Mechanistic Insight into Nanomolar Binding of Multivalent Neoglycopeptides to Wheat Germ Agglutinin

Abstract

Multivalent carbohydrate-protein interactions are frequently involved in essential biological recognition processes. Accordingly, multivalency is often also exploited for the design of high-affinity lectin ligands aimed at the inhibition of such processes. In a previous study (D. Schwefel et al., J. Am. Chem. Soc. 2010, 132, 8704-8719) we identified a tetravalent cyclopeptide-based ligand with nanomolar affinity to the model lectin wheat germ agglutinin (WGA). To unravel the structural features of this ligand required for high-affinity binding to WGA, we synthesized a series of cyclic and linear neoglycopeptides that differ in their conformational freedom as well as the number of GlcNAc residues. Combined evidence from isothermal titration calorimetry (ITC), enzyme-linked lectin assays (ELLA), and dynamic light scattering (DLS) revealed different binding modes of tetra- and divalent ligands and that conformational preorganization of the ligands by cyclization is not a prerequisite for achieving high binding affinities. The high affinities of the tetravalent ligands rather stem from their ability to form crosslinks between several WGA molecules. The results illustrate that binding affinities and mechanisms are strongly dependent on the used multivalent system which offers opportunities to tune and control binding processes.

Bacterial expression, purification and biophysical characterization of wheat germ agglutinin and its four hevein‐like domains

“…The fitting is consistent with the presence of 4 active binding sites per monomer for both agglutinins ( n = 4.1 ± 0.1 and 3.9 ± 0.0 for rWGA and wWGA, respectively). This binding stoichiometry is in agreement with ligand occupancies seen in recently solved crystallographic structures as well as in solution binding studies with designed multivalent neoglycopeptides exhibiting nanomolar affinity for WGA . In an early microcalorimetric study, Bains et al characterized the interaction of wWGA with several chitin‐derived oligosaccharides.…”

Revealing biomedically relevant cell and lectin type-dependent structure–activity profiles for glycoclusters by using tissue sections as an assay platform

“…The mechanism(s) underlying the inhibition of lectin binding will involve intra-and/or intermolecular bridging, and, "depending on the size and geometry of proteins and ligands, several binding modes may be possible and effective in parallel and in varying proportions." 68 The comparison of the data sets for the two leguminous lectins discloses large disparities in affinity already at the monosaccharide level, fully in line with published data. 43,45 These results emphasise that simple extrapolations between related proteins are not possible, a strong caveat when considering activity on members of families of tissue lectins such as C-type lectins or galectins.…”

“…Overall, structural design of each type of glycocluster accounts for differences in inhibitory potency on this hexavalent lectin ( Table 3 ), indicative of the assumed mutual complementarity. The mechanism(s) underlying the inhibition of lectin binding will involve intra- and/or intermolecular bridging, and, “depending on the size and geometry of proteins and ligands, several binding modes may be possible and effective in parallel and in varying proportions.” 68 …”

Multivalent Thiosialosides and Their Synergistic Interaction with Pathogenic Sialidases

“…We anticipated that polymer 9 could also act as a potent inhibitor of sialoside‐binding lectins. We selected wheat germ agglutinin (WGA) as a model lectin, and it was previously shown to be successfully inhibited by multivalent N ‐acetylglucosamine derivatives . As seen in Table , WGA can also accommodate sialosides such as Neu5Ac and 1 with affinities (m m ) in a similar range as those of NanA and NanA‐L .…”

“…We selected wheat germ agglutinin (WGA)a samodel lectin, and it was previously shown to be successfully inhibited by multivalent N-acetylglucosamine derivatives. [14,38] As seen in Table 1. Binding inhibition against WGA, NanA and NanA-L determined on achip.…”