Covalent crosslinking of mucins: From biomaterial design to tailored functions

Mucins are high-molecular-weight glycoproteins essential for the hydration, lubrication, and protective barrier functions of epithelial surfaces. Beyond these physical properties, their dense O-glycan brushes serve as potent multivalent ligands that regulate immune communication through interactions with sialic acid-binding immunoglobulin-like lectins (Siglecs) and other glycan-binding receptors. The translation of native mucins into functional biomaterials is often hindered by the mechanical instability of noncovalent assemblies, and investigations of mucin biology can require well-defined model mucin materials with controlled properties. This chapter presents a detailed protocol for domain-specific covalent crosslinking, a strategy to engineer mucin-based hydrogels with controlled architecture and bioactivity. By selectively targeting either the protein backbone (via carbodiimide/NHS coupling of bioorthogonal handles) or the glycan side chains (via mild periodate oxidation and oxyamine or reductive amination conjugation), the spatial arrangement and mesh architecture of the resulting hydrogel network can be dictated. The two crosslinking approaches yield materials with distinct gelation kinetics, protease susceptibility, pore structure, and immune-modulatory properties, while preserving the bioactive sialic acid residues critical for receptor engagement. The protocol covers the full workflow from mucin dissolution and chemical modification through purification, gelation, and characterization by NMR, HPLC-PAD, and rheometry. By providing a detailed roadmap for manipulating mucin macromolecular architecture, this chapter enables both the design of instructive biomaterials for drug delivery and tissue engineering, and the construction of well-defined model glycoprotein systems for fundamental research on glycan-mediated biology.