Global and Site-Specific Analysis Revealing Unexpected and Extensive Protein S‑GlcNAcylation in Human Cells

Protein glycosylation
is highly diverse and essential for mammalian
cell survival. Heterogeneous glycans may be bound to different amino
acid residues, forming multiple types of protein glycosylation. In
this work, unexpected protein S-GlcNAcylation on cysteine residues
was observed to extensively exist in human cells through global and
site-specific analysis of protein GlcNAcylation by mass spectrometry.
Three independent experiments produced similar results of many cysteine
residues bound to N-acetylglucosamine (GlcNAc). Among
well-localized S-GlcNAcylation sites, several motifs with an acidic
amino acid around the sites were identified, which strongly suggests
that a particular type of enzyme is responsible for this modification.
Clustering results show that glycoproteins modified with S-GlcNAc
are mainly involved in cell–cell adhesion and gene expression.
For the first time, we found that proteins were extensively bound
to GlcNAc through the side chains of cysteine residues in human cells,
and the current discovery further advances our understanding of protein
glycosylation.