Galectins are carbohydrate-binding proteins that act as extracellular signaling molecules in various normal and pathological processes. Galectin bioactivity is mediated by specific non-covalent interactions with cell-surface and extracellular matrix (ECM) glycoproteins, which can enhance or inhibit signaling events that influence various cellular behaviors, including adhesion, proliferation, differentiation, and apoptosis. Here, we developed a materials approach to modulate galectin bioactivity by mimicking natural galectin-glycoprotein interactions. Specifically, we created a variant of a peptide that self-assembles into β-sheet nanofibers under aqueous conditions, QQKFQFQFEQQ (Q11), which has an asparagine residue modified with the monosaccharide N-acetylglucosamine (GlcNAc) at its N-terminus (GlcNAc-Q11). GlcNAc-Q11 self-assembled into β-sheet nanofibers under similar conditions as Q11. Nanofibrillar GlcNAc moieties were efficiently converted to the galectin-binding disaccharide N-acetyllactosamine (LacNAc) via the enzyme β-1,4-galactosyltransferase and the sugar donor UDP-galactose, while retaining β-sheet structure and nanofiber morphology. LacNAc-Q11 nanofibers bound galectin-1 and -3 in a LacNAc concentration-dependent manner, although nanofibers bound galectin-1 with higher affinity than galectin-3. In contrast, galectin-1 bound weakly to GlcNAc-Q11 nanofibers, while no galectin-3 binding to these nanofibers was observed. Galectin-1 binding to LacNAc-Q11 nanofibers was specific because it could be inhibited by excess soluble β-lactose, a galectin-binding carbohydrate. LacNAc-Q11 nanofibers inhibited galectin-1-mediated apoptosis of Jurkat T cells in a LacNAc concentration-dependent manner, but were unable to inhibit galectin-3 activity, consistent with galectin-binding affinity of the nanofibers. We envision that glycopeptide nanofibers capable of modulating galectin-1 bioactivity will be broadly useful as biomaterials for various medical applications, including cancer therapeutics, immunotherapy, tissue regeneration, and viral prophylaxis.

No abstract

Glycosylation alters protein form and function by establishing intermolecular forces that mediate specific interactions while preventing non-specific aggregation. Self-assembled peptide nanofibers modified with carbohydrates are increasingly used as biomaterials to mimic glycosylated protein function, yet the influence of carbohydrate conjugates on nanofiber structure remains poorly defined. Here we show that a dense carbohydrate surface layer can facilitate hierarchical organization of peptide nanofibers into anisotropic networks. Glycosylated peptide nanofibers remain dispersed in dilute conditions, whereas non-glycosylated nanofibers tend to aggregate. In crowded conditions, some glycosylated nanofibers laterally associate and align. This behavior depends on carbohydrate chemistry, particularly hydroxyls, suggesting involvement of short-range attractive forces. Macroscopic gels fabricated from densely glycosylated peptide nanofibers are resistant to non-specific interactions with proteins, mammalian cells, and bacteria, yet selectively bind lectins, analogous to natural lowfouling mucosal barriers. Collectively, these observations demonstrate that glycosylation can inform structure in addition to endowing function to peptide-based supramolecular biomaterials.

Success of enzymes as drugs requires that they persist within target tissues over therapeutically effective time frames. Here we report a general strategy to anchor enzymes at injection sites via fusion to galectin-3 (G3), a carbohydrate-binding protein. Fusing G3 to luciferase extended bioluminescence in subcutaneous tissue to ~7 days, whereas unmodified luciferase was undetectable within hours. Engineering G3-luciferase fusions to self-assemble into a trimeric architecture extended bioluminescence in subcutaneous tissue to 14 days, and intramuscularly to 3 days. The longer local half-life of the trimeric assembly was likely due to its higher carbohydrate-binding affinity compared to the monomeric fusion. G3 fusions and trimeric assemblies lacked extracellular signaling activity of wild-type G3 and did not accumulate in blood after subcutaneous injection, suggesting low potential for deleterious off-site effects. G3-mediated anchoring to common tissue glycans is expected to be broadly applicable for improving local pharmacokinetics of various existing and emerging enzyme drugs.

Carbohydrate-modified biomaterials are attractive candidates for disrupting natural protein-glycan binding events because they present ligands in multivalent arrangements that can address the weak affinity of monovalent protein-carbohydrate interactions. However, protein binding depends on physical aspects of immobilized carbohydrate display, such as density and valency, which are often difficult to predict and can vary for different types of biomaterials. Here, we report on protein interactions with β-sheet peptide nanofibers with tunable immobilized carbohydrate content, which were prepared by co-assembling QQKFQFQFEQQ (Q11) with a glycosylated variant modified with N-acetylglucosamine (GQ11) at different molar ratios. The rate of protein binding increased as carbohydrate density decreased, with nanofibers having a GQ11 : Q11 molar ratio of 1 : 3 reaching equilibrium faster than formulations with a GQ11 mole fraction of 1. Larger proteins demonstrated a lower extent of binding than smaller proteins; however, the optimal range of carbohydrate densities was independent of the protein size. Nanofibers with the highest apparent protein binding affinity inhibited T cell death induced by wheat germ agglutinin (WGA) more effectively than did sub-optimal formulations, because they bound more protein within biologically relevant time frames (min to h). Collectively, these observations suggest that tuning carbohydrate density via co-assembly of glycosylated and non-glycosylated Q11 variants can maximize multivalent avidity effects while minimizing steric penalties. We anticipate that this approach will enable rapid iterative development of biomaterials with optimal activity for inhibiting the protein-glycan interactions implicated in disease progression.