A Chemoenzymatic Method To Systematically Quantify Core Fucosylation Stoichiometry of Glycoproteins and Reveal Its...

Core fucosylation of N-glycoproteins plays pivotal roles in regulating many cellular events such as receptor-ligand binding and cell adhesion. Here, we developed a chemoenzymatic method combining selective enrichment, enzymatic reactions, and multiplexed proteomics to systematically quantify the core fucosylation stoichiometries of glycoproteins in human cells. The results demonstrated that the core fucosylation stoichiometries vary dramatically in different subcellular compartments with the lowest in the lysosome and the highest in the extracellular matrix. Different core fucosylation stoichiometries were observed among glycosylation sites in various protein domains, and more aromatic and hydrophobic residues neighboring glycosylation sites are associated with lower core fucosylation stoichiometry. The method was applied to quantify the core fucosylation stoichiometry changes in the epithelial-to-mesenchymal transition (EMT), and some glycoproteins involved in extracellular matrix organization and ligand recognition displayed marked stoichiometry changes. Furthermore, the core fucosylation stoichiometries in embryonic human kidney cells (HEK293T) were compared with those in kidney cancer cells (A498). The average stoichiometry in HEK293T cells was much higher than that of A498 cells, indicating that core fucosylation may be a critical regulator in embryonic development. Without any sample restriction, this method can be extensively applied to investigate core fucosylation changes in various biological samples.