Role of O-Linked N-Acetylglucosamine Protein Modification in Cellular (Patho) Physiology

REVIEW ARTICLE ROLE OF O-LINKED N-ACETYLGLUCOSAMINE PROTEIN MODIFICATION IN CELLULAR (PATHO) PHYSIOLOGY

AUTHORS O-GlcNAc and Cellular function John C. Chatham, Jianhua Zhang, Circadian Transcriptional Adam R. Wende regulation Regulation Epigenetics

Mitochondrial Cellular Stress function Response CORRESPONDENCE

CH OH 2 [email protected] O OGT Metabolic OH Intracellular regulation HO OH G signaling OH Protein Protein Learning and Memory Contractility Neurodegeneration Cardiac hypertrophy KEY WORDS OGA calcium; cancer; diabetes; genetics; metabolism

Muscle fiber type Cancer/Tumor Cell survival Proliferation, Metastasis

Metabolic homeostasis Insulin resistance O-GlcNAc and (patho)physiology

CLINICAL HIGHLIGHTS The modification of proteins by sugars is one of the most common posttranslational modifications of proteins. Such modifications were believed to occur only on extracellular and secreted proteins and to consist of large branching structures comprising different sugar molecules. In the mid-1980s, a new modification was identified which consisted of a single N-acetylglucosamine moiety (O-GlcNAc) attached to serine and threonine residues of nuclear and cytoplasmic proteins. Since its discovery, O-GlcNAc modification of proteins has been shown to affect numerous cellular functions, and changes in O-GlcNAc levels have been implicated in a wide variety of diseases. The goal of this review is to summarize our current knowledge of O-GlcNAc biology and its contribution to normal physiology and disease.

CHATHAM ET AL., 2021, Physiol Rev 101: 427–493 July 30, 2020; Copyright © 2021 the American Physiological Society https://doi.org/10.1152/physrev.00043.2019 Physiol Rev 101: 427–493, 2021 First published July 30, 2020; doi:10.1152/physrev.00043.2019

REVIEW ARTICLE ROLE OF O-LINKED N-ACETYLGLUCOSAMINE PROTEIN MODIFICATION IN CELLULAR (PATHO) PHYSIOLOGY

John C. Chatham, Jianhua Zhang, and Adam R. Wende Division of Molecular and Cellular Pathology, Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama; and Birmingham Veterans Affairs Medical Center, Birmingham, Alabama

Abstract In the mid-1980s, the identification of serine and threonine residues on nuclear and cytoplasmic proteins modified by a N-acetylglucosamine moiety (O-GlcNAc) via an O-linkage overturned the widely held assumption that glycosylation only occurred in the endoplasmic reticulum, Golgi apparatus, and secretory pathways. In contrast to traditional glycosylation, the O-GlcNAc modification does not lead to complex, branched glycan structures and is rapidly cycled on and off proteins by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Since its discovery, O-GlcNAcylation has been shown to contribute to numerous cellular functions, including signaling, protein localization and stability, transcription, chromatin remodeling, mitochondrial function, and cell survival. Dysregulation in O-GlcNAc cycling has been implicated in the progression of a wide range of diseases, such as diabetes, diabetic complications, cancer, cardiovascular, and neurodegenerative diseases. This review will outline our current understanding of the processes involved in regulating O-GlcNActurnover,theroleofO-GlcNAcylation in regulating cellular physiology, and how dysregulation in O-GlcNAc cycling contributes to pathophysiological processes.

calcium; cancer; diabetes; genetics; metabolism

CLINICAL HIGHLIGHTS 1. INTRODUCTION 427 2. REGULATION OF O-GLCNACYLATION 434 The modification of proteins by sugars is one of the most common fi fi 3. O-GLCNACYLATION AND ... 449 posttranslational modi cations of proteins. Such modi cations were believed to occur only on extracellular and secreted proteins and to 4. O-GLCNACYLATION IN ... 461 consist of large branching structures comprising different sugar mol- 5. CONCLUSIONS 470 ecules. In the mid-1980s, a new modification was identified which consisted of a single N-acetylglucosamine moiety (O-GlcNAc) attached to serine and threonine residues of nuclear and cytoplasmic proteins. Since its discovery, O-GlcNAc modification of proteins 1. INTRODUCTION has been shown to affect numerous cellular functions, and changes in O-GlcNAc levels have been implicated in a wide variety of diseases. The goal of this review is to summarize our current knowl- 1.1. Brief History of O-GlcNAc edge of O-GlcNAc biology and its contribution to normal physiology and disease. The modification of proteins by carbohydrates, oth- erwise known as protein glycosylation, is the most However, in 1984, Torres and Hart (2)designedastudyto common posttranslational modification of proteins characterize terminal N-acetylglucosamine (GlcNAc) resi- and occurs in all cells and organisms (1). In the early dues on the surface of lymphocytes. Unexpectedly, they 1900s, there was considerable speculation that car- demonstrated that the majority of these terminal residues bohydrates were important parts of the structure of were localized inside the cell, and that rather than part of proteins, but the technology was lacking to provide extended glycan structures, they existed as a single definitive evidence. It was not until the 1960s and O-linked GlcNAc monosaccharide. 1970s that a better understanding of the structure Two years later, Holt and Hart (3) characterized the and function of these complex glycans on proteins cellular distribution of O-GlcNAc-modified proteins in rat started to emerge. Through the mid-1980s, the consen- liver cells, demonstrating that while they were found in sus was that protein glycosylation was restricted to nearly every cellular compartment, they were particu- extracellular proteins that originated in the endoplasmic larly enriched in the cytoplasm and nucleus. In 1987, a reticulum (ER), Golgi apparatus, and secretory pathway (1). monoclonal antibody to rat liver nuclear pore complex

(clone RL2), appeared to primarily recognize O-linked of pathophysiological processes, such as diabetes, dia- O-GlcNAc groups (4). Hanover et al. (5)alsoreportednu- betic complications, cancer, cardiovascular, and neuro- clear pore proteins were modified by O-GlcNAc; however, degenerative diseases (19–24). the functional consequence of this modification was unclear at that time. During the same period, Holt et al. (6) 1.2. Differences between O-GlcNAc and identified both serine (Ser) and threonine (Thr) residues as Traditional Glycosylation the primary residues modified by O-GlcNAc. The fact that O-GlcNAcylated proteins were especially enriched in the Until the paradigm-changing study by Torres and Hart (2), nucleus raised the possibility that the modification could protein glycosylation was thought to be limited to extracel- be involved in protein transport into the nucleus; however, lular and excreted proteins. These proteins are processed the observation that cytoskeletal proteins in erythrocytes, via the ER-Golgi pathway, which contains a large number which lack a nucleus, were modified by O-GlcNAc sug- of glycosyltransferases that are responsible for creating gested other functions for this modification (7). In 1992, the N-andO-linked glycan structures. N-glycans are attached protein responsible for adding O-GlcNAc to proteins, O- to proteins as asparagine residues via an N-glycosidic GlcNAc transferase (OGT) was purified (8), but it was not bond; whereas, O-glycans are attached to Ser or Thr resi- until 1997 that the gene encoding OGT was identified, dues. These proteins are subject to processing and matu- revealing a glycosyltransferase that was unrelated to any ration by numerous glycosyltransferases, leading to stable other previously known glycosyltransferases (9). elongated and branched structures comprising a number In contrast to traditional protein glycosylation, it was of different monosaccharides. Glycosyltransferases are quickly established that O-GlcNAc modifications occurred estimated to account for at least 2% of the human ge- rapidly and reversibly (10), suggesting the existence of an nome (25).Theimportanceofthetightregulationofthis N-acetyl-glucosaminidase(s) responsible for its removal process is highlighted by the fact that mutations in genes from proteins. Dong et al. (11)purified an O-GlcNAcase related to glycosylation are associated with more than 100 that was distinctly different from lysosomal hexosamini- human genetic diseases, which are frequently associated dases, in that it was localized in the cytosol and was opti- with intellectual disabilities, as well as abnormalities in mally active at a neutral, rather than acidic, pH. In 2001, most organ systems (25). O-GlcNAcase was cloned and recognized to be identical Key distinguishing features of the O-GlcNAc modifica- to a previously known hexosaminidase C of unknown tion are that 1) with few exceptions, it occurs primarily on function, which specifically cleaved O-GlcNAc but not nuclear and cytoplasmic proteins; 2) it consists of a single O-linked N-acetylgalactosamine (O-GalNAc) from glyco- monosaccharide; 3) it is dynamic and rapidly reversible; 4) peptides (12). O-GlcNAcase was subsequently shown to it is catalyzed by a single unique O-GlcNAc transferase, have an identical sequence to a previously identified pro- and 5) it is removed by a glycohydrolase that is specificfor tein from meningioma patients called meningioma the removal of O-GlcNAc. It is of note that only very expressed antigen 5 (MGEA5) (13). recently have mutations in the OGT gene been linked to Over the decades since its discovery, O-GlcNAc modi- human disease, such as intellectual disability (26–29). fied proteins have been identified in all metazoans, HowmutationsofOGTleadtodiseaseandwhatspecific some bacteria, protozoa, and viruses, but to date, not in functions are perturbed remain to be determined. In light yeast (14). Recent studies have suggested that in yeast, of the growing recognition of the importance of O-GlcNAc- the addition of O-linked mannose (O-Man) on nuclear modified proteins in regulating cellular homeostasis—and and cytoplasmic proteins might play a similar role to that it is likely as abundant a modification as phosphoryla- O-GlcNAc (15). In support for the necessity of O-GlcNAc tion—it may seem surprising that it had not been identified signaling in mammals, germline deletion of OGT in mice earlier. One reason for this is that proteins modified by has been shown to be embryonically lethal (16), and de- O-GlcNAc do not exhibit changes in mobility during gel letion of OGA results in perinatal mortality in mice (17). electrophoresis, because of their low molecular weight Furthermore, mammalian OGT and OGA are ubiqui- and because unlike O-phosphate, it is uncharged. tously expressed, and proteins of every functional class Moreover, despite its abundance, it exhibits low stoichiom- have been shown to be subject to O-GlcNAcylation etry, estimated to be 5–10% of a specific site. In addition, (FIGURE 1A)(18). Since its discovery, O-GlcNAcylation thepresenceofhexosaminidasescanremoveO-GlcNAc has been shown to contribute to numerous cellular func- from proteins unless they are specifically inhibited. tions, including signaling, protein localization and stability, transcription, chromatin remodeling, mitochondrial 1.3. Identification of O-GlcNAcylated Proteins function, and cell survival (FIGURE 1B)(14). Given its diverse roles, it is not surprising that dysregulation in The most widely used approach to detect O-Glc- O-GlcNAcylation has been implicated in a wide range NAcylated proteins are pan-specific O-GlcNAc antibodies,

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A O-GlcNAc protein distribution 2% Cell Cycle 7% Proteins Nuclear Pore Proteins 7% 26% Protein Transcription & Processing Translation

8% Signaling

11% Stress Proteins 14% Structural Proteins FIGURE 1. A: O-linked N-acetylglucosamine acy- 12% lated (O-GlcNAcylated) proteins belong to many dif- Other/Unknown ferent classes of proteins responsible for regulating 13% diverse cellular processes. Some of the largest Metabolic classes of proteins include those in regulating me- Proteins tabolism, transcription, and translation, as well as B O-GlcNAc cellular distribution structural proteins. B: O-GlcNAcylated proteins are present in numerous cellular compartments, including the nucleus, cytosol, and mitochondria. Cytosolic domains of membrane proteins are also O-GlcNA- cylated, as well as proteins involved in autophagy and proteosomal degradation of proteins, chaperone pro- Cytoplasmic domain Cytoskeletal teins, vesicle proteins, and numerous cytosolic pro- of receptors & proteins teins and enzymes. This ﬁgure is based, in part, on ion channels Chaperone information presented in Chapter 19, The O-GlcNAc proteins Mitochondrial Cytosolic Modiﬁcation, Essentials in Glycobiology,3rded.(14). proteins enzymes ER, endoplasmic reticulum.

Proteosomal Vesicle Autophagosome proteins proteins

Autophagic & Oncogenes Chromatin & lysosomal Tumor suppressor RNA polymerase proteins Cytoplasmic domain of ER membrane proteins Autolysosome Nuclear Pore proteins RNA processing Transcription factors Viral proteins

the most commonly being RL2 and CTD110.6, but there fact that they have epitope specificity, and their selectivity are also a number of other commercially available O- for high- versus low-abundance proteins. Furthermore, a GlcNAc antibodies as listed in TABLE 1 These can be continuing limitation in the study of O-GlcNAcylated pro- used to provide a semiquantitative assessment of overall teins is the lack of commercially available site-specific O- changes in O-GlcNAc levels via immunoblot or distribution GlcNAc antibodies, although several have been devel- of O-GlcNAc via immunohistochemistry in tissues and cells oped for specificstudies(79–82). Because of the lack of (FIGURE 2). The limitations of these antibodies include the site-specific antibodies to determine whether a protein of

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Table 1. Tools for use in studying O-GlcNAc levels in cells and tissues

Commercial AntibodiesÃ Characteristics and Limitations Availability

CTD110.6 (IgM) (30) Monoclonal antibody raised against RNA polymerase II subunit 1C terminal domain. Reportedly Multiple sources less dependent on protein structure than other antibodies, thereby, recognizing more proteins. Relatively low-binding afﬁnity, therefore, biased toward more highly abundant proteins.

HGAC39 (IgG) (31–33) Monoclonal antibody raised against streptococcal group A carbohydrate (GAC) demonstrated to No recognize O-GlcNAcylated proteins.

HGAC85 (IgG) (33) Monoclonal antibody raised against streptococcal group A carbohydrate (GAC) demonstrated to Multiple sources recognize O-GlcNAcylated proteins. Not as widely used as CTD110.6 or RL2. Some of the most recent studies have used it in ChIP-chip assays.

MY95 (IgG) (34,35) Originally generated as an antinuclear pore complex antibody, subsequently shown to recognize No O-GlcNAc modiﬁcation.

RL2 (IgG) (4) Because of epitope speciﬁcity, having been raised against nuclear pore protein, it recognizes Multiple sources only a subset of O-GlcNAc proteins. Relatively low binding afﬁnity; therefore, it is biased toward more highly abundant proteins. This is the case for all pan-O-GlcNAc antibodies.

1F5.D6(14) (IgG) (36) Appears to recognize a wider range of O-GlcNAcylated proteins, including those below 50 kDa Millipore that are not usually identiﬁed by RL2 or CTD110.6.

9D1.E4(10) (IgG) (36) Appears to recognize a wider range of O-GlcNAcylated proteins, including those below 50 kDa, Millipore which are not usually identiﬁed by RL2 or CTD110.6.

18B10.C7(3) (IgG) (36) Appears to recognize a wider range of O-GlcNAcylated proteins, including those below 50 kDa, Millipore which are not usually identiﬁed by RL2 or CTD110.6.

Other Identification Commercial Methods Characteristics and Limitations Availability

Agrocybe aegerita AANL, also reported as AAL2, is a useful tool for enrichment and identiﬁcation of O- Not known GlcNAc-speciﬁc lectin GlcNAcylated proteins and peptides. (AANL) (37)

Click-IT O-GlcNAc Chemoenzymatic labeling of proteins resulting in O-GlcNAc residues being replaced by Multiple sources Enzymatic Labeling azido-modified galactose (GalNAz), followed by chemoselective ligation azide. System (38–40) Resulting proteins can be detected via Western blot using a using a variety of alkyne- modified chemical probes. This approach is not epitope specific, which has advan- tages over pan-O-GlcNAc antibodies; however, more sample processing steps are required.

Galactosyl transferase/ Results in incorporation of 3H into terminal GlcNAc residues on proteins allowing for detec- Multiple sources [3H]-galactose (41) tion by autoradiography. Can be very time intensive because of low sensitivity of 3H.

Wheat germ agglutinin Both WGA and sWGA can be used for immunoblotting. WGA identifies all terminal Multiple sources (WGA) and succiny- GlcNAc residues, as well as sialic acid. sWGA reduces affinity for sialic acid. Lack of lated WGA (sWGA) (41) specific for O-GlcNAc requires careful interpretation.

Enrichment Commercial Strategies Characteristics and Limitations Availability

b-elimination followed BEMAD relies on the b-elimination of phosphate or O-GlcNAc under basic conditions fol- by Michael addition lowed by Michael addition using dithiothreitol or a biotin-thiol probe. of dithiothreitol (BEMAD) (42)

Chemi-enzymatic Using the same approach described for BEMAD, it replaces O-GlcNAc with GalNAz, com- labeling (43) bined with a biotin- or streptavidin-cleavable linker can be used to enrich O-GlcNAcylated peptides. A range of linkers have been developed that have different properties.

Continued

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Table 1.—Continued

Enrichment Commercial Strategies Characteristics and Limitations Availability

Immunoprecipitation The most widely used antibodies, RL2 and CTD110.6, do not perform well for immuno- (36, 44) puriﬁcation. Although not as widely used, the Millipore antibodies listed above have been reported to be effective for IP.

WGA-agarose (45) Because of the lack of speciﬁcity for O-GlcNAc, other glycoproteins will be copuriﬁed. Multiple sources This can be minimized by pretreatment to remove the N- and O-linked glycans.

Commercial GFAT Inhibitors Characteristics and Limitations Availability

Azaserine (46) Glutamine analog used to decrease HBP ﬂux by inhibiting GFAT; however, it lacks speci- Multiple sources ﬁcity as it can inhibit other pathways that utilize glutamine (47). In the absence of any other small molecule GFAT inhibitors, it continues to be widely used, but interpretation of results needs to be cautious.

6-diazo-5-oxo-L-nor- Glutamine analog used to decrease HBP ﬂux by inhibiting GFAT; however, it lacks speci- Multiple sources leucine (DON) (46) ﬁcity as it can inhibit other pathways that utilize glutamine (48). In the absence of any other small molecule GFAT inhibitors, it continues to be widely used, but interpretation of results needs to be cautious.

Commercial OGA Inhibitors Characteristics and Limitations Availability a-GlcNAc thiolsulfonate OGA inhibitor exhibiting selectivity of short OGA compared to long OGA. No (49)

Gluco-nagstatin (50,51) Based on natural product, nagstatin, which is a potent inhibitor of b-hexosaminidase. No Inhibits OGA but is more effective for of b-hexosaminidase.

GlcNAcstatins (50, Family of OGA inhibitors, with high degree of potency and selectivity. Abmole Bioscience, Inc. 52,53)

NAG-thiazoline (50, 54) Inhibitor of OGA with much greater selectivity over other hexosaminidases than No PUGNAc.

NButGT (50, 55,56) OGA inhibitor based on NAG-thiazoline scaffold. Less potent that NAG-thiazoline but No even more selective over other hexosaminidases.

PUGNAc (57) Widely used cell permeable OGA inhibitor, but also inhibits hexosaminidase A and B in- Multiple sources hibitor. Limited aqueous solubility; usually dissolved in DMSO prior to dilution in aqueous solutions for biological studies.

Streptozotocin (50, 54, A GlcNAc analog initially thought to inhibit OGA. Has major off target effects and more Multiple sources 58,59) recent studies have reported that it does not inhibit OGA. Not recommended for use.

Thiamet-G (50, 60) Derivative of NButGT, with greater stability, water soluble, and orally available. Multiple sources

Commercial OGT Inhibitors Characteristics and Limitations Availability

Alloxan (61,62) A weak OGT inhibitor with numerous off target effects. Requires millimolar concentra- Multiple sources tions to lower cellular O-GlcNAc levels. Not recommended for use.

Benzyl-2-acetamido-2- Although BADGP decreases O-GlcNAc levels in millimolar range it lacks speciﬁcity for Sigma deoxy-a-D-galacto- OGT. Inhibits other O-Glycosyltransferases. Not recommended for use. pyranoside (BADGP) (63,64)

L01 (63) Effective in reducing cellular O-GlcNAc levels in 10–100-mM range. No

Continued

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Table 1.—Continued

Commercial OGT Inhibitors Characteristics and Limitations Availability

OSMI-1 (65–68) Reduces O-GlcNAc levels in cells at 50 mM. Shown to be speciﬁc for OGT compared Sigma with other glycosyltranserases.

OSMI-3, OSMI-4 (69) On the basis of OSMI-1 scaffold, but with greater potency. Reduces cellular O-GlcNAc ProbeChem levels in 5-mM range.

ST045849 (TT04) (63, Identiﬁed via high throughput screen. Speciﬁcity for OGT compared to other glycosyl- TimTech 66, 70–73) transerases unclear.

ST060266 (70, 74) Identiﬁed via high-throughput screen. Speciﬁcity for OGT compared to other glycosyl- TimTech transerases unclear.

ST078925 (70, 72, 75) Identiﬁed via high-throughput screen. Speciﬁcity for OGT compared to other glycosyl- TimTech transerases unclear.

UDP-5SGlcNAc; (61, 76) Effective in vitro inhibitor of OGT but lacks cell permeability. No

2-deoxy-2-N-hexana- Shown to be effective in lowering tissue O-GlcNAc levels following in vivo No mide-5-thio-d-gluco- administration. pyranoside (5SGlcNHex); (77)

4-methoxyphenyl 6- A cell permeable, irreversible inhibitor of OGT. No acetyl-2-oxo-2,3-dihy- dro-1,3-benzoxazole- 3-carboxylate (78)

5-thioglucosamine The peracetylated form of 5SGlcNAc crosses the cell membrane and converts to UDP- No (5SGlcNAc) (76) 5SGlcNAc, which binds to the active site of OGT. Reduces O-GlcNAc levels in cells in the 10-100-mM range.

AAL2, Agrocybe aegerita GlcNAc-speciﬁc lectin; CTD, COOH-terminal domain; GFAT, D-fructose-6-phosphate amidotransferase; GlcNAc, N-acetylglucosamine moiety; HBP, hexosamine biosynthesis pathway; IP, immunoprecipitation; NAG, N-acetylglucosamine; O-GlcNAc, O-linked N-acetylglucosamine; OGT, O-GlcNAc transferase; RL2, rat liver nuclear pore complex; UDP, uridine diphosphate-azido-modiﬁed galactose.

interest is an O-GlcNAc target, the simplest approach is to significantly enhanced the identification of O-GlcNAc immunoprecipitate the protein of interest followed by an modification sites, since ETD does not usually result in O-GlcNAc immunoblot; however, because of differences the loss of the O-GlcNAc moiety from the peptide; how- in epitope specificities, a negative result with a single anti- ever, the problems of ion suppression and low stoichiom- body does not preclude the possibility that a protein is etry remain. To overcome these limitations, it is modified. Moreover, because of the possibility of cross- necessary to enrich the sample for O-GlcNAcylated pep- reactivity with other sugars, if a positive result is obtained, tides (88). One approach is to use a traditional immuno- a number of additional control experiments should be con- precipitation with a single anti-O-GlcNAc antibody, as sidered, such as preincubation with free GlcNAc to out- described in several studies (36, 89). On the other hand, compete the antibody. Additionally, their cross-reactivity because of the limitations of epitope specificity, there is a with N-linked modificationsvariesbydetectionconditions concern that only a subset of O-GlcNAc proteins will be (83). A number of methodological reviews on different identified. One way to overcome that limitation is to use a approaches for studying O-GlcNAcylated proteins have combination of antibodies; for example, Lee et al. (90) been published that provide valuable technical details developed a G5-lectibody resin column that consisted of (44,45, 84,85). four different O-GlcNAc antibodies and the lectin wheat Another challenge in the identification and characteri- germ agglutinin (WGA) (FIGURE 2). Another enrichment zation of O-GlcNAcylated proteins is that O-GlcNAc-modi- approach is chemoenzymatic labeling (38, 44, 91), which fied peptides are frequently not detected using traditional involves using uridine diphosphate-azido-modified galac- collision-induced dissociation mass spectrometry (MS), as tose (UDP-GalNAz) and a mutant galactosyltransferase to O-GlcNAc is very labile and is usually lost during collision- tag O-GlcNAc moieties with a reactive azide group. This induced fragmentation. The development of electron is followed by the addition of a biotin group attached to a transfer dissociation (ETD) MS techniques (86,87) cleavable linker, using a copper-based azide-alkene

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Identification of O-GlcNAcylated proteins

Galactosyltransferase-labeling

Treat sample with UDP-[3H]Gal Treat with PNGaseF to SDS-PAGE and and galactosyltransferase remove N-Glycans Detect with autoradiography

IP against protein of interest

O-GlcNAc Immunoblot Use GlcNAc to confirm O-GlcNAc specificity

Chemoenzymatic labeling LC-MS/MS to identify O-GlcNAc modification sites IP with O-GlcNAc antibody or lectin affinity column

Pre-treat sample with hexosaminidase to remove O-GlcNAc to confirm O-GlcNAc Immunoblot with protein of interest Pre-treat sample with PNGaseF to remove N-Glycans Global Chemoenzymatic LC-MS/MS labeling

Trypsin digestion of Chemoenzymatic labeling Enrich for LC-MS/MS protein lysate O-GlcNAc peptides

FIGURE 2. Overview of different approaches for the identification of O-linked N-acetylglucosamine acylated (O-GlcNAcylated) proteins, including galactosyltransferase labeling; immunopurification and chemoenzymatic labeling were combined with LC-MS/MS. Further details are found in TABLE 1.IP, immunoprecipitation; UDP, uridine diphosphate-azido-modified galactose. cycloaddition (84). The resulting modified proteins or pep- 225 (or more than the first 20 years combined). As tides can then be affinity purifiedusinganavidincolumn. this becomes a more widely recognized area of biol- A number of different linkers have been described, ogy, it is likely to grow more rapidly, given the including a UV-cleavable linker (92,93)and,more increasing appreciation of its importance in regulat- recently, a hydrazine-sensitive linker (94). A key advant- ing key physiological processes combined with its age of the latter approach is the resulting tag at the O- contributions to the development of diverse patholo- GlcNAc site allows for more efficient fragmentation. gies. Several thousands of proteins have been identi- There are several different versions of this technique, and fied as O-GlcNAc targets (44), and the number refinements continue to be developed focused on continues to increase, as new techniques are devel- improving the efficiency of the release of proteins/pep- oped. Many new O-GlcNAc sites are identified via tides from the avidin column, as well as simplifying the high-throughput MS; consequently, the biological underlying chemistry (43, 95,96). function of the modification is often not known. In Compared with most other PTMs, our understand- TABLE 2, we provide a list of some key proteins, di- ing of how O-GlcNAcylation regulates protein func- vided via cellular location or function, which are vali- tion and cellular physiology remains limited, although dated O-GlcNAc targets in which the specific our knowledge is rapidly growing. The development modification site has been identified. In TABLE 3,the of increasingly selective and specific small-molecule physiological effects of gain or loss of function of inhibitors of OGT and OGA, as summarized in TABLE OGT and OGA in mammals are summarized. 1, have helped advance our knowledge of the role of In the following sections, we have provided an over- O-GlcNAc in cellular function. These new tools have view of our current understanding of the regulation of helped stimulate research in O-GlcNAc biology, as O-GlcNAcylation, its role in regulating cellular physiol- illustrated in FIGURE 3. About 20 years ago, there ogy, as well as its potential role in pathophysiology of were less than 20 articles published per year, but this diseases, including diabetes, cardiovascular disease, has been steadily increasing, and in 2018, it reached and cancer.

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250 OGA KO mouse

200 hOGTcrystal structure OGT protease activity OGA inhibition is cardioprotective

150 ETD Mass spec for O-GlcNAcmapping

100 Thiazolinebased OGA inhibitors described Increased O-GlcNAccytoprotective Chemoezymaticapproach to tag O-GlcNAcproteins OGA cloned CTD 110.6 antibody available Generation of OGT floxedmouse OGA purified EMeg32 KO embryonically lethal OGT sequenced Number of Papers Per Year O-GlcNAc discovered RL2 antibody available Ser/Thrresidues are O-GlcNAc sites OGT purified 50

1983 1988 1993 1998 2003 2008 2013 2018 Years

FIGURE 3. Number of O-linked N-acetylglucosamine acylated (O-GlcNAc) publications by year annotated by key events in O-GlcNAc biology from its initial discovery. Relevant citations are all included in the main text. CTD, COOH-terminal domain; ETD, electron dissociation transfer hOGA, human O- GlcNAcase; hOGT, human O-GlcNAc transferase; KO, knockout; OGA, O-GlcNAcase; OGT, O-GlcNAc transferase; RL2, rat liver nuclear pore complex.

2. REGULATION OF O-GlcNACYLATION predominates over GFAT1, whereas, in most other tissues, GFAT1 is more highly expressed (214). A splice var- 2.1. Hexosamine Biosynthesis Pathway iant of GFAT1, known as GFAT1Alt, has been identified, and it appears to be predominantly expressed in skele- Uridine diphosphate-GlcNAc (UDP-GlcNAc) is the com- tal muscle and exhibits a higher Km for fructose-6-phos- mon substrate for all amino sugars involved in the syn- phate and a lower Ki for UDP-GlcNAc than those for thesis of glycoproteins and proteoglycans. As the end GFAT1 (213, 215). In mammals, GFAT exists as a tetramer, product of the hexosamine biosynthesis pathway (HBP), and its activity is highly dependent on the availability of UDP-GlcNAc integrates multiple metabolic pathways both glutamine and glucose (213). Both glucosamine-6- and has long been considered an important nutrient sig- phosphate and UDP-GlcNAc are potent allosteric inhibi- naling pathway partially regulated by substrate availabil- tors of mammalian GFAT (FIGURE 5)(214). Up to 20 dif- ity (FIGURE 4)(210–212). L-glutamine: D-fructose-6- ferent phosphorylation sites have been identified in phosphate transaminase (GFPT, EC 2.6.1.16), often GFAT; however, the responsible kinases and function(s) referred to as L-glutamine: D-fructose-6-phosphate ami- of the majority of them are unknown. GFAT1 and GFAT2 dotransferase (GFAT), is the rate limiting enzyme of the are regulated by cAMP-dependent protein kinase (PKA) HBP, catalyzing the transfer of the amide group from phosphorylation on Ser-205 and Ser-235, respectively glutamine to fructose 6-phosphate, leading to the syn- (213, 216,217); both AMPK and calcium/calmodulin-de- thesis of glucosamine 6-phosphate (213). In mice and pendent kinase (CaMK) II phosphorylate GFAT1 at Ser- humans, there are two different isoforms of GFAT 243 (FIGURE 5)(129, 218). There are contradictory (GFAT1, GFAT2) encoded by two different genes, reports on the effects of phosphorylation on GFAT activ- located on different chromosomes, and each isoform ity, which may be due to isoform-specific differences. In has markedly different tissue distribution (213, 214). In tis- the heart, a number of studies indicate that AMPK phos- sue from the central nervous system, GFAT2 expression phorylation of GFAT1 reduces its activity (219); however,

434 Physiol Rev VOL 101 APRIL 2021 www.prv.org O-GlcNACYLATION AND (PATHO)PHYSIOLOGY

Table 2. List of selected O-GlcNAcylated proteins and their modiﬁcation sites

Transcription Factors and Transcriptional Regulators Protein Symbol O-GlcNAc Modification SitesÃ Effects on Protein Function Citations

C/EBPb Ser-180, Ser-181 Regulates both the phosphorylation and DNA binding ac- (97) tivity of C/EBPb. cMyc Thr-58 Reduces phosphorylation of Ser-62 and Thr-58 and (98,99) increases protein stability.

CREB Ser-40 Represses both basal and activity-dependent (100) transcription.

ERa Ser-10, Thr-50, Thr-575 May regulate protein turnover. (101,102)

ERb Ser-16 May regulate protein turnover. (103)

ERRc Ser-317, Ser-319 Enhances receptor activity. (104)

FOXO1 Thr-317, Ser-550, Thr-648, Ser-654 Increases target gene transcription. (105)

FXR Ser-62 Enhances FXR gene expression and protein stability. (106)

KEAP1 Ser-104 O-GlcNAc is required for the efﬁcient ubiquitination and (107) degradation of Nrf2.

Sp1 Ser-491, Ser-612, Thr-640, Ser-641, Ser-698, Inhibits transcriptional activation. (108,109) Ser-702

LXRa/b Ser-49 Increases transactivation. (110–112)

Oct4 Thr-116, Thr-225, Ser-236, Ser-288/889/890, O-GlcNAc increases transcriptional activity. (113) Ser-335, Ser-349, Thr-351, Thr-352, Ser-355, Ser-359

P27 Ser-2 Suppresses protein interactions. (114) p53 Ser-139 Reduces phosphorylation and stabilizes protein. (115)

Per2 Ser-566, Ser-580, Ser-653, Ser-662, Ser-668, O-GlcNAc increases its suppressor activity. (116) Ser-671, Thr-734, Thr-965, Ser-983, Thr-1180

PGC-1a Ser-334, Ser-333 Enhances stability and upregulated downstream genes. (117,118)

PPARc Thr-54 Reduces its transcriptional activity. (119)

SIRT1 Ser-549 O-GlcNAcylation increases deacetylase activity, promotes (120) cytoprotection under stress.

Insulin Signaling and Other Metabolic Proteins Protein Symbol O-GlcNAc Modification SitesÃ Effects on Protein Function Citations

Akt1/2 Ser-126, Ser-129, Thr-305, Thr-308, Thr-312, Decreases phosphorylation and activity. (121–126) Ser-473

CRTC2 Ser-70, Ser-171 Increased O-GlcNAc leads to nuclear translocation and (127) promotes gluconeogenesis.

Continued

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Table 2.—Continued

Insulin Signaling and Other Metabolic Proteins Protein Symbol O-GlcNAc Modification SitesÃ Effects on Protein Function Citations

GAPDH Thr-227 Increases nuclear translocation. (128)

GFAT1 Ser-243 AMPK phosphorylation of Ser-243 reduced GFAT (129,130) activity.

GSK3b Ser-9, Thr-38, Thr-39, Thr-43 Decreased activity. (125, 131–133)

G6PDH Ser-84 Increases activity. (134)

IRS1 Ser-914, Ser-1009, Ser-1036, Ser-1041 Attenuates insulin-mediated phosphorylation of IRS1. (135,136)

PDH (E1) Ser-13, Ser-15, Ser-134, Ser-232 Higher O-GlcNAc levels associated with greater activity. (137,138)

PDH (E2) Ser-7, Ser-15, Ser-239, Ser-411 Remains to be determined. (137–139)

PDK2 Ser-110 Remains to be determined. (138)

PFK1 Ser-529 O-GlcNAc levels increased in hypoxia and decreases (140) activity.

PKM2 Thr-405, Ser-406 Reduces activity and increases nuclear translocation. (141)

Contractile and Cytoskeletal Proteins Protein Symbol O-GlcNAc Modification SitesÃ Effects on Protein Function Citations

aB-crystallin Thr-170 Regulates stress induced translocation. (142,143)

b-Actin (Skeletal Ser-52, Ser-155, Ser-199, Ser-232, Ser-323, O-GlcNAcylation of Ser-199 may regulate elongation of (144–147) Muscle) Ser-368 actin ﬁlaments.

Keratin18 Ser-29, Ser-30, Ser-48 Increased O-GlcNAc promotes cell survival by activating (148,149) Akt.

Myosin heavy chain Ser-1097, Ser-1299, Ser-1708, Ser-1920 Reduced myosin Ca21 sensitivity. (145,146) (Skeletal Muscle)

Myosin heavy 6 Thr-35, Thr-60, Ser-172, Ser-179, Ser-196 Ser- Reduced myosin Ca21 sensitivity. (147, 150) (Cardiac) 392, Ser-622, Ser-626, Ser-644, Ser-645, Ser-749. Ser-880, Ser-1038, Ser-1148, Ser- 1189, Ser-1200, Ser-1308, Ser-1336, Ser-1470, Ser-1597, Thr-1600, Thr-1606, Ser-1638, Thr- 1697, Ser-1711, Ser-1777, Ser-1838, Ser-1916

MYL1 Ser-45, Thr-93, Thr-164 Remains to be determined. (147, 150)

MYL2 Ser-15 Remains to be determined. (147)

Synapsin I Ser-55, Thr-56, Thr-87, Ser-436, Ser-516, Thr- Regulation of synaptogenesis.O-GlcNAc of Thr-87 regu- (151,152) 524, Thr-562, and Ser-576 lates localization of synapsin I.

Tau Ser-208, Ser-238, Ser-400, Ser-692 Loss of O-GlcNAc induces hyperphosphorylation. (153–155)

TnI Ser-150 Remains to be determined. (147)

Continued

436 Physiol Rev VOL 101 APRIL 2021 www.prv.org O-GlcNACYLATION AND (PATHO)PHYSIOLOGY

Table 2.—Continued

Contractile and Cytoskeletal Proteins Protein Symbol O-GlcNAc Modification SitesÃ Effects on Protein Function Citations

TnT Ser-190 Increased O-GlcNAc reduces Ser208 phosphorylation. (156)

Tropomyosin a1 Ser-87, Ser-123, Ser-186, Ser-206 Remains to be determined. (150)

Oxidative Phosphorylation and Other Mitochondrial Proteins Protein Symbol O-GlcNAc Modification SitesÃ Effects on Protein Function Citations

ATP5A Ser-76, Thr-432 Decreased activity. (138, 157,158)

ATP5B Ser-106, Ser-128 Decreased activity. (138)

DRP1 Thr-585, Thr-586 Associated with increased mitochondrial fragmentation (159) and decreased mitochondrial membrane potential.

Milton Ser-447, Ser-829, Ser-830, Ser-938 Attenuates mitochondrial motility. (160)

NDUFA9 Ser-156, Ser-230 Impaired complex activity. (138, 161)

Prohibitin Ser-121 Decreases phosphorylation. (138,139, 162)

VDAC1 Thr-2, Ser-240, Ser-260 Increased O-GlcNAc attenuates mitochondrial Ca21 (138, 163) uptake.

VDAC2 Ser-2 Increased O-GlcNAc contributes to mitochondrial dysfunc- (139, 164) tion and apoptosis.

Kinases, Phosphatases, and Other Signaling Molecules Protein Symbol O-GlcNAc Modification SitesÃ Effects on Protein Function Citations

CaMKII Ser-279, Ser-280 Activates enzyme.Increases NOX2 ROS production (165)(166)

CaMKIV Thr-57, Ser-58, Ser-137, Ser-189, Ser-344, Ser- Activates enzyme. (167) 345, Ser-356

CDK5 Ser-46, Thr-245, Thr-246, and Ser-247 Decreases activity. (168)

CK2a Ser-347 Attenuates interaction with Pin1 and facilitates proteaso- (131, 169,170) mal degradation.Reduces CKII phosphorylation decreasing its stability. May also affect substrate selectivity.

GRASP55 Ser-389, Ser-390,Thr-403, Thr-404,Thr-413 O-GlcNAcylation attenuates autophagy. (171)

IKKb Ser-733 Increases activity. (172)

PKC-a, b,d, ɛ, h, f Numerous (see citations for details) Function not well deﬁned and could be isoform speciﬁc. In (173–175) some cases, likely competes with phosphorylation and decreases enzyme activity.

PTP1B Ser-104, Ser-201, Ser-386 Lower O-GlcNAc levels leads to lower phosphatase (176) activity.

RACK1 Ser-122 Promotes stability and interaction with PKCbII. (177,178)

Continued

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Table 2.—Continued

Kinases, Phosphatases, and Other Signaling Molecules Protein Symbol O-GlcNAc Modification SitesÃ Effects on Protein Function Citations

RIPK3 Thr-467 Prevents dimerization and limits inﬂammation. (179)

SNAP29 Ser-2, Ser-61, Thr-130, Ser-153 O-GlcNAcylation attenuates autophagy. (180)

ULK1,2 Thr-613, Thr-635, Thr-726, Thr-754, O-GlcNAc increases AMPK-mediated phosphorylation of (92, 181,182) ULK, leading to increased activity.

YAP1 Ser-109, Thr-241 Ser-109 O-GlcNAc Disrupts interaction with upstream ki- (183,184) nase LATS1. Thr-241 O-GlcNAc increases activity by improving stability.

Miscellaneous

Protein Symbol O-GlcNAc Modiﬁcation Sites Effects on Protein Function Citations

a-synuclein Thr-64, Thr-72, Thr-75, Thr-81, Ser-87 Increased O-GlcNAcylation associated with decrease in (185) aggregation; however, this has primarily been identiﬁed in in vitro studies.

b-catenin Ser-23, Thr-40, Thr-41, Thr-112 Promotes its recruitment to the plasma membrane and its (186,187) binding to E-cadherin and regulates stability.

CREB-binding Ser-147, Ser-2360 Remains to be determined. (188) protein

eIF4A Ser-322, Ser-323 Regulates formation of translation initiation complex. (189)

eNOS Ser-1177 Impairs activity. (190)

FNIP1 Ser-938 Attenuates phosphorylation and increases proteasomal (191) degradation.

HCF-1 Thr-490, Ser-569, Ser-620, Ser-622, Ser-623, O-GlcNAcylation is a signal for its proteolytic processing (188, 192) Thr-625, Ser-685, Thr-726, Thr-779 Thr-801, Thr-861, Thr-1143, Thr-1273, Thr-1335, Thr- 1743, Thr-1238, Thr-1241

HSP90 Ser-434, Ser-452, Ser-461 Remains to be determined (193)

PLB Ser-16 Inhibits phosphorylation. (194)

TAB1 Ser-395 Required for full activation of TAK1 (81)

TAB2 Ser-166, Ser-350, Ser-354, Thr-456 May control the activity of IL-1 signaling pathway (95, 188)

TAB3 Ser-408 Mediates cell migration via activation of NF-κB, (195)

Ã The proteins included in this table were selected primarily on the basis of their inclusion within the main body of the article. In addition to the citations listed in Table 2, other resources that were used included PhosphoSite Plus, www.phosphosite.org (196), Dias et al. (197), Ma et al. (138,139). CKII, casein kinase II; FXR, farnesoid X receptor; GFAT, L-glutamine: D-fructose-6-phosphate amidotransferase; IRS1, insulin receptor substrate 1; LATS1, large tumor suppressor kinase 1; NOX2, NADPH phagocyte oxidase isoform; Nrf2, nuclear factor E2–related factor-2; ROS, reactive oxygen species; O-GlcNAc, O- linked N-acetylglucosamine; TAK1, transforming growth factor-b activated kinase 1; ULK, Unc-51-like autophagy activating kinase 1.

it remains to be determined whether this is also the leading to loss of activity and reduced O-GlcNAc lev- case in other cells and tissues. GFAT is also tran- els have been linked to neuromuscular transmission scriptionally regulated (FIGURE 5)byspeciﬁcity pro- defects (222). Despite reports of several putative tein 1 (Sp1) (220) and activating transcription factor 4 GFAT inhibitors (213, 223, 224), the regulation of (ATF4) (221). Multiple GFAT1 missense mutations HBP ﬂux via small-molecule inhibitors of GFAT has

438 Physiol Rev VOL 101 APRIL 2021 www.prv.org O-GlcNACYLATION AND (PATHO)PHYSIOLOGY Table 3. Physiological roles of OGT and OGA in mammals

Genotype Phenotype Citations

Human

Ogt human mutation L254F or E1974H Intellectual disability. (26,27, 29, 198)

Rat

Oga Goto-Kakizaki (GK) rat mutation Diabetes. (199)

Systemic inhibition of OGA by thiamet Impaired novel place recognition. (200) G in wildtype rat

Mouse

AgRP-cre::Ogtf/f Ogt ablation in AgRP neurons. Obese due to lack of browning of white fat. (201)

CaMKII-cre::Ogtf/f Constitutive Ogt ablation in forebrain neurons. Loss of body and brain weight (202) and signiﬁcant neurodegeneration almost as early as birth, decreased lifespan.

CaMKII-creER::Ogtf/f Inducible Ogt ablation in forebrain neurons. Obese due to overeating after (203) acute deletion of Ogt.

c-F-crystallin-rtTA:: dnOGA OGA overexpression in the eye. Premature cataracts. (204)

MCK-rtTA::dnOGA OGA overexpression in the skeletal muscle. Skeletal muscle atrophy. (205)

MMTV-cre::Ogaf/f Constitutive Oga ablation in mammary gland. Obesity, in females. (17)

MHC-cre::Ogtf/f Constitutive Ogt ablation in cardiomyocytes. Postnatal lethality, dilated (206) hearts, and signs of heart failure.

Oga-/- Germline knockout. Perinatal lethality. (17, 207)

Oga1/- Germline heterozygote. Lean not due to overeating or higher energy ex- (207) penditure, associated with higher RER.

Ogt-/- Germline knockout. Embryonic stem cell lethality. (16, 208)

Systemic inhibition of OGA by thiamet Decrease pathology. (60, 209) GinAb and Tau overexpression models

AgRP, Agouti-related protein. been limited to the glutamine analogs azaserine and phosphorylated by N-acetylglucosamine kinase to gener- 6-diazo-5-oxo-L-norleucine (DON), which are limited ate N-acetylglucosamine 6-phosphate. A second path- because of their lack of speciﬁcity (TABLE 1). way involves the conversion of N-acetyl-galactosamine to Glucosamine 6-phosphate N-acetyltransferase (GNPNAT, N-acetylgalactosamine-1-phosphate and UDP-N-acetylga- GNA1), known as Emeg32 in mice, uses acetyl-CoA to con- lactosamine, with subsequent conversion by an epimerase vert glucosamine 6-phosphate to N-acetylglucosamine to UDP-GlcNAc (FIGURE 4).Therelativecontribut- 6-phosphate (225), which is subsequently isomerized by ion of the salvage pathways to total UDP-GlcNAc syn- phosphoglucomutase (PGM) to N-acetylglucosamine-L-phos- thesis is not known. That deletion of GNPNAT gene is phate. The ﬁnalstepintheHBPisconversionofN-acetyl- embryonically lethal (228)suggeststhatdenovosyn- glucosamine-1-phosphate to UDP-GlcNAc, which is cata- thesis is likely the predominant pathway. On the lyzed by UDP-N-acetylglucosamine pyrophosphorylase other hand, while deletion of EMeg32 substantially (UAP1), also known as UDP-N-acetylhexosamine pyro- decreased UDP-GlcNAc levels, they were not elimi- phosphorylase (226). In addition to its de novo synthesis, nated. Further, the loss of EMeg32 did not lead to UDP-GlcNAc can also be generated by two salvage path- major changes in N-andO-glycosylation, whereas, ways (FIGURE 4)(227). In one pathway, GlcNAc is O-GlcNAc levels were markedly suppressed, suggesting

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Glucose

Glycogen Glycolysis Synthesis

HEX PGI Glucose Gluc-6P Fruc-6P PKA Glutamine GFAT AMPK Glutamate CaMKII GlcN-6P Glucosamine Acetyl-CoA CoA EMeg32 NAGK GlcNAc GlcNAc-6P

Agm1

GlcNAc-1P UTP N-and O-linked PPi Uap1 glycosylation EPI UDP-GalNAc UDP-GlcNAc IRS1

Uap1 AMPK Protein OGT GalNAc-1P UDP GSK3β

GALK2 CHK1 CaMKII Protein-O-GlcNAc GalNAc

OGA CK2

Protein + GlcNAc+ H2O

FIGURE 4. Schematic of UDP-GlcNAc and O-GlcNAc synthesis. Glucose enters the cell via the glucose transporter system where it is rapidly phosphorylated by hexokinase (HK) and converted to fructose-6-phosphate by phosphoglucoseisomerase (PGI). Fructose-6-phosphate is subsequently metabolized to glucosamine-6-phosphate by L-Glutamine: D-fructose-6-phosphate amidotransferase (GFAT), which requires glutamine. Glucosamine- 6-phosphate is converted to N-acetylglucosamine-6-phosphate by glucosamine 6-phosphate N-acetyltransferase (Emeg32), utilizing acetyl-CoA. Phosphoacetylglucosamine mutase (Agm1) converts N-acetylglucosamine-6-phosphate to N-acetylglucosamine-1-phosphate. The synthesis of uridine- diphosphate-N-acetylglucosamine (UDP-GlcNAc) is catalyzed by UDP-N-acetylglucosamine pyrophosphorylase (Uap1), which consumes uridine tri- phosphate (UTP). UDP-GlcNAc is the substrate for (O-GlcNAc transferase (OGT) leading to the formation of O-linked b-N-acetylglucosamine (O- GlcNAc)-modiﬁed proteins. b-N-acetylglucosaminidase (OGA) catalyzes the removal of O-GlcNAc from the proteins. GlcNAc can reenter the HBP via two salvage pathways: 1)viaN-acetylglucosamine kinase (NAGK) to generate N-acetylglucosamine 1-phosphate and 2) involving the conversion by N- acetylgalactosamine kinase (GALK2) of N-acetyl-galactosamine to N-acetylgalactosamine 1-phosphate and UDP-N-acetylgalactosamine, with subsequent conversion by an epimerase to UDP-GlcNAc. Glucosamine, which enters the cell via the glucose transport system and can be phosphorylated by hexokinase (HK) to form glucosamine 6-phosphate thereby bypassing GFAT. The kinases that have been identiﬁed as regulating GFAT, OGT, and OGA are indicated; additional details may be found in the text (see FIGURE 6 and TABLE 3). AMPK, AMPK-activated protein kinase; CaMKII, calcium/ calmodulin (Ca21/CaM) dependent protein kinase II; CHK1, checkpoint kinase-1; CK2, casein kinase 2; EPI, epimerase; GalNAc, glucosamine fructose-6- phosphate amidotransferase; GFAT, glucosamine fructose-6-phosphate amidotransferase; GlcNAc, N-acetylglucosamine; GlcNAc-1P, N-acetylglucosamine-1-phosphate; GSK3b, glycogen synthase kinase 3b; HEX, hexokinase; IRS1, insulin receptor substrate-1; PPi, pyrophosphate; UDP-GalNAc, uridine diphosphate N-acetylgalactosamine.

that salvage pathways were sufficient to maintain ER- and in which metabolic flux through glycolysis and the HBP Golgi-mediated glycosylation, but not O-GlcNAcylation was not directly measured, and the fraction of glucose (228). Alternatively, under conditions in which UDP- metabolized by the HBP was inferred from other meas- GlcNAc levels are limiting, N-andO-glycosylation are pri- urements (46). Moreover, energy metabolism, including oritized over O-GlcNA-cylation. glycolysis, varies widely from quiescent cells in culture It is frequently stated that 2–5% of glucose entering a to high-energy demands of the heart and brain. cell is metabolized via GFAT and the HBP. The origin of Consequently, the relative flux of glucose via glycolysis this statement is from a study using cultured adipocytes and HBP will also vary widely. There are very few studies

440 Physiol Rev VOL 101 APRIL 2021 www.prv.org O-GlcNACYLATION AND (PATHO)PHYSIOLOGY

fl 14 ubiquitination sites 20 phosphorylation sites glycolytic, not HBP, ux rates. This illustrates the limita- fl O O O tion of evaluating HBP ux as a fraction of glycolysis, as fl Ub C NH Lys HO P O OH well as the assumption that HBP ux will be similar NH OH 2 across biological systems with widely varying metabolic demands. While, much remains to be understood about Glucose PKA Ser-205/235 of the regulation of HBP, the implementation of new Glutamine fl GFAT AMPK techniques will enable direct measures of HBP ux in UDP-GlcNAc Ser-243 diverse biological systems. CaMKII GlcN-6-P 2.2. O-GlcNAc Transferase ATF4, Sp1, XBP1s O-GlcNAc transferase (OGT; uridine diphospho-N-acetyl- H N HN COO– glucosamine:polypeptide b-N-acetylglucosaminyltransfe- O O rase; EC 2.4.1.255) is a soluble glycosyltransferase primarily located in the cytoplasm and nucleus, which is respon- Acetylation at Succinylation at sible for using UDP-GlcNAc to modify proteins with K114, 547, 650 K529 O-GlcNAc (211, 231–234). OGT is located on the X-chro- FIGURE 5. Regulation of glucosamine fructose-6-phosphate ami- mosome and encodes a multidomain protein containing dotransferase (GFAT). GFAT activity is regulated at several levels multiple tetratriopeptide repeats (TPRs) in the NH2-termi- including substrate availability, feedback inhibition by uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) and glucosamine- nal domain and two catalytic domains in the COOH-termi- 6-phosphate (GlcN-6-P), as well as post-translational modifications nal region (FIGURE 6A)(211, 231–234). OGT is highly by phosphorylation, acetylation, succinylation, and ubiquitination. conserved, across all metazoans, and with the exception The only kinases that have been identified as phosphorylating of zebrafish (237, 238), a single gene encodes OGT. GFAT are PKA, AMPK, and calcium/calmodulin (Ca21/CaM) dependent protein kinase II (CaMKII). Sp1, ATF4, and XBP1s have been Alternative splicing results in three mammalian iso- shown to regulate GFAT at the transcriptional level. The database forms of OGT, which differ primarily by the number of phosphosite.org and the citations contained therein were used to TPRs. A form of OGT, epidermal growth factor do- identify known posttranslational modification sites of GFAT (196). main-specific OGT (EOGT), has also been identified ATF4, activating transcription factor 4; Lys, lysine; Sp1, specificity protein 1; XBP1s, X-box binding protein 1. in the ER, but it shares little homology with other forms of OGT. In contrast to OGT, it can also elongate O-GlcNAc into complex glycans (239), as reviewed in detail elsewhere (240, 241). that have directly measured the flux of glucose via the The full length or nucleocytoplasmic OGT (ncOGT; 110 HBP, relative to other glucose-utilizing pathways, using kDa) and short OGT (sOGT; 78 kDa) have up to 13.5 and radio- or stable-isotope techniques, either in cell culture 4 TPRs, respectively; the precise number of TPRs is spe- 13 or intact organs. In 2017, Gibb et al. (229), using C6-glu- cies specific(232). The mitochondrial OGT (mOGT; 90 cose labeling in cultured neonatal cardiomyocytes, con- kDa) has a mitochondrial targeting sequence at the NH2 cluded that glucose is metabolized more by the HBP terminus of the protein and 9 TPRs. All three OGT iso- than the pentose phosphate pathway, suggesting that forms have the same two catalytic domains, as well as a glucose utilization via the HBP could be considerably putative phosphatidylinositol (3,4,5)-trisphosphate (PIP3)- greater than previous estimates; however, this was binding domain (242); however, studies of the crystal under the low-energy demands of cell culture, as well as structure of human OGT-UDP-peptide complex were being in neonatal cardiomyocytes which would have dif- unable to confirm the presence of the PIP3 domain (90). ferent energetic demands from those in an adult heart. OGT is a member of the GT-B glycosyltransferase super- Olsen et al. (230) have recently developed an LC/MS family, and it is the only glycosyl transferase to have a technique using 13C-labeledsubstratestomeasurethe 120 amino acid sequence in the middle of the catalytic rate of glucose metabolism via the HBP at the same domain, known as the intervening domain (Int-D) (232, time as glycolytic flux. In the isolated perfused working 234, 243). The function of this region of the protein heart, an HBP flux of 2.5 nmol/g protein/min was meas- remains unclear, although it contains a large number of ured, which represented 0.003–0.006% of the glycolytic basic residues, suggesting contacts with negatively flux. This is several orders of magnitude lower than ear- charged partners, which may affect cellular localization lier estimates, most likely because of the high metabolic or protein-protein interactions (232, 243). rate of the heart. Moreover, when increasing glucose The catalytic domain of OGT represents less than half concentration from 5 to 25 mM, changes in HBP flux rel- of the total molecular weight of the protein with the re- ative to glycolysis occurred as a result of changes in mainder of the protein primarily consisting of the TPRs

Physiol Rev VOL 101 APRIL 2021 www.prv.org 441 CHATHAM ET AL.

A hOGT 1 466 496 697 827 997 1046 TPR

123456789101112 13 NLS CD I Int-D CD II

T1043 S 20 K 168 K 385 T 454 K 718 K 742 Y 976 K 991 390-426 S 3-4 CHK1 Phosphorylation GSK3β AMPK IRS1 CaMKII GlcNAcylation Acetylation

All reported OGT phosphorylation sites

S3 S4 S11T15 S20 S74 S78 T325S369 T444 T454 S491 Y844 T857 Y976 Y989 S1004 S1045

Caspase 3 B hOGA cleavage

160 366 393 555 715 916 Asp-413 LC N-acetyl glucosaminidase LC Stalk Pseudo-AT

S 405 K 599 All reported OGA phosphorylation sites

S6 T121 S268 S354 S364 T370 Y374 S375 S405 S509 S511 S515 S522 T709 S712 S859 S867 S873 S915

FIGURE 6. Structure and posttranslational modifications of human OGT (A)andOGA(B). Phosphorylation sites with identified kinases are shown as red circles, and all known phosphorylation sites are listed below the figures. O-GlcNAc sites are shown as blue squares, and acetylation sites are shown as green circles. AT, acetyl transferase; CaMKII, calcium/calmodulin (Ca21/CaM) dependent protein kinase II; CD, catalytic domain; CHK1, checkpoint kinase-1; GSK3b,glycogensynthasekinase3b; hOGT, human O-GlcNAc transferase; hOGA, huma O-GlcNAcase; Int-D, intervening domain; IRS1, insulin receptor substrate-1; LC, low complexity region; NLS, nuclear localization sequence; TPR, tetratricopeptide repeats. The databases https://www. phosphosite.org and http://www.phosphonet.ca/, and the citations contained therein were used to for known phosphorylation sites and acetylation modification sites(196). Citations for sites where kinases have been identified are included in the text; additional resources include Lundby, et al. (235), Levine and Walker (232) and Roth et al. (236). Other PTMs not shown include ubiquitination and sumoylation.

(234). TPRs comprise 34 amino acid motifs clustered in TPR 6–7 region prevent OGT dimerization, as well as repeats that form a-helical superspirals and have been reduce O-GlcNAc levels of nuclear pore glycoprotein proposed to play a key role in determining OGT interac- p62 (Nup62) (244). tions with target substrate proteins (234, 244). The im- Adefinitive consensus sequence for O-GlcNAcylation portance of the TPR domain in regulating OGT substrate has not been identified; consequently, although our selectivity was demonstrated by the observation that knowledge of OGT structure and its molecular interac- modification of only two aspartate residues in the TPR tions with substrates has improved, our knowledge of substantially changed OGT substrate preference (245). the regulation of OGT function and the mechanisms Although most OGT substrates require the presence of underlying its substrate specificity remains limited (231). the TPR domains for O-GlcNAcylation to occur, some Reports demonstrating that interactions between UDP- proteins only interact with the catalytic region (131, 232, GlcNAc and the peptide play a role in orientation of the 234). OGT exists as a homodimer (243, 244, 246), and peptide relative to the active site suggest that the active the TPR regions are also required for dimerization; how- site contributes to substrate selection (248). Moreover, ever, in vitro disruption of this interaction did not change preference by OGT for Ser/Thr residues that have pro- OGT activity (247). The function of OGT dimerization is lines and branched-chain amino acids in close proximity, unclear; however, it could serve to stabilize interactions resulting in an extended peptide orientation, also sug- with specific substrates. For example, mutations in the gests that the active site imposes some degree of

442 Physiol Rev VOL 101 APRIL 2021 www.prv.org O-GlcNACYLATION AND (PATHO)PHYSIOLOGY sequence constraint and, thus, substrate selection (249). recruit substrates on OGT. For example, in the liver dur- However, perhaps the most widely accepted view is that ing fasting host cell factor 1 (HCF1) targets OGT to peroxi- OGT substrate selection is largely determined by bind- some proliferator-activated receptor c coactivator 1a ing to the TPR domains. This is supported by the fact (PGC-1a), where increased O-GlcNAcylation increases that the TPR domains are essential for O-GlcNAcylation PGC-1a stability and upregulates gluconeogenic genes of most proteins, as well as the fact that specific sub- (118). Other such interactions include p38 MAPK, which strates interact with specific TPR regions. For example, recruits OGT to neurofilament-heavy polypeptide (NFH) in brain tissue, ataxin-10 (Atx-10) binds to TPRs 6–8 (256), REV-ERBa, which prevents OGT degradation (65), (246), whereas mSIN3A, ten-eleven translocation (TET) and OGA, which increases interactions between OGT 2/3, and trafficking kinesin protein 1 (TRAK1) all require and pyruvate kinase isoform M2 (PKM2) (257). The TPRs 1–6(232). Moreover, mutations of just two aspar- potential for an OGT-OGA interaction raises the possibil- tate residues in the TPR domain changed OGT activity ity of an “O-GlcNAczyme” complex, which would be as well as target specificity (245). Structural studies have consistent with rapid and reversible changes in revealed a hinge region around TPRs 12–13, which could O-GlcNAcylation (258). Insulin treatment results in trans- influence the access of protein substrates to the active location of OGT from the nucleus to the cytosol and site of OGT (232, 234). There is also evidence that under plasma membrane, and changes in nutrient availability some conditions, sOGT can act as a negative regulator also lead to redistribution of OGT between the nucleus of ncOGT (233, 246). The importance of the TPR domain and cytosol (242, 251). The three OGT isoforms also ex- is reflected in the observations that missense mutations hibit differences in their subcellular localization, and in this region result in decreased OGT function and neu- along with differences in the TPR regions between the rodevelopmental abnormalities (29, 250). isoforms, different proteome subsets can result; how- OGT activity and substrate recognition can also be ever, this has only been demonstrated with in vitro stud- regulated by phosphorylation of OGT on Ser, Thr, and ty- ies (169). rosine (Tyr) residues (233). Almost 20 different phospho- In addition to its role as a glycosyl transferase, OGT rylation sites have been reported on OGT, many also exhibits protease activity, although, to date, only identified by large-scale proteomic studies (FIGURE 6A). one proteolytic substrate, the transcriptional coregulator Although the function of many of these sites remains HCF1, has been identified (259, 260). To fully function as unknown, as do the kinase(s) responsible for their phos- a coregulator HCF1 is required to undergo proteolytic phorylation, a few have been characterized. For example, cleavage;however,themechanismbywhichthis insulin increases Tyr phosphorylation of OGT via activa- occurred remained elusive until 2011 when two reports tion of the insulin receptor (IR), resulting in increased OGT demonstrated that OGT played a key role in this process activity (251). Glycogen synthase kinase (GSK)-3b phos- (259, 260). These studies demonstrated that both UDP- phorylates OGT on Ser-3/4, leading to increased OGT ac- GlcNAc and OGT were required for HCF1 proteolysis, tivity (116), and AMPK phosphorylates Thr-444, resulting in consistent with the notion that O-glycosylation of HCF1 changes in subcellular localization and substrate binding was necessary. It was initially thought that OGT had a targets (252). In addition, OGT is phosphorylated on Ser- specific protease active site; however, subsequent stud- 20 by checkpoint kinase 1 (Chk1), leading to stabilization ies demonstrated that the COOH-terminal of HCF1 binds of OGT, which is required for cytokinesis (253). Ser-20 on to the TPR domain of OGT, and the cleavage region in OGT is also a target for CaMKII, which increases its activ- the glycosyltransferase active site was similar to that for ity (181). O-GlcNAcylation of Ser-389, located in the TPR regular glycosylation substrates (261). Moreover, these domain regulates OGT nuclear localization (254). Serines studies also showed that UDP-5SGlcNAc, which binds 3 and 4 on OGT are also sites of O-GlcNAcylation (116); to the OGT-active site in the same manner as UDP- however, the function of this modification remains to be GlcNAc, but is resistant to glycosyltransferase, inhibited determined. Recent work that has focused on some of HCF1 proteolysis. The role of OGT mediated cleavage of these phosphorylation sites has identified that in sOGT, HCF1 remains unclear; however, it could represent a link mutation of either Thr-12 or Ser-56 to an alanine signifi- between cellular metabolism and the regulation of cell cantly altered substrate binding by over 500 proteins cycle, which is supported by reports that the OGT/HCF1 (255). OGT is also acetylated on multiple residues complex itself is an important regulator of glucose me- (FIGURE 6A)(235). While the effects of this modification tabolism (118). are not known, the fact that two of the sites occur within one of the catalytic domains suggests they could regulate 2.3. O-GlcNAcase OGT activity in some manner. Targeting of specific proteins by OGT can also occur O-GlcNAcase (OGA, EC 3.2.1.169) is a hexosaminidase via its interaction with adaptor or scaffold proteins, which that was first characterized in 1994 by Dong and Hart (11)

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following purification from rat spleen; it has also been interactions with different peptide substrates, which referred to as NCOAT, nuclear cytoplasmic O-GlcNA- has implications for differential regulation of O-GlcNAc- case and acetyltransferase (262), GCA (263), MEA5, and ylation on different proteins (278,279). While the active MGEA5 (264). Dong and Hart (11) demonstrated that it site of OGA is now better characterized than ever was distinct from lysosomal hexosaminidases by virtue before, other regions, including the low-complexity of its subcellular localization to the cytosol and nucleus, region and the pseudo-AT domain remain poorly under- its neutral pH optimum, and its selectivity for GlcNAc stood (266). over GalNAc. Subsequent sequence identification and Similar to OGT, OGA is also subject to both phospho- cloning demonstrated that OGA was identical to a previ- rylation and O-GlcNAcylation. At least 20 different Ser, ously identified protein, MGEA5, found in meningioma Thr, and Tyr phosphorylation sites have been mapped patients (264). The human OGA gene is located on chro- by MS in both the glycosyl hydrolase and pseudo-HAT mosome 10 and encodes a protein with an NH2-terminal domains (FIGURE 6B); however, the effects of these glycosyl hydrolase domain and a COOH-terminal do- modifications on OGA activity have not been deter- main that demonstrates sequence homology to histone mined. Interestingly, the Ser-405 O-GlcNAcylation site acetyl transferase (AT) proteins (FIGURE 6B). The glyco- on OGA, is in the central low-complexity region, which is syl hydrolase domain has two low-complexity or disor- also the region where OGA interacts with OGT (266). dered regions [i.e., repeats of single amino acids or Thus, O-GlcNAcylation of this residue plays a role in the short amino acid motifs (265)], on either side. The larger regulation of the interaction between OGT and OGA. low-complexity region is followed by the stalk domain, OGA is also acetylated in the stalk domain at Lys-599 which connects to the COOH-terminal domain (231, (235). 266). Between the catalytic domain and the COOH-terminal region is a noncanonical caspase cleavage site 2.4. Maintenance of O-GlcNAc Homeostasis (267). It has been reported that OGA exhibited AT activity (199, 268); however, this has not been replicated by Changes in cellular O-GlcNAc levels occur in response to others (269). Recent structural studies found that human a diverse array of physiological and pathological stimuli, OGA (hOGA) lacks the residues necessary for binding to and dysregulation of O-GlcNAc homeostasis has been acetyl-CoA and described this region as a “catalytically linked to a wide array of diseases, as discussed in detail incompetent pseudo-AT domain” (270). Alternative splic- later. Deletion of the OGT gene is embryonically lethal ing results in the generation of a short OGA (sOGA), (208), and in cell culture, mouse embryonic fibroblasts which lacks the pseudo-AT region and has a different 15- (MEFs) die around 4–5daysafterOGTisknockedout amino acid COOH-terminal region (271). sOGA has lower (280). Although OGA knockout mice survive to birth, the hexosaminidase activity than hOGA in vitro (56, 272)and majoritydiebeforeweaning(17). Chronic increases in O- appears primarily localized to the surface of lipid droplets GlcNAc levels induced by overexpression of a dominant (273), although its specific function remains to be eluci- negative OGA (dnOGA) in a tissue-specificmannerresults dated. in apoptosis in skeletal muscle (205) and altered metabo- For many years, full-length hOGA proved resistant to lism in the heart (281). Therefore, it is evident that mainte- crystallization, consequently, most of the initial structural nance of O-GlcNAc homeostasis is essential for the and mechanistic information were derived from bacterial normal physiological function of cells and tissues. This homologs of OGA (274). In 2017, three independent has led to the concept that there is an optimal range of studies reported the crystal structure of hOGA using dif- O-GlcNAcylation that supports normal physiological proc- ferent, catalytically functional, truncated versions of the esses and that outside that range, either too high or too protein (236, 275, 276). A key and unexpected finding in low, cellular dysfunction results. Consequently, it has all three studies was that hOGA formed an obligate been proposed that to keep within this optimal range, homodimer in which the stalk domain of one monomer OGT and OGA work together to form a “buffering” is positioned over the catalytic site of the other mono- system that can respond to moderate changes in mer (266). One consequence of this arrangement is the O-GlcNAcylation (257). creation of a substrate binding site, comprising con- The prevailing wisdom for many years was that the served hydrophobic residues, which supports the notion primary mechanism regulating cellular O-GlcNAc levels that the dimer is the active form of OGA. Further struc- was nutrient availability, so that under conditions of nu- tural analysis suggests OGA may preferentially remove trient excess, such as hyperglycemia, O-GlcNAc levels O-GlcNAc from certain sites, suggesting that OGA may increased, and if glucose levels dropped O-GlcNAc lev- be an equal partner with OGT in regulating O-GlcNAc els would decrease. This concept is consistent with the turnover (277). Moreover, it has also been reported that fact that glucosamine, which bypasses GFAT, leads to a number of specific residues on OGA contribute to its uncontrolled synthesis of UDP-GlcNAc and increased O-

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GlcNAc levels (24, 271, 282, 283). This also enables glu- mechanism by which leptin stimulated GFAT expression cosamine to be used pharmacologically to increase and O-GlcNAc levels was not identified. Ghrelin, a hor- UDP-GlcNAc synthesis and O-GlcNAc levels. Acute mone that is released in response to fasting, increases changes in glucose availability either by increasing ex- O-GlcNAc levels in hypothalamic appetite-stimulating ogenous glucose or via insulin-stimulated increases in agouti-related protein (AgRP) neurons, leading to their glucose uptake can have little or no effect on cellular increased firing rate (201). In the liver, glucagon, which O-GlcNAc levels (284). This is also consistent with the increases in response to fasting, increased OGT phos- report that a five-fold increase in glucose had no effect phorylation and O-GlcNAc levels in a CaMKII-dependent on HBP flux in the isolated perfused heart (230). manner (181). However, such responses likely vary greatly, depending In addition to nutrient-dependent hormones, G pro- on cell type and duration of treatments (285). In addition, tein-coupled receptor agonists have also been shown to OGT activity is very sensitive to UDP-GlcNAc concentra- increase cellular O-GlcNAc levels. For example, activa- tions, exhibiting multiple apparent Km values for UDP- tion of the endothelin (ET)A receptor with ET-1 resulted in GlcNAc under varying UDP-GlcNAc concentrations a time-dependent increase in O-GlcNAc levels in vascu- (247). Consequently, changes in UDP-GlcNAc concen- lar cells, and the subsequent downstream signaling by trations can directly influenceglobalaswellasregional ET-1 was dependent on the increase in O-GlcNAc levels OGT activities and O-GlcNAc levels. (296, 297). Phenylephrine (PE), primarily an a-receptor However, while nutrient availability as a regulatory agonist, also increased O-GlcNAc levels in cardiomyo- mechanism is a valuable concept, it does not address cytes, and the increase in O-GlcNAc was required for the mechanisms that underlie widely differing rates of subsequent activation of PE-mediated signaling path- changes in O-GlcNAcylation that occur in response to ways (298, 299). One explanation for PE-dependent different stimuli. For example, depolarization of neuro- increases in O-GlcNAc levels was an increase in GFAT blastoma cells dramatically increased OGT activity, expression (298, 299). Another study suggested that reaching a peak within 1 min, leading to increased the increase was mediated by the Ca21 dependent O-GlcNAc levels (286), and stimulation of human neutro- CaMKII/calcineurin pathway (298). phils leads to an approximately five fold increase in The transcriptional regulation of OGT and OGA has O-GlcNAc within 30 s, returning to normal levels over been understudied; however, there are a number of the next 5–10 min (287). On the other hand, stress, such reports indicating that they regulate each other and that as ischemia (288) or heat shock occur over periods of O-GlcNAcylation itself might contribute to their transcrip- minutes to hours, induced increases in O-GlcNAc levels tional regulation (257), further discussed in Sect. IIIA and (289). Conversely, in certain disease states, such as can- IIIB. For example, in OGT knockout MEFs the time cer (21, 290), diabetes (283, 290), or cardiac hypertro- course of loss of OGT was paralleled by a decrease in phy (291,292), O-GlcNAc levels are chronically elevated, OGA (280). Increasing O-GlcNAc levels via OGA in- and the mechanisms that lead to this resetting of hibition demonstrated that OGA transcription was steady-state O-GlcNAc levels outside of the normal O-GlcNAc dependent (300). Other studies have range remains poorly understood (293). As discussed reported that low O-GlcNAc levels contribute to above, GFAT, OGT, and OGA are all modified by phos- increased OGT transcription (68). The strongest evi- phorylation, indicating that these posttranslational modi- dence to date of mutual regulation of OGT and OGA fications may contribute to the dynamic regulation of was by Qian et al. (301), who reported that overexpres- O-GlcNAcylation. sion of OGA resulted in increased OGT transcription, Given that HBP flux and O-GlcNAc levels are modu- whereas knockdown OGA with siRNA significantly lated, in part, by nutrient availability, it is perhaps not sur- decreased OGT levels. Using promoter luciferase prising that O-GlcNAc levels can also be regulated by reporters for both OGT and OGA, they clearly demon- nutrient-regulating hormones. Insulin is probably the strated reciprocal transcriptional regulation (301). They most studied of these, and it has been shown that insulin also showed that OGA cooperated with p300m, a his- treatment recruits OGT from the nucleus to the plasma tone acetyltransferase and the transcription factor membrane, where it is phosphorylated by the IR (242, CCAAT/enhancer-binding protein-b (C/EBP-b)topro- 251, 294). This phosphorylation increases OGT activity mote OGT transcription (301). E2F transcription factor 1 and leads to O-GlcNAcylation of insulin receptor sub- (E2F1), which contributes to the activation of many strate-1 (IRS1) and AKT, resulting in attenuation of insulin genes, was found to be a repressor of both OGT and signaling. Treatment of HepG2 cells with the adipokine OGA (302). It has been shown that E2F1 activity itself leptin resulted in an approximately twofold increase might be regulated by O-GlcNAcylation, illustrating in overall O-GlcNAc levels within 15 min with parallel another mechanism by which O-GlcNAc can regulate increases in GFAT protein levels (295). However, the the transcription of OGT and OGA (303). The OGT

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promoter contains a TATA box, which likely facilitates Ca21 was required for this increase in O-GlcNAc levels, OGT transcription, whereas the OGA transcription is not and inhibition of CaMKII blunted the response to glu- dependent on a TATA box (302). In macrophages, Cullin cose deprivation (312). 3 (CUL3), an E3 ubiquitin ligase, has been reported to Historically, nutrient availability was considered to downregulate OGT expression in a nuclear factor E2- be the primary regulator of cellular O-GlcNAc homeo- related factor-2 (Nrf2)-dependent manner (304). stasis; however, it is increasingly evident that it is Another potential mechanism for maintaining only one of many factors. Our knowledge of tran- O-GlcNAc homeostasis is alternative splicing of both scriptional regulation is growing, but it remains lim- OGT and OGA, resulting in intron retention, which is ited, and the role of phosphorylation in regulating regulated by changes in O-GlcNAc levels (68, 305). GFAT and OGT activity is improving; however, the Consequently, when O-GlcNAc levels are high, nuclear role of phosphorylation in regulating OGA activity is retention of OGT due to intron retention increases, underexplored. Several lines of evidence suggest 1 whereas low-O-GlcNAc levels decrease this process; that Ca2 -mediated activation of CaMKII contributes thereby, decreasing or increasing OGT protein, respec- to O-GlcNAc homeostasis, which would be consistent tively (68, 305). Conversely, low O-GlcNAc levels with rapid agonist-induced increases in O-GlcNAc increase nuclear retention of OGA (305). It is noteworthy being independent of nutrient availability or tran- that these changes occur relatively rapidly and, as such, scriptional regulation of GFAT1, OGT, and OGA. represent a potentially important mechanism in O- GlcNAc-mediated regulation of O-GlcNAc homeostasis 2.5. Cross Talk Between O-GlcNAcylation and (305). A number of microRNAs (miRs), including miR-101, Phosphorylation 200a/b, 423-5p, 501-3p, 539, and 619-3p, have also been shown to regulate O-GlcNAc levels by targeting As the dynamic nature of O-GlcNAcylation was recog- OGT (306–309)orOGA(310). Consequently, although nized, there was speculation that it might play a regula- our knowledge of the transcriptional regulation of OGT tory role in protein function. Once it was recognized that and OGA is improving, the physiological role of these O-GlcNAc modified Ser and Thr residues, which are also pathways remains to be determined. potential sites of phosphorylation, there was increasing One of the more puzzling aspects of O-GlcNAc home- consideration about possible interactions between ostasis is that glucose deprivation leads to a marked O-GlcNAc and phosphorylation on proteins (211, 293). increase in overall cellular O-GlcNAc levels. This was One concept that gained early popularity was the possi- first reported in HepG2 cells where it was observed that bility of reciprocity between O-GlcNAcylation and phos- 12 h following the removal of glucose, there was approxi- phorylation. In other words, that a specific residue on a mately eight-fold increase in O-GlcNAc levels, which protein could be modified by either O-GlcNAc or phos- was accompanied by a 40% decrease in UDP-GlcNAc phorylation became more widely known as the “Ying- levels, suggesting that increased HBP flux was not con- Yang” hypothesis (315). There are, indeed, several pro- tributing to the elevated O-GlcNAcylation (311). A later teins that support this concept, such as cMyc (Thr-58) study came to a similar conclusion, as the addition (98), estrogen receptor (ER)-b (Ser-16), and endothelial of 50–100 mM glucosamine blocked the O-GlcNAc nitric oxide synthase (eNOS) (Ser-1177) (190). increase, resulting from glucose deprivation (312). Alternatively, modification of adjacent sites can nega- Contrary to this finding was a report that glycogen deg- tively interact with each other, for example, histone radation triggered by glucose deprivation provided the deacetylase 4 (HDAC4) is O-GlcNAcylated at Ser-642, substrate for O-GlcNAcylation (313). Moreover, ATF4, a and this blocks CaMKII-mediated phosphorylation of regulator of the unfolded protein response (UPR), Ser-632 (316). Consequently, it is becoming increasingly increased GFAT1 expression in response to glucose de- clear that the cross talk between O-GlcNAc and phos- privation, and ATF4 inhibition or knockdown prevented phorylation is much more complex than first thought the increase in O-GlcNAc and GFAT1 (221). They also (FIGURE 7). A high-throughput proteomic analysis esti- suggested that ATF4, along with another UPR-related mated that in only 8% of O-GlcNAcylated proteins was protein X-box binding protein 1 (XBP1), mediated the thesameresiduemodified by phosphorylation (182). steady-state levels of GFAT1 (221). This is consistent with Some proteins are modified by both O-GlcNAc and phos- an earlier report demonstrating that stress-indu- phate, but not at the same sites; for example, increases in ced increases in O-GlcNAc were mediated via XBP1 phosphorylation of Thr-200 of CaMKIV decreases overall increases in GFAT1 protein levels (314). Glucose depriva- O-GlcNAcylation at several sites, including Ser-189, and tion increased mRNA levels of both OGT and OGA; how- conversely, prevention of Thr-200 phosphorylation ever, this was not associated with an increase in the increased CaMKIV O-GlcNAcylation (167). In myosin light levels of either protein (312). Interestingly, extracellular chain 1, the O-GlcNAc-modified sites are Thr-93 and Thr-

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A B

i) i) O-GlcNAc O-PO O-GlcNAc OGT 3 O-PO3 OGA PPT Kinase OGA Kinase Kinase Kinase OGT Kinase PPT OGT OGA Increased Low activity Inactive Activity e.g. Akt or GSK3

ii) O-GlcNAc ii) OGA O-PO O-GlcNAc PPT 3 OGA Low activity Kinase Kinase Kinase Inactive OGT OGT Kinase O-GlcNAc

O-PO 3 PPT OGA PPT OGA O-PO 3 OGT Kinase OGT Kinase O-PO3

C O-GlcNAc OGA O-PO3 O-GlcNAc O-PO 3 Kinase O-PO3 O-PO 3 O-PO OGA PPT Low activity Kinase Kinase 3 PPT PPT OGT OGT PPT Active OGT Kinase OGT Low Increased Low Increased activity Activity activity Activity

e.g. PTP1B e.g. CaMKII or GSK3

FIGURE 7. Extensive crosstalk between phosphorylation and O-GlcNAcylation. A, i: example of phosphorylation and O-GlcNAcylation of the same or adjacent sites that prevent simultaneous modifications. and A, ii: modification by both phosphorylation and O-GlcNAcylation, as well as O- GlcNAcylation enhancing phosphorylation via for example increased binding to adaptor proteins. B, i: example of a kinase that is activated when phosphorylated and inactivated by O-GlcNAcylation. B, ii: a kinase that can be modified by both O-GlcNAc and phosphorylation involving complex interactions between OGT/OGA and kinases/phosphatases. C: O-GlcNAcylation also regulates phosphorylation via modification of phosphatases, and OGT activity is directly regulated by kinase phosphorylation. GSK3b, glycogen synthase kinase 3b;OGA,O-GlcNAcase; OGT, O-GlcNAc transferase; PPT, protein phosphatase.

164, whereas the phosphorylation sites are at Thr-69 and two posttranslational modifications is complex, and it is Ser-200 (147). currently not possible to predict a priori how they The intricate relationship between O-GlcNAcylation will interact on individual proteins. However, specific and phosphorylation was demonstrated by a study motifs have been identified, which when phosphoryl- in which GSK3b was inhibited and O-GlcNAcylation lev- ated, strongly inhibit O-GlcNAcylation and vice versa; els of specific proteins were quantified. Of the 45 whereas, sites that are phosphorylated by proline- O-GlcNAcylated proteins identified, 10 exhibited an directed kinases do not appear to be subject to increase in O-GlcNAcylation, whereas 19 showed a O-GlcNAcylation (321). decrease (317). In another study in which O-GlcNAc lev- Recent studies have demonstrated that OGT and els were increased by inhibition of OGA, increased OGA form complexes with both kinases and phospha- phosphorylation was observed on 148 sites and lower tases (322–324)demonstratingthatchangesin phosphorylation was observed at 280 sites (318). A com- O-GlcNAcylation and phosphorylation could be occur- parison between wild-type and OGT-null cells identified ring simultaneously on a target protein (FIGURE 7A). 232 phosphosites that were upregulated and 133 phos- There is also a growing list of kinases that have phosites that were downregulated 36 h after OGT dele- been shown to be O-GlcNAcylated and that this modi- tion (319). Interestingly O-GlcNAc modifications have fication directly affects their function (see TABLE 2 been reported to occur in clusters (182), a phenome- and FIGURE 7B). An analysis of glycoproteomic data non that is also observed with phosphorylation (320). sets reported that more than 100 kinases contain - Therefore, it is evident that the cross talk between these identified O-GlcNAcylation sites, emphasizing the

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importance of O-GlcNAc in regulating kinase function phosphorylation leads to changes in OGT cellular local- (325). A study of synaptic proteins found that kinases ization and substrate specificity (252). Interestingly, were more frequently a target for O-GlcNAcylation although OGA has not been shown to be a target for than other proteins; however, the specificmodification AMPK,deletionoftheAMPKa2isoformresultsinlower sites were frequently outside the catalytic domain of OGA protein levels and increased O-GlcNAc levels, with- the kinases in question (182). Thus, protein O- out changes in either OGT or GFAT (299). Bullen et al. GlcNAcylation can alter phosphorylation either via (252), demonstrated that all a-andc-subunits of AMPK direct modification of phosphorylated proteins, but are O-GlcNAcylated and further that activation of AMPK also by regulation of kinases that are responsible for increased O-GlcNAcylation of the c1-subunit. They also phosphorylation. Although there is less known about reported that inhibition of OGA attenuated physiological O-GlcNAcylation of phosphatases, protein tyrosine and pharmacological activation of AMPK. phosphatase 1B (PTP1B) has been shown to be O- Although Ser and Thr residues are often the focus of GlcNAcylated at Ser-104, Ser-201, and Ser-386, result- discussions of cross talk between O-GlcNAcylation and ing in increased enzymatic activity (176). phosphorylation, it is also clear that Tyr phosphorylation One specific example of the complex regulation and O-GlcNAcylation also influence each other (326). An between kinases and O-GlcNAcylation is CaMKIV, analysis of a small set of O-GlcNAcylated proteins con- which is also known to activate OGT (286)(FIGURE cluded that >60% of them were also Tyr phosphorylated 7C). Activation/deactivation of CaMKIV is rapid and (326). The potential importance of interactions between tightly regulated beginning with displacement of pro- O-GlcNAc and Tyr phosphorylation was first recognized, tein phosphatase 2a (PP2A) and subsequent phos- when it was demonstrated that insulin stimulates Tyr phorylation of Thr-200 by CaMKK; subsequent phosphorylation of OGT via the IR, thereby increasing inactivation, involves the reassociation with PP2A OGT activity (251). A subsequent study revealed that and reduction in phosphorylation. Dias et al. (167) increased O-GlcNAc levels reduced Tyr phosphorylation found that CaMKIV contained at least five O-GlcNAc of IRS-1 (327). Studies on prohibitin, reported that O- modification sites and that during its activation, O- GlcNAcylation attenuated its Tyr phosphorylation (328). GlcNAc levels rapidly decreased as the interaction Using peptides that contained residues that could be with OGA increased. Following inactivation, CaMKIV both Tyr phosphorylated and O-GlcNAcylated, Ande et O-GlcNAc levels returned to normal, suggesting that al. (328)showedthatwhileO-GlcNAcylation reduced OGT was recruited to CaMKIV; however, a direct Tyr phosphorylation, an increase in Tyr phosphorylation interaction was not identified (167). Mutation of Thr- actually enhanced O-GlcNAcylation. Another study 200 to alanine, thereby, preventing phosphorylation, using peptide microarrays concluded that Tyr phospho- resulted in an increase in CaMKIV O-GlcNAcylation; rylation may have a greater effect on the regulation of conversely, mutation to glutamate to mimic phospho- O-GlcNAcylation than O-GlcNAcylation on phosphoryla- rylation led to lower O-GlcNAc levels, demonstrating tion (329). a direct interaction between Thr-200 and O- Here, the focus has been on interactions between GlcNAcylation. Of the five O-GlcNAc sites identified, phosphorylation and O-GlcNAcylation; however, O- three Ser-189, Thr-57, and Ser-58, when mutated to GlcNAcylation also interacts with other PTMs, although alanine to prevent O-GlcNAcylation, markedly these are less widely studied (257). For example, both reduced Thr-200 phosphorylation. The double-mu- OGT and OGA have been found to be ubiquitinated, tant T57A/S58A resulted in no measurable kinase ac- and O-GlcNAc has been shown to stabilize proteins by tivity (167). Conversely, the S189A mutant, not only inhibiting their ubiquitination (330, 331). RelA/p65, a dramatically reduced O-GlcNAc levels, but also member of the nuclear factor κ-light-chain enhancer of increased basal kinase activity. O-GlcNAcylation of activated B cells (NF-κB) family of transcription factors, CaMKIIhasalsobeenshowntoincreaseitskinase is both O-GlcNAcylated (Thr-305, Ser-319, Ser-337, activity (165). Thr-352, and Ser-374) and acetylated (lysine-310). Ace- AMPK is another key example of a kinase that both reg- tylation is required for full transcriptional activity of RelA/ ulates O-GlcNAc levels, as well as being regulated by O- p65, and O-GlcNAcylation at Thr-305 and Thr-315 pro- GlcNAc itself, as reviewed in detail (219). As discussed motes its acetylation at lysine-310 (332, 333). In addition, earlier, GFAT is phosphorylated on Ser-243 by AMPK, both OGT and OGA are also acetylated, although the resulting in decreased GFAT activity and lower O-GlcNAc effect of this modification on their function is not known levels (130, 218). Moreover, activation of AMPK leads to (FIGURE 6)(235). Of note, HDAC4 has been shown to increased GFAT phosphorylation and decreased O- be modified by O-GlcNAc on Ser-642, providing further GlcNAc levels (299). AMPK also targets OGT by phospho- evidence of direct interactions between O-GlcNA cyla- rylating Thr-444; however, rather than alter its activity, this tion and acetylation (316). As further discussed in the

448 Physiol Rev VOL 101 APRIL 2021 www.prv.org O-GlcNACYLATION AND (PATHO)PHYSIOLOGY next section, cross talk between acetylation and O- clues as to how diabetes, for example, could disrupt cel- GlcNAcylation has also been implicated in epigenetic lular processes of signaling via decreased Sp1 transcrip- regulation (334). tional activation. In the context of diabetes, O-GlcNAc modification of Sp1 was further shown to modify transcriptional activity because of a dynamic interplay with 3. O-GlcNACYLATION AND CELLULAR its phosphorylation status, which further alters Sp1 sub- FUNCTION cellular compartmentalization and activity (338) and may impact transcriptional regulation of mitochondrial func- In the prior sections, we discussed the discovery of O- tion (339). Additionally, Sp1 O-GlcNAcylation may simply GlcNAcylation, its unique place in glycobiology, and the interfere with its interaction with other transcription fac- numerous pathways that impact its regulation. In this tors, such as Elf-1 (340), NF-Y (341), Oct1 (342), as well as section, we will focus on the cellular functions of regula- Sp3 and Sp4 (343), highlighting the diverse set of mech- tion by O-GlcNAcylation, including modulation of gene anisms by which O-GlcNAcylation of transcription factors expression at both the levels of transcription, as well as mediate gene expression. via epigenetic mechanisms, followed by specific exam- Although much of the early work was done on O- ples of how O-GlcNAcylation impacts cellular signaling GlcNAc modification of Sp1, a number of subsequent (e.g., insulin and calcium), metabolism (e.g., mitochon- studies have identified a continuously growing list of dria), and survival (e.g., autophagy). transcription factors with a direct O-GlcNAc modification and functional consequence. This includes additional 3.1. Transcription key transcriptional regulators of insulin signaling and metabolism, such as brain and muscle ARNT-like 1 Gene expression is controlled at a number of levels (BMAL1) (344), carbohydrate-responsive element-bind- (FIGURE 8). First, at the level of DNA sequence are ing protein (ChREBP) (345), Forkhead Box O1 (FOXO1) response elements that recruit transcriptional regulators (105, 346), liver X receptor (LXR)a (111, 347), PGC-1a (117), that include numerous families of transcription factors. and peroxisome proliferator-activated receptors c However, the regulation of gene expression is intricately (PPARc)(119), as well as a growing number of transcrip- controlled beyond proper recruitment of transcriptional tion factors involved in cancer: GLI (348), HIC1 (349), κ machinery to the promoter regions of genes that contain LXRa/b (350), and NF- B(333), and many others (TABLE these response elements. In particular, the direct modifi- 2). In the case of ChREBP (FIGURE 8A, II), O- cation of transcription factors and the transcriptional ma- GlcNAcylation can again have both activating, as well as chinery can be either inhibitory or activating in altering inhibitory roles (345). In the context of high glucose con- RNA levels. One of the first molecular functions to show ditions, parallel phosphorylation enhances O-GlcNAc biological relevance of O-GlcNAc-mediated regulation levels to maintain transcriptional activity, which was con- is modification of these transcription factors (210). sistentwithearlierfindings showing higher hepatic The early studies started with an initial focus on O- ChREBP O-GlcNAcylation and transcriptional activity in GlcNAcylation of the ubiquitously expressed Sp1, a zinc diabetic mice (351). However, under normal glucose finger transcription factor that binds GC-rich motifs of conditions ChREBP is O-GlcNAc modified at a different many promoters (FIGURE 8A, I). Work by Jackson and Ser residue, which increases its interaction with other Tjian (335) found that increased O-GlcNAcylation on Sp1 factors, such as 14-3-3, resulting in nuclear exclusion and from either Drosophila or human cells increased its tran- decreased transcriptional activity (345). These findings scriptional activity. The mechanism of this transcriptional demonstrate the dynamic, and sometimes opposing, activation was identified by Kudlow and colleagues roles that O-GlcNAcylation can have on transcriptional (336, 337), who reported that the O-GlcNAc modification regulation. Although not comprehensive, these exam- protected Sp1 from proteasomal degradation. In turn, ples, and numerous others, solidify our knowledge that this allowed O-GlcNAc modification of Sp1 to act as a modification of transcription factors by O-GlcNAcylation is nutritional checkpoint for times of inadequate nutrients, responsible for changing transcriptional activity through hypoglycosylation, and resource sparing through dec- altered DNA binding, localization, stability, and interaction reased transcription (336). Subsequent studies on the with other coregulators (352). mechanisms of O-GlcNAc action on transcriptional regu- The second level of transcriptional regulation is on the lation also found that within this same factor, Sp1 could transcriptional machinery itself. Although the earliest be O-GlcNAcylated at its activation domain to suppress studies were focused on the modification of the tran- transcriptional activity (109), suggesting multiple and scription factors, it was also recognized by the early opposing roles for this single modification depending on 1990s that RNA polymerase II (RNAP II) is a direct target context. These mechanistic insights provided important of protein O-GlcNAcylation at its COOH-terminal domain

Physiol Rev VOL 101 APRIL 2021 www.prv.org 449 CHATHAM ET AL.

A Transcriptional regulation G

G G UDP– G O NPC U Glucose G 14-3-3 G IV G RNAP II I II III

Sp1 G ChREBP

Sp1 G ChREBP Proteasome O PP G Glucose

B Epigenetic regulation

OGT IncRNA III G EZH2 OGT ncRNA

OGT OGT miR OGT M G miR-200a/b SIN3a G TET XIST

A OGA 5hmC miR-539 5mC OGA

I Histone Modifications II DNA Modifications

FIGURE 8. O-linked b-N-acetylglucosamine (O-GlcNAc) regulation of gene expression and epigenetics. A: examples of transcriptional regulation mediated by O-GlcNAc with additional details and references highlighted in the text. These examples include both inhibitory and activating roles of O- GlcNAc (blue square with G). A, I: for the transcription factor Sp1, one O-GlcNAc modification blocks an activation site to inhibit transcription, while a different O-GlcNAc site inhibits proteasomal degradation increasing transcription. A, II: a separate set of examples of this dual role of O-GlcNAc-mediated regulation is at ChREBP, which, under normal glucose levels, leads to O-GlcNAc modification (blue square) to enhance 14-3-3 binding decreasing transcriptional activity, while under high glucose levels, additional posttranslational regulation by phosphorylation (red circle with P) leads to a different O- GlcNAc modification and augments activity. A, III: multiple roles are shown for both direct O-GlcNAc modification of RNA polymerase II (RNAP II), as well as the contribution of uridine diphosphate-azido-modified galactose (UDP)-GlcNAc hydrolysis to enhance transcriptional activity. A, IV:multiple roles are shown for different O-GlcNAc modifications to either inhibit ubiquitination (gray square with U) to increase complex stability or other modifications that may impact nuclear cytoplasmic import/export of cargo (e.g., RNA). B: examples of epigenetic regulation mediated by O-GlcNAc with additional details and references highlighted in the text. B, I: highlights the multiple direct (i.e., O-GlcNAc of histone proteins) and indirect (i.e. interaction and modification of other histone modifiers), such as EZH2 to impact histone methylation (black square with M) and SIN3a to impact histone acetylation (purple square with A). B, II: highlights the interaction of O-GlcNAc transferase (OGT) with the ten-eleven translocation (TET) enzymes impacting DNA 5 hydroxymethylation (5hmC) which, in turn, can lead to DNA demethylation. B, III: a few examples are highlighted from the text of noncoding RNA [ncRNA; e.g., long-noncoding RNA (lncRNA) and microRNA (miR)] regulation of O-GlcNAc enzymes.

(CTD) (353). Subsequently, this observation was cycling on RNAP II as a critical regulatory circuit of the expanded into the idea that RNAP II posttranslational assembly of the transcriptional preinitiation complex regulation occurred as mutually exclusive states of (PIC), a mechanism that was further extended to suggest phosphorylation and O-GlcNAcylation to establish differ- that the hydrolysis of UDP-GlcNAc may actually serve as ent functional states of this transcriptional regulator a high-energy donor to facilitate PIC formation and the (354)(FIGURE 8A, III). Later, Ranuncolo et al. (355), elongation step (356,357). Owing to the repetitive na- deﬁned the functional signiﬁcance of this O-GlcNAc ture of the CTD domain of RNAP II, the O-GlcNAcylation

450 Physiol Rev VOL 101 APRIL 2021 www.prv.org O-GlcNACYLATION AND (PATHO)PHYSIOLOGY of RNAP II additionally allows for a series of highly heter- of histones in mammalian cells (368), a robust and grow- ogeneous glycoforms, providing the potential to regu- ing number of studies have mapped histone O- late transcription in response to fluctuating cellular GlcNAcylation (80, 369–373). The role of this histone conditions (358). This mechanism has subsequently modification is still being determined, with recent evi- been suggested to perform a nutrient-sensing role to dence suggesting that it mediates cell cycle progression buffer transcriptional activity to match environmental by suppressing histone H3 phosphorylation (369, 373). metabolic demands, as well as developmental mile- Alternatively, O-GlcNAcylation of histone H2B appears stones (32). to facilitate ubiquitin ligase binding, and further histone Other transcriptional machinery shown to be modified modification in a mechanism proposed to initiate tran- by O-GlcNAcylation include TATA-binding proteins (TBP) scriptional activation (80). Additional sites of O- (359), topoisomerase I (Topo I) (360), and nucleoporins GlcNAcylation have been identified on histone H2B with (NUPs) of the nuclear pore complex (NPC) (361)(FIGURE unknown consequences (370). While still other histone 8A, IV). Although the roles of O-GlcNAcylation on TBP or variants have been identified with O-GlcNAcylation sites, Topo I are generally understudied, the presence of O- including histone H2A (371,372), a modification that is in GlcNAc on NUPs is extensive and has been widely stud- parallel with H2A phosphorylation and inverse to H2A ied (5,6, 362). In the case of TBP, it has been suggested acetylation. Clearly, more work in this area needs to be that O-GlcNAc directs the cycling of this protein on and completed as O-GlcNAcylation has been identified on off promoter regions during the regulation of transcrip- all four core histone proteins (374). tion. While the regulation of Topo I by O-GlcNAc appears Another interesting component of the influence O- to directly mediate DNA relaxation and, therefore, tran- GlcNAcylation has on histones, is its interaction with other scriptional accessibility to genes. On the other hand, posttranslational mechanisms of histone modification modification of the NPC by O-GlcNAcylation has growing (FIGURE 8B, I). Specifically, it was shown that OGT evidence to support a direct role in regulation of nucleo- directly interacts with the histone deacetylase and tran- cytoplasmic transport (363). Furthermore, the relatively scriptional corepressor, SIN3a (375). This and other high steady-state levels of O-GlcNAcylation on compo- observations directly connected histone O-GlcNAcylation nents of the NPC appear to preserve the integrity of the with acetylation, an observation that was subsequently complex by preventing its ubiquitination and degradation shown to be regulated by both pathological conditions, (364).Extendingbeyondthisroleintrafficking, compo- as well as physiological stresses such as exercise (376). A nents of both UDP-GlcNAc synthesis and protein O- second potential link between these two posttranslational GlcNAcylation appear to involve the NPC toward regula- modifications came from the identification of a putative tion of speckle and paraspeckle formation, suggesting histone acetyltransferase activity in OGA (199), an obser- additional roles in gene expression (365), as it relates to vation that has subsequently come under debate (270). chromatin structure. However, the link between these two modifications continues to be explored with a more recent study finding 3.2. Epigenetics and Chromatin Remodeling that OGT interacts with the histone acetyltransferase non- specific lethal (NSL) to alter histone H4 acetylation Epigenetics is defined as the changes that occur above through complex stabilization (377). The influence of O- the genome to alter transcriptional regulation in a man- GlcNAc on other posttranslational modifications contin- ner responsive to other events (366); in this way, it is a ues to expand and provide novel insights into transcrip- logical continuation of the O-GlcNAc-mediated control tional regulation with its interaction with protein of gene expression described above. More specifically, methylation. Specifically, OGT can O-GlcNAcylate the his- epigenetics encompasses three areas (FIGURE 8B): 1) tone methyltransferase enhancer of Zeste homolog 2 histone modifications (e.g., O-GlcNAcylation and acety- [EZH2, (378)],which,inturn,regulatesgeneexpression lation), 2) DNA modifications (e.g., 5-mC and 5-hmC), involved in skeletal muscle insulin sensitivity (379), tumor and 3) noncoding RNA (e.g., miR, lncRNA). Direct O- suppression (380), as well as neuronal memory formation GlcNAc modification of histones combined with the (381). It is clear that we are only at the beginning of uncov- cross talk between epigenetics and the machinery ering the cross-talk between O-GlcNAcylation and the involved in O-GlcNAcylation (e.g., OGT, OGA) have histone code. revealed this to be another important level of O-GlcNAc DNA modification by 5-methylcytosine was one of the regulation as discussed here. first epigenetic modifications recognized in the early Sakabe and colleagues (367)werethefirst to demon- 50s (382), with direct enzymatic regulation identified strate that the histone proteins themselves were directly by the early 60s (383). We now have a relatively robust O-GlcNAcylated. Despite some evidence questioning understanding of the addition of methylation to the relative abundance of direct O-GlcNAc modification CpG sites within the genome (384); however, DNA

Physiol Rev VOL 101 APRIL 2021 www.prv.org 451 CHATHAM ET AL.

demethylation is more diverse and less well understood as being induced. This miR induction, in turn, sup- (385). In 2009, two articles were published on the pressed OGA protein levels, resulting in higher total pro- discovery and characterization of TET, methylcytosine- tein O-GlcNAcylation. However, in hepatocarcinoma dioxygenase, enzymes responsible for active DNA cells, miR-24-1 was shown to be lower in those cell lines hydroxymethylation (5hmC) and demethylation (386, with higher metastasis potential (395). The authors 387).Subsequently,itwasshownthatOGTinteracted showed that this partly occurs though binding of miR- with TET protein providing a connection between DNA 24-1 to the 3'-UTR of the transcript for OGT, which regu- modifications and O-GlcNAcylation (388–391)(FIGURE lates levels of O-GlcNAcylation and stability of the onco- 8B, II). The role by which this interaction influences gene protein c-Myc. In contrast, high glucose decreases expression varies by the context and TET family member. levels of miR-200a and miR-200b in endothelial cells, For example, TET2 interacts with OGT to mediate O- resulting in less binding of these miRs to OGT mRNA GlcNAcylation of histone H2B (388). However, when and increased OGT protein levels associated with OGT interacts with TET1, DNA 5hmC appears to increase increased O-GlcNAcylation (308). Further, treating dia- (391). Interestingly, these interactions can be part of a betic mice (db/db) with miR-200a and miR-200b mimics larger complex, including TET2/TET3/OGT and HCF1, fur- leads to decreased OGT levels that could reduce O- ther bringing in regulation of histone methylation to medi- GlcNAcylation and inflammation. A final example of the ate transcriptional activation (389). OGT, in turn, can O- link between miR and O-GlcNAc came from a screen GlcNAc modify each of the three TET enzymes, reduc- that identified miR-501-3p for its potential to reduce ing phosphorylation, which could alter their activity. OGT protein levels, decrease infectivity of hepatitis C vi- Therefore, this interaction between O-GlcNAc and DNA rus, and decrease liver disease progression and devel- modifications appears to be an important additional area opment of cancer (306). of study that is beginning to provide important insight link- ing a number of epigenetic mechanisms. 3.3. Insulin Signaling The last, and least explored, area connecting O- GlcNAcylation and epigenetics is that of noncoding RNA In the late 1980s, it was established that insulin resist- (ncRNA) (FIGURE 8B, III). Although ncRNA was ance induced by different interventions was associated described for decades as a having a passive role as a with a decrease in translocation of the insulin-sensitive messenger between DNA and protein, this idea has glucose transporter, GLUT4. In cultured adipocytes, it been proven incorrect, as ncRNAs are now well known was shown that neither elevated glucose nor insulin to play active roles in transcriptional regulation (392). A alone could reduce insulin sensitivity; however, to- unique mechanism by which O-GlcNAc machinery is gether, they resulted in a marked decrease in maximal directly regulated in an epigenetic manner is through insulin response (46). Additional studies demonstrated long noncoding RNA (lncRNA). The first example of this that glutamine was required for the development relates to the location of the OGT gene on the X chro- of decreased insulin sensitivity, and subsequently, mosome. Specifically, the XIST lncRNA is localized to Marshall et al. (46) demonstrated that increased flux the inactive X chromosome (Xi), and its presence regu- through the HBP was a factor in this process. Prolonged lates OGT levels in females but not males (393). As men- treatment with glucosamine in the presence of insulin, tioned above, it was recently shown that a novel splice decreased basal and insulin-stimulated glucose uptake, variant of OGT, nuclear OGT retained-intron (OGT-RI), and reduced plasma membrane GLUT4 levels, providing may function as a nuclear ncRNA to regulate O-Glc- further evidence that products from the HBP regulated NAc homeostasis (68). In addition to regulating O- insulin signaling (396). This was supported by Patti et al. GlcNAcylation, lncRNA can also be regulated by O- (397), who found that glucosamine infusion in rats GlcNAcylation. For example, it was shown that under resulted in impaired insulin-stimulated glucose uptake high-glucose conditions O-GlcNAcylation of p65 can and glycogen synthesis in skeletal muscle. In the same activate the lncRNA for hyaluronan synthase 2 (HAS2), a study, they showed that this was associated with a glu- naturally occurring antisense transcript (HAS2-AS1), cosamine-dependent increase in O-GlcNAc levels of leading to increased HAS2 to regulate hyaluronan syn- IRS1/2. Moreover, overexpression of GFAT in skeletal thesis (394). This mechanism provides an additional muscle and adipocytes resulted in peripheral insulin re- example by which O-GlcNAcylation can regulate, as well sistance (398). Together, these studies provided the first as be regulated by ncRNA. indications that insulin signaling may be regulated via an At the other end of the spectrum, miRs have also HBP-mediated increase in O-GlcNAcylation. been implicated in the regulation of O-GlcNAcylation. Increasing overall O-GlcNAc levels in 3T3-L1 adipo- One of the first examples of this was in a study of failing cytes, by inhibiting OGA with PUGNAc, resulted in heart in which Methusamy et al. (310)identified miR-539 impaired insulin-stimulated glucose uptake, with no

452 Physiol Rev VOL 101 APRIL 2021 www.prv.org O-GlcNACYLATION AND (PATHO)PHYSIOLOGY changes in insulin-mediated increase in IR-b or IRS2 sustained activation of insulin signaling is suppressed in phosphorylation (133). On the other hand, insulin- an insulin-dependent manner. induced increases of AKT and GSK3b phosphorylation One limitation of many studies examining the role of were attenuated following PUGNAc treatment. While O-GlcNAc in insulin signaling is that they have been AKT and GSK3b were not found to be O-GlcNAcylated, mostly in cultured adipocytes; consequently, it may differ IRS1 and b-catenin were both modified in a PUGNAc-de- in other insulin-sensitive cells and tissues. Nevertheless, pendent manner (133). Subsequent studies have shown there is no doubt that key elements of the insulin signal- that AKT is also a target for O-GlcNAcylation and this ing pathway, including IRS1/2, PDK1, AKT, and GSK3b attenuates its function (121, 126). Several studies have (See TABLE 2) are all targets for O-GlcNAcylation and questioned the role of O-GlcNAcylation in the develop- that in all cases, increasing O-GlcNAcylation suppresses ment of insulin resistance. For example, lowering O- their activities (290). GlcNAc levels via overexpression of OGA or knockdown of OGT did not mitigate hyperglycemia-induced insulin 3.4. Calcium Signaling resistance in adipocytes (399). In addition, the use of more specific OGA inhibitors than PUGNAc, such as There is growing recognition of reciprocal regulation NBuGt and 6-Ac-Cas (599, 600), failed to recapitulate between Ca21 signaling and protein O-GlcNAcylation, earlier observations of insulin resistance seen with perhaps best exemplified by the regulation of CaMKIV PUGNAc, although they did confirm that IRS-1 was a tar- activity via O-GlcNAcylation and OGT activity regulated get for O-GlcNAcylation (600). Buse et al. (399)con- by CaMKIV-mediated phosphorylation (FIGURE 9B) cluded that increased O-GlcNAcylation was just one of (167). Depolarization of neuroblastoma cells resulted in a many factors involved in insulin resistance and was not rapid increase in OGT activity and O-GlcNAc levels that necessarily required. On the other hand, in the setting of was shown to be mediated by CaMKIV phosphorylation normoglycemia, a reduction of O-GlcNAc in the liver in of OGT (286). An early indication of the potential for vivo via overexpression of O-GlcNAcase significantly Ca21/O-GlcNAc cross talk was the identification of increased AKT activity (400). seven O-GlcNAc modification sites on synapsin I that Although the precise role of increased O-GlcNAc were clustered around its regulatory phosphorylation in contributing to cellular insulin resistance remains sites, and O-GlcNAcylation of these sites reduced the af- unclear, there is consensus over the fact that insulin finity of CaMKII for synapsin I (151). In the heart, CaMKII treatment stimulates Tyr phosphorylation of OGT has also been shown to be O-GlcNAcylated on Ser-279, (FIGURE 9A)(242, 251). Insulin stimulation of adipocytes and this results in autonomous activation of CaMKII, resulted in marked increased in Tyr phosphorylation of which, in the setting of diabetes, was linked to increased OGT, resulting in an increase in OGT activity and an arrhythmias (165). In liver, similar to the observations association between OGT and IR. Insulin treatment also in neuroblastoma cells, CaMKII phosphorylated OGT, resulted in translocation of OGT from the nucleus to the increasing O-GlcNAc levels, which subsequently acti- cytoplasm (251). Another study reported that insulin trig- vated autophagy (181). Given the diverse array of cellular gered the translocation of OGT from the nucleus to the functions that are regulated by the CaMK family of pro- plasma membrane, which was facilitated by PIP3 binding teins (406), it is likely that many more links with O- to OGT (242). As a result, it was proposed that OGT con- GlcNAcylation remain to be discovered. tained a PIP3-binding domain; however, structural stud- The nuclear factor of activated T cells (NFAT) family of ies have not been able to confirm the presence of such transcription factors, are widely distributed and contrib- a domain (90). The recruitment of OGT to the plasma ute to the regulation of numerous processes, including membrane, resulted in increased OGT phosphorylation the immune system, cardiac and skeletal muscle, and and activity, with subsequent increases in O- brain. Ca21-dependent activation of calmodulin, acti- GlcNAcylation of IRS1, AKT, and other downstream tar- vates the phosphatase calcineurin which rapidly gets of insulin signaling (242). PTP1B, has long been dephosphorylates NFAT, resulting in its nuclear translo- considered to be a major mechanism in attenuating in- cation and activation. In neonatal cardiomyocytes NFAT sulin signaling through the dephosphorylation of Tyr res- translocation is initiated by hypertrophic agonists, such idues in the activation loop of the insulin receptor (405). as angiotensin II (ANG II) or PE, and hyperglycemia was Interestingly, PTP1B has been reported to be O-Glc- found to inhibit this translocation in an HBP-dependent NAcylated, leading an increase in enzyme activity, manner (407). A subsequent study demonstrated that thereby potentially contributing to a reduction in insulin increasing O-GlcNAc levels attenuated the ANG II- signaling (FIGURE 9A). These findings suggest that induced increase in cytosolic Ca21 (408). More recently, OGT translocation and O-GlcNAcylation of target pro- it was reported that activation of O-GlcNAc signaling teins are part of a feedback mechanism in which was required for NFAT translocation in cardiomyocytes,

Physiol Rev VOL 101 APRIL 2021 www.prv.org 453 CHATHAM ET AL.

A Insulin signaling Initiation of insulin signaling

Insulin

PIP PIP3 2 P AKT P P PDK1 P PI3K

P IRS1 P FOXO P P P Glucose uptake GSK3 Glycolysis Glyconeogenesis Glycogen OGT Lipogenesis synthesis Protein synthesis

Termination of insulin signaling

Insulin

PIP G OGT PIP2 3 AKT P P P PDK1 X G P G PI3K X X G X IRS1 G OGT FOXO P P G OGT GSK3

OGT

B Calcium signaling

Ca2+

STIM1 2+ P mediated Ca ? Ca2+ Ca2+ Ca2+-CaM OGT Ca2+ Ca2+

PLB Ca2+ SERCA STIM1 G Ca2+ CaMKII/IV G Ca2+ P IP R G G G 3 Calcineurin P Contractile proteins ?

GG OGT Reduced Ca2+ Ion currents sensitivity SR/ER Ca2+ handling OGT Transcription Protein synthesis Voltage gated Ca2+ channels Mitochondrial Ca2+ homeostasis Ca2+ Attenuates Mito -adrenergic signaling Autophagy Ca2+ Overload Mitochondrial dynamics Transcription OGT

G CaMKII P MCU

454 Physiol Rev VOL 101 APRIL 2021 www.prv.org O-GlcNACYLATION AND (PATHO)PHYSIOLOGY and this could be blocked with the calcineurin inhibitor increases in O-GlcNAc levels reduced Ca21 sensitivity, cyclosporin A (298). Thus, O-GlcNAcylation may have consistent with the earlier skeletal muscle study (146). regulatory roles in both the initiation of NFAT signaling, Uptake of cytosolic Ca21 into the ER/SR is an impor- as well as its inhibition (FIGURE 9B). Another protein tant mechanism for regulating Ca21 signaling, as well as that has been established as a key player in Ca21-de- muscle contraction, and this is controlled by sarcoplas- pendent activation of NFAT, particularly in the immune mic/endoplasmic reticulum calcium ATPase (SERCA) system, is stromal interacting molecule 1 (STIM1), via its and its inhibitor phospholamban (PLB). Phosphorylation role in regulating the store operated calcium entry path- of PLB attenuates its inhibitory effect, thereby, facilitating way (SOCE) (409). STIM1 is positively and negatively more rapid uptake of Ca21 into the ER/SR by SERCA. regulated by phosphorylation, and increases in O- PLB is a target for O-GlcNAcylation, and Ser-16 was GlcNAc increased basal phosphorylation, but attenuated found to be the most likely target (194), although to date, activation-dependent increases in phosphorylation this has not been confirmed by MS. Increased PLB O- (401). STIM1, is a highly conserved protein, considered to GlcNAc levels, either by inhibition of OGA or under con- be a core component of mammalian Ca21 signaling ditions of hyperglycemia, reduced PKA-mediated phos- (409); consequently, its functional regulation by O- phorylation of PLB Ser-16 (194); conversely, knockdown GlcNAc could have far reaching consequences. of OGT significantly increased PLB phosphorylation In skeletal and cardiac muscle, Ca21 also plays a cen- (FIGURE 9B). Increased O-GlcNAc levels, were associ- tral role in contraction of the myofilaments, via Ca21 bind- ated with lower SERCA activity and a greater association ing to key proteins, as well as regulating Ca21 release between SERCA and PLB (194). It was concluded that and uptake by the ER/SR (FIGURE 9B)(410). Skeletal the increase in PLB O-GlcNAcylation could be a factor muscle myosin was the first contractile protein that was in the slower reuptake of Ca21 by the ER/SR in the shown to be O-GlcNAcylated, with all isoforms being heart that occurs with diabetes. SERCA expression modified (411). Subsequent studies identified actin and in the heart, has been shown to be decreased under myosin light chain (MLC) 1,2 as O-GlcNAc targets and conditions of increased O-GlcNAc levels, and this was found that in skinned muscle fibers, acute increases in O- attributed to an increase in O-GlcNAcylation of the tran- GlcNAc levels reduced Ca21 sensitivity, suggesting a scription factor Sp1 (263). While Yokoe et al. (194), possible role for O-GlcNAc in regulating skeletal muscle reported that SERCA was not an O-GlcNAc target, contractility (146). Myofilament proteins from cardiac mus- others have shown that it is O-GlcNAcylated (412, 413), clewerealsofoundtobeO-GlcNAcylated, and in addi- although the functional consequence of this modification to those identified in skeletal muscle, troponin I (TnI) tion remains to be identified. was modified at Ser-150, which is a phosphorylation site Another important contributor to cellular Ca21 signal- 21 that regulates Ca sensitivity (147). Pharmacological ing is the inositol 1,4,5-trisphosphate (InsP3)-receptor,

FIGURE 9. Examples of O-linked b-N-acetylglucosamine (O-GlcNAc) regulation of cellular signaling pathways. A: Insulin signaling. On initiation, in response to insulin, there is autophosphorylation of the insulin receptor (IR) and subsequent phosphorylation and activation of Akt, FOXO and GSK3b and their downstream signaling pathways. On termination, insulin triggers rapid translocation of O-GlcNAc transferase (OGT) from the nucleus to the plasma membrane, where it is phosphorylated by the IR, which increases OGT activity, leading to the subsequent O-GlcNAcylation of insulin receptor substrate 1 (IRS1), AKT, FOXO, and GSK3b, thereby attenuating activity at multiple steps in the insulin signaling cascade. The protein tyrosine phosphatase 1B (PTP1B) is responsible for decreasing phosphorylation of IR and attenuating insulin signaling. It is also O-GlcNAcylated, which increases its activ-

ity contributing to a further reduction in insulin signaling. It has been reported that translocation of OGT to the plasma membrane is via binding to PIP3 (242), which is generated in response to activation of insulin signaling; however, to date, structural studies have not revealed a phosphatidylinositol-3- 21 phosphate (PIP3)-binding motif in OGT(90). B: Calcium signaling. Intracellular Ca increases in response to diverse number of agonists, either as a result of Ca21 release from ER/SR alone or via activation of plasma membrane Ca21 channels as a result of ER/SR Ca21 release (store-operated Ca21 entry, SOCE). This increase in Ca21 leads to activation of calmodulin (CaM), resulting in the phosphorylation and activation of both CaMKII and IV, which are responsible for regulation of numerous cellular processes. CaMKII also phosphorylates OGT, increasing its activity, and in a feedback manner, OGT O-GlcNAcylates CaMKII/IV reduces their activities. Ca21-CaM also activates the calcium-dependent phosphatase calcineurin, which is responsible for regulating diverse cellular processes, and it has been reported that increased OGT expression and O-GlcNAcylation is sufficient to activate calcineurin- mediated transcription pathways (298). Influx of extracellular Ca21 has been shown to contribute to the stress-induced increases in O-GlcNAc levels (312). Therefore, it is possible, that Ca21-CaM and/or calcineurin regulate OGT (or OGA) activities; however, this has yet to be demonstrated experimen- tally. Multiple contractile proteins are O-GlcNAc modified (TABLE 2), and the increases in O-GlcNAc levels that can occur in diseases, such as diabetes, reduces Ca21 sensitivity of some contractile proteins (147, 150). Phospholamban (PLB) and SERCA are responsible, in part, for the reuptake of Ca21 into the sarcoplasmic reticulum (SR), contributing to muscle relaxation. Increases in O-GlcNAc levels decrease the activities of both PLB and SERCA directly or indirectly, which could be a contributing factor to impaired myocardial relaxation (i.e., diastolic function) that occurs with diabetes. The endoplasmic reticulum (ER)/SR transmembrane protein STIM1, which plays a key role in regulating SOCE, is also a target for O-GlcNAcylation and increases in O-GlcNAc levels impairs its function and attenuates SOCE (401). Increasing O-GlcNAc levels attenuates mitochondrial Ca21 overload, although the precise mechanisms are unclear(163, 402). CaMKII phosphorylation of the mitochondrial Ca21 uniporter (MCU) potentiates mitochondrial Ca21 overload (403); therefore, as O-GlcNAcylation of CaMKII decreases its activity, this may represent a potential protective mechanism. However, others have questioned the role of CaMKII in the regulation of mitochondrial Ca21 uptake by the MCU (404).

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which is localized to the ER/SR membrane and is acti- linked to greater fatty acid oxidation, potentially via vated by InsP3 generated in response to a variety of O-GlcNAcylation of the fatty acid transporter, CD36 extracellular stimuli. In C2C12 myotubes, increased O- (419,420). Similarly, in adipocytes, activation of the HBP GlcNAc levels attenuated bradykinin-induced produc- and increasing O-GlcNAc levels also stimulated fatty acid 21 tion of IP3 and associated InsP3R-mediated Ca oxidation (421). Interestingly, a splice variant of OGA, release, which was associated with O-GlcNAcylation of sOGA, is associated with lipid droplets, further supporting PLC-b1(414). The InsP3 receptor type 1 (InsP3R-1) itself is a connection between O-GlcNAcylation and the regula- O-GlcNAcylated under basal conditions and this could tion of lipid metabolism (273). In addition to direct regula- be increased following treatment with the OGA inhibitor tion of metabolic fluxes, O-GlcNAcylation also contributes PUGNAc (415). Moreover, decreases in basal O-GlcNAc to the transcriptional regulation of metabolism via O- levels resulted in a marked increase in channel opening GlcNAcylation of multiple transcription factors, including probability, and conversely, increasing O-GlcNAc levels PGC-1a,FoxO3,andCREB(100, 105, 117, 598). reduced channel opening probability. These findings The identification of a splice variant of OGT, with a mi- demonstrated a potential role for O-GlcNAc in regulating tochondrial targeting sequence (422), suggested that mi- InsP3R-1 under physiological conditions (FIGURE 9B) tochondrial proteins may be targets for O-GlcNAcylation. (415). A subsequent study from the same group found However, early reports indicated that there was little, if that InsP3R-2 was not O-GlcNAcylated, and its function any, O-GlcNAcylation in mitochondria. It was proposed was unaltered by global changes in O-GlcNAc levels that the lack of mitochondrial O-GlcNAc was due to the (416). Interestingly, however, they found that InsP3R-3 lack of substrate, as there was no clear mechanism was O-GlcNAcylated, but that changes in its O-GlcNAc for UDP-GlcNAc transport into the mitochondria (423). levels had the opposite effect to those observed with Nevertheless, a number of studies suggested that altera- InsP3R-1. InsP3R is ubiquitously expressed, although the tions in cellular O-GlcNAc levels could alter mitochondrial different isoforms exhibit tissue-specific differences in function and particularly their response to stress. For their function (417); consequently, there is a clear need example, increasing O-GlcNAc levels in cardiomyocytes for a better understanding of the role of O-GlcNAc in with PUGNAc attenuated the loss of mitochondrial mem- their regulation. brane potential induced by oxidative stress, and this was It is clear that O-GlcNAcylation provides a key link associated with O-GlcNAcylation of voltage-dependent 1 between nutrient and Ca2 signaling contributing to the anion channel (VDAC)1 (163). Both glucosamine treatment 21 regulation of the majority of key Ca signalling path- and OGT overexpression increased O-GlcNAc levels and ways (FIGURE 9B). What is also becoming increasingly reduced ischemia-reperfusion-induced injury in isolated 21 evident is that O-GlcNAc levels are regulated in a Ca - cardiomyocytes (402).Thesametreatmentsattenuated dependent manner, as exemplified by CaMKII/IV-medi- hydrogen peroxide-induced mitochondrial membrane ated phosphorylation of OGT, resulting in increased ac- potential and enhanced recruitment of the anti-apoptotic tivity and high O-GlcNAc levels (181, 286). Stress protein BCL2 to the mitochondria (402). Subsequent induced increases in cellular O-GlcNAc levels have also studies suggested BCL2 itself might be a target for O- 21 been shown to be dependent on extracellular Ca and GlcNAcylation (424). 21 activation of CaMKII (312). The role of Ca in regulating Although acute activation of O-GlcNAc levels pro- GFAT activity is poorly understood, even though it can tected mitochondria from oxidative stress, increa- be phosphorylated by CaMKII, and to date, the role of ses in O-GlcNAcylation associated with hyperglycemia 21 Ca in regulating OGA activity is unknown. were found to have adverse effects on mitochondrial function. For example, in pancreatic b-cells, hyperglyce- 3.5. Metabolism and Mitochondrial Function mia increased O-GlcNAc levels on the chaperone heat shock protein (HSP) 60, interfering with its binding to As discussed in sect. 2.1, substrate availability plays a key the proapoptotic factor BCL2 Associated X, Apoptosis role in regulating HBP flux and O-GlcNAcylation; how- Regulator (BAX), resulting in BAX translocation to the mi- ever, what is less well known is that O-GlcNAcylation con- tochondria and subsequent cytochrome-c release (425). tributes to the regulation of metabolism at multiple levels. Hyperglycemia resulted in impaired activity of mitochon- For example, almost every enzyme in glycolysis has been drial complexes in neonatal cardiomyocytes, which shown to be O-GlcNAc modified from GLUT4 to pyruvate could be reversed by overexpression of OGA. High glu- dehydrogenase (FIGURE 10A)(418). In addition, O- cose levels also increased O-GlcNAcylation on subunits GlcNAcylation of glucose-6-phosphate dehydrogenase of mitochondrial complexes, which was reduced follow- regulates pentose phosphate pathway activity, and ing overexpression of OGA (161). Hyperglycemia also GSK3b O-GlcNAcylation regulates glycogen synthesis. In increased O-GlcNAcylation of dynamin-related protein 1 the heart, increased flux through the HBP has been (DRP1), increasing mitochondrial fragmentation and

456 Physiol Rev VOL 101 APRIL 2021 www.prv.org O-GlcNACYLATION AND (PATHO)PHYSIOLOGY decreasing mitochondrial membrane potential (159). In increased AKT O-GlcNAcylation and decreased AKT neurons, hyperglycemia impaired mitochondrial motility phosphorylation (124). Hyper-O-GlcNAcylation is also evi- in an O-GlcNAc-dependent manner, due to O- dent in response to cerebral ischemia (124). In a model of GlcNAcylation of MILTON1 (also known as TRAK1), a traf- diabetic retinopathy, increased O-GlcNAcylation of NF-κB ficking protein that is essential for mitochondrial move- was associated with retinal ganglion cell death (430). ment (160). 8-oxoguanine DNA glycosylase (Ogg1), is Recent studies also have shown that O-GlcNAcylation on involved in mitochondrial DNA repaired and is O- Ser-56 and Ser-57 of c-Fos is increased in 5XFAD mouse GlcNAcylated in response to hyperglycemia, decreasing model of Alzheimer’sdisease,aswellasneuroblastoma its activity and possibly contributing to increased mtDNA cells exposed to amyloid beta (Ab), this increased its sta- damage (426). bility and interaction with c-JUN, increased target gene In 2009, Hu et al. (161), identified O-GlcNAcylation on Bim expression, and promoted cell death (431). Increased a number of mitochondrial oxidative phosphorylation O-GlcNAc levels have also been reported to exacerbate complex proteins, which was associated with decreased acetaminophen-induced liver injury, whereas lower O- complex I, II, and IV activity in mitochondrial of cardiomy- GlcNAc levels, resulting from liver-specific OGT knockout ocytes exposed to high glucose. Cao et al. (157) identi- reduced the degree of injury (432). Increased cardiomyo- fied 11 O-GlcNAcylated proteins from rat liver mitoc- cyte apoptosis observed in a rat model of diabetes was hondria, which included enzymes in the TCA cycle, as linked to an overall increase in O-GlcNAc levels, as well well as those involved in ATP synthesis (FIGURE 10B). It as increased O-GlcNAcylation and reduced phosphoryla- remained unclear, however, as to how mitochondrial tion of the proapoptotic protein BCL2-associated agonist proteins could be modified by O-GlcNAc. In 2015, trans- of cell death (or BAD) (433). port of 3H-UDP-GlcNAc into cardiac mitochondria was In contrast to the increase in O-GlcNAc levels, leading reported and the pyrimidine nucleotide carrier (PNC1) to cell death, Zachara et al. (289), demonstrated that ex- was identified as the UDP-GlcNAc mitochondrial trans- posure of cells to a variety of stressors induced an porter (427). Immunogold labeling and live cell imaging increase in O-GlcNAc that they showed was cytoprotec- were also used to demonstrate the presence of OGA tive and represented an endogenous cell survival localized to the mitochondria, demonstrating for the first response. Subsequent studies revealed a link between time that an active O-GlcNAc cycle was present in the the increase in O-GlcNAc and cell survival with the mitochondria (427). Subsequent proteomics studies greater induction of heat shock protein expression via identified 86 mitochondrial proteins as O-GlcNAc tar- inactivation of G3K3b (280). A number of proteins gets that were involved in a diverse array of mitochon- involved in the DNA repair pathway have also been drial functions, including the TCA cycle, oxidative shown to be regulated either directly or indirectly by O- phosphorylation, fatty acid oxidation, and calcium regu- GlcNAcylation (89, 319, 434–436). O-GlcNAcylation is lation (FIGURE 10C)(138, 139). Others have been unable required for the normal regulation of DNA damage path- to detect mOGT isoform in cells or tissues, and ncOGT ways, and OGT is recruited to sites of DNA damage was reported to be the isoform responsible for (434). Additional studies found that pharmacologically O-GlcNAcylation of mitochondrial proteins (428). It has increasing O-GlcNAc levels was cytoprotective, particu- also been suggested that mOGT regulates mitochon- larly in the setting of acute oxidative stress or ischemia- drial stress responses, while ncOGT is responsible for reperfusion (163, 402, 437). There have been several the regulating mitochondrial bioenergetics (429). Thus, reports demonstrating a strong correlation between O- additional work remains to elucidate the fundamental GlcNAc levels and tissue injury, with low O-GlcNAc lev- mechanisms regulating O-GlcNAc cycling in the mito- els associated with increased injury (437–439). The spe- chondria, as well as understanding the physiological cific mechanisms by which the increase in O-GlcNAc is role of O-GlcNAcylation in regulating mitochondrial protective remains to be determined; however, there function. are indications that it attenuates Ca21 overload, protects mitochondria against oxidative stress, and attenuates 3.6. Cell Survival/Autophagy proinflammatory responses (163, 402, 440–443). Autophagy is an important cell survival mechanism, Pharmacological studies of OGT or OGA inhibition in INS-1 and a role for O-GlcNAc in regulating autophagy is start- and bTC-6 cells have linked high O-GlcNAcylation with ing to emerge. Recent studies have shown Unc-51-like cell death and increased O-GlcNAcylation with reduced autophagy activating kinase 1 (ULK1) (444), mammalian phosphorylation of Ser-473 on AKT1 as the potential target of rapamycin (mTOR) (445), hypoxia-inducible fac- mechanism (122). In human embryonic kidney 293 and tor 1a (HIF1 a)(446), synaptosome-associated protein 29 HeLa cells, hyper-O-GlcNAcylation by overexpression of (SNAP29), tubulin polymerization-promoting protein OGT induced apoptosis, which was associated with (TPPP), Golgi reassembly-stacking protein of 55 kDa

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(GRASP55) (180, 447–450), all of which play import- increased O-GlcNAc levels and O-GlcNAcylation of ant roles in the regulation of autophagy, are modif- BCL2 and Beclin 1 (424). Arsenic-induced inhibition ied by O-GlcNAcylation. Increased O-GlcNAcylation of of autophagic ﬂux was shown to be mediated by O- SNAP29 and GRASP55 resulted in decreased autoph- GlcNAcylation of SNAP29, and this was prevented by agy (FIGURE 11)(180, 450). In C. elegans, an ogt muta- reducing O-GlcNAc levels by knockdown of OGT tion elevated autophagy during development and (451). Transfection of SNAP29 containing an O- knockdown of OGT in mammalian cells also promo- GlcNAc site mutant attenuated arsenic inhibition of ted autophagy (180). Reduced OGT was associated with autophagy, further substantiating the role of SNAP29 increased formation of the SNARE complex, which O-GlcNAcylation in regulating autophagy (451). includes SNAP29 and increased O-GlcNAcylation of A mutagenesis study has shown that single mutations SNAP29-inhibited autophagy at the step of autophago- of GRASP55 at Ser-389, Ser-390, Thr-403, Thr-404, and some maturation (180). Impaired autophagic sig- Thr-413 decreased its O-GlcNAcylation, while a single naling in the diabetic heart was associated with mutation at Ser-380 increased its O-GlcNAcylation,

A Gucose

GLUT4 G

Glucose G HEX UDP-GP PGM UDP-G G1P G6P G G G GS PGI G6PDH Pentose GP F6P Phosphate Glucose G Pathway PFK1 GFAT Hexosamine Biosynthesis F2,6P F1,6P Pathway PFK2 G G ALD

GA3P G GAPDH G G PGK PGM 1,3BPG 3PG 2PG G ENO

PEP G G PK PDK TCA Acetyl-CoA Pyruvate

LDH

Lactate– +H+

FIGURE 10. Metabolism and mitochondrial O-GlcNAcylation. A: majority of enzymes in glycolysis and glucose metabolism are targets for O- GlcNAcylation (418). ALD, aldolase; ENO, enolase; GAPDH, glyceraldehyde-6-phosphate dehydrogenase; GFAT, glutamine fructose-6phosphate amidotransferase; GP, glycogen phosphorylase; G6PDH, glucose-6-phosphate dehydrogenase; GS, glycogen synthase; HEX, hexokinase; LDH, lactate dehydrogenase; PFK, phosphofructokinase; PDC, pyruvate dehydrogenase complex; PGI, phosphoglucoisomerase; PGK, phosphoglycerate kinase; PGM phosphoglucomutase; PK, pyruvate kinase; UDP-GP, UDP-glucose pyrophosphorylase. B: wide range of different types of mitochondrial proteins that are O-GlcNAcylated. C: map of the O-GlcNAc modiﬁcation sites on proteins that are central to the regulation of energy metabolism and mitochondrial function. Data largely based on a number of recent proteomic studies (138,139). Blue squares denote O-GlcNAc, whereas numbers inside blue squares indicate number of O-GlcNAc sites on the protein.

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B 3% 2% Translation 3% Pyrimidine Amino Acid Metabolism Metabolism 3% Redox 3% Protein Homeostasis 5% Pyruvate Carboxylation 36% Respiratory Chain 8% TCA cycle

16% ATP 21% sythase Fatty acid metabolism

Pyruvate Fatty Acid C 4 7 1 Milton 3 Miro CPT1 PCrCKMT2 Cr SAM50 CPT2

Pyruvate Acetyl-CoA 3 4 2 PDH KCT ACAD 2 VADC1/2 Acetyl-CoA 3 2 HADH1 ECH

? DRP1 2 HSP60 2 Citrate Malate Synthase Dehydrogenase 4 1 ? Aconitase SOD2 Fumarase ? 2 PRDX Isocitrate Dehydrogenase 3 Succinate Dehydrogenase -Ketoglutarate 1 Dehydrogenase Succinyl-CoA 1 Synthase

4 29 26 8 CV ATP 3 4 SLC25A4 CI CII CIII CIV

1 SLC25A3 Pi

FIGURE 10. Continued

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