Use of N-glycanase to release asparagine-linked oligosaccharides for structural analysis |
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| Authors: | S Hirani R J Bernasconi J R Rasmussen |
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| Affiliation: | 1. TIPs CP 165/67, Université libre de Bruxelles, 50 avenue Franklin-Roosevelt, 1050 Bruxelles, Belgium;2. Service de Microbiologie Appliquée, Université libre de Bruxelles (ULB), c/o Institut de Recherches Microbiologiques Jean-Marie Wiame (IRMW), 1 avenue Emile Gryson, 1070 Bruxelles, Belgium;3. Institut de Recherches Microbiologiques Jean-Marie Wiame (IRMW), 1 avenue Emile Gryson, 1070 Bruxelles, Belgium;1. Department of Anatomy and Neuroscience, University College Cork, Ireland;2. APC Microbiome Institute, University College Cork, Ireland;1. Henan Key Laboratory for Pharmacology of Liver Diseases, Henan Institute of Medical and Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450052, China;2. Department of Ophthalmology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China |
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| Abstract: | ![]()
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. |
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