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.
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Author contributions: J.C.M., A.L.B., and K.F.M. designed research; J.C.M. performed research; J.C.M. and K.F.M. analyzed data; J.C.M. and K.F.M. wrote the paper.
This work was supported by NIH grant NIGMS 8P41GM103481, the Howard Hughes Medical Institute and the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
