Radiolabelled oligosaccharides as substrates for the estimation of sulfamidase and the detection of the Sanfilippo type A syndrome.

(1) A series of tritiated oligosaccharides, 2-sulfamino-2-deoxy-D-[1-14C]glucose (GlcNS) and [sulfamino-34S]heparin were evaluated as substrates for sulfamidase present in cultured human skin fibroblasts. (2) The following radiolabelled disaccharides were prepared from heparin: O-(alpha-2-sulfamino-2-deoxy-D-glucopyranosyl)-(1 leads to 3)-L-[6,3H]idonic acid (GlcNS-IdOA) and O-(alpha-2-sulfamino-2-deoxy-D-glucopyranosyl)-(1 leads to 3)-2,5 anhydro-L-[6,3H]idonic acid (HlcNS-anIdOA). Other radiolabelled oligosaccharides evaluated as sulfamidase substrates were the disaccharides O-(alpha-2-sulfamino-2-deoxy-D-glucopyranosyl)-(1 leads to 4)-L-[6,3H]idose (GlcNS-Ido) and O-(alpha-2-sulfamino-2-deoxy-D-glucopyranosyl)-)1 leads to 4)-L-[6,3H]idose 2-sulfate (GlcNS-Ido(OS)) and a preparation containing the tetrasaccharide GlcNS-UA-GlcNS-l-idonic acid, GlcNS-UA-GlcNS-anhydro-L-idonic acid and GlcNS-UA-GlcNS-L-gulonic acid. (3) Sulfamidase activity assessed with GlcNS-IdOA and GlcNS-anIdOA were approximately equal and up to 4, 8 and 800 times higher than the value obtained using [sulfamino-35S]heparin, GlcNS-Ido(OS) and GlcNS-Ido respectively. Under the assay conditions used GlcNS was not de-N-sulfated. These results demonstrate that C6 carboxyl and C2 sulfate ester groups on the adjacent residue to the sulfaminoglucosamine moiety are important structural requirements in the mechanism of action or binding of sulfamidase toward N-sulfated disaccharides. The results obtained for a partially characterized mixture of tetrasaccharides suggest that they are degraded four times faster than their disaccharide structural counterparts. (4) No detectable sulfamidase activity toward [sulfamino-35S]heparin, monosaccharide, disaccharide or tetrasaccharide substrates could be detected using homogenates of fibroblast cultures from Sanfilippo A patients (sulfamidase deficient). (5)sulfamidase activity measured with GlcNS-IdOA exhibited a pH optimum at 4.5 to 5.5, an apparent Km of approximately 220 mumol/l and potent inhibition by sulfate ions.