title: Overexpression of the human lysosomal enzyme alpha-L-iduronidase in Chinese hamster ovary cells.
abstract: We developed a Chinese hamster ovary (CHO) cell line that produces and secretes large quantities of recombinant human alpha-L-iduronidase, the lysosomal hydrolase deficient in mucopolysaccharidosis I (Hurler, Hurler-Scheie, and Scheie syndromes). The alpha-L-iduronidase cDNA was introduced into a vector containing the cytomegalovirus immediate early gene promoter/enhancer, a murine immunoglobulin C alpha region intron, and the bovine growth hormone polyadenylation signal. Following cotransfection with a plasmid containing the neomycin resistance gene, stably transfected lines were selected with G-418. The highest expressing CHO cell line contained 1400-6000 units of alpha-L-iduronidase per milligram of protein, or 0.6-2.4% of total cell protein. Secreted alpha-L-iduronidase was 3000- to 7000 fold increased, with about 5000 units accumulating in 24 h per 10(7) cells. The activity and distribution of five other lysosomal glycosidases were not significantly affected. Metabolic labeling showed that half of the newly synthesized alpha-L-iduronidase was secreted, but generally less was recovered due to its instability in the medium. It was post-translationally processed as previously shown for alpha-L-iduronidase of human fibroblasts. Recombinant alpha-L-iduronidase was efficiently endocytosed by Hurler fibroblasts utilizing a mannose 6-phosphate-dependent mechanism (half maximal uptake at 0.7 nM) and was "corrective" for abnormal glycosaminoglycan accumulation (half-maximal correction at 0.7 pM). The half-life of the recombinant enzyme was 5 days following uptake into Hurler fibroblasts. Production in a 5-liter microcarrier culture system permitted the collection of 15 mg or more per day.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Pediatrics, Harbor-UCLA Medical Center, Torrance 90502.
authors.forename: E D
authors.initials: ED
authors.lastname: Kakkis
authors.forename: A
authors.initials: A
authors.lastname: Matynia
authors.forename: A J
authors.initials: AJ
authors.lastname: Jonas
authors.forename: E F
authors.initials: EF
authors.lastname: Neufeld
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D003001:Cloning, Molecular; D006224:Cricetinae; D015870:Gene Expression; D006025:Glycosaminoglycans / Q000096:biosynthesis; D006801:Humans; D007068:Iduronidase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D008059:Mucopolysaccharidosis I / Q000201:enzymology; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014162:Transfection
chemical_list: D006025:Glycosaminoglycans; D011994:Recombinant Proteins; D013431:Sulfates; D013462:Sulfur Radioisotopes; D007068:Iduronidase