Human  -N-acetylgalactosaminidase: site occupancy and structure of N-linked oligosaccharides (original) (raw)

Human α-N-acetylgalactosaminidase (α-GalNAc; also known as α-galactosidase B) is the lysosomal exoglycohydrolase that cleaves α-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 α-2,3-linked sialic acid residues. The majority of hybrid type oligosaccharides were GalGlcNAcMan 6 GlcNAc-Fuc 0-1 GlcNAc. 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, Man 5-7 GlcNAc 2 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 α-GalNAc and αgalactosidase A, an evolutionary-related and highly homologous exoglycosidase, indicated that α-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.