Glycobiology, 2000, Vol. 10, No. 3 251-261
© 2000 Oxford University Press
Human 
-N-acetylgalactosaminidase: site occupancy and structure of N-linked oligosaccharides
Department of Biotechnology, Fukuyama University, Fukuyama, Hiroshima 729–0292, Japan, 2Department of Human Genetics, Mount Sinai School of Medicine, Fifth Avenue and 100th Street, New York, NY 10029–6574
Human 
-N-acetylgalactosaminidase (-GalNAc; also known as -galactosidase B) is the lysosomal exoglycohydrolase that cleaves -N-acetylgalactosaminyl moieties in glycoconjugates. Mutagenesis studies indicated that the first five (N124, N177, N201, N359, and N385) of the six potential N-glycosylation sites were occupied. Site 3 occupancy was important for enzyme function and stability. Characterization of the N-linked oligosaccharide structures on the secreted enzyme overexpressed in Chinese hamster ovary cells revealed highly heterogeneous structures consisting of complex (~53%), hybrid (~12%), and high mannose-type (~33%) oligosaccharides. The complex structures were mono-, bi-, 2,4-tri-, 2,6-tri-, and tetraantennary, among which the biantennary structures were most predominant (~53%). Approximately 80% of the complex oligosaccharides had a core-region fucose and 50% of the complex oligosaccharides were sialylated exclusively with -2,3-linked sialic acid residues. The majority of hybrid type oligosaccharides were GalGlcNAcMan6GlcNAcFuc0–1GlcNAc. Approximately 54% of the hybrid oligosaccharide were phosphorylated and one-third of these structures were further sialylated, the latter representing unique phosphorylated and sialylated structures. Of the high mannose oligosaccharides, Man5-7GlcNAc2 were the predominant species (~90%) and about 50% of the high mannose oligosaccharides were phosphorylated, exclusively as monoesters whose positions were determined. Comparison of the oligosaccharide structures of -GalNAc and -galactosidase A, an evolutionary-related and highly homologous exoglycosidase, indicated that -GalNAc had more completed complex chains, presumably due to differences in enzyme structure/domains, rate of biosynthesis, and/or aggregation of the overexpressed recombinant enzymes.
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