Density variant glycan microarray for evaluating cross-linking of mucin-like glycoconjugates by lectins

Abstract

Interactions of mucin glycoproteins with cognate receptors are dictated by the structures and spatial organization of glycans that decorate the mucin polypeptide backbone. The glycan-binding proteins, or lectins, that interact with mucins are often oligomeric receptors with multiple ligand binding domains. In this work, we employed a microarray platform comprising synthetic glycopolymers that emulate natural mucins arrayed at different surface densities to evaluate how glycan valency and spatial separation affect the preferential binding mode of a particular lectin. We evaluated a panel of four lectins (Soybean agglutinin (SBA), Wisteria floribunda lectin (WFL), Vicia villosa-B-4 agglutinin (VVA), and Helix pomatia agglutin (HPA)) with specificity for α-N-acetylgalactosamine (α-GalNAc), an epitope displayed on mucins overexpressed in many adenocarcinomas. While these lectins possess the ability to agglutinate A(1)-blood cells carrying the α-GalNAc epitope and cross-link low valency glycoconjugates, only SBA showed a tendency to form intermolecular cross-links among the arrayed polyvalent mucin mimetics. These results suggest that glycopolymer microarrays can reveal discrete higher-order binding preferences beyond the recognition of individual glycan epitopes. Our findings indicate that glycan valency can set thresholds for cross-linking by lectins. More broadly, well-defined synthetic glycopolymers enable the integration of glycoconjugate structural and spatial diversity in a single microarray screening platform.

Figure 1

Schematic representation of two major binding modes between mucins and multidomain lectin receptors. (A) The oligomeric macrophage galactose-type lectins (MGLs) of dendritic cells form discrete adhesion complexes with MUC1 overexpressed on cancer cells. (B) Galectin-1 cross-linking of the mucin-type glycoprotein CD43 and CD7 triggers apoptosis in T-cells.

Figure 2

Schematic of mucin mimetic glycopolymer arrays. (A) Traditional glycan arrays rely on two-dimensional arrangements of monovalent glycans with very little control over spatial organization. (B) Glycan presentation on polymeric scaffolds more closely mimics that in native mucins. (C) Left: serine- and threonine-rich domains decorated with branched glycans initiating with a core sugar, α-N-acetylgalactosamine (α-GalNAc), are the hallmark of mucins. Right: a mucin-mimetic domain generated by oxime ligation of α-aminooxy-GalNAc to a poly(methylvinyl ketone) backbone.

Scheme 1. Synthesis of Mucin Mimetics

Figure 3

Determination of cross-linking by lectins in mucin mimetic arrays. (A) Cross-linking by lectin does not occur and the observed dissociation constant should be independent of glycolpolymer surface density. (B) Cross-linking is a contributing factor and increasing separation between neighboring mucin mimetic molecules should result in weaker binding (K_d,high and K_d,low denote apparent dissociation constants determined for a lectin in a high and a low glycopolymer surface density array, respectively).

Figure 4

Control of glycopolymer surface density in microarrays and a binding profile of SBA in the lowest density array. (A) Printing with solutions of polymers 6 at concentrations of 75, 150, and 400 nM afforded arrays with average polymer spacing (Δ) of ∼35, 25, and 13 nm, respectively. (B) Image of a portion of the lowest surface density array before (Cy3-channel) and after incubation with SBA-AF647 (100 nM in buffer). (C) Biding isotherms for SBA-AF647 to polymers 6 in the lowest surface density array. (D) Apparent K_d’s obtained for SBA-AF647 in the lowest density array plotted against GalNAc valency in 6. (**p < 0.01; p-value refers to a comparison of K_d’s for polymers 6a and 6e).

Figure 5

Determination of lectin cross-linking in variable ligand density arrays. (A) A drop in apparent K_d’s for binding of SBA to polymers 6a–c in the highest surface density array (lowest Δ) corresponds to avidity enhancements due to cross-linking by SBA. No SBA cross-linking was observed for polymers 6d and e. Binding profiles for WFL (B), VVA (C), and HPA (D) indicate no cross-linking of polymers 6. A decrease in avidity for VVA and HPA in the highest density array is likely the result of steric interference between lectin molecules bound to proximal ligands (*p < 0.05, **p < 0.01, ⧫p > 0.05; p-values refer to comparison of K_d’s for each polymer in the lowest and highest density arrays).

Figure 6

Cross-linking by lectins in density variant arrays of low-valency glycopolymer 9. The lectins show different levels of avidity enhancements resulting from decreased interligand spacing (Δ) indicating their unique intrinsic ability to cross-link polymer 9. No measurable binding was observed for SBA in the lowest density array. RWFL, a reduced nonagglutinating form of WFL, showed no cross-linking activity (p-values refer to comparison of K_d’s for each lectin at the lowest and highest polymer density).

Figure 7

Quantitative precipitation of SBA and HPA by soluble glycopolymers 6b, 6e, and 9 with valencies of 92, 170, and 17 GalNAc residues, respectively. (A) Plot of glycopolymer concentrations (P_1/2) necessary to affect half-maximal lectin precipitation as a function of glycopolymer valency. (B) Plot of the number of GalNAc residues per lectin molecule bound in precipitates at P_1/2.

Figure 8

Crystal structures of SBA (A) and HPA (B) lectins in complex with GalNAc-containing ligands (left) and their proposed interactions with mucin mimetics (right). (A) A “bind-and-slide” mode has previously been proposed for the interaction of the tetrameric SBA lectin with mucin-type polyvalent glycoconjugates. (B) The strong, valency-independent association of HPA with mucin mimetics 6 is likely to occur through a “face-to-face” binding mechanism.

Figure 9

A mechanistic rationale for distinct cross-linking activities of SBA and HPA. (A) The reversible “bind-and-slide” mechanism allows for dynamic assembly of SBA along the polymer scaffold while maximizing binding interactions through cross-linking. (B) Strong “face-to-face” interactions of HPA with the glycopolymers may lead to the formation of kinetically trapped species with a limited number of unbound GalNAc residues available for cross-linking.