Substitutions in the N-Glycan Core as Regulators of Biorecognition:  The Case of Core-Fucose and Bisecting GlcNAc Moieties

Abstract

Core fucosylation and the bisecting N-acetylglucosamine residue are prominent natural substitutions of the N-glycan core. To address the issue of whether these two substituents can modulate ligand properties of complex-type biantennary N-glycans, we performed chemoenzymatic synthesis of the respective galactosylated and alpha2,3/6-sialylated N-glycans. Neoglycoproteins were then produced to determine these glycans' reactivities with sugar receptors in solid-phase assays and with tumor cells in vitro as well as their in vivo biodistribution profiles in mice. Slight protein-type-dependent changes were noted in lectin binding, including adhesion/growth-regulatory galectins as study objects, when the data were related to properties of N-glycans without or with only one core substituent. Monitoring binding in vitro revealed cell-type-dependent changes. They delimited the ligand activity of this glycan type from that of chains with un- and monosubstituted cores. A markedly prolonged serum half-life was conferred to the neoglycoprotein by the galactose-terminated N-glycan, which together with increased organ retention of all three neoglycoproteins underscores the conspicuous relevance for glycoengineering of pharmaproteins. The predominant presentation of the two branches in the disubstituted N-glycan as extended (alpha1,3-antenna) and backfolded (alpha1,6-antenna) forms, revealed by molecular dynamics simulations, can underlie the measured characteristics. These results obtained by a combined strategy further support the concept of viewing N-glycan core substitutions as non-random additions which exert a modulatory role on ligand properties. Moreover, our data inspire us to devise new, non-natural modifications to realize the full potential of glycoengineering for diagnostic and therapeutic purposes.

Small angle neutron scattering as sensitive tool to detect ligand-dependent shape changes in a plant lectin with β-trefoil folding and their dependence on the nature of the solvent

“…In line with literature data [13,20,31] and our gel filtration analysis of aliquots run in parallel the lectin consistently formed (AB) 2 dimers (A = toxin subunit, B = lectin subunit) under these conditions. This confirms that the dimer structure seen in crystals [13,14] was not due to a crystal packing force.…”

“…Analyses of purity and activity were performed by gel filtration, oneand two-dimensional gel electrophoresis, haemagglutination, solid-phase and cell binding/growth assays [20,[23][24][25]. Prior to SANS measurements, H 2 O of freshly prepared VAA solutions in phosphate buffer (PB; 20 mM, pH 7.2, H 2 O) was exchanged to D 2 O by adding PB in D 2 O (20 mM, pD 7.2), followed by a step using a Millipore centrifugal concentrator with a 10 kDa MW cut-off.…”

“…So far, its ligand binding had been analyzed by atomic force microscopy, Scatchard analysis, surface plasmon resonance and titration calorimetry [18][19][20]. We herein resolve the question as to whether ligand binding has a bearing on the shape of this lectin with the β-trefoil fold.…”

Sequential Double “Clicks” toward Structurally Well‐Defined Heterogeneous N‐Glycoclusters: The Importance of Cluster Heterogeneity on Pattern Recognition In Vivo

“…In studies of N ‐glycan‐related compounds, Gabius and co‐workers demonstrated that immobilizing a few N ‐glycans onto 125 I‐labeled bovine serum albumin (BSA) moderately regulated the biodistributions and serum stabilities of the proteins in mice 25, 26, 27, 42. The presence of sialic acid residues in the glycoalbumin stabilized the molecules in serum, consistent with the receptor‐mediated excretion mechanism associated with asialoglycoprotein 43, 44, 45.…”

Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins

“…It has been reported that core fucosylation affects the conformations of N-glycans and modulates glycan-lectin interactions and antibody-Fc receptor recognition (41)(42)(43)(44)(45). Moreover, core fucosylation is associated with cancer progression, suggesting that core-fucosylated N-glycans and glycopeptides could serve as novel biomarkers for cancer diagnosis (46,47).…”