Background

Following entry of a nascent polypeptide into the ER, it is subjected to several post-translational modifications, which are crucial for folding, maturation and quality control processes. The addition of the core oligosaccharide, Glc₃Man₉GlcNAc₂ to produce N-linked glycoproteins is a very common modification and the first to occur (Benyair et al., 2011). Processing of the precursor N-glycan directs the glycoproteins to maturation and quality control machineries by creating recognition tags while in early secretory compartments (Tannous et al., 2015). At later stages, throughout the secretory pathway the core of the oligosaccharide serves as a platform for expansion of the sugar chains into complex glycans, the structures of which relate to the trafficking and function of the glycoproteins (Kamiya et al., 2012). Because the early N-linked glycan modifications reflect glycoprotein biosynthesis and quality control, the oligosaccharide processing has been the subject of many studies (Avezov et al., 2010; Hosokawa et al., 2010; Ninagawa et al., 2014; Ogen-Shtern et al., 2016). Glycoproteomic methods have greatly improved N-glycan characterization, but they do not allow the study of the dynamics of glycan processing in the early secretory pathway.

Here we describe a simplified pulse-chase method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The method includes radioactive labeling by [2-³H]Man followed by enzymatic removal of oligosaccharides by endo-beta-N-acetylglucosaminidase H (Endo H). Then, N-linked oligosaccharide isolation by molecular filtration and separation by high-performance liquid chromatography (HPLC), which discriminates between high-mannose glycan structures depending on their number of monosaccharide residues. The protocol allows analysis of the dynamics of N-linked glycan modification under different conditions, e.g., after drug treatment or modification of protein levels by overexpression or knockdown. Trimming to shorter species, Man_5-6GlcNAc₂, is a requirement for glycoprotein targeting to endoplasmic reticulum-associated degradation (ERAD) (Frenkel et al., 2003). Man1A (α1,2 mannosidase 1A ) appears to be involved in endoplasmic reticulum (ER) quality control and required for this trimming, as we present in an example. This is a surprising finding considering that the enzyme was thought to be located in the Golgi complex (Igdoura et al., 1999; Herscovics, 2001); a recent reevaluation locates it in quality control vesicles (Ogen-Shtern et al., 2016).

To conclude, the protocol presented here enables the study of the dynamics of N-linked high-mannose glycan modifications, which has an important role in glycoprotein quality control and trafficking. The advantages of the method are its simplicity, high sensitivity of detection and unique information on the dynamics of N-glycan processing in the early secretory pathway.