Emerging roles of O-GlcNAcylation in protein trafficking and secretion

Abstract

The emerging roles of O-GlcNAcylation, a distinctive post-translational modification, are increasingly recognized for their involvement in the intricate processes of protein trafficking and secretion. This modification exerts its influence on both conventional and unconventional secretory pathways. Under healthy and stress conditions, such as during diseases, it orchestrates the transport of proteins within cells, ensuring timely delivery to their intended destinations. O-GlcNAcylation occurs on key factors, like coat protein complexes (COPI and COPII), clathrin, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), and GRASP55 (Golgi reassembly stacking protein of 55 kDa) that control vesicle budding and fusion in anterograde and retrograde trafficking and unconventional secretion. The understanding of O-GlcNAcylation offers valuable insights into its critical functions in cellular physiology and the progression of diseases, including neurodegeneration, cancer, and metabolic disorders. In this review, we summarize and discuss the latest findings elucidating the involvement of O-GlcNAc in protein trafficking and its significance in various human disorders.

Overview of O-GlcNAcylation, a unique glycosylation

The human genome contains approximately 20,000 to 25,000 encoding genes, and the complexity of the human proteome is at least two orders of magnitude greater than the genome. The high complexity of the proteome arises from several factors, including alternative mRNA splicing; protein synthesis, folding, and turnover; cell/tissue-specific expression; protein–protein interactions and complex formation; and, in particular, protein trafficking and subcellular localization, along with post-translational modifications (PTMs). For example, compared with distinct promoter sequences and alternative splicing of mRNA molecules that contribute to proteome complexity and increase it by about 5-fold, protein PTMs amplify proteome complexity by more than 100-fold (1). Over 350 distinct PTMs have been identified (2). Protein glycosylation is the most prevalent PTM, involving more than 200 glycosyltransferases and glycan-processing enzymes (3), which are compartmentalized in the endoplasmic reticulum (ER) and the Golgi apparatus (4). These enzymes determine which proteins become glycoproteins, dictate the positions of glycans on those proteins, and orchestrate the assembly of the glycan and protein structures. Protein glycosylation plays a crucial role in protein folding, trafficking, and degradation. In fact, more than 85% secretory proteins undergo protein glycosylation in a sequential and concerted order within the ER and Golgi apparatus (4).

Protein glycosylation can be broadly classified into two types, N-linked glycosylation and O-linked glycosylation. N-glycosylation involves the attachment of GlcNAc to the nitrogen atom of an Asn side chain at Asn-x-Ser/Thr motifs, whereas O-linked glycosylation entails the attachment of the glycan to the oxygen atom of the amino acid Ser or Thr side chains (5, 6). The N-glycosylation pathway initiates in the ER, where a preformed high-mannose oligosaccharide precursor is added to nascent polypeptide chain, accounting for over 90% of all glycosylation events. Furthermore, protein O-glycosylation can be classified based on the attached sugar type, including O-GalNAcylation, O-GlcNAcylation, O-fucosylation, O-mannosylation, and O-glucosylation (7, 8). Two most abundant types of O-glycosylation in proteins are the GalNAc type, primarily occurring in the Golgi apparatus and often referred to as mucin-type O-glycosylation because of its enrichment in mucin proteins, and the GlcNAc type, which occurs in the nucleus, cytoplasm, and mitochondria (9, 10).

O-GlcNAcylation is a prevalent PTM and a noncanonical glycosylation (10). Over 15,000 proteins across 43 species have been identified with O-GlcNAc modifications (11). O-GlcNAcylation modulates protein synthesis, localization, stability, and protein–protein interaction at the molecular level. Crosstalk between O-GlcNAcylation and other PTMs significantly increases the complexity of protein regulation. For example, O-GlcNAcylation and phosphorylation often occur reciprocally or sequentially on the same or neighboring residues of numerous proteins, as both modify Ser and Thr residues (12, 13, 14).

A distinct feature that differs O-GlcNAcylation from most other PTMs is that a single pair of enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), are responsible for adding and removing the monosaccharide O-GlcNAc on protein substrates (10, 15). O-GlcNAcylation serves as a nutrient-sensing mechanism in cells. When cellular glucose levels are elevated, the flux of glucose through the hexosamine biosynthetic pathway increases. This pathway produces the precursor molecule UDP-GlcNAc, which is essential for O-GlcNAcylation. As a result, higher glucose concentrations lead to an enhanced supply of UDP-GlcNAc, increasing O-GlcNAcylation of proteins (10). The nutrient-sensing aspect of O-GlcNAcylation is particularly significant because it allows cells to rapidly adapt to fluctuations in nutrient availability (16, 17). This PTM enables cells to sense nutrient abundance and make real-time adjustments in various cellular processes (14). In addition to nutrient sensing, O-GlcNAcylation participates in a variety of biological processes, including gene transcription, epigenetic modifications, signal transduction, stress response, metabolic homeostasis, and immune response (10, 11).

The conventional and unconventional secretory pathways

Protein trafficking and secretion, fundamental processes within cells that contribute to the increased complexity and functional specificity of the human proteome, are pivotal for maintaining cellular homeostasis and facilitating various physiological functions (18). Protein trafficking involves the movement of proteins within the cell, from their site of synthesis (e.g., ER) to various cellular destinations such as specific organelles like endosomes and lysosomes (19). Conversely, protein secretion entails the controlled release of proteins from the cell to the extracellular environment. Furthermore, proteins located at the cell surface or in the extracellular environment can also be subject to endocytosis. Proper protein trafficking, secretion, and endocytosis are vital because they govern critical cellular functions, including cell signaling, immune responses, enzymatic activities, and structural integrity. Dysregulation in these processes can lead to a wide range of diseases, including neurodegenerative disorders, cancer, and metabolic conditions (20, 21, 22).

There are two major pathways governing protein secretion, primarily determined by whether the process involves the Golgi and vesicle formation. In the classical secretory pathway, secretory and membrane proteins are synthesized in the ER and subsequently transported by coat protein complex II (COPII) vesicles to the Golgi, where the cargo molecules are processed, concentrated, and packaged for delivery to various destinations, including endosomes and lysosomes, or secreted into the extracellular space (ER→Golgi→plasma membrane) (Fig. 1) (20). During this process, cargo molecules are shuttled between different membrane structures by membrane-coated vesicles (23, 24). This intricate process ensures the accurate sorting, modification, and transportation of proteins to their designated locations. Secretory proteins, such as antibodies, hormones, growth factors, digestive enzymes, and extracellular matrix proteins, play vital roles in cellular physiology.

Figure 1.

O-GlcNAcylation regulates ER–Golgi trafficking in the conventional secretory pathway. Three components of the COPII coat (Sec31, Sec23, and Sec24) are modified with O-GlcNAc, and this modification affects COPII vesicle budding at the ER exit site (ERES) and ER-to-Golgi trafficking. The COPγ1 subunit of the COPI coat is also O-GlcNAcylated, but its role in Golgi-to-ER trafficking requires further investigation. COPI, coat protein complex I; COPII, coat protein complex II; ER, endoplasmic reticulum.

In contrast to conventional protein secretion, some proteins can be secreted via a Golgi-independent unconventional protein secretion pathway (25, 26, 27, 28, 29). Certain cytoplasmic proteins are synthesized in the cytosol and, because of the lack of an ER signal sequence, do not enter the conventional secretory pathway. Instead, they are secreted directly across the plasma membrane or via a Golgi-independent, autophagy-dependent, and stress-induced unconventional protein secretion pathway (cytosol→autophagosomes→extracellular space) (30). This latter pathway involves the transport of cytoplasmic proteins from the cytosol to autophagosomes and, ultimately, to the extracellular space. Numerous cytosolic proteins have been shown to be secreted via this pathway, including Acb1 in yeast (31, 32), Udp2 in Drosophila fat cells (33), IL-1β, IL-18, TGFB1, and HMGB1 in mammalian macrophages (34, 35), and insulin-degrading enzyme in Alzheimer’s disease (AD) neurons (36). In addition, some aggregative proteins associated with neurodegeneration, such as huntingtin, SOD1, α-synuclein, tau, and TDP-43 (TAR DNA–binding protein 43), are secreted via this pathway (Fig. 2) (37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48).

Figure 2.

De-O-GlcNAcylation of GRASP55 and SNAP-29 facilitates autophagosome–lysosome fusion under starvation. Under growth condition, the Golgi stacking protein GRASP55, but not its homolog GRASP65, is O-GlcNAcylated. Upon starvation, GRASP55 becomes de-O-GlcNAcylated and relocates from the Golgi to autophagosome–lysosome interface, where the de-O-GlcNAcylated form of GRASP55 acts as a membrane tether, facilitating autophagosome–lysosome fusion by bridging LC3 and LAMP2. In addition, SNAP-29 also undergoes dynamic O-GlcNAcylation, and when de-GlcNAcylated, it promotes the formation of SNARE complexes, thereby facilitating the fusion of autophagosomes with lysosomes. Whether there is any communication between GRASP55 and SNAP-29 under stressful conditions remains an intriguing and unanswered question. GRASP55, Golgi reassembly stacking protein of 55 kDa.

Recent remarkable findings reveal that O-GlcNAcylation plays an important role in regulating both conventional and unconventional secretory pathways. O-GlcNAcylation occurs directly on some key subunits of the protein complexes involved in generating COPII, COPI, and clathrin-coated vesicles (CCVs) (12). Furthermore, some proteins in autophagy and unconventional secretory pathways are reported to be regulated by O-GlcNAcylation. Considering that the level of O-GlcNAcylation is directly influenced by the available energy in cells, alterations in O-GlcNAcylation reflect a stress response in which cells must adjust their activities, including intracellular trafficking, which plays essential roles in cell physiology.

In this review, we summarize recent advances in the role of O-GlcNAcylation in protein trafficking and secretion as well as its implications in diseases. Instead of providing a comprehensive list of all proteins known to be modified by O-GlcNAc, which can be readily accessed in online databases and proteomic studies (13, 49), and extensively reviewed elsewhere (12, 15), we concentrate on a select set of key proteins and their functions in both conventional and unconventional secretion.

Novel roles of O-GlcNAcylation in conventional protein trafficking

In this section, we delve into well-established examples of O-GlcNAcylation significantly influencing essential components of intracellular vesicle transport systems, particularly within the classical exocytic and endocytic pathways involving COPI, COPII, and clathrin-coated vesicles. Furthermore, we investigate how this modification extends its influence on specific cargo proteins, illuminating the multifaceted realm of O-GlcNAcylation in cellular processes (Fig. 1).

O-GlcNAcylation of COPII coat proteins regulates ER-to-Golgi trafficking

Since the first description of O-GlcNAcylation of intracellular proteins in 1984 by Torres and Hart (50), O-GlcNAc has been widely observed on cytosolic proteins or nuclear proteins, with limited information available regarding its role in membrane proteins and proteins involved in membrane trafficking. In the conventional or biosynthetic secretory pathway, membrane and secreted proteins are synthesized in the ER and transported through the Golgi to their targeted destinations. The functional role of O-GlcNAc on protein trafficking in the conventional secretory pathway remains largely unknown, but increasing evidence shows that many proteins responsible for protein trafficking are modified and regulated by O-GlcNAcylation (Fig. 1).

The first compelling evidence that O-GlcNAcylation plays a role in regulating protein trafficking came from the work of Paccaud et al. in 2004 (51). They discovered that a crucial component of the COPII vesicle coat, Sec24, undergoes cytosolic O-GlcNAcylation during interphase, a characteristic that diminishes during mitosis. The COPII coat machinery consists of five cytosolic proteins: Sar1, Sec23, and Sec24 (inner coat), Sec13 and Sec31 (outer coat) (52, 53). In this study, no similar modification was observed for other COPII components, such as Sec23, Sec13, Sec31, or Sar1. This mitotic deglycosylation of Sec24 was found to be associated with Sec24 phosphorylation, resulting in an increase in the apparent molecular weight of these molecules on SDS-PAGE. Remarkably, these modifications were correlated with changes in the membrane-binding properties of Sec24. These findings suggest that as cells enter mitosis, the COPII component Sec24p undergoes simultaneous deglycosylation and phosphorylation, which likely inhibits COPII coat assembly and contributes to the observed block in ER-to-Golgi trafficking during mitosis (51).

In recent research, all five core components of COPII vesicles were systematically examined, revealing that three of them, Sec23, Sec31, and Sec24, are subject to O-GlcNAcylation (54, 55, 56). Notably, O-GlcNAcylation induced by Thiamet-G, an OGA inhibitor, accelerated the trafficking of GFP-tagged tsVSV-G, a temperature-sensitive mutant of the vesicular stomatitis virus glycoprotein tagged with green fluorescent protein, through the secretory pathway. Mechanistically, it was discovered that O-GlcNAcylation of Sec31A on Ser964 enhances tsVSV-G trafficking by promoting COPII vesicle formation at the ER exit site, a specific ER domain on which COPII vesicles form (54). Furthermore, O-GlcNAc modification of Sec31A reduces its interaction with ALG-2, a calcium-binding protein known to interact with Sec31 and inhibit COPII vesicle formation (57, 58). The study indicated that O-GlcNAcylation of Sec31A effectively decreases its binding with ALG-2, thereby facilitating COPII vesicle formation and ER-to-Golgi trafficking in mammalian cells. However, in a parallel study conducted by Boyce et al. (55), it was shown that O-GlcNAcylation induced by Thiamet-G markedly delayed COPII-dependent tsVSV-G-eGFP trafficking. These two studies used HeLa cells transiently transfected with tsVSV-G-eGFP and COS7 cells stably expressing tsVSV-G-eGFP, respectively, which may account for the observed discrepancy in the role of O-GlcNAcylation in ER-to-Golgi trafficking. Therefore, a systematic investigation of the effects of O-GlcNAcylation on ER-to-Golgi trafficking using different cargoes in various cell lines may help resolve this discrepancy.

In mammalian cells, the Sec23 protein family consists of two isoforms, Sec23A and Sec23B, whereas the Sec24 protein family is composed of four isoforms, Sec24A, Sec24B, Sec24C, and Sec24D. Boyce et al. (55) mapped O-GlcNAcylated sites of overexpressed proteins by mass spectrometry identified at least 26, 11, and 10 O-GlcNAc sites on Sec23A, Sec24C, and Sec31A, respectively. Importantly, they established that O-GlcNAcylation of Sec23A is essential for its proper functioning in both human cells and vertebrate development. Mutations affecting these O-GlcNAcylation sites impaired collagen trafficking and skeletogenesis in a zebrafish model (55), underscoring the conservation and significance of O-GlcNAc as a regulatory modification in the vertebrate COPII-dependent trafficking pathway. Furthermore, a combination of O-GlcNAc- and GlcNAc-specific antibodies and lectins (G5-lectibody column), along with stable isotope labeling using amino acids in cell culture labeling, expanded the list of O-GlcNAcylated proteins within the COPII coat proteins, including Sec23A, Sec23B, Sec24A, Sec24B, Sec24C, Sec24D, Sec13, and Sec31A (59).

Boyce et al. (56) further investigated the role of O-GlcNAcylation in the COPII complex and identified new O-GlcNAcylation sites on core COPII components, particularly Sec24C, Sec24D, and Sec31A. This study yielded some interesting findings. For example, rapamycin, an inhibitor of the mammalian target of rapamycin complexes known for inducing autophagy, was found to induce O-GlcNAcylation of Sec24C. It was also observed that LC3 cleavage was increased in Sec24C knockout cells, suggesting a potential interaction between Sec24 O-GlcNAcylation and the autophagic process. These findings imply that site-specific glycosylation within the COPII complex can influence nutrient-sensitive regulatory pathways, revealing a previously unknown connection between cellular metabolism and vesicle trafficking modulated by O-GlcNAcylation (56). Mutation of Sec31A O-GlcNAcylation site Ser1202 reduces Sec31A and Sec13 interaction, suggesting that Sec31A O-GlcNAcylation at Ser964 and Ser1202 may have similar effects in COPII formation at the ER exit site. Unexpected, Sec31A O-GlcNAcylation was substantially increased under low glucose condition, which is quite different from most O-GlcNAcylation events and requires more future exploration.

O-GlcNAcylation of COPI coat protein regulates retrograde trafficking

COPI vesicles mediate retrograde transport of proteins within the Golgi apparatus and trafficking from the Golgi apparatus to the ER. The COPI coat is composed of seven subunits (α-, β-, β′-, γ-, δ-, ϵ-, and ζ-COP subunits) (Fig. 1). Unlike COPII vesicles, the COPI pathway does not seem to be directly involved in transporting large-size protein cargoes (60). To investigate whether O-GlcNAcylation also regulates COPI-mediated trafficking, Boyce et al. (61) employed a novel glycoproteomics approach they developed, utilizing chemical biology and mass spectrometry to profile stimulus-induced changes in O-GlcNAcylated proteins.

In contrast to the widespread O-GlcNAcylation observed in multiple COPII subunits, only one subunit of COPI, COPγ1, undergoes O-GlcNAcylation. Furthermore, COPγ1 is found to be O-GlcNAcylated at 11 sites under normal conditions, and this glycosylation is significantly reduced under brefeldin A (BFA) treatment (61). BFA disrupts COPI coat assembly and halts COPI-mediated trafficking. These findings suggest that O-GlcNAcylation may regulate intra-Golgi and/or retrograde Golgi-to-ER protein trafficking within the COPI pathway, akin to its established roles in COPII-mediated anterograde ER-to-Golgi trafficking. Unlike other studies that often rely on glucose starvation or OGA inhibition, the use of BFA should not significantly affect the cellular levels of UDP-GlcNAc or OGA activity, thus enabling further investigations into the functional consequences of COPγ1 O-GlcNAcylation on COPI coat assembly. While the exact impact of COPγ1 O-GlcNAcylation on COPI coat assembly, its regulation, and effects on intracellular trafficking remain to be elucidated, these discoveries shed light on the potential role of O-GlcNAcylation in governing various protein trafficking processes within the cell, expanding its known impact from COPII- to COPI-mediated pathways.

Based on the O-GlcNAcome databases (The O-GlcNAc Database v1.3 and O-GlcNAcAtalas_3.0), apart from COPγ1, several other proteins involved in COPI-mediated trafficking also undergo O-GlcNAc modification. These proteins encompass ArfGAP1, ArfGAP3, and GBF1, which are implicated in COPI vesicle formation through the regulation of Arf1 activity (12). In addition, Sec22B, a vesicle-associated soluble N-ethylmaleimide-sensitive factor attachment protein receptor (v-SNARE) protein that facilitates both anterograde and retrograde vesicular transport between the ER and Golgi apparatus, is also subject to O-GlcNAcylation (12). However, comprehensive investigations regarding the validation, regulation, and biological significance of O-GlcNAcylation on these proteins are still pending.

O-GlcNAcylation of endosomal sorting complex required for transport proteins regulates endosome trafficking

The role of O-GlcNAcylation extends beyond the secretory pathway, reaching into the endocytic pathway and endosome trafficking, where it exerts a significant impact on cellular processes. One excellent example of this influence is its regulation of the epidermal growth factor receptor (EGFR), a prototypic receptor tyrosine kinase with a pivotal role in mediating cellular responses to extracellular cues. Upon ligand stimulation, EGFR initiates signaling cascades that control fundamental cellular processes, including cell growth, proliferation, and survival. Dysregulation of EGFR signaling is closely associated with various diseases, notably cancer (62).

A crucial aspect of EGFR regulation involves its internalization and subsequent lysosomal degradation, serving as a key mechanism to terminate EGFR signaling. Interestingly, O-GlcNAcylation regulates EGFR intracellular trafficking and signaling. The degradation process of EGFR is orchestrated by the endosomal sorting complex required for transport (ESCRT). A pivotal player within ESCRT-0 is hepatocyte growth factor–regulated tyrosine kinase substrate (HGS). Remarkably, HGS, a key component in EGFR intracellular sorting, is dynamically modified by O-GlcNAc. This modification exerts profound effects on EGFR trafficking within the cell (63). Specifically, O-GlcNAcylation of HGS impedes the efficient trafficking of EGFR from endosomes to lysosomes, leading to an accumulation of EGFR within the endosomal system and thus prolonging EGFR signaling (63). Furthermore, this modification increases HGS ubiquitination and reduces its protein stability, resulting in the accumulation of EGFR and prolonged signaling. In addition, this study demonstrates that HGS glycosylation promotes tumor growth and chemoresistance, emphasizing the impact of O-GlcNAcylation on various cellular processes (63).

Another example is PD-L1, where O-GlcNAcylation impairs the lysosomal degradation of PD-L1 and promotes tumor evasion. PD-L1 is a key molecule in immune regulation. Like EGFR, internalized PD-L1 from the cell surface is also degraded by lysosomes. O-GlcNAcylation of HGS disrupts its interaction with internalized PD-L1, impairing the lysosomal degradation of PD-L1. This ultimately leads to sustained PD-L1 expression in cancer cells and promotes immune evasion by tumor cells (64). Inhibiting O-GlcNAcylation can activate T-cell-mediated antitumor immune responses, and this approach, when combined with PD-L1 antibody therapy, synergistically enhances antitumor immune responses (64).

These findings collectively underscore the profound implications of O-GlcNAcylation in the context of endosome trafficking, revealing its critical role in cellular processes and its potential as a therapeutic target in various diseases. By modulating the interplay between endosomal proteins, O-GlcNAcylation plays a vital role in controlling the fate of proteins like EGFR and PD-L1, with far-reaching implications in cellular physiology and diseases.

O-GlcNAcylation of clathrin adaptor protein complex-1

Adaptor protein complex-1 (AP-1) is a vital player in cellular cargo sorting, particularly in the formation of CCVs at the interface between the trans-Golgi network and endosomes. AP-1 efficiently packages specific membrane proteins into CCVs for their further processing. Two intriguing proteins, γ-synergin and p34, have been identified as key AP-1 binding partners through a yeast two-hybrid screening (65). Of particular interest is γ-synergin, which binds directly to γ-adaptin and is highly concentrated in CCVs. This protein was identified as a target of O-GlcNAcylation through mass spectrometry–based assays (66). In addition to γ-synergin, both clathrin light chain B and clathrin heavy chain 1 are also O-GlcNAcylated (12, 67). While this modification is intriguing, the implications of O-GlcNAcylation of these proteins in the formation and trafficking of CCVs remain to be fully explored (65).

O-GlcNAcylation of cargo proteins

In addition to COPI, COPII coats, and other trafficking proteins, some cargo molecules are also modified by O-GlcNAc. One example is alanine, serine, cysteine transporter 2 (ASCT2), also known as SLC1A5. ASCT2 is a vital transporter responsible for the uptake of glutamine, a crucial nutrient in various cellular processes. A study conducted by Wang et al. (68) revealed that ASCT2 is dynamically modified by O-GlcNAc at Thr122, which significantly impacts its stability and trafficking to the cell surface. The importance of this finding is underscored by its role in liver fibrogenesis. Liver fibrosis, often triggered by excessive activation of hepatic stellate cells (HSCs), can have severe consequences. In fibrotic mouse livers and activated HSCs, ASCT2 interacts with OGT, leading to elevated ASCT2 O-GlcNAcylation at Thr122, which stabilizes ASCT2 and contributes to HSC activation. This modification also plays a role in mediating the trafficking of ASCT2 within HSCs, ultimately contributing to liver fibrogenesis (68).

Many other cargo molecules are also modified by O-GlcNAc. For example, the cell adhesion molecule E-cadherin is O-GlcNAcylated in thapsigargin-induced apoptosis, blocking its cell surface transport and resulting in reduced intercellular adhesion (69). Another example is the glutamate receptor AMPAR (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor); O-GlcNAcylation regulates its subcellular distribution and modulates hippocampal synaptic transmission (70). In addition, decreasing O-GlcNAc level stimulates AMPAR translocation of GluA2 from the cytosol toward the plasma membrane (71). While numerous cargo proteins have been shown to undergo O-GlcNAcylation, further investigations are needed to fully understand the functional impact of this PTM.

O-GlcNAcylation of synapsin I modulates synaptic plasticity

Synaptogenesis is the complex process of synapse formation, a pivotal mechanism in establishing connections between neurons within the brain, spinal cord, and between neurons and muscle cells. Synapsin I, one of the most abundant neuronal phosphoproteins, plays a crucial role in regulating synaptogenesis and subsequently the release of neurotransmitters from mature nerve terminals by tethering synaptic vesicles to the actin cytoskeleton, forming a reserve pool (72). Traditionally, it was well known that the binding of synapsin I to actin and synaptic vesicles was primarily regulated by phosphorylation. Two studies provided insights into the effects of O-GlcNAcylation on synapsin I. Cole and Hart (73) identified seven O-GlcNAcylation sites on synapsin I. These O-GlcNAc sites cluster around synapsin I's five phosphorylation sites in domains B and D. This close proximity suggests that O-GlcNAcylation might modulate phosphorylation, thereby affecting synapsin I interactions. While further studies are required to fully elucidate the precise role of O-GlcNAcylation, the existing evidence suggests that it plays a direct and critical role in the intricate network of interactions involving synapsin I (73).

Subsequently, Skorobogatko et al. (74) discovered that O-GlcNAcylation of synapsin I during hippocampal synaptogenesis in rats had a profound impact on its function. The study identified three novel O-GlcNAc sites on synapsin I, two of which coincided with known Ca²⁺/calmodulin-dependent protein kinase II phosphorylation sites. These sites were found within the regulatory regions of synapsin I. To demonstrate the significance of this modification, the researchers mutated a single O-GlcNAc site, Thr87, to alanine in primary hippocampal neurons. This mutation led to a dramatic increase in the localization of synapsin I at synapses, a higher density of synaptic vesicle clusters along axons, and an increase in the size of the reserve pool of synaptic vesicles. These results suggest that O-GlcNAcylation of synapsin I at Thr87 plays a crucial role in modulating presynaptic plasticity, particularly by influencing synapsin I localization and function (74).

Collectively, these studies underscore the significant role of O-GlcNAcylation in regulating the intricate machinery of synaptogenesis, highlighting its potential in modulating synaptic plasticity and brain function.

O-GlcNAcylation, a coordinator between Golgi structure formation, autophagy, and unconventional secretion

In this section, we explore the evolving landscape of O-GlcNAcylation and its increasingly recognized roles in two vital cellular processes: autophagy and unconventional secretion. The intricate interplay between O-GlcNAcylation, Golgi structure formation, and autophagosome–lysosome fusion, as well as conventional and unconventional secretion (Fig. 2), is the key focus of this section. These cellular mechanisms highlight the multifaceted nature of O-GlcNAcylation's regulatory influence within the cell. By delving into the connection between O-GlcNAcylation, autophagy, and unconventional secretion, we aim to gain deeper insights into how this dynamic modification orchestrates fundamental aspects of cellular homeostasis and intercellular communication.

GRASP55 senses glucose deprivation through O-GlcNAcylation to promote autophagosome–lysosome fusion

The Golgi apparatus plays a pivotal role in fundamental cellular functions, such as membrane trafficking, processing, and sorting (75). In mammalian cells, it comprises a series of flattened membrane-bound sacs known as cisternae, which are often laterally linked together to form a continuous ribbon-like structure (75). This distinctive Golgi structure is essential for its proper functioning in intracellular trafficking and the modification of proteins and lipids within the conventional secretory pathway. While the exact molecular mechanisms underlying Golgi stack formation remain enigmatic, the Golgi reassembly stacking proteins, GRASP65 (Golgi reassembly stacking protein of 65 kDa) and GRASP55 (Golgi reassembly stacking protein of 55 kDa), are the only proteins known to participate in Golgi stacking to date (76, 77, 78, 79, 80, 81, 82, 83). Depletion of GRASP proteins leads to Golgi cisternal unstacking and accelerated membrane trafficking (18, 78, 84, 85, 86, 87). Such Golgi structural perturbations result in inaccurate N- and O-glycosylation (85, 88), mis-sorting and secretion of lysosomal enzymes (85), alterations in cellular glycolipid profiles (78), and impact various cellular activities like cell attachment, migration, and growth (89, 90).

Recent research has unveiled a fascinating aspect of GRASP55. Under normal growth conditions, GRASP55 undergoes O-GlcNAcylation, whereas glucose starvation leads to its de-O-GlcNAcylation. This de-O-GlcNAcylation prompts GRASP55 to relocate from the Golgi to the interface between autophagosomes and lysosomes (91, 92, 93, 94). In this new location, GRASP55 plays a pivotal role by interacting with LC3-II and LAMP2, acting as a membrane tether between autophagosomes and lysosomes to facilitate their fusion (Fig. 2) (92). Furthermore, GRASP55 also interacts with Beclin-1, promoting the assembly and membrane association of the PI3K complex II (94), a crucial facilitator of autophagosome maturation. As a result, depletion of GRASP55 leads to the accumulation of autophagosomes, indicating its role in mediating autophagosome maturation, which is regulated by nutrient availability. This study thus reveals an unexpected dual role of GRASP55, serving as both an energy sensor through O-GlcNAcylation, and a membrane tether, effectively promoting autophagosome–lysosome fusion (Fig. 2) (92).

De-O-GlcNAcylation of SNAP-29 facilitates autophagosome–lysosome fusion

The fusion between autophagosomes and lysosomes, a crucial step in the autophagic process, is orchestrated by a group of proteins known as SNAREs. These include syntaxin 17, SNAP-29, and VAMP8 or VAMP7. Notably, SNAP-29, a pivotal component within this fusion machinery, undergoes O-GlcNAcylation (95). Under growth conditions, SNAP-29 is O-GlcNAcylated at multiple sites, including Ser2, Ser61, Thr130, and Ser153. However, in both mammalian cells and Caenorhabditis elegans, the level of SNAP-29 O-GlcNAcylation is significantly reduced during nutrient starvation. When de-O-GlcNAcylated, SNAP-29 undergoes conformational change, forming a more robust and stable 4-helix bundle with Syntaxin 17 and VAMP8 (95). This structural change is significant because it enhances the efficiency of autophagosome–lysosome fusion (Fig. 2). In essence, de-O-GlcNAcylated SNAP-29 becomes a more proficient mediator, contributing to the seamless fusion of autophagosomes and lysosomes (95). In addition to SNAP-29, Rab11-interacting protein 5 (RABFIP5), another protein essential for endosomal trafficking through its interaction with the Rab11 GTPase, is also subjected to O-GlcNAcylation (96). However, the biological significance of this PTM has not been thoroughly investigated.

Interestingly, it seems that energy deprivation enhances autophagosome–lysosome fusion by inducing the deglycosylation of both GRASP55 and SNAP-29. However, whether GRASP55 interacts with SNAP-29 under conditions of growth or energy deprivation remains a compelling subject for future investigation.

De-O-GlcNAcylation of SNAP-23 promotes exosome release

Exosomes, small membrane-bound vesicles released by various cell types into the extracellular environment, serve as crucial messengers in cell-to-cell communication. They transport an array of molecules, including proteins, nucleic acids (such as DNA, RNA, and microRNAs), and lipids. Exosome release, a fundamental cellular process, is orchestrated by SNARE proteins, which include SNAP-23, a target-SNARE primarily located at the plasma membrane. SNAP-23 plays a pivotal role in fusing cellular membrane vesicles with the plasma membrane, releasing their cargo into the extracellular space. Intriguingly, SNAP-23 undergoes O-GlcNAc modification. Notably, reduced O-GlcNAcylation of SNAP-23 promotes the formation of a complex involving SNAP-23, syntaxin 4 (Stx4), and VAMP8, leading to an increase in exosome release (Fig. 3). This enhanced exosome release holds particular significance in the context of cisplatin resistance, a phenomenon observed in ovarian cancer (97). These exosomes facilitate the efflux of cisplatin from cancer cells, ultimately contributing to chemoresistance. The mechanism by which cisplatin is selectively secreted by exosomes and the implications of this process are not yet understood. However, the decreased intracellular concentration of cisplatin contributes to its reduced effectiveness in fighting cancer in this context (97).

Figure 3.

O-GlcNAcylation modulates exosome secretion. In this illustration, we depict the dual role of O-GlcNAcylation in modulating exosome secretion. On one hand, de-O-GlcNAcylated SNAP-23 accelerates exosome release through the formation of the VAMP8–SNAP-23–STX-4 SNARE complex, influencing the process at a global level. Conversely, O-GlcNAcylation of cargo proteins, including αB-crystallin and hnRNPA2B1, facilitates their exosome secretion. The precise mechanisms by which O-GlcNAcylation governs exosome secretion in different cellular contexts or scenarios warrant further investigation to elucidate the seemingly contrasting effects.

O-GlcNAcylation promotes exosome release of cargo proteins

α-Crystallin, a major lens protein comprising up to 40% of total lens proteins, is also known as a member of the small heat shock protein family and expressed in many nonlens tissues. In the lens, α-crystallin consists of αA to αB subunits at a ratio of 3:1. Hart et al. (98) identified that αB-crystallin was modified by O-GlcNAc at Thr170. Because of the lack of a classic ER signal peptide, αB-crystallin is secreted into extracellular space by exosome (99). The exosome packaging and secretion of αB-crystallin in glioma cells relies on both O-GlcNAcylation and dephosphorylation (Fig. 3) (100). O-GlcNAcylation is also necessary for the exosome release of the heterogeneous nuclear ribonucleoprotein hnRNPA2B1. In brief, O-GlcNAcylation of hnRNPA2B1 induced by oxidative stress altered miRNA binding repertoire, enhanced its interaction with phosphorylated caveolin-1, and promoted exosome release of hnRNPA2B1 and caveolin-1 complex (Fig. 3) (101).

It seems controversial that de-O-GlcNAcylation of SNAP-23 promotes exosome release by facilitating SNARE complex formation, whereas O-GlcNAcylation accelerated exosome packaging and release of αB-crystallin and hnRNPA2B1. It is possible that different cell lines may contribute to the opposite conclusions because de-O-GlcNAcylation of SNAP-23 was performed in ovarian cancer cells, whereas exosome packaging and release of αB-crystallin and hnRNPA2B1 were conducted in glioma and lung epithelial cells, respectively. To resolve this issue, it is necessary to test both SNARE complex formation and cargo packaging into exosomes in the same system. In addition, to better understand how O-GlcNAcylation regulates exosome-mediated secretion, more substrates need to be investigated.

O-GlcNAcylation modulates unconventional secretion of galectins

Galectins, a class of cytoplasmic proteins known for their specific binding to cell surface glycans, are crucial mediators of extracellular functions, particularly clathrin-independent endocytosis. While there are a total of 15 galectins found in mammals, humans possess 12 distinct galectin proteins, and these glycan-binding molecules are present across various organisms (102). An intriguing connection emerges between O-GlcNAcylation and galectin secretion, a nonclassical secretory process that is not yet fully understood.

Dynamic O-GlcNAcylation, manipulated by altering OGA or OGT levels in HeLa cells, or through the use of OGA knockout mice, has been shown to modulate galectin-3 secretion, influencing clathrin-independent endocytosis (16). In these studies, clathrin-independent endocytosis of CD59 was found to increase upon disrupting O-GlcNAc cycling by knockdown of OGT or OGA, whereas clathrin-mediated endocytosis of transferrin remained unaffected. In addition, in HL-60 cells, exposure to all-trans retinoic acid and the GFPT inhibitor 6-diazo-5-oxo-l-norleucine, both of which induce cellular differentiation while concurrently decreasing the O-GlcNAcylation of intracellular proteins, results in elevated secretion of galectin-1, -9, and -10 (103).

Although the underlying mechanisms remain unknown, these findings demonstrate that O-GlcNAcylation actively regulates the unconventional secretion of galectins. These glycan-binding proteins play a pivotal role in the intricate interplay between nutrient sensing and cellular responses. These studies further underscore the multifaceted role of O-GlcNAcylation in cellular physiology (16, 103).

Aberrant O-GlcNAcylation in diseases and potential therapeutical strategies

Aberrant O-GlcNAcylation has emerged as a significant player in the pathogenesis of various diseases. Dysregulation of O-GlcNAc cycling has been linked to numerous conditions, including diabetes, cancer, neurodegenerative disorders, and cardiovascular diseases. The precise molecular mechanisms and specific protein targets underlying these associations are subjects of ongoing research, with the potential to uncover critical insights into disease progression and open new therapeutic avenues. Understanding how O-GlcNAcylation contributes to disease states offers a unique opportunity for the development of targeted interventions aimed at restoring the delicate balance of O-GlcNAcylation in afflicted tissues. In this context, exploring potential therapeutic strategies to manipulate O-GlcNAcylation levels presents an exciting frontier in biomedical research with the promise of novel treatments and improved disease management.

O-GlcNAcylation inhibits tau aggregation in AD

In recent years, there has been a growing number of research focus on the role of O-GlcNAcylation in the context of AD. Numerous studies have explored how this PTM influences key proteins, including the amyloid precursor protein (APP), which is involved in the development of AD. AD is characterized by the presence of two classes of highly insoluble and densely packed filament structures in patient brains: extracellular amyloid plaques of amyloid-β (Aβ) peptides and intraneuronal neurofibrillary tangles of tau (104). Aβ peptides are proteolytic fragments of the transmembrane protein APP, whereas tau is a brain-specific and axon-enriched microtubule-associated protein. Notably, both APP and tau are modified by O-GlcNAc moieties (104).

In AD, tau is hyperphosphorylated compared with normal adult brain tau. Bovine brain tau, isolated in 1996, was reported to have multiple O-GlcNAc sites (105). A critical point of interest is the concept of site-specific or stoichiometric changes in O-GlcNAcylation. The stoichiometry and occupancy of O-GlcNAcylation sites on tau are not yet fully defined. While tau splicing variants are observed to be multiply O-GlcNAcylated, the number of sites occupied appears to exceed 12, implying substoichiometric occupancy at any given site (105). This is an intriguing aspect of O-GlcNAcylation's regulation and warrants further investigation. Understanding the site-specific nature of O-GlcNAcylation on tau, as well as how it is orchestrated across different sites and variants, may offer deeper insights into its functional implications in neurodegenerative diseases.

Certain research suggests that O-GlcNAcylation may serve a protective function by inhibiting the hyperphosphorylation of tau protein (105, 106). This protective role aligns with the notion that increased O-GlcNAcylation might mitigate tau hyperphosphorylation and, consequently, reduce neurofibrillary tangle formation, which contributes to cognitive decline in AD (105, 106, 107, 108, 109). Furthermore, studies indicate that modulation of O-GlcNAcylation can yield beneficial effects in animal models of neurodegenerative diseases. For instance, elevating O-GlcNAcylation in a tau transgenic mouse model has been associated with the prevention of tau aggregation and reduced neuronal cell loss (110, 111, 112, 113, 114). These findings suggest that O-GlcNAcylation may play a role in mitigating the progression of tauopathies.

A recent proteomic analysis of mouse brain tissue treated with Thiamet-G unveiled 278 O-GlcNAcylated proteins in the cortex, with 65 proteins displaying over 1.5-fold increased O-GlcNAcylation levels in response to Thiamet-G treatment (115). This diverse landscape of O-GlcNAcylated proteins emphasizes the broad-reaching potential of O-GlcNAcylation in the realm of neurodegenerative disorders like AD. However, it remains uncertain how O-GlcNAcylation exerts these effects at the molecular level and whether it directly influences the aggregation of tau or involves downstream pathways. Furthermore, besides tau, other neurogenerative proteins, including TDP-43 (116) and α-synuclein (117), are also known to be modified by O-GlcNAc. Additional research is essential to delineate these mechanisms, ultimately help us understand the therapeutic potential of O-GlcNAcylation in the context of AD and other neurodegenerative disorders.

O-GlcNAcylation inhibits APP amyloidogenic processing in AD

APP is a transmembrane protein that plays a central role in the pathogenesis of AD. In the brain, APP is cleaved by enzymes in two main pathways: the amyloidogenic pathway and the nonamyloidogenic pathway. The amyloidogenic pathway results in the production of Aβ peptides, known for their aggregation into toxic oligomers and plaques in the AD-affected brain. In contrast, the nonamyloidogenic pathway produces soluble and less harmful APP fragments. Several groups have studied the intricate relationship between O-GlcNAcylation and APP processing. O-GlcNAcylation emerges as a potential guardian against neurotoxic Aβ production by reducing APP amyloidogenic processing and inhibition of APP endocytosis from the plasma membrane (118, 119, 120, 121). These findings indicate that O-GlcNAcylation restrains endocytosis of APP by translocating it from lipid raft microdomains to nonraft regions in the plasma membrane (122). Increasing O-GlcNAcylation, either through pharmacological inhibition of OGA or insulin treatment, promotes nonamyloidogenic APP processing, thereby reducing AD pathology (122). This regulation, triggered by insulin signaling, offers a novel mechanism to reduce Aβ production.

Furthermore, researchers are also interested in understanding the interplay between tau protein and APP O-GlcNAcylation. The hyperphosphorylation of tau, another prominent feature of AD that is discussed previously, is known to be influenced by O-GlcNAcylation. It is suggested that increased O-GlcNAcylation might mitigate tau hyperphosphorylation, and this, in turn, may influence APP processing. This intricate web of interactions between tau, APP, and O-GlcNAcylation raises compelling questions about their collective role in AD pathogenesis (110). However, the exact mechanisms underlying how O-GlcNAcylation regulates APP trafficking and processing remain a subject of ongoing research.

De-O-GlcNAcylated GRASP55 facilitates the unconventional secretion of mutant huntingtin

Follow-up research on GRASP55 O-GlcNAcylation has elucidated the pivotal role of GRASP55 in governing autophagy-dependent unconventional secretion and the proteotoxic aggregation of cytosolic proteins, as exemplified by mutant huntingtin (mHtt) (37). Unconventional secretion of mHtt is GRASP55 and autophagy dependent and is enhanced under stress conditions such as glucose starvation and ER stress. Depletion of GRASP55 reduced mHtt secretion, which can be rescued by expressing GRASP55 in GRASP55 knockout (55KO) cells (92). Expression of WT GRASP55 increased mHtt secretion in 55KO cells; whereas F37A, a mutant that is defective in autophagy, failed to do so, indicating that both GRASP55 and autophagy function in the same pathway in mHtt secretion. The O-GlcNAcylation-deficient mutant 5A (S389A, S390A, T403A, T404A, and T413A) (92) also rescued mHtt secretion, indicating that de-O-GlcNAcylation of GRASP55 is required for unconventional secretion.

Mechanistically, GRASP55 facilitates mHtt secretion by tethering autophagosomes to lysosomes to promote autophagosome maturation and subsequent lysosome secretion (Fig. 2) and by stabilizing p23/TMED10, a channel for translocation of cytoplasmic proteins into the lumen of the ER–Golgi intermediate compartment (37). Moreover, GRASP55 levels are upregulated by various stresses to facilitate unconventional secretion, whereas inhibition of mHtt secretion by GRASP55 KO enhances mHtt aggregation and toxicity. In conclusion, this study defines the pathway of GRASP55-mediated unconventional protein secretion and provides important insights into the progression of Huntington’s disease (37).

This dual role of GRASP55, both as a sensor responding to nutrient availability through O-GlcNAcylation and as a vital tether facilitating Golgi cisternal stacking and autophagosome–lysosome fusion, underscores the intricate interplay between GRASP55, Golgi structure, autophagy, and unconventional secretion (29). These findings hold substantial implications for unraveling the mechanisms underpinning neurodegenerative diseases, with a particular focus on conditions like Huntington's disease, characterized by protein misfolding and aggregation as defining features. Future investigations should delve into whether the secretion and aggregation of other neurodegenerative proteins, such as tau, TDP-43, and α-synuclein, are similarly regulated by deglycosylated GRASP55.

O-GlcNAcylation and cancer

The interplay between O-GlcNAcylation and cancer is a multifaceted area of research encompassing various aspects of tumorigenesis and progression. One intriguing facet of this relationship is the role of O-GlcNAcylation in promoting tumor immune evasion, a fundamental mechanism that cancer cells employ to evade the host immune system. Studies have shown that O-GlcNAcylation facilitates this immune evasion by inhibiting the lysosomal degradation of PD-L1. The stabilization of PD-L1 via O-GlcNAcylation enables cancer cells to avoid immune surveillance, thereby fostering tumor growth (64). This mechanism illustrates how O-GlcNAcylation, a seemingly subtle modification of proteins, can have profound implications in the context of cancer progression.

On the flip side, O-GlcNAcylation has also been implicated in the development of resistance to chemotherapy, a significant challenge in cancer treatment. For instance, in ovarian cancer, reduced O-GlcNAcylation of SNAP-23 has been linked to cisplatin resistance (97). The reduction in O-GlcNAcylation levels appears to induce exosome secretion, leading to the extracellular release of factors that support drug resistance. This dual role of O-GlcNAcylation, promoting immune evasion while potentially inducing chemoresistance, underscores its complex and context-dependent nature in the landscape of cancer.

Moreover, O-GlcNAcylation's influence on various signaling pathways, transcription factors, and oncogenes has sparked interest in its role as an oncogenic driver (for more detailed reviews, see Refs. (123, 124, 125, 126, 127)). This modification's impact on gene expression and signaling cascades can contribute to the proliferation, survival, and metastatic potential of cancer cells. Researchers are actively investigating potential therapeutic interventions aimed at targeting O-GlcNAcylation to enhance immune responses against cancer cells and sensitize them to chemotherapy. Understanding the intricate web of O-GlcNAcylation's involvement in cancer is essential for advancing our knowledge of cancer biology and developing novel therapeutic strategies in the ongoing battle against this complex disease.

O-GlcNAcylation and metabolic disorders

The relationship between O-GlcNAcylation and metabolic disorders is a subject of increasing interest in the field of medical research. O-GlcNAcylation plays a pivotal role in regulating glucose and lipid metabolism. Dysregulation of this process is closely associated with various metabolic disorders, including diabetes and obesity (128, 129).

One significant aspect of O-GlcNAcylation's involvement in metabolic disorders is its influence on insulin signaling (130, 131). Proper insulin signaling is essential for maintaining glucose homeostasis in the body. Studies have revealed that elevated O-GlcNAcylation of insulin signaling proteins, including insulin receptor substrate and protein kinase B (Akt), can lead to insulin resistance (132). This impairs the ability of cells to respond to insulin, ultimately resulting in elevated blood glucose levels, a hallmark of diabetes. Therefore, O-GlcNAcylation can act as a molecular switch, where increased levels tip the balance toward insulin resistance and contribute to the development of type 2 diabetes (133).

Furthermore, O-GlcNAcylation has been linked to the regulation of key metabolic enzymes, such as AMP-activated protein kinase and peroxisome proliferator–activated receptor gamma (134, 135, 136, 137). AMP-activated protein kinase is a cellular energy sensor that helps maintain energy balance by promoting glucose uptake and fatty acid oxidation. In contrast, peroxisome proliferator–activated receptor gamma is a transcription factor that plays a crucial role in adipocyte differentiation and lipid storage. Dysregulated O-GlcNAcylation of these enzymes can disrupt metabolic processes, potentially leading to imbalances in glucose and lipid metabolism. This disruption is highly relevant in the context of obesity, where abnormal lipid accumulation and insulin resistance are common features (128).

Conclusion and perspectives

The emerging roles of O-GlcNAcylation in protein trafficking and secretion have expanded our understanding of this intricate PTM and its multifaceted impact on cellular processes. O-GlcNAcylation of key factors like COPI, COPII, clathrin, SNAREs, and GRASP55 underscore its pivotal role in protein trafficking. Mounting evidence indicates that O-GlcNAcylation acts as a pivotal regulator of protein trafficking, secretion, exosome release, and endocytosis, impacting both conventional and unconventional pathways.

In the conventional trafficking pathway, O-GlcNAcylation finely tunes protein transport by modifying COPI, COPII, AP-1, and SNARE proteins, optimizing their functions in vesicular trafficking, secretion, and endocytosis. By influencing these critical molecular effectors, O-GlcNAcylation plays a central role in ensuring the timely delivery of cargo proteins to their designated destinations in response to different cellular stress and stimuli. The significance of this regulation also extends to the endocytic pathways, where O-GlcNAcylation participates in the internalization of cell surface receptors and the regulation of downstream signaling events. Its influence on canonical secretory routes, such as Golgi dynamics, vesicular transport, exocytosis, and endocytosis, highlights its central role in maintaining cellular homeostasis.

Simultaneously, the role of O-GlcNAcylation in unconventional trafficking pathways sparks growing interest. Here, its interaction with GRASP55, a Golgi stacking protein, modulates unconventional protein secretion. Similarly, de-O-GlcNAcylation of SNAP-29 also promotes autophagosome–lysosome fusion. In addition, the role of O-GlcNAcylation in regulating exosome secretion highlights its involvement in intercellular communication, with potential implications in tissue repair and disease progression.

The understanding of O-GlcNAcylation offers insights into its critical functions in both normal cellular physiology and pathological conditions, including neurodegenerative disorders, cancer, and metabolic diseases. Notably, the modification appears to have both protective and detrimental effects in human diseases. For instance, it plays a protective role in AD by inhibiting APP amyloidogenic processing and tau aggregation. Conversely, in cancer, O-GlcNAcylation can promote tumor immune evasion while potentially inducing chemoresistance. In metabolic disorders, it contributes to insulin resistance and imbalances in glucose and lipid metabolism.

Looking ahead, there are several paths that encourage further investigation into how O-GlcNAcylation regulates protein trafficking and secretion. First, further elucidation of the precise molecular mechanisms and pathways underlying the effects of O-GlcNAcylation is imperative. Second, the therapeutic potential of targeting O-GlcNAcylation in the treatment of diseases linked to protein trafficking abnormalities holds promise. However, this potential must be balanced with a comprehensive understanding of the global impact of O-GlcNAcylation on other cellular processes.

In conclusion, the roles of O-GlcNAcylation in protein trafficking and secretion represent a dynamic area of research with far-reaching implications for human health. As our understanding of this PTM continues to evolve, it offers exciting possibilities for advancements in the fields of biology, medicine, and therapeutics.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Reviewed by members of the JBC Editorial Board. Edited by Robert Haltiwanger