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Published in final edited form as: Curr Opin Chem Biol. 2024 Jun 10;81:102476. doi: 10.1016/j.cbpa.2024.102476

The non-catalytic domains of O-GlcNAc cycling enzymes present new opportunities for function-specific control

Chia-Wei Hu 1, Ke Wang 1, Jiaoyang Jiang 1,*
PMCID: PMC11323188  NIHMSID: NIHMS2002963  PMID: 38861851

Abstract

O-GlcNAcylation is an essential protein glycosylation governed by two O-GlcNAc cycling enzymes: O-GlcNAc transferase (OGT) installs a single sugar moiety N-acetylglucosamine (GlcNAc) on protein serine and threonine residues, and O-GlcNAcase (OGA) removes them. Aberrant O-GlcNAcylation has been implicated in various diseases. However, the large repertoire of more than 1,000 O-GlcNAcylated proteins and the elusive mechanisms of OGT/OGA in substrate recognition present significant challenges in targeting the dysregulated O-GlcNAcylation for therapeutic development. Recently, emerging evidence suggested that the non-catalytic domains play critical roles in regulating the functional specificity of OGT/OGA via modulating their protein interactions and substrate recognition. Here, we discuss recent studies on the structures, mechanisms, and related tools of the OGT/OGA non-catalytic domains, highlighting new opportunities for function-specific control.

Keywords: O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), Non-catalytic domain, Function-specific modulator

Introduction

O-linked-N-acetylglucosaminylation (O-GlcNAcylation) is an intracellular glycosylation essential for numerous cellular functions [1], including epigenetics [2,3] and metabolism [4-6]. In response to nutrient and environmental changes, this post-translational modification (PTM) dynamically modulates protein activity, stability, and protein-protein interactions (PPIs), among others [7,8]. Aberrant O-GlcNAcylation has been implicated in many life-threatening diseases such as diabetes, cancer, neurodegeneration, and cardiovascular dysfunction [9,10]. Intriguingly, only two enzymes catalyze this reversible O-GlcNAcylation on thousands of proteins [11]: O-GlcNAc transferase (OGT) transfers the GlcNAc moiety from sugar donor UDP-GlcNAc to the serine and threonine residues of protein substrates [12,13], whereas O-GlcNAcase (OGA) hydrolyzes this modification (Figure 1a) [14-16]. Interestingly, OGT can associate with and O-GlcNAcylate OGA in cells, further complicating their functional regulations [11,17]. Over the past two decades, many small-molecule active-site inhibitors of OGT and OGA have been developed and a few of them become powerful tools for investigating O-GlcNAcylation in biological and disease models [18,19]. However, these active-site inhibitors perturb the O-GlcNAc homeostasis at a global scale, concomitantly disturbing various cellular functions and raising concerns on undesired side effects. Hence, new strategies are much needed to overcome these limitations.

Figure 1.

Figure 1.

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The enzymatic reactions and domain organizations of O-GlcNAc cycling enzymes (OGT and OGA). (a) Schematic of reversible O-GlcNAcylation catalyzed by OGT and OGA. The chemical structure of UDP-GlcNAc is also shown. (b) Domain architecture of full-length OGT (13.5 TPR repeats) and the construct of the reported crystal structure OGT4.5. TPR, tetratricopeptide repeat. N-Cat and C-Cat, two lobes of OGT’s catalytic domain. Int-D, intervening domain. (c) Domain architecture of full-length OGA and the construct of the reported crystal structure OGAcryst. IDR, intrinsically disordered region. pHAT, pseudo histone acetyltransferase domain.

Recently, emerging data has shown that OGT/OGA could regulate particular biological functions through their non-catalytic domains [20-22]. For instance, instead of its catalytic domain, OGT’s non-catalytic tetratricopeptide repeat (TPR) domain was found responsible for osmotic stress response in C. elegans [23]. Another study reported that the non-catalytic C-terminal region of OGA participated in DNA damage repair [24]. Moreover, the non-catalytic domains modulate diverse protein interactions of OGT and/or OGA in cells [22,25]. Collectively, these new findings support the non-catalytic domains of OGT/OGA as key elements in conferring substrate and functional specificity for these essential O-GlcNAc cycling enzymes. With a focus on the studies reported in the past two years, here, we highlight the recent advances in structural and mechanistic investigations, and the related chemical biology tools of OGT/OGA non-catalytic domains. We anticipate this work will promote O-GlcNAc research and inspire the rational design of function-specific modulators of OGT/OGA via targeting their non-catalytic domains for biomedical applications.

Domain architecture of O-GlcNAc cycling enzymes

Human OGT comprises an N-terminal TPR domain (13.5 TPRs for the most abundant isoform) and a C-terminal catalytic region, the latter of which is separated into two lobes (N-Cat and C-Cat) by an intervening domain (Int-D) (Figure 1b) [26,27]. OGT’s catalytic region was first revealed by the crystal structure of a truncated construct (OGT4.5 in Figure 1b), illustrating a GT-B glycosyltransferase catalytic domain consisting of two Rossmann folds, which flank a unique Int-D with unknown function (see below) [28]. Human OGA is composed of an N-terminal catalytic domain, a stalk domain involving an intrinsically disordered region (IDR), and a C-terminal pseudo histone acetyltransferase (pHAT) domain (Figure 1c) [27]. While the full-length OGA structure is currently unavailable, the crystal structures of truncated OGA constructs (e.g., OGAcryst in Figure 1c) have been reported. These structures consistently show that OGA’s catalytic domain folds into a (β/α)8 barrel, classical for the glycoside hydrolase 84 (GH84) family of enzymes [29-31]. Beyond the highly conserved catalytic site in GT-B (for OGT) or GH84 (for OGA) family, these O-GlcNAc cycling enzymes carry unique structural features in their non-catalytic domains (discuss below), presenting underexplored mechanisms for functional regulation.

Non-catalytic TPR domain of OGT

TPR is a tandem repeat of 34-residue motif typically serving as a protein interaction scaffold [32]. OGT’s TPR domain folds into a superhelix containing multiple ladder-like asparagine and aspartate residues for interacting with various binding partners [33-37], as revealed by the OGT4.5 complex structures with different peptide substrates [27]. Two recent studies have reported the structures of full-length OGT (5.3 Å and 3.69 Å) using cryogenic electronic microscopy (cryo-EM) approach, showing that OGT homodimerizes via TPRs 6 and 7 (Figure 2a) [38,39]. Dimerization enhances the structural complexity of OGT, expanding its PPI landscape in macromolecular assembly. An example is shown in the recently reported cryo-EM structure of OGT dimer bound with monomeric OGA (Figure 2b) [39]. In this structure, OGA’s catalytic domain and a partial stalk domain are visible and largely interact with OGT’s TPR region. OGA’s catalytic domain is anchored on the convex surface of OGT TPR 11-13, occluding OGA’s active site. Meanwhile, a segment of IDR from OGA’s stalk domain binds in OGT’s catalytic pocket, revealing the conformation of OGA’s major O-GlcNAcylation site (S405 in IDR). The OGA IDR further extends into the TPR lumen of OGT and displays a series of backbone and side-chain interactions with the ladder-like asparagine residues in the TPR domain. These asparagine residues, especially the ones in the N-terminal TPRs, are essential for the OGT-OGA interaction. This structure offers the first view of the multisite interactions of full-length OGT with a polypeptide substrate and may represent a popular binding mode of OGT with unstructured protein regions. Consistent with a previous biochemical study of OGT and OGA [40], the observed side-chain interactions and the asymmetric impacts of different TPR regions on OGA binding are promising factors contributing to the substrate selectivity of OGT. Future efforts on obtaining more complete OGT complex structures with full-length OGA, as well as other protein partners, are expected to uncover new OGT binding modes and important interaction sites for specific targeting.

Figure 2.

Figure 2.

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The new binding modes and modulators of OGT non-catalytic domains (TPR and Int-D). (a) The cryo-EM structure (PDB 7YEA) of full-length OGT dimer with TPR (dark blue), N-Cat and C-Cat (gray), and Int-D (yellow) highlighted in different colors. (b) The cryo-EM structure of full-length OGT dimer in complex with monomeric OGA polypeptides (PDB 7YEH). OGA’s N-terminal catalytic domain and part of the stalk domain are visible in one monomer. OGT domains are colored as the structure displayed in (a). (c) Top, the crystal structure of OGT4.5 in complex with a motif-containing SMG9 peptide bound in the Int-D exosite (PDB 8FE7). Zoom-in view shows the hydrophobic and polar interactions between the OGT Int-D and SMG9 peptide residues. UDP-GlcNAc is shown in red surface. Bottom, the biological functions regulated by the OGT Int-D exosite. PxYxI represents the general Int-D peptide binding motif. (d) Cartoon representation of the mechanisms of OGT exosite inhibitors in modulating protein O-GlcNAcylation. (e) Dual-specificity aptamer achieves protein-specific O-GlcNAcylation in cells by simultaneously targeting the endogenous protein substrate β-catenin and OGT TPR domain.

Non-catalytic intervening domain (Int-D) of OGT

OGT Int-D adopts a unique fold that is not found in other reported protein structures. Intriguingly, this non-catalytic Int-D is conserved among vertebrate OGTs, but its function is just beginning to unfold. Recently, using phage screening and structural analysis, two groups independently reported similar OGT binding peptide motifs: PxYx[I/L] and [Y/F]-x-P-x-Y-x-[I/M/F] [41,42]. Surprisingly, the crystal structures of OGT4.5 in complex with a few motif-containing peptides revealed that these peptides bound similarly in an extended conformation on a surface grove of OGT’s Int-D (Figure 2c). Interestingly, single mutations of the Int-D interacting residues did not alter the intrinsic catalytic activity of OGT, but significantly abrogated the binding with motif-containing peptides in vitro, unraveling a novel OGT exosite (ligand-binding site beyond the active site of enzyme). One of the studies applied the Proteomic Peptide Phage Display (ProP-PD) to identify OGT binders and provided the initial hints at Int-D exosite functions [41]. The ProP-PD library consists of peptides derived from the IDRs of human proteome, enabling the efficient profiling of biologically relevant target-binding modules [43,44]. This strategy is particularly valuable for screening OGT-peptide interactions since O-GlcNAcylation often occurs in the IDRs of substrate proteins [45]. The ProP-PD screening-identified peptide motif PxYx[I/L], structurally and biochemically characterized as an Int-D exosite ligand, has been found in ~223 intracellular human proteins (e.g., SMG9 and ZNF831). Notably, approximately 57% and 85% of these motif-containing proteins can be O-GlcNAcylated and tyrosine phosphorylated (pTyr), respectively, indicating an important role of the OGT’s Int-D exosite in regulating these two essential PTMs [41]. Further, around 1/3 of the motif-containing proteins can be phosphorylated at the motif tyrosine residue (motif pTyr), and majority of them are also known O-GlcNAcylated proteins. For example, SMG9 protein carrying the motif pTyr (Y147) also contains a single major O-GlcNAcylation site (T114) located in proximity. At the molecular level, motif pTyr was found to preclude the SMG9 peptide from binding to OGT, likely due to the unfavorable charge repulsion and/or steric hindrance of the phosphate group with the Int-D exosite residues (Figure 2c). In cells, a single mutation at the SMG9 motif or OGT Int-D exosite (N791A) impeded the binding and O-GlcNAcylation of SMG9 protein. The same Int-D mutant also dysregulated O-GlcNAcylation and tyrosine phosphorylation in response to nutrient deprivation, supporting the OGT Int-D exosite acting as a nutrient and stress sensor. Taken together, this work offered key insights into the unique biological functions of the OGT Int-D exosite, making it distinct from other glycosyltransferases.

From both recent reports on OGT Int-D, it is worth noting that a significant number of motif-containing proteins in the human proteome are not O-GlcNAcylated, suggesting the potential interactions of Int-D with non-substrate proteins for functional regulations [41,42]. Along with the aforementioned TPR studies, decoding the coordination between Int-D and TPR domain should be accentuated to uncover new exosite binding and protein recognition modes that attribute to OGT’s multi-functional roles in cells.

Chemical biology tools targeting the non-catalytic domains of OGT

The broad implications of O-GlcNAcylation in biology and disease have raised substantial interests in developing chemical tools to regulate and manipulate O-GlcNAcylation in cells. As the investigations of O-GlcNAc cycling enzymes advanced, their non-catalytic domains have emerged as promising targets for developing function-specific or protein-selective modulators. Due to the limited space, we will highlight representative works reported recently that have not been extensively discussed. For comprehensive summaries of O-GlcNAc toolbox, please refer to other excellent reviews [18,19,46,47].

Exosite inhibitors

Ligands binding to a regulatory exosite can modulate the PPIs and catalytic activity of a protein target (Figure 2d) [48]. This information is particularly useful for designing function- specific modulators of proteins with various binding partners like OGT. In the study that discovered the Int-D exosite binding motif [Y/F]-x-P-x-Y-x-[I/M/F], extending both ends of the motif-containing peptides created non-competitive inhibitors of OGT in vitro (IC50 = 5-7 μM using HCF-1 peptide substrate) [42]. Interestingly, their binding affinities or inhibitory efficiency toward OGT could be tuned by the N- and C-terminal peptide residues, as well as the residues immediately adjacent to and within the motif.

Besides the Int-D, the TPR domain provides additional promising exosites for regulating OGT-protein interactions. Although targeting the OGT TPR domain is challenging due to its structural similarity with many other TPR-containing proteins, small-molecule and peptide probes of OGT TPRs have been recently reported. A natural product, oleanolic acid derivative (CDDO), was shown to inhibit OGT’s activity in the test with CKII peptide substrate (IC50 = 6.6 μM) [49]. The following mass spectrometric and molecular docking analyses indicated that CDDO binds to the OGT TPRs. On the other hand, exploiting the random non-standard peptides integrated discovery (RaPID) system, one study discovered a series of thioether-linked macrocyclic peptides targeting the OGT TPR domain [50]. Although the exact binding site requires further investigation, optimization of this class of molecules has led to a peptide ligand (D3-15) with an exceptional inhibition potency toward OGT in vitro (IC50 = 3.3 nM using HCF-1 peptide substrate).

Currently, the Int-D and TPR exosite inhibitors are not yet suitable for cellular use. Future developments, such as improving the inhibitor properties and/or obtaining the complex structures of full-length OGT with exosite inhibitors, would greatly facilitate our understanding about the allosteric regulation of OGT, inspiring the rational design of next generation exosite modulators for broader applications.

Protein-specific O-GlcNAcylation in cells

Typically, manipulating the O-GlcNAcylation level on a target protein in cells can be achieved by mutating the O-GlcNAcylation sites. However, mutations may affect other PTMs on the same or neighboring residues. As an alternative approach, the non-catalytic domains of O-GlcNAc cycling enzymes can be conjugated with a nanobody to induce protein-specific O-GlcNAcylation or deglycosylation [51,52]. A similar concept has been applied to design bifunctional small molecules for targeted protein O-GlcNAcylation [53]. These approaches are straightforward, but it should be noted that the nanobody conjugation and bifunctional small molecules might induce altered PPIs or generate artificial neo-O-GlcNAcylation sites. Recently, RNA aptamers have been reported to selectively induce O-GlcNAcylation of OGT substrate β-catenin [54] (Figure 2e), a critical transcription factor controlling cell proliferation and differentiation [55]. Aptamers are single-stranded oligonucleotides that bind various biomolecules [56]. They exhibit advantages such as small size and accessibility of synthetic or chemical modifications [56]. In the report, two RNA aptamers recognizing either the OGT TPR domain or β-catenin were connected by a linker for simultaneous targeting (Figure 2e). The resulting dual-specificity aptamer, when expressed in cells, elevated the β-catenin O-GlcNAcylation by inducing its proximity with endogenous OGT. Advantageously, this approach eliminates the need to engineer endogenous proteins and is applicable for various OGT substrates. The extremely short half-life of the dual-specificity aptamer (minutes) also precludes the aptamer from prolonged target association that may influence downstream PPIs and global O-GlcNAcylation. The expression of dual-specificity aptamer could be further controlled by coupling with a riboswitch or inducible RNA antidote. Aside from the many notable advantages of this strategy, a few factors should be also taken into consideration: 1) the availability of substrate-binding aptamers; 2) the linker design that affects the targeting efficiency of aptamers through altered binding conformation or orientation; 3) the molar ratio of aptamer to its binding protein that affects the cellular efficacy and specificity; 4) like the nanobody-conjugation strategy, this dual-specificity aptamer cannot achieve site-specific O-GlcNAcylation; and 5) it may not be applicable for protein substrates that bind to the same site on OGT as the TPR aptamer. Nevertheless, these non-catalytic domain-based tools hold promise for broad applications in investigating OGT’s multifaceted roles, enlightening future innovations in developing function-specific or protein-selective modulators.

Non-catalytic stalk and pHAT domains of OGA

The investigation of OGA’s non-catalytic domains has been proceeding at a slower pace than OGT, primarily due to the missing information about the full-length OGA structure. However, recent biochemical and structural analyses on truncated OGAs have denoted the significance of non-catalytic domains in OGA functions.

While it remains challenging to attain the structure of full-length OGA, the crystal structures of truncated OGAs involving the catalytic domain and stalk domain excluding the IDR have been reported (e.g., OGAcryst in Figures 1c and 3) [29-31]. Unlike the OGA polypeptides shown in the full-length OGT complex (Figure 2b), these OGA structures, along with various in-solution analyses, consistently show that the crystallizable OGA constructs, as well as the full-length human OGA, are tightly homodimerized [29,31]. A series of truncated OGA structures in complex with distinct glycopeptide substrates [29,57], including a recent report using nonhydrolyzable glycopeptide (S-GlcNAc on a cysteine residue) [58], illustrate that the sugar binding conformations in OGA’s active site are nearly identical. On the other hand, the non-catalytic OGA stalk domain folds into a special α-helix bundle and collaborates with the catalytic domain to create “substrate-binding clefts” in the OGA dimer (Figure 3). This unique architecture not only enhances the dimerization and stability of OGA, but also allows distinct glycopeptides to form different sets of interactions with the “cleft” surface residues (Figure 3), revealing a potential mechanism of OGA in substrate discrimination. More interestingly, a cancer-derived single mutation on the OGA’s stalk domain was recently reported to dysregulate specific protein deglycosylation and fuel cell malignancy [59]. The extensive IDR in the same stalk domain also consists of potential hotspots and short liner motifs for PPIs [44,60]. Besides, the non-catalytic OGA pHAT domain was found essential for the interaction and deglycosylation of OGA substrates in cancer progression and DNA damage repair [22,24]. These discoveries imply OGA’s non-catalytic domains serving as important protein binding sites for regulating OGA’s diverse roles. While efforts are underway to investigate the OGA’s non-catalytic domains, studies on the full-length OGA and its protein complexes are expected to uncover new binding conformations and exosite regulations that will illuminate future development of function-specific modulators of OGA.

Figure 3.

Figure 3.

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The crystal structure of OGAcryst dimer in complex with O-GlcNAcylated p53 peptide (glycopeptide) substrate (PDB 5UN8). Zoom-in view shows the OGA substrate-binding cleft surface residues (highlighted in blue and magenta) in contact with p53 glycopeptide. The peptide part of the substrate is highlighted in orange and the GlcNAc modification is shown in red. The OGA’s catalytic domain and stalk domain are shown in pink and cyan, respectively.

Conclusion

O-GlcNAc cycling enzymes dynamically regulate the functions of thousands of proteins and their multifaceted roles are context dependent. The associations of various protein binders with OGT or OGA, especially through their non-catalytic domains, could be functionally specific. However, our understanding of how OGT and OGA harness these binding modules to elicit specific functions is still in the infancy. Recent discoveries about the new OGT binding modes and exosite modulators supported the unique features of OGT’s non-catalytic domains for functional regulation. As for OGA, its stalk domain- and pHAT domain-dependent PPIs or deglycosylation can be potentially leveraged for controlling distinct OGA functions. It is expected that complementary strategies integrating biochemistry, structure, proteomics, chemical biology, and bioinformatics will accelerate the mechanistic dissections of OGT and OGA in achieving their functional specificity. We envision that the new knowledge of PPI interface and allosteric regulation would facilitate the development of function-specific modulators of OGT/OGA for biological and pathophysiological studies.

Acknowledgement

We thank Drs. Jacques Lowe and Ziyong Hong in the Jiang lab for their helpful suggestions on the manuscript. This work was supported by National Institutes of Health (NIH) R01GM121718 and R01GM152998.

Footnotes

Declaration of competing interest

The authors declare no competing interests.

Data availability

No data was used for the research described in the article.

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