Introduction: Carbohydrates are prevalent within extracellular microenvironments, where they play an important role in regulating cell behavior in both normal and pathological processes via their non-covalent interaction with soluble and transmembrane carbohydrate-binding proteins (i.e. “lectins”). Thus, there is increasing interest in therapeutics that can harness, recapitulate, or interfere with carbohydrate-lectin binding to modulate cell behavior. Current efforts to modulate carbohydrate-lectin binding are often challenged by the low affinity of soluble, monovalent carbohydrates for lectins. Within living systems, lectin-carbohydrate affinity is often enhanced via avidity effects conferred by high-density display of immobilized carbohydrates on the cell surface or within the extracellular matrix, which is referred to as the “glycocluster effect”. Here, we describe a materials-based approach to create synthetic lectin-binding glycoclusters [1]. Our platform is based on carbohydrate-modified peptides (i.e. “glycopeptides”) that can self-assemble into beta-sheet nanofibers under aqueous conditions. Carbohydrate type along the nanofiber can be varied via the action of glycosyltransferase enzymes, while carbohydrate density can be tuned via co-assembly of glycosylated and non-glycosylated peptides at different molar ratios. Independent tuning of carbohydrate type and density yields glycoclusters with optimized lectin-binding affinity and specificity, which outperform conventional inhibitors. This simple and versatile platform is likely to lead to new therapeutics that can modulate lectin bioactivity for various biomedical applications.
Materials and Methods: Peptides, Q11 (QQKFQFQFEQQ) and its glycosylated analog n-acetylglucosamine-Q11 (GlcNAc-Q11), were synthesized using standard solid-phase peptide synthesis protocols. Q11 and GlcNAc-Q11 were assembled into nanofibers by dissolving dry peptides in water, followed by dilution in neutral aqueous buffer. GlcNAc content of the nanofibers was varied via co-assembly of Q11 and GlcNAc-Q11 at different molar ratios. GlcNAc moieties along the nanofiber were converted to the disaccharide, n-acetyllactosamine (LacNAc), by the enzyme beta-1,4-galactosyltransferase (GalT) in the presence of UDP-galactose (UDP-gal). Glycopeptide nanofiber binding to lectins (wheat germ agglutinin (WGA) and galectin-1) was analyzed via co-precipitation experiments. Glycocluster efficacy for inhibiting lectin bioactivity was assessed using a Jurkat T cell apoptosis assay, in which cells treated with galectin-1 demonstrate reduced metabolic activity.
Results: GlcNAc-Q11 self-assembled into nanofibers under aqueous conditions, and the concentration of GlcNAc integrated into the nanofibers was tuned by varying the molar ratio of GlcNAc-Q11 to Q11 present during assembly. GlcNAc moieties along the nanofiber were converted to LacNAc in high yield and purity via GalT and UDP-gal. GlcNAc nanofibers bound WGA, a GlcNAc-binding lectin, with affinity that correlated with nanofiber GlcNAc content (Fig. 1a). In contrast, LacNAc nanofibers failed to bind to WGA, but bound to galectin-1, a LacNAc-binding lectin, with affinity that correlated with nanofiber LacNAc content (Fig. 1a-b). Nanofibers having highest affinity for galectin-1 inhibited T cell apoptosis more effectively than thiodigalactoside, a small molecule galectin inhibitor (Fig. 2).
Conclusion: Self-assembly of glycopeptides into nanofibers provides glycoclusters with tunable lectin binding affinity and selectivity that can be optimized to inhibit lectin bioactivity. Thus, self-assembled glycopeptide nanofibers may ultimately lead to therapeutics that can more effectively modulate cell function in response to lectins for various biomedical applications, including cancer immunotherapy, autoimmunity treatment, tissue regeneration, and infection prophylaxis.
National Science Foundation Career Award (DMR-1455201)
References:
[1] Restuccia, A., Tian, Y.F., Collier, J.H., Hudalla, G.A. Cellular and Molecular Bioengineering, 8(3):471-487