Sulfatases contain a unique posttranslational modification in their active site, a formylglycine residue generated from a cysteine or a serine residue. The formylglycine residue is part of a sequence that is highly conserved among sulfatases, suggesting that it might direct the generation of this unique amino acid derivative. In the present study residues 68-86 flanking formylglycine 69 in arylsulfatase A were subjected to an alanine/glycine scanning mutagenesis. The mutants were analyzed for the conversion of cysteine 69 to formylglycine and their kinetic properties. Only cysteine 69 turned out to be essential for formation of the formylglycine residue, while substitution of leucine 68, proline 71, and alanine 74 within the heptapeptide LCTPSRA reduced the formylglycine formation to about 30-50%. Several residues that are part of or directly adjacent to an alpha-helix presenting the formylglycine 69 at the bottom of the active site pocket were found to be critical for catalysis. A surprising outcome of this study was that a number of residues fully or highly conserved between all known eukaryotic and prokaryotic sulfatases turned out to be essential neither for generation of formylglycine nor for catalysis.

Mucopolysaccharidosis type VI (MPSVI, Maroteaux-Lamy syndrome) is a lysosomal storage disease for which multiple clinical phenotypes have been described. A deficiency of the enzyme arylsulfatase B (ASB, N-acetylgalactosamine-4-sulfatase) is the cause of this autosomal recessively inherited disorder. The genotypes of two patients with an intermediate form of MPSVI have been determined by polymerase chain reaction (PCR) amplification of the entire open reading frame of the ASB gene and subsequent direct sequencing of both strands of the PCR fragments by an automated nonradioactive approach. In patient A, a C to T transition in allele I resulting in an exchange of the Arg codon 160 for a premature stop codon (R160*, exon 2), and a G to A transition in allele II leading to a Gln to Arg160 substitution (R160Q, exon 2) were detected. Patient B exhibited a 7-bp deletion in exon 1 of allele I resulting in a frame shift and a premature stop codon 33 triplets 3' of the site of deletion (delta G237-C243), and a C to T transition in allele II giving rise to a Trp to Arg152 substitution (R152W, exon 2). None of these four mutant alleles was present among 60 alleles of the ASB gene in unrelated controls, indicating that the former are not polymorphisms. These results emphasize the broad molecular heterogeneity of Maroteaux-Lamy syndrome and contribute to the establishment of a genotype/phenotype correlation in this disease.

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Cultured arterial fibroblasts were used for a quantitative study on adsorption, uptake and degradation of [ 35 S]proteoglycans derived from secretions of cultured arterial or skin fibroblasts. The following results were obtained: 1) Proteoglycans added to the culture medium are integrated into the pool of cell membraneassociated (trypsin-removable) glycosaminoglycans by a saturable process, which depends on time and temperature.2) Up to 17% of the added proteoglycans are taken up by the cells within 24 h. The uptake exhibits saturation kinetics, characteristic for adsorptive pinocytosis. Proteoglycan concentrations required for half-maximum uptake are higher than for haii'-maximum saturation of the glycosaminoglycan pool associated with the cell membrane.3) After a lag phase, inorganic 3S S0 4 appears in the culture medium as a degradation product of the internalized proteoglycans. Pinocytosed proteoglycans are catabolized more rapidly than proteoglycans which remain inside the cell after their biosynthesis. 4) Pinocytosis exhibits specificity, the individual proteoglycans being internalized at different rates. The highest rate of uptake was measured for a dermatan-sulfate-rich proteoglycan. No competition of uptake between a dermatan-sulfate-rich and a heparan-sulfate-rich proteoglycan was observed. 5) Optimum pinocytosis requires an intact protein moiety and, presumably, undegraded carbohydrate chains of the proteoglycans. Abbreviations: CS, Chondroitin sulfate; DS, dermatan sulfate; HS, heparan sulfate. Nomenclature: Glycosaminoglycans (CS, DS, HS) are defined as [ 35 S]sulfate-containing polysaccharides characterized by the NaCl molarity by which they are eluted from Dowex 1X2 as compared with authentic glycosaminoglycan preparations and by their susceptibility to Chondroitin lyases. A covalent linkage of glycosaminoglycans to a protein or polypeptide moiety is not excluded. The term proteoglycan is used for glycosaminoglycan-containing macromolecules which are excluded from Sepharose 4B gel or are eluted from it with a volume distinctly smaller than that of glycosaminoglycans.

Purified urinary α‐N‐acetylglucosaminidase acts as an exoglycosidase. The enzyme removes from heparan sulfate exclusively α‐glycosidically linked N‐acetylglucosamine residues. The pH optimum of around 4.4 towards heparan sulfate and heparin is similar to that towards synthetic arylglycosides. Urinary α‐N‐acetylglucosaminidase can be separated by isoelectric focusing into multiple forms with pI values between 3.3 and 6.0. The multiple forms differ in their recognition and endocytosis by cultivated skin fibroblasts. Forms with pI values of 4.8 ± 0.3 respond best to endcytosis. From these forms up to 0.8 × 106 molecules may be recognized and taken up in an hour by a single cell. Sodium periodate treatment reduces the α‐N‐acetylglucosaminidase recognition by fibroblasts and suggests that the recognition sites on the enzyme are associated with its carbohydrate moiety. Attempts to modify the recognition of α‐N‐acetylglucosaminidase by pretreatment with purified glycosidases failed.