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Purification and characterization of recombinant murine sulfamidase
B L Gliddon1, G Yogalingam, J J Hopwood
1Lysosomal Diseases Research Unit, Department of Genetic Medicine, Women's and Children's Hospital, North Adelaide, SA 5006, Australia. briony.gliddon@adelaide.edu.au
Abstract:
Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder caused by a deficiency in the lysosomal enzyme sulfamidase, which is required for the degradation of heparan sulfate. The disease is characterized by neurological dysfunction but relatively mild somatic manifestations. A naturally occurring mouse model to MPS IIIA exhibits a similar disease progression to that observed in patients. Disease in the mice results from a base substitution at codon 31 in the sulfamidase gene, altering an aspartic acid to an asparagine (D31N). This aspartic 31 is involved in binding of the divalent metal ion needed for catalytic function, and as such reduces the specific activity of the enzyme to about 3% of that of wild-type. The mutant protein has decreased stability and shows increased degradation over a 24 h chase period when compared to wild-type mouse sulfamidase. Mouse sulfamidase that was purified using a two-step ion exchange procedure was shown to have similar kinetic properties to that of purified human sulfamidase. Recombinant murine sulfamidase was able to correct the storage phenotype of MPS IIIA fibroblasts after endocytosis via the mannose-6-phosphate receptor.
Insights
Mucopolysaccharidosis type IIIA (MPS IIIA) is a rare genetic disorder. A mouse model with a specific sulfamidase gene mutation shows reduced enzyme activity, impacting heparan sulfate breakdown and causing neurological issues.
Area of Science:
- Biochemistry
- Genetics
- Lysosomal Storage Disorders
Background:
- Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder resulting from sulfamidase deficiency.
- Heparan sulfate degradation is impaired, leading to neurological dysfunction and milder somatic symptoms.
- A naturally occurring mouse model mirrors human MPS IIIA progression.
Purpose of the Study:
- To characterize the molecular defect in the MPS IIIA mouse model.
- To investigate the biochemical properties of the mutant sulfamidase enzyme.
- To assess the potential of recombinant sulfamidase for therapeutic intervention.
Main Methods:
- Genetic analysis of the sulfamidase gene in the MPS IIIA mouse model.
- Enzyme kinetics and stability assays of wild-type and mutant mouse sulfamidase.
- Cellular uptake and functional correction studies using recombinant murine sulfamidase in MPS IIIA fibroblasts.
Main Results:
- A D31N mutation in the sulfamidase gene was identified as the cause of MPS IIIA in mice.
- The mutant enzyme exhibited significantly reduced specific activity (approx. 3% of wild-type) and decreased stability.
- Purified mouse sulfamidase showed similar kinetics to human sulfamidase.
- Recombinant murine sulfamidase corrected the storage phenotype in MPS IIIA fibroblasts via mannose-6-phosphate receptor-mediated endocytosis.
Conclusions:
- The D31N mutation impairs sulfamidase catalytic function and stability, explaining MPS IIIA pathogenesis in mice.
- The mouse model accurately reflects human MPS IIIA biochemical defects.
- Recombinant sulfamidase holds promise for treating MPS IIIA by restoring lysosomal enzyme activity.
