BACKGROUND Mucin-type O-glycosylation (referred to as O-GalNAc glycosylation) is the most abundant O-glycosylation on membrane and secretory proteins. Recently several evidences suggest that nuclear or cytoplasmic proteins might also have O-GalNAc glycosylation. However, what nucleocytoplasmic proteins are O-GalNAc glycosylated and what the biological function of this modification in cells are still poorly understood. Previously, we reported the tumor suppressor p53 could be O-GalNAc glycosylated in vitro. To investigate the existence and function of O-GalNAc glycosylation on nucleocytoplasmic proteins in cell, p53 as a representative nucleocytoplasmic protein was studied. METHODS Using lectin blotting with GalNAc specific lectins, enzymatic treatments with O-GlcNAcase, core 1 β1, 3-galactosyltransferase and O-glycosidase, and metabolic labeling with un-O-acetylated GalNAz in UDP-Gal/UDP-GalNAc 4-epimerase (GALE) knockout cells, we validated the O-GalNAc glycosylation on p53. Using mass spectrometry analysis and site-directed mutagenesis, we identified the glycosylated sites and studied the functions of O-GalNAc glycosylation on p53. RESULTS The p53 was O-GalNAc glycosylated in cells. Ser121 residue was one of the glycosylated sites on p53. The O-GalNAc glycosylation at Ser121 was associated with the stability and activity of p53. CONCLUSIONS These results revealed that the O-GalNAc glycosylation was a novel modification on p53. GENERAL SIGNIFICANCE Our study provided a pilot evidence that the O-GalNAc glycosylation existed on nucleocytoplasmic protein.

中文翻译：

---

O-联的N-乙酰半乳糖胺修饰存在于肿瘤抑制因子p53上。

背景技术粘蛋白型O-糖基化（称为O-GalNAc糖基化）是膜和分泌蛋白上最丰富的O-糖基化。最近，一些证据表明核蛋白或胞质蛋白也可能具有O-GalNAc糖基化作用。然而，仍然不清楚O-GalNAc被糖基化的是什么胞质蛋白，以及这种修饰在细胞中的生物学功能是什么。以前，我们报道肿瘤抑制因子p53可能在体外被O-GalNAc糖基化。为了研究O-GalNAc糖基化在细胞核蛋白上的存在和功能，研究了以p53为代表的核蛋白。方法：使用GalNAc特异性凝集素进行凝集素印迹，用O-GlcNAcase，核心1 β1、3-半乳糖基转移酶和O-糖苷酶进行酶处理，并在敲除UDP-Gal / UDP-GalNAc 4-表异构酶（GALE）的细胞中用非O-乙酰化GalNAz进行代谢标记，我们验证了p53上的O-GalNAc糖基化。使用质谱分析和定点诱变，我们确定了糖基化的位点，并研究了O-GalNAc糖基化对p53的功能。结果p53在细胞中被O-GalNAc糖基化。Ser121残基是p53上糖基化的位点之一。Ser121处的O-GalNAc糖基化与p53的稳定性和活性有关。结论这些结果表明，O-GalNAc糖基化是对p53的新修饰。一般意义我们的研究提供了一个初步证据，表明核质蛋白上存在O-GalNAc糖基化。我们验证了p53的O-GalNAc糖基化。使用质谱分析和定点诱变，我们确定了糖基化的位点，并研究了O-GalNAc糖基化对p53的功能。结果p53在细胞中被O-GalNAc糖基化。Ser121残基是p53上糖基化的位点之一。Ser121处的O-GalNAc糖基化与p53的稳定性和活性有关。结论这些结果表明，O-GalNAc糖基化是对p53的新修饰。一般意义我们的研究提供了一个初步证据，表明核质蛋白上存在O-GalNAc糖基化。我们验证了p53上的O-GalNAc糖基化。使用质谱分析和定点诱变，我们确定了糖基化的位点，并研究了O-GalNAc糖基化对p53的功能。结果p53在细胞中被O-GalNAc糖基化。Ser121残基是p53上糖基化的位点之一。Ser121处的O-GalNAc糖基化与p53的稳定性和活性有关。结论这些结果表明，O-GalNAc糖基化是对p53的新修饰。一般意义我们的研究提供了一个初步证据，表明核质蛋白上存在O-GalNAc糖基化。结果p53在细胞中被O-GalNAc糖基化。Ser121残基是p53上糖基化的位点之一。Ser121处的O-GalNAc糖基化与p53的稳定性和活性有关。结论这些结果表明，O-GalNAc糖基化是对p53的新修饰。一般意义我们的研究提供了一个初步证据，表明核质蛋白上存在O-GalNAc糖基化。结果p53在细胞中被O-GalNAc糖基化。Ser121残基是p53上糖基化的位点之一。Ser121处的O-GalNAc糖基化与p53的稳定性和活性有关。结论这些结果表明，O-GalNAc糖基化是对p53的新修饰。一般意义我们的研究提供了一个初步证据，表明核质蛋白上存在O-GalNAc糖基化。