Explorations of N- and O-linked Glycosylation on Extracellular and Intracellular Cell Surfaces

Explorations of N- and O-linked Glycosylation on Extracellular and Intracellular Cell Surfaces

N- and O-linked glycosylation is often the key to inter- and intracellular signaling, as well as the entry of pathogens and toxins. Mass spectrometry and complementary approaches are powerful tools for the determination of the locations and specificity of such interactions. This can help to understand normal and aberrant pathways, and guide development of therapeutic interventions. Representative examples, including the following, will be discussed.

Mutation of one N-glycosylation site near the Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) ligand binding site quickly amplified its ligand-dependent phosphorylation (activation) at specific sites, and resulted in higher levels of phospho-Src compared to wild type VEGFR-2, which may have implications for endothelial cell proliferation and migration, whereas mutations at nearby N-glycosylation sites did not appear to impact ligand-mediated activation of VEGFR-2.

At specific N-glycosylation sites, Epidermal Growth Factor Receptor (EGFR) from indolent CAL27 cells had highly fucosylated N-glycans, whereas the corresponding N-linked glycans on EGFR from metastatic HSC-3 cells displayed much lower levels of fucosylation. Our MS/MS data showed that treatment with a drug candidate promoted modification of HSC-3 glycans with terminal fucose, potentially inhibiting EGFR signaling. The results suggest inhibition of β-catenin/CBP signaling as a therapeutic approach to downregulate EGFR pro-tumorigenic activity.

Surprising results obtained during recent studies revealed that highly fucosylated proteins are present adjacent to the nucleus of cells in the pathogens Cryptosporidium and T. gondii and related organisms. These unusual protein modifications, which apparently arose via the kidnapping of plant genes, may represent targets for drug development.

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