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.

O-GlcNAc Transferase – An Auxiliary Factor or a Full-blown Oncogene?

“…To test this, cancer-associated OGT mutations can be modeled to existing crystal structures to select the most interesting once for functional studies using in vitro enzyme assays. The ultimate experiment to test if OGT mutations promote tumorigenesis requires replacement of the endogenous OGT with a mutant version, which could be achieved using the degron-tagged OGT cell lines recently described by (66). Systematic analysis of protein-protein interaction surfaces revealed that the interface between OGT and HCF-1 is a mutation hotspot (67).…”

“…Overall, forced overexpression of OGT promotes the malignant phenotype of cancer cells but in the molecular level, it is not known what the key mediators of this response are. In many instances, OGT behaves similar to metabolic genes: it is critical for tumor growth but may also be essential for the functions of the normal metabolically active cells for example by affecting mitochondrial respiration (66,84,85). When evaluating these results, it is important to note possible context-dependency.…”

A broadly active fucosyltransferase LmjFUT1 whose mitochondrial localization and catalytic activity is essential in the parasitic protozoan Leishmania

“…Fucosyltransferase activity of FUT1 is required for the essential function. We next tested whether the essential function of FUT1 required fucosyltransferase activity, perhaps through a structural role (50). We mutated the first arginine to alanine (R297A) in the FUT1 motif I (Fig.…”

O-GlcNAcylation reduces phase separation and aggregation of the EWS N-terminal low complexity region

“…O -linked β-D- N -acetylglucosaminylation (‘ O -GlcNAc’ylation) is a form of glycosylation occurring in the nucleus, mitochondria and cytoplasm of higher eukaryotic cells 11, 12 . Distinct from the often polymeric and structurally complex extracellular glycans, intracellular O -GlcNAcylation entails the reversible modification of serine and threonine O -hydroxyl groups with a single GlcNAc moiety.…”