Characterization of the N-linked glycosylation site of recombinant pectate lyase.
Recombinant pectate lyase from Aspergillus niger was overexpressed in Aspergillus nidulans. The two recombinant proteins produced differed in molecular mass by 1200 Da, which suggested that the larger molecular weight protein was glycosylated. The deduced amino acid sequence was searched for potential N-linked glycosylation sites, and one potential site was identified at residue 64. The proteins were analyzed for their ability to bind various lectins as an assay for the presence of carbohydrates. The proteins were then digested with trypsin to facilitate the isolation of the potential glycosylation site. The resulting digestion products were subsequently analyzed by liquid chromatography/mass spectrometry using in-source collision induced dissociation to detect glycopeptides. Once the glycopeptide had been identified, treatment with an endoglycosidase both verified the location of glycosylation and identified the mass of the glycan. The Complex Carbohydrate Structural Database was searched for possible N-linked structures with the same mass, and the suggested primary sequence was confirmed by an exoglycosidase digestion. The data demonstrated that the larger recombinant protein contained a high mannose N-linked structure (Man(5)GlcNAc(2)) attached to N-64, while this site was not occupied in the smaller protein.
Colangelo
J
J
Complex Carbohydrate Research Center and Departments of Biochemistry and Molecular Biology, and Chemistry, University of Georgia, 220 Riverbend Road, Athens, GA 30602-4712, USA.
Licon
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Benen
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Visser
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Bergmann
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Orlando
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D001233:Aspergillus nidulans; D001234:Aspergillus niger / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003001:Cloning, Molecular; D006020:Glycopeptides / Q000737:chemistry*; D006026:Glycoside Hydrolases; D006031:Glycosylation; D008970:Molecular Weight; D011133:Polysaccharide-Lyases / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014357:Trypsin
D006020:Glycopeptides; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D014357:Trypsin; D011133:Polysaccharide-Lyases; C023911:pectate lyase