From structural to functional glycomics: core substitutions as molecular switches for shape and lectin affinity of N-glycans

Abstract

Glycan epitopes of cellular glycoconjugates act as versatile biochemical signals (sugar coding). Here, we test the hypothesis that the common N-glycan modifications by core fucosylation and introduction of the bisecting Nacetylglucosamine moiety have long-range effects with functional consequences. Molecular dynamics simulations indicate a shift in conformational equilibria between linear extension or backfolding of the glycan antennae upon substitution. We also present a new fingerprint-like mode of presentation for this multi-parameter system. In order to delineate definite structure-function relationships, we strategically combined chemoenzymatic synthesis with bioassaying cell binding and the distribution of radioiodinated neoglycoproteins in vivo. Of clinical relevance, tailoring the core region affects serum clearance markedly, e.g., prolonging circulation time for the neoglycoprotein presenting the N-glycan with both substitutions. a2,3-Sialylation is another means toward this end, similarly seen for type II branching in triantennary N-glycans. This discovery signifies that rational glycoengineering along the given lines is an attractive perspective to optimize pharmacokinetic behavior of glycosylated pharmaproteins. Of general importance for the concept of the sugar code, the presented results teach the fundamental lesson that N-glycan core substitutions convey distinct characteristics to the concerned oligosaccharide relevant for cis and trans biorecognition processes. These modifications are thus molecular switches.

Conformational flexibility of N-glycans in solution studied by REMD simulations

“…Extensive conformational sampling is required to access the multiple conformers of N-glycans. For this reason, past conventional MD simulations in explicit solvent have been limited to small fragments like mono-, di-, or trisaccharides (Sayers and Prestegaud 2000;Kim et al 2009;Kirschner and Woods 2001;Almond 2005;Perić-Hassler et al 2010;Corzana et al 2004;Landström and Widmalm 2010), or larger fragments with short sampling (Naidoo et al 1997;Woods et al 1998;André et al 2009). …”

“…They are readily comparable to experimental NMR data. André and co-workers proposed a single-plot fingerprint-like presentation in polar coordinates for describing the mutual dependence among the many glycosidic angles involved (André et al 2009). …”

“…The currently accepted view is that chemical modification changes binding, by way of either switching the major conformation from one to another or inducing a unique bioactive conformation, rather than by making an additional unique binding site. Past studies, including NMR-based-analysis of model oligosaccharides (Homans et al 1987a), FRET experiments (Stubbs et al 1996), and molecular modeling followed by systematic bioassays with neoglycoproteins carrying synthetic biantennary N-glycans (André et al 2009), indicate a switch-like change in the shape of the glycans upon chemical modification. Figures 3 and 4 show the results of REMD simulations of four N-glycans with and without chemical modification (Nishima et al 2012).…”

“…Additional branches (antennae) complicate the structures. The number of pairs of dihedral glycosidic angles rapidly increases; 22 parameters for typical bianntenary complex-type N-glycan (André et al 2009). Actually, the number of different conformers of glycans is less than that predicted from the flexibilities of all the individual linkages (Woods et al 1998).…”

Glycosylation Affects Ligand Binding and Function of the Activating Natural Killer Cell Receptor 2B4 (CD244) Protein

“…It can act as a biochemical signal recognized by lectins but also have structural influence in receptor-ligand recognition because of its bulky shape caused by branched side chains (20). Carbohydrates are linked to proteins by N-and O-glycosidic bonds.…”