Writing and erasing O-GlcNAc from target proteins in cells

O-linked N-acetylglucosamine (O-GlcNAc) is a widespread reversible modification on nucleocytoplasmic proteins that plays an important role in many biochemical processes and is highly relevant to numerous human diseases. The O-GlcNAc modification has diverse functional impacts on individual proteins and glycosites, and methods for editing this modification on substrates are essential to decipher these functions. Herein, we review recent progress in developing methods for O-GlcNAc regulation, with a focus on methods for editing O-GlcNAc with protein- and site-selectivity in cells. The applications, advantages, and limitations of currently available strategies for writing and erasing O-GlcNAc and future directions are also discussed. These emerging approaches to manipulate O-GlcNAc on a target protein in cells will greatly accelerate the development of functional studies and enable therapeutic interventions in the O-GlcNAc field.

Generation of an Unbiased Interactome for the Tetratricopeptide Repeat Domain of O-GlcNAc Transferase Indicates a Role for the Enzyme in Intellectual Disability

The O-GlcNActransferase (OGT) is localized to the nucleus and cytoplasm where it regulates nucleocytoplasmic proteins by modifying serine and threonine residues with a non-extended monosaccharide, b-N-Acetyl-Glucosamine (O-GlcNAc). With thousands ofknown O-GlcNAcmodifiedproteinsbut only oneOGTencoded in the mammalian genome, a prevailing question is howOGTselects its substrates. Prior work has indicated that theN-terminaltetratricopeptide repeat (TPR) domain of OGT, rather than itsC-terminalcatalytic domain, is responsible forsubcellular targeting andsubstrate selection.An additional impetus for exploring the OGT TPR domain interactome is the fact that missense mutations inOGTassociated with X-linked intellectual disability (XLID) are primarily localized to the TPR domain without substantial impact on activity or stability of the enzyme.Therefore, we adapted theBioIDlabeling method to identify interactors of a TPR-BirA* fusion protein in HeLa cells. We identified 115high confidenceinteractors representing both known and novel O-GlcNAcmodified proteins and OGT interactors. The TPR interactors are highly enriched in processes in which OGT has a known role (e.g. chromatin remodeling, cellular survival of heat stress, circadian rhythm), as well as processesin which OGT has yet to be implicated (e.g. pre-mRNA processing). Importantly,the identified TPR interactors are involved in several disease states but most notably are highly enriched in pathologies featuring intellectual disability.Theseproteinsrepresent candidateinteractors that may underlie the mechanismby which mutations in OGT lead to XLID. Furthermore, the identified interactors provide additional evidence of the importance of the TPR domain for OGT targeting and/or substrate selection.Thus, this defined interactome for the TPR domain of OGT serves as ajumping off point for future researchexploringthe role of OGT, the TPR domain, and its protein interactorsin multiple cellular processes and disease mechanisms, including intellectual disability.

Increased O-GlcNAcylation of c-Myc Promotes Pre-B Cell Proliferation

O-linked β-N-acetylglucosamine (O-GlcNAc) modification regulates the activity of hundreds of nucleocytoplasmic proteins involved in a wide variety of cellular processes, such as gene expression, signaling, and cell growth; however, the mechanism underlying the regulation of B cell development and function by O-GlcNAcylation remains largely unknown. Here, we demonstrate that changes in cellular O-GlcNAc levels significantly affected the growth of pre-B cells, which rapidly proliferate to allow expansion of functional clones that express successfully rearranged heavy chains at the pro-B stage during early B cell development. In our study, the overall O-GlcNAc levels in these proliferative pre-B cells, which are linked to the glucose uptake rate, were highly induced when compared with those in pro-B cells. Thus, pharmacologically, genetically, or nutritionally, inhibition of O-GlcNAcylation in pre-B cells markedly downregulated c-Myc expression, resulting in cell cycle arrest via blockade of cyclin expression. Importantly, the population of B cells after the pro-B cell stage in mouse bone marrow was severely impaired by the administration of an O-GlcNAc inhibitor. These results strongly suggest that O-GlcNAcylation-dependent expression of c-Myc represents a new regulatory component of pre-B cell proliferation, as well as a potential therapeutic target for the treatment of pre-B cell-derived leukemia.

Contribution of O-GlcNAc modification of NF-κB p65 in the attenuation of diabetes-induced haptoglobin expression in rat liver

Haptoglobin (Hp) is a hemoglobin-binding protein that prevents free hemoglobin-induced tissue oxidative damage. In streptozotocin-induced diabetic rats, the initial elevation of Hp expression in the serum and liver tends to decrease with diabetes progression, contributing to increased oxidative stress. Glucose toxicity and diabetic complications are closely related to increased modification of nucleocytoplasmic proteins by O-linked-N-acetylglucosamine (O-GlcNAc). We examined the contribution of O-GlcNAcylation of NF-?B p65 to changes in liver Hp expression in diabetic rats. WGA-affinity chromatography revealed a progressive increase in O-GlcNAcylation in nuclear NF-?B p65 during eight weeks of diabetes. DNA-affinity chromatography followed by immunoblot analysis revealed that decreased Hp expression at 4 and 8 weeks of diabetes was accompanied by the absence of Hp gene hormone-responsive element (HRE) occupancy with NF-?B p65, low occupancy with glucocorticoid receptor (GR), and almost no changes in STAT3 occupancy compared to 2 weeks, when Hp expression was highest. Coimmunoprecipitation experiments indicate that these events were the result of impaired NF-?B p65/STAT3 and GR/STAT3 interactions. Results suggest that the attenuation of Hp expression associated with diabetes was at least in part the result of O-GlcNAcylation of NF-?B p65, which prevents the formation of an effective transcription initiation complex on the Hp gene promoter.

Catalytic deficiency of O-GlcNAc transferase leads to X-linked intellectual disability

O-GlcNAc transferase (OGT) is an X-linked gene product that is essential for normal development of the vertebrate embryo. It catalyses the O-GlcNAc posttranslational modification of nucleocytoplasmic proteins and proteolytic maturation of the transcriptional coregulator Host cell factor 1 (HCF1). Recent studies have suggested that conservative missense mutations distal to the OGT catalytic domain lead to X-linked intellectual disability in boys, but it is not clear if this is through changes in the O-GlcNAc proteome, loss of protein–protein interactions, or misprocessing of HCF1. Here, we report an OGT catalytic domain missense mutation in monozygotic female twins (c. X:70779215 T > A, p. N567K) with intellectual disability that allows dissection of these effects. The patients show limited IQ with developmental delay and skewed X-inactivation. Molecular analyses revealed decreased OGT stability and disruption of the substrate binding site, resulting in loss of catalytic activity. Editing this mutation into the Drosophila genome results in global changes in the O-GlcNAc proteome, while in mouse embryonic stem cells it leads to loss of O-GlcNAcase and delayed differentiation down the neuronal lineage. These data imply that catalytic deficiency of OGT could contribute to X-linked intellectual disability.

Recognition of a glycosylation substrate by the O-GlcNAc transferase TPR repeats

O-linked N -acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification found on hundreds of nucleocytoplasmic proteins in metazoa. Although a single enzyme, O-GlcNAc transferase (OGT), generates the entire cytosolic O-GlcNAc proteome, it is not understood how it recognizes its protein substrates, targeting only a fraction of serines/threonines in the metazoan proteome for glycosylation. We describe a trapped complex of human OGT with the C-terminal domain of TAB1, a key innate immunity-signalling O-GlcNAc protein, revealing extensive interactions with the tetratricopeptide repeats of OGT. Confirmed by mutagenesis, this interaction suggests that glycosylation substrate specificity is achieved by recognition of a degenerate sequon in the active site combined with an extended conformation C-terminal of the O-GlcNAc target site.

Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast

Dynamic cycling of N-Acetylglucosamine (GlcNAc) on serine and threonine residues (O-GlcNAcylation) is an essential process in all eukaryotic cells except yeast, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. O-GlcNAcylation modulates signaling and cellular processes in an intricate interplay with protein phosphorylation and serves as a key sensor of nutrients by linking the hexosamine biosynthetic pathway to cellular signaling. A longstanding conundrum has been how yeast survives without O-GlcNAcylation in light of its similar phosphorylation signaling system. We previously developed a sensitive lectin enrichment and mass spectrometry workflow for identification of the human O-linked mannose (O-Man) glycoproteome and used this to identify a pleothora of O-Man glycoproteins in human cell lines including the large family of cadherins and protocadherins. Here, we applied the workflow to yeast with the aim to characterize the yeast O-Man glycoproteome, and in doing so, we discovered hitherto unknown O-Man glycosites on nuclear, cytoplasmic, and mitochondrial proteins in S. cerevisiae and S. pombe. Such O-Man glycoproteins were not found in our analysis of human cell lines. However, the type of yeast O-Man nucleocytoplasmic proteins and the localization of identified O-Man residues mirror that of the O-GlcNAc glycoproteome found in other eukaryotic cells, indicating that the two different types of O-glycosylations serve the same important biological functions. The discovery opens for exploration of the enzymatic machinery that is predicted to regulate the nucleocytoplasmic O-Man glycosylations. It is likely that manipulation of this type of O-Man glycosylation will have wide applications for yeast bioprocessing.

β-N-Acetylglucosamine (O-GlcNAc) Is a Novel Regulator of Mitosis-specific Phosphorylations on Histone H3

O-Linked β-N-acetylglucosamine, or O-GlcNAc, is a dynamic post-translational modification that cycles on and off serine and threonine residues of nucleocytoplasmic proteins. The O-GlcNAc modification shares a complex relationship with phosphorylation, as both modifications are capable of mutually inhibiting the occupation of each other on the same or nearby amino acid residue. In addition to diabetes, cancer, and neurodegenerative diseases, O-GlcNAc appears to play a significant role in cell growth and cell cycle progression, although the precise mechanisms are still not well understood. A recent study also found that all four core nucleosomal histones (H2A, H2B, H3, and H4) are modified with O-GlcNAc, although no specific sites on H3 were reported. Here, we describe that histone H3, a protein highly phosphorylated during mitosis, is modified with O-GlcNAc. Several biochemical assays were used to validate that H3 is modified with O-GlcNAc. Mass spectrometry analysis identified threonine 32 as a novel O-GlcNAc site. O-GlcNAc was detected at higher levels on H3 during interphase than mitosis, which inversely correlated with phosphorylation. Furthermore, increased O-GlcNAcylation was observed to reduce mitosis-specific phosphorylation at serine 10, serine 28, and threonine 32. Finally, inhibiting OGA, the enzyme responsible for removing O-GlcNAc, hindered the transition from G2 to M phase of the cell cycle, displaying a phenotype similar to preventing mitosis-specific phosphorylation on H3. Taken together, these data indicate that O-GlcNAcylation regulates mitosis-specific phosphorylations on H3, providing a mechanistic switch that orchestrates the G2-M transition of the cell cycle.

RNA-related nuclear functions of human Pat1b, the P-body mRNA decay factor

The evolutionarily conserved Pat1 proteins are P-body components recently shown to play important roles in cytoplasmic gene expression control. Using human cell lines, we demonstrate that human Pat1b is a shuttling protein whose nuclear export is mediated via a consensus NES sequence and Crm1, as evidenced by leptomycin B (LMB) treatment. However, not all P-body components are nucleocytoplasmic proteins; rck/p54, Dcp1a, Edc3, Ge-1, and Xrn1 are insensitive to LMB and remain cytoplasmic in its presence. Nuclear Pat1b localizes to PML–associated foci and SC35-containing splicing speckles in a transcription-dependent manner, whereas in the absence of RNA synthesis, Pat1b redistributes to crescent-shaped nucleolar caps. Furthermore, inhibition of splicing by spliceostatin A leads to the reorganization of SC35 speckles, which is closely mirrored by Pat1b, indicating that it may also be involved in splicing processes. Of interest, Pat1b retention in these three nuclear compartments is mediated via distinct regions of the protein. Examination of the nuclear distribution of 4E-T(ransporter), an additional P-body nucleocytoplasmic protein, revealed that 4E-T colocalizes with Pat1b in PML-associated foci but not in nucleolar caps. Taken together, our findings strongly suggest that Pat1b participates in several RNA-related nuclear processes in addition to its multiple regulatory roles in the cytoplasm.

Glucosamine cardioprotection in perfused rat hearts associated with increased O-linked N-acetylglucosamine protein modification and altered p38 activation

We have shown that, in the perfused heart, glucosamine improved functional recovery following ischemia and that this appeared to be mediated via an increase in O-linked N-acetylglucosamine ( O-GlcNAc) levels on nucleocytoplasmic proteins. Several kinase pathways, specifically Akt and the mitogen-activated protein kinases (MAPKs) p38 and ERK1/2, which have been implicated in ischemic cardioprotection, have also been reported to be modified in response to increased O-GlcNAc levels. Therefore, the goals of this study were to determine the effect of ischemia on O-GlcNAc levels and to evaluate whether the cardioprotection resulting from glucosamine treatment could be attributed to changes in ERK1/2, Akt, and p38 phosphorylation. Isolated rat hearts were perfused with or without 5 mM glucosamine and were subjected to 5, 10, or 30 min of low-flow ischemia or 30 min of low-flow ischemia and 60 min of reperfusion. Glucosamine treatment attenuated ischemic contracture and improved functional recovery at the end of reperfusion. Glucosamine treatment increased flux through the hexosamine biosynthesis pathway and increased O-GlcNAc levels but had no effect on ATP levels. Glucosamine did not alter the response of either ERK1/2 or Akt to ischemia-reperfusion; however, it significantly attenuated the ischemia-induced increase in p38 phosphorylation and paradoxically increased p38 phosphorylation at the end of reperfusion. These data support the notion that O-GlcNAc may play an important role as an internal stress response and that glucosamine-induced cardioprotection may be mediated via the p38 MAPK pathway.