RECOMBINANT ALPHA-L-IDURONIDASE - CHARACTERIZATION OF THE PURIFIED ENZYME AND CORRECTION OF MUCOPOLYSACCHARIDOSIS TYPE-I FIBROBLASTS

Mucopolysaccharidosis type I (MPS I, Hurler and Scheie syndromes) is a n autosomal recessive lysosomal storage disorder that results from a d eficiency of the hydrolase alpha-L-iduronidase (IDUA) which is involve d in the lysosomal degradation of both heparan sulphate (HS) and derma tan sulphate (DS). Patients with MPS I store and excrete large amounts of partially degraded HS and DS. In order to evaluate enzyme replacem ent therapy for MPS I patients we have expressed human IDUA cDNA in Ch inese Hamster Ovary (CHO)-K1 cells utilizing a plasmid vector that pla ces the cDNA under the transcriptional control of the human polypeptid e-chain-elongation factor I alpha gene promoter. A clonal cell-line th at secreted recombinant IDUA in a precursor form at approximately 2.2 mu g/10(6) cells per day was identified. This enzyme was shown to be e ndocytosed into cultured MPS I fibroblasts via mannose-6-phosphate rec eptors and to correct the storage phenotype of these cells by enabling the lysosomal-digestion of accumulated sulphated glycosaminoglycans. The recombinant IDUA had on SDS/PAGE a molecular mass of 85 kDa and wa s processed to 74 kDa and smaller forms following its uptake by fibrob lasts. Milligram quantities of the recombinant IDUA were immunopurifie d and the enzyme was shown to have pH optimum and kinetic parameters d iffering from those of the mature enzyme purified from human liver. Th e specific activity of the recombinant enzyme was shown to increase on dilution and on incubation with reducing agents. This was in contrast to the mature IDUA form (74 kDa) which did not have its activity stim ulated by reducing agents or dilution.