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Glycosylation effects: Core contributions

Schematic structure of the disialylated, core-disubstituted N-glycan (attached to bovine serum albumin, BSA). For symbol meaning see here

Protein–protein binding can be influenced by amino acids distant from the interaction region. But do carbohydrates remote from the glycan terminal position also influence protein binding? Sugar residues attached to the N-glycan core — a chain of two initial N-acetylglucosamines (GlcNAc) and three mannoses that branch into two antennae — are known to influence the enzymatic transfer of carbohydrate residues to the periphery of N-glycans. André et al. now show in Biochemistry that carbohydrate substitutions at the N-glycan core can indeed influence the protein binding properties of N-glycans in a biologically significant way.

Using recently developed chemoenzymatic strategies, the authors synthesized N–glycans containing a fucose substitution at the initial GlcNAc residue and/or a GlcNAc substitution at the mannose residue at the base of the glycan antennae. The antennae of each of the synthesized N-glycans were elongated with N-acetyllactosamines. Two further glycans were synthesized by adding either 2,3- or 2,6-linked sialic acid to the disubstituted glycans. To test lectin affinity alterations, the synthesized glycans were transferred to bovine serum albumin (BSA) and the resulting BSA-neoglycoproteins were adsorbed to a plastic plate, thereby mimicking a cell surface. André et al. tested a panel of plant and medically relevant human lectins specific for the terminal sugars and observed a difference of up to five-fold in glycan–lectin binding affinity. This has provided the first evidence of a role for N–glycan core substitution in lectin binding at the glycan periphery.

The authors used flow cytometry to analyze the binding of the synthesized neoglycoproteins to tumor cell surface lectins. There was a large degree of variation in the number of cells binding to the neoglycoproteins, indicating the influence of distal carbohydrates. When the BSA-neoglycoproteins were injected into mice, André et al. found that neoglycoproteins containing the disubstituted N-glycans exhibited strongly retarded serum clearance and organ retention. Computational chemistry confirmed the hypothetical glycan conformational changes induced by the fucose and GlcNAc substitutions in the core region. The orientation of the two antennae was changed depending on the core substitution, leading to a structural distortion of the glycan.

Taken together, the results of André et al. extend our knowledge about how glycan structures can influence protein binding and cell–cell recognition. Core substitutions can alter glycan ligand properties through conformational changes which act as molecular switches for target affinity. In addition, the in vivo studies point to a use for glycan engineering in drug research. Prolonged serum presence of neoglycoproteins can be achieved using core–substituted glycans which alter glycan binding to hepatocyte asialoglycoprotein receptors that mediate endocytosis and degradation of glycoproteins. Further studies are needed to determine whether the observed distal effects of glycan substitution are also valid for O-glycans.

Author: Mirko von Elstermann