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Expression of five iduronate-2-sulfatase site-directed mutations
G R Villani1, A Daniele, N Balzano
1Department of Biochemistry and Medical Biotechnologies, Medical School, University of Naples 'Federico II', Via S. Pansini 5, 80131, Naples, Italy.
Biochimica Et Biophysica Acta
|June 6, 2000
Summary
Mutations in the iduronate-2-sulfatase (IDS) gene impact enzyme function in Hunter syndrome patients. Specific mutations affect enzyme activity and lysosomal transport, crucial for disease understanding.
Area of Science:
- Biochemistry
- Genetics
- Molecular Biology
Background:
- Hunter syndrome is a rare genetic disorder caused by mutations in the iduronate-2-sulfatase (IDS) gene.
- Understanding the functional consequences of these mutations is crucial for developing effective therapies.
Purpose of the Study:
- To investigate the functional impact of five specific IDS gene mutations (R88H, R88P, T118I, 959delT, R468Q) found in Italian Hunter patients.
- To assess the effects of these mutations on IDS enzyme activity, protein processing, and intracellular localization.
Main Methods:
- Expression of mutant IDS variants in COS cells.
- Enzyme activity assays and kinetic analysis (K(m) determination).
- Pulse-chase labeling and subcellular fractionation to evaluate protein maturation and localization.
Main Results:
- R88H mutation resulted in 13.7% residual enzyme activity with increased substrate affinity.
- R88P mutation led to a complete loss of enzyme activity.
- Missense mutations (R88H, R88P, T118I, R468Q) showed normal maturation but impaired transport to lysosomes.
- Residues 88, 118, and 468 are important for IDS conformation, not essential for processing.
Conclusions:
- Specific IDS mutations significantly alter enzyme function and cellular trafficking.
- Impaired lysosomal transport is a key consequence of these mutations, contributing to Hunter syndrome.
- Findings provide insights into the structural importance of key IDS residues for enzyme function and localization.