Detection of the Sanfilippo type B syndrome using radiolabelled oligosaccharides as substrates for the estimation of α-N-acetylglucosaminidase

1. 1. The following radiolabelled disaccharides were prepared from heparin and evaluated as substrates for α-N-acetylglucosaminidase present in cultured skin fibroblasts: O-(α-2-acetamido-2-deoxy-d-glucopyranosyl)-(l → 4)-l-[6,³H]idose (GlcNAc-Ido), O-(α-2-acetamido-2-deoxy-d-glucopyranosyl)-(l '→ 4)-1,6 anhydro-l-6,³H] idose (GlcNAc-anldo), O-(α-2-acetamido-2-deoxy-d-glucopyranosyl)-(1→ 4)-L-[6,³H] idose 2-sulfate (GlcNAc-Ido(OS)), O-(α-2-acetamido-2-deoxy-d-glucopyranosyl)-(l → 3)-l-[6,³H] idonic acid (GlcNAc-IdOA). 2. 2. α-N-Acetylglucosaminidase activity assessed with GlcNAc-IdOA was 12 times higher than the values obtained using GlcNAc-Ido, GlcNAc-anldo and GlcNAc-Ido(OS). Less than 5% of normal activity resulted when these substrates were incubated with fibroblasts from Sanfilippo B patients. These results demonstrate that a C6 carboxyl group on the adjacent residue to the N-acetylglucosaminide moiety is an important structural requirement in the mechanism of action or binding of α-N-acetylglucosaminidase toward α-linked N-acetylglucosaminide residues. The presence of a C2 sulfate group on the adjacent residue had no effect on enzyme activity. 3. 3. α-N-Acetylglucosaminidase activity in leucocyte and fibroblast homogenates assayed using GlcNAc-IdOA as substrate clearly distinguished Sanfilippo B patients from normal controls, and Sanfilippo A, C and D patients.