O-linked N-acetylgalactosamine modification is present on the tumor suppressor p53

• Published in: Biochimica et biophysica acta. General subjects Vol. 1864; no. 8; p. 129635
• Main Authors: Xu, Zhijue, Ku, Xin, Tomioka, Azusa, Xie, Wenxian, Liang, Tao, Zou, Xia, Cui, Yalu, Sato, Takashi, Kaji, Hiroyuki, Narimatsu, Hisashi, Yan, Wei, Zhang, Yan
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2020
• Subjects: Acetylgalactosamine - metabolism; Apoptosis - genetics; Cells, Cultured; enzymatic treatment; GalNAz-metabolic labeling; Glycosylation; HEK293 Cells; Humans; lectins; Mass Spectrometry; neoplasms; Nucleocytoplasmic protein; O-GalNAc glycosylation; p53; Polysaccharides - analysis; Polysaccharides - metabolism; site-directed mutagenesis; transferases; Tumor Suppressor Protein p53 - genetics; Tumor Suppressor Protein p53 - metabolism; UDP-Gal/UDP-GalNAc 4-epimerase; GalNAz-metabolic labeling; VVA; O-GalNAc glycosylation; PNA; GalNAz; DSS; OGA; Dox; UDP-Gal/UDP-GalNAc 4-epimerase; p53; GalNAc; CHX; GALE; ppGalNAc-T; Gal; C1GalT1; CRISPR-Cas9; HPA; GlcNAc; TBTA; Nucleocytoplasmic protein
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2020.129635

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. •The O-GalNAc glycosylation was a novel modification on the tumor suppressor p53.•Ser121 residue was one of O-GalNAc glycosylated sites on p53 and was associated with the stability and activity of p53.•This study provided a pilot evidence that O-GalNAc glycosylation existed on nucleocytoplasmic protein.