Changes in glycogen and glycosaminoglycan levels in hepatocytes of iduronate-2-sulfatase knockout mice before and after recombinant iduronate-2-sulfatase supplementation.

PURPOSE: Mucopolysaccharidosis II (MPS II) is a lysosomal storage disorder caused by a deficiency of iduronate-2 sulfatase (IdS), which is involved in the degradation of glycosaminoglycan (GAG). In this study, the frequency of fasting hypoglycemia in patients with MPS II was investigated and changes in accumulation of glycogen and GAG in the hepatocytes of IdS-knockout (KO) mice were evaluated before and after recombinant IdS enzyme replacement therapy (ERT). MATERIALS AND METHODS: Plasma glucose levels were evaluated after an 8-hour fast in 50 patients with MPS II. The IdS-KO mice were divided into three groups (group 2; saline, group 3; 0.15 mg/kg of IdS, and group 4; 0.5 mg/kg of IdS); wild-type mice were included as controls (group 1). ERT was initiated intravenously at four weeks of age, and continued every week until 20 weeks of age. RESULTS: The mean glucose level after an 8-hour fast was 94.1 ± 23.7 mg/dL in the patients with MPS II. Two (4%) out of 50 patients had fasting hypoglycemia. For the mice, GAG in the lysosomes nearly disappeared and glycogen particles in the cytoplasm were restored to the normal range in group 4. CONCLUSION: Glucose metabolism in patients with MPS II appeared to function well despite hepatocytic GAG accumulation and hypothetical glycogen depletion. A higher dose of IdS infusion in MPS II mice led to disappearance of lysosomal GAG and restoration of glycogen to the cytoplasm of hepatocytes.

Role of iduronate-2-sulfatase in glucose-stimulated insulin secretion by activation of exocytosis.

Iduronate-2-sulfatase (IDS) is a lysosomal enzyme expressed in pancreatic islets responsible for the degradation of proteoglycans such as perlecan and dermatan sulfate. Previous findings of our group demonstrated the involvement of IDS in the normal pathway of lysosomal degradation of secretory peptides, suggesting a role of this enzyme in beta-cell secretory functionality. The present study was undertaken to characterize the effect of IDS overexpression on insulin release. INS1E cells were transiently transfected with a construct encoding human IDS (hIDS). hIDS overexpression was associated with a gain of function detected by a reduction in heparan sulfate content. hIDS potentiated the glucose-stimulated insulin secretory response compared with controls (61%) with no changes in insulin mRNA levels or insulin peptide content. Results on quantification of the exocytotic process showed a significant increase in hIDS-transfected cells compared with controls. Furthermore, ultramorphological analysis demonstrated an increase in the number of granules in the immediate vicinity of the plasma membrane in hIDS-transfected cells and a decrease in total vesicles per square micrometer. hIDS overexpression induced phosphorylation of protein kinase C (PKC) alpha and its newly myristoylated alanine-rich C kinase substrate, MARCKS. We conclude that IDS has a role in glucose-stimulated insulin secretion via a mechanism that involves the activation of exocytosis through phosphorylation of PKCalpha and MARCKS.

Characterization of iduronate sulphatase mutants affecting N-glycosylation sites and the cysteine-84 residue.

Iduronate sulphatase (IDS) is responsible for mucopolysaccharidosis type II, a rare recessive X-linked lysosomal storage disease. The aim of this work was to evaluate the functional importance of each N-glycosylation site, and of the cysteine-84 residue. IDS mutant cDNAs, lacking one of the eight potential N-glycosylation sites, were expressed in COS cells. Although each of the potential sites was used, none of the eight glycosylation sites appeared to be essential for lysosomal targeting. Another important sulphatase co- or post-translational modification for generating catalytic activity involves the conversion of a cysteine residue surrounded by a conserved sequence C-X-P-S-R into a 2-amino-3-oxopropionic acid residue [Schmidt, Selmer, Ingendoh and von Figura (1995) Cell 82, 271-278]. This conserved cysteine, located at amino acid position 84 in IDS, was replaced either by an alanine (C84A) or by a threonine (C84T) using site-directed mutagenesis. C84A and C84T mutant cDNAs were expressed either in COS cells or in human lymphoblastoid cells deleted for the IDS gene. C84A had a drastic effect both for IDS processing and for catalytic activity. The C84T mutation produced a small amount of mature forms but also abolished enzyme activity, confirming that the cysteine residue at position 84 is required for IDS activity.