Heterogeneity of the complex N-linked oligosaccharides at specific glycosylation sites of two secreted carrot glycoproteins.
The N-linked glycans from the 52/54-kDa medium protein and cell wall beta-fructosidase, two glycoproteins secreted by carrot suspension culture cells, were characterized. Carrot cells were labelled with [3H]glucosamine or [3H]fucose. The 52/54-kDa medium protein was isolated from the culture medium and beta-fructosidase from cell walls. The purified proteins were digested with trypsin and glycopeptides were isolated and sequenced. Glycans obtained from individual glycopeptides were separated by gel filtration chromatography and characterized by concanavalin A chromatography, by treatments with exoglycosidases and by sugar composition analysis. The 52/54-kDa medium protein and cell wall beta-fructosidase have one high-mannose-type glycan similar to those from yeast and animal glycoproteins. In addition, the 52/54-kDa medium protein has three complex-type and cell wall beta-fructosidase two complex-type glycans per polypeptide. The complex-type glycans isolated from individual glycosylation sites are fairly large and very heterogeneous. The smallest of these glycans has the structure [Xyl](Man)3[Fuc](GlcNAc]2Asn (square brackets indicating branching) whereas the larger ones carry additional sugars like terminal N-acetylglucosamine and possibly rhamnose and arabinose in the case of the 52/54-kDa medium protein and only arabinose in the case of cell wall beta-fructosidase. These terminal sugars are linked to the alpha-mannose residues of the glycan cores. The 52/54-kDa medium protein is secreted with large and homogeneous complex glycans, their heterogeneity originates from slow processing after secretion. The complex glycans from cell wall beta-fructosidase are processed before the enzyme is integrated into the cell wall.
Sturm
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Friedrich Miescher Institute, Basel, Switzerland.
D000595:Amino Acid Sequence; D002473:Cell Wall / Q000201:enzymology; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006020:Glycopeptides / Q000235:genetics; D006023:Glycoproteins / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism*; D010449:Peptide Mapping; D010940:Plant Proteins / Q000378:metabolism; D014357:Trypsin; D014675:Vegetables / Q000378:metabolism*; D043324:beta-Fructofuranosidase
D006020:Glycopeptides; D006023:Glycoproteins; D009844:Oligosaccharides; D010940:Plant Proteins; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D014357:Trypsin