Removal of N-sulfated glucosamine residues during degradation of heparan sulfate is accomplished by the sequential action of three enzymes. Action of sulfamidase resuits in the formation of a-glucosaminide residues. Removal of these groups requires conversion to a-N-acetylglucosaminide by the action of an acetyltransferase in the presence of acetylCoA, followed by hydrolysis by a-N-acetylglucosaminidase. In

In human fibroblasts, the recognition of lysosomal enzymes by cell surface receptors is mediated by mannose 6-phosphate residues located on oligosaccharides that can be cleaved by endo-p-N-acetylglucosaminidase H. About half of these oligosaccharides, as isolated frm -hexosaminidase and cathepsin D secreted by human skin fibroblasts, are anionic. Most of these are resistant to alkaline phosphatase. The resistance is due to a-N-acetylglucosamine residues linked to mannose 6phosphate by a phosphodiester bond. The major phosphorylated oligosaccharides contain one and two and possibly three phosphate groups blocked by N-acetylglucosamine. Besides the blocked phosphate groups these oligosaccharides contain a common inner core consisting of Manal,6-(Manal,3)Manal,f(Manal,3)ManBGlcNAc and either one or two al,21linked mannose residues.Lysosomal enzymes share an oligosaccharide recognition marker, which plays an important role in the transfer of these enzymes into lysosomes.(1). In 1977 Kaplan et al. (2) suggested, primarily on the basis of competition experiments, that phosphorylated mannose residues were the principal component of the recognition marker of f3-glucuronidase. The same conclusion was reached for other enzymes and in other laboratories (3)(4)(5). Later on phosphate groups were found in several lysosomal glycoproteins (6-10) and were localized on carbon 6 of mannose (7-9).To study the structure of the oligosaccharide bearing the phosphorylated group, we used procedures developed (9, 11) to isolate mannose 6-phosphate residues from lysosomal enzymes. We labeled cultured human fibroblasts in the presence of NH4Cl, collected the secretions, immunoprecipitated precursors of f,-hexosaminidase and of cathepsin D, and isolated the oligosaccharides that were cleaved by endo-f3-N-acetylglucosaminidase H. We have observed that the phosphate groups are to a large extent buried in a diester with N-acetylglucosamine and that the oligosaccharides may contain up to three such phosphodiester residues.During the preparation of this manuscript Tabas and Kornfeld reported that (3-glucuronidase from mouse lymphoma cells contains high-mannose oligosaccharides with phosphate residues blocked by N-acetylglucosamine (12).MATERIALS AND METHODS Labeling and Immunoprecipitation of Lysosomal Enzymes. Human skin fibroblasts were maintained at 370C in 5% CO2 in Eagle's minimal essential medium supplemented with antibiotics, nonessential amino acids, and 10% fetal calf serum (Flow Laboratories, Bonn, Federal Republic of Germany) as described (13). Conditions of labeling cells with [2-3H]mannose and 32P, and immunoprecipitation of f3-hexosaminidase and cathepsin D were those described (11) with the following modifications. The labeling medium was prepared without glucose and supplemented with 10 mM NH4Cl and with 0.5% fetal calf serum that had been incubated at pH 10.4 for 30 min at 37°C to destroy acid hydrolases. NH4Cl has been included in the medium to increase the yield of secreted precursors of lysosomal enzymes (11). Labe...

Summaryiduronide 2-sulfate sulfatase, sulfamidase, and arylsulfatase B (Nacetylgalactosamine 4-sulfate sulfatase) were assayed in four muFibroblasts of four patients affected with mucosulfatidosis (mul-cosulfatidosis fibroblast lines using natural substrates.tiple sulfatase deficiency, Austin variant of metachromatic leukodystrophy) were assayed for activities of the five sulfatases known to degrade mucopolysaccharides. These were iduronide 2-sulfate sulfatase, sulfamidase, N-acetyl-galactosamine 6-sulfate sulfatase, (specific activity 27 mCi/mmole) from hen oviduct (lo), the monosulfated tri-saccharide N-acetylgalactosamine 6-sulfate Speculation (GlcNAC (6s)) D-glucuronic acid (G1cu~)-[1-3H]anhydromannit01 (specific activity 68.8 mCi/mmole) prepared from heparin Mucosulfatidosis is a disease characterized by the deficiency of sulfate (9), and the disulfated trisaccharide GalNAc(6S)-GlcUAmultiple sulfatases in cultured fibroblasts in contrast to the defi-~-acet~l-[l-~H]galactosaminitol(6S) (specific activity 24.9 mCi/ ciencies of singe sulfatases that are known for the five mucopoly-mmole) prepared from chondroitin 6-sulfate (12) were obtained saccharide degrading sulfatases and arylsulfatase A. The genes as described. responsible for the expression of the sulfatases are located both on autosomes and the X-chromosomes. Mucosulfatidosis fibroblasts should provide an experimental model for the study of a PATIENTS hitherto unknown common mechanism responsible for the exPresSkin biopsies or fibroblast cultures from patients with mucosulsion of sulfatase activities. fatidosis were kindly provided by Drs. J. Couchot (Reims), cell line 1, U. Wiesmann (Bern), cell line 2, and M. Niermeijer Mucosulfatidosis is a lysosomal storage disease characterized by (Rotterdam), line 3. line (GM 2407)9 was from the storage of sulfatides, glycosaminoglycans, and steroid sulfates. the Human Genetic Mutant Camden* N.J.The storage is caused by the deficiency of multiple sulfatases (for review, see refs. I and 2). In fibroblasts and tissues so far the METHODS deficiency of arylsulfatases A, B, and C, cholesteryl sulfatase and dehydroepiandrosterone sulfatase are known (3,4). It is, however, Cell Culture. Human skin fibroblasts from normal individuals still unclear whether activities of arylsulfatase C and the steroid and from patients of various genotypes were cultured in 25 cm2 sulfatases reside in the same enzyme or whether different enzymes Falcon plastic flasks with Eagle's Minimal Essential Medium (18) catalyze these reactions (2). Furthermore, mucosulfatidosis fibro-containing 10% fetal calf serum (LS-Labor Service, Miinchen), blasts do not correct the deranged mucopolysaccharide catabolism nonessential amino acids, and antibiotics as described previously in fibroblasts derived from patients affected with Hunter or Sari-(14). Cultures grown to confluency were harvested by trypsinizafilippo A syndrome which implies that mucosulfatidosis fibre-tion, the cells were washed twice with 0.15 M NaCI, and cell blasts are deficient in iduron...

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A new genetic variant of the Sanfilippo syndrome due to deficiency of acetyl CoA: alpha-glucosaminide N-acetyltransferase, was recently demonstrated in four patients. The clinical findings of these patients are reported here. Differential diagnosis from other types of the Sanfilippo syndrome on clinical and routine laboratory criteria is difficult and enzyme assay is necessary to reach the diagnosis. Since two of the patients reported are females and consanguinity was present in one case, autosomal recessive inheritance is most probable.