Radiolabelled
disaccharide substrates for
alpha-L-iduronidase, beta-D-
glucuronidase, and
sulfoiduronate sulfatase have been prepared from
dermatan sulfate by application in sequence of N-deacetylation, deaminative cleavage, and reduction with NaBT4. The yield of
disaccharides was approximately 87% of the total
oligosaccharide fraction. Five
disaccharides were isolated and tentatively identified. The major
disaccharide, O-(alpha-L-idopyranosyluronic
acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]
talitol 4-sulfate (IdoA-anT4S), represented approximately 75% of the total
disaccharide fraction. The other
disaccharides were O-(alpha-L-idopyranosyluronic
acid 2-
sulfate)-(1 leads to 3)-2,5-anhydro-D-[1-3H]
talitol 4-sulfate (IdoA2S-anT4S), O-(beta-D-glucopyranosyluronic
acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]
talitol 4-sulfate (GlcA-anT4S), O-(beta-D-glucopyranosyluronic
acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]
talitol 6-sulfate (GlcA-anT6S), and O-(alpha-L-idopyranosyluronic
acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]
talitol (IdoA-anT), which represented approximately 4.5, 11.2, 1.0, and 1.8%, respectively, of the total
disaccharide fraction. When incubated with cultured skin-fibroblasts from normal controls,
IdoA-anT4S was shown to be a sensitive substrate for
alpha-L-iduronidase to produce
2,5-anhydro-D-talitol 4-sulfate (anT4S). Activity toward
IdoA-anT4S was not observed with fibroblast homogenates from
alpha-L-iduronidase-deficient patients (
Mucopolysaccharidosis Type I). Similarly, normal-fibroblast homogenates degraded GlcA-anT6S to anT6S, and GlcA-anT4S to anT4S, at a rate 6 to 8 times greater than found for fibroblasts from beta-D-
glucuronidase-deficient patients (
Mucopolysaccharidosis Type VII).
IdoA-anT4S was hydrolysed by
alpha-L-iduronidase at a rate 365 times greater than that for IdoA-anT. Sulfation of the anhydro-D-[1-3H]
talitol residues is an important structural determinant in the mechanism of action of
alpha-L-iduronidase on
disaccharide substrates. IdoA2S-anT4S was degraded to
IdoA-anT4S and then to anT4S by normal-fibroblast homogenates, whereas fibroblasts from
alpha-L-iduronidase-deficient and
sulfoiduronate sulfatase-deficient (
Mucopolysaccharidosis Type II) patients produced considerably decreased levels of anT4s and
IdoA-anT4S (and anT4S), respectively.