Human alpha-N-acetylgalactosaminidase: site occupancy and structure of N-linked oligosaccharides.
Human alpha-N-acetylgalactosaminidase (alpha-GalNAc; also known as alpha-galactosidase B) is the lysosomal exoglycohydrolase that cleaves alpha-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 (approximately 53%), hybrid (approximately 12%), and high mannose-type (approximately 33%) oligosaccharides. The complex structures were mono-, bi-, 2,4-tri-, 2,6-tri-, and tetraantennary, among which the biantennary structures were most predominant (approximately 53%). Approximately 80% of the complex oligo-saccharides had a core-region fucose and 50% of the complex oligosaccharides were sialylated exclusively with alpha-2,3-linked sialic acid residues. The majority of hybrid type oligo-saccharides 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 (approximately 90%) and about 50% of the high mannose oligosaccharides were phosphorylated, exclusively as monoesters whose positions were determined. Comparison of the oligosaccharide structures of alpha-GalNAc and alpha-galactosidase A, an evolutionary-related and highly homologous exoglycosidase, indicated that alpha-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.
Ohta
M
M
Department of Biotechnology, Fukuyama University, Fukuyama, Hiroshima 729-0292, Japan,
Ohnishi
T
T
Ioannou
Y A
YA
Hodgson
M E
ME
Matsuura
F
F
Desnick
R J
RJ
D000818:Animals; D019556:COS Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D006596:Hexosaminidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D048809:alpha-N-Acetylgalactosaminidase
D009844:Oligosaccharides; C016735:sialooligosaccharides; D006596:Hexosaminidases; C482722:NAGA protein, human; D048809:alpha-N-Acetylgalactosaminidase