Use of N-glycanase to release asparagine-linked oligosaccharides for structural analysis.
An enzymatic procedure for releasing asparagine-linked oligosaccharides from glycoproteins by treatment with N-glycanase (peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase) has been investigated. Ribonuclease B, transferrin, fetuin, and alpha 1-acid glycoprotein were treated with N-glycanase and the released oligosaccharides were radiolabeled with NaB3H4. Lectin staining of the N-glycanase-treated proteins indicated that the deglycosylation reactions had proceeded to completion. The labeled carbohydrate chains were analyzed by HPLC on Micro-Pak AX-5 and AX-10 columns. The proportion of high-mannose and bi-, tri-, and tetraantennary complex chains obtained from each glycoprotein was in agreement with literature values. These results demonstrate that N-glycanase provides a simple method to release all common classes of asparagine-linked oligosaccharides from a glycoprotein in a form that can be radiolabeled directly for structural analysis.
D001894:Borohydrides; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis; D006026:Glycoside Hydrolases; D007553:Isotope Labeling; D009439:Neuraminidase; D009844:Oligosaccharides / Q000302:isolation & purification*; D014316:Tritium
D001894:Borohydrides; D006023:Glycoproteins; D009844:Oligosaccharides; D014316:Tritium; C025364:sodium borohydride; D006026:Glycoside Hydrolases; C020668:glycanase; D009439:Neuraminidase