By Taylor Woetzel
Glycosylation is a widespread post-translational modification in eukaryotic cells. It serves a variety of crucial roles, broadly grouped as structural, metabolic, and information carrying.1 Several main forms of glycosylation exist, with certain linkages predominating among glycoconjugates, which are proteins or lipids linked to a sugar structure. N-linked glycans are oligosaccharide structures linked via a glycosidic bond to the amide group of an asparagine residue within a specific motif (Asn-X-Ser/Thr, where X is any amino acid except proline). On the other hand, O-linked glycans are those linked to the hydroxyl group of a serine or threonine residue.2 The vast majority of glycoconjugates are located at the cell surface, in extracellular space, or in luminal spaces, as glycosylation occurs in the secretory pathway where components travel en route to their destinations at these locations. However, in 1984, an unexpected discovery of nucleocytoplasmic glycosylation was made, shattering what had previously been assumed to be a tenet of glycobiology.3
O-linked glycosylation typically begins in the Golgi apparatus.2 Eight currently identified possible core structures arise from the addition of an initial N-acetylgalactosamine (GalNAc) residue onto a serine or threonine residue by the sugar-transferring activity of an N-acetylgalactoaminyltransferase enzyme. The nucleocytoplasmic form of glycosylation discovered, however, was found to begin with an N-acetylglucosamine (GlcNAc) residue, despite also being O-linked. This previously unseen form of glycosylation begat the question of its biosynthesis, as an amino acid-GlcNAc linkage had previously only been associated with N-linked glycosylation. Further research revealed two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), as the machinery responsible for the addition and removal of this form of glycosylation, referred to as O–GlcNAcylation.
OGT is the sole enzyme responsible for generating O-GlcNAc modifications.4 The enzyme contains two characteristic domains: an N-terminal tetratricopeptide repeat (TPR) domain and a C-terminal catalytic domain, which transfers a GlcNAc residue from a nucleotide sugar donor, UDP-GlcNAc, onto target proteins. Three OGT isoforms are encoded by the OGT gene, which differ only in the number of TPR domains. The importance of the two shorter isoforms is debated, and catalytic activity has generally been attributed to the full-length form which contains 13.5 TPRs. Unlike typically membrane-bound glycosyltransferases, OGT primarily resides in the nucleus and cytoplasm,5 and singlehandedly modifies thousands of proteins. Working alongside OGT is OGA, which alone is responsible for the removal of O-GlcNAc modifications. Human OGA exists in two isoforms, a short and long form, both of which contain N-terminal glycoside hydrolase domains.
Several aspects of O-GlcNAcylation enable it to act as a widespread regulator, one of which is the ability to further modulate protein activity through increased levels of regulatory control. The coordinated activity of OGT and OGA generate the cycling of O-GlcNAc modifications, much like how phosphorylation is controlled by the activity of nucleocytoplasmic kinases and phosphorylases. This further differentiates O-GlcNAcylation as a form of glycosylation and positions it as a regulatory mechanism that parallels phosphorylation.6 In fact, the similarities between these post-translational modifications extend beyond this; O–GlcNAcylation and phosphorylation often occur on the same or similar sites, and are highly responsive to nutrients as their donor substrates are products of cellular metabolism. Moreover, the proteins modified by O-GlcNAcylation and phosphorylation have high levels of overlap, with most O-GlcNAc modified proteins identified also known to be modified by phosphorylation. Several examples of crosstalk between these modifications demonstrate the functional importance of them. Take Akt, for instance, a key serine/threonine kinase involved in cell signalling, cellular proliferation, survival, and metabolism. This protein kinase is fully activated by phosphorylation at two sites, one of which, Thr 308, is in close proximity to O-GlcNAcylation sites, Thr 305 and 312.7 O-GlcNAcylation downregulates the activity of Akt by inhibiting its phosphorylation at Thr 308, thus maintaining optimal Akt signalling and enabling further regulatory control beyond that provided by phosphorylation and dephosphorylation alone. As such, this provides a prime example of the role O-GlcNAc has in regulating protein activity via crosstalk with phosphorylation.
The substrate specificity of OGT also contributes to its broad regulatory roles. Amongst thousands of possible substrates, OGT is able to narrow substrate specificity through several components including TPR domains, partner proteins, post-translational modifications of OGT itself, and UDP-GlcNAc sensing.4 The TPR domains of OGT are known to be essential in OGT catalytic activity and in substrate specificity, although the exact mechanisms by which it confers specificity is unclear. However, residues forming an ‘asparagine ladder’ and ‘aspartate ladder’ have been implicated in directing specific substrates to the catalytic site. Furthermore, a latch between TPR domains in full-length OGT has been found to allow the highly mobile conformation of OGT which allows activity on a variety of substrates. TPR domains have also been found to bind with partner proteins which induce OGT to interact with specific substrate proteins. As for the role of UDP-GlcNAc, the sugar donor for O-GlcNAcylation, O-GlcNAc levels are reflective of glucose levels in a cell as the synthesis of UDP-GlcNAc is dependent on the hexosamine biosynthetic pathway, which branches from glycolysis. This makes O-GlcNAcylation an excellent nutrient sensor, exemplified in an experiment where periventricular OGT knockout mice were shown to lose their natural ability to regulate satiation.8 Moreover, OGT varies in substrate specificity depending on levels of UDP-GlcNAc present, enabling it to modify different targets appropriate for varying cellular circumstances. As such, the several levels of specificity in OGT clearly enable the broad regulatory activity of O-GlcNAcylation across crucial functions in multicellular organisms.
It is evident that O-GlcNAcylation plays a crucial regulatory role in multicellular organisms in influencing protein activity and cellular functions. The interplay between O-GlcNAcylation and phosphorylation, enabled by the dynamic activity of OGT and OGA, underscores its importance in creating greater levels of regulatory control. The levels of possible substrate-specifying influencers of OGT enable specific activity depending on circumstance, despite the thousands of possible OGT substrates. Moreover O-GlcNAcylation not only regulates protein functions but also responds to cellular metabolic states, acting as a nutrient sensor. Looking to the future, continued research into the specific mechanisms of O-GlcNAcylation is likely to further reveal its regulatory roles, continuing the elucidation of this unique and vital form of glycosylation.
References
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6. Butkinaree C, Park K, Hart GW. O-linked β-N-acetylglucosamine (O-GlcNAc): Extensive crosstalk with phosphorylation to regulate signaling and transcription in response to nutrients and stress. Biochimica et Biophysica Acta (BBA) – General Subjects. 2010 Feb 1;1800(2):96–106.
7. Wang S, Huang X, Sun D, Xin X, Pan Q, Peng S, et al. Extensive Crosstalk between O-GlcNAcylation and Phosphorylation Regulates Akt Signaling. PLOS ONE. 2012 May 22;7(5):e37427.
8. Lagerlöf O, Slocomb JE, Hong I, Aponte Y, Blackshaw S, Hart GW, et al. The nutrient sensor OGT in PVN neurons regulates feeding. Science. 2016 Mar 18;351(6279):1293–6.
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