Expression and characterization of wild type and mutant recombinant human sulfamidase - Implications for Sanfilippo (mucopolysaccharidosis IIIA) syndrome

Mucopolysaccharidosis IIIA (MPS-IIIA) is an autosomal recessive lysosomal s torage disorder caused by the deficiency of sulfamidase (NS; EC 3.10.1.1), resulting in defective degradation and storage of heparan sulfate. This pap er reports the production and characterization of monoclonal and polyclonal antibodies against recombinant human sulfamidase (rhNS) to quantitate and characterize normal and mutant sulfamidase produced from the wild type NS e xpression vector. Glycosylation and phosphorylation studies of immunoprecip itated rhNS show that all five potential glycosylation sites are utilized, with three high mannose/hybrid oligosaccharides and two simpler chains, wit h at least one functional mannose B-phosphate group. An NS quantification s ystem was developed to determine the effect of the three most common and se vere patient mutations: S66W (Italy), R74C (Poland), and R245H (The Netherl ands), The quantity and specific activity of expressed mutant rhNS was sign ificantly lower than expressed normal rhNS, with 0.3, 0.2, and 0.05% of nor mal rhNS produced and 15, 17, and 83% of normal specific activity for S66W, R74C, and R245H observed, respectively. The recent structural elucidation of N-acetylgalactosamine-4-sulfatase was utilized to postulate the effect o n the structure-function relationship of NS, The characterization of normal and mutated rhNS has relevance for efficient diagnosis and therapeutic dev elopments for MPS-IIIA patients.