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Recombinant human sulphamidase: expression, amplification, purification and characterization

J Bielicki¹, J J Hopwood, E L Melville

¹Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women's and Children's Hospital, 72 King William Road, North Adelaide, South Australia 5006, Australia.

The Biochemical Journal

|February 14, 1998

Summary

Researchers developed a stable, high-level expression system for recombinant human sulphamidase to treat Mucopolysaccharidosis type IIIA (MPS IIIA). This enzyme replacement therapy shows promise for correcting the storage defect in MPS IIIA cells.

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Area of Science:

Biochemistry
Genetics
Cell Biology

Background:

Mucopolysaccharidosis type IIIA (MPS IIIA), or Sanfilippo A syndrome, is a severe lysosomal storage disease characterized by profound neurological decline.
The condition results from a deficiency in the lysosomal enzyme sulphamidase, which is essential for breaking down heparan sulfate.

Purpose of the Study:

To establish a robust expression system for recombinant human sulphamidase to support enzyme replacement therapy development for MPS IIIA.
To achieve high-level and stable production of functional sulphamidase in Chinese hamster ovary (CHO) cells.

Main Methods:

Utilized a methotrexate-resistant dihydrofolate reductase (DHFR) gene for gene amplification in CHO cells, enabling increased sulphamidase expression.
Purified recombinant human sulphamidase from both unamplified and amplified cell lines for characterization.

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Assessed enzyme kinetics and its ability to correct the MPS IIIA cellular phenotype in patient-derived fibroblasts.

Main Results:

Achieved a significant increase in sulphamidase expression, from <1 mg/l to approximately 15 mg/l, using gene amplification.
The recombinant human sulphamidase was purified and characterized as a 115 kDa dimer with 62 kDa subunits.
The recombinant enzyme exhibited similar kinetic properties to the native enzyme and successfully corrected the storage defect in MPS IIIA fibroblasts via mannose-6-phosphate receptor-mediated endocytosis.

Conclusions:

A stable, high-yield expression system for recombinant human sulphamidase was successfully developed in CHO cells.
The characterized recombinant enzyme holds potential for enzyme replacement therapy in Mucopolysaccharidosis type IIIA.
The findings support the feasibility of using recombinant sulphamidase to address the underlying biochemical defect in MPS IIIA.