Haltiwanger R S, Grove K, Philipsberg G A
Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, State University of New York, Stony Brook, New York 11794-5215, USA.
J Biol Chem. 1998 Feb 6;273(6):3611-7. doi: 10.1074/jbc.273.6.3611.
O-Linked N-acetylglucosamine (O-GlcNAc) is a ubiquitous and abundant post-translational modification found on nuclear and cytoplasmic proteins and is thought to be a dynamically regulated modification much like phosphorylation. In this study we have demonstrated that O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbama te (PUGNAc), a potent in vitro inhibitor of the enzyme responsible for the removal of O-GlcNAc from proteins (peptide O-GlcNAc-beta-N-acetylglucosaminidase), can be used to increase O-GlcNAc levels on nuclear and cytoplasmic proteins in vivo. Overall, PUGNAc caused approximately a 2-fold increase in O-GlcNAc levels in the human colon cancer cells, HT29, although the effects on individual 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 affected by the treatment. O-GlcNAc levels in other cell lines tested (NIH 3T3, CV-1, and HeLa) were also affected by PUGNAc, although the effects on HeLa cells were minimal. At the concentrations tested, PUGNAc was non-toxic and had no affect on the growth rate of any of the cell lines examined. Interestingly, we demonstrated that an increase in O-GlcNAc levels on the transcription factor Sp1 resulted in a reciprocal decrease in its level of phosphorylation, supporting the hypothesis that O-GlcNAc competes with phosphate on some proteins. These studies demonstrate that PUGNAc is an effective inhibitor of O-GlcNAc turnover within cells and can be used to selectively alter the extent of O-GlcNAc on cellular proteins.
O-连接的N-乙酰葡糖胺（O-GlcNAc）是一种在核蛋白和胞质蛋白上普遍存在且含量丰富的翻译后修饰，被认为是一种像磷酸化一样受到动态调节的修饰。在本研究中，我们证明了O-(2-乙酰氨基-2-脱氧-D-吡喃葡糖亚基)氨基-N-苯基氨基甲酸酯（PUGNAc），一种负责从蛋白质上去除O-GlcNAc的酶（肽O-GlcNAc-β-N-乙酰葡糖胺酶）的有效体外抑制剂，可用于在体内增加核蛋白和胞质蛋白上的O-GlcNAc水平。总体而言，PUGNAc使人类结肠癌细胞HT29中的O-GlcNAc水平增加了约2倍，尽管对个别蛋白质的影响有所不同。这种增加似乎是肽O-GlcNAc-β-N-乙酰葡糖胺酶直接受到抑制的结果，因为O-GlcNAc转移酶和UDP-GlcNAc水平均未受到该处理的影响。测试的其他细胞系（NIH 3T3、CV-1和HeLa）中的O-GlcNAc水平也受到PUGNAc的影响，尽管对HeLa细胞的影响最小。在所测试的浓度下，PUGNAc无毒，对所检查的任何细胞系的生长速率均无影响。有趣的是，我们证明转录因子Sp1上O-GlcNAc水平的增加导致其磷酸化水平的相应降低，支持了O-GlcNAc在某些蛋白质上与磷酸竞争的假说。这些研究表明，PUGNAc是细胞内O-GlcNAc周转的有效抑制剂，可用于选择性地改变细胞蛋白质上O-GlcNAc的程度。
