Abstract
Biological functions of nuclear proteins are regulated by post-translational modifications (PTMs) that modulate gene expression and cellular physiology. However, the role of O-linked glycosylation (O-GalNAc) as a PTM of nuclear proteins in the human cell has not been previously reported. Here, we examined in detail the initiation of O-GalNAc glycan biosynthesis, representing a novel PTM of nuclear proteins in the nucleus of human cells, with an emphasis on HeLa cells. Using soluble nuclear fractions from purified nuclei, enzymatic assays, fluorescence microscopy, affinity chromatography, MS, and FRET analyses, we identified all factors required for biosynthesis of O-GalNAc glycans in nuclei: the donor substrate (UDP-GalNAc), nuclear polypeptide GalNAc -transferase activity, and a GalNAc transferase (polypeptide GalNAc-T3). Moreover, we identified O-GalNAc glycosylated proteins in the nucleus and present solid evidence for O-GalNAc glycan synthesis in this organelle. The demonstration of O-GalNAc glycosylation of nuclear proteins in mammalian cells reported here has important implications for cell and chemical biology.
Highlights
The nucleus is one of the most important structures of eukaryotic cells
OGT acts as an epigenetic “writer,” whereas O-GlcNAcase acts as an “eraser” of this posttranslational modifications (PTMs) by modulating biological activity of relevant nuclear proteins such as histones, RNA polymerase II, and transcription factors
We examined O-GalNAc glycan biosynthesis in the nucleus of human cells as a PTM of nuclear proteins that may play an important role in regulating their functions
Summary
To examine nuclear localization of several molecules, we purified HeLa cell nuclei as described previously [15]. To examine the presence of this sugar donor in the nucleus, we developed an enzymatic assay for measurement of UDP-GalNAc substrate, in which the standard curve showed an appropriate linear concentration range (Fig. S1A). High specificity of this enzymatic method for UDP-GalNAc was demonstrated by comparison with UDP-GlcNAc, which was found not to participate in the reaction. When nuclei were added with UDP-GalNAc, WB analysis with detection by WGA did not recognize an increase in O-glycosylation level These findings indicate a clear distinction between initiation of O-GalNAc versus O-GlcNAc glycosylation of proteins in purified nuclei.
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