Yaohao Li

A concise divergent strategy affords 10 core-1 type O -linked glycoamino acid building blocks. Their availability enables homogeneous GHRH glycoform preparation and reveals that Thr ⁷ O -glycosylation improves proteolytic stability against DPP-IV.

Post-translational modifications such as clustered O-glycosylation are frequently enriched in intrinsically disordered regions (IDRs), yet their roles in shaping molecular behavior and functional specificity remain poorly understood. Here, we addressed this challenge through an integrative, stepwise strategy that combines chemical synthesis, experimental characterization, and molecular dynamics (MD) simulations, employing the extensively O-mannosylated linker of Trichoderma reesei Family 7 cellobiohydrolase as a model IDR. Using synthetically accessible linker glycoforms bearing monomannose residues, we showed that unglycosylated and partially glycosylated linkers strongly prefer lignin over cellulose, driven by electrostatic interactions with the negatively charged lignin surface. Increasing glycan density or glycan size, such as through the introduction of dimannose, weakens lignin binding and partially shifts binding preference toward cellulose. Importantly, MD simulations informed and validated by these experimental data enabled prediction of currently synthetically inaccessible glycoforms, revealing that negatively charged glycans, such as phosphorylated mannose, effectively abolish lignin affinity while largely preserving cellulose binding. This work provides a generalizable and predictive framework for elucidating how complex glycosylation patterns regulate IDR-ligand interactions and identifies charge-mediated modulation as a key mechanism through which clustered O-glycosylation tunes IDR function.

Protein and peptide aggregation poses substantial challenges in disease pathology and therapeutic development. While natural glycosylation may mitigate aggregation, its efficacy and underlying mechanisms remain poorly understood due to limited access to homogeneous samples with complex glycans. This study addresses these knowledge gaps by investigating the natural glycosylation of islet amyloid polypeptide (IAPP), a peptide with therapeutic potential for type 2 diabetes but problematic aggregation. An optimized chemical synthesis enabled preparation of diverse IAPP glycoforms with complex glycan structures, allowing systematic evaluation of their effects on aggregation, cytotoxicity, and solubility. Sialylated glycans at Thr ³⁰ completely inhibited IAPP aggregation, eliminated cytotoxicity toward pancreatic β cells, and enhanced solubility by up to 280-fold. Replica exchange molecular dynamics simulations revealed that glycosylation impedes adoption of a four-stranded β-sheet conformation in IAPP dimers. These findings advance the understanding of the role of natural glycosylation in aggregation and highlight its potential as an evolutionarily inspired strategy to enhance the therapeutic utility of IAPP.

Glycosylation plays a critical role in modulating protein and peptide properties, yet the impact of recently discovered natural mucin-type O-glycosylation on therapeutic peptides like glucagon remains underexplored due to challenges in obtaining homogeneous research samples. Here, we address this challenge by developing a streamlined multistep synthesis-one purification protocol, enabling the production of 21 glucagon glycoforms with systematically varied glycosylation patterns. Investigation of these glycoforms revealed a remarkable impact of natural glycosylation on two properties critical for glucagon: solubility increased by over 870-fold, and fibrillation was completely inhibited, even under stringent conditions, while biological activity in elevating blood glucose levels was preserved. Computational analyses indicated that sialylated glycans disrupt intermolecular associations in the solid state, enhancing solubility, and shift secondary structures from β-sheets to α-helices, mitigating fibrillation. These findings suggest that glycosylation may have evolved as a natural mechanism to optimize undesirable properties, offering valuable insights for the development of therapeutics based on glucagon and other proteins and peptides with similar challenges.

This study focused on the CDMBI-mediated synthesis of GlcNAc oxazoline.

Therapeutic proteins have unique advantages over small-molecule drugs in the treatment of various diseases, such as higher target specificity, stronger pharmacological efficacy and relatively low side effects. These advantages make them increasingly valued in drug development and clinical practice. However, although highly valued, the intrinsic limitations in their physical, chemical and pharmacological properties often restrict their wider applications. As one of the most important post-translational modifications, glycosylation has been shown to exert positive effects on many properties of proteins, including molecular stability, and pharmacodynamic and pharmacokinetic characteristics. Glycoengineering, which involves changing the glycosylation patterns of proteins, is therefore expected to be an effective means of overcoming the problems of therapeutic proteins. In this review, we summarize recent efforts and advances in the glycoengineering of erythropoietin and IgG monoclonal antibodies, with the goals of illustrating the importance of this strategy in improving the performance of therapeutic proteins and providing a brief overview of how glycoengineering is applied to protein-based drugs.

Improved understanding of the effect of protein glycosylation is expected to provide the foundation for the design of protein glycoengineering strategies. In this study, we examine the impact of O-glycosylation on the binding selectivity of a model Family 1 carbohydrate-binding module (CBM), which has been shown to be one of the primary sub-domains responsible for non-productive lignin binding in multi-modular cellulases. Specifically, we examine the relationship between glycan structure and the binding specificity of the CBM to cellulose and lignin substrates. We find that the glycosylation pattern of the CBM exhibits a strong influence on the binding affinity and the selectivity between both cellulose and lignin. In addition, the large set of binding data collected allows us to examine the relationship between binding affinity and the correlation in motion between pairs of glycosylation sites. Our results suggest that glycoforms displaying highly correlated motion in their glycosylation sites tend to bind cellulose with high affinity and lignin with low affinity. Taken together, this work helps lay the groundwork for future exploitation of glycoengineering as a tool to improve the performance of industrial enzymes.