MODULATION OF O-LINKED N-ACETYLGLUCOSAMINE LEVELS ON NUCLEAR AND CYTOPLASMIC PROTEINS IN-VIVO USING THE PEPTIDE O-GLCNAC-BETA-N-ACETYLGLUCOSAMINIDASE INHIBITOR EOXY-D-GLUCOPYRANOSYLIDENE)AMINO-N-PHENYLCARBAMATE

O-Linked N-acetylglucosamine (O-GlcNAc) is a ubiquitous and abundant p ost-translational modification found on nuclear and cytoplasmic protei ns and is thought to be a dynamically regulated modification much like phosphorylation. In this study we have demonstrated that eoxy-D-gluco pyranosylidene)amino-N-phenylcarbamate (PUGNAc), a potent in vitro inh ibitor of the enzyme responsible for the removal of O-GlcNAc from prot eins (peptide O-GlcNAc-beta-N-acetylglucosaminidase), can be used to i ncrease O-GlcNAc levels on nuclear and cytoplasmic proteins in vivo. O verall, PUGNAc caused approximately a 2-fold increase in O-GlcNAc leve ls in the human colon cancer cells, HT29, although the effects on indi vidual proteins varied, The increase appeared to be the result of the direct inhibition of the peptide O-GlcNAc-beta-N-acetylglucosaminidase since neither the O-GlcNAc transferase nor UDP-GlcNAc levels were aff ected by the treatment. O-GlcNAc levels in other cell lines tested (NI H 3T3, CV-1, and HeLa) were also affected by PUGNAc, although the effe cts on HeLa cells were minimal. At the concentrations tested, PUGNAc w as non-toxic and had no affect on the growth rate of any of the cell l ines examined, Interestingly, we demonstrated that an increase in O-Gl cNAc levels on the transcription factor Sp1 resulted in a reciprocal d ecrease in its level of phosphorylation, supporting the hypothesis tha t O-GlcNAc competes with phosphate on some proteins, These studies dem onstrate that PUGNAc is an effective inhibitor of O-GlcNAc turnover wi thin cells and can be used to selectively alter the extent of O-GlcNAc on cellular proteins.