O-GlcNAc-specific antibody CTD110.6 cross-reacts with N-GlcNAc2-modified proteins induced under glucose deprivation.
O-GlcNAc-specific antibody CTD110.6 cross-reacts with N-GlcNAc2-modified proteins induced under glucose deprivation.
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DOI：
10.1371/journal.pone.0018959
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发表时间：
2011-04-19
期刊：
影响因子：
3.7
通讯作者：
Isono T
中科院分区：
文献类型：
--
作者：
Isono T
O-连接 β-N-乙酰氨基葡萄糖 (O-GlcNAc) 糖基化对蛋白质中丝氨酸和苏氨酸残基的修饰是许多细胞对营养状态和应激反应的一个特征。 O-GlcNAc 修饰受 O-连接 β-N-乙酰氨基葡萄糖转移酶 (OGT) 和 β-D-N-乙酰氨基葡萄糖酶 (O-GlcNAcase) 可逆调节。蛋白质的 O-GlcNAc 修饰取决于尿苷 5'-二磷酸-N-乙酰氨基葡萄糖 (UDP-GlcNAc) 的浓度，UDP-GlcNAc 是 OGT 的底物，通过己糖胺生物合成途径合成。使用 O-GlcNAc 特异性抗体 CTD110.6 进行的免疫印迹分析表明，葡萄糖剥夺会增加某些癌细胞中蛋白质 O-GlcNAc 的酰化。这种矛盾现象的机制仍不清楚。在这里，我们表明，由葡萄糖剥夺诱导并由 CTD110.6 抗体检测到的糖基化增加实际上是由 N-GlcNAc2 修饰，而不是由 O-GlcNAc 修饰。我们发现，与典型的 O-GlcNAc 化不同，这种诱导的糖基化不受 OGT 和 O-GlcNAcase 的调节，并且可以通过用衣霉素（一种 N-糖基化抑制剂）处理来抑制。蛋白质组学分析表明，通过这种诱导糖基化修饰的蛋白质是 N-GlcNAc2 修饰的糖蛋白。此外，CTD110.6 抗体与 N-GlcNAc2 修饰的糖蛋白反应，该糖蛋白由具有 ALG1 ts 突变体的酵母菌株产生，该突变体无法向 dolichol-PP-GlcNAc2 添加甘露糖残基。我们的结果表明，N-GlcNAc2 修饰的糖蛋白在葡萄糖剥夺下被诱导，并且它们与 O-GlcNAc 特异性抗体 CTD110.6 发生交叉反应。因此，我们建议必须重新检查之前根据其与 CTD110.6 抗体的反应性分类为 O-GlcNAc 修饰蛋白的蛋白的糖基化状态。我们还认为，由于 N-GlcNAc2 修饰蛋白水平的增加而抑制成熟的 N 连接糖蛋白是新认识的在压力和剥夺条件下有效利用糖的途径。需要进一步的研究来阐明 N-GlcNAc2 修饰蛋白的生理和病理作用。
Modification of serine and threonine residues in proteins by O-linked β-N-acetylgulcosamine (O-GlcNAc) glycosylation is a feature of many cellular responses to the nutritional state and to stress. O-GlcNAc modification is reversibly regulated by O-linked β-N-acetylgulcosamine transferase (OGT) and β-D-N-acetylgulcosaminase (O-GlcNAcase). O-GlcNAc modification of proteins is dependent on the concentration of uridine 5′-diphospho-N-acetylgulcosamine (UDP-GlcNAc), which is a substrate of OGT and is synthesized via the hexosamine biosynthetic pathway. Immunoblot analysis using the O-GlcNAc-specific antibody CTD110.6 has indicated that glucose deprivation increases protein O-GlcNAcylation in some cancer cells. The mechanism of this paradoxical phenomenon has remained unclear. Here we show that the increased glycosylation induced by glucose deprivation and detected by CTD110.6 antibodies is actually modification by N-GlcNAc2, rather than by O-GlcNAc. We found that this induced glycosylation was not regulated by OGT and O-GlcNAcase, unlike typical O-GlcNAcylation, and it was inhibited by treatment with tunicamycin, an N-glycosylation inhibitor. Proteomics analysis showed that proteins modified by this induced glycosylation were N-GlcNAc2-modified glycoproteins. Furthermore, CTD110.6 antibodies reacted with N-GlcNAc2-modified glycoproteins produced by a yeast strain with a ts-mutant of ALG1 that could not add a mannose residue to dolichol-PP-GlcNAc2. Our results demonstrated that N-GlcNAc2-modified glycoproteins were induced under glucose deprivation and that they cross-reacted with the O-GlcNAc-specific antibody CTD110.6. We therefore propose that the glycosylation status of proteins previously classified as O-GlcNAc-modified proteins according to their reactivity with CTD110.6 antibodies must be re-examined. We also suggest that the repression of mature N-linked glycoproteins due to increased levels of N-GlcNAc2-modifed proteins is a newly recognized pathway for effective use of sugar under stress and deprivation conditions. Further research is needed to clarify the physiological and pathological roles of N-GlcNAc2-modifed proteins.
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