Detection of Morquio a Syndrome Using Radiolabelled Substrates Derived from Keratan Sulphate for the Estimation of Galactose 6-Sulphate Sulphatase

Abstract

1. The following radiolabelled disaccharide and tetrasaccharide were prepared from keratan sulphate and evaluated as substrates for galactose 6-sulphate sulphatase present in cultured human skin fibroblasts: O-(β-d-6-sulphogalactosyl)−(1 → 4)−2,5-anhydro-d-[1-³H] mannitol 6-sulphate (Gal6S-anM6S) and the tetrasaccharide equivalent Gal6S-GlcNAc6S-Gal6S-anM6S. Radiolabelled trisaccharide substrates, N-acetylgalactosamine 6-sulphate (β, 1 → 4)-glucuronic acid-(β 1→3)-N-acetyl[l-³H]galactosaminitol 6-sulphate (GalNAc6S-GlcA-GalitolNAc6S), and N-acetylgalactosamine 6-sulphate (β, 1 → 4)-glucose-(β, 1 → 3)-N-acetyl-[1-³H]galactosaminitol 6-sulphate (GalNAc6S-GlcGalitol-NAc6S), were prepared from chondroitin 6-sulphate and used to assay N-acetylgalactosamine 6-sulphate sulphatase. 2. Sulphatase activity obtained with Gal6S-anM6S was approximately the same, 100 and 30–50 times less than values obtained with Gal6S-GlcNAc6S-Gal6S-anM6S, GalNAc6S-GlcA-Galitol-NAc6S and GalNAc6S-Glc-GalitolNAc respectively. Less than 5% of normal activity resulted when these substrates were incubated with fibroblasts from Morquio A patients. These results demonstrate that the trisaccharide substrates derived from chondroitin 6-sulphate although de-O-sulphated at a considerably higher rate than was observed for the de-O-sulphation of the disaccharide and tetrasaccharide derived from keratan sulphate, are acted upon by the same sulphatase. 3. Galactose 6-sulphate sulphatase activity in fibroblast homogenates assayed with Gal6S-anM6S as substrate clearly distinguished Morquio A patients from normal controls, and Morquio B and Sanfilippo D patients. 4. We recommend the use of both the radiolabelled disaccharide derived from keratan sulphate and the trisaccharide derived from chondroitin 6-sulphate to assess sulphatase activity in fibroblasts of atypical Morquio patients.