Discovering Glycosylation-Dependent Protein Function by Thermal Proteome Profiling.

Protein glycosylation regulates essential cellular processes including protein folding, stability, and cell-cell interactions; however, how aberrant glycosylation impacts protein function and interaction networks remains poorly understood. Here we combine mass spectrometry-based proteomics, chemical glycobiology, and molecular dynamics simulations to systematically investigate glycosylation dependent protein stability and function. By perturbing the secretory pathway at defined steps, we generated proteins with distinct glycan structures and analyzed their functional consequences using thermal proteome profiling. This approach revealed that cells mount convergent stress responses to diverse glycosylation perturbations, characterized by coordinated trafficking reorganization. This reorganization effectively redirects protein flux from secretion toward degradation. We further identified that perturbing terminal glycan modifications, particularly sialylation and fucosylation, exerts the most profound effects on cell surface protein function. We studied in detail how loss of fucosylation restricts the conformational dynamics of key domains in integrin alpha 4, reducing VCAM-1 binding affinity and suggesting a potential therapeutic strategy for multiple sclerosis. Overall, our systematic approach uncovered extensive complexity in how glycosylation regulates protein function beyond simple glycoform identification and provides a resource for dissecting essential glycan-protein relationships.