n-acetylglucosamine-6-sulfate and Mucopolysaccharidoses

Sulphated N-acetylhexosamines have been isolated from human urine and tentatively identified as N-acetylglucosamine 6-sulphate (GlcNAc6S), N-acetylgalactosamine 6-sulphate (GalNAc6S), N-acetylgalactosamine 4-sulphate (GalNAc4S) and N-acetylgalactosamine 4,6-disulphate (GalNAc4,6diS). Urine from mucopolysaccharidosis-Type-IIID, -IVA and -VI patients compared with that from normal individuals contains elevated levels of GlcNAc6S (380-fold), GalNAc6S (180-fold) and GalNAc4S (420-fold) respectively. Urine from mucopolysaccharidosis-Type-VI patients also contain more than 600 times the normal level of GalNAc4,6diS. Urine from a mucolipidosis-Type-II and a multiple-sulphatase-deficient patient, and, in general, all mucopolysaccharidosis patients studied, contain at least 5-10-fold elevations of sulphated N-acetylhexosamines over the levels detected in urine from normal controls and a alpha-mannosidosis patient. Urine from patients with clinically mild phenotypes contains less sulphated N-acetylhexosamines than isolated from urine of clinically severe mucopolysaccharidosis patients. The source of the four sulphated N-acetylhexosamines is not known. However, incubation of a series of oligosaccharide substrates, derived from keratan sulphate and chondroitin 6-sulphate and containing non-reducing-end beta-linked 6-sulphated N-acetylhexosamine residues, with homogenates of cultured human skin fibroblasts has indirectly been shown to release GlcNA6S and GalNAc6S respectively. Release of GalNAc4S could not be demonstrated in similar incubations of oligosaccharide substrates derived from chondroitin 4-sulphate and containing non-reducing-end beta-linked GalNAc4S residues. We propose that some, if not all, of the sulphated N-acetylhexosamine present in human urine is derived from the action of beta-N-acetylhexosaminidase on sulphated GlcNAc or GalNAc residues at the non-reducing end of keratan sulphate, dermatan sulphate or chondroitin sulphate.

Topics: Acetylgalactosamine; Acetylglucosamine; Amino Acids; Cells, Cultured; Chromatography, Paper; Fibroblasts; Galactosamine; Humans; Mucopolysaccharidoses; Oligosaccharides; Structure-Activity Relationship

N-Acetylglucosamine 6-sulfate (GlcNAc6S) has been isolated from human urine and shown to be present at levels of approximately 0.02 and 14 mg/mmole creatinine in urine from normal individuals and a mucopolysaccharidosis type IIID (MPS IIID) patient respectively. We propose that the greater than 500-fold elevation of GlcNAc6S in urine from the MPS IIID patient indicates that this sulfated monosaccharide is also a substrate for the sulfatase deficient in MPS IIID patients. We further propose that part, if not all, of the GlcNAc6S found in urine may be produced from the cleavage by beta-N-acetylhexosaminidase A of non-reducing end beta-linked GlcNAc6S residues of keratan sulfate and/or sulfated glycoproteins.

Topics: Acetylglucosamine; Chromatography; Galactosamine; Glucosamine; Heparitin Sulfate; Humans; Keratan Sulfate; Mucopolysaccharidoses; Mucopolysaccharidosis III; Mucopolysaccharidosis VI

The first step of the degradation of p-nitrophenyl-6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside and of keratan sulfate-derived oligosaccharides bearing N-acetylglucosamine-6-sulfate residues at the nonreducing end was considered to be accomplished by the action of a specific sulfatase (Kresse, H., Paschke, E., von Figura, K., Gilberg, W., and Fuchs W. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6822-6826). In purification from human placenta, however, this activity co-chromatographed with isoenzyme A of beta-N-acetylhexosaminidase and had the same electrophoretic mobility as the latter enzyme. The activity was precipitated by a specific antiserum against beta-N-acetylhexosaminidase. A pronounced enzyme deficiency was found in Tay-Sachs and Sandhoff fibroblasts. The purified enzyme released p-nitrophenol from the chromogenic substrate as well as a second product which contained equimolar amounts of hexosamine and sulfate. This product had the same electrophoretic and chromatographic behavior as sulfated N-acetylglucosamine. It could be degraded by periodate to a smaller charged fragment. Incubation of keratan sulfate-derived oligosaccharides with beta-N-acetylhexosaminidase A analogously resulted in the liberation of N-acetylglucosamine-6-sulfate. The enzyme showed the highest affinity towards a trisulfated tetrasaccharide and exhibited a similar Km for the sulfated and the unsulfated p-nitrophenyl derivative.

Topics: Acetylglucosamine; beta-N-Acetylhexosaminidases; Cells, Cultured; Fibroblasts; Glucosamine; Hexosaminidases; Humans; Kinetics; Mucopolysaccharidoses; Sandhoff Disease; Skin; Substrate Specificity; Sulfur Radioisotopes; Tay-Sachs Disease; Tritium

[1-3H]Galactitol-6-sulfate, N- [1-3H]acetylgalactosaminitol-6-sulfate, N-[1-3H]acetylglucosaminitol-6-sulfate, N-acetylglucosamine-6-sulfate, and 6-sulfated tetrasaccharides from chondroitin-6-sulfate have been used for the measurement of 6-sulfatase activity of extracts of normal skin fibroblasts and of fibroblasts cultured from patients with genetic mucopolysaccharidoses. With these substrates, extracts of fibroblasts derived from Morquio patients lack or have greatly reduced activities for galactitol-6-sulfate, N-acetylgalactosaminitol-6-sulfate, and 6-sulfated tetrasaccharides but have normal activity for N-acetylglucosamine-6-sulfate and its alditol; those derived from a patient with a newly discovered mucopolysaccharidosis have greatly reduced activity for N-acetylglucosamine-6-sulfate and its alditol but normal activity for galactitol-6-sulfate, N-acetylgalactosaminitol-6-sulfate, and the 6-sulfated tetrasaccharides. These findings demonstrate the existence of two different hexosamine-6-sulfate sulfatases, specific for the glucose or galactose configuration of their substrates. Their respective deficiencies, causing inability to degrade keratan sulfate and heparan sulfate in one case and keratan sulfate and chondroitin-6-sulfate in the other, are responsible for different clinical phenotypes.

Topics: Acetylgalactosamine; Acetylglucosamine; Cells, Cultured; Child, Preschool; Chondroitin Sulfates; Chondroitinsulfatases; Fibroblasts; Galactitol; Heparitin Sulfate; Humans; Hydrogen-Ion Concentration; Keratan Sulfate; Male; Mucopolysaccharidoses; Mucopolysaccharidosis III; Mucopolysaccharidosis IV; Skin; Substrate Specificity; Sulfatases