. Polyclonal antibodies raised against the s form immunoprecipitated activities of the s form of arylsulfatase C and steroid sulfatase but not the f form of arylsulfatase C. Therefore, we conclude that only the s form of arylsulfatase C is immunologically related to steroid sulfatase so that arylsulfatase C per se is not necessarily identical with steroid sulfatase. In addition, a novel form of genetic heterogeneity of isozymes in human fibroblasts is demonstrated.     

GM1-gangliosidosis: Accumulation of ganglioside GM1 in cultured skin fibroblasts and correlation with clinical types

Summary Uptake of radioactivity from ¹⁴C-galactose into gangliosides by cultured skin fibroblasts was studied. G_M3 was the major ganglioside in control human fibroblasts. An increase of G_M1 was demonstrated in G_M1-gangliosidosis fibroblasts. The degree of G_M1 accumulation was correlated with the clinical types of this disease. The fibroblasts from an infantile-type patient showed a marked increase of G_M1. In late-onset types the amount of total gangliosides was only slightly increased, but the distribution of individual gangliosides was definitely abnormal; a relative increase of G_M1 was demonstrated in these cases. G_M1 -galactosidase activities were not detectable in either infantile or late-onset cases.     

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.     

An 11-bp deletion in the arylsulfatase A gene of a patient with late infantile metachromatic leukodystrophy

Summary Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulfatase A. Examination of the arylsulfatase A gene in a patient suffering from late infantile metachromatic leukodystrophy revealed an 11-bp deletion in exon 8. Although this allele produces normal amounts of ASA mRNA, no arylsulfatase A cross-reacting material could be detected in cultured fibroblasts from the patient. The patient was found to be a compound heterozygote, the other allele is also known to generate no ASA polypeptides. This patient is another example where absence of ASA polypeptides correlates with the severe late infantile form of metachromatic leukodystrophy.     

Glutamic Acid Decarboxylase and γ-Aminobutyric Acid in Huntington''s Disease Fibroblasts and Other Cultured Cells, Determined by a [3H]Muscimol Radioreceptor Assay

A sensitive and reproducible [3H]muscimol radioreceptor assay was developed for measuring low levels of both glutamic acid decarboxylase activity and gamma-aminobutyric acid. By using this technique, endogenous gamma-aminobutyric acid and glutamic acid decarboxylase activity were detected in two rat neuroblastomas, B35 and B50, a human medulloblastoma cell line, TE671, and cultured human skin fibroblasts. Glutamic acid decarboxylase activities and gamma-aminobutyric acid levels were compared for human skin fibroblasts obtained from patients with Huntington's disease and their controls in a well-controlled, blind study. However, no significant difference was found to either measure between Huntington and control cells. Glutamic acid decarboxylase activity was relatively low in all cell types examined except for the TE671 cells, which had more than four times the activity found in the other cells. This human medulloblastoma cell line appears to be a good model for studying gamma-aminobutyric acid metabolism and the control of glutamic acid decarboxylase expression.     

Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome). An intermediate clinical phenotype caused by substitution of valine for glycine at position 137 of arylsulfatase B.

The Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI) is a lysosomal storage disease with autosomal recessive inheritance caused by deficiency of the enzyme arylsulfatase B. Severe, intermediate, and mild forms of the disease have been described. The molecular correlate of the clinical heterogeneity is not known at present. To identify the molecular defect in a patient with the intermediate form of the disease, arylsulfatase B mRNA from his fibroblasts was reverse-transcribed, amplified by the polymerase chain reaction, and subcloned. Three point mutations were detected by DNA sequence analysis, two of which, a silent A to G transition at nucleotide 1191 and a G to A transition at nucleotide 1126 resulting in a methionine for valine 376 substitution, were polymorphisms. A G to T transversion at nucleotide 410 causing a valine for glycine 137 substitution (G137V) was identified as the mutation underlying the Maroteaux-Lamy phenotype of the patient, who was homozygous for the allele. The kinetic parameters of the mutant arylsulfatase B enzyme toward a radiolabeled trisaccharide substrate were normal excluding an alteration of the active site. The G137V mutation did not affect the synthesis but severely reduced the stability of the arylsulfatase B precursor. While the wild type precursor is converted by limited proteolysis in late endosomes or lysosomes to a mature form, the majority of the mutant precursor was degraded presumably in a compartment proximal to the trans Golgi network and only a small amount escaped to the lysosomes accounting for the low residual enzyme activity in fibroblasts of a patient with the juvenile form of the disease.     

Placental steroid deficiency: association with arylsulfatase A deficiency

A family with an obstetric history consistent with placental sulfatase deficiency has X-linked ichthyosis. Steroid sulfatase deficiency was confirmed in placenta, leukocytes, and cultured skin fibroblasts of affected males; arylsulfatase A diminution was also observed in these tissues of both affected males and 2 generations of related females. No symptoms of metachromatic leukodystrophy are present in any family members. In this family, placental sulfatase deficiency, and arylsulfatase A pseudodeficiency are nonallelic.     

Genetic control of heat sensitivity and activity level of murine arylsulfatase B

BALB/c male mice possess twofold higher kidney p-nitrocatechol-SO4 arylsulfatase B than do A/HeJ male mice; however, their liver arylsulfatase activities are comparable. Twentyfold-purified kidney arylsulfatases B from these two strains have similar Michaelis constants, electrophoretic mobilities, pH optima, and inhibitor profiles; however, the BALB/c enzyme is more heat stable than the A/HeJ enzyme. BALB/c, C3H/HeJ, DBA/2J, and SWR/J mice share an autosomal allele, As-1a, which apparently determines the heat-stable arylsulfatase B, while A/HeJ and C57BL/6J mice possess the As-1b allele, which determines the heat-sensitive enzyme. A second autosomal locus, Asr-1, determines liver arylsulfatase B activity. C57BL/6J mice carry the Asr-1a allele, which results in high liver activities, while C3H/HeJ mice are homozygous for the low-activity allele, Asr-1b. Male mice generally have 30-40% higher kidney activities than females; however, female kidney arylsulfatase activities rise and actually surpass those of males during late pregnancy and lactation.     

Regulatory role of insulin in the degradation of low density lipoprotein by cultured human skin fibroblasts.

The degradation of 125I-labeled low density lipoprotein by cultured human skin fibroblasts was enhanced 25% by preincubation of cells with insulin. This effect of insulin appeared to be mediated via stimulation of low density lipoprotein binding to its cell surface receptor, since binding and subsequent internalization of low density lipoprotein were stimulated to a similar extent as was degradation. In addition, insulin enhanced binding of low density lipoprotein at 4 degrees C, at which temperature internalization of the lipoprotein does not occur. A similar effect of insulin on the interaction of very low density lipoprotein with cultured fibroblasts was observed. Insulin-induced changes in the degradation of low density lipoprotein and very low density lipoprotein appeared to be a function of the change in lipoprotein binding. Thus, insulin may play a role in the regulation of low density lipoprotein and very low density lipoprotein degradation by peripheral cells by influencing the receptor-mediated transport of these lipoproteins.     

Monoamine oxidase and catechol-O-methyltransferase activities in cultured human skin fibroblasts

Human fibroblasts obtained from normal male children and children with the Lesch-Nyhan syndrome were found to contain both the A and B forms of monoamine oxidase, with the A form predominating. Both forms of monoamine oxidase showed decreased activities in Lesch-Nyhan, as compared to normal cells; while catechol-O-methyltrans-ferase activities were similar. This study demonstrates the usefulness of fibroblasts cultured from human skin biopsies in analyses of alterations in catecholamine catabolism associated with inherited neurologic diseases.     

Quantitative correlation between the residual activity of β-hexosaminidase A and arylsulfatase A and the severity of the resulting lysosomal storage disease

Summary A previously suggested model for the correlation between residual activity of a lysosomal enzyme and the turnover rate of its substrate(s) has been extended to a discussion of substrate accumulation rates in individual cells and whole organs. With these considerations, much of the observed variability in age of onset and clinical phenotype, as well as the phenomenon of pseudodeficiency, can be understood as the consequences of small differences in the residual activity of the affected enzyme. In order to experimentally verify the basic assumptions on which this model rests, studies were performed in cell culture. The radiolabeled substrates ganglioside G_M2 and sulfatide were added to cultures of skin fibroblasts with different activities of -hexosaminidase A or arylsulfatase A, respectively, and their uptake and turnover measured. In both series of experiments, the correlation between residual enzyme activity and the turnover rate of the substrate was essentially as predicted: degradation increased steeply with residual activity, to reach the control level at a residual activity of approximately 10–15% of normal. All cells with an activity above this critical threshold had a normal turnover. Comparison of the results of these feeding studies with the clinical status of the donor of each cell line basically confirmed our notions but also revealed the limitations of the cell culture approach.     

Ascorbic acid-2-sulfate sulfhohydrolase activity of human arylsulfatase A.

Pure human arylsulfatase A (EC 3.1.6.1) was found to hydrolyze ascorbic acid 2-sulfate to ascorbic acid and inorganic sulfate at rates from 200 to 2000 mumol/mg per h depending on the method of assay. This rate was lower than that observed with the synthetic substrate 4-nitrocatechol sulfate, but higher than that seen with the physiological substrate cerebroside sulfate. Extracts of cultured fibroblasts from normal subjects were also shown to hydrolyze ascorbic acid 2-sulfate; extracts of fibroblasts from patients with metachromatic leukodystrophy, known to be deficient in arylsulfatase A, did not. Similarly, hydrolysis of ascorbic acid 2-sulfate was not observed when a partially purified preparation of human arylsulfatase B was tested under a variety of conditions. Thus, in the human, arylsulfatase A appears to be the major, if not the only, ascorbic acid-2-sulfate sulfohydrolase.     

Sphingomyelinase activities in cultured skin fibroblasts from patients with Niemann-Pick disease

Summary Sphingomyelinase activity in cultured skin fibroblasts from a fetus affected with infantile-type Niemann-Pick disease was 0.5% of control activity; the activities in cells from two patients with adult-type disease (Cases 2 and 3) were 5.0% and 59.0%.Sphingomyelinase activity was separated into three peaks (I–III) by isoelectric focusing. The isoelectric points were 4.5, 4.9, and 5.2 for peaks I, II, and III, respectively. The three peaks in the Case 2 cells were drastically reduced; only a very small peak could be distinguished (pI of 4.7). On the other hand, three peaks were observed in the Case 3 cells. Peak I had a pI of 4.4, peak II a pI of 4.7, and peak III a pI of 5.2. Peak I was found at near normal level, but both peaks II and III were markedly reduced.Sphingomyelinase in the peak I fraction obtained from isoelectric focusing in Case 3 cells was found to have the same Km value as that in control cells.