A Chemoenzymatic Method To Systematically Quantify Core Fucosylation Stoichiometry of Glycoproteins and Reveal Its Roles in EMT and Embryonic Development

TL;DR

This paper introduces a new method to measure core fucosylation levels in glycoproteins and shows how these levels vary in different cell compartments and during development and cancer.

Contribution

A chemoenzymatic method to systematically quantify core fucosylation stoichiometry in glycoproteins.

Findings

Core fucosylation levels vary significantly across subcellular compartments, with the lowest in lysosomes and highest in the extracellular matrix.

Glycosylation sites with more aromatic and hydrophobic residues show lower core fucosylation stoichiometry.

Core fucosylation changes during EMT and is higher in embryonic kidney cells compared to kidney cancer cells.

Abstract

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.

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