Hunter's syndrome: a deficiency of L-idurono-sulfate sulfatase

[³⁵SO₄] Dermatan sulfate, isolated from normal Hurler and Hunter fibroblasts was degraded by chondroitinase A, B, C to yield mono-and disaccharides. The products were separated by ion exchange chromatography and those arising from the non-reducing terminus were characterized by paper electrophoresis. The position of sulfate substituents was established by periodate oxidation and partial acid hydrolysis. Normal dermatan sulfate terminates with GalN-SO₄ whereas IdUA-SO₄ was a prominent terminus in Hunter dermatan sulfate but not in Hurler dermatan sulfate. It is concluded that Hunter's syndrome is due to a deficiency of L-idurono-sulfate sulfatase.     

Glycosaminoglycan synthesis by cultured human skin fibroblasts after transformation with simian virus 40.

The synthesis of glycosaminoglycans by human skin fibroblasts derived from normal subjects, Hurler and Marfan patients before and after transformation by SV40 virus has been studied. Virus transformation results in a marked increase in hyaluronic acid synthesis in normal and Hurler fibroblasts and, to a lesser extent, in Marfan fibroblasts which show augmented synthesis of this polysaccharide before transformation. There is also an increase in heparan sulfate synthesis but a moderate decrease in dermatan sulfate synthesis on transformation. Incubation of transformed fibroblasts with 4-methylumbelliferyl-beta-D-xyloside results in a marked increase in synthesis of free chondroitin sulfate chains. The synthesis of hyaluronic acid, but not of dermatan sulfate, is inversely proportional to cell density in normal fibroblasts but not in transformed fibroblasts.     

The Hurler syndrome: treatment of cultured Hurler fibroblasts with normal human serum.

Prolonged replacement of fetal calf serum by normal human serum for the enrichment of medium during tissue culture of Hurler fibroblasts resulted in increased acid mucopolysaccharides in the cells and in the medium. The predominant intracellular mucopolysaccharide had the characteristics of dermatan sulfate when Hurler cells were treated with either serum. Normal human serum contains a nonspecific coreective factor capable of augmenting the loss of 35SO4-AMPS from Hurler cells, but not from normal cells. Fetal calf serum and Hurler serum have similar corrective factor activity for labeled Hurler cells. The corrective factor activity of all three sera was recovered from reconstituted dialyzed ammonium sulfate precipitates. The corrective factor of normal human serum did not increase degradation of mucopolysaccharide, but increased secretion of macromolecular and large oligosaccharide components. Failure of the corrective factor of normal human serum to effectively decrease the dermatan sulfate content of Hurler cells during prolonged exposure may be a quantitative phenomenon due partly to the brief duration of corrective factor activity and partly to increased synthesis of mucopolysaccharide.     

Maroteaux-Lamy disease (mucopolysaccharidosis VI), subtype A: deficiency of a N-acetylgalactosamine-4-sulfatase

The non-reducing terminal moiety of ³⁵SO₄-dermatan sulfate accumulating in fibroblasts cultured from the skin of patients with one form of Maroteaux-Lamy disease was found to be N-acetylgalactosamine-4-sulfate. This end group accounted for about 3 % of the total radioactivity. Using both ³⁵SO₄- and ¹⁴C-N-acetylgalactosamine-labeled dermatan sulfates from the patients fibroblasts as substrates, it was found that homogenates of Maroteaux-Lamy fibroblasts, but not of normal, Hurler and Sandhoff fibroblasts fail to cleave inorganic sulfate from the non-reducing termini. We conclude, that deficiency of N-acetylgalactosamine-4-sulfatase is the biochemical basis for this form of Maroteaux-Lamy disease.     

Properties of α-l-iduronidase in cultured skin fibroblasts from α-l-iduronidase-deficient patients

Summary On DEAE cellulose column chromatography, -l-iduronidase in cultured skin fibroblasts was resolved into two distinct components, forms A and B. They had similar K_m values for 4-methylumbelliferyl--l-iduronide, but differed in pH optima and thermal stability. Form B was more heat-stable than form A.Residual -l-iduronidase activity in Hurler fibroblasts was heat-stable, while that in Scheie fibroblasts was heat-labile, and moreover, that in Hurler-Scheie compound fibroblasts lay intermediate between Hurler and Scheie syndromes. These findings demonstrated that Hurler syndrome, Scheie syndrome and Hurler-Scheie compound were enzymatically distinguishable.     

The relationship of alpha-L-iduronidase and Hurler corrective factor.

Hurler corrective factor (the protein that normalizes the faulty mucopolysaccharide catabolism of fibroblasts derived from Hurler patients) was previously identified as the enzyme α-l-iduronidase. However, not all α-l-iduronidase functions as Hurler corrective factor. The corrective and noncorrective forms of the human urinary enzyme have been separated from each other by chromatography on Sepharose-bound heparin. They have similar catalytic properties, some difference in ability to bind to the galactose-specific lectin of castor bean, and a significant difference in molecular weight (87,000 and 67,000 for the corrective and noncorrective forms, respectively). Only the corrective form is efficiently internalized by Hurler fibroblasts.     

N-acetylglucosamine 6-sulfate residues in keratan sulfate and heparan sulfate are desulfated by the same enzyme

We have prepared a series of oligosaccharides to assess the substrate specificity of exo sulfatase activity in cultured human skin fibroblasts toward N-acetylglucosamine-6-sulfate residues present in keratan sulfate (KS) and heparan sulfate (HS). Non-reducing end alpha-GlcNAc-6-SO4 residues (derived from HS) were desulfated by a specific sulfatase that when deficient leads to the accumulation of HS and the expression of mucopolysaccharidosis type IIID (Sanfilippo D). Under the in vitro conditions studied there are two pathways for the degradation of oligosaccharides containing non-reducing end beta-GlcNAc-6-SO4 residues (derived from KS). In one pathway beta-N-acetylglucosaminidase produces GlcNAc-6-SO4 which is then desulfated. In the other pathway the beta-GlcNAc-6-SO4 residue is desulfated and then cleaved by the action of an beta-N-acetylglucosaminidase activity. There was no detectable beta-N-acetylglucosaminidase activity in fibroblasts from a Tay-Sachs patient to produce GlcNAc-6-SO4 from beta-GlcNAc-6-SO4 residues in KS of oligosaccharides. There was approximately 10% of this normal beta-N-acetylglucosaminidase activity in fibroblasts from a Sandhoff patient, suggesting the A and S forms may be involved in this reaction. Desulfation of GlcNAc-6-SO4 residues in KS, HS and the monosaccharide GlcNAc-6-SO4 was considerably reduced or not detected in fibroblasts from a Sanfilippo D patient. As KS was not detected in the urine of a Sanfilippo D patient we propose that KS degradation in these patients proceeds by the action of a beta-N-acetylglucosaminidase activity to produce GlcNAc-6-SO4 which is not further degraded.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycosaminoglycan composition of human amniotic fluid

The glycosaminoglycan composition of human amniotic fluid between 12–21 weeks gestation has been studied by Dowex column chromatography coupled with enzymatic analyses of the specific glycosaminoglycan in each column fraction. The total uronic acid recovered from the columns consisted of “glycopeptides” (7%), hyaluronic acid (34%), nonsulfated chondroitin (14%), chondroitin-4-sulfate (13%), chondroitin-6-sulfate (20%), dermatan sulfate (5%), and heparan sulfate (6%). Based on these studies a simple screening procedure was devised to detect increased quantities of heparan sulfate and dermatan sulfate in 5–10-ml samples of amniotic fluid and tested in the antenatal diagnosis of Hurler and Hunter's syndrome. A false negative result was recorded in a Hunter fluid obtained early gestation and a false positive result recorded in a normal fluid obtained at weeks. These data suggest that the time in gestation when amniotic fluid is sampled for chemical analysis is an important variable affecting glycosaminoglycan composition in both normal and pathological pregnancies.     

Protamine increases the affinity of 3′-phosphoadenosine 5′-phosphosulfate toward a sulfotransferase from chicken embryo epiphyseal cartilage

A 3′-phosphoadenosine 5′-phosphosulfate (PAPS):chondroitin sulfate sulfotransferase from chicken embryo epiphyseal cartilage, which was partially purified, exhibited a molecular mass of 150 kDa. The enzymatic sulfation of totally desulfated chondroitin was activated up to 12-fold by protamine while the sulfation of partially sulfated chondroitin was activated only 3-fold. Protamine increased the affinity of the enzyme for PAPS about 4-fold when partially desulfated chondroitin was used as sulfate acceptor. The S_0.5 for the totally desulfated chondroitin was not affected by protamine, while high PAPS concentration slightly increased the affinity of the enzyme for the same sulfate acceptor. The possible role of these substances in the regulation of the sulfation of chondroitin sulfate is discussed.     

The disorder of hyaluronic acid metabolism in cultured skin fibroblasts derived from a patient with the Hurler syndrome

The metabolism of hyaluronic acid in cultured skin fibroblasts derived from a patient with the Hurler syndrome and from a normal subject was examined. 1. An increased net incorporation of [(3)H]glucose into the hyaluronic acid fraction of the Hurler-syndrome cells occurred when compared with normal cells. 2. During a ;chase' period, approx. 35% of the radioactivity derived from glucose was lost from the hyaluronic acid fraction of the Hurler-syndrome cells, whereas the normal cells retained all their radioactivity. 3. Although the Hurler-syndrome cells contained a ninefold greater amount of hyaluronic acid than normal cells, simultaneous determination of the specific radioactivity derived from the label revealed a value for the Hurler-syndrome cells one-half that of normal cells. These results are taken to indicate that the Hurler cells synthesize hyaluronic acid de novo at a higher rate than do normal cells. 4. Exposure of Hurler-syndrome cultured fibroblasts to a crude urine corrective-factor preparation (Neufeld & Cantz, 1971), now known to contain alpha-l-iduronidase, the specific Hurler-syndrome corrective factor (Bach et al., 1972), decreased the hyaluronic acid content to near-normal values before any effect was observed on [(3)H]glucose incorporation into the hyaluronic acid fraction. 5. In addition, the hyaluronic acid content of the normal cells decreased after exposure to the corrective factor of urine. 6. The mobilization of hyaluronic acid in Hurler-syndrome and normal cells exposed to the crude corrective-factor preparation of urine caused a decrease in specific radioactivity in the ;corrected' Hurler-syndrome cells and an increase in specific radioactivity in the ;corrected' normal cells.     

High-performance liquid chromatographic identification of eight constitutional disaccharides from heparan sulfate isomers digested with heparitinases

Identification with specific heparan sulfate-lyases, heparitinase I and heparinase of the constitutional unsaturated disaacharide (ΔDi-S_HS) derived from heparan sulfate (HS) isomers and heparin was achieved using high-performance liquid chromatography (HPLC) with a sulfonated styrene-divinylbenzene copolymer. Eight ΔDi-S_HS products derived from HS isomers were identified. Enzymatic digestion with heparitinase I and heparinase converts heterogeneous sulfated HS isomers and heparin into different ΔDi-S_HS. The practical application of these enzymes was examined using specific enzymes and HPLC. In a patient with Hurler syndrome, eight individual Δi-S_HS were identified in urinary HS isomers.     

Mucopolysaccharidosis 3 A (Sanfilippo A disease): deficiency of a heparin sulfamidase in skin fibroblasts and leucocytes

Cultured skin fibroblasts and peripheral leucocytes from patients with Sanfilippo A disease are strikingly deficient in sulfamidase activity (sulfamatase, EC 3.1.6.?), as measured with heparin - N³⁵SO₄. A partial sulfamidase deficiency was found in the cells of the heterozygote carriers. Since Sanfilippo A fibroblasts have normal sulfate ester hydrolase activities towards oligosaccharides prepared from ³⁵SO₄-labelled heparan sulfate by nitrous acid treatment, the basic defect in Sanfilippo A disease is considered to be the inactivity of a heparin (heparan sulfate) sulfamidase.     

Alpha-L-iduronidase activity in cultured skin fibroblasts and amniotic fluid cells

Using phenyl-α-l-iduronide as substrate, we have examined the level of α-l-iduronidase activity in homogenates of fibroblasts derived from normal individuals, from patients affected with α-l-iduronidase deficiency disorders (Hurler syndrome, Scheie syndrome, and a disease of intermediate severity presumed to be a Hurler/ Scheie compound) and from parents of such patients. Extracts derived from the affected individuals had no detectable α-l-iduronidase activity, whereas those derived from heterozygotes varied between 20% and 95% of the normal mean. Overlap between normal and heterozygous levels was reduced if the α-l-iduronidase activity was expressed on the basis of the β-galactosidase activity in the same homogenate. Cultured amniotic fluid cells from normal pregnancies had less than half as much α-l-iduronidase activity as fibroblasts from normal adults; this might cause problems in distinguishing a heterozygous fetus from an affected one by the enzyme assay alone.     

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.     

In vitro correction of Hurler fibroblasts with bovine testicular hyaluronidase.

Bovine testicular hyaluronidase (endo-beta-N-acetyl hexosaminidase) has a significant corrective effect on cultured Hurler fibroblasts. Nonspecificity of this effect is indicated by its equally strong corrective effect on Hunter fibroblasts. Although all specimens of hyaluronidase also possessed iduronidase activity, a separate corrective effect could be attributed to the endo-N-acetyl hexosaminidase activity of at least one hyaluronidase (Wyeth M-151) for four reasons: (i) its very low content of iduronidase activity; (ii) a decrease in intracellular macromolecular mucopolysaccharides (believed to be largely dermatan sulfate) with a corresponding increase in intracellular and extracellular oligosaccharides; (iii) no measurable increase in iduronidase activity of hyaluronidase-treated cells despite near maximal correction; (iv) direct correlation between Hurler cell correction and hyaluronidase activity when enzymes of different strength were used at less than maximal correction.     

Relationship between binding activity of 67Ga and low sulfated acid glycosaminoglycans

The sulfate content of acid glycosaminoglycan (AGAG) extracted from granuloma which had been produced by turpentine oil was inversely proportional to the amount of ⁶⁷Ga accumulation in the granuloma. Additionally, the lowest sulfation occurred in granuloma at a peak of inflammation when the uptake of ⁶⁷Ga had reached a maximum. On the basis of electrophoretic pattern, sulfate content, and specific optical rotation, it was concluded that acid glycosaminoglycans obtained from granuloma are mainly composed of chondroitin sulfate-A, -B, and desulfated heparin, while heparan sulfate was a minor component. From in vitro assays, desulfated acid glycosaminoglycans, especially desulfated-heparin and desulfated-heparan sulfate, were found to have a high affinity to ⁶⁷Ga. These results suggest that low- or de-sulfation of AGAG is related to the accumulation of ⁶⁷Ga in inflammatory lesions such as granuloma. Moreover, these results suggest that ⁶⁷Ga does not bind to glycosaminoglycans via sulfuric acid residues.     

Cytochemistry of the skin of patients with mucopolysaccharidoses.

The distribution of complex carbohydrates has been investigated at the light and electron microscope levels in sweat glands of normal subjects and patients with Hurler's or Hunter's disease. Normal sweat glands examined with a battery of light microscopic histochemical methods revealed sulphated complex carbohydrate in secretory granules of the dark cells. These granules lacked affinity for dialysed iron (DI) at the light and electron microscope levels. The DI method demonstrated acid complex carbohydrates ultrastructurally on the surface of the intercellular canaliculi and central lumen in normal sweat glands. DI-reactive acidic material, presumably of mucopolysaccharide nature, surrounded and extended between collagen bundles in the stroma of normal skin, but was absent from the band which ensheathed the sweat gland and consisted of individual rather than bundled collagen fibrils. DI-reactive mucopolysaccharide lined and partially filled vacuoles of dark cells showing a laminar distribution in vacuoles of clear cells in sweat glands of a Hunter patient. The DI method also visualized mucopolysaccharide distributed throughout vacuoles in fibroblasts of this patient. DI-reactive acid material covered the luminal surface of the sweat gland, coated collagen bundles in the stroma and spared the periglandular collagenous sheath in skin from Hurler and Hunter patients as in that from normal controls. Acid phosphatase was localized ultrastructually in vacuoles and nearby cytoplasm and on plasmalemmae of clear cells, dark cells and myoepithelial cells of sweat glands from Hurler and Hunter patients. Vacuoles of dermal fibroblasts and Schwann cells in these specimens also exhibited strong acid phosphatase activity.     

Comparative structural studies of urinary glycosaminoglycans in the Hurler and Hunter syndromes.

Some hitherto undetected differences in chemical and macromolecular structure between both dermatan sulphates and heparan sulphates excreted in the Hurler and Hunter syndromes are demonstrated. 1. Of Hunter dermatan sulphate, 37-43% is resistant to periodate oxidation, as opposed to 25% of the corresponding Hurler material. It is likely that the resistance is conferred by the presence of sulphate groups on carbon atoms 2 or 3 of the iduronate residues, correlating with the recently established deficiency of a sulphoiduronate sulphatase in Hunter fibroblasts. 2. Two distinct electrophoretic species of dermatan sulphate are found in Hunter urine, but only one in Hurler preparations. 3. Ion-exchange chromatography and gel filtration reveal that Hurler dermatan sulphate is more heterogeneous with respect to molecular weight distribution than the other. The dermatan sulphates were degraded by hyaluronidase to a limited extent. 4. Hurler heparan sulphate contains a higher proportion of sulphoamino-glucose than material from Hunter urine. Similar high levels in Sanfilippo patients, representing 65-78% of the total glucosamine suggest a direct correlation with mental deficiency.     

MUCOPOLYSACCHARIDES SYNTHESIZED BY CULTURED GLIAL CELLS DERIVED FROM A PATIENT WITH SANFILIPPO A SYNDROME

—Glial cells were cultured from brain tissue obtained at autopsy of a patient with Sanfilippo A syndrome. Mucopolysaccharides were labeled by culturing the cells in the presence of [³⁵S]sulfate. After proteolysis, intracellular and media-elaborated mucopolysaccharides were fractionated by Dowex 1 chromatography. One fraction, identified as heparan sulfate by chromatographic, electrophoretic, and enzyme susceptibility properties, accumulated in Sanfilippo glial cells in greater amounts than in controls. Heparan sulfate was also excreted into the culture media by both Sanfilippo and normal cultures, and it constituted a major fraction of the sulfated mucopolysaccharides synthesized by glial cells. Sanfilippo and normal fibroblasts were also included in these studies for comparative purposes. Sanfilippo fibroblasts accumulated significantly increased amounts of heparan sulfate as compared to normal fibroblasts. Heparan sulfate was excreted into the culture media by Sanfilippo and normal fibroblasts in equivalent amounts, but in contrast to glial cells, it was only a minor component of the sulfated mucopolysaccharides produced. Cultured glial cells should provide a useful system for investigating the role of heparan sulfate in glial cell function.     

Human kidney alpha-L-iduronidase: purification and characterization.

α-l-Iduronidase has been purified 25,000-fold from the soluble proteins of human kidney by chromatography on heparin-Sepharose, hydroxylapatite, and Bio-Gel P-100. The α-l-iduronidase activity is associated with 80% of the protein in the most purified preparation. It has a molecular weight of 60,000 ± 6500, determined by sedimentation equilibrium, and can be dissociated by reduction into subunits of molecular weight 31,000 ± 6500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate in the presence of dithiothreitol. It contains glucosamine and binds to concanavalin A. The pH optimum, K_m and V_max for two substrates, phenyl iduronide and [³H]anhydromannitol iduronide, were found to be 4.0, 1.05 mm, 16 μmol/mg protein/min, and 4·5, 9 mm and 270 μmol/mg protein/min, respectively. The enzyme is of the low uptake, noncorrective form with respect to fibroblasts cultured from the skin of patients with Hurler syndrome. It is inhibited by 10⁶ m p-chloromercuribenzoate and 10^?3 m Cu²⁺, but is not significantly affected by other divalent cations, EDTA, or sulfhydryl compounds. Antibodies to α-l-iduronidase have been raised in goats.