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Recombinant canine alpha-l-fucosidase: expression, purification, and characterization
J Bielicki1, V Muller, M Fuller
1Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women's and Children's Hospital, 72 King William Road, North Adelaide, S.A. 5006, Australia.
Molecular Genetics and Metabolism
|February 3, 2000
Summary
Canine fucosidosis serves as a model for human lysosomal storage disorders. Researchers produced recombinant canine alpha-l-fucosidase (rcFUC), showing its potential for treating central nervous system diseases.
Area of Science:
- Biochemistry
- Genetics
- Veterinary Medicine
Background:
- Canine fucosidosis is a valuable large animal model for human lysosomal storage disorders (LSDs) and central nervous system (CNS) pathology.
- Bone marrow transplantation in this model demonstrated successful modification of CNS disease and identified key treatment parameters.
Purpose of the Study:
- To evaluate alternative treatment routes for CNS diseases in LSDs.
- To produce and characterize recombinant canine alpha-l-fucosidase (rcFUC) for therapeutic potential.
Main Methods:
- Recombinant canine alpha-l-fucosidase (rcFUC) was expressed in mammalian cells (CHO and MDCK) and purified using affinity chromatography.
- Enzyme kinetics and subunit composition of rcFUC were analyzed.
- The efficacy of rcFUC in correcting cellular storage phenotypes and degrading storage products was assessed.
Main Results:
- rcFUC was produced at high levels (2-13 mg/L) and purified to homogeneity.
- The enzyme consists of 50 kDa subunits, forming a 156 kDa homotrimer, with kinetics similar to native enzymes.
- rcFUC effectively corrected the storage phenotype in human fucosidosis cells via mannose-6-phosphate receptor-mediated endocytosis and degraded storage products from affected dog brains.
Conclusions:
- The availability of large quantities of functional rcFUC opens avenues for enzyme replacement therapy (ERT) in CNS lysosomal storage disorders.
- The fucosidosis dog model is crucial for exploring ERT strategies targeting CNS pathology.