Abstract
Intracellular GlcNAcylation of Ser and Thr residues is a well-known and widely investigated post-translational modification. This post-translational modification has been shown to play a significant role in cell signaling and in many regulatory processes within cells. O-GlcNAc transferase is the enzyme responsible for glycosylating cytosolic and nuclear proteins with a single GlcNAc residue on Ser and Thr side-chains. Here we report that the same enzyme may also be responsible for S-GlcNAcylation, i.e. for linking the GlcNAc unit to the peptide by modifying a cysteine side-chain. We also report that O-GlcNAcase, the enzyme responsible for removal of O-GlcNAcylation does not appear to remove the S-linked sugar. Such Cys modifications have been detected and identified in mouse and rat samples. This work has established the occurrence of 14 modification sites assigned to 11 proteins unambiguously. We have also identified S-GlcNAcylation from human Host Cell Factor 1 isolated from HEK-cells. Although these site assignments are primarily based on electron-transfer dissociation mass spectra, we also report that S-linked GlcNAc is more stable under collisional activation than O-linked GlcNAc derivatives.
Highlights
Protein glycosylation is one of the most frequently occurring post-translational modifications
O-glycoproteins belong to two distinct categories: GlcNAcylation of Ser and Thr residues occurs within the cytoplasm or nucleus [3, 5]; proteins destined for secretion or presentation on the cell membrane are modified in the ER and the Golgi, where GalNAc, Xyl, Fuc, Glc, or Man may be directly attached onto hydroxy amino acid side-chains [3]
S-linked GlcNAcylation seems to be more stable than O-linked GlcNAcylation as illustrated with the fragmentation of an O-GlcNAcylated peptide of identical amino acid sequence (Fig. 2B)
Summary
Purification and Proteolytic Digestion of Mouse Synaptic Membranes—Isolation and proteolytic digestion of the mouse synaptosome was performed as described previously [20]. Glycopeptides collected from all 10 mg of total peptides were eluted with 50% acetonitrile 0.1% formic acid in a single 500 l fraction. The required mass accuracy was 20 ppm for precursor ions, and 30 ppm or 0.8 Da for HCD and ETD fragments, respectively. A new mouse synaptosome mixture was analyzed after glycopeptide enrichment in LC/MS EThcD mode on an Orbitrap Fusion Lumos Tribrid Mass Spectrometer in 1 h gradient fractionation/ data acquisition. In this experiment both the precursor and fragment ions were measured in the Orbitrap. The database searches were performed as described above but with a 10 ppm and 20 ppm mass accuracy requirement for the precursors and fragments, respectively. When generating the XIC chromatogram for the comparison of the relative amounts of S- and OGlcNAcylated sequences the monoisotopic masses Ϯ20 ppm were extracted from the MS1 data
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