Human -N-acetylgalactosaminidase: site occupancy and structure of N-linked oligosaccharides
| Autor: | Tomomi Ohnishi, Yiannis A. Ioannou, Masaya Ohta, Robert J. Desnick, Mark E. Hodgson, Fumito Matsuura |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
chemistry.chemical_classification
Glycoconjugate Stereochemistry Chinese hamster ovary cell Molecular Sequence Data Oligosaccharides Oligosaccharide Biochemistry Chromatography Affinity Enzyme structure Fucose alpha-N-Acetylgalactosaminidase Sialic acid carbohydrates (lipids) chemistry.chemical_compound Hexosaminidases Enzyme Carbohydrate Sequence chemistry Exoglycosidase COS Cells Carbohydrate Conformation Mutagenesis Site-Directed Animals Humans |
| Zdroj: | Glycobiology. 10:251-261 |
| ISSN: | 1460-2423 0959-6658 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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