Recent Advances in the Chemical Biology of N-Glycans

Abstract

Asparagine-linked N-glycans on proteins have diverse structures, and their functions vary according to their structures. In recent years, it has become possible to obtain high quantities of N-glycans via isolation and chemical/enzymatic/chemoenzymatic synthesis. This has allowed for progress in the elucidation of N-glycan functions at the molecular level. Interaction analyses with lectins by glycan arrays or nuclear magnetic resonance (NMR) using various N-glycans have revealed the molecular basis for the recognition of complex structures of N-glycans. Preparation of proteins modified with homogeneous N-glycans revealed the influence of N-glycan modifications on protein functions. Furthermore, N-glycans have potential applications in drug development. This review discusses recent advances in the chemical biology of N-glycans.

Keywords: N-glycan; NMR; chemical biology; glycan array; glycoprotein; lectin.

Figure 1

Structures of N-glycans. Complex-type N-glycans have diverse structures with/without sialic acid, poly-N-acetyl-lactosamine, bisecting GlcNAc, core fucose and so on. High-mannose-type N-glycan is composed of 14 residues (Glc₃Man₉GlcNAc₂) containing 3 glucoses, 9 mannoses, and 2 GlcNAc. Hybrid-type N-glycans have both high-mannose and complex-type structures.

Figure 2

The chemical biology study of homogeneous N-glycans. Chemical synthesis, enzymatic synthesis, and isolation of diverse, pure N-glycans enable their functional analysis at the molecular level. Interaction analysis using various N-glycans revealed the significance of complex N-glycan structures—for example, distinction of each branch and multivalent interaction in lectin recognition. Functional analysis using proteins with homogeneous glycoforms clarifies glycan function on each protein. Application of N-glycans for drug development is also investigated.

Figure 3

Mechanism of Saturation transfer difference nuclear magnetic resonance (STD-NMR). “Off resonance” experiment gives a reference spectrum. Under “on resonance” conditions, the saturation is transferred from protein to ligand by spin diffusion through intermolecular nuclear Overhauser effects (NOEs). The closer the protons are to the protein, the stronger the STD signals that are observed.

Figure 4

Analysis of glycan‒lectin interaction using glycan arrays. Interaction analysis using small fragments, such as disaccharides and trisaccharides, revealed the epitope required for lectin recognition, whereas interaction analysis using whole structures of N-glycans revealed the significance of complexity of N-glycan structures; these analyses provided the insights into the differences in the lectin recognition of each branch, the improvement of affinity due to the inclusion of multiple recognition units (multivalent effect), the influence of chain length on affinity, and remote (heterovalent) recognition.

Figure 5

Conformation analysis of N-glycan using pseudocontact shift (PCS). Chelation with paramagnetic metals can induce PCS to give geometric information of N-glycan.

Figure 6

Recognition of bisecting GlcNAc containing N-glycan by Calsepa and Phaseolus vulgaris erythroagglutinin (PHA-E). Attachment of bisecting GlcNAc enhances back-fold conformation, which is recognized by Calsepa and PHA-E.

Figure 7

Protein quality control utilizing high-mannose-type N-glycan as a tag. UDP-glucose:glycoprotein glucosyltransferase (UGGT) complex distinguishes misfolded glycoproteins to transfer glucose to the nonreducing end of the high-mannose glycan. This monoglycosylation serves as a marker for misfolded glycoproteins and the chaperone proteins calnexin/calreticulin (CNT/CRT) promotes folding.

Figure 8

Glycan editing of immunoglobulin (IgG) antibodies. N-Glycans at Asn297 of IgG affect their activity. Core fucose reduces the antibody-dependent cellular cytotoxicity (ADCC) activity, whereas bisecting GlcNAc enhances the ADCC activity. N-Glycan editing using Endo-β-N-acetylglucosaminidase (ENGase) can give IgG as a homogeneous glycoform or can be applied for the preparation of Antibody–drug conjugates (ADCs).

Figure 9

In vivo reaction using artificial glycosylated albumin metalloenzymes. Specific N-glycan conjugated albumin is specifically uptaken into the specific organs. Thus, the albumins conjugated with N-glycans were used as carriers of metal catalysts to realize chemical reactions for the activation of prodrug at the desired organ.