CARBOHYDRATE STRUCTURES OF RECOMBINANT HUMAN ALPHA-L-IDURONIDASE SECRETED BY CHINESE-HAMSTER OVARY CELLS

alpha-L-Iduronidase is a lysosomal hydrolase that is deficient in Hurl er syndrome and clinically milder variants. Recombinant human alpha-L- iduronidase, isolated from secretions of an overexpressing Chinese ham ster ovary cell line, is potentially useful for replacement therapy of these disorders. Because of the importance of carbohydrate residues f or endocytosis and lysosomal targeting, we examined the oligosaccharid es of recombinant alpha-L-iduronidase at each of its six N-glycosylati on sites. Biosynthetic radiolabeling showed that three or four of the six oligosaccharides of the secreted enzyme were cleaved by endo-beta- N-acetylglucosaminidase H, with phosphate present on the sensitive oli gosaccharides and L-fucose on the resistant ones. For structural analy sis, tryptic and chymotryptic glycopeptides were isolated by lectin bi nding and reverse phase high pressure liquid chromatography; their mol ecular mass was determined by matrix-assisted laser desorption-time of flight mass spectrometry before and after treatment with endo- or exo glycosidases or with alkaline phosphatase. Identification of the pepti des was assisted by amino-or carboxyl-terminal sequence analysis. The major oligosaccharide structures found at each site were as follows: A sn-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 glycopep tide was unexpectedly resistant to digestion by N-glycanase unless fir st dephosphorylated, but it was sensitive to endo-beta-N-acetylglucosa minidase 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 ha mster ovary cells.