Isolation and use of a new natural substrate for the diagnosis of MPS VII

Abstract

Mucopolysaccharidosis Type VII is characterized by a severe deficiency of the enzyme β-D-glucuronidase. As with other mucopolysaccharidoses, this inherited enzyme disorder results in a wide range of phenotypes. from extremely mild to severe. Diagnosis of the enzyme deficiency has usually been made using artificial substrates such as 4-methylumbelliferyl-β-D-glucuronide (MuGlcUA), and p-nitrophenyl-β-D-glucuronide. We now report the isolation of a tritium-labelled disaccharide, namely glucuronosyl anhydromannitol 6-sulphate (GMs) derived from the controlled degradation of heparin. Under optimal assay conditions for normal skin fibroblast homogenates, this natural substrate gives β-D-glucuronidase activities of 0-2.4, 9 17 and 38 56 pmol/min/mg protein for homozygote affected, heterozygote and normal cell lines, respectively. Compared with the artificial substrate MuGlcUA, normal activity measured with GMs demonstrates a similar pH profile and time course, but a changed response to protein levels, greater heat sensitivity, and also lower Km and Vmax values. GMs has a similar structure to iduronosyl anhydromannitol 6-sulphate (IMs), a natural substrate of α-L-iduronidase. However, in contrast to α-L-iduronidase, β-D-glucuromdase is inhibited only slightly by SO 4 2 or Cu2+ ions; while NaCl is a much more effective inhibitor at lower pH values. This suggests that β-D-glucuronidase may-lack the specific SO 4 2 and Cu2+ binding sites postulated for α-L-iduronidase. Mucopolysaccharidosis Type VII is characterized by a severe deficiency of the enzyme β-D-glucuronidase. As with other mucopolysaccharidoses, this inherited enzyme disorder results in a wide range of phenotypes. from extremely mild to severe. Diagnosis of the enzyme deficiency has usually been made using artificial substrates such as 4-methylumbelliferyl-β-D-glucuronide (MuGlcUA), and p-nitrophenyl-β-D-glucuronide. We now report the isolation of a tritium-labelled disaccharide, namely glucuronosyl anhydromannitol 6-sulphate (GMs) derived from the controlled degradation of heparin. Under optimal assay conditions for normal skin fibroblast homogenates, this natural substrate gives β-D-glucuronidase activities of 0-2.4, 9 17 and 38 56 pmol/min/mg protein for homozygote affected, heterozygote and normal cell lines, respectively. Compared with the artificial substrate MuGlcUA, normal activity measured with GMs demonstrates a similar pH profile and time course, but a changed response to protein levels, greater heat sensitivity, and also lower Km and Vmax values. GMs has a similar structure to iduronosyl anhydromannitol 6-sulphate (IMs), a natural substrate of α-L-iduronidase. However, in contrast to α-L-iduronidase, β-D-glucuromdase is inhibited only slightly by SO 4 2 or Cu2+ ions; while NaCl is a much more effective inhibitor at lower pH values. This suggests that β-D-glucuronidase may-lack the specific SO 4 2 and Cu2+ binding sites postulated for α-L-iduronidase.