Characterization of the glycosylation of recombinant endopolygalacturonase I from Aspergillus niger.
The carbohydrate chains of recombinant endopolygalacturonase I (EPG I) from Aspergillus niger were characterized using a combination of mass spectrometric techniques. High performance liquid chromatography (HPLC) in conjunction with electrospray ionization mass spectrometry was used to separate the components of EPG I liberated by trypsin digestion. In-source collision-induced dissociation (CID) was utilized to fragment the digestion products entering the mass spectrometer, and the generation of carbohydrate fragment ions allowed for the identification of glycopeptides. The masses of the resulting glycans were calculated and entered into a carbohydrate database to search for possible structures. The primary sequences of the carbohydrate chains were confirmed by digesting aliquots of the intact glycopeptide with endo- and exoglycosidases and then analyzing the digestion products using matrix-assisted laser desorption/ionization mass spectrometry. These experiments demonstrated that one of the two N-linked sites of EPG I was occupied by a series of high-mannose structures, the second N-linked site was not occupied, and no O-linked sites were detected.
Complex Carbohydrate Research Center and Departments of Biochemistry and Molecular Biology, and Chemistry, University of Georgia, 220 Riverbend Road, Athens, GA 30602-4712, USA.
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Colangelo
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Licon
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Benen
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Visser
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Bergmann
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Orlando
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000737:chemistry* / Q000201:enzymology; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D011096:Polygalacturonase / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D005656:Fungal Proteins; D011994:Recombinant Proteins; D011096:Polygalacturonase