Key Laboratory of Systems Biomedicine (Ministry of Education) and Collaborative Innovation Center of Systems Biomedicine, Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China.
Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8568, Japan.
Biochim Biophys Acta Gen Subj. 2020 Aug;1864(8):129635. doi: 10.1016/j.bbagen.2020.129635. Epub 2020 May 14.
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.
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.
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.
These results revealed that the O-GalNAc glycosylation was a novel modification on p53.
Our study provided a pilot evidence that the O-GalNAc glycosylation existed on nucleocytoplasmic protein.
粘蛋白型 O-糖基化（简称 O-GalNAc 糖基化）是膜蛋白和分泌蛋白上最丰富的 O-糖基化。最近有几项证据表明，核内或细胞质蛋白也可能有 O-GalNAc 糖基化。然而，哪些核质蛋白被 O-GalNAc 糖基化，以及这种修饰在细胞中的生物学功能仍知之甚少。此前，我们报道肿瘤抑制因子 p53 在体外可以被 O-GalNAc 糖基化。为了研究细胞中核质蛋白上 O-GalNAc 糖基化的存在和功能，我们以 p53 作为代表性核质蛋白进行了研究。
使用 GalNAc 特异性凝集素的凝集素印迹、O-GlcNAcase、核心 1 β1,3-半乳糖基转移酶和 O-糖苷酶的酶处理、以及在 UDP-Gal/UDP-GalNAc 4-差向异构酶（GALE）敲除细胞中用未-O-乙酰化的 GalNAz 进行代谢标记，我们验证了 p53 上的 O-GalNAc 糖基化。通过质谱分析和定点突变，我们确定了糖基化位点，并研究了 p53 上 O-GalNAc 糖基化的功能。
p53 在细胞中被 O-GalNAc 糖基化。Ser121 残基是 p53 上的糖基化位点之一。p53 上 Ser121 的 O-GalNAc 糖基化与 p53 的稳定性和活性有关。
这些结果表明，O-GalNAc 糖基化是 p53 上的一种新型修饰。
我们的研究提供了一个初步证据，表明 O-GalNAc 糖基化存在于核质蛋白上。
