Density Variant Glycan Microarray for Evaluating Cross-Linking of Mucin-like Glycoconjugates by Lectins

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 A1-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.


Determination of Mw of lectin monomers by SDS-PAGE.
4. Determination of apparent Mw of all lectins by FPLC.

Mucin mimetic array characterization.
A) Images of a low-density array with polymers 6a-e. B) Printing parameters. C) Derivation of equation 2 for the determination of average polymer spacing.
6. Images of a density variant array of polymer 9 and a plot of average polymer spacing.
7. Plot of average GalNAc density as a function of glycopolymer spacing.

Tables S4-8. Apparent K d values for individual lectins.
Chart S1. Apparent K d 's for lectins obtained from the lowest surface density array plotted against GalNAc valency in polymers 6. Table S9. Apparent K d and K rel values for lectins toward polymer 9 in a density variant array. Table S10. SBA and HPA precipitation by soluble glycopolymers 6b, 6e, and 9.

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6. Images of a density variant array of polymer 9 and a plot of average polymer spacing.
S15 7. Plot of average GalNAc density as a function of glycopolymer spacing.

HPA
2.5 µM → 5 x dilution → 32 pM 0.10/hexamer   p values refer to a comparison of K d 's between 6a and each of the other glycopolymers (t-test, two-tailed distribution, equal variance). 2 p values refer to a comparison of K d 's for the same polymer at the lowest and highest surface densities (t-test, two-tailed distribution, equal variance).  Chart S1. Apparent K d 's for lectins obtained from the lowest surface density array plotted against GalNAc valency in polymers 6.   9.5 ± 0.4 0.024 1.6 ± 0.1 11 ± 1 1 p values refer to a comparison of P 1/2 values for glycopolymers 6 and 9 (t-test, two-tailed distribution, equal variance). 2 p values refer to a comparison of P 1/2 values for the precipitation of both lectins by the same glycopolymer (t-test, two-tailed distribution, equal variance). 3 lectin to polymer ratio in the precipitate at P 1/2 . 4 p values refer to a comparison of GalNAc/lectin ratios for polymers 6e and 6b. Lectin stoichiometries and GalNAc per lectin ratios were determined as described in reference 33.