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Volume 272, Number 36, Issue of September 5, 1997 pp. 22758-22765
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Carbohydrate Structures of Recombinant Human alpha -L-Iduronidase Secreted by Chinese Hamster Ovary Cells

(Received for publication, April 10, 1997, and in revised form, June 11, 1997)

Ke-Wei Zhao , Kym F. Faull Dagger , Emil D. Kakkis § and Elizabeth F. Neufeld

From the Department of Biological Chemistry and Molecular Biology Institute and the Dagger  Department of Psychiatry & Biobehavioral Sciences and Chemistry & Biochemistry Center for Molecular and Medical Sciences Mass Spectrometry, UCLA, Los Angeles, California 90095 and the § Department of Pediatrics, Harbor-UCLA Medical Center, Torrance, California 90502

alpha -L-Iduronidase is a lysosomal hydrolase that is deficient in Hurler syndrome and clinically milder variants. Recombinant human alpha -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 alpha -L-iduronidase 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-beta -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 (P2Man7GlcNAc2); Asn-372, high mannose (mainly Man9GlcNAc2, some of which was monoglucosylated); Asn-415, mixed high mannose and complex; Asn-451, bisphosphorylated (P2Man7GlcNAc2). The Asn-451 glycopeptide was unexpectedly resistant to digestion by N-glycanase unless first dephosphorylated, but it was sensitive to endo-beta -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.


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