Characterization of protein glycoforms with N-linked neutral and phosphorylated oligosaccharides: studies on the glycosylation of endoglucanase 1 (Cel7B) from Trichoderma reesei.
Using anion-exchange chromatography the catalyticdomain of endoglucanase 1 (Cel7B) from Trichoderma reesei was resolved in multiple fractions with different isoelectric points, presumably related to different glycoforms of the enzyme. The protein fractions were analysed using lectins and electrospray MS. Isolated N-glycans were analysed by fluorophore-assisted carbohydrate electrophoresis and amine-adsorption HPLC. The results show that this particular preparation contained at least 14 different glycoforms. The major isoform contained only one GlcNAc, presumably N-linked, and one mannose, most probably O-linked to serine/threonine at a separate site. Except for a small population containing Man(5)GlcNAc(2)+1-2 Man, the rest of the protein had negatively charged phosphate-containing N-glycans. All glycoforms contained at least one O-linked mannose residue. The increased negative charge of the protein, introduced by oligosaccharide phosphorylation, is the most probable reason for the different isoelectric points and the occurrence of multiple peaks during purification.
GlycoLab, Carbohydrates Department, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana, Cuba.
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García
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Cremata
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Quintero
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Montesino
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Benkestock
J
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Ståhlberg
D002236:Carbohydrate Conformation; D002480:Cellulase / Q000302:isolation & purification / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D007526:Isoelectric Point; D037102:Lectins / Q000737:chemistry; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011134:Polysaccharides / Q000032:analysis; D020033:Protein Isoforms / Q000737:chemistry; D014242:Trichoderma / Q000201:enzymology / Q000378:metabolism*
D006023:Glycoproteins; D037102:Lectins; D009844:Oligosaccharides; D011134:Polysaccharides; D020033:Protein Isoforms; C028569:oligomannoside; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase