Carbohydrate Structures of Recombinant Human α-l-Iduronidase Secreted by Chinese Hamster Ovary Cells

Abstract

␣-L-Iduronidase is a lysosomal hydrolase that is deficient in Hurler syndrome and clinically milder variants. Recombinant human ␣-L-iduronidase, isolated from secretions of an overexpressing Chinese hamster ovary cell line, is potentially useful for replacement therapy of these disorders. Because of the importance of carbohydrate residues for endocytosis and lysosomal targeting, we examined the oligosaccharides of recombinant ␣-Liduronidase at each of its six N-glycosylation sites. Biosynthetic radiolabeling showed that three or four of the six oligosaccharides of the secreted enzyme were cleaved by endo-␤-N-acetylglucosaminidase H, with phosphate present on the sensitive oligosaccharides and L-fucose on the resistant ones. For structural analysis, tryptic and chymotryptic glycopeptides were isolated by lectin binding and reverse phase high pressure liquid chromatography; their molecular mass was determined by matrix-assisted laser desorption-time of flight mass spectrometry before and after treatment with endo-or exoglycosidases or with alkaline phosphatase. Identification of the peptides was assisted by amino-or carboxyl-terminal sequence analysis. The major oligosaccharide structures found at each site were as follows: Asn-110, complex; Asn-190, complex; Asn-336, bisphosphorylated (P 2 Man 7 GlcNAc 2 ); Asn-372, high mannose (mainly Man 9 GlcNAc 2 , some of which was monoglucosylated); Asn-415, mixed high mannose and complex; Asn-451, bisphosphorylated (P 2 Man 7 GlcNAc 2 ). The Asn-451 glycopeptide was unexpectedly resistant to digestion by N-glycanase unless first dephosphorylated, but it was sensitive to endo-␤-N-acetylglucosaminidase H and to glycopeptidase A. The heterogeneity of carbohydrate structures must represent the accessibility of the glycosylation sites as well as the processing capability of the overexpressing Chinese hamster ovary cells.

Biosynthesis, Glycosylation, and Enzymatic Processingin Vivo of Human Tripeptidyl-peptidase I

“…1C). Thus, the observed difference in mass was caused by dissimilar oligosaccharide structures on secreted and intracellular proenzyme of hTPP I, a finding that was reported for other overexpressed mature acid hydrolases (24,25) or their proforms (26). CHO cells also secreted very small amounts of mature enzyme, which could be visualized only after prolonged exposure of autoradiograms and immunoblots following immunoprecipitation (see below).…”

Reprogramming erythroid cells for lysosomal enzyme production leads to visceral and CNS cross-correction in mice with Hurler syndrome

“…The enzyme is normally targeted to the lysosome via the cation-independent mannose 6-phosphate (M6P) receptor (MPR) (19). To test if this endogenous uptake pathway remains effective for IDUA protein released by erythroid cells, lymphoblastoid cells derived from an MPS I patient were exposed to medium preconditioned by induced MEL-KIiG (Fig.…”

Lipoprotein Receptor Binding, Cellular Uptake, and Lysosomal Delivery of Fusions between the Receptor-associated Protein (RAP) and α-l-Iduronidase or Acid α-Glucosidase

“…Uptake of enzyme from the blood following intravenous administration requires specific oligosaccharides on the enzyme itself and corresponding oligosaccharide receptors on target cells. Examples include the binding of phosphorylated high-mannose oligosaccharides on ␣-L-iduronidase by the cation-independent mannose 6-phosphate receptor (MPR) 1 and binding of high-mannose oligosaccharides on glucocerebrosidase by the mannose receptor (2,3). The former system is the basis for treatment of patients with Hurler syndrome, the latter for Gaucher syndrome.…”