Glycoengineering strategies for constructing defined Mucin O-glycans

Mucins are densely O-glycosylated proteins that establish mucosal barriers, mediate immune recognition, and serve as ligands for host lectins and microbes. However, mucin O-glycan structure-function relationships remain poorly defined due to inherent glycoform heterogeneity. Glycoengineering has emerged as a suite of methodologies enabling construction of mucins and mucin-type glycoconjugates with defined O-glycan structures suitable for rigorous biochemical and biophysical investigation. Here, we evaluate three glycoengineering approaches from a practical methodology perspective, emphasizing analytical validation requirements and method selection criteria. Cellular glycoengineering exploits genetic reconstruction of glycosylation pathways to produce simplified glycoforms in living cells, offering scalability and native protein context but limited homogeneity. Chemoenzymatic synthesis employs purified or cell-free-derived glycosyltransferases for sequential in vitro assembly with precise regiochemical and stereochemical control, though scalability remains constrained. Synthetic mucin mimetics utilize polymer chemistry to decouple glycan presentation from protein backbone constraints, enabling systematic variation of valency, density, and spacing. We discuss analytical validation workflows for each approach, and highlight emerging tools that may address current limitations in scalability and structural complexity. Together, we provide a decision framework that aligns experimental objective with the most appropriate glycoengineering platform.