Asparagine (N)-linked glycosylation is one of the most common co-and post-translational modifications of both intra-and extracellularly distributing proteins, which directly affects their biological functions, such as protein folding, stability and intercellular traffic. Production of the structural well-defined homogeneous N-glycans contributes to comprehensive investigation of their biological roles and molecular basis. Among the various methods, chemo-enzymatic approach serves as an alternative to chemical synthesis, providing high stereoselectivity and economic efficiency. This review summarizes some recent advances in the chemo-enzymatic methods for the production of N-glycans, including the preparation of substrates and sugar donors, and the progress in the glycosyltransferases characterization which leads to the diversity of N-glycan synthesis. We discuss the bottle-neck and new opportunities in exploiting the chemo-enzymatic synthesis of N-glycans based on our research experiences. In addition, downstream applications of the constructed N-glycans, such as automation devices and homogeneous glycoproteins synthesis are also described.

N-glycosylation starts with the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER). Alg2 mannosyltransferase adds both the α1,3- and α1,6-mannose (Man) onto ManGlcNAc2-pyrophosphate-dolichol (M1Gn2-PDol) in either order to generate the branched M3Gn2-PDol product. The well-studied yeast Alg2 interacts with ER membrane through four hydrophobic domains. Unexpectedly, we show that Alg2 structure has diverged between yeast and humans. Human Alg2 (hAlg2) associates with the ER via a single membrane-binding domain and is markedly more stable in vitro. These properties were exploited to develop a liquid chromatography-mass spectrometry quantitative kinetics assay for studying purified hAlg2. Under physiological conditions, hAlg2 prefers to transfer α1,3-Man onto M1Gn2 before adding the α1,6-Man. However, this bias is altered by an excess of GDP-Man donor or an increased level of M1Gn2 substrate, both of which trigger production of the M2Gn2(α-1,6)-PDol. These results suggest that Alg2 may regulate the LLO biosynthetic pathway by controlling accumulation of M2Gn2 (α-1,6) intermediate.

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N-glycosylation starts with the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum. Alg2 mannosyltransferase adds both the α1,3- and α1,6-Man onto ManGlcNAc2-pyrophosphate-dolichol (M1Gn2-PDol) in either order to generate the branched M3Gn2-PDol product. The well-studied yeast Alg2 interacts with ER membrane through four hydrophobic domains. Unexpectedly, we show that Alg2 structure has diverged significantly between yeast and humans. Human Alg2 (hAlg2) associates with the ER via a single membrane-binding domain and is markedly more stable in vitro. These properties were exploited to develop an LC-MS quantitative kinetic assay for studying purified hAlg2. Under physiological conditions, hAlg2 prefers to transfer α1,3-Man on to M1Gn2 before adding the α1,6-Man. However, this bias is altered by an excess of GDP-Man donor or an increased level of M1Gn2 substrate, both of which trigger production of the M2Gn2 (α-1,6)-PDol. These results suggest that Alg2 may regulate the LLO biosynthetic pathway by controlling accumulation of M2Gn2 (α-1,6) intermediate.