# An overview of tools to decipher O-GlcNAcylation from historical approaches to new insights

Thomas Dupas, Charlotte Betus, Angélique Blangy-Letheule, Thomas Pelé,

Antoine Persello, Manon Denis, Benjamin Lauzier

► To cite this version:

Thomas Dupas, Charlotte Betus, Angélique Blangy-Letheule, Thomas Pelé, Antoine Persello, et al. An overview of tools to decipher O-GlcNAcylation from historical approaches to new insights. International Journal of Biochemistry and Cell Biology, 2022, 151, pp.106289. 10.1016/j.biocel.2022.106289. hal-04590700

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Submitted on 28 May 2024

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# An overview of tools to decipher O-GlcNAcylation from historical approaches to new insights

Thomas Dupas, Charlotte Betus, Angélique Blangy-Letheule, Thomas Pelé,

Antoine Persello, Manon Denis, Benjamin Lauzier

<figure><img src="image_1.png" /></figure>

## To cite this version:

Thomas Dupas, Charlotte Betus, Angélique Blangy-Letheule, Thomas Pelé, Antoine Persello, et al. An overview of tools to decipher O-GlcNAcylation from historical approaches to new insights. International Journal of Biochemistry and Cell Biology, 2022, 151, pp.106289. 10.1016/j.biocel.2022.106289. hal-04590700

HAL Id: hal-04590700

https://hal.science/hal-04590700v1

Submitted on 28 May 2024

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>
## Review article

# An overview of tools to decipher O-GlcNAcylation from historical approaches to new insights

Thomas Dupas<sup>a, *</sup>, Charlotte Betus<sup>a,b,c</sup>, Angélique Blangy-Letheule<sup>a</sup>, Thomas Pelé<sup>a</sup>, Antoine Persello<sup>a</sup>, Manon Denis<sup>a,b,c</sup>, Benjamin Lauzier<sup>a</sup>

<figure><img src="image_2.png" /></figure>

Check for
updates

### ARTICLE INFO

**Keywords:**

O-GlcNAcylation
New approaches
O-GlcNAcylomic
Biochemistry

### ABSTRACT

O-GlcNAcylation is a post-translational modification which affects approximately 5000 human proteins. Its involvement has been shown in many if not all biological processes. Variations in O-GlcNAcylation levels can be associated with the development of diseases. Deciphering the role of O-GlcNAcylation is an important issue to (i) understand its involvement in pathophysiological development and (ii) develop new therapeutic strategies to modulate O-GlcNAc levels. Over the past 30 years, despite the development of several approaches, knowledge of its role and regulation have remained limited. This review proposes an overview of the currently available tools to study O-GlcNAcylation and identify O-GlcNAcylated proteins. Briefly, we discuss pharmacological modulators, methods to study O-GlcNAcylation levels and approaches for O-GlcNAcylomic profiling. This review aims to contribute to a better understanding of the methods used to study O-GlcNAcylation and to promote efforts in the development of new strategies to explore this promising modification.

## 1. Background

O-linked-β-N-acetyl glucosaminylation, more commonly referred to as O-GlcNAcylation, is a post-translational modification (PTM) consisting in adding monosaccharide (β-D-N-acetylglucosamine) on serine and threonine residues (Torres and Hart, 1984; Yang and Qian, 2017). Two enzymes control O-GlcNAcylation cycling: the O-linked N-acetylglucosamine transferase (OGT) and the protein O-GlcNAcase (OGA) which adds and removes the GlcNAc moiety, respectively. Glucosamine-fructose-6-phosphate aminotransferase (GFAT) uses glutamine and fructose-6-phosphate to form UDP-GlcNAc, the OGT substrate, via the hexosamine biosynthesis pathway (HBP) (Fig. 1). Cytosolic, nuclear, mitochondrial and extracellular O-GlcNAcylated proteins (estimated at approximately 5000 in humans in 2021) are involved in many, if not all, cellular processes (Ma and Hart, 2014; Wulff-Fuentes et al., 2021; Bond and Hanover, 2015). O-GlcNAcylation can impact the properties of proteins and a variation in O-GlcNAc protein levels can be associated with a variety of diseases (e.g. cardiovascular, cancer, neurodegenerative diseases and diabetes) (Bond and Hanover, 2015; Zachara et al., 2011a, 2017; Ferron et al., 2019a).

Deciphering the role of the O-GlcNAcylation is an important issue in the development of new pharmacological approaches, notably to better understand the involvement of this PTM in pathophysiological situations and identify new O-GlcNAcylated proteins. While O-GlcNAcylation is well conserved over all phyla, ubiquitous and largely described over the past 30 years, progress on its roles or its regulation has remained poor. This can be notably explained by (i) the lack of tools and techniques available to study this PTM on specific proteins and (ii) the particularities associated with GlcNAc moiety. In fact, added sugar does not modify molecular weight or the isoelectric point of O-GlcNAcylated proteins which renders the usual techniques such as gel electrophoresis, two-dimensional gel and high-pressure liquid chromatography (HPLC) infeasible (Zachara et al., 2011a, 2004; Roquemore et al., 1994; Whelan and Hart, 2006). Conventional mass spectrometry methods do not detect O-GlcNAcylated peptides since the high lability of the β-glycosidic bond is associated with a loss in GlcNAc residue owing to the electrospray step (Greis and Hart, 1998; Wang et al., ). In addition, the signal of ions derived from O-GlcNAcylated peptides is limited compared with the ion signal derived from unmodified peptides, even when O-GlcNAcylated peptides are more present (Wang et al., ). Study of O-GlcNAcylation and

<footnote><sup>a</sup> Nantes Université, CHU Nantes, CNRS, INSERM, l'Institut du thorax, F-44000 Nantes, France</footnote>

<footnote><sup>b</sup> Department of Pharmacology and Physiology, University of Montreal, Montreal, QC H3T 1C5, Canada</footnote>

<footnote><sup>c</sup> CHU Sainte-Justine Research Center, Montreal, QC H3T 1C5, Canada</footnote>

<footnote>* Correspondence to: 8 Quai Moncousu, 44007 Nantes, France.</footnote>

<footnote>E-mail address: thomas.dupas@univ-nantes.fr (T. Dupas).</footnote>
Review article

# An overview of tools to decipher O-GlcNAcylation from historical approaches to new insights

Thomas Dupas<sup>a, *</sup>, Charlotte Betus<sup>a,b,c</sup>, Angélique Blangy-Letheule<sup>a</sup>, Thomas Pelé<sup>a</sup>, Antoine Persello<sup>a</sup>, Manon Denis<sup>a,b,c</sup>, Benjamin Lauzier<sup>a</sup>

<figure><img src="image_3.png" /></figure>

Check for
updates

## ARTICLE INFO

### Keywords:

O-GlcNAcylation
New approaches
O-GlcNAcylomic
Biochemistry

## ABSTRACT

O-GlcNAcylation is a post-translational modification which affects approximately 5000 human proteins. Its involvement has been shown in many if not all biological processes. Variations in O-GlcNAcylation levels can be associated with the development of diseases. Deciphering the role of O-GlcNAcylation is an important issue to (i) understand its involvement in pathophysiological development and (ii) develop new therapeutic strategies to modulate O-GlcNAc levels. Over the past 30 years, despite the development of several approaches, knowledge of its role and regulation have remained limited. This review proposes an overview of the currently available tools to study O-GlcNAcylation and identify O-GlcNAcylated proteins. Briefly, we discuss pharmacological modulators, methods to study O-GlcNAcylation levels and approaches for O-GlcNAcylomic profiling. This review aims to contribute to a better understanding of the methods used to study O-GlcNAcylation and to promote efforts in the development of new strategies to explore this promising modification.

## 1. Background

O-linked-β-N-acetyl glucosaminylation, more commonly referred to as O-GlcNAcylation, is a post-translational modification (PTM) consisting in adding monosaccharide (β-D-N-acetylglucosamine) on serine and threonine residues (Torres and Hart, 1984; Yang and Qian, 2017). Two enzymes control O-GlcNAcylation cycling: the O-linked N-acetylglucosamine transferase (OGT) and the protein O-GlcNAcase (OGA) which adds and removes the GlcNAc moiety, respectively. Glucosamine-fructose-6-phosphate aminotransferase (GFAT) uses glutamine and fructose-6-phosphate to form UDP-GlcNAc, the OGT substrate, via the hexosamine biosynthesis pathway (HBP) (Fig. 1). Cytosolic, nuclear, mitochondrial and extracellular O-GlcNAcylated proteins (estimated at approximately 5000 in humans in 2021) are involved in many, if not all, cellular processes (Ma and Hart, 2014; Wulff-Fuentes et al., 2021; Bond and Hanover, 2015). O-GlcNAcylation can impact the properties of proteins and a variation in O-GlcNAc protein levels can be associated with a variety of diseases (e.g. cardiovascular, cancer, neurodegenerative diseases and diabetes) (Bond and Hanover, 2015; Zachara et al., 2011a, 2017; Ferron et al., 2019a).

Deciphering the role of the O-GlcNAcylation is an important issue in the development of new pharmacological approaches, notably to better understand the involvement of this PTM in pathophysiological situations and identify new O-GlcNAcylated proteins. While O-GlcNAcylation is well conserved over all phyla, ubiquitous and largely described over the past 30 years, progress on its roles or its regulation has remained poor. This can be notably explained by (i) the lack of tools and techniques available to study this PTM on specific proteins and (ii) the particularities associated with GlcNAc moiety. In fact, added sugar does not modify molecular weight or the isoelectric point of O-GlcNAcylated proteins which renders the usual techniques such as gel electrophoresis, two-dimensional gel and high-pressure liquid chromatography (HPLC) infeasible (Zachara et al., 2011a, 2004; Roquemore et al., 1994; Whelan and Hart, 2006). Conventional mass spectrometry methods do not detect O-GlcNAcylated peptides since the high lability of the β-glycosidic bond is associated with a loss in GlcNAc residue owing to the electrospray step (Greis and Hart, 1998; Wang et al., ). In addition, the signal of ions derived from O-GlcNAcylated peptides is limited compared with the ion signal derived from unmodified peptides, even when O-GlcNAcylated peptides are more present (Wang et al., ). Study of O-GlcNAcylation and

<footnote><sup>a</sup> Nantes Université, CHU Nantes, CNRS, INSERM, l'Institut du thorax, F-44000 Nantes, France</footnote>

<footnote><sup>b</sup> Department of Pharmacology and Physiology, University of Montreal, Montreal, QC H3T 1C5, Canada</footnote>

<footnote><sup>c</sup> CHU Sainte-Justine Research Center, Montreal, QC H3T 1C5, Canada</footnote>

<footnote>* Correspondence to: 8 Quai Moncousu, 44007 Nantes, France.
E-mail address: thomas.dupas@univ-nantes.fr (T. Dupas).</footnote>
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Hexosamine biosynthesis pathway, O-GlcNAcylation and pharmacological tools. DON: 6-diazo-5-oxo-L-norleucine; BZX: benzoxazolinones; BADGP: benzyl-2-acetymidode-2-deoxy- α-D-galactopyranoside. Adapted from Yang and Qian (2017).</figcaption></figure>

O-GlcNAcylated proteins by mass spectrometry requires the adaption of methods and the development of new enrichment approaches. Special consideration vis-à-vis tissue-sample preparation must be taken to protect GlcNAc residues from OGA (Dupas et al., 2020). The aim of this review was to provide an overview of the pharmacological approaches used to modulate GlcNAc levels and define the biochemical tools used to modify, detect, quantify, identify and map O-GlcNAcylation.

## 2. Development of biochemical approaches to study O-GlcNAc levels

### 2.1. Quantification methods

#### 2.1.1. O-GlcNAc antibodies

Like other post-translational modifications, antibodies are a classic approach used to study O-GlcNAcylation by western blot and immunoprecipitation. Several antibodies have been described to recognize GlcNAc residues such as RL2 (Snow et al., 1987), CTD110.6 (Comer et al., 2001) and others (Turner et al., 1990; Matsuoka et al., 2002; Tashima and Stanley, 2014; Teo et al., 2010) (Table 1). However, the

poor immunogenicity of GlcNAc residue, which is a neutral sugar, and the lack of knowledge about O-GlcNAcylated sequences are limits to massive development of selective antibodies. Considering that antibodies are developed to recognize a unique GlcNAc epitope and the potential cross reactivity with other sugar moieties, the profile of immunoprecipitated peptides is strictly different between antibodies (Teo et al., 2010; Okuda, 2017; Isono, 2011), thus limiting the information.

With the appearance of consensus sequences for both serine and threonine O-GlcNAc modifications (Wulff-Fuentes et al., 2021; Malard et al., 2021), new antibodies have been developed to increase the bank of antibodies and identify more O-GlcNAcylated proteins (Song et al., 2019). Moreover, some site-specific O-GlcNAc antibodies have been developed (Pathak et al., 2012; Cameron et al., 2013; Hirosawa et al., 2016; Shan et al., 2018). Despite the relatively large number of available antibodies, their different selectivity and their poor specificity are a limitation to detect all O-GlcNAcylated proteins and validate results obtained in mass spectrometry (MS) (Mueller et al., 2021). Development of site-specific O-GlcNAc antibodies is a challenge to study the impact of O-GlcNAcylation on protein individually.

#### 2.1.2. O-GlcNAc lectins

Lectins are largely used in glycobiology (Cummings and Etzler, 2009) and can therefore be used to study O-GlcNAcylation. The wheat germ agglutinin (WGA) has a particular affinity to GlcNAc moiety and is an interesting tool owing to its resistance to several protein denaturing techniques such as acidic environments, chaotropic agents and high temperature (Wright, 1987, 1992; Rodríguez-Romero et al., 1989; Chavelas et al., 2004). However, WGA has an affinity for other epitopes such as sialic acid and GlcNAc moiety presents on branched N-glycans and GlcNAc residue recognition requires close spatial proximity for high affinity binding (Zachara et al., 2011b). This lack of specificity is the argument used by Comer et al. to explain differences obtained in western blot between WGA-HRP (Horseradish peroxidase) and CTD110.6 (Comer et al., 2001). To improve its selectivity, WGA can be chemically modified with the addition of a succinyl group. Succinylated WGA (sWGA) selectively binds to GlcNAc but not to sialic acid (Monsigny et al., 1979).

Although it is the most widely used, WGA is not the only lectin with an affinity for the GlcNAc motif. Agrocybe aegerita lectin 2 (AAL2) has been reported to specifically bind to terminal non-reducing GlcNAc moiety with higher selectivity than WGA or griffonia simplicifolia lectin

<table><caption>**Table 1**
Antibodies used to detect O-GlcNAc.</caption><thead><tr><th rowspan="2">Name</th><th rowspan="2">Isotype</th><th rowspan="2">Epitope</th><th colspan="2">Specificity</th><th rowspan="2">Commercial availability</th><th rowspan="2">Reference</th></tr><tr><th>O-GlcNAc</th><th>β-GlcNAc</th></tr></thead><tbody><tr><td>RL2</td><td>IgG</td><td>Pore complex-lamina fraction isolated from rat liver nuclear envelopes</td><td>+</td><td>-</td><td>+</td><td>(Snow et al., 1987)</td></tr><tr><td>CTD110.6</td><td>IgM</td><td>Synthetic peptide (serine and threonine O-GlcNAcylation)</td><td>-</td><td>+</td><td>+</td><td>(Comer et al., 2001)</td></tr><tr><td>HGAC39</td><td>IgG</td><td rowspan="3">GlcNAc residues of streptococcal group A carbohydrate</td><td>-</td><td>+</td><td>-</td><td rowspan="3">(Turner et al., 1990)</td></tr><tr><td>HGAC49</td><td>IgM</td><td>-</td><td>+</td><td>-</td></tr><tr><td>HGAC85</td><td>IgG</td><td>-</td><td>+</td><td>+</td></tr><tr><td>MY95</td><td>IgG</td><td rowspan="2">N-acetylglucosamine</td><td>-</td><td>-</td><td>-</td><td>(Matsuoka et al., 2002)</td></tr><tr><td>10D8</td><td>IgM</td><td>-</td><td>-</td><td>-</td><td>(Tashima and Stanley, 2014)</td></tr><tr><td>18B10. C7</td><td>IgG</td><td rowspan="3">TLR2 ligand/helper T-epitope / CK II B-epitope (containing O-GlcNAc serine)</td><td>+</td><td>-</td><td>+</td><td rowspan="3">(Teo et al., 2010)</td></tr><tr><td>9D1. E4</td><td>IgG</td><td>-</td><td>+</td><td>+</td></tr><tr><td>1F5. D6</td><td>IgG</td><td>+</td><td>-</td><td>+</td></tr><tr><td>PTM-952</td><td>IgG</td><td>Serine and threonine O-GlcNAcylation</td><td>-</td><td>-</td><td>+</td><td>(Song et al., 2019)</td></tr><tr><td>TAB1 S395</td><td>IgG</td><td>Synthetic peptide (containing 389–403 TAB1 residues)</td><td>-</td><td>-</td><td>+</td><td>(Pathak et al., 2012)</td></tr><tr><td>Otau(S400)</td><td>IgG</td><td>Synthetic peptide (containing serine 400 of tau)</td><td>-</td><td>-</td><td>-</td><td>(Cameron et al., 2013)</td></tr><tr><td>20B2</td><td>IgG</td><td>Synthetic peptide (several putative O-GlcNAc histones target sequences)</td><td>-</td><td>-</td><td>-</td><td>(Hirosawa et al., 2016)</td></tr><tr><td>SIRT1-549-O</td><td>IgG</td><td>Synthetic peptide (containing serine 549 of SIRT1)</td><td>-</td><td>-</td><td>-</td><td>(Shan et al., 2018)</td></tr></tbody></table>
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Hexosamine biosynthesis pathway, O-GlcNAcylation and pharmacological tools. DON: 6-diazo-5-oxo-L-norleucine; BZX: benzoxazolinones; BADGP: benzyl-2-acetymido-2-deoxy- α-D-galactopyranoside. Adapted from Yang and Qian (2017).</figcaption></figure>

O-GlcNAcylated proteins by mass spectrometry requires the adaption of methods and the development of new enrichment approaches. Special consideration vis-à-vis tissue-sample preparation must be taken to protect GlcNAc residues from OGA (Dupas et al., 2020). The aim of this review was to provide an overview of the pharmacological approaches used to modulate GlcNAc levels and define the biochemical tools used to modify, detect, quantify, identify and map O-GlcNAcylation.

## 2. Development of biochemical approaches to study O-GlcNAc levels

### 2.1. Quantification methods

#### 2.1.1. O-GlcNAc antibodies

Like other post-translational modifications, antibodies are a classic approach used to study O-GlcNAcylation by western blot and immunoprecipitation. Several antibodies have been described to recognize GlcNAc residues such as RL2 (Snow et al., 1987), CTD110.6 (Comer et al., 2001) and others (Turner et al., 1990; Matsuoka et al., 2002; Tashima and Stanley, 2014; Teo et al., 2010) (Table 1). However, the

poor immunogenicity of GlcNAc residue, which is a neutral sugar, and the lack of knowledge about O-GlcNAcylated sequences are limits to massive development of selective antibodies. Considering that antibodies are developed to recognize a unique GlcNAc epitope and the potential cross reactivity with other sugar moieties, the profile of immunoprecipitated peptides is strictly different between antibodies (Teo et al., 2010; Okuda, 2017; Isono, 2011), thus limiting the information.

With the appearance of consensus sequences for both serine and threonine O-GlcNAc modifications (Wulff-Fuentes et al., 2021; Malard et al., 2021), new antibodies have been developed to increase the bank of antibodies and identify more O-GlcNAcylated proteins (Song et al., 2019). Moreover, some site-specific O-GlcNAc antibodies have been developed (Pathak et al., 2012; Cameron et al., 2013; Hirosawa et al., 2016; Shan et al., 2018). Despite the relatively large number of available antibodies, their different selectivity and their poor specificity are a limitation to detect all O-GlcNAcylated proteins and validate results obtained in mass spectrometry (MS) (Mueller et al., 2021). Development of site-specific O-GlcNAc antibodies is a challenge to study the impact of O-GlcNAcylation on protein individually.

#### 2.1.2. O-GlcNAc lectins

Lectins are largely used in glycobiology (Cummings and Etzler, 2009) and can therefore be used to study O-GlcNAcylation. The wheat germ agglutinin (WGA) has a particular affinity to GlcNAc moiety and is an interesting tool owing to its resistance to several protein denaturing techniques such as acidic environments, chaotropic agents and high temperature (Wright, 1987, 1992; Rodríguez-Romero et al., 1989; Chavelas et al., 2004). However, WGA has an affinity for other epitopes such as sialic acid and GlcNAc moiety presents on branched N-glycans and GlcNAc residue recognition requires close spatial proximity for high affinity binding (Zachara et al., 2011b). This lack of specificity is the argument used by Comer et al. to explain differences obtained in western blot between WGA-HRP (Horseradish peroxidase) and CTD110.6 (Comer et al., 2001). To improve its selectivity, WGA can be chemically modified with the addition of a succinyl group. Succinylated WGA (sWGA) selectively binds to GlcNAc but not to sialic acid (Monsigny et al., 1979).

Although it is the most widely used, WGA is not the only lectin with an affinity for the GlcNAc motif. Agrocybe aegerita lectin 2 (AAL2) has been reported to specifically bind to terminal non-reducing GlcNAc moiety with higher selectivity than WGA or griffonia simplicifolia lectin

<table><caption>**Table 1**
Antibodies used to detect O-GlcNAc.</caption><thead><tr><th rowspan="2">Name</th><th rowspan="2">Isotype</th><th rowspan="2">Epitope</th><th colspan="2">Specificity</th><th rowspan="2">Commercial availability</th><th rowspan="2">Reference</th></tr><tr><th>O-GlcNAc</th><th>β-GlcNAc</th></tr></thead><tbody><tr><td>RL2</td><td>IgG</td><td>Pore complex-lamina fraction isolated from rat liver nuclear envelopes</td><td>+</td><td>-</td><td>+</td><td>(Snow et al., 1987)</td></tr><tr><td>CTD110.6</td><td>IgM</td><td>Synthetic peptide (serine and threonine O-GlcNAcylation)</td><td>-</td><td>+</td><td>+</td><td>(Comer et al., 2001)</td></tr><tr><td>HGAC39</td><td>IgG</td><td rowspan="3">GlcNAc residues of streptococcal group A carbohydrate</td><td>-</td><td>+</td><td>-</td><td rowspan="3">(Turner et al., 1990)</td></tr><tr><td>HGAC49</td><td>IgM</td><td>-</td><td>+</td><td>-</td></tr><tr><td>HGAC85</td><td>IgG</td><td>-</td><td>+</td><td>+</td></tr><tr><td>MY95</td><td>IgG</td><td rowspan="2">N-acetylglucosamine</td><td>-</td><td>-</td><td>-</td><td>(Matsuoka et al., 2002)</td></tr><tr><td>10D8</td><td>IgM</td><td>-</td><td>-</td><td>-</td><td>(Tashima and Stanley, 2014)</td></tr><tr><td>18B10. C7</td><td>IgG</td><td rowspan="3">TLR2 ligand/helper T-epitope / CK II B-epitope (containing O-GlcNAc serine)</td><td>+</td><td>-</td><td>+</td><td rowspan="3">(Teo et al., 2010)</td></tr><tr><td>9D1. E4</td><td>IgG</td><td>-</td><td>+</td><td>+</td></tr><tr><td>1F5. D6</td><td>IgG</td><td>+</td><td>-</td><td>+</td></tr><tr><td>PTM-952</td><td>IgG</td><td>Serine and threonine O-GlcNAcylation</td><td>-</td><td>-</td><td>+</td><td>(Song et al., 2019)</td></tr><tr><td>TAB1 S395</td><td>IgG</td><td>Synthetic peptide (containing 389–403 TAB1 residues)</td><td>-</td><td>-</td><td>+</td><td>(Pathak et al., 2012)</td></tr><tr><td>Otau(S400)</td><td>IgG</td><td>Synthetic peptide (containing serine 400 of tau)</td><td>-</td><td>-</td><td>-</td><td>(Cameron et al., 2013)</td></tr><tr><td>20B2</td><td>IgG</td><td>Synthetic peptide (several putative O-GlcNAc histones target sequences)</td><td>-</td><td>-</td><td>-</td><td>(Hirosawa et al., 2016)</td></tr><tr><td>SIRT1-549-O</td><td>IgG</td><td>Synthetic peptide (containing serine 549 of SIRT1)</td><td>-</td><td>-</td><td>-</td><td>(Shan et al., 2018)</td></tr></tbody></table>
II (GSL-II) (Jiang et al., 2012). Psathyrella velutina lectin (PVL), another fungal lectin which has a close sequence and selectivity to AAL2 (Jiang et al., 2012; Kochibe and Matta, 1989; Ren et al., 2015), recognizes GlcNAc moiety but also N-acetylneuraminic acid (Ueda et al., 1999). Other natural lectins such as Boletopsis leucomelas lectin, Solanum lycopersicum lectin, Helix pomatia agglutinin and recombinant rAAL2 are described as GlcNAc-binding lectins but no study has been specifically performed on GlcNAc residue (Jiang et al., 2012).

In 2009, Ferrand et al. produced synthetic lectin which specifically recognized β-GlcNAc moiety (Ferrand et al., 2009), thereby enabling the development of new lectins derived from natural forms. In 2017, for example, Machon et al. reported a new lectin called Psathyrella velutina lectin (PVL). They observed that the recombinant form of PVL (rPVL) has an affinity to GlcNAc moiety and N-sialylated oligosaccharides as well as glycoproteins with a lower affinity but still to be considered when this lectin is used. By comparison with RL2 antibody, detection of GlcNAc moiety by HRP-rPVL and the signal/background ratio is identical but at a lower concentration (HRP-rPVL: 0.2 µg.ml⁻¹; RL2: 0.66 µg. ml⁻¹), suggesting that this lectin is a good and potentially cheaper alternative to antibody (Machon et al., 2017; Machon and Varrot, 2020). One perspective, as described in the study, is associated with the capacity to conjugate rPVL with different molecules such as Alexa Fluor, FITC, rhodamine, ferritin to use in ELISA, confocal and electronic microscopy, immunoprecipitation and immunohistology.

In addition to the possibility of coupling them to several molecules, lectins can be added in widely used techniques. In 2017, Kubota et al. described a new approach based on WGA affinity gel electrophoresis. In this method, sWGA is co-polymerized with acrylamide in the separating part of the gel. Based on the capacity of sWGA to bind GlcNAc residues, O-GlcNAcylated proteins are retained during migration which makes it possible to separate O-GlcNAcylated proteins from unmodified proteins (Kubota et al., 2017). One disadvantage associated with this technique is the potential difficulty to co-polymerize the lectin/acrylamide mixture. In fact, some contaminants such as the heavy metals present in WGA powder can impact the polymerization of the gel.

More recently, Gao et al. used a surface plasmon resonance (SPR) as biosensor to quantify O-GlcNAc levels on samples. They used a gold film associated with a construct of gold nanoparticles (AuNPS)/WGA to increase the signal. They showed that the addition of AuNPS/WGA construct amplified O-GlcNAc detection and that SPR intensity was linearly related to O-GlcNAc-CREB concentration. They also used this method to detect and quantify O-GlcNAc levels on α-crystallin protein and on human blood samples (Gao et al., 2018).

In summary, lectins are a promising complementary method to antibodies for the study of O-GlcNAcylation (Maynard and Chalkley, 2021). However, their selectivity could be increased to make this approach even more interesting.

## 2.2. Labeling methods

Labeling methods make it possible to (i) quantify O-GlcNAc protein levels and (ii) identify O-GlcNAcylated proteins (Ma and Hart, 2014; Zhu and Yi, 2021).

### 2.2.1. Chemoenzymatic labeling

O-GlcNAcylation was discovered and detected in 1984 through the use of bovine β1-4-galactosyltransferase 1 (GalT) which catalyzes the addition of a radioactive labeled galactose from UDP-[³H]galactose to terminal GlcNAc moiety (Torres and Hart, 1984). Despite a major breakthrough associated with the discovery of a new post-translational modification, this radioactivity approach which is associated with expensive material, drastic protection and exposure times from day to month, is now barely used (Khidekel et al., 2003).

GalT can be mutated into position 289 (Y289L) which increases its substrate range (e.g. UDP-GalNAc, unnatural ketone body (Keto-Gal), azido-modified UDP-galactose (UDP-GalNaz)) but also to stop using

radioactive substrate (Ramakrishnan and Qasba, 2002). By using this approach, Khidekel et al. detected α-crystallin O-GlcNAcylation which is known as being difficult to detect, thus validating the sensitivity of the method. Ultimately, ketone can be associated with biotin which can be detected in western blot or by fluorescence (Khidekel et al., 2003).

In 2018, Darabedian et al (Darabedian et al., 2018; Darabedian and Pratt, 2019), and Thompson et al (Thompson et al., 2018), described the copper-catalyzed azide-alkyne cycloaddition (CuAAC, more commonly known as click chemistry) and strain-promoted-azide-alkyne-cycloaddition (SPAAC) chemistry. They described the protocols for these techniques which make it possible to stabilize and label O-GlcNAc residues by using GalT (Y289L), UDP-GalNaz and alkyne-probe (alkyne-biotin, alkyne-TAMRA, alkyne-PEG, alkyne-Dde-biotin) and also to study O-GlcNAcylated proteins by mass spectrometry (Wang et al., ; Dupas et al., 2020; Denis et al., 2021).

In 2014, Wu et al. developed a new approach based on the principle that sulfation with a radioisotope ³⁵S, via two N-acetylglucosamine-specific sulfotransferases (carbohydrate sulfotransferase 2 (CHST2) and 4 (CHST4)) can detect GlcNAc moiety on proteins in vitro and identify O-GlcNAcylated proteins via MS (Wu et al., 2014).

As described, chemoenzymatic labeling approaches can be used to quantify O-GlcNAcylation and detect O-GlcNAcylated proteins but also to study protein interactions. Yu et al. used a combined genetic and chemical approach which enabled the introduction of diazirine photo-crosslinker onto O-GlcNAcylated proteins (Tanaka and Kohler, 2008; Bond et al., 2011; Yu et al., 2012). They mutated UDP-GlcNAc phosphorylase (an enzyme involved in the formation of UDP-GlcNAc) and synthetized GlcNDAz which efficiently produces UDP-GlcNDAz. UV light stimulation activates the crosslinker and produces covalent links between O-GlcNAcylated proteins and neighboring peptides or molecules, which can be identified via MS approach (Yu et al., 2012).

All of these approaches share a common drawback since non-specific labeling requires a supplemental step consisting of treatment with peptide: N-glycosidase F (PNGase F) to remove N-linked glycans which contain GlcNAc residues (Boeggeman et al., 2007).

### 2.2.2. Metabolic labeling

Contrary to chemoenzymatic labeling, which is only available in vitro, metabolic labeling is available both in vitro and in vivo. For example, [³H]glucosamine which is metabolized to UDP-[³H]GlcNAc throughout the HBP is an alternative method to UDP-[³H]galactose utilization (Roquemore et al., 1994; Kearse and Hart, 1991). The utilization of stable isotopes could be an alternative. For example, by using ¹³C-labeled substrates ([U-¹³C₆] glucosamine or [U-¹³C₆] glucose) combined with LC-MS, Olson et al. developed a new method to quantify both absolute HBP flux and HBP flux relative to glycolysis in perfused isolated murine working hearts. With this approach, they demonstrated that HBP flux represents 0.003–0.006 % of glycolysis (Olson et al., 2020).

Owing to the utilization of cell machinery to label O-GlcNAc modified proteins, metabolic labeling does not require the addition of other enzymes. N-azidoacetylglucosamine (GlcNAz) is the most widely used substrate but N-azidoacetylgalactosamine (GalNAz), a GlcNAc analogue, can also be incorporated in cells after its epimerization in GlcNAz and can also be used by hexosamine salvage enzymes to form UDP-GlcNAz (Vocadlo et al., 2003; Boyce et al., 2011). Once bound with proteins, a variety of probes can be coupled to the GlcNAz via Staudinger ligation or click chemistry (Sprung et al., 2005; Nandi et al., 2006; Gurcel et al., 2008; Hahne et al., 2013; Cm et al., 2015). Another GlcNAc analogue, alkynyl-modified GlcNAc (GlcNAlk) which is more selective for O-GlcNAc modification can be used. By coupling GlcNAlk with click chemistry, Zaro et al. identified 374 O-GlcNAcylated proteins, 279 of them undescribed (Zaro et al., 2011). However, cellular enzymes used for metabolic labeling promote their natural substrates compared with non-natural ones which can explain why the obtained signals are weak. Moreover, a recent study which compared metabolic (GlcNAz) and
II (GSL-II) (Jiang et al., 2012). Psathyrella velutina lectin (PVL), another fungal lectin which has a close sequence and selectivity to AAL2 (Jiang et al., 2012; Kochibe and Matta, 1989; Ren et al., 2015), recognizes GlcNAc moiety but also N-acetylneuraminic acid (Ueda et al., 1999). Other natural lectins such as Boletopsis leucomelas lectin, Solanum lycopersicum lectin, Helix pomatia agglutinin and recombinant rAAL2 are described as GlcNAc-binding lectins but no study has been specifically performed on GlcNAc residue (Jiang et al., 2012).

In 2009, Ferrand et al. produced synthetic lectin which specifically recognized β-GlcNAc moiety (Ferrand et al., 2009), thereby enabling the development of new lectins derived from natural forms. In 2017, for example, Machon et al. reported a new lectin called Psathyrella velutina lectin (PVL). They observed that the recombinant form of PVL (rPVL) has an affinity to GlcNAc moiety and N-sialylated oligosaccharides as well as glycoproteins with a lower affinity but still to be considered when this lectin is used. By comparison with RL2 antibody, detection of GlcNAc moiety by HRP-rPVL and the signal/background ratio is identical but at a lower concentration (HRP-rPVL: 0.2 µg.ml⁻¹; RL2: 0.66 µg. ml⁻¹), suggesting that this lectin is a good and potentially cheaper alternative to antibody (Machon et al., 2017; Machon and Varrot, 2020). One perspective, as described in the study, is associated with the capacity to conjugate rPVL with different molecules such as Alexa Fluor, FITC, rhodamine, ferritin to use in ELISA, confocal and electronic microscopy, immunoprecipitation and immunohistology.

In addition to the possibility of coupling them to several molecules, lectins can be added in widely used techniques. In 2017, Kubota et al. described a new approach based on WGA affinity gel electrophoresis. In this method, sWGA is co-polymerized with acrylamide in the separating part of the gel. Based on the capacity of sWGA to bind GlcNAc residues, O-GlcNAcylated proteins are retained during migration which makes it possible to separate O-GlcNAcylated proteins from unmodified proteins (Kubota et al., 2017). One disadvantage associated with this technique is the potential difficulty to co-polymerize the lectin/acrylamide mixture. In fact, some contaminants such as the heavy metals present in WGA powder can impact the polymerization of the gel.

More recently, Gao et al. used a surface plasmon resonance (SPR) as biosensor to quantify O-GlcNAc levels on samples. They used a gold film associated with a construct of gold nanoparticles (AuNPS)/WGA to increase the signal. They showed that the addition of AuNPS/WGA construct amplified O-GlcNAc detection and that SPR intensity was linearly related to O-GlcNAc-CREB concentration. They also used this method to detect and quantify O-GlcNAc levels on α-crystallin protein and on human blood samples (Gao et al., 2018).

In summary, lectins are a promising complementary method to antibodies for the study of O-GlcNAcylation (Maynard and Chalkley, 2021). However, their selectivity could be increased to make this approach even more interesting.

## 2.2. Labeling methods

Labeling methods make it possible to (i) quantify O-GlcNAc protein levels and (ii) identify O-GlcNAcylated proteins (Ma and Hart, 2014; Zhu and Yi, 2021).

### 2.2.1. Chemoenzymatic labeling

O-GlcNAcylation was discovered and detected in 1984 through the use of bovine β1-4-galactosyltransferase 1 (GalT) which catalyzes the addition of a radioactive labeled galactose from UDP-[³H]galactose to terminal GlcNAc moiety (Torres and Hart, 1984). Despite a major breakthrough associated with the discovery of a new post-translational modification, this radioactivity approach which is associated with expensive material, drastic protection and exposure times from day to month, is now barely used (Khidekel et al., 2003).

GalT can be mutated into position 289 (Y289L) which increases its substrate range (e.g. UDP-GalNAc, unnatural ketone body (Keto-Gal), azido-modified UDP-galactose (UDP-GalNaz)) but also to stop using

radioactive substrate (Ramakrishnan and Qasba, 2002). By using this approach, Khidekel et al. detected α-crystallin O-GlcNAcylation which is known as being difficult to detect, thus validating the sensitivity of the method. Ultimately, ketone can be associated with biotin which can be detected in western blot or by fluorescence (Khidekel et al., 2003).

In 2018, Darabedian et al (Darabedian et al., 2018; Darabedian and Pratt, 2019), and Thompson et al (Thompson et al., 2018), described the copper-catalyzed azide-alkyne cycloaddition (CuAAC, more commonly known as click chemistry) and strain-promoted-azide-alkyne-cycloaddition (SPAAC) chemistry. They described the protocols for these techniques which make it possible to stabilize and label O-GlcNAc residues by using GalT (Y289L), UDP-GalNaz and alkyne-probe (alkyne-biotin, alkyne-TAMRA, alkyne-PEG, alkyne-Dde-biotin) and also to study O-GlcNAcylated proteins by mass spectrometry (Wang et al., ; Dupas et al., 2020; Denis et al., 2021).

In 2014, Wu et al. developed a new approach based on the principle that sulfation with a radioisotope ³⁵S, via two N-acetylglucosamine-specific sulfotransferases (carbohydrate sulfotransferase 2 (CHST2) and 4 (CHST4)) can detect GlcNAc moiety on proteins in vitro and identify O-GlcNAcylated proteins via MS (Wu et al., 2014).

As described, chemoenzymatic labeling approaches can be used to quantify O-GlcNAcylation and detect O-GlcNAcylated proteins but also to study protein interactions. Yu et al. used a combined genetic and chemical approach which enabled the introduction of diazirine photo-crosslinker onto O-GlcNAcylated proteins (Tanaka and Kohler, 2008; Bond et al., 2011; Yu et al., 2012). They mutated UDP-GlcNAc phosphorylase (an enzyme involved in the formation of UDP-GlcNAc) and synthetized GlcNDAz which efficiently produces UDP-GlcNDAz. UV light stimulation activates the crosslinker and produces covalent links between O-GlcNAcylated proteins and neighboring peptides or molecules, which can be identified via MS approach (Yu et al., 2012).

All of these approaches share a common drawback since non-specific labeling requires a supplemental step consisting of treatment with peptide: N-glycosidase F (PNGase F) to remove N-linked glycans which contain GlcNAc residues (Boeggeman et al., 2007).

### 2.2.2. Metabolic labeling

Contrary to chemoenzymatic labeling, which is only available in vitro, metabolic labeling is available both in vitro and in vivo. For example, [³H]glucosamine which is metabolized to UDP-[³H]GlcNAc throughout the HBP is an alternative method to UDP-[³H]galactose utilization (Roquemore et al., 1994; Kearse and Hart, 1991). The utilization of stable isotopes could be an alternative. For example, by using ¹³C-labeled substrates ([U-¹³C₆] glucosamine or [U-¹³C₆] glucose) combined with LC-MS, Olson et al. developed a new method to quantify both absolute HBP flux and HBP flux relative to glycolysis in perfused isolated murine working hearts. With this approach, they demonstrated that HBP flux represents 0.003–0.006 % of glycolysis (Olson et al., 2020).

Owing to the utilization of cell machinery to label O-GlcNAc modified proteins, metabolic labeling does not require the addition of other enzymes. N-azidoacetylglucosamine (GlcNAz) is the most widely used substrate but N-azidoacetylgalactosamine (GalNAz), a GlcNAc analogue, can also be incorporated in cells after its epimerization in GlcNAz and can also be used by hexosamine salvage enzymes to form UDP-GlcNAz (Vocadlo et al., 2003; Boyce et al., 2011). Once bound with proteins, a variety of probes can be coupled to the GlcNAz via Staudinger ligation or click chemistry (Sprung et al., 2005; Nandi et al., 2006; Gurcel et al., 2008; Hahne et al., 2013; Cm et al., 2015). Another GlcNAc analogue, alkynyl-modified GlcNAc (GlcNAlk) which is more selective for O-GlcNAc modification can be used. By coupling GlcNAlk with click chemistry, Zaro et al. identified 374 O-GlcNAcylated proteins, 279 of them undescribed (Zaro et al., 2011). However, cellular enzymes used for metabolic labeling promote their natural substrates compared with non-natural ones which can explain why the obtained signals are weak. Moreover, a recent study which compared metabolic (GlcNAz) and
chemoenzymatic (GalNAz) labeling demonstrated that GlcNAz was preferentially incorporated into N- and O- glycans rather than in O-GlcNAcylated proteins, raising a significant limit to this method (Cieniewski-Bernard et al., 2014).

Despite their limitations, labeling methods are commonly used for the purification process of O-GlcNAcylated proteins with mass spectrometry (Maynard and Chalkley, 2021).

### 2.2.3. New probes

Many labeling methods have been developed based on Y289L GalT, UDP-GalNaz, alkyne-biotin-linkers and click chemistry. However, several linkers require a large spacer between the alkyne and the biotin group which increases the O-GlcNAcylated protein mass and could make its identification difficult. Moreover, the conditions to disrupt biotin-streptavidin interaction can hydrolyze GlcNAc residue (Szychowski et al., 2010). In 2016, Griffin et al. developed a new cleavable probe based on 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) functional group largely used as a protective group in peptide synthesis. Hydrazine treatment cleaves the Dde linker and generates a positively-charged aminomethytriazolyl group on O-GlcNAc modified sites with minimal modification of mass and the linker is compatible with rigorous washing steps. With this technique, they validated O-GlcNAc sites known on OGT and also managed to describe a new one, suggesting new ways to regulate OGT activity (Griffin et al., 2016).

In 2018, Qin et al. developed a new probe with an isotope-tagged cleavable linker (isoTCL) to study O-GlcNAcylation (Qin et al., 2018a). In order to quantify O-GlcNAcylation, they designed a pair of alkyne-biotin tags containing an acid-cleavable dialkoxydiphenylsilane (DADPS) linker which is isotopically labeled. They also described “light” alkyne-L-AC-biotin and “heavy” alkyne-H-AC-biotin. Both linkers can react with modified azido-GlcNAc proteins and enrichment can therefore be performed by streptavidin approach. Acid treatment makes it possible to cleave DADPS and liberate “heavy” or a “light” molecule with a difference of 6.0138 Da. O-GlcNAc sites can then be identified by comparing light/heavy ratios. Using this approach, they identified 259 and 317 O-GlcNAcylation sites from male and female mouse placenta, respectively, with 127 common sites.

### 2.2.4. New enzyme-based methods

In 2017, Aguilar et al. described a new chemoenzymatic histology method based on the utilization of a new alkyne donor called UDP-N-pentynylgalactosamine (UDP-GalNA1) to label O-GlcNAcylated proteins. UDP-GalNA1 is accepted by (Y289L) GalT and can be conjugated to a biotin azide via click chemistry. With this approach and using neutravidin-HRP, they observed O-GlcNAc distribution in murine organ samples but they also demonstrated that healthy brain tissue express higher O-GlcNAc levels than brain tissue from patients with Alzheimer's disease (Aguilar et al., 2017). Functionally, GalT (Y289L) possesses nonspecific activity with binds to terminal N-acetylglucosamine residues in N-glycans. A pretreatment with PNGase F to remove N-glycans is necessary to avoid false positives and to enhance selectivity.

More recently, Wu et al. used tandem glycan labeling based on (Y289L) GalT and α-2,6-sialyltransferase (ST6Gal1) enabling highly sensitive detection of the O-GlcNAcylation. The GlcNAc moiety is first galactosylated and then recognized by ST6Gal1 which added a fluorophore conjugated to sialic acid. They validated the method with identification of known O-GlcNAcylated proteins such as pyruvate kinase or sterol regulatory element binding protein-c1 but they also identified hypoxia-inducible factor 1-alpha as O-GlcNAcylated (Wu et al., 2020).

Several methods detect and quantify O-GlcNAcylable sites which are O-GlcNAcylated (closed sites) but few methods study O-GlcNAcylable sites non-O-GlcNAcylated (open sites). In 2018, Wu et al. developed a method based on two glycosyltransferases: OGT which incorporates GlcNAz in open sites and Beta-1,3-N-Acetylgalactosaminyltransferase 2 which incorporates GalNAz in closed sites. By coupling this approach

with click chemistry, they probed open and closed sites on proteins and observed variations in western blot migration or signal intensity in cellular extracts exclusively associated with O-GlcNAcylation (OGA treatment removes the signal). Ultimately, they conjugated biotinylated sugar with streptavidin-Alexa antibody to map O-GlcNAc sites in Chinese hamster ovary cells and identified that open sites are in the nucleocytoplasmic compartment while closed sites are enriched in the nucleus (Wu et al., 2018).

As described above, fluorescence is a powerful multistep-approach to localize and quantify O-GlcNAcylation. In 2018, Tan et al. developed direct one-step fluorescent labeling of O-GlcNAc modified with glucosamine-nitrobenzoxadiazole (GlcN-NBD) residue. They showed that a metabolic precursor (Ac3GlcN-β-Ala-NBD-α-1-P(Ac-SATE)2) can be integrated in the last step of HBP and labeled O-GlcNAcylated proteins in two cell models (mouse embryonic fibroblasts and HeLa). They also demonstrated that OGT tolerates the unnatural analogues of UDP-GlcNAc such as UDP-GlcN-β-Ala-NBD (Tan et al., 2018). One perspective associated with this technique is to incorporate distinct fluorochromes to perform two color pulse-chase experiments. The other possibility is to conjugate this approach with fluorescence lifetime imaging (FLIM) and follow in real-time the spatiotemporal action of OGT. However, despite a lower efficiency to transfer UDP-GlcN-β-Ala-NBD compared with OGT, other enzymes can transfer the metabolic precursor and potentially interfere with the signal.

Recently, per-O-acetylation of monosaccharides and their alkyne/azide analogues have been described as reacting with cysteine residue via S-glycosylation and therefore be at the origin of false positives (Qin et al., 2018b). In 2019, Hao et al. developed new unnatural monosaccharides to detect O-GlcNAcylation (Hao et al., 2019). They demonstrated that both 1,3-Ac2GalNAz and 1,3-Pr2GalNAz, two protected GalNAz, significantly improve labeling efficiency compared with unprotected GalNAz and without reaction with S-glycosylated cysteine. Interestingly, they found twice as many O-GlcNAcylated sites with 1, 3-Pr2GalNAz compared with GalNAz in HeLa cells (699 vs. 312, respectively, 272 of which are common). They identified that steroid hormone receptor ERR2, a transcription factor involved in pluripotency, is O-GlcNAcylated and that its O-GlcNAcylation is important for mouse embryonic stem cell properties (self-renewal and pluripotency).

## 3. Pharmacological tools

The lack of adapted biochemical tools to study O-GlcNAcylation has promoted the development of pharmacological approaches to decipher the role of O-GlcNAcylation and develop new potential therapeutic strategies. In this regard, several molecules used for pharmacological regulation of GFAT, OGT or OGA have been developed (Figs. 1, 2 and Table 2).

### 3.1. Decrease O-GlcNAcylation levels by GFAT or OGT inhibition

Cancer and diabetes are two pathologies where O-GlcNAc levels are increased. Pharmacological decrease in O-GlcNAc levels by GFAT or OGT inhibition may be a promising therapeutic strategy (Ma and Hart, 2013; Ferrer et al., 2016).

#### 3.1.1. GFAT inhibitors

There are few GFAT inhibitors (Walter). The most commonly used are DON and azaserine, two structural analogs of glutamine (Stock et al., 1954; Coffey et al., 1956) which can decrease O-GlcNAc levels. In 2011, Qian et al. discovered the compound RO0509347 as a reversible GFAT inhibitor (Qian et al., 2011). However, a recent study demonstrated that the compound RO0503947 reduces in vitro O-GlcNAcylation levels only at high concentrations (50 µM) limiting its use in vivo (Walter et al., 2020).

Considering (i) the pleiotropic effects due to the involvement of UDP-GlcNAc in many cellular processes such as N- and O-linked glycosylation
chemoenzymatic (GalNAz) labeling demonstrated that GlcNAz was preferentially incorporated into N- and O- glycans rather than in O-GlcNAcylated proteins, raising a significant limit to this method (Cieniewski-Bernard et al., 2014).

Despite their limitations, labeling methods are commonly used for the purification process of O-GlcNAcylated proteins with mass spectrometry (Maynard and Chalkley, 2021).

### 2.2.3. New probes

Many labeling methods have been developed based on Y289L GalT, UDP-GalNaz, alkyne-biotin-linkers and click chemistry. However, several linkers require a large spacer between the alkyne and the biotin group which increases the O-GlcNAcylated protein mass and could make its identification difficult. Moreover, the conditions to disrupt biotin-streptavidin interaction can hydrolyze GlcNAc residue (Szychowski et al., 2010). In 2016, Griffin et al. developed a new cleavable probe based on 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) functional group largely used as a protective group in peptide synthesis. Hydrazine treatment cleaves the Dde linker and generates a positively-charged aminomethytriazolyl group on O-GlcNAc modified sites with minimal modification of mass and the linker is compatible with rigorous washing steps. With this technique, they validated O-GlcNAc sites known on OGT and also managed to describe a new one, suggesting new ways to regulate OGT activity (Griffin et al., 2016).

In 2018, Qin et al. developed a new probe with an isotope-tagged cleavable linker (isoTCL) to study O-GlcNAcylation (Qin et al., 2018a). In order to quantify O-GlcNAcylation, they designed a pair of alkyne-biotin tags containing an acid-cleavable dialkoxydiphenylsilane (DADPS) linker which is isotopically labeled. They also described “light” alkyne-L-AC-biotin and “heavy” alkyne-H-AC-biotin. Both linkers can react with modified azido-GlcNAc proteins and enrichment can therefore be performed by streptavidin approach. Acid treatment makes it possible to cleave DADPS and liberate “heavy” or a “light” molecule with a difference of 6.0138 Da. O-GlcNAc sites can then be identified by comparing light/heavy ratios. Using this approach, they identified 259 and 317 O-GlcNAcylation sites from male and female mouse placenta, respectively, with 127 common sites.

### 2.2.4. New enzyme-based methods

In 2017, Aguilar et al. described a new chemoenzymatic histology method based on the utilization of a new alkyne donor called UDP-N-pentynylgalactosamine (UDP-GalNA1) to label O-GlcNAcylated proteins. UDP-GalNA1 is accepted by (Y289L) GalT and can be conjugated to a biotin azide via click chemistry. With this approach and using neutravidin-HRP, they observed O-GlcNAc distribution in murine organ samples but they also demonstrated that healthy brain tissue express higher O-GlcNAc levels than brain tissue from patients with Alzheimer's disease (Aguilar et al., 2017). Functionally, GalT (Y289L) possesses nonspecific activity with binds to terminal N-acetylglucosamine residues in N-glycans. A pretreatment with PNGase F to remove N-glycans is necessary to avoid false positives and to enhance selectivity.

More recently, Wu et al. used tandem glycan labeling based on (Y289L) GalT and α-2,6-sialyltransferase (ST6Gal1) enabling highly sensitive detection of the O-GlcNAcylation. The GlcNAc moiety is first galactosylated and then recognized by ST6Gal1 which added a fluorophore conjugated to sialic acid. They validated the method with identification of known O-GlcNAcylated proteins such as pyruvate kinase or sterol regulatory element binding protein-c1 but they also identified hypoxia-inducible factor 1-alpha as O-GlcNAcylated (Wu et al., 2020).

Several methods detect and quantify O-GlcNAcylable sites which are O-GlcNAcylated (closed sites) but few methods study O-GlcNAcylable sites non-O-GlcNAcylated (open sites). In 2018, Wu et al. developed a method based on two glycosyltransferases: OGT which incorporates GlcNAz in open sites and Beta-1,3-N-Acetylgalactosaminyltransferase 2 which incorporates GalNAz in closed sites. By coupling this approach

with click chemistry, they probed open and closed sites on proteins and observed variations in western blot migration or signal intensity in cellular extracts exclusively associated with O-GlcNAcylation (OGA treatment removes the signal). Ultimately, they conjugated biotinylated sugar with streptavidin-Alexa antibody to map O-GlcNAc sites in Chinese hamster ovary cells and identified that open sites are in the nucleocytoplasmic compartment while closed sites are enriched in the nucleus (Wu et al., 2018).

As described above, fluorescence is a powerful multistep-approach to localize and quantify O-GlcNAcylation. In 2018, Tan et al. developed direct one-step fluorescent labeling of O-GlcNAc modified with glucosamine-nitrobenzoxadiazole (GlcN-NBD) residue. They showed that a metabolic precursor (Ac3GlcN-β-Ala-NBD-α-1-P(Ac-SATE)2) can be integrated in the last step of HBP and labeled O-GlcNAcylated proteins in two cell models (mouse embryonic fibroblasts and HeLa). They also demonstrated that OGT tolerates the unnatural analogues of UDP-GlcNAc such as UDP-GlcN-β-Ala-NBD (Tan et al., 2018). One perspective associated with this technique is to incorporate distinct fluorochromes to perform two color pulse-chase experiments. The other possibility is to conjugate this approach with fluorescence lifetime imaging (FLIM) and follow in real-time the spatiotemporal action of OGT. However, despite a lower efficiency to transfer UDP-GlcN-β-Ala-NBD compared with OGT, other enzymes can transfer the metabolic precursor and potentially interfere with the signal.

Recently, per-O-acetylation of monosaccharides and their alkyne/azide analogues have been described as reacting with cysteine residue via S-glycosylation and therefore be at the origin of false positives (Qin et al., 2018b). In 2019, Hao et al. developed new unnatural monosaccharides to detect O-GlcNAcylation (Hao et al., 2019). They demonstrated that both 1,3-Ac2GalNAz and 1,3-Pr2GalNAz, two protected GalNAz, significantly improve labeling efficiency compared with unprotected GalNAz and without reaction with S-glycosylated cysteine. Interestingly, they found twice as many O-GlcNAcylated sites with 1, 3-Pr2GalNAz compared with GalNAz in HeLa cells (699 vs. 312, respectively, 272 of which are common). They identified that steroid hormone receptor ERR2, a transcription factor involved in pluripotency, is O-GlcNAcylated and that its O-GlcNAcylation is important for mouse embryonic stem cell properties (self-renewal and pluripotency).

## 3. Pharmacological tools

The lack of adapted biochemical tools to study O-GlcNAcylation has promoted the development of pharmacological approaches to decipher the role of O-GlcNAcylation and develop new potential therapeutic strategies. In this regard, several molecules used for pharmacological regulation of GFAT, OGT or OGA have been developed (Figs. 1, 2 and Table 2).

### 3.1. Decrease O-GlcNAcylation levels by GFAT or OGT inhibition

Cancer and diabetes are two pathologies where O-GlcNAc levels are increased. Pharmacological decrease in O-GlcNAc levels by GFAT or OGT inhibition may be a promising therapeutic strategy (Ma and Hart, 2013; Ferrer et al., 2016).

#### 3.1.1. GFAT inhibitors

There are few GFAT inhibitors (Walter). The most commonly used are DON and azaserine, two structural analogs of glutamine (Stock et al., 1954; Coffey et al., 1956) which can decrease O-GlcNAc levels. In 2011, Qian et al. discovered the compound RO0509347 as a reversible GFAT inhibitor (Qian et al., 2011). However, a recent study demonstrated that the compound RO0503947 reduces in vitro O-GlcNAcylation levels only at high concentrations (50 µM) limiting its use in vivo (Walter et al., 2020).

Considering (i) the pleiotropic effects due to the involvement of UDP-GlcNAc in many cellular processes such as N- and O-linked glycosylation
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Chemical structure of GFAT, OGT and OGA inhibitors. DON: 6-diazo-5-oxo-L-norleucine; BZX: benzoxazolinones (Phenyl 5-chloro-2-oxobenzo[d]oxazole-3 (2 H)-carboxylate); BADGP: benzyl-2-acetymido-2-deoxy- α-D-galactopyranoside.</figcaption></figure>

<table><caption>**Table 2**
Pharmacological tools to modulate O-GlcNAcylation levels.</caption><thead><tr><th>Name</th><th>IC50</th><th>Ki</th><th>Selectivity</th><th>Half-time clearance</th><th>Reference</th></tr></thead><tbody><tr><td colspan="6"><strong>GFAT inhibitors</strong></td></tr><tr><td>DON</td><td>18 µM</td><td></td><td></td><td></td><td>(Walter)</td></tr><tr><td>Azaserine</td><td>0.1–80 µM<sup>†</sup></td><td>0.77 mM</td><td></td><td></td><td>(Walter)</td></tr><tr><td>RO0509347</td><td>1 µM</td><td>0.3 µM</td><td></td><td></td><td>(Qian et al., 2011)</td></tr><tr><td colspan="6"><strong>OGT inhibitors</strong></td></tr><tr><td>Ac-5SGlcNAc</td><td>5 µM</td><td></td><td></td><td></td><td>(Trapannone et al., 2016)</td></tr><tr><td>OSMI-1</td><td>2.7 µM</td><td></td><td></td><td></td><td>(Ortiz-Meoz et al., 2015)</td></tr><tr><td>Alloxan</td><td>18 µM</td><td></td><td></td><td>1.5 min<sup>#</sup></td><td>(Trapannone et al., 2016), (Lenzen and Panten, 1988)</td></tr><tr><td>BZX</td><td>10 µM</td><td></td><td></td><td></td><td>(Trapannone et al., 2016)</td></tr><tr><td>BADGP</td><td>2–5 mM<sup>†</sup></td><td></td><td></td><td></td><td>(Trapannone et al., 2016)</td></tr><tr><td>L01</td><td>21.8 µM</td><td></td><td></td><td></td><td>(Liu et al., 2017)</td></tr><tr><td>5SGlcNHex</td><td>50–300 mg.kg<sup>-1</sup>†</td><td></td><td></td><td></td><td>(Liu et al., 2018a)</td></tr><tr><td colspan="6"><strong>OGA inhibitors</strong></td></tr><tr><td>PUGNAc</td><td>8.6 nM</td><td>46 nM</td><td>0.8</td><td></td><td>(Macauley et al., 2005), (Rao et al., 2006)</td></tr><tr><td>GlcNAcstatin</td><td>4 nM (GlcNAcstatin G)</td><td>0.42–8500 nM</td><td>190 to > 900,000</td><td></td><td>(Dorfmueller et al., 2009), (Dorfmueller et al., 2010)</td></tr><tr><td>Thiamet G</td><td>30 nM</td><td>21 nM</td><td>37,000</td><td></td><td>(Yuzwa et al., 2008)</td></tr><tr><td>NAG-thiazoline</td><td>50 µM to 4,5 mM<sup>†</sup></td><td>70 nM</td><td>1</td><td></td><td>(Macauley et al., 2005)</td></tr><tr><td>NButGT</td><td>8 µM</td><td>230 nM</td><td>1200</td><td>30 min<sup>*</sup></td><td>(Macauley et al., 2010a), (Macauley et al., 2008)</td></tr><tr><td>6-Ac-Cas</td><td>9 µM</td><td>300 nM</td><td></td><td></td><td>(Macauley et al., 2010b)</td></tr><tr><td>MK-8719</td><td>52.7 nM</td><td>7.9 nM</td><td></td><td>1.9 h<sup>*</sup></td><td>(Selnick et al., 2019), (Wang et al., 2020)</td></tr></tbody></table>

DON: 6-diazo-5-oxo-L-norleucine; BZX: benzoxazolinones (Phenyl 5-chloro-2-oxobenzo[d]oxazole-3(2 H)-carboxylate); BADGP: benzyl-2-acetymido-2-deoxy- α-D-galactopyranoside; IC50: concentration of inhibitor required to achieve 50 % inhibition; Ki: dissociation constant describing the affinity between the inhibitor and the enzyme; †: concentrations used in experiments; #: in vitro; *: in vivo.

(Akella et al., 2019) and (ii) the associated side effects (Ferron et al., 2019a), GFAT inhibitors are rarely used and OGT inhibitors appear to be a more promising strategy.

### 3.1.2. OGT inhibitors

OGT inhibitors (the most described in the literature: Alloxan, BZX, Ac-5SGlcNAc, BADGP and OSMI-1) (Trapannone et al., 2016;
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Chemical structure of GFAT, OGT and OGA inhibitors. DON: 6-diazo-5-oxo-L-norleucine; BZX: benzoxazolinones (Phenyl 5-chloro-2-oxobenzo[d]oxazole-3 (2 H)-carboxylate); BADGP: benzyl-2-acetymido-2-deoxy- α-D-galactopyranoside.</figcaption></figure>

<table><caption>**Table 2**
Pharmacological tools to modulate O-GlcNAcylation levels.</caption><thead><tr><th>Name</th><th>IC50</th><th>Ki</th><th>Selectivity</th><th>Half-time clearance</th><th>Reference</th></tr></thead><tbody><tr><td colspan="6"><strong>GFAT inhibitors</strong></td></tr><tr><td>DON</td><td>18 µM</td><td></td><td></td><td></td><td>(Walter)</td></tr><tr><td>Azaserine</td><td>0.1–80 µM<sup>†</sup></td><td>0.77 mM</td><td></td><td></td><td>(Walter)</td></tr><tr><td>RO0509347</td><td>1 µM</td><td>0.3 µM</td><td></td><td></td><td>(Qian et al., 2011)</td></tr><tr><td colspan="6"><strong>OGT inhibitors</strong></td></tr><tr><td>Ac-5SGlcNAc</td><td>5 µM</td><td></td><td></td><td></td><td>(Trapannone et al., 2016)</td></tr><tr><td>OSMI-1</td><td>2.7 µM</td><td></td><td></td><td></td><td>(Ortiz-Meoz et al., 2015)</td></tr><tr><td>Alloxan</td><td>18 µM</td><td></td><td></td><td>1.5 min<sup>#</sup></td><td>(Trapannone et al., 2016), (Lenzen and Panten, 1988)</td></tr><tr><td>BZX</td><td>10 µM</td><td></td><td></td><td></td><td>(Trapannone et al., 2016)</td></tr><tr><td>BADGP</td><td>2–5 mM<sup>†</sup></td><td></td><td></td><td></td><td>(Trapannone et al., 2016)</td></tr><tr><td>L01</td><td>21.8 µM</td><td></td><td></td><td></td><td>(Liu et al., 2017)</td></tr><tr><td>5SGlcNHex</td><td>50–300 mg.kg<sup>-1</sup> <sup>†</sup></td><td></td><td></td><td></td><td>(Liu et al., 2018a)</td></tr><tr><td colspan="6"><strong>OGA inhibitors</strong></td></tr><tr><td>PUGNAc</td><td>8.6 nM</td><td>46 nM</td><td>0.8</td><td></td><td>(Macauley et al., 2005), (Rao et al., 2006)</td></tr><tr><td>GlcNAcstatin</td><td>4 nM (GlcNAcstatin G)</td><td>0.42–8500 nM</td><td>190 to > 900,000</td><td></td><td>(Dorfmueller et al., 2009), (Dorfmueller et al., 2010)</td></tr><tr><td>Thiamet G</td><td>30 nM</td><td>21 nM</td><td>37,000</td><td></td><td>(Yuzwa et al., 2008)</td></tr><tr><td>NAG-thiazoline</td><td>50 µM to 4,5 mM<sup>†</sup></td><td>70 nM</td><td>1</td><td></td><td>(Macauley et al., 2005)</td></tr><tr><td>NButGT</td><td>8 µM</td><td>230 nM</td><td>1200</td><td>30 min<sup>*</sup></td><td>(Macauley et al., 2010a), (Macauley et al., 2008)</td></tr><tr><td>6-Ac-Cas</td><td>9 µM</td><td>300 nM</td><td></td><td></td><td>(Macauley et al., 2010b)</td></tr><tr><td>MK-8719</td><td>52.7 nM</td><td>7.9 nM</td><td></td><td>1.9 h<sup>*</sup></td><td>(Selnick et al., 2019), (Wang et al., 2020)</td></tr></tbody></table>

DON: 6-diazo-5-oxo-L-norleucine; BZX: benzoxazolinones (Phenyl 5-chloro-2-oxobenzo[d]oxazole-3(2 H)-carboxylate); BADGP: benzyl-2-acetymido-2-deoxy- α-D-galactopyranoside; IC50: concentration of inhibitor required to achieve 50 % inhibition; Ki: dissociation constant describing the affinity between the inhibitor and the enzyme; †: concentrations used in experiments; #: in vitro; *: in vivo.

(Akella et al., 2019) and (ii) the associated side effects (Ferron et al., 2019a), GFAT inhibitors are rarely used and OGT inhibitors appear to be a more promising strategy.

### 3.1.2. OGT inhibitors

OGT inhibitors (the most described in the literature: Alloxan, BZX, Ac-5SGlcNAc, BADGP and OSMI-1) (Trapannone et al., 2016;
Ortiz-Meoz et al., 2015; Lenzen and Panten, 1988; Rj et al., 2002; Kang et al., 2008; Pantaleon et al., 2010; Gloster et al., 2011; Jiang et al., 2011; Lenzen and Munday, 1991) induce a decrease in O-GlcNAc levels. Unfortunately, OGT inhibitors are poorly compatible with biological environments (some of them are solubilized in DMSO) and their lack of selectivity makes them unsuitable therapeutic strategies (Ferron et al., 2019a). For example, the lack of selectivity induces toxicity and reduces cell viability even with the most recent compound OSMI-1 and alloxan (Trapannone et al., 2016; Ortiz-Meoz et al., 2015; Lee et al., 2006).

In response to these drawbacks, Liu et al. identified via structure-
based virtual screening analysis a new specific natural-product OGT
inhibitor (L01) (Liu et al., 2017). L01 inhibits OGT in vitro by interacting
with residues in the UDP-binding site of OGT without toxicity in cellular
and zebrafish models. However, L01 also blocks mucin-type O-glyco-
sylation, another protein modification involved in protein secretion,
processing and function (Tran and Ten Hagen, 2013). In 2018, another
team synthesized a new compound (5SGlcNHex) based on 5SGlcNAc
structure. The addition of the hydrophobic N-acyl groups provides a
balance between hydrophilicity and lipophilicity making it
water-soluble yet able to diffuse into cells. They demonstrated that
5SGlcNHex can reduce O-GlcNAc levels in vitro and in vivo in a
dose-dependent manner (Liu et al., 2018a). Contrary to Ac-5SGlcNAc
which can impact other forms of glycosylation, this compound does
not have weakness an effect on other types of glycosylation even at
higher doses.

The development of new OGT inhibitors remains a major challenge
to (i) decipher the roles of O-GlcNAcylation and (ii) develop new po-
tential therapeutic molecules (Zhang et al., 2018; Kim, 2020; Weiss
et al., 2021).

3.2. Increase O-GlcNAcylation levels by OGA inhibition

Acute increase protein O-GlcNAc levels have been reported to be
beneficial in many different models and situations (e.g. ischemia-
reperfusion, heart failure, hemorrhagic and septic shock) (Champatta-
nachai et al., 2007; Nöt et al., 2010; Watson et al., 2010; Ferron et al.,
2019b). An increase in UDP-GlcNAc by addition of glutamine or
glucosamine (Marshall et al., 2004) or inhibition of OGA (PUGNAc,
NButGT, Thiamet G, NAG-thiazoline and GlcNAcstatin) (Macauley et al.,
2005, 2010a; Rao et al., 2006; Dorfmueller et al., 2009, 2006, 2010;
Yuzwa et al., 2008; Macauley and Vocadlo, 2009; Kong et al., 2015,
2008, 2010b) efficiently increase O-GlcNAc levels. Despite in vitro and
in vivo utilization, OGA inhibitors are associated with low selectivity
(PUGNAc), lack of stability in vivo (NButGT) and high production costs
(GlcNAcstatin). One of the most promising compounds is MK-8719, an
OGA inhibitor developed by Selnick et al. with very low off-target effects
on lysosomal hexosaminidase A or B. Moreover, they validate ADME-tox
for the molecule which is in phase I clinical trial for Tauopathies (Selnick
et al., 2019; Wang et al., 2020). Also in favor of the therapeutic potential
of O-GlcNAcylation, another industrial company has developed an OGA
inhibitor (LY3372689), currently in phase 2 clinical trial for Alzheimer's
disease (Shcherbinin et al., 2020).

Improved knowledge of substrate recognition by OGA is an avenue to
develop new inhibitors (Elbatrawy et al., 2020). Considering the
drawbacks of chemical products, the development of new inhibitors
with higher selectivity, solubility, stability and lower toxicity is a major
issue. Moreover, pharmacological tools to modulate O-GlcNAc levels are
insufficient to decipher the role of this post-translational modification.
In response, complementary techniques have been developed over the
past decade to quantify O-GlcNAc levels (e.g., antibodies, lectins and
labeling-method approaches) and also to identify O-GlcNAcylated
proteins.

3.3. RNA interference approaches

RNA interference (RNAi) is a commonly used approach for the

understanding of any given protein function and can be an alternative
strategy when inhibitors are associated with strong side effects.

GFAT exists in two isoforms (75 % amino acid identity in humans and mice), GFAT1 and GFAT2, encoded by two different genes: Gfpt1 and Gfpt2. GFAT1 mRNA is ubiquitously expressed while GFAT2 mRNA is mainly expressed in the central nervous system and the heart (Oki et al., 1999). Unlike GFAT inhibitors which do not distinguish the two isoforms, the use of RNAi makes it possible to specifically target one of the two isoforms. In a mouse model of pancreatic tumor, Ying et al. demonstrated that GFAT1 RNAi decreases efficiently both GFAT1 protein expression and O-GlcNAc levels (Ying et al., 2012). Another study on human keratinocytes validated that RNAi against GFAT1 reduced GFAT1 mRNA and UDP-GlcNAc amount (Oikari et al., 2016). Interestingly, a recent study demonstrated despite a decrease in GFAT2 protein expression that GFAT2 RNAi was not associated with decreased O-GlcNAc levels in neonatal rat ventricular cardiomyocytes (Ishikita et al., 2021). Considering the time- and tissue-dependent GFAT1 and GFAT2 expression and the proximity of both isoform sequences, the use of RNAi directed against GFAT1 or GFAT2 at the same time should be used to be effective (Dupas et al., 2020). Moreover, GFAT knock-down is generally incomplete and leads to a slight decrease in UDP-GlcNAc which can maintain O-GlcNAc levels even at low concentrations (Boehmelt et al., 2000). Moreover, considering that the UDP-GlcNAc is involved in N- and O-linked glycosylation (Akella et al., 2019), GFAT RNAi can be associated with side effects.

Faced with the lack of existing OGT inhibitors, the development and use of RNAi has expanded greatly. O-GlcNAc transferase exists in three isoforms resulting from alternative splicing: nucleocytoplasmic OGT (ncOGT), mitochondrial OGT (mOGT) and short OGT (sOGT) which differs by the number of tetratricopeptide repeat motifs (Lazarus et al., 2006). As proof that RNAi is a high selectivity tool, a recent study demonstrated that RNAi against mOGT decreased its expression without affecting ncOGT expression (Sacoman et al., 2017). However, depending on the experimental conditions, OGT RNAi does not always change O-GlcNAc levels (Lima et al., 2011; Runager et al., 2014).

OGA exists in two splice isoforms: long OGA and short OGA which
differ by the presence of a HAT domain (IOGA) or not (sOGA). As for
GFAT and OGT, the RNAi approach makes it possible to target only one
isoform (Keembiyehetty et al., 2011). As described above for the other
targets, RNAi directed against OGA does not always change O-GlcNAc
levels (Runager et al., 2014; Keembiyehetty et al., 2011; Decourcelle
et al., 2019). However, owing to the existence of effective inhibitors
against OGA, the development of RNAi targeting it is a less important
issue.

In summary, RNAi is a highly selective complementary technique as a pharmacological tool to modulate O-GlcNAc levels but also to unravel the role of different isoforms. Despite the interesting aspect of being able to independently target the different isoforms of each enzyme, the RNAi approach has drawbacks such as RNAi stability (notably owing to RNAse sensitivity), poor pharmacokinetic behavior and potential off-target effects (Hu et al., 2020). Development of chemically modified RNAi could help to limit these drawbacks.

4. Development of enrichment approaches to study O-GlcNAc
using mass spectrometry

Mass spectrometry has long been used to identify proteins and their
potential PTM. However, the high lability of the GlcNAc moiety remains
a major issue to the use of this technique for O-GlcNAcylation study
(Greis and Hart, 1998; Wang et-al.). In order to limit O-GlcNAc loss
during fragmentation (Vosseller et-al.; Mirgorodskaya et al., 1999;
Hahne and Kuster, 2011), adapted fragmentation methods for mass
spectrometry such as electron capture dissociation (ECD), electron
transfer dissociation (ETD), higher energy C-trap dissociation (HCD)
have been developed. For details regarding these approaches, see the
different reference articles (Ma and Hart, 2014, 2017; Ma et al., 2019).
Ortiz-Meoz et al., 2015; Lenzen and Panten, 1988; Rj et al., 2002; Kang et al., 2008; Pantaleon et al., 2010; Gloster et al., 2011; Jiang et al., 2011; Lenzen and Munday, 1991) induce a decrease in O-GlcNAc levels. Unfortunately, OGT inhibitors are poorly compatible with biological environments (some of them are solubilized in DMSO) and their lack of selectivity makes them unsuitable therapeutic strategies (Ferron et al., 2019a). For example, the lack of selectivity induces toxicity and reduces cell viability even with the most recent compound OSMI-1 and alloxan (Trapannone et al., 2016; Ortiz-Meoz et al., 2015; Lee et al., 2006).

In response to these drawbacks, Liu et al. identified via structure-
based virtual screening analysis a new specific natural-product OGT
inhibitor (L01) (Liu et al., 2017). L01 inhibits OGT in vitro by interacting
with residues in the UDP-binding site of OGT without toxicity in cellular
and zebrafish models. However, L01 also blocks mucin-type O-glyco-
sylation, another protein modification involved in protein secretion,
processing and function (Tran and Ten Hagen, 2013). In 2018, another
team synthesized a new compound (5SGlcNHex) based on 5SGlcNAc
structure. The addition of the hydrophobic N-acyl groups provides a
balance between hydrophilicity and lipophilicity making it
water-soluble yet able to diffuse into cells. They demonstrated that
5SGlcNHex can reduce O-GlcNAc levels in vitro and in vivo in a
dose-dependent manner (Liu et al., 2018a). Contrary to Ac-5SGlcNAc
which can impact other forms of glycosylation, this compound does
not have weakness an effect on other types of glycosylation even at
higher doses.

The development of new OGT inhibitors remains a major challenge to (i) decipher the roles of O-GlcNAcylation and (ii) develop new potential therapeutic molecules (Zhang et al., 2018; Kim, 2020; Weiss et al., 2021).

3.2. Increase O-GlcNAcylation levels by OGA inhibition

Acute increase protein O-GlcNAc levels have been reported to be
beneficial in many different models and situations (e.g. ischemia-
reperfusion, heart failure, hemorrhagic and septic shock) (Champatta-
nachai et al., 2007; Nöt et al., 2010; Watson et al., 2010; Ferron et al.,
2019b). An increase in UDP-GlcNAc by addition of glutamine or
glucosamine (Marshall et al., 2004) or inhibition of OGA (PUGNAc,
NButGT, Thiamet G, NAG-thiazoline and GlcNAcstatin) (Macauley et al.,
2005, 2010a; Rao et al., 2006; Dorfmueller et al., 2009, 2006, 2010;
Yuzwa et al., 2008; Macauley and Vocadlo, 2009; Kong et al., 2015,
2008, 2010b) efficiently increase O-GlcNAc levels. Despite in vitro and
in vivo utilization, OGA inhibitors are associated with low selectivity
(PUGNAc), lack of stability in vivo (NButGT) and high production costs
(GlcNAcstatin). One of the most promising compounds is MK-8719, an
OGA inhibitor developed by Selnick et al. with very low off-target effects
on lysosomal hexosaminidase A or B. Moreover, they validate ADME-tox
for the molecule which is in phase I clinical trial for Tauopathies (Selnick
et al., 2019; Wang et al., 2020). Also in favor of the therapeutic potential
of O-GlcNAcylation, another industrial company has developed an OGA
inhibitor (LY3372689), currently in phase 2 clinical trial for Alzheimer's
disease (Shcherbinin et al., 2020).

Improved knowledge of substrate recognition by OGA is an avenue to
develop new inhibitors (Elbatrawy et al., 2020). Considering the
drawbacks of chemical products, the development of new inhibitors
with higher selectivity, solubility, stability and lower toxicity is a major
issue. Moreover, pharmacological tools to modulate O-GlcNAc levels are
insufficient to decipher the role of this post-translational modification.
In response, complementary techniques have been developed over the
past decade to quantify O-GlcNAc levels (e.g., antibodies, lectins and
labeling-method approaches) and also to identify O-GlcNAcylated
proteins.

3.3. RNA interference approaches

RNA interference (RNAi) is a commonly used approach for the

understanding of any given protein function and can be an alternative
strategy when inhibitors are associated with strong side effects.

GFAT exists in two isoforms (75 % amino acid identity in humans and mice), GFAT1 and GFAT2, encoded by two different genes: Gfpt1 and Gfpt2. GFAT1 mRNA is ubiquitously expressed while GFAT2 mRNA is mainly expressed in the central nervous system and the heart (Oki et al., 1999). Unlike GFAT inhibitors which do not distinguish the two isoforms, the use of RNAi makes it possible to specifically target one of the two isoforms. In a mouse model of pancreatic tumor, Ying et al. demonstrated that GFAT1 RNAi decreases efficiently both GFAT1 protein expression and O-GlcNAc levels (Ying et al., 2012). Another study on human keratinocytes validated that RNAi against GFAT1 reduced GFAT1 mRNA and UDP-GlcNAc amount (Oikari et al., 2016). Interestingly, a recent study demonstrated despite a decrease in GFAT2 protein expression that GFAT2 RNAi was not associated with decreased O-GlcNAc levels in neonatal rat ventricular cardiomyocytes (Ishikita et al., 2021). Considering the time- and tissue-dependent GFAT1 and GFAT2 expression and the proximity of both isoform sequences, the use of RNAi directed against GFAT1 or GFAT2 at the same time should be used to be effective (Dupas et al., 2020). Moreover, GFAT knock-down is generally incomplete and leads to a slight decrease in UDP-GlcNAc which can maintain O-GlcNAc levels even at low concentrations (Boehmelt et al., 2000). Moreover, considering that the UDP-GlcNAc is involved in N- and O-linked glycosylation (Akella et al., 2019), GFAT RNAi can be associated with side effects.

Faced with the lack of existing OGT inhibitors, the development and use of RNAi has expanded greatly. O-GlcNAc transferase exists in three isoforms resulting from alternative splicing: nucleocytoplasmic OGT (ncOGT), mitochondrial OGT (mOGT) and short OGT (sOGT) which differs by the number of tetratricopeptide repeat motifs (Lazarus et al., 2006). As proof that RNAi is a high selectivity tool, a recent study demonstrated that RNAi against mOGT decreased its expression without affecting ncOGT expression (Sacoman et al., 2017). However, depending on the experimental conditions, OGT RNAi does not always change O-GlcNAc levels (Lima et al., 2011; Runager et al., 2014).

OGA exists in two splice isoforms: long OGA and short OGA which
differ by the presence of a HAT domain (IOGA) or not (sOGA). As for
GFAT and OGT, the RNAi approach makes it possible to target only one
isoform (Keembiyehetty et al., 2011). As described above for the other
targets, RNAi directed against OGA does not always change O-GlcNAc
levels (Runager et al., 2014; Keembiyehetty et al., 2011; Decourcelle
et al., 2019). However, owing to the existence of effective inhibitors
against OGA, the development of RNAi targeting it is a less important
issue.

In summary, RNAi is a highly selective complementary technique as a pharmacological tool to modulate O-GlcNAc levels but also to unravel the role of different isoforms. Despite the interesting aspect of being able to independently target the different isoforms of each enzyme, the RNAi approach has drawbacks such as RNAi stability (notably owing to RNAse sensitivity), poor pharmacokinetic behavior and potential off-target effects (Hu et al., 2020). Development of chemically modified RNAi could help to limit these drawbacks.

4. Development of enrichment approaches to study O-GlcNAc
using mass spectrometry

Mass spectrometry has long been used to identify proteins and their
potential PTM. However, the high lability of the GlcNAc moiety remains
a major issue to the use of this technique for O-GlcNAcylation study
(Greis and Hart, 1998; Wang et-al.). In order to limit O-GlcNAc loss
during fragmentation (Vosseller et-al.; Mirgorodskaya et al., 1999;
Hahne and Kuster, 2011), adapted fragmentation methods for mass
spectrometry such as electron capture dissociation (ECD), electron
transfer dissociation (ETD), higher energy C-trap dissociation (HCD)
have been developed. For details regarding these approaches, see the
different reference articles (Ma and Hart, 2014, 2017; Ma et al., 2019).
In addition to its high lability, ion suppression occurs in O-GlcNAc
modified peptides compared with unmodified ones, even when both
occur at similar concentrations, making it difficult to observe (Wang
et-al.). Given that, enrichment methods have been developed to protect
and stabilize the GlcNAc moiety in order to improve the signal from
O-GlcNAcylated peptides.

4.1. Enrichment of the proteins by direct capture

Direct capture is based on direct recognition of the GlcNAc moiety and uses either O-GlcNAc antibodies (with the limitations discussed above) or O-GlcNAc-binding lectin.

In the conventional antibody-based enrichment approach, RL2 and CTD 110.6 are the most commonly used. However, the low affinity of antibodies and cross-reactivity may interfere with the enrichment of the O-GlcNAcylated proteins for their identification (Tashima and Stanley, 2014). In order to increase the number of enriched O-GlcNAcylated proteins, monoclonal antibodies (e.g. 18B10. C7; 9D1. E4; 1F5. D6; PTM-952) can be used (Teo et al., 2010; Song et al., 2019). However due to O-GlcNAc epitope recognition, immunoprecipitated proteins are different with each antibody, suggesting that (i) the use of several antibodies is mandatory to identify more O-GlcNAcylated proteins and (ii) some O-GlcNAcylated proteins are not enriched.

To increase the number of enriched O-GlcNAc proteins, a low-affinity
lectin chromatography (LWAC) enrichment method was developed in
2006. In this approach, a lectin, such as WGA, is coupled with agarose
and packed into high performance liquid chromatography (HPLC)
(Vosseller et al., ). Using LWAC, Vosseller et al. enriched 145 unique
O-GlcNAc modified peptides (Vosseller et al., ). More recently, using
LWAC combined with ETD/HCD-MS/MS, Kim et al. identified
123 O-GlcNAcylated proteins from rat sciatic nerve. Among them, they
demonstrated the importance of the periaxin O-GlcNAcylation for
myelin maintenance (Kim et al., 2016). However, this approach favors
enrichment of high-abundance proteins or those with multiple clustered
O-GlcNAc residues. Low abundance proteins with single or separate
O-GlcNAcylated sites are difficult to purify using this tool since their
binding to solid affinity support is completed with more abundant
O-GlcNAcylated proteins. Moreover, LWAC suffers from low affinity
with the GlcNAc moiety. A large amount of protein sample (around tens
of milligrams) is therefore required for O-GlcNAc profiling (Shen et al.,
2017a).

In 2017, in order to decrease the amount of protein sample used to
detect GlcNAc motif, Liu et al. developed a new enrichment method
based on Agrocybe aegerita lectin 2 (AAL2). AAL2 was conjugated with
resin (Sepharose 4B or POROS) and packed in an affinity column. With
this technique, they identified 17 O-linked HexNAc-modified proteins
(Liu et al., 2018b), suggesting that AAL2 is a new and useful tool for
enrichment of O-GlcNAcylated proteins.

In order to improve the number of identified, quantified proteins and
the number of O-GlcNAcylated sites mapped, Lee et al. performed an
enrichment method based on a novel G5-lectibody. This approach cor-
responded to a combination of 4 O-GlcNAc specific antibodies
(CTD110.6, RL2, HGAC39 and HGAC85) and the lectin WGA. Coupled
with LC-MS/MS, they identified 990 O-GlcNAcylated proteins and they
demonstrated that oxidative stress is associated with a modulation of O-
GlcNAc levels (Lee et al., 2016). This study suggests that G5-lectibody
enrichment provides greater coverage of the O-GlcNAc sub-proteome.
However, like other lectins, WGA, AAL2 and rPVL also have an affin-
ity to GlcNAc terminal residues on N-glycans. Specificity could be
improved via treatment with PNGase F prior to enrichment.

In 2017, to increase the enrichment efficiency of O-GlcNAc peptides, Shen et al. performed a novel strategy for global mapping of O-GlcNAcylated proteins using hydrophilic interaction liquid metabolic chemical reporter chromatography (HILIC) combined with selective deglycosylation. In that study, most of the N- and O-linked oligosaccharides were removed by treatment with PNGase F, sialidase and O-glycosidase

(Endo-α-N-Acetylgalactosaminidase). This step was followed by HILIC
enrichment using hydrophilic polymer modified-silica microparticles
and mass spectrometry analysis (pPEGMA-SMs). A combination of these
two steps enabled efficient O-GlcNAcylated protein enrichment even in
the presence of 500-fold more abundant unmodified proteins. Combined
with LC-MS, they identified 457 O-GlcNAcylated proteins from human
urine suggesting that this simple and robust method could be available
for other biological samples (tissues, blood, etc.) (Shen et al., 2017b).

In the same year, Selvan et al. developed a novel enrichment method based on mutant O-GlcNAcase (Selvan et al., 2017). In this study, a catalytically inactive bacterial O-GlcNAcase mutant (Clostridium perfringens O-GlcNAcase (CpOGA-D298N)) was used to trap O-GlcNAcylated proteins. Conjugated to HaloLink beads, CpOGA is used to pull down O-GlcNAcylated proteins to enable their enrichment. This approach provided identification of 2358 proteins from Drosophila embryos which were significantly enriched compared with the control pull-down. However, CpOGA (D298N) has a higher affinity to Ser-O-GlcNAc compared with Thr-O-GlcNAc peptides which limit O-GlcNAcylated enrichment (Mariappa et al., 2015).

4.2. Protein enrichment by labeling methods

In contrast to direct capture, labeling enrichment is a broad category
of indirect enrichment techniques. Labeling enrichment methods are
distinguished in two distinct categories: enrichment by replacement and
enrichment by chemical functionalization.

4.2.1. Enrichment by replacement

β-elimination Michael addition (BEMA) is an approach leading to the replacement of the GlcNAc moiety by a new group with a stronger link and stable enough to withstand fragmentation throughout mass spectrometry (Wells et al., 2002; Greis et al., 1996). In BEMA, GlcNAc residue is eliminated via basic conditions and produces an α, β-unsaturated carbonyl group. This group can be modified using a sulfhydryl nucleophile such as dithiothreitol (DTT), deuterated DTT or biotinylated cystamine/pentylamine which enables generation of a unique mass label (Wells et al., 2002; Overath et al., 2012). Modified proteins can then be enriched via thiol-capture resin or streptavidin-conjugated beads. In addition to being an enrichment method, BEMA can be a quantification method by using labeled DTT. This approach can facilitate identification of the modified sites by monitoring the BEMA label. The technical weakness associated with BEMA is cross-reactivity such as with phosphorylated residues or alkylated cysteines. However, pretreatment with phosphatase can improve selectivity. Despite its common use across studies, the low efficacy of the BEMA reaction will be a limiting factor for large-scale profiling O-GlcNAcylation studies.

The hydrazide reaction is a well-known technique for N-glycoprotein
enrichment. This approach makes it possible to enrich O-GlcNAcylated
proteins by oxidizing the GlcNAc moiety to its aldehyde derivative.
Proteins are then captured by hydrazide solid support and released by
hydroxylamine treatment (Klement et al., 2010). The major disadvan-
tage associated with this technique is the difficulty to produce aldehyde
derivative from GlcNAc moiety which limits O-GlcNAcylated protein
enrichment (Chen et al., 2021). A modification of the GlcNAc group with
the addition of a Gal moiety prior to its oxidation could limit the
problem (Chen et al., 2021; Xu et al., 2020).

4.2.2. Enrichment by chemical functionalization

Instead of replacing the GlcNAc moiety, a chemical reporter can be
incorporated with the protein of interest by chemoenzymatic or meta-
bolic labeling (Darley-Usmar et al., 2012).

4.2.2.1. *Chemoenzymatic labeling.* One common tool used in chemo-
enzymatic approaches is Y289L GalT. Used in combination with the
lectin Ricinus communis agglutinin I (RCA I) which recognizes
In addition to its high lability, ion suppression occurs in O-GlcNAc
modified peptides compared with unmodified ones, even when both
occur at similar concentrations, making it difficult to observe (Wang
et-al.). Given that, enrichment methods have been developed to protect
and stabilize the GlcNAc moiety in order to improve the signal from
O-GlcNAcylated peptides.

4.1. Enrichment of the proteins by direct capture

Direct capture is based on direct recognition of the GlcNAc moiety and uses either O-GlcNAc antibodies (with the limitations discussed above) or O-GlcNAc-binding lectin.

In the conventional antibody-based enrichment approach, RL2 and CTD 110.6 are the most commonly used. However, the low affinity of antibodies and cross-reactivity may interfere with the enrichment of the O-GlcNAcylated proteins for their identification (Tashima and Stanley, 2014). In order to increase the number of enriched O-GlcNAcylated proteins, monoclonal antibodies (e.g. 18B10. C7; 9D1. E4; 1F5. D6; PTM-952) can be used (Teo et al., 2010; Song et al., 2019). However due to O-GlcNAc epitope recognition, immunoprecipitated proteins are different with each antibody, suggesting that (i) the use of several antibodies is mandatory to identify more O-GlcNAcylated proteins and (ii) some O-GlcNAcylated proteins are not enriched.

To increase the number of enriched O-GlcNAc proteins, a low-affinity
lectin chromatography (LWAC) enrichment method was developed in
2006. In this approach, a lectin, such as WGA, is coupled with agarose
and packed into high performance liquid chromatography (HPLC)
(Vosseller et al., ). Using LWAC, Vosseller et al. enriched 145 unique
O-GlcNAc modified peptides (Vosseller et al., ). More recently, using
LWAC combined with ETD/HCD-MS/MS, Kim et al. identified
123 O-GlcNAcylated proteins from rat sciatic nerve. Among them, they
demonstrated the importance of the periaxin O-GlcNAcylation for
myelin maintenance (Kim et al., 2016). However, this approach favors
enrichment of high-abundance proteins or those with multiple clustered
O-GlcNAc residues. Low abundance proteins with single or separate
O-GlcNAcylated sites are difficult to purify using this tool since their
binding to solid affinity support is completed with more abundant
O-GlcNAcylated proteins. Moreover, LWAC suffers from low affinity
with the GlcNAc moiety. A large amount of protein sample (around tens
of milligrams) is therefore required for O-GlcNAc profiling (Shen et al.,
2017a).

In 2017, in order to decrease the amount of protein sample used to
detect GlcNAc motif, Liu et al. developed a new enrichment method
based on Agrocybe aegerita lectin 2 (AAL2). AAL2 was conjugated with
resin (Sepharose 4B or POROS) and packed in an affinity column. With
this technique, they identified 17 O-linked HexNAc-modified proteins
(Liu et al., 2018b), suggesting that AAL2 is a new and useful tool for
enrichment of O-GlcNAcylated proteins.

In order to improve the number of identified, quantified proteins and
the number of O-GlcNAcylated sites mapped, Lee et al. performed an
enrichment method based on a novel G5-lectibody. This approach cor-
responded to a combination of 4 O-GlcNAc specific antibodies
(CTD110.6, RL2, HGAC39 and HGAC85) and the lectin WGA. Coupled
with LC-MS/MS, they identified 990 O-GlcNAcylated proteins and they
demonstrated that oxidative stress is associated with a modulation of O-
GlcNAc levels (Lee et al., 2016). This study suggests that G5-lectibody
enrichment provides greater coverage of the O-GlcNAc sub-proteome.
However, like other lectins, WGA, AAL2 and rPVL also have an affin-
ity to GlcNAc terminal residues on N-glycans. Specificity could be
improved via treatment with PNGase F prior to enrichment.

In 2017, to increase the enrichment efficiency of O-GlcNAc peptides, Shen et al. performed a novel strategy for global mapping of O-GlcNAcylated proteins using hydrophilic interaction liquid metabolic chemical reporter chromatography (HILIC) combined with selective deglycosylation. In that study, most of the N- and O-linked oligosaccharides were removed by treatment with PNGase F, sialidase and O-glycosidase

(Endo-α-N-Acetylgalactosaminidase). This step was followed by HILIC
enrichment using hydrophilic polymer modified-silica microparticles
and mass spectrometry analysis (pPEGMA-SMs). A combination of these
two steps enabled efficient O-GlcNAcylated protein enrichment even in
the presence of 500-fold more abundant unmodified proteins. Combined
with LC-MS, they identified 457 O-GlcNAcylated proteins from human
urine suggesting that this simple and robust method could be available
for other biological samples (tissues, blood, etc.) (Shen et al., 2017b).

In the same year, Selvan et al. developed a novel enrichment method based on mutant O-GlcNAcase (Selvan et al., 2017). In this study, a catalytically inactive bacterial O-GlcNAcase mutant (Clostridium perfringens O-GlcNAcase (CpOGA-D298N)) was used to trap O-GlcNAcylated proteins. Conjugated to HaloLink beads, CpOGA is used to pull down O-GlcNAcylated proteins to enable their enrichment. This approach provided identification of 2358 proteins from Drosophila embryos which were significantly enriched compared with the control pull-down. However, CpOGA (D298N) has a higher affinity to Ser-O-GlcNAc compared with Thr-O-GlcNAc peptides which limit O-GlcNAcylated enrichment (Mariappa et al., 2015).

4.2. Protein enrichment by labeling methods

In contrast to direct capture, labeling enrichment is a broad category
of indirect enrichment techniques. Labeling enrichment methods are
distinguished in two distinct categories: enrichment by replacement and
enrichment by chemical functionalization.

4.2.1. Enrichment by replacement

β-elimination Michael addition (BEMA) is an approach leading to the replacement of the GlcNAc moiety by a new group with a stronger link and stable enough to withstand fragmentation throughout mass spectrometry (Wells et al., 2002; Greis et al., 1996). In BEMA, GlcNAc residue is eliminated via basic conditions and produces an α, β-unsaturated carbonyl group. This group can be modified using a sulfhydryl nucleophile such as dithiothreitol (DTT), deuterated DTT or biotinylated cystamine/pentylamine which enables generation of a unique mass label (Wells et al., 2002; Overath et al., 2012). Modified proteins can then be enriched via thiol-capture resin or streptavidin-conjugated beads. In addition to being an enrichment method, BEMA can be a quantification method by using labeled DTT. This approach can facilitate identification of the modified sites by monitoring the BEMA label. The technical weakness associated with BEMA is cross-reactivity such as with phosphorylated residues or alkylated cysteines. However, pretreatment with phosphatase can improve selectivity. Despite its common use across studies, the low efficacy of the BEMA reaction will be a limiting factor for large-scale profiling of O-GlcNAcylation studies.

The hydrazide reaction is a well-known technique for N-glycoprotein
enrichment. This approach makes it possible to enrich O-GlcNAcylated
proteins by oxidizing the GlcNAc moiety to its aldehyde derivative.
Proteins are then captured by hydrazide solid support and released by
hydroxylamine treatment (Klement et al., 2010). The major disadvan-
tage associated with this technique is the difficulty to produce aldehyde
derivative from GlcNAc moiety which limits O-GlcNAcylated protein
enrichment (Chen et al., 2021). A modification of the GlcNAc group with
the addition of a Gal moiety prior to its oxidation could limit the
problem (Chen et al., 2021; Xu et al., 2020).

4.2.2. Enrichment by chemical functionalization

Instead of replacing the GlcNAc moiety, a chemical reporter can be
incorporated with the protein of interest by chemoenzymatic or meta-
bolic labeling (Darley-Usmar et al., 2012).

4.2.2.1. *Chemoenzymatic labeling.* One common tool used in chemo-
enzymatic approaches is Y289L GalT. Used in combination with the
lectin Ricinus communis agglutinin I (RCA I) which recognizes
specifically galactosylated GlcNAc, Y289L GalT can be used for O-GlcNAc protein enrichment. O-GlcNAc peptides are incubated with UDP-galactose (UDP-GalNAc) and Y289L GalT which catalyzes the addition of galactose on GlcNAc moiety. RCA I has a higher affinity than other lectins such as WGA but PNGase treatment is required to remove N-linked GlcNAc-terminating sugars on proteins/peptides and to improve specificity. However, few studies have used this method and its feasibility for large-scale application has not yet been evaluated (Hayes et al., 1995; Haynes and Aebersold, 2000; Kim et al., 2011).

In addition to adding galactose on GlcNAc moiety, Y289 GalT can also transfer a Keto-Gal or GalNAz moiety onto O-GlcNAcylated proteins. Once incorporated, chemoselective reaction between the functional group and an appropriate probe makes it possible to tag and enrich the proteins. In 2004, Khidekel et al. used GlcNAc keto-Gal proteins and enriched them using a biotin-aminooxy reagent followed by streptavidin-conjugated beads (Khidekel et al., 2004). Combined with LC-MS/MS, they identified 25 O-GlcNAcylated proteins in brain tissue from rats.

Biotin is a frequently used tag in chemical functionalization approaches. However, biotin is fragmented during MS/MS fragmentation, leading to spectral analysis difficulties (Hu et al., 2010). Moreover, the strong bond between biotin and streptavidin requires harsh conditions to be disrupted and can lead to the alteration of O-GlcNAcylated proteins resulting in poor product recovery. To overcome this problem, a recent study has used an alternative approach based on the biotin/streptavidin-agarose bead method. Direct trypsin on-beads digestion enables removal of the unconjugated peptides and improves identification of O-GlcNAcylated proteins. Coupled with mass spectrometry approach, 342 proteins were identified as O-GlcNAcylated from C2C12 myotube cell lines (Deracinois et al., 2018). Chemoenzymatic attachment can also be used for visualization of affinity tags to O-GlcNAc modifications but requires several manipulation steps that may lead to false identification and should be carefully validated.

#### 4.2.2.2. Metabolic labeling.
A chemical reporter can also be incorporated using metabolic labeling. Some studies have exploited the ability of GalNAz to be epimerized to GlcNAk for use as a chemical reporter for GlcNAc moiety in living cells. GlcNAk is another GlcNAc analog which can be incorporated with proteins. Via click-chemistry and adapted biotin probes (with alkyne or azide group) and enrichment with streptavidin beads coupled with LC-MS/MS, Gurcel et al. identified 32 O-GlcNAc-azido tagged proteins (14 were previously unreported) in MCF-7 cells, while Zaro et al. identified 374 O-GlcNAc alkyned proteins (279 previously undescribed) in a COS-7 cell model (Gurcel et al., 2008; Zaro et al., 2011). The major drawback of this approach is that the enzymes in the cell preferably use natural substrate, resulting in relatively low levels of labeling.

As described above, in order to improve identification of O-GlcNAcylated proteins with the biotin/streptavidin method, on-beads digestion is also applicable in metabolic labeling. Coupled with LC-MS/MS, this method enabled the Kuster team to identify approximately 1500 O-GlcNAcylated proteins from a single cell line (Hahne et al., 2013). The other possibility is utilization of cleavable tags. In 2010, Wang et al. applied this strategy using a photocleavable PEG-biotin-alkyne reagent. In that study, the keto-galactose label was replaced by an azido-galactose and an alkyne-biotin probe was used for enrichment. After protein enrichment, UV light exposition terminal biotin group can be cleaved and the released peptides carry a basic aminomethytriazolyl acetylgalactosamine group in the GlcNAc site. This group is associated with a specific pattern under ETD MS (Syka et al., 2004). This approach enabled the author to map 8 O-GlcNAc sites from tau-enriched preparation (Wang et al.).

In 2022, Xu et al. developed a new approach to simultaneously identify and distinguish O-GlcNAc and O-GalNAc proteins. After having labeled (Ac₄GalNAz) and enriched (click-chemistry) the glycoproteins,

they treated the proteins with galactose oxidase to specifically oxidize O-GalNAc, thereby generating a difference in molecular weight visible in mass spectrometry (Xu et al., 2022).

### 4.2.3. Other enrichment approaches

Stable isotope labeling by amino acids in cell culture (SILAC) method can be a strategy which makes it possible to quantify O-GlcNAc levels, identify O-GlcNAcylated proteins and site-specific O-GlcNAc (Ma and Hart, 2017; Hu et al., 2010; Wang et al., 2007; Cox et al., 2018). This enrichment approach coupled with click chemistry, UV cleavage and ETD-MS/MS made it possible to map, quantify and compare relative site occupancy for approximately 140 previously unknown O-GlcNAcylated sites (Wang et al., 2010). This approach can also be used in combination with other enrichment approaches such as BEMA/CID-MS/MS for O-GlcNAc site mapping and quantitative analysis of O-GlcNAc modification (Khidekel et al., 2007). While in SILAC, cells must be cultured in two separate mediums, isobaric tag for relative and absolute quantification (iTRAQ), a method to determine the number of proteins is realizable in one single experiment. This approach is based on the utilization of isobaric tagging reagents which link with lysine side chain and N-terminus group of peptides (Ross et al., 2004). Like iTRAQ reagents, isotope dimethyl can link with lysine side chain and N-terminus group of peptides (Hsu et al., 2003; Boersema et al., 2009) and be used to study O-GlcNAcylation with chemoenzymatic enrichment (Khidekel et al., 2007). The iTRAQ method is compatible with ion trap MS and makes it possible, combined with a chemoenzymatic method, to map site-specific O-GlcNAc and quantify O-GlcNAcylated proteins (Wang et al., 2009; Skorobogatko et al., 2011). However, all studies with the iTRAQ method are performed in vitro whereas SILAC is validated in vivo and with tissue and could enable the study of changes in O-GlcNAc levels in tissues or biological fluids from animal models (Krüger et al., 2008; Geiger et al., 2010).

## 5. Other approaches

### 5.1. Study of OGT/OGA activity

Specific approaches must be designed for specific areas of research and expertise. OGT and OGA adds and removes the GlcNAc moiety, respectively. A modification of their activity can notably be associated with alteration of embryogenesis (Muha et al., 2021) or X-linked intellectual disability (Pravata et al., 2019). However, just one commercial assay is available to evaluate the activity of each enzyme (Basehore et al., 2021). In order to respond to this low offer, Carrillo et al. adapted O-GlcNAcylation to a technique used for other post-translational modifications – the fluorescence resonance energy transfer (FRET). The construct, called O-GlcNAc sensor, is composed of a cyan fluorescent protein, an O-GlcNAc binding domain (GafD, a lectin from Escherichia coli), an O-GlcNAc substrate (from casein kinase II) and a yellow fluorescent protein variant Venus (Carrillo et al., ). In 2011, they improved the technique by i) modifying the linker position and ii) adding a localization sequence to the nucleus or plasma membrane. They also demonstrated that during serum-stimulated signal transduction in COS-7 cells, O-GlcNAc transferase activity is increased in the nucleus and in plasma membrane while it is decreased in cytoplasm (Carrillo et al., 2011). In 2017, Groussaud et al. adapted the FRET construct developed by Carrillo in 2006 to a BRET (bioluminescence resonance energy transfer) by replacing cyan fluorescent protein by with Rlc8 (Groussaud et al., 2017). Using this strategy, they demonstrated that the viral Tax oncoprotein interacts with OGT and OGA and increasing Tax expression improves the affinity between OGT and OGA, resulting in a decrease in OGA activity. In a recent study (2020), Al-Mukh et al. used plasma membrane, cytosol or nucleus-targeted BRET O-GlcNAc biosensors. They demonstrated that lipopolysaccharide stimulation is associated with an increase in O-GlcNAc levels in RAW264.7 macrophages (Al-Mukh et al., 2020).
specifically galactosylated GlcNAc, Y289L GalT can be used for O-GlcNAc protein enrichment. O-GlcNAc peptides are incubated with UDP-galactose (UDP-GalNAc) and Y289L GalT which catalyzes the addition of galactose on GlcNAc moiety. RCA I has a higher affinity than other lectins such as WGA but PNGase treatment is required to remove N-linked GlcNAc-terminating sugars on proteins/peptides and to improve specificity. However, few studies have used this method and its feasibility for large-scale application has not yet been evaluated (Hayes et al., 1995; Haynes and Aebersold, 2000; Kim et al., 2011).

In addition to adding galactose on GlcNAc moiety, Y289 GalT can also transfer a Keto-Gal or GalNAz moiety onto O-GlcNAcylated proteins. Once incorporated, chemoselective reaction between the functional group and an appropriate probe makes it possible to tag and enrich the proteins. In 2004, Khidekel et al. used GlcNAc keto-Gal proteins and enriched them using a biotin-aminooxy reagent followed by streptavidin-conjugated beads (Khidekel et al., 2004). Combined with LC-MS/MS, they identified 25 O-GlcNAcylated proteins in brain tissue from rats.

Biotin is a frequently used tag in chemical functionalization approaches. However, biotin is fragmented during MS/MS fragmentation, leading to spectral analysis difficulties (Hu et al., 2010). Moreover, the strong bond between biotin and streptavidin requires harsh conditions to be disrupted and can lead to the alteration of O-GlcNAcylated proteins resulting in poor product recovery. To overcome this problem, a recent study has used an alternative approach based on the biotin/streptavidin-agarose bead method. Direct trypsin on-beads digestion enables removal of the unconjugated peptides and improves identification of O-GlcNAcylated proteins. Coupled with mass spectrometry approach, 342 proteins were identified as O-GlcNAcylated from C2C12 myotube cell lines (Deracinois et al., 2018). Chemoenzymatic attachment can also be used for visualization of affinity tags to O-GlcNAc modifications but requires several manipulation steps that may lead to false identification and should be carefully validated.

#### 4.2.2.2. Metabolic labeling.
A chemical reporter can also be incorporated using metabolic labeling. Some studies have exploited the ability of GalNAz to be epimerized to GlcNAk for use as a chemical reporter for GlcNAc moiety in living cells. GlcNAk is another GlcNAc analog which can be incorporated with proteins. Via click-chemistry and adapted biotin probes (with alkyne or azide group) and enrichment with streptavidin beads coupled with LC-MS/MS, Gurcel et al. identified 32 O-GlcNAc-azido tagged proteins (14 were previously unreported) in MCF-7 cells, while Zaro et al. identified 374 O-GlcNAc alkyned proteins (279 previously undescribed) in a COS-7 cell model (Gurcel et al., 2008; Zaro et al., 2011). The major drawback of this approach is that the enzymes in the cell preferably use natural substrate, resulting in relatively low levels of labeling.

As described above, in order to improve identification of O-GlcNAcylated proteins with the biotin/streptavidin method, on-beads digestion is also applicable in metabolic labeling. Coupled with LC-MS/MS, this method enabled the Kuster team to identify approximately 1500 O-GlcNAcylated proteins from a single cell line (Hahne et al., 2013). The other possibility is utilization of cleavable tags. In 2010, Wang et al. applied this strategy using a photocleavable PEG-biotin-alkyne reagent. In that study, the keto-galactose label was replaced by an azido-galactose and an alkyne-biotin probe was used for enrichment. After protein enrichment, UV light exposition terminal biotin group can be cleaved and the released peptides carry a basic aminomethytriazolyl acetylgalactosamine group in the GlcNAc site. This group is associated with a specific pattern under ETD MS (Syka et al., 2004). This approach enabled the author to map 8 O-GlcNAc sites from tau-enriched preparation (Wang et al.).

In 2022, Xu et al. developed a new approach to simultaneously identify and distinguish O-GlcNAc and O-GalNAc proteins. After having labeled (Ac₄GalNAz) and enriched (click-chemistry) the glycoproteins,

they treated the proteins with galactose oxidase to specifically oxidize O-GalNAc, thereby generating a difference in molecular weight visible in mass spectrometry (Xu et al., 2022).

### 4.2.3. Other enrichment approaches

Stable isotope labeling by amino acids in cell culture (SILAC) method can be a strategy which makes it possible to quantify O-GlcNAc levels, identify O-GlcNAcylated proteins and site-specific O-GlcNAc (Ma and Hart, 2017; Hu et al., 2010; Wang et al., 2007; Cox et al., 2018). This enrichment approach coupled with click chemistry, UV cleavage and ETD-MS/MS made it possible to map, quantify and compare relative site occupancy for approximately 140 previously unknown O-GlcNAcylated sites (Wang et al., 2010). This approach can also be used in combination with other enrichment approaches such as BEMA/CID-MS/MS for O-GlcNAc site mapping and quantitative analysis of O-GlcNAc modification (Khidekel et al., 2007). While in SILAC, cells must be cultured in two separate mediums, isobaric tag for relative and absolute quantification (iTRAQ), a method to determine the number of proteins is realizable in one single experiment. This approach is based on the utilization of isobaric tagging reagents which link with lysine side chain and N-terminus group of peptides (Ross et al., 2004). Like iTRAQ reagents, isotope dimethyl can link with lysine side chain and N-terminus group of peptides (Hsu et al., 2003; Boersema et al., 2009) and be used to study O-GlcNAcylation with chemoenzymatic enrichment (Khidekel et al., 2007). The iTRAQ method is compatible with ion trap MS and makes it possible, combined with a chemoenzymatic method, to map site-specific O-GlcNAc and quantify O-GlcNAcylated proteins (Wang et al., 2009; Skorobogatko et al., 2011). However, all studies with the iTRAQ method are performed in vitro whereas SILAC is validated in vivo and with tissue and could enable the study of changes in O-GlcNAc levels in tissues or biological fluids from animal models (Krüger et al., 2008; Geiger et al., 2010).

## 5. Other approaches

### 5.1. Study of OGT/OGA activity

Specific approaches must be designed for specific areas of research and expertise. OGT and OGA adds and removes the GlcNAc moiety, respectively. A modification of their activity can notably be associated with alteration of embryogenesis (Muha et al., 2021) or X-linked intellectual disability (Pravata et al., 2019). However, just one commercial assay is available to evaluate the activity of each enzyme (Basehore et al., 2021). In order to respond to this low offer, Carrillo et al. adapted O-GlcNAcylation to a technique used for other post-translational modifications – the fluorescence resonance energy transfer (FRET). The construct, called O-GlcNAc sensor, is composed of a cyan fluorescent protein, an O-GlcNAc binding domain (GafD, a lectin from Escherichia coli), an O-GlcNAc substrate (from casein kinase II) and a yellow fluorescent protein variant Venus (Carrillo et al., ). In 2011, they improved the technique by i) modifying the linker position and ii) adding a localization sequence to the nucleus or plasma membrane. They also demonstrated that during serum-stimulated signal transduction in COS-7 cells, O-GlcNAc transferase activity is increased in the nucleus and in plasma membrane while it is decreased in cytoplasm (Carrillo et al., 2011). In 2017, Groussaud et al. adapted the FRET construct developed by Carrillo in 2006 to a BRET (bioluminescence resonance energy transfer) by replacing cyan fluorescent protein by with Rlc8 (Groussaud et al., 2017). Using this strategy, they demonstrated that the viral Tax oncoprotein interacts with OGT and OGA and increasing Tax expression improves the affinity between OGT and OGA, resulting in a decrease in OGA activity. In a recent study (2020), Al-Mukh et al. used plasma membrane, cytosol or nucleus-targeted BRET O-GlcNAc biosensors. They demonstrated that lipopolysaccharide stimulation is associated with an increase in O-GlcNAc levels in RAW264.7 macrophages (Al-Mukh et al., 2020).
## 5.2. Protein-specific O-GlcNAcylation

One of the most promising tools developed to decipher O-GlcNAcylation is definitely site-protein specific O-GlcNAcylation. Recently, Ramirez et al. described a new method consisting of an association of OGT with a nanobody. They showed that the OGT-nanobody can O-GlcNAcylate proteins in a selective and via proximity-directed OGT manner, targeted recombinant-protein or in vitro (Ramirez et al., 2020). Based on these positive results, the group has developed a nanobody-fused split OGA (Ge et al., 2021). The limits associated with this construct is the lifetime of the nanobody and the lack of information concerning potential off-target effects.

As stated before, the main difficulty associated with the study of O-GlcNAcylation is the high lability of GlcNAc residue. One possibility is to replace O-GlcNAc by a thio-linked GlcNAc (S-GlcNAc) which is resistant to OGA activity and mimics O-GlcNAc in vitro (De Leon et al., 2017; Tegl et al., 2019). S-GlcNAcylation on cysteine is a new post-translational modification and OGT is able to add this modification (Maynard et al., 2016). In 2019, Gorelik et al. developed a site-specific installation of S-GlcNAc compatible with CRISPR-Cas9 genome editing (Gorelik et al., 2019). They showed that by using CRISPR-Cas9, OGA S405C mutation enables S-GlcNAc cysteine in a site-specific manner. Using this strategy, they showed that O-GlcNAcylation in position 405 of OGA affects its stability. The advantage associated with this approach is compatibility of the usual techniques to study O-GlcNAcylation (e.g., antibodies, galactosyltransferase labeling) with the S-GlcNAc moiety. Moreover, utilization of CRISPR-Cas9 genome editing enables the study of O-GlcNAcylation in both cells and animal models.

An innovative approach has recently been developed to control O-GlcNAcylation levels. By replacing an essential lysine for OGT activity by a photocaged lysine, He et al. developed a light-sensitive OGT. They demonstrated that illumination at 365 nm reactivates the enzyme by decaging it and makes it possible to increase O-GlcNAc levels in vitro (He et al., 2022). One of the most promising issues is certainly the ability to control stimulation of O-GlcNAcylation in a tissue-specific manner in vivo.

## 6. Conclusion

Since the discovery of O-GlcNAcylation in 1984, knowledge of this post-translational modification has evolved slowly owing to the lack of adapted methods. However, considering the involvement of O-GlcNAcylation in many pathophysiological situations, several tools have recently been described to decipher O-GlcNAc and potentially develop new therapeutic strategies. Pharmacological tools make it possible to visualize the impact of O-GlcNAc stimulation or inhibition in cell or animal models while biochemical strategies such as enrichment methods and mass spectrometry make it possible to identify and map O-GlcNA-cylated proteins. Despite scientific and technical efforts, O-GlcNAcylation remains a complicated protein modification to study – notably due to the lack of sequence consensus and the low stoichiometry of modification (occupancy at a particular site is estimated from 5 % to 10 %) (Klein et al., 2009). All new strategies developed today converge at site-specific O-GlcNAc characterizations. New adapted methods of O-GlcNAc suppression such as CRISPR-Cas9, combined with the use of non-hydrolysable O-GlcNAc analogues (Gorelik et al., 2019; Li et al., 2016; Wang et al., 2019) can be interesting, in particular to understand, site by site, the role of this modification. Fig. 3 comprehensively summarizes all of the current possibilities to study O-GlcNAcylation (Fig. 3).

## 7. Perspectives

Identification of serine and threonine targeted by O-GlcNAcylation and prediction of O-GlcNAcylation sites are the main future challenges. In 2011, dbOGAP, the first database of O-GlcNAcylated proteins reported approximately 800 proteins, mainly human, with approximately 400 identified O-GlcNAcylated sites (Wang et al., 2011). A decade later, with the development of new approaches, including the feasibility of mass spectrometry, more than 16,000 proteins have been reported as O-GlcNAcylated (half of them in humans) and nearly 14,000 O-GlcNAc site locations have been identified across 43 species on “The O-GlcNAc DataBase v1.2” (Wulff-Fuentes et al., 2021; Malard et al., 2021). To our knowledge, YinOYang, O-GlcNApred II and OGTsite are the three best

<figure><img src="image_5.png" /><figcaption>Fig. 3. An overview of tools to decipher O-GlcNAcylation.</figcaption></figure>
## 5.2. Protein-specific O-GlcNAcylation

One of the most promising tools developed to decipher O-GlcNAcylation is definitely site-protein specific O-GlcNAcylation. Recently, Ramirez et al. described a new method consisting of an association of OGT with a nanobody. They showed that the OGT-nanobody can O-GlcNAcylate proteins in a selective and via proximity-directed OGT manner, targeted recombinant-protein or in vitro (Ramirez et al., 2020). Based on these positive results, the group has developed a nanobody-fused split OGA (Ge et al., 2021). The limits associated with this construct is the lifetime of the nanobody and the lack of information concerning potential off-target effects.

As stated before, the main difficulty associated with the study of O-GlcNAcylation is the high lability of GlcNAc residue. One possibility is to replace O-GlcNAc by a thio-linked GlcNAc (S-GlcNAc) which is resistant to OGA activity and mimics O-GlcNAc in vitro (De Leon et al., 2017; Tegl et al., 2019). S-GlcNAcylation on cysteine is a new post-translational modification and OGT is able to add this modification (Maynard et al., 2016). In 2019, Gorelik et al. developed a site-specific installation of S-GlcNAc compatible with CRISPR-Cas9 genome editing (Gorelik et al., 2019). They showed that by using CRISPR-Cas9, OGA S405C mutation enables S-GlcNAc cysteine in a site-specific manner. Using this strategy, they showed that O-GlcNAcylation in position 405 of OGA affects its stability. The advantage associated with this approach is compatibility of the usual techniques to study O-GlcNAcylation (e.g., antibodies, galactosyltransferase labeling) with the S-GlcNAc moiety. Moreover, utilization of CRISPR-Cas9 genome editing enables the study of O-GlcNAcylation in both cells and animal models.

An innovative approach has recently been developed to control O-GlcNAcylation levels. By replacing an essential lysine for OGT activity by a photocaged lysine, He et al. developed a light-sensitive OGT. They demonstrated that illumination at 365 nm reactivates the enzyme by decaging it and makes it possible to increase O-GlcNAc levels in vitro (He et al., 2022). One of the most promising issues is certainly the ability to control stimulation of O-GlcNAcylation in a tissue-specific manner in vivo.

## 6. Conclusion

Since the discovery of O-GlcNAcylation in 1984, knowledge of this post-translational modification has evolved slowly owing to the lack of adapted methods. However, considering the involvement of O-GlcNA-cylation in many pathophysiological situations, several tools have recently been described to decipher O-GlcNAc and potentially develop new therapeutic strategies. Pharmacological tools make it possible to visualize the impact of O-GlcNAc stimulation or inhibition in cell or animal models while biochemical strategies such as enrichment methods and mass spectrometry make it possible to identify and map O-GlcNA-cylated proteins. Despite scientific and technical efforts, O-GlcNAcylation remains a complicated protein modification to study – notably due to the lack of sequence consensus and the low stoichiometry of modification (occupancy at a particular site is estimated from 5 % to 10 %) (Klein et al., 2009). All new strategies developed today converge at site-specific O-GlcNAc characterizations. New adapted methods of O-GlcNAc suppression such as CRISPR-Cas9, combined with the use of non-hydrolysable O-GlcNAc analogues (Gorelik et al., 2019; Li et al., 2016; Wang et al., 2019) can be interesting, in particular to understand, site by site, the role of this modification. Fig. 3 comprehensively summarizes all of the current possibilities to study O-GlcNAcylation (Fig. 3).

## 7. Perspectives

Identification of serine and threonine targeted by O-GlcNAcylation and prediction of O-GlcNAcylation sites are the main future challenges. In 2011, dbOGAP, the first database of O-GlcNAcylated proteins reported approximately 800 proteins, mainly human, with approximately 400 identified O-GlcNAcylated sites (Wang et al., 2011). A decade later, with the development of new approaches, including the feasibility of mass spectrometry, more than 16,000 proteins have been reported as O-GlcNAcylated (half of them in humans) and nearly 14,000 O-GlcNAc site locations have been identified across 43 species on “The O-GlcNAc DataBase v1.2” (Wulff-Fuentes et al., 2021; Malard et al., 2021). To our knowledge, YinOYang, O-GlcNApred II and OGTsite are the three best

<figure><img src="image_6.png" /><figcaption>Fig. 3. An overview of tools to decipher O-GlcNAcylation.</figcaption></figure>
known prediction tools available in August 2022 (Gupta and Brunak, 2002; Jia et al., 2018; Kao et al., 2015). However, a recent study demonstrated the inability of these tools to predict O-GlcNAcylated site location with a precision exceeding 9 % (Mauri et al., 2021). Faced with this failure of the current prediction tools and the constant increase in the number of proteins identified as O-GlcNAcylated, the development of new and more efficient prediction software is required. Based on the fact that OGT partners can impact the substrate spectrum, one promising track is characterization of OGT interactome.

## Funding

This work was supported by "Société Française d'Anesthésie et de Réanimation" (Paris, France), "Fondation d'Entreprises Genavie" (Nantes, France), "Fédération Française de Cardiologie" (Paris, France), "Agence Nationale de la Recherche" (20ASTC003201hErOiSmE) (Paris, France) and "Direction Générale de l'Armement" (Paris, France). T.D. is a PhD student supported by grants from Direction Générale de l'Armement and Région des Pays de la Loire.

## Author contributions

Idea for the article: Thomas Dupas and Benjamin Lauzier; Literature search and data analysis: Thomas Dupas; Writing – original draft preparation: Thomas Dupas; Benjamin Lauzier; Writing – review and editing: Thomas Dupas, Charlotte Betus, Angelique Blangy—Letheule, Thomas Pelé, Antoine Persello, Manon Denis, Benjamin Lauzier. All authors have read and approved the final manuscript.

## Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

## Acknowledgments

The authors thank the Olivier-Van Stichelen lab for "The O-GlcNAc Database v1.2."

## References

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Akella, N.M., Ciraku, L., Reginato, M.J., 2019. Fueling the fire: emerging role of the hexosamine biosynthetic pathway in cancer. BMC Biol. 17, 1–14. https://doi.org/10.1186/s12915-019-0671-3.

Al-Mukh, H., Baudoin, L., Bouaboud, A., et al., 2020. LPS induces GFAT2 expression to promote O-GlcNAcylation and attenuate inflammation in macrophages. bioRxiv. https://doi.org/10.1101/2020.03.22.002303.

Basehore, S.E., Bohlman, S., Weber, C., et al., 2021. Laminar flow on endothelial cells suppresses eNOS O-GlcNAcylation to promote eNOS activity. Circ. Res 129, 1054–1066. https://doi.org/10.1161/CIRCRESAHA.121.318982.

Boeggeman, E., Ramakrishnan, B., Kilgore, C., et al., 2007. Direct identification of nonreducing GlcNAc residues on N-Glycans of glycoproteins using a novel chemoenzymatic method. Bioconjug. Chem. 18, 806–814. https://doi.org/10.1021/bc060341n.

Boehmelt, G., Wakeham, A., Elia, A., et al., 2000. Decreased UDP-GlcNAc levels abrogate proliferation control in EMeg32-deficient cells. EMBO J. 19, 5092–5104. https://doi.org/10.1093/emboj/19.19.5092.

Boersema, P.J., Raijmakers, R., Lemeer, S., et al., 2009. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat. Protoc. 4, 484–494. https://doi.org/10.1038/nprot.2009.21.

Bond, M.R., Hanover, J.A., 2015. A little sugar goes a long way: the cell biology of O-GlcNAc. J. Cell Biol. 208, 869–880. https://doi.org/10.1083/jcb.201501101.

Bond, M.R., Zhang, H., Kim, J., et al., 2011. Metabolism of diazirine-modified N-Acetylmannosamine analogues to photo-cross-linking sialosides. Bioconjug. Chem. 22, 1811–1823. https://doi.org/10.1021/bc2002117.

Boyce, M., Carrico, I.S., Ganguli, A.S., et al., 2011. Metabolic cross-talk allows labeling of O-linked β-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc. Natl. Acad. Sci. USA 108, 3141–3146. https://doi.org/10.1073/pnas.1010045108.

Cameron, A., Giacomozzi, B., Joyce, J., et al., 2013. Generation and characterization of a rabbit monoclonal antibody site-specific for tau O-GlcNAcylated at serine 400. FEBS Lett. 587, 3722–3728. https://doi.org/10.1016/j.febslet.2013.09.042.

Carrillo, L.D., Krishnamoorthy, L., Mahal, L.K., 2006. A Cellular FRET-Based Sensor for β-O-GlcNAc, A Dynamic Carbohydrate Modification Involved in Signaling. J Am Chem Soc 128, 14768–14769. https://doi.org/10.1021/ja065835+.

Carrillo, L.D., Froemming, J.A., Mahal, L.K., 2011. Targeted in Vivo O-GlcNAc sensors reveal discrete compartment-specific dynamics during signal transduction. J. Biol. Chem. 286, 6650–6658. https://doi.org/10.1074/jbc.M110.191627.

Champattanachai, V., Marchase, R.B., Chatham, J.C., 2007. Glycosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein-associated O-GlcNAc. Am. J. Physiol., Cell Physiol. 292, C178–C187. https://doi.org/10.1152/ajpcell.00162.2006.

Chavelas, E.A., Beltrán, A.P., Pérez-Hernández, G., García-Hernández, E., 2004. Spectroscopic characterization of the thermal unfolding of wheat germ agglutinin. Rev. De. la Soc. Quím. De. México 48, 279–282.

Chen, Y., Qin, H., Yue, X., et al., 2021. Highly efficient enrichment of O-GlcNAc glycopeptides based on chemical oxidation and reversible hydrazide chemistry. Anal. Chem. 93, 16618–16627. https://doi.org/10.1021/acs.analchem.1c04031.

Cieniewski-Bernard, C., Dupont, E., Deracinois, B., et al., 2014. Multiplexed detection of O-GlcNAcome, phosphoproteome, and whole proteome within the same gel. Front. Endocrinol. 5. https://doi.org/10.3389/fendo.2014.00184.

Cm, W., At, I., Dr, S., et al., 2015. Isotope-targeted glycoproteomics (IsoTag): a mass-independent platform for intact N- and O-glycopeptide discovery and analysis. Nat. Methods 12, 561–567. https://doi.org/10.1038/nmeth.3366.

Coffey, G.L., Ehrlich, J., Fisher, M.W., et al., 1956. 6-Diazo-5-oxo-L-norleucine, a new tumor-inhibitory substance. I. Biologic studies. Antibiot. Chemother. 6, 487–497.

Comer, F.I., Vosseller, K., Wells, L., et al., 2001. Characterization of a mouse monoclonal antibody specific for O-Linked N-Acetylglucosamine. Anal. Biochem. 293, 169–177. https://doi.org/10.1006/abio.2001.5132.

Cox, N.J., Luo, P.M., Smith, T.J., et al., 2018. A novel glycoproteomics workflow reveals dynamic O-GlcNAcylation of COPγ1 as a candidate regulator of protein trafficking. Front. Endocrinol. 9. https://doi.org/10.3389/fendo.2018.00606.

Cummings, R.D., Etzler, M.E., 2009. Antibodies and Lectins in Glycan Analysis. Cold Spring Harbor Laboratory Press.

Darabedian, N., Pratt, M.R., 2019. Chapter Thirteen - Identifying potentially O-GlcNAcylated proteins using metabolic labeling, bioorthogonal enrichment, and Western blotting. In: Shukla, A.K. (Ed.), Methods in Enzymology. Academic Press, pp. 293–307.

Darabedian, N., Thompson, J.W., Chuh, K.N., et al., 2018. Optimization of chemoenzymatic mass tagging by Strain-Promoted Cycloaddition (SPAAC) for the determination of O-GlcNAc atoichiometry by western blotting. Biochemistry 57, 5769–5774. https://doi.org/10.1021/acs.biochem.8b00648.

Darley-Usmar, V.M., Ball, L.E., Chatham, J.C., 2012. Protein O-linked β-N-acetylglucosamine: a novel effector of cardiomyocyte metabolism and function. J. Mol. Cell. Cardiol. 52, 538–549. https://doi.org/10.1016/j.yjmcc.2011.08.009.

De Leon, C.A., Levine, P.M., Craven, T.W., Pratt, M.R., 2017. The sulfur-linked analogue of O-GlcNAc (S-GlcNAc) is an enzymatically stable and reasonable structural surrogate for O-GlcNAc at the peptide and protein levels. Biochemistry 56, 3507–3517. https://doi.org/10.1021/acs.biochem.7b00268.

Decourcelle, A., Loison, I., Baldini, S., et al., 2019. Evidence of a compensatory regulation of colonic O-GlcNAc transferase and O-GlcNAcase expression in response to disruption of O-GlcNAc homeostasis. Biochem. Biophys. Res. Commun. 521, 125–130. https://doi.org/10.1016/j.bbrc.2019.10.090.

Denis, M., Dupas, T., Persello, A., et al., 2021. An O-GlcNAcylomic approach reveals ACLY as a potential target in sepsis in the young rat. IJMS 22, 9236. https://doi.org/10.3390/ijms22179236.

Deracinois, B., Camoin, L., Lambert, M., et al., 2018. O-GlcNAcylation site mapping by (azide-alkyne) click chemistry and mass spectrometry following intensive fractionation of skeletal muscle cells proteins. J. Proteom. 186, 83–97. https://doi.org/10.1016/j.jprot.2018.07.005.

Dorfmueller, H.C., Borodkin, V.S., Schimpl, M., et al., 2006. GlcNAcstatin: a picomolar, selective O-GlcNAcase inhibitor that modulates intracellular O-glcNAcylation levels. J. Am. Chem. Soc. 128, 16484–16485. https://doi.org/10.1021/ja066743n.

Dorfmueller, H.C., Borodkin, V.S., Schimpl, M., van Aalten, D.M.F., 2009. GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation. Biochem J. 420, 221–227. https://doi.org/10.1042/BJ20090110.

Dorfmueller, H.C., Borodkin, V.S., Schimpl, M., et al., 2010. Cell-Penetrant, nanomolar O-GlcNAcase inhibitors selective against lysosomal hexosaminidases. Chem. Biol. 17, 1250–1255. https://doi.org/10.1016/j.chembiol.2010.09.014.

Dupas, T., Denis, M., Dontaine, J., et al., 2020. Protein O-GlcNAcylation levels are regulated independently of dietary intake in a tissue and time-specific manner during rat postnatal development. Acta Physiol. (Oxf.), e13566. https://doi.org/10.1111/apha.13566.

Elbatrawy, A.A., Kim, E.J., Nam, G., 2020. O-GlcNAcase: emerging mechanism, substrate recognition and small-molecule inhibitors. ChemMedChem 15, 1244–1257. https://doi.org/10.1002/cmdc.202000077.

Ferrand, Y., Klein, E., Barwell, N.P., et al., 2009. A synthetic lectin for O-Linked β-N-Acetylglucosamine. angew. Chem. Int Ed. 48, 1775–1779. https://doi.org/10.1002/anie.200804905.

Ferrer, C.M., Sodi, V.L., Reginato, M.J., 2016. O-GlcNAcylation in cancer biology: linking metabolism and signaling. J. Mol. Biol. 428, 3282–3294. https://doi.org/10.1016/j.jmb.2016.05.028.

Ferron, M., Denis, M., Persello, A., et al., 2019a. Protein O-GlcNAcylation in cardiac pathologies: past, present, future. Front. Endocrinol. 9. https://doi.org/10.3389/fendo.2018.00819.
known prediction tools available in August 2022 (Gupta and Brunak, 2002; Jia et al., 2018; Kao et al., 2015). However, a recent study demonstrated the inability of these tools to predict O-GlcNAcylated site location with a precision exceeding 9 % (Mauri et al., 2021). Faced with this failure of the current prediction tools and the constant increase in the number of proteins identified as O-GlcNAcylated, the development of new and more efficient prediction software is required. Based on the fact that OGT partners can impact the substrate spectrum, one promising track is characterization of OGT interactome.

## Funding

This work was supported by "Société Française d'Anesthésie et de Réanimation" (Paris, France), "Fondation d'Entreprises Genavie" (Nantes, France), "Fédération Française de Cardiologie" (Paris, France), "Agence Nationale de la Recherche" (20ASTC003201hErOiSmE) (Paris, France) and "Direction Générale de l'Armement" (Paris, France). T.D. is a PhD student supported by grants from Direction Générale de l'Armement and Région des Pays de la Loire.

## Author contributions

Idea for the article: Thomas Dupas and Benjamin Lauzier; Literature search and data analysis: Thomas Dupas; Writing – original draft preparation: Thomas Dupas; Benjamin Lauzier; Writing – review and editing: Thomas Dupas, Charlotte Betus, Angelique Blangy—Letheule, Thomas Pelé, Antoine Persello, Manon Denis, Benjamin Lauzier. All authors have read and approved the final manuscript.

## Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

## Acknowledgments

The authors thank the Olivier-Van Stichelen lab for "The O-GlcNAc Database v1.2."

## References

Aguilar, A.L., Hou, X., Wen, L., et al., 2017. A chemoenzymatic histology method for o-GlcNAc detection. ChemBioChem 18, 2416–2421. https://doi.org/10.1002/cbic.201700515.

Akella, N.M., Ciraku, L., Reginato, M.J., 2019. Fueling the fire: emerging role of the hexosamine biosynthetic pathway in cancer. BMC Biol. 17, 1–14. https://doi.org/10.1186/s12915-019-0671-3.

Al-Mukh, H., Baudoin, L., Bouaboud, A., et al., 2020. LPS induces GFAT2 expression to promote O-GlcNAcylation and attenuate inflammation in macrophages. bioRxiv. https://doi.org/10.1101/2020.03.22.002303.

Basehore, S.E., Bohlman, S., Weber, C., et al., 2021. Laminar flow on endothelial cells suppresses eNOS O-GlcNAcylation to promote eNOS activity. Circ. Res 129, 1054–1066. https://doi.org/10.1161/CIRCRESAHA.121.318982.

Boeggeman, E., Ramakrishnan, B., Kilgore, C., et al., 2007. Direct identification of nonreducing GlcNAc residues on N-Glycans of glycoproteins using a novel chemoenzymatic method. Bioconjug. Chem. 18, 806–814. https://doi.org/10.1021/bc060341n.

Boehmelt, G., Wakeham, A., Elia, A., et al., 2000. Decreased UDP-GlcNAc levels abrogate proliferation control in EMeg32-deficient cells. EMBO J. 19, 5092–5104. https://doi.org/10.1093/emboj/19.19.5092.

Boersema, P.J., Raijmakers, R., Lemeer, S., et al., 2009. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat. Protoc. 4, 484–494. https://doi.org/10.1038/nprot.2009.21.

Bond, M.R., Hanover, J.A., 2015. A little sugar goes a long way: the cell biology of O-GlcNAc. J. Cell Biol. 208, 869–880. https://doi.org/10.1083/jcb.201501101.

Bond, M.R., Zhang, H., Kim, J., et al., 2011. Metabolism of diazirine-modified N-Acetylmannosamine analogues to photo-cross-linking sialosides. Bioconjug. Chem. 22, 1811–1823. https://doi.org/10.1021/bc2002117.

Boyce, M., Carrico, I.S., Ganguli, A.S., et al., 2011. Metabolic cross-talk allows labeling of O-linked β-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc. Natl. Acad. Sci. USA 108, 3141–3146. https://doi.org/10.1073/pnas.1010045108.

Cameron, A., Giacomozzi, B., Joyce, J., et al., 2013. Generation and characterization of a rabbit monoclonal antibody site-specific for tau O-GlcNAcylated at serine 400. FEBS Lett. 587, 3722–3728. https://doi.org/10.1016/j.febslet.2013.09.042.

Carrillo, L.D., Krishnamoorthy, L., Mahal, L.K., 2006. A Cellular FRET-Based Sensor for β-O-GlcNAc, A Dynamic Carbohydrate Modification Involved in Signaling. J Am Chem Soc 128, 14768–14769. https://doi.org/10.1021/ja065835+.

Carrillo, L.D., Froemming, J.A., Mahal, L.K., 2011. Targeted in Vivo O-GlcNAc sensors reveal discrete compartment-specific dynamics during signal transduction. J. Biol. Chem. 286, 6650–6658. https://doi.org/10.1074/jbc.M110.191627.

Champattanachai, V., Marchase, R.B., Chatham, J.C., 2007. Glycosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein-associated O-GlcNAc. Am. J. Physiol., Cell Physiol. 292, C178–C187. https://doi.org/10.1152/ajpcell.00162.2006.

Chavelas, E.A., Beltrán, A.P., Pérez-Hernández, G., García-Hernández, E., 2004. Spectroscopic characterization of the thermal unfolding of wheat germ agglutinin. Rev. De. la Soc. Quím. De México 48, 279–282.

Chen, Y., Qin, H., Yue, X., et al., 2021. Highly efficient enrichment of O-GlcNAc glycopeptides based on chemical oxidation and reversible hydrazide chemistry. Anal. Chem. 93, 16618–16627. https://doi.org/10.1021/acs.analchem.1c04031.

Cienskiwi-Bernard, C., Dupont, E., Deracinois, B., et al., 2014. Multiplexed detection of O-GlcNAcome, phosphoproteome, and whole proteome within the same gel. Front. Endocrinol. 5. https://doi.org/10.3389/fendo.2014.00184.

Cm, W., At, I., Dr, S., et al., 2015. Isotope-targeted glycoproteomics (IsoTag): a mass-independent platform for intact N- and O-glycopeptide discovery and analysis. Nat. Methods 12, 561–567. https://doi.org/10.1038/nmeth.3366.

Coffey, G.L., Ehrlich, J., Fisher, M.W., et al., 1956. 6-Diazo-5-oxo-L-norleucine, a new tumor-inhibitory substance. I. Biologic studies. Antibiot. Chemother. 6, 487–497.

Comer, F.I., Vosseller, K., Wells, L., et al., 2001. Characterization of a mouse monoclonal antibody specific for O-Linked N-Acetylglucosamine. Anal. Biochem. 293, 169–177. https://doi.org/10.1006/abio.2001.5132.

Cox, N.J., Luo, P.M., Smith, T.J., et al., 2018. A novel glycoproteomics workflow reveals dynamic O-GlcNAcylation of COPγ1 as a candidate regulator of protein trafficking. Front. Endocrinol. 9. https://doi.org/10.3389/fendo.2018.00606.

Cummings, R.D., Etzler, M.E., 2009. Antibodies and Lectins in Glycan Analysis. Cold Spring Harbor Laboratory Press.

Darabedian, N., Pratt, M.R., 2019. Chapter Thirteen - Identifying potentially O-GlcNAcylated proteins using metabolic labeling, bioorthogonal enrichment, and Western blotting. In: Shukla, A.K. (Ed.), Methods in Enzymology. Academic Press, pp. 293–307.

Darabedian, N., Thompson, J.W., Chuh, K.N., et al., 2018. Optimization of chemoenzymatic mass tagging by Strain-Promoted Cycloaddition (SPAAC) for the determination of O-GlcNAc atoichiometry by western blotting. Biochemistry 57, 5769–5774. https://doi.org/10.1021/acs.biochem.8b00648.

Darley-USmar, V.M., Ball, L.E., Chatham, J.C., 2012. Protein O-linked β-N-acetylglucosamine: a novel effector of cardiomyocyte metabolism and function. J. Mol. Cell. Cardiol. 52, 538–549. https://doi.org/10.1016/j.yjmcc.2011.08.009.

De Leon, C.A., Levine, P.M., Craven, T.W., Pratt, M.R., 2017. The sulfur-linked analogue of O-GlcNAc (S-GlcNAc) is an enzymatically stable and reasonable structural surrogate for O-GlcNAc at the peptide and protein levels. Biochemistry 56, 3507–3517. https://doi.org/10.1021/acs.biochem.7b00268.

Decourcelle, A., Loison, I., Baldini, S., et al., 2019. Evidence of a compensatory regulation of colonic O-GlcNAc transferase and O-GlcNAcase expression in response to disruption of O-GlcNAc homeostasis. Biochem. Biophys. Res. Commun. 521, 125–130. https://doi.org/10.1016/j.bbrc.2019.10.090.

Denis, M., Dupas, T., Persello, A., et al., 2021. An O-GlcNAcylomic approach reveals ACLY as a potential target in sepsis in the young rat. IJMS 22, 9236. https://doi.org/10.3390/ijms22179236.

Deracinois, B., Camoin, L., Lambert, M., et al., 2018. O-GlcNAcylation site mapping by (azide-alkyne) click chemistry and mass spectrometry following intensive fractionation of skeletal muscle cells proteins. J. Proteom. 186, 83–97. https://doi.org/10.1016/j.jprot.2018.07.005.

Dorfmueller, H.C., Borodkin, V.S., Schimpl, M., et al., 2006. GlcNAcstatin: a picomolar, selective O-GlcNAcase inhibitor that modulates intracellular O-glcNAcylation levels. J. Am. Chem. Soc. 128, 16484–16485. https://doi.org/10.1021/ja066743n.

Dorfmueller, H.C., Borodkin, V.S., Schimpl, M., van Aalten, D.M.F., 2009. GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation. Biochem. J. 420, 221–227. https://doi.org/10.1042/BJ20090110.

Dorfmueller, H.C., Borodkin, V.S., Schimpl, M., et al., 2010. Cell-Penetrant, nanomolar O-GlcNAcase inhibitors selective against lysosomal hexosaminidases. Chem. Biol. 17, 1250–1255. https://doi.org/10.1016/j.chembiol.2010.09.014.

Dupas, T., Denis, M., Dontaine, J., et al., 2020. Protein O-GlcNAcylation levels are regulated independently of dietary intake in a tissue and time-specific manner during rat postnatal development. Acta Physiol. (Oxf.), e13566. https://doi.org/10.1111/apha.13566.

Elbatrawy, A.A., Kim, E.J., Nam, G., 2020. O-GlcNAcase: emerging mechanism, substrate recognition and small-molecule inhibitors. ChemMedChem 15, 1244–1257. https://doi.org/10.1002/cmdc.202000077.

Ferrand, Y., Klein, E., Barwell, N.P., et al., 2009. A synthetic lectin for O-Linked β-N-Acetylglucosamine. angew. Chem. Int Ed. 48, 1775–1779. https://doi.org/10.1002/anie.200804905.

Ferrer, C.M., Sodi, V.L., Reginato, M.J., 2016. O-GlcNAcylation in cancer biology: linking metabolism and signaling. J. Mol. Biol. 428, 3282–3294. https://doi.org/10.1016/j.jmb.2016.05.028.

Ferron, M., Denis, M., Persello, A., et al., 2019a. Protein O-GlcNAcylation in cardiac pathologies: past, present, future. Front. Endocrinol. 9. https://doi.org/10.3389/fendo.2018.00819.
Ferron, M., Cadiet, J., Persello, A., et al., 2019b. O-GlcNAc stimulation: a new metabolic approach to treat septic shock. Sci. Rep. 9, 18751. https://doi.org/10.1038/s41598-019-55381-7.

Gao, L., Zhao, R., Wang, Y., et al., 2018. Surface plasmon resonance biosensor for the accurate and sensitive quantification of O-GlcNAc based on cleavage by β-D-N-acetylglucosaminidase. Anal. Chim. Acta 1040, 90–98. https://doi.org/10.1016/j.aca.2018.07.058.

Ge, Y., Ramirez, D.H., Yang, B., et al., 2021. Target protein deglycosylation in living cells by a nanobody-fused split O-GlcNAcase. Nat. Chem. Biol. 17, 593–600. https://doi.org/10.1038/s41589-021-00757-y.

Geiger, T., Cox, J., Ostiniewicz, P., et al., 2010. Super-SILAC mix for quantitative proteomics of human tumor tissue. Nat. Methods 7, 383–385. https://doi.org/10.1038/nmeth.1446.

Gloster, T.M., Zandberg, W.F., Heinonen, J.E., et al., 2011. Hijacking a biosynthetic pathway yields a glycosyltransferase inhibitor within cells. Nat. Chem. Biol. 7, 174–181. https://doi.org/10.1038/nchembio.520.

Gorelik, A., Galan Bartual, S., Borodkin, V.S., et al., 2019. Genetic recoding to dissect the roles of site-specific protein O-GlcNAcylation. Nat. Struct. Mol. Biol. 26, 1071–1077. https://doi.org/10.1038/s41594-019-0325-8.

Greis, K.D., Hart, G.W., 1998. Analytical Methods for the Study of O-GlcNAc Glycoproteins and Glycopeptides. In: Hounsell, E.F. (Ed.), Glycoanalysis Protocols. Humana Press, Totowa, NJ, pp. 19–33.

Greis, K.D., Hayes, B.K., Comer, F.I., et al., 1996. Selective detection and site-analysis of O-GlcNAc-modified glycopeptides by beta-elimination and tandem electrospray mass spectrometry. Anal. Biochem 234, 38–49. https://doi.org/10.1006/abio.1996.0047.

Griffin, M.E., Jensen, E.H., Mason, D.E., et al., 2016. Comprehensive mapping of O-GlcNAc modification sites using a chemically cleavable tag. Mol. BioSyst. 12, 1756–1759. https://doi.org/10.1039/C6MB00138F.

Groussaud, D., Khair, M., Tollenaere, A.I., et al., 2017. Hijacking of the O-GlcNAcZYME complex by the HTLV-1 Tax oncoprotein facilitates viral transcription. PLoS Pathog. 13. https://doi.org/10.1371/journal.ppat.1006518.

Gupta, R., Brunak, S., 2002. Prediction of glycosylation across the human proteome and the correlation to protein function. Pac. Symp. Biocomput 310–322.

Gurcel, C., Vercoutter-Edouart, A.-S., Fonbonne, C., et al., 2008. Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Anal. Bioanal. Chem. 390, 2089–2097. https://doi.org/10.1007/s00216-008-1950-y.

Hahne, H., Kuster, B., 2011. A novel two-stage tandem mass spectrometry approach and scoring scheme for the identification of O-GlcNAc modified peptides. J. Am. Soc. Mass Spectrom. 22, 931–942. https://doi.org/10.1007/s13361-011-0107-y.

Hahne, H., Sobotzki, N., Nyberg, T., et al., 2013. Proteome wide purification and identification of O-GlcNAc-modified proteins using click chemistry and mass spectrometry. J. Proteome Res. 12, 927–936. https://doi.org/10.1021/pr300967y.

Hao, Y., Fan, X., Shi, Y., et al., 2019. Next-generation unnatural monosaccharides reveal that ESRRB O-GlcNAcylation regulates pluripotency of mouse embryonic stem cells. Nat. Commun. 10, 1–13. https://doi.org/10.1038/s41467-019-11942-y.

Hayes, B.K., Greis, K.D., Hart, G.W., 1995. Specific isolation of O-Linked N-Acetylglucosamine glycopeptides from complex mixtures. Anal. Biochem. 228, 115–122. https://doi.org/10.1006/abio.1995.1322F.

Haynes, P.A., Aebersold, R., 2000. Simultaneous detection and identification of O-GlcNAc-Modified glycoproteins using liquid chromatography–tandem mass spectrometry. Anal. Chem. 72, 5402–5410. https://doi.org/10.1021/ac000512w.

He, J., Fan, Z., Tian, Y., et al., 2022. Spatiotemporal activation of protein O-GlcNAcylation in living cells. J. Am. Chem. Soc. JACS. https://doi.org/10.1021/jacs.lc11041.

Hirosawa, M., Hayakawa, K., Yoneda, C., et al., 2016. Novel O-GlcNAcylation on Ser40 of canonical H2A isoforms specific to viviparity. Sci. Rep. 6. https://doi.org/10.1038/srep31785.

Hsu, J.-L., Huang, S.-Y., Chow, N.-H., Chen, S.-H., 2003. Stable Isotope dimethyl labeling for quantitative proteomics. Anal. Chem. 75, 6843–6852. https://doi.org/10.1021/ac0348625.

Hu, B., Zhong, L., Weng, Y., et al., 2020. Therapeutic siRNA: state of the art. Signal Transduct. Target Ther. 5, 1–25. https://doi.org/10.1038/s41392-020-0207-x.

Hu, P., Shimoji, S., Hart, G.W., 2008. Site-specific interplay between O-GlcNAcylation and phosphorylation in cellular regulation. FEBS Lett. 584, 2526–2538. https://doi.org/10.1016/j.feblets.2010.04.044.

Ishikita, A., Matsushima, S., Ikeda, S., et al., 2021. GFAT2 mediates cardiac hypertrophy through HBP-O-GlcNAcylation-Akt pathway. iScience 24, 103517. https://doi.org/10.1016/j.isci.2021.103517.

Isono, T., 2011. O-GlcNAc-Specific antibody CTD110.6 cross-reacts with N-GlcNAc2-Modified proteins induced under glucose deprivation. PLoS One 6, e18959. https://doi.org/10.1371/journal.pone.0018959.

Jia, C., Zuo, Y., Zou, Q., 2018. O-GlcNAcPRED-II: an integrated classification algorithm for identifying O-GlcNAcylation sites based on fuzzy undersampling and a K-means PCA oversampling technique. Bioinformatics 34, 2029–2036. https://doi.org/10.1093/bioinformatics/bty039.

Jiang, J., Lazarus, M.B., Pasquina, L., et al., 2011. A neutral diphosphate mimic crosslinks the active site of human O-GlcNAc transferase. Nat. Chem. Biol. 8, 72–77. https://doi.org/10.1038/nchembio.711.

Jiang, S., Chen, Y., Wang, M., et al., 2012. A novel lectin from Agrocybe aegerita shows high binding selectivity for terminal N-acetylglucosamine. Biochem. J. 443, 369–378. https://doi.org/10.1042/BJ20112061.

Kang, E.-S., Han, D., Park, J., et al., 2008. O-GlcNAc modulation at Akt1 Ser473 correlates with apoptosis of murine pancreatic β cells. Exp. Cell Res. 314, 2238–2248. https://doi.org/10.1016/j.yexcr.2008.04.014.

Kao, H.-J., Huang, C.-H., Bretaña, N., et al., 2015. A two-layered machine learning method to identify protein O-GlcNAcylation sites with O-GlcNAc transferase substrate motifs. BMC Bioinforma. 16, S10. https://doi.org/10.1186/1471-2105-16-S18-S10.

Kearse, K.P., Hart, G.W., 1991. Lymphocyte activation induces rapid changes in nuclear and cytoplasmic glycoproteins. PNAS 88, 1701–1705. https://doi.org/10.1073/pnas.88.5.1701.

Keembiyehetty, C.N., Krzeslak, A., Love, D.C., Hanover, J.A., 2011. A lipid-droplet-targeted O-GlcNAcase isoform is a key regulator of the proteasome. J. Cell Sci. 124, 2851–2860. https://doi.org/10.1242/jcs.083287.

Khidekel, N., Arndt, S., Lamarre-Vincent, N., et al., 2003. A Chemoenzymatic approach toward the rapid and sensitive detection of O-GlcNAc posttranslational modifications. J. Am. Chem. Soc. 125, 16162–16163. https://doi.org/10.1021/ja038545r.

Khidekel, N., Ficarro, S.B., Peters, E.C., Hsieh-Wilson, L.C., 2004. Exploring the O-GlcNAc proteome: direct identification of O-GlcNAc-modified proteins from the brain. Proc. Natl. Acad. Sci. 101, 13132–13137. https://doi.org/10.1073/pnas.0403471101.

Khidekel, N., Ficarro, S.B., Clark, P.M., et al., 2007. Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics. Nat. Chem. Biol. 3, 339–348. https://doi.org/10.1038/nchembio881.

Kim, E.J., 2020. O-GlcNAc transferase: structural characteristics, catalytic mechanism and small-molecule inhibitors. ChemBioChem 21, 3026–3035. https://doi.org/10.1002/cbic.202000194.

Kim, S., Maynard, J.C., Sasaki, Y., et al., 2016. Schwann cell O-GlcNAc glycosylation is required for myelin maintenance and axon integrity. J. Neurosci. 36, 9633–9646. https://doi.org/10.1523/JNEUROSCI.1235-16.2016.

Kim, Y.-C., Udeeshi, N.D., Balsbaugh, J.L., et al., 2011. O-GlcNAcylation of the plum pox virus capsid protein catalyzed by SECRET AGENT: characterization of O-GlcNAc sites by electron transfer dissociation mass spectrometry. Amino Acids 40, 869–876. https://doi.org/10.1007/s00726-010-0706-0.

Klein, A.L., Berkaw, M.N., Buse, M.G., Ball, L.E., 2009. O-Linked N-Acetylglucosamine modification of insulin receptor substrate-1 occurs in close proximity to multiple SH2 domain binding motifs. Mol. Cell Proteom. 8, 2733–2745. https://doi.org/10.1074/mcp.M900207-MCP200.

Klement, E., Lipinszki, Z., Kupihár, Z., et al., 2010. Enrichment of O-GlcNAc modified proteins by the periodate oxidation – hydrazide resin capture approach. J. Proteome Res. 9, 2200–2206. https://doi.org/10.1021/pr900984h.

Kochibe, N., Matta, K.L., 1989. Purification and properties of an N-acetylglucosamine-specific lectin from Psathyrella velutina mushroom. J. Biol. Chem. 264, 173–177.

Kong, H., Chen, W., Lu, H., et al., 2015. Synthesis of NAG-thiazoline-derived inhibitors for β-N-acetyl-d-hexosaminidases. Carbohydr. Res. 413, 135–144. https://doi.org/10.1016/j.carres.2015.06.004.

Krüger, M., Moser, M., Ussar, S., et al., 2008. SILAC mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function. Cell 134, 353–364. https://doi.org/10.1016/j.cell.2008.05.033.

Kubota, Y., Fujioka, K., Takekawa, M., 2017. WGA-based lectin affinity gel electrophoresis: a novel method for the detection of O-GlcNAc-modified proteins. PLoS One 12. https://doi.org/10.1371/journal.pone.0180714.

Lazarus, B.D., Love, D.C., Hanover, J.A., 2006. Recombinant O-GlcNAc transferase isoforms: identification of O-GlcNAcase, yes tyrosine kinase, and tau as isoform-specific substrates. Glycobiology 16, 415–421. https://doi.org/10.1093/glycob/cwj078.

Lee, A., Miller, D., Henry, R., et al., 2016. Combined antibody/Lectin enrichment identifies extensive changes in the O-GlcNAc sub-proteome upon oxidative stress. J. Proteome Res. 15, 4318–4336. https://doi.org/10.1021/acs.proteome.6b00369.

Lee, T.N., Alborn, W.E., Knierman, M.D., Konrad, R.J., 2006. Alloxan is an inhibitor of O-GlcNAc-selective N-acetyl-β-d-glucosaminidase. Biochem. Biophys. Res. Commun. 350, 1038–1043. https://doi.org/10.1016/j.bbrcc.2006.09.155.

Lenzen, S., Munday, R., 1991. Thiol-group reactivity, hydrophilicity and stability of alloxan, its reduction products and its N-methyl derivatives and a comparison with ninhydrin. Biochem. Pharm. 42, 1385–1391. https://doi.org/10.1016/0006-2952(91)90449-f.

Lenzen, S., Panten, U., 1988. Alloxan: history and mechanism of action. Diabetologia 31, 337–342. https://doi.org/10.1007/BF02341500.

Li, J., Wang, J., Wen, L., et al., 2016. An OGA-Resistant probe allows specific visualization and accurate identification of O-GlcNAc-Modified proteins in cells. ACS Chem. Biol. 11, 3002–3006. https://doi.org/10.1021/acs.chembio.6b00678.

Lima, V.V., Giachini, F.R., Carneiro, F.S., et al., 2011. O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of the RhoA/Rho-kinase pathway. Cardiovasc. Res. 89, 614–622. https://doi.org/10.1093/cvrr/cvq338.

Liu, T.-W., Zandberg, W.F., Gloster, T.M., et al., 2018a. Metabolic inhibitors of O-GlcNAc transferase that act in vivo implicate decreased O-GlcNAc levels in leptin-mediated nutrient sensing. Angew. Chem. Int. Ed. 57, 7644–7648. https://doi.org/10.1002/anie.201803254.

Liu, W., Han, G., Yin, Y., et al., 2018b. AANL (Agrocybe aegerita lectin 2) is a new facile tool to probe for O-GlcNAcylation. Glycobiology 28, 363–373. https://doi.org/10.1093/glycob/cwy029.

Liu, Y., Ren, Y., Cao, Y., et al., 2017. Discovery of a low toxicity O-GlcNAc transferase (OGT) inhibitor by structure-based virtual screening of natural products. Sci. Rep. 7, 1–11. https://doi.org/10.1038/s41598-017-12522-0.

Ma, J., Hart, G.W., 2013. Protein O-GlcNAcylation in diabetes and diabetic complications. Expert Rev. Proteom. 10, 365–380. https://doi.org/10.1586/14789450.2013.820536.

Ma, J., Hart, G.W., 2014. O-GlcNAc profiling: from proteins to proteomes. Clin. Proteom. 11, 8. https://doi.org/10.1186/1559-0275-11-8.
Ferron, M., Cadiet, J., Persello, A., et al., 2019b. O-GlcNAc stimulation: a new metabolic approach to treat septic shock. Sci. Rep. 9, 18751. https://doi.org/10.1038/s41598-019-55381-7.

Gao, L., Zhao, R., Wang, Y., et al., 2018. Surface plasmon resonance biosensor for the accurate and sensitive quantification of O-GlcNAc based on cleavage by β-D-N-acetylglucosaminidase. Anal. Chim. Acta 1040, 90–98. https://doi.org/10.1016/j.aca.2018.07.058.

Ge, Y., Ramirez, D.H., Yang, B., et al., 2021. Target protein deglycosylation in living cells by a nanobody-fused split O-GlcNAcase. Nat. Chem. Biol. 17, 593–600. https://doi.org/10.1038/s41589-021-00757-y.

Geiger, T., Cox, J., Ostiniewicz, P., et al., 2010. Super-SILAC mix for quantitative proteomics of human tumor tissue. Nat. Methods 7, 383–385. https://doi.org/10.1038/nmeth.1446.

Gloster, T.M., Zandberg, W.F., Heinonen, J.E., et al., 2011. Hijacking a biosynthetic pathway yields a glycosyltransferase inhibitor within cells. Nat. Chem. Biol. 7, 174–181. https://doi.org/10.1038/nchembio.520.

Gorelik, A., Galan Bartual, S., Borodkin, V.S., et al., 2019. Genetic recoding to dissect the roles of site-specific protein O-GlcNAcylation. Nat. Struct. Mol. Biol. 26, 1071–1077. https://doi.org/10.1038/s41594-019-0325-8.

Greis, K.D., Hart, G.W., 1998. Analytical Methods for the Study of O-GlcNAc Glycoproteins and Glycopeptides. In: Hounsell, E.F. (Ed.), Glycoanalysis Protocols. Humana Press, Totowa, NJ, pp. 19–33.

Greis, K.D., Hayes, B.K., Comer, F.I., et al., 1996. Selective detection and site-analysis of O-GlcNAc-modified glycopeptides by beta-elimination and tandem electrospray mass spectrometry. Anal. Biochem 234, 38–49. https://doi.org/10.1006/abio.1996.0047.

Griffin, M.E., Jensen, E.H., Mason, D.E., et al., 2016. Comprehensive mapping of O-GlcNAc modification sites using a chemically cleavable tag. Mol. BioSyst. 12, 1756–1759. https://doi.org/10.1039/C6MB00138F.

Groussaud, D., Khair, M., Tollenaere, A.I., et al., 2017. Hijacking of the O-GlcNAcZYME complex by the HTLV-1 Tax oncoprotein facilitates viral transcription. PLoS Pathog. 13. https://doi.org/10.1371/journal.ppat.1006518.

Gupta, R., Brunak, S., 2002. Prediction of glycosylation across the human proteome and the correlation to protein function. Pac. Symp. Biocompt 310–322.

Gurcel, C., Vercoutter-Edouart, A.-S., Fonbonne, C., et al., 2008. Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Anal. Bioanal. Chem. 390, 2089–2097. https://doi.org/10.1007/s00216-008-1950-y.

Hahne, H., Kuster, B., 2011. A novel two-stage tandem mass spectrometry approach and scoring scheme for the identification of O-GlcNAc modified peptides. J. Am. Soc. Mass Spectrom. 22, 931–942. https://doi.org/10.1007/s13361-011-0107-y.

Hahne, H., Sobotzki, N., Nyberg, T., et al., 2013. Proteome wide purification and identification of O-GlcNAc-modified proteins using click chemistry and mass spectrometry. J. Proteome Res. 12, 927–936. https://doi.org/10.1021/pr300967y.

Hao, Y., Fan, X., Shi, Y., et al., 2019. Next-generation unnatural monosaccharides reveal that ESRRB O-GlcNAcylation regulates pluripotency of mouse embryonic stem cells. Nat. Commun. 10, 1–13. https://doi.org/10.1038/s41467-019-11942-y.

Hayes, B.K., Greis, K.D., Hart, G.W., 1995. Specific isolation of O-Linked N-Acetylglucosamine glycopeptides from complex mixtures. Anal. Biochem. 228, 115–122. https://doi.org/10.1006/abio.1995.1322F.

Haynes, P.A., Aebersold, R., 2000. Simultaneous detection and identification of O-GlcNAc-Modified glycoproteins using liquid chromatography-tandem mass spectrometry. Anal. Chem. 72, 5402–5410. https://doi.org/10.1021/ac000512w.

He, J., Fan, Z., Tian, Y., et al., 2022. Spatiotemporal activation of protein O-GlcNAcylation in living cells. J. Am. Chem. Soc. JACS. https://doi.org/10.1021/jacs.1c11041.

Hirosawa, M., Hayakawa, K., Yoneda, C., et al., 2016. Novel O-GlcNAcylation on Ser40 of canonical H2A isoforms specific to viviparity. Sci. Rep. 6. https://doi.org/10.1038/srep31785.

Hsu, J.-L., Huang, S.-Y., Chow, N.-H., Chen, S.-H., 2003. Stable Isotope dimethyl labeling for quantitative proteomics. Anal. Chem. 75, 6843–6852. https://doi.org/10.1021/ac0348625.

Hu, B., Zhong, L., Weng, Y., et al., 2020. Therapeutic siRNA: state of the art. Signal Transduct. Target Ther. 5, 1–25. https://doi.org/10.1038/s41392-020-0207-x.

Hu, P., Shimoji, S., Hart, G.W., 2008. Site-specific interplay between O-GlcNAcylation and phosphorylation in cellular regulation. FEBS Lett. 584, 2526–2538. https://doi.org/10.1016/j.feblets.2010.04.044.

Ishikita, A., Matsushima, S., Ikeda, S., et al., 2021. GFAT2 mediates cardiac hypertrophy through HBP-O-GlcNAcylation-Akt pathway. iScience 24, 103517. https://doi.org/10.1016/j.isci.2021.103517.

Isono, T., 2011. O-GlcNAc-Specific antibody CTD110.6 cross-reacts with N-GlcNAc2-Modified proteins induced under glucose deprivation. PLoS One 6, e18959. https://doi.org/10.1371/journal.pone.0018959.

Jia, C., Zuo, Y., Zou, Q., 2018. O-GlcNAcPRED-II: an integrated classification algorithm for identifying O-GlcNAcylation sites based on fuzzy undersampling and a K-means PCA oversampling technique. Bioinformatics 34, 2029–2036. https://doi.org/10.1093/bioinformatics/bty039.

Jiang, J., Lazarus, M.B., Pasquina, L., et al., 2011. A neutral diphosphate mimic crosslinks the active site of human O-GlcNAc transferase. Nat. Chem. Biol. 8, 72–77. https://doi.org/10.1038/nchembio.711.

Jiang, S., Chen, Y., Wang, M., et al., 2012. A novel lectin from Agrocybe aegerita shows high binding selectivity for terminal N-acetylglucosamine. Biochem. J. 443, 369–378. https://doi.org/10.1042/BJ20112061.

Kang, E.-S., Han, D., Park, J., et al., 2008. O-GlcNAc modulation at Akt1 Ser473 correlates with apoptosis of murine pancreatic β cells. Exp. Cell Res. 314, 2238–2248. https://doi.org/10.1016/j.yexcr.2008.04.014.

Kao, H.-J., Huang, C.-H., Bretaña, N., et al., 2015. A two-layered machine learning method to identify protein O-GlcNAcylation sites with O-GlcNAc transferase substrate motifs. BMC Bioinforma. 16, S10. https://doi.org/10.1186/1471-2105-16-S18-S10.

Kearse, K.P., Hart, G.W., 1991. Lymphocyte activation induces rapid changes in nuclear and cytoplasmic glycoproteins. PNAS 88, 1701–1705. https://doi.org/10.1073/pnas.88.5.1701.

Keembiyehetty, C.N., Krzeslak, A., Love, D.C., Hanover, J.A., 2011. A lipid-droplet-targeted O-GlcNAcase isoform is a key regulator of the proteasome. J. Cell Sci. 124, 2851–2860. https://doi.org/10.1242/jcs.083287.

Khidekel, N., Arndt, S., Lamarre-Vincent, N., et al., 2003. A Chemoenzymatic approach toward the rapid and sensitive detection of O-GlcNAc posttranslational modifications. J. Am. Chem. Soc. 125, 16162–16163. https://doi.org/10.1021/ja038545r.

Khidekel, N., Ficarro, S.B., Peters, E.C., Hsieh-Wilson, L.C., 2004. Exploring the O-GlcNAc proteome: direct identification of O-GlcNAc-modified proteins from the brain. Proc. Natl. Acad. Sci. 101, 13132–13137. https://doi.org/10.1073/pnas.0403471101.

Khidekel, N., Ficarro, S.B., Clark, P.M., et al., 2007. Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics. Nat. Chem. Biol. 3, 339–348. https://doi.org/10.1038/nchembio881.

Kim, E.J., 2020. O-GlcNAc transferase: structural characteristics, catalytic mechanism and small-molecule inhibitors. ChemBioChem 21, 3026–3035. https://doi.org/10.1002/cbic.202000194.

Kim, S., Maynard, J.C., Sasaki, Y., et al., 2016. Schwann cell O-GlcNAc glycosylation is required for myelin maintenance and axon integrity. J. Neurosci. 36, 9633–9646. https://doi.org/10.1523/JNEUROSCI.1235-16.2016.

Kim, Y.-C., Udeeshi, N.D., Balsbaugh, J.L., et al., 2011. O-GlcNAcylation of the plum pox virus capsid protein catalyzed by SECRET AGENT: characterization of O-GlcNAc sites by electron transfer dissociation mass spectrometry. Amino Acids 40, 869–876. https://doi.org/10.1007/s00726-010-0706-0.

Klein, A.L., Berkaw, M.N., Buse, M.G., Ball, L.E., 2009. O-Linked N-Acetylglucosamine modification of insulin receptor substrate-1 occurs in close proximity to multiple SH2 domain binding motifs. Mol. Cell Proteom. 8, 2733–2745. https://doi.org/10.1074/mcp.M900207-MCP200.

Klement, E., Lipinszki, Z., Kupihár, Z., et al., 2010. Enrichment of O-GlcNAc modified proteins by the periodate oxidation – hydrazide resin capture approach. J. Proteome Res. 9, 2200–2206. https://doi.org/10.1021/pr900984h.

Kochibe, N., Matta, K.L., 1989. Purification and properties of an N-acetylglucosamine-specific lectin from Psathyrella velutina mushroom. J. Biol. Chem. 264, 173–177.

Kong, H., Chen, W., Lu, H., et al., 2015. Synthesis of NAG-thiazoline-derived inhibitors for β-N-acetyl-d-hexosaminidases. Carbohydr. Res. 413, 135–144. https://doi.org/10.1016/j.carres.2015.06.004.

Krüger, M., Moser, M., Ussar, S., et al., 2008. SILAC mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function. Cell 134, 353–364. https://doi.org/10.1016/j.cell.2008.05.033.

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Ma, J., Wang, W.-H., Li, Z., et al., 2019. O-GlcNAc site mapping by using a combination of chemoenzymatic labeling, copper-free click chemistry, reductive cleavage, and electron-transfer dissociation mass spectrometry. Anal. Chem. 91, 2620–2625. https://doi.org/10.1021/acs.analchem.8b05688.

Macauley, M.S., Vocadlo, D.J., 2009. Enzymatic characterization and inhibition of the nuclear variant of human O-GlcNAcase. Carbohydr. Res. 344, 1079–1084. https://doi.org/10.1016/j.carres.2009.04.017.

Macauley, M.S., Whitworth, G.E., Debowski, A.W., et al., 2005. O-GlcNAcase uses substrate-assisted catalysis: kinetic analysis and development of highly selective mechanism-inspired inhibitors. J. Biol. Chem. 280, 25313–25322. https://doi.org/10.1074/jbc.M413819200.

Macauley, M.S., Bubb, A.K., Martinez-Fleites, C., et al., 2008. Elevation of global O-GlcNAc levels in 3T3-L1 adipocytes by selective inhibition of O-GlcNAcase does not induce insulin resistance. J. Biol. Chem. 283, 34687–34695. https://doi.org/10.1074/jbc.M804525200.

Macauley, M.S., Shan, X., Yuzwa, S.A., et al., 2010a. Elevation of global O-GlcNAc in rodents using a selective O-GlcNAcase inhibitor does not cause insulin resistance or perturb glucomethoestasis. Chem. Biol. 17, 949–958. https://doi.org/10.1016/j.chembiol.2010.07.005.

Macauley, M.S., He, Y., Glaster, T.M., et al., 2010b. Inhibition of O-GlcNAcase using a potent and cell-permeable inhibitor does not induce insulin resistance in 3T3-L1 adipocytes. Chem. Biol. 17, 937–948. https://doi.org/10.1016/j.chembiol.2010.07.006.

Machon, O., Varrot, A., 2020. Expression, purification, and applications of the recombinant lectin PVL from <Emphasis Type="Italic">>Psathyrella velutina</> <Emphasis> specific for terminal <Emphasis Type="Italic">>N</Emphasis> <Emphasis>>-Acetyl- Glucosamine. Lectin Purif. Anal. 421–436. https://doi.org/10.1007/978-1-0716- 0430-4_41.

Machon, O., Baldini, S.F., Ribeiro, J.P., et al., 2017. Recombinant fungal lectin as a new tool to investigate O-GlcNAcase processes. Glycobiology 27, 123–128. https://doi.org/10.1093/glycob/cww105.

Malard, F., Wulff-Fuentes, E., Berendt, R.R., et al., 2021. Automatization and self-maintenance of the O-GlcNAcome catalog: a smart scientific database. Database 2021, baab039. https://doi.org/10.1093/database/baab039.

Mariappa, D., Selvan, N., Borodkin, V.S., et al., 2015. A mutant O-GlcNAcase as a probe to reveal global dynamics of protein O-GlcNAcylation during Drosophila embryonic development. Biochem. J. 470, 255–262. https://doi.org/10.1042/BJ20150610.

Marshall, S., Nadeau, O., Yamasaki, K., 2004. Dynamic actions of glucose and glucosamine on hexosamine biosynthesis in isolated adipocytes: differential effects on glucosamine 6-phosphate, UDP-N-acetylglucosamine, and ATP levels. J. Biol. Chem. 279, 35313–35319. https://doi.org/10.1074/jbc.M404133200.

Matsuoka, Y., Matsuoka, Y., Shibata, S., et al., 2002. Identification of Ewing's sarcoma gene product as a glycoprotein using a monoclonal antibody that recognizes an immunodetoxifying N-acetylglucosamine moiety. Hybridoma 21, 233–236. https://doi.org/10.1089/153685902760213831.

Mauri, T., Menu-Bouaouiche, L., Bardor, M., et al., 2021. O-GlcNAcylation prediction: an unattained objective. AABC Volume 14, 87–102. https://doi.org/10.2147/AABC.S294867.

Maynard, J.C., Chalkley, R.J., 2021. Methods for enrichment and assignment of N-acetylglucosamine modification sites. Mol. Cell. Proteom. 20, 100031 https://doi.org/10.1074/mcp.R120.002206.

Maynard, J.C., Burlingame, A.L., Medzhirodszky, K.F., 2016. Cysteine S-linked N-acetylglucosamine (S-GlcNAcylation), a new post-translational modification in mammals. Mol. Cell Proteom. 15, 3405–3411. https://doi.org/10.1074/mcp.M116.061549.

Mirgorodskaya, E., Roepstorff, P., Zubarev, R.A., 1999. Localization of O-Glycosylation sites in peptides by electron capture dissociation in a fourier transform mass spectrometer. Anal. Chem. 71, 4431–4436. https://doi.org/10.1021/ac990578v.

Monsigny, M., Sene, C., Obrenovitch, A., et al., 1979. Properties of succinylated wheat-germ agglutinin. Eur. J. Biochem. 98, 39–45. https://doi.org/10.1111/j.1432-1033.1979.tb13157.x.

Müeller, T., Ouyang, X., Johnson, M.S., et al., 2021. New insights into the biology of protein O-GlcNAcylation: approaches and observations. Front. Aging 1, 620382. https://doi.org/10.3389/fragi.2020.620382.

Muha, V., Authier, F., Szoke-Kovacs, Z., et al., 2021. Loss of O-GlcNAcase catalytic activity leads to defects in mouse embryogenesis. J. Biol. Chem. 296, 100439 https://doi.org/10.1016/j.jbc.2021.100439.

Nandi, A., Sprung, R., Barma, D.K., et al., 2006. Global identification of O-GlcNAc-modified proteins. Anal. Chem. 78, 452–458. https://doi.org/10.1021/ac051207j.

Nöt, L.G., Brocks, C.A., Vámhidy, L., et al., 2010. Increased O-linked beta-N-acetylglucosamine levels on proteins improves survival, reduces inflammation and organ damage 24 h after trauma-hemorrhage in rats. Crit. Care Med. 38, 562–571. https://doi.org/10.1097/CCM.0b013e3181cb10b3.

Oikari, S., Makkonen, K., Deen, A.J., et al., 2016. Hexosamine biosynthesis in keratinocytes: roles of GFAT and GNPDA enzymes in the maintenance of UDP-GlcNAc content and hyaluronan synthesis. Glycobiology 26, 710–722. https://doi.org/10.1093/glycob/cww019.

Oki, T., Yamazaki, K., Kuromitsu, J., et al., 1999. cDNA cloning and mapping of a novel subtype of glutamine:fructose-6-phosphate amidotransferase (GFAT2) in human and mouse. Genomics 57, 227–234. https://doi.org/10.1006/geno.1999.5785.

Okuda, T., 2017. Western blot data using two distinct anti-O-GlcNAc monoclonal antibodies showing unique glycosylation status on cellular proteins under 2-deoxy-d-glucose treatment. Data Brief. 10, 449–453. https://doi.org/10.1016/j.dib.2016.12.001.

Olson, A.K., Bouchard, B., Zhu, W.Z., et al., 2020. First characterization of glucose flux through the hexosamine biosynthesis pathway (HBP) in ex vivo mouse heart. J. Biol. Chem. 295, 2018–2033. https://doi.org/10.1074/jbc.RA119.010565.

Ortiz-Meoz, R.F., Jiang, J., Lazarus, M.B., et al., 2015. A small molecule that inhibits OGT activity in cells. ACS Chem. Biol. 10, 1392–1397. https://doi.org/10.1021/acsbiochem.5b00004.

Overah, T., Kuckelkorn, U., Henklein, P., et al., 2012. Mapping of O-GlcNAc sites of 20 S proteasome subunits and Hsp90 by a novel biotin-cystatin tag. Mol. Cell Proteom. 11, 467–477. https://doi.org/10.1107/mcp.M111.015966.

Pantaleon, M., Tan, H.Y., Kafer, G.R., Kaye, P.L., 2010. Toxic effects of hyperglycemia are mediated by the hexosamine signaling pathway and O-Linked glycosylation in early mouse embryos. Biol. Reprod. 82, 751–758. https://doi.org/10.1095/biolreprod.109.076661.

Pathak, S., Borodkin, V.S., Albarbarawi, O., et al., 2012. O-GlcNAcylation of TAB1 modulates TAK1-mediated cytokine release. EMBO J. 31, 1394–1404. https://doi.org/10.1038/emboj.2012.8.

Pravata, V.M., Muha, V., Gundogdu, M., et al., 2019. Catalytic deficiency of O-GlcNAc transferase leads to X-linked intellectual disability. PNAS 116, 14961–14970. https://doi.org/10.1073/pnas.1900065116.

Qian, Y., Ahmad, M., Chen, S., et al., 2011. Discovery of 1-arylcarbonyl-6,7-dimethoxy-yisoquinoline derivatives as glutamine fructose-6-phosphate amidotransferase (GFAT) inhibitors. Bioorg. Med. Chem. Lett. 21, 6264–6269. https://doi.org/10.1016/j.bmcl.2011.09.009.

Qin, K., Zhu, Y., Qin, W., et al., 2018a. Quantitative profiling of protein O-GlcNAcylation sites by an isotope-tagged cleavable linker. ACS Chem. Biol. 13, 1983–1989. https://doi.org/10.1021/acsbiochem.8b000414.

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Ma, J., Wang, W.-H., Li, Z., et al., 2019. O-GlcNAc site mapping by using a combination of chemoenzymatic labeling, copper-free click chemistry, reductive cleavage, and electron-transfer dissociation mass spectrometry. Anal. Chem. 91, 2620–2625. https://doi.org/10.1021/acs.analchem.8b05688.

Macauley, M.S., Vocadlo, D.J., 2009. Enzymatic characterization and inhibition of the nuclear variant of human O-GlcNAcase. Carbohydr. Res. 344, 1079–1084. https://doi.org/10.1016/j.carres.2009.04.017.

Macauley, M.S., Whitworth, G.E., Debowski, A.W., et al., 2005. O-GlcNAcase uses substrate-assisted catalysis: kinetic analysis and development of highly selective mechanism-inspired inhibitors. J. Biol. Chem. 280, 25313–25322. https://doi.org/10.1074/jbc.M413819200.

Macauley, M.S., Bubb, A.K., Martinez-Fleites, C., et al., 2008. Elevation of global O-GlcNAc levels in 3T3-L1 adipocytes by selective inhibition of O-GlcNAcase does not induce insulin resistance. J. Biol. Chem. 283, 34687–34695. https://doi.org/10.1074/jbc.M804525200.

Macauley, M.S., Shan, X., Yuzwa, S.A., et al., 2010a. Elevation of global O-GlcNAc in rodents using a selective O-GlcNAcase inhibitor does not cause insulin resistance or perturb glucomethoestasis. Chem. Biol. 17, 949–958. https://doi.org/10.1016/j.chembiol.2010.07.005.

Macauley, M.S., He, Y., Glaster, T.M., et al., 2010b. Inhibition of O-GlcNAcase using a potent and cell-permeable inhibitor does not induce insulin resistance in 3T3-L1 adipocytes. Chem. Biol. 17, 937–948. https://doi.org/10.1016/j.chembiol.2010.07.006.

Machon, O., Varrot, A., 2020. Expression, purification, and applications of the recombinant lectin PVL from <Emphasis Type="Italic">>Psathrella velutina</> / <Emphasis> specific for terminal <Emphasis Type="Italic">>N</Emphasis> / <Emphasis> >Acetyl- Glucosamine. Lectin Purif. Anal. 421–436. https://doi.org/10.1007/978-1-0716- 0430-4.41.

Machon, O., Baldini, S.F., Ribeiro, J.P., et al., 2017. Recombinant fungal lectin as a new tool to investigate O-GlcNAcase processes. Glycobiology 27, 123–128. https://doi.org/10.1093/glycob/cww105.

Malard, F., Wulff-Fuentes, E., Berendt, R.R., et al., 2021. Automatization and self-maintenance of the O-GlcNAcome catalog: a smart scientific database. Database 2021, baab039. https://doi.org/10.1093/database/baab039.

Mariappa, D., Selvan, N., Borodkin, V.S., et al., 2015. A mutant O-GlcNAcase as a probe to reveal global dynamics of protein O-GlcNAcylation during Drosophila embryonic development. Biochem. J. 470, 255–262. https://doi.org/10.1042/BJ20150610.

Marshall, S., Nadeau, O., Yamasaki, K., 2004. Dynamic actions of glucose and glucosamine on hexosamine biosynthesis in isolated adipocytes: differential effects on glucosamine 6-phosphate, UDP-N-acetylglucosamine, and ATP levels. J. Biol. Chem. 279, 35313–35319. https://doi.org/10.1074/jbc.M404133200.

Matsuoka, Y., Matsuoka, Y., Shibata, S., et al., 2002. Identification of Ewing's sarcoma gene product as a glycoprotein using a monoclonal antibody that recognizes an immunodetoxifying N-acetylglucosamine moiety. Hybridoma 21, 233–236. https://doi.org/10.1089/153685902760213831.

Mauri, T., Menu-Bouaouiche, L., Bardor, M., et al., 2021. O-GlcNAcylation prediction: an unattained objective. AABC Volume 14, 87–102. https://doi.org/10.2147/AABC.S294867.

Maynard, J.C., Chalkley, R.J., 2021. Methods for enrichment and assignment of N-acetylglucosamine modification sites. Mol. Cell. Proteom. 20, 100031 https://doi.org/10.1074/mcp.R120.002206.

Maynard, J.C., Burlingame, A.L., Medzhirodszky, K.F., 2016. Cysteine S-linked N-acetylglucosamine (S-GlcNAcylation), a new post-translational modification in mammals. Mol. Cell Proteom. 15, 3405–3411. https://doi.org/10.1074/mcp.M116.061549.

Mirgorodskaya, E., Roepstorff, P., Zubarev, R.A., 1999. Localization of O-Glycosylation sites in peptides by electron capture dissociation in a fourier transform mass spectrometer. Anal. Chem. 71, 4431–4436. https://doi.org/10.1021/ac990578v.

Monsigny, M., Sene, C., Obrenovitch, A., et al., 1979. Properties of succinylated wheat-germ agglutinin. Eur. J. Biochem. 98, 39–45. https://doi.org/10.1111/j.1432-1033.1979.tb13157.x.

Müeller, T., Ouyang, X., Johnson, M.S., et al., 2021. New insights into the biology of protein O-GlcNAcylation: approaches and observations. Front. Aging 1, 620382. https://doi.org/10.3389/fragi.2020.620382.

Muha, V., Authier, F., Szoke-Kovacs, Z., et al., 2021. Loss of O-GlcNAcase catalytic activity leads to defects in mouse embryogenesis. J. Biol. Chem. 296, 100439 https://doi.org/10.1016/j.jbc.2021.100439.

Nandi, A., Sprung, R., Barma, D.K., et al., 2006. Global identification of O-GlcNAc-modified proteins. Anal. Chem. 78, 452–458. https://doi.org/10.1021/ac051207j.

Nöt, L.G., Brocks, C.A., Vámhidy, L., et al., 2010. Increased O-linked beta-N-acetylglucosamine levels on proteins improves survival, reduces inflammation and organ damage 24 h after trauma-hemorrhage in rats. Crit. Care Med. 38, 562–571. https://doi.org/10.1097/CCM.0b013e3181cb10b3.

Oikari, S., Makkonen, K., Deen, A.J., et al., 2016. Hexosamine biosynthesis in keratinocytes: roles of GFAT and GNPDA enzymes in the maintenance of UDP-GlcNAc content and hyaluronan synthesis. Glycobiology 26, 710–722. https://doi.org/10.1093/glycob/cww019.

Oki, T., Yamazaki, K., Kuromitsu, J., et al., 1999. cDNA cloning and mapping of a novel subtype of glutamine:fructose-6-phosphate amidotransferase (GFAT2) in human and mouse. Genomics 57, 227–234. https://doi.org/10.1006/geno.1999.5785.

Okuda, T., 2017. Western blot data using two distinct anti-O-GlcNAc monoclonal antibodies showing unique glycosylation status on cellular proteins under 2-deoxy-d-glucose treatment. Data Brief. 10, 449–453. https://doi.org/10.1016/j.dib.2016.12.001.

Olson, A.K., Bouchard, B., Zhu, W.Z., et al., 2020. First characterization of glucose flux through the hexosamine biosynthesis pathway (HBP) in ex vivo mouse heart. J. Biol. Chem. 295, 2018–2033. https://doi.org/10.1074/jbc.RA119.010565.

Ortiz-Meoz, R.F., Jiang, J., Lazarus, M.B., et al., 2015. A small molecule that inhibits OGT activity in cells. ACS Chem. Biol. 10, 1392–1397. https://doi.org/10.1021/acsbiochem.5b00004.

Overah, T., Kuckelkorn, U., Henklein, P., et al., 2012. Mapping of O-GlcNAc sites of 20 S proteasome subunits and Hsp90 by a novel biotin-cystatin tag. Mol. Cell Proteom. 11, 467–477. https://doi.org/10.1107/mcp.M111.015966.

Pantaleon, M., Tan, H.Y., Kafer, G.R., Kaye, P.L., 2010. Toxic effects of hyperglycemia are mediated by the hexosamine signaling pathway and O-Linked glycosylation in early mouse embryos. Biol. Reprod. 82, 751–758. https://doi.org/10.1095/biolreprod.109.076661.

Pathak, S., Borodkin, V.S., Albarbarawi, O., et al., 2012. O-GlcNAcylation of TAB1 modulates TAK1-mediated cytokine release. EMBO J. 31, 1394–1404. https://doi.org/10.1038/emboj.2012.8.

Pravata, V.M., Muha, V., Gundogdu, M., et al., 2019. Catalytic deficiency of O-GlcNAc transferase leads to X-linked intellectual disability. PNAS 116, 14961–14970. https://doi.org/10.1073/pnas.1900065116.

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