Recent Progress in Chemo-Enzymatic Methods for the Synthesis of N-Glycans

Abstract

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.

Keywords: N-glycosylation; biomarker; chemo-enzymatic synthesis; glycosidase; glycosyltransferases; glycosynthase; homogeneous glycoprotein.

Figure 1

Structures and symbols of common monosaccharides in N-glycans.

Figure 2

Typical glycosyltransferases and glycosidases for the enzymatic synthesis of high-mannose, hybrid and complex type N-glycans.

Figure 3

(A) The dolichol-linked oligosaccharide (DLO) biosynthesis pathway. (B) The schematic diagram of Man₉GlcNAc₂-PP-Phy enzymatic synthesis pathway in vitro.

Figure 4

(A) The function of Golgi endo-α-1,2-mannosidase, which can cleave terminal Glc-Man moiety from GlcMan₉GlcNAc₂. (B) The function of endo-β-N-acetylglucosaminidase (ENGase), which can hydrolyze the N-glycan structure from glycoproteins.

Figure 5

Chemo-enzymatic synthesis of asymmetric branched N-glycans with “stop and go” strategy. Transformation of GlcNTFA into GlcNH2 or GlcN3 (Stop); conversion of GlcNH₂ or GlcN₃ into GlcNAc (Go).

Figure 6

Chemo-enzymatic synthesis of high-mannose type N-glycan library by the orthogonal liberation of protection moieties in A, B and C arms from a non-natural tetradecasaccharide.

Figure 7

Core Synthesis/Enzymatic Extension (CSEE) strategy for N-glycan synthesis. (A) Chemo-enzymatic synthesis of core-fucosylated asymmetrical triantennary complex type N-glycans. (B) Construction of an N-glycan library by the enzymatic extension of 8 N-glycan core structures which are chemically synthesized from 5 building blocks.

Figure 8

Enzyme-mediated assembly of N-glycans through the automated platform. (A) Schematic illustration of the automated oligosaccharide synthesizer. (B) Automated chemo-enzymatic synthesis of asymmetrical N-glycan from a naturally derived oligosaccharide precursor.

Figure 9

Microarray using chemo-enzymatically synthesized N-glycans. (A) Preparation of different naturally occurring complex type N-glycans by chemo-enzymatic methods from a common precursor obtained from egg yolk. (B) Binding specificity analyses of the lectins using N-glycan microarray.

Figure 10

Chemo-enzymatic approach to prepare homogeneous antibodies. (A) The schematic diagram of synthesizing the homogeneous glycoproteins by ENGase-derived glycosynthases and glycan oxazolines. (B) Three identified Endo-S2 mutants (D226Q, D182Q, and T138Q) could be applied to transfer high-mannose, hybrid, bi- and tri-antennary complex type N-glycans to IgG. (C) The FcγIIIA binding affinity and ADCC activities of homogeneous Rtx-variants with different N-glycan structures.