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Use of N-glycanase to release asparagine-linked oligosaccharides for structural analysis

1987; Elsevier BV; Volume: 162; Issue: 2 Linguagem: Inglês

10.1016/0003-2697(87)90424-6

1096-0309

Shirish Hirani, Richard Bernasconi, James R. Rasmussen,

Infant Nutrition and Health

An enzymatic procedure for releasing asparagine-linked oligosaccharides from glycoproteins by treatment with N-glycanase (peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase) has been investigated. Ribonuclease B, transferrin, fetuin, and α1-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.