Mammalian cell proliferation requires noncatalytic functions of O-GlcNAc transferase

Abstract

O-GlcNAc transferase (OGT), found in the nucleus and cytoplasm of all mammalian cell types, is essential for cell proliferation. Why OGT is required for cell growth is not known. OGT performs two enzymatic reactions in the same active site. In one, it glycosylates thousands of different proteins, and in the other, it proteolytically cleaves another essential protein involved in gene expression. Deconvoluting OGT’s myriad cellular roles has been challenging because genetic deletion is lethal; complementation methods have not been established. Here, we developed approaches to replace endogenous OGT with separation-of-function variants to investigate the importance of OGT’s enzymatic activities for cell viability. Using genetic complementation, we found that OGT’s glycosyltransferase function is required for cell growth but its protease function is dispensable. We next used complementation to construct a cell line with degron-tagged wild-type OGT. When OGT was degraded to very low levels, cells stopped proliferating but remained viable. Adding back catalytically inactive OGT rescued growth. Therefore, OGT has an essential noncatalytic role that is necessary for cell proliferation. By developing a method to quantify how OGT’s catalytic and noncatalytic activities affect protein abundance, we found that OGT’s noncatalytic functions often affect different proteins from its catalytic functions. Proteins involved in oxidative phosphorylation and the actin cytoskeleton were especially impacted by the noncatalytic functions. We conclude that OGT integrates both catalytic and noncatalytic functions to control cell physiology.

Truncation of the TPR domain of OGT alters substrate and glycosite selection

“…These data could serve to increase glycosylation on desired substrates and influence glycosite selection while minimizing perturbations to the cell for O-GlcNAc engineering, in addition to the biological implications arising from differential substrate and site selection of the OGT TPR isoforms. The relationship between the TPR isoforms of OGT and biological function is a growing area of investigation as interactions and substrate selection through the TPR domain may be of particular relevance to normal O-GlcNAc regulation [6,10,31,48]. Comparison of glycoproteins in the presence of RFP(13) to a recently determined TPR interactome of ncOGT via a proximity-labeling approach [24] shows enrichment for similar biological processes, indicating that the TPR interactome and the enriched O-GlcNAcylated proteome correspond.…”

“…Due to the essential requirement of OGT for substrate O-GlcNAcylation [10], deciphering the basis of OGT's substrate selection mechanisms has attracted much interest [2,11,12]. The canonical nucleocytoplasmic form of OGT (ncOGT) is composed of two domains: an N-terminal tetratricopeptide repeat (TPR) domain and a C-terminal catalytic domain (Fig.…”

“…1A) [16][17][18]. Truncation of the TPR domain affects the OGT interactome [19][20][21][22][23][24] which may provide a molecular basis for observed cellular signaling and proliferation effects [10,25,26] or for human diseases associated with mutations in the TPR domain, such as X-linked intellectual disability (XLID) [6,[27][28][29][30]. The relationship of these OGT isoforms to substrate and glycosite selection has been explored in vitro, where variation of the TPR domain alters substrate selectivity and truncation of the TPR domain as in sOGT limits the resulting glycosyltransferase activity [11,18,23].…”

Diabetic Embryopathy Susceptibility in Mice Is Associated with Differential Dependence on Glucosamine and Modulation of High Glucose-Induced Oxidative Stress

“…There have been some reports that stimulation of O -GlcNAc transferase, which catalyzes O -linked glycosylation of serine and threonine residues with UDP-N-Acetyl-GlcN, by GlcN promotes proliferation of murine ESC [ 30 , 31 , 32 ]. There was also a recent report that proliferation of mouse embryo fibroblasts (MEFs) requires noncatalytic functions of O -GlcNAc transferase [ 33 ]. However, the ESC and MEF lines used in those studies were isolated under conventional high glucose conditions and would not express GLUT2, and the ESC were treated with GlcN at concentrations that would be transported by GLUT1 [ 17 ].…”

The Interplay Between Prostate Cancer Genomics, Metabolism, and the Epigenome: Perspectives and Future Prospects

“…However, that alone is unlikely to provide an understanding of crosstalk; we need to establish more clearly how OGT selects protein substrates, and we also need to account for a second enzymatic activity ascribed to OGT, its proteolytic function. A recent study has indicated that in some cell types, OGT can sustain cell proliferation through a non-catalytic function that needs to be fully characterised (109). This in turn also needs to be put into a spatial context, since OGT can function as different isoforms in distinct organelles within the cell, mitochondria, and nuclei being principal examples.…”