Biosynthesis of O-N-acetylgalactosamine glycans in the human cell nucleus

Abstract

Biological functions of nuclear proteins are regulated by posttranslational 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. The nucleus is one of the most important structures of eukaryotic cells. This complex organelle stores the chromosomes and also regulates their duplication, segregation, repair, and expression through a series of specific processes. The cell's biological information is saved and transferred within the nucleus by three types of biopolymer molecules: DNA, RNA, and proteins (1). Proteins play crucial roles in nuclear scaffolding, DNA assembly, replication, transcription, and transport of molecules. The biological activity of proteins is directly modulated by their conformation, and changes in protein conformation are controlled mainly by post-translational modifications (PTMs). 3 The common PTMs of nuclear proteins are acetyla-This work was supported by CONICET Grant PIP 11220150100226 and SeCyT, UNC Grant 05/C422 (to F. J. I.). The authors declare that they have no conflicts of interest with the contents of this article. This work is dedicated to the memory of Mafalda C. Pellegrini-Irazoqui. This article contains Figs. S1-S5 and Table S1.

Can Glycosylation Mask the Detection of MHC Expressing p53 Peptides by T Cell Receptors?

“…Clearly these studies suggest that glycosylation of epitopes can affect immune recognition, albeit in different ways. Nuclear proteins, such as p53, are known to be glycosylated, particularly with the monosaccharide N-Acetylglucosamine (GlcNAc) [19][20][21][22] and N-Acetylgalactosamine (or GalNAc) [23]. However, we could not find any reports on the role of glycosylation in the presentation and recognition of p53 peptide epitopes.…”

Expression Dynamics of the O-Glycosylated Proteins Recognized by Amaranthus leucocarpus Lectin in T Lymphocytes and Its Relationship With Moesin as an Alternative Mechanism of Cell Activation

“…Under this hypothesis, other interesting questions arise, like if O -moesin is synthesized de novo and directed to the cell membrane, or if the glycan addition to the protein occurs at the cytosol redirecting its translocation towards the cell membrane. The latter would be a rare example where the O -glycosylation of the T antigen type is synthesized in a place other than the endoplasmic reticulum ( 47 ); the answers to these questions are beyond the scope of this work but are proposed as thought-provoking perspectives.…”

A validated collection of mouse monoclonal antibodies to human glycosyltransferases functioning in mucin-type O-glycosylation

“…We therefore suggest that it is likely that most of the pAbs to GalNAc-Ts developed by the Human Protein Atlas consortium, despite the specificity analyses performed, do not reliably identify these GalNAc-Ts in cells and tissues by the applied IC/IH methods. Use of such antibodies may have consequences and provide conflicting results, as recently illustrated by an interesting study suggesting localization of the GalNAc-T3 isoenzyme in the nucleus of HeLa cells (Cejas et al 2019). Although additional data to support the presence of enzyme activity and O-glycoproteins in nucleocytosolic preparations were presented in this study, we note that the mAb to GalNAc-T3 included in our collection has only produced Golgi-like staining in cells including in HeLa (Rottger et al 1998).…”