Recombinant α-l-iduronidase: characterization of the purified enzyme and correction of mucopolysaccharidosis type I fibroblasts

Abstract

Mucopolysaccharidosis type I (MPS I, Hurler and Scheie syndromes) is an autosomal recessive lysosomal storage disorder that results from a deficiency of the hydrolase alpha-L-iduronidase (IDUA) which is involved in the lysosomal degradation of both heparan sulphate (HS) and dermatan sulphate (DS). Patients with MPS I store and excrete large amounts of partially degraded HS and DS. In order to evaluate enzyme replacement therapy for MPS I patients we have expressed human IDUA cDNA in Chinese Hamster Ovary (CHO)-K1 cells utilizing a plasmid vector that places the cDNA under the transcriptional control of the human polypeptide-chain-elongation factor I alpha gene promoter. A clonal cell-line that secreted recombinant IDUA in a precursor form at approximately 2.2 micrograms/10(6) cells per day was identified. This enzyme was shown to be endocytosed into cultured MPS I fibroblasts via mannose-6-phosphate receptors 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 was processed to 74 kDa and smaller forms following its uptake by fibroblasts. Milligram quantities of the recombinant IDUA were immunopurified and the enzyme was shown to have pH optimum and kinetic parameters differing from those of the mature enzyme purified from human liver. The 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 stimulated by reducing agents or dilution.

Mild Feline Mucopolysaccharidosis Type VI

“…BslI restriction analysis of each PCR product was used to identify f4S clones containing the D520N mutation. Mutant f4S clones were sequenced with a fmol sequencing kit (Promega), and a clone that contained the D520N mutation and no other changes was then excised from pBluescript SK Ϫ with EcoRI and cloned into the mammalian expression vector pEFNeo (pEFNeoD520N) (12). Restriction analysis was used to identify recombinants containing the 4S construct in the correct orientation.…”

Uptake of a Recombinant Human .ALPHA.-L-Iduronidase (laronidase) by Cultured Fibroblasts and Osteoblasts

“…However, there have been few reports concerning such studies. 6) In this study, we investigated the uptake of laronidase by cultured fibroblasts from a patient with MPS I, and examined its morphological effect. Furthermore, we compared the uptake of laronidase by cultured mouse osteoblasts with that by cultured mouse fibroblasts.…”

“…Barsϭ50 mm. 6) In this study, we examined the effect of laronidase, which is now available for therapy for MPS I, on cultured MPS I fibroblasts biochemically and morphologically.…”

Feline Mucopolysaccharidosis Type VI

“…Expression of the f4S cDNA-The f4S cDNA was cloned into the expression vector pEFNeo (12). Restriction analysis was used to identify recombinants containing the f4S cDNA in the correct orientation.…”

“…HaeIII restriction analysis of each PCR product was used to identify f4S clones containing the mutant Pro 476 allele. Mutant f4S clones were sequenced with Sequenase Version 2 (Amersham Corp.), and a clone that contained the Pro 476 allele and no other changes was then excised from pBluescript SK Ϫ with EcoRI and cloned into the mammalian expression vector pEFNeo (pEFNeoL476P) (12). Restriction analysis was used to identify recombinants containing the mutated construct in the correct orientation.…”