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O-GlcNAc: a regulatory post-translational modification
BBRC Biochemical and Biophysical Research Communications 302 (2003) 435–441 www.elsevier.com/locate/ybbrc
Breakthroughs and Views
O-GlcNAc: a regulatory post-translational modification Lance Wells, Stephen A. Whelan, and Gerald W. Hart* Department of Biological Chemistry, Johns Hopkins School of Medicine, 517 WBSB, 725 N. Wolfe St., Baltimore, MD 21205, USA Received 21 January 2003
Abstract b-N-Acetylglucosamine (O-GlcNAc) is a regulatory post-translational modification of nuclear and cytosolic proteins. The enzymes for its addition and removal have recently been cloned and partially characterized. While only about 80 mammalian proteins have been identified to date that carry this modification, it is clear that this represents just a small percentage of the modified proteins. O-GlcNAc has all the properties of a regulatory modification including being dynamic and inducible. The modification appears to modulate transcriptional and signal transduction events. There are also accruing data that O-GlcNAc plays a role in apoptosis and neurodegeneration. A working model is emerging that O-GlcNAc serves as a metabolic sensor that attenuates a cellÕs response to extracellular stimuli based on the energy state of the cell. In this review, we will focus on the enzymes that add/remove O-GlcNAc, the functional impact of O-GlcNAc modification, and the current working model for O-GlcNAc as a nutrient sensor. Ó 2003 Published by Elsevier Science (USA). Keywords: O-GlcNAc; Post-translational modification; Transcription; Signal transduction; Apoptosis; Neurodegeneration; Nutrient sensing; Diabetes; AlzheimerÕs disease; Glycosylation
In 1984, Torres and Hart described the presence of O-glycosidically linked GlcNAc monosaccharides on cell-surface proteins and suggested that the majority of O-GlcNAc modified proteins appeared to be within the cell [1]. A couple of years later, two independent laboratories described the existence of O-GlcNAc modified nucleocytoplasmic proteins [2,3]. Further work demonstrated that this post-translational modification (PTM) was both abundant and dynamic and occurred on a myriad of nucleocytoplasmic proteins (reviewed in [4–6], see Table 1). Thus, the dogma that glycosylated proteins were only secreted or associated with biological membranes was disproved [7]. In recent years, the nucleocytoplasmic enzymes for the addition (O-GlcNAc transferase, OGT) and removal (neutral b-N-acetylglucosaminidase, O-GlcNAcase) of O-GlcNAc have been cloned and characterized [8–13]. Recent work has not only expanded the list of modified proteins (see Table 1) but has begun to elucidate functions for O-GlcNAc (reviewed in [4–6], see Fig. 1). In this review, we will focus on the enzymes that add and remove O-GlcNAc and the * Corresponding author. Fax: 1-410-614-8804. E-mail address: [email protected] (G.W. Hart).
impact of O-GlcNAc modification on mammalian protein properties and functions.
The enzymes A soluble O-GlcNAc transferase (OGT) was first purified and characterized in 1990 [14] but it would be another seven years before the enzyme was cloned [8,9]. The 110 kDa polypeptide has two domains: an N-terminus with 11.5 tetratricopeptide repeats (TPRs) and a putative catalytic C-terminus. TPRs are known protein–protein association domains [15]. The enzyme functions as a trimer with the polypeptide chains interacting via the TPRs [10]. The enzyme transfers N-acetylglucosamine from UDP-GlcNAc to the hydroxyl oxygen of serine and threonines in a b confirmation [14]. Initial kinetic data showed that OGT is able to respond to a wide range of concentrations, including the known physiological range, of UDP-GlcNAc [10]. One possible explanation for these nearly unsaturatable UDP-GlcNAc kinetics is a PingPong mechanism for the enzyme, even though this hypothesis has yet to be tested. It was also established that the enzyme is tyrosine phosphorylated as well as being
0006-291X/03/$ - see front matter Ó 2003 Published by Elsevier Science (USA). doi:10.1016/S0006-291X(03)00175-X
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Table 1 Identified mammalian O-GlcNAc modified proteins Protien
Reference
Proof
Protein
Reference
Proof
OGT Sp-1 HNF-1 HCF EWS NF-jB p53 EIF2aAp67 EF1 Nup62 Nup54 K8/18/13 Annexin I Piccolo b-Synuclein APP Talin Neurofilament H,M,L Band 4.1 AnkyrinG Nucleophosmin Pyruvate Kinase Enolase UDPGP aB-Crystallin HSP90 ProteasomeC2 UCH homologue PYPp65 i2pp2a b-Catenin eNOS OIP-106 Glut-1
[8] [65] [67] [63] [63] [52] [68] [30] [63] [3] [63] [69] [63] [63] [39] [33] [70] [34] [71] [73] [63] [63] [63] [63] [60] [63] [63] [63] [75] [63] [50] [48] [17] [77]
b,c,e,h,k a–h e k k f b,e,g b–e,g,l k a–k k a–e k k b–e,i b,e,f b,c a–e a–e b–f k k k k a–e k k k c,e k e,f,i f,i f b,f
RNA Pol II SRF c-myc Oct1 ERa&b hhSEC23 EF-2D EIF4A1 40SrpS24 Nup155 Ran Synapsin I E-cadherin Dynein LC1 CRMP-2 Tau Clathrin AP-3 MAPS Cofillin a-Tubulin hnRNP p43 GAPDH PGK UGP-1 HSC70 PPI UCH-L1 GSK3b CKII RHO-GDI-a IRSs GRIF-1 p85PI3K GS
[64] [66] [25] [63] [23] [63] [63] [63] [63] [63] [63] [38] [50] [63] [39] [32] [36] [35] [63] [72] [74] [63] [63] [63] [63] [63] [39] [11] [10] [63] [76] [17] [49] [78]
a–i a, b a–i,k,l k a–i k k k k a,c,f k a–h e k b–e,i a–h b,c,e b,c k f b,e,f k k k k k b–e,i h a–h,k k f,k,i f f b,e,f,i
a, sites mapped; b, galactose labeling; c, b-elimination; d, PNGaseF resistant; e, WGA binding/blot; f, antibody blot; g, hexosaminidase sensitivity; h, in vitro OGT labeling; i, GlcNAc modulation; j, mass shift; k, O-GlcNAc antibody precipitation; l, protein/site-specific O-GlcNnAc antibody western. In several cases, several papers describe O-GlcNAc on a particular protein and we chose to cite only the initial manuscript. Abbreviations used (not described in the text) are the following: SRF, serum response factor; HCF, human factor C1; HNF, hepatocyte nuclear factor; EWS, EwingÕs Sarcoma protein; hhSEC23, human homologue of SEC23; ER, estrogen receptor; 40SRPS24, 40S ribosomal protein S24; NUP, nuclear pore protein; K8/18/13, keratins; EIF, elongation initiation factor; HSP, heat-shock protein; UCH, ubiquitin carboxy-hydrolase; APP, b-amyloid precursor protein; PYP, protein tyrosine phosphatase; i2pp2a, inhibitor-2 of protein phosphatase 2A; IRS, insulin-receptor substrate; GRIF, GABA receptor interacting factor; OIP, OGT interacting protein; UGP, UDP-Glucose pyrophosphorylase; GAPDH, glyceraldyde3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; Glut, glucose transporter; GS, glycogen synthase.
modified by O-GlcNAc itself [8]. The TPRs suggested that the enzyme might be interacting with other proteins and it was recently shown that OGT has several binding partners, including mSin3A, GRIF-1, and OIP106 which may be involved in targeting the enzyme to discrete intracellular locations [16,17]. The importance of OGT was confirmed by the fact that ablation of the gene is lethal in mouse embryonic stem cells [18]. This work also suggested that there was only one OGT in mammals. By analogy to RNA polymerase II that is regulated by its binding partners and state of modification, we postulate that OGT is regulated by localization, binding partners, and PTMs. Future work will focus on establishing the identity of other binding partners and PTMs and how they modulate OGT activity, stability, and localization.
A neutral, nucleocytoplasmic hexosaminidase activity was described in 1976 [19]. A nucleocytoplasmic, neutral, b-N-acetylglucosaminidase (O-GlcNAcase) that appears to be responsible for the previously described activity was purified and cloned in 2002 [12]. Unlike hexosaminidase A or B, O-GlcNAcase is localized to the cytosol and to a lesser degree the nucleus, has a neutral pH optimum, and does not catalyze the removal of nor is inhibited by GalNAc. Overexpression of O-GlcNAcase has been shown to lower global O-GlcNAc levels [13]. Interestingly, O-GlcNAcase is a substrate for the executioner apoptotic caspase-3 [13] and is cleaved during the induction of apoptosis in cells by treatment with cytotoxic lymphocyte granules. Surprisingly, cleavage of full-length 130 kDa O-GlcNAcase into 2
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Fig. 1. Functional impact O-GlcNAc modification. O-GlcNAc is transiently found on a multitude of various nuclear and cytoplasmic proteins resulting in diverse functions. The interaction of O-GlcNAc containing proteins in addition to phosphorylation increases the complexity of the cellular signaling events. O-GlcNAc may be serving as a metabolic sensor through the hexosamine pathway based on the availability of nutritional metabolites such as glucose and glucosamine. High levels of glucose and glucosamine results in elevated levels of UDP-GlcNAc and O-GlcNAc modification of proteins. Elevated O-GlcNAc modification of proteins in the insulin signaling pathway result in insulin resistance and inhibition of insulin-stimulated translocation of Glut4 to the plasma membrane. On the other hand, O-GlcNAc modification on tau may protect it from aggregating into neurofibrillary tangles. The O-GlcNAc modification on c-myc and Sp-1 appears to stabilize these transcription factors. Also, OGT activity and association with the mSinA corepressor complex is necessary for the maximum gene silencing. Note: Boxed G represents O-GlcNAc modification in figure.
approximately 65 kDa fragments has no effect on in vitro O-GlcNAcase activity. It is of interest to note that sequence analysis reveals that half of O-GlcNAcase has weak homology to histone acetyltransferases though no such activity for O-GlcNAcase has yet to be reported.
Transcription/translation A large number of transcription factors are modified by O-GlcNAc as well as the C-terminal domain (CTD) of RNA polymerase II [20]. Glycosylation of the CTD induces a conformational change in the CTD that could have a variety of functional consequences [21]. Recent data have shown that in vitro glycosylation of the CTD of RNA polymerase II prevents the required phosphor-
ylation for elongation [22]. Thus it has been proposed that O-GlcNAc may modify RNA polymerase II that is in the preinitiation complex or a storage form. On estrogen receptors a and b, c-myc, and Sp-1, glycosylation appears to stabilize the transcription factor [23–26]. Furthermore, Kudlow and co-workers [27] recently showed that increased glycosylation of Sp-1 inhibits its transactivation capability. Further, it has been reported that Sp-1 is O-GlcNAc modified in a cell-type specific manner. OGT was found to associate with the mSin3A corepressor complex and OGT activity is necessary for maximal gene silencing [16]. OGT expression and elevated levels of UDP-GlcNAc also seem to be involved in increased leptin gene transcription [28,29]. Thus, a model is emerging for O-GlcNAc regulating gene transcription (see Fig. 1) but the details of the mechanism and the
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impact on various promoters, transcription factors, and the regulation of this system remain to be investigated. There is also evidence to suggest that glycosylation plays a role in regulating translation via glycosylation of the eIF-2 associated p67 (eIF-2A) protein. O-GlcNAc modification of eIF-2A appears to protect the protein from degradation and promotes translation by binding eIF-2 and impeding the inhibitory phosphorylation of eIF-2 [30,31]. Interestingly, this system appears to be sensitive to the nutrient state of the cell and starvation results in the loss of glycosylated eIF-2A that culminates in the inhibition of translation [30,31].
Neurodegenration There is considerable indirect evidence that O-GlcNAc may play a role in neurodegenerative disorders. It is well established that glucose metabolism is reduced in the aging neurons. A reduction in glucose flux results in lower UDP-GlcNAc levels and presumably lower levels of O-GlcNAc modified proteins. There are a variety of O-GlcNAc modified proteins that are enriched in brain neurons including tau, b-amyloid precursor protein, neurofilaments, microtubule-associated proteins, clathrin assembly proteins (AP3 and AP180), synapsin I, collapsin response mediator protein-2 (CRMP-2), ubiquitin carboxyl hydrolase-L1 (UCH-L1), and bsynuclein [32–39]. Interestingly, OGT maps to the Xlinked Parkinson dystonia locus [18] and O-GlcNAcase maps to the AlzheimerÕs disease locus on chromosome 10 [13,40]. Also, several groups have demonstrated both a global as well as protein specific yin-yang relationship between phosphorylation and O-GlcNAc modification (that is when phosphorylation is elevated O-GlcNAc is reduced and vice versa, reviewed in [4,41]). The microtubule-associated protein tau is hyperphosphorylated when in neurofibrillary tangles [42], suggesting that the O-GlcNAc modification may protect tau from aggregation. Understanding the impact of O-GlcNAc on tau and the b-amyloid precursor protein as well as establishing any possible links between disease states and the enzymes OGT and O-GlcNAcase are questions that are currently being pursued by several laboratories.
Signal transduction While the attractive model of O-GlcNAc participating in signal transduction events has been proposed for more than a decade [41], only recently have data emerged implicating O-GlcNAc in specific signal transduction cascades. The hexosamine biosynthetic pathway (HSP), which converts fructose-6-phosphate to UDPGlcNAc (see Fig. 1), the donor sugar nucleotide for OGT, has been implicated in type II diabetes, specifi-
cally in insulin resistance and glucose toxicity [43,44]. Very compelling evidence for the HSP being involved in insulin resistance was generated in 1991 when Marshall et al. [43] showed that inhibition of this pathway prevented hyperglycemia-induced insulin resistance in peripheral tissue. Further work in 1998 by Yki-Jarvinen et al. [45] demonstrated that mice made insulin resistant had elevated O-GlcNAc levels in skeletal muscle. Thus, a clear correlation was established between elevated O-GlcNAc levels and insulin resistance. Using the 3T3L1 adipocyte cell line, we demonstrated that elevation of O-GlcNAc levels via inhibition of O-GlcNAcase with PUGNAc [46] resulted in a defect in insulin-stimulated glucose uptake [47]. Further, we were able to demonstrate that elevated O-GlcNAc levels inhibited the insulin-stimulated phosphorylation of AKT. This work was further supported a few months later when McClain et al. [29] demonstrated that transgenic mice overexpressing OGT in skeletal muscle and adipose tissue were mildly diabetic. Thus, there are both pharmacological and genetic evidence to suggest that elevation of O-GlcNAc levels results in insulin resistance associated with a defect in AKT activation. Lauro and colleagues also demonstrated a defect in insulin signaling and endothelial nitric oxide synthase (eNOS) activation attributed to elevated O-GlcNAc [48,49], further supporting a role for O-GlcNAc in the insulin signaling cascade. There have also been tantalizing hints that O-GlcNAc may be involved in other cascades such as the E-cadherin, b-catenin, PKA, and NF-jB pathways [47,50–52]. There is compelling evidence for O-GlcNAc being involved in glucose toxicity and apoptosis pathways. For example, O-GlcNAcase is cleaved by caspase3 [13], elevation/reduction of O-GlcNAc levels inhibits/ enhances activation of the anti-apoptotic AKT [47,53], and the HSP has been implicated in b-cell death and retinal neuron degeneration [54,55]. Future work will be aimed at not only determining what pathways O-GlcNAc is involved in but also in elucidating the molecular consequences of site-specific O-GlcNAc modification.
Working model and future directions The current nutritional sensor model of O-GlcNAc has been reviewed elsewhere [56]. Briefly, the model proposes that cells are not blindly responding to extracellular stimuli but instead are taking into account their own energy stores. O-GlcNAc, which appears to be highly responsive to nutrient state, modifies signaling components, cytoskeletal components, and the transcriptional and translational machinery. Thus, O-GlcNAc modification could be modulating the proteins that are present and their post-translational state and localization, so that they respond in an appropriate way to extracellular cues.
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Recent advancements in the field, including the development of general [57] and site-specific [58] O-GlcNAc antibodies, the cloning of OGT [8,9] and O-GlcNAcase [12], and the development of mass spectrometry-based and other techniques for protein identification and site-mapping [59–63], should accelerate the field. Future work will take advantage of genetic, biochemical, and pharmacological approaches to elucidate the impact of O-GlcNAc modification on specific proteins and pathways. The emerging picture is that O-GlcNAc, as well as other PTMs in addition to phosphorylation, are playing important dynamic roles in the function of proteins and the general biology of the cell.
Acknowledgments We thank members of the Hart laboratory and Karen M. Wells for critical reading of the manuscript. The ‘‘O-GlcNAc field’’ is rapidly expanding and thus it is likely that we failed to acknowledge the contributions of some of our colleagues (for this we apologize). This work was supported by NIH Grants CA42486, DK38418, and HD13563 to GWH.
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Breakthroughs and Views
O-GlcNAc: a regulatory post-translational modification Lance Wells, Stephen A. Whelan, and Gerald W. Hart* Department of Biological Chemistry, Johns Hopkins School of Medicine, 517 WBSB, 725 N. Wolfe St., Baltimore, MD 21205, USA Received 21 January 2003
Abstract b-N-Acetylglucosamine (O-GlcNAc) is a regulatory post-translational modification of nuclear and cytosolic proteins. The enzymes for its addition and removal have recently been cloned and partially characterized. While only about 80 mammalian proteins have been identified to date that carry this modification, it is clear that this represents just a small percentage of the modified proteins. O-GlcNAc has all the properties of a regulatory modification including being dynamic and inducible. The modification appears to modulate transcriptional and signal transduction events. There are also accruing data that O-GlcNAc plays a role in apoptosis and neurodegeneration. A working model is emerging that O-GlcNAc serves as a metabolic sensor that attenuates a cellÕs response to extracellular stimuli based on the energy state of the cell. In this review, we will focus on the enzymes that add/remove O-GlcNAc, the functional impact of O-GlcNAc modification, and the current working model for O-GlcNAc as a nutrient sensor. Ó 2003 Published by Elsevier Science (USA). Keywords: O-GlcNAc; Post-translational modification; Transcription; Signal transduction; Apoptosis; Neurodegeneration; Nutrient sensing; Diabetes; AlzheimerÕs disease; Glycosylation
In 1984, Torres and Hart described the presence of O-glycosidically linked GlcNAc monosaccharides on cell-surface proteins and suggested that the majority of O-GlcNAc modified proteins appeared to be within the cell [1]. A couple of years later, two independent laboratories described the existence of O-GlcNAc modified nucleocytoplasmic proteins [2,3]. Further work demonstrated that this post-translational modification (PTM) was both abundant and dynamic and occurred on a myriad of nucleocytoplasmic proteins (reviewed in [4–6], see Table 1). Thus, the dogma that glycosylated proteins were only secreted or associated with biological membranes was disproved [7]. In recent years, the nucleocytoplasmic enzymes for the addition (O-GlcNAc transferase, OGT) and removal (neutral b-N-acetylglucosaminidase, O-GlcNAcase) of O-GlcNAc have been cloned and characterized [8–13]. Recent work has not only expanded the list of modified proteins (see Table 1) but has begun to elucidate functions for O-GlcNAc (reviewed in [4–6], see Fig. 1). In this review, we will focus on the enzymes that add and remove O-GlcNAc and the * Corresponding author. Fax: 1-410-614-8804. E-mail address: [email protected] (G.W. Hart).
impact of O-GlcNAc modification on mammalian protein properties and functions.
The enzymes A soluble O-GlcNAc transferase (OGT) was first purified and characterized in 1990 [14] but it would be another seven years before the enzyme was cloned [8,9]. The 110 kDa polypeptide has two domains: an N-terminus with 11.5 tetratricopeptide repeats (TPRs) and a putative catalytic C-terminus. TPRs are known protein–protein association domains [15]. The enzyme functions as a trimer with the polypeptide chains interacting via the TPRs [10]. The enzyme transfers N-acetylglucosamine from UDP-GlcNAc to the hydroxyl oxygen of serine and threonines in a b confirmation [14]. Initial kinetic data showed that OGT is able to respond to a wide range of concentrations, including the known physiological range, of UDP-GlcNAc [10]. One possible explanation for these nearly unsaturatable UDP-GlcNAc kinetics is a PingPong mechanism for the enzyme, even though this hypothesis has yet to be tested. It was also established that the enzyme is tyrosine phosphorylated as well as being
0006-291X/03/$ - see front matter Ó 2003 Published by Elsevier Science (USA). doi:10.1016/S0006-291X(03)00175-X
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Table 1 Identified mammalian O-GlcNAc modified proteins Protien
Reference
Proof
Protein
Reference
Proof
OGT Sp-1 HNF-1 HCF EWS NF-jB p53 EIF2aAp67 EF1 Nup62 Nup54 K8/18/13 Annexin I Piccolo b-Synuclein APP Talin Neurofilament H,M,L Band 4.1 AnkyrinG Nucleophosmin Pyruvate Kinase Enolase UDPGP aB-Crystallin HSP90 ProteasomeC2 UCH homologue PYPp65 i2pp2a b-Catenin eNOS OIP-106 Glut-1
[8] [65] [67] [63] [63] [52] [68] [30] [63] [3] [63] [69] [63] [63] [39] [33] [70] [34] [71] [73] [63] [63] [63] [63] [60] [63] [63] [63] [75] [63] [50] [48] [17] [77]
b,c,e,h,k a–h e k k f b,e,g b–e,g,l k a–k k a–e k k b–e,i b,e,f b,c a–e a–e b–f k k k k a–e k k k c,e k e,f,i f,i f b,f
RNA Pol II SRF c-myc Oct1 ERa&b hhSEC23 EF-2D EIF4A1 40SrpS24 Nup155 Ran Synapsin I E-cadherin Dynein LC1 CRMP-2 Tau Clathrin AP-3 MAPS Cofillin a-Tubulin hnRNP p43 GAPDH PGK UGP-1 HSC70 PPI UCH-L1 GSK3b CKII RHO-GDI-a IRSs GRIF-1 p85PI3K GS
[64] [66] [25] [63] [23] [63] [63] [63] [63] [63] [63] [38] [50] [63] [39] [32] [36] [35] [63] [72] [74] [63] [63] [63] [63] [63] [39] [11] [10] [63] [76] [17] [49] [78]
a–i a, b a–i,k,l k a–i k k k k a,c,f k a–h e k b–e,i a–h b,c,e b,c k f b,e,f k k k k k b–e,i h a–h,k k f,k,i f f b,e,f,i
a, sites mapped; b, galactose labeling; c, b-elimination; d, PNGaseF resistant; e, WGA binding/blot; f, antibody blot; g, hexosaminidase sensitivity; h, in vitro OGT labeling; i, GlcNAc modulation; j, mass shift; k, O-GlcNAc antibody precipitation; l, protein/site-specific O-GlcNnAc antibody western. In several cases, several papers describe O-GlcNAc on a particular protein and we chose to cite only the initial manuscript. Abbreviations used (not described in the text) are the following: SRF, serum response factor; HCF, human factor C1; HNF, hepatocyte nuclear factor; EWS, EwingÕs Sarcoma protein; hhSEC23, human homologue of SEC23; ER, estrogen receptor; 40SRPS24, 40S ribosomal protein S24; NUP, nuclear pore protein; K8/18/13, keratins; EIF, elongation initiation factor; HSP, heat-shock protein; UCH, ubiquitin carboxy-hydrolase; APP, b-amyloid precursor protein; PYP, protein tyrosine phosphatase; i2pp2a, inhibitor-2 of protein phosphatase 2A; IRS, insulin-receptor substrate; GRIF, GABA receptor interacting factor; OIP, OGT interacting protein; UGP, UDP-Glucose pyrophosphorylase; GAPDH, glyceraldyde3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; Glut, glucose transporter; GS, glycogen synthase.
modified by O-GlcNAc itself [8]. The TPRs suggested that the enzyme might be interacting with other proteins and it was recently shown that OGT has several binding partners, including mSin3A, GRIF-1, and OIP106 which may be involved in targeting the enzyme to discrete intracellular locations [16,17]. The importance of OGT was confirmed by the fact that ablation of the gene is lethal in mouse embryonic stem cells [18]. This work also suggested that there was only one OGT in mammals. By analogy to RNA polymerase II that is regulated by its binding partners and state of modification, we postulate that OGT is regulated by localization, binding partners, and PTMs. Future work will focus on establishing the identity of other binding partners and PTMs and how they modulate OGT activity, stability, and localization.
A neutral, nucleocytoplasmic hexosaminidase activity was described in 1976 [19]. A nucleocytoplasmic, neutral, b-N-acetylglucosaminidase (O-GlcNAcase) that appears to be responsible for the previously described activity was purified and cloned in 2002 [12]. Unlike hexosaminidase A or B, O-GlcNAcase is localized to the cytosol and to a lesser degree the nucleus, has a neutral pH optimum, and does not catalyze the removal of nor is inhibited by GalNAc. Overexpression of O-GlcNAcase has been shown to lower global O-GlcNAc levels [13]. Interestingly, O-GlcNAcase is a substrate for the executioner apoptotic caspase-3 [13] and is cleaved during the induction of apoptosis in cells by treatment with cytotoxic lymphocyte granules. Surprisingly, cleavage of full-length 130 kDa O-GlcNAcase into 2
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Fig. 1. Functional impact O-GlcNAc modification. O-GlcNAc is transiently found on a multitude of various nuclear and cytoplasmic proteins resulting in diverse functions. The interaction of O-GlcNAc containing proteins in addition to phosphorylation increases the complexity of the cellular signaling events. O-GlcNAc may be serving as a metabolic sensor through the hexosamine pathway based on the availability of nutritional metabolites such as glucose and glucosamine. High levels of glucose and glucosamine results in elevated levels of UDP-GlcNAc and O-GlcNAc modification of proteins. Elevated O-GlcNAc modification of proteins in the insulin signaling pathway result in insulin resistance and inhibition of insulin-stimulated translocation of Glut4 to the plasma membrane. On the other hand, O-GlcNAc modification on tau may protect it from aggregating into neurofibrillary tangles. The O-GlcNAc modification on c-myc and Sp-1 appears to stabilize these transcription factors. Also, OGT activity and association with the mSinA corepressor complex is necessary for the maximum gene silencing. Note: Boxed G represents O-GlcNAc modification in figure.
approximately 65 kDa fragments has no effect on in vitro O-GlcNAcase activity. It is of interest to note that sequence analysis reveals that half of O-GlcNAcase has weak homology to histone acetyltransferases though no such activity for O-GlcNAcase has yet to be reported.
Transcription/translation A large number of transcription factors are modified by O-GlcNAc as well as the C-terminal domain (CTD) of RNA polymerase II [20]. Glycosylation of the CTD induces a conformational change in the CTD that could have a variety of functional consequences [21]. Recent data have shown that in vitro glycosylation of the CTD of RNA polymerase II prevents the required phosphor-
ylation for elongation [22]. Thus it has been proposed that O-GlcNAc may modify RNA polymerase II that is in the preinitiation complex or a storage form. On estrogen receptors a and b, c-myc, and Sp-1, glycosylation appears to stabilize the transcription factor [23–26]. Furthermore, Kudlow and co-workers [27] recently showed that increased glycosylation of Sp-1 inhibits its transactivation capability. Further, it has been reported that Sp-1 is O-GlcNAc modified in a cell-type specific manner. OGT was found to associate with the mSin3A corepressor complex and OGT activity is necessary for maximal gene silencing [16]. OGT expression and elevated levels of UDP-GlcNAc also seem to be involved in increased leptin gene transcription [28,29]. Thus, a model is emerging for O-GlcNAc regulating gene transcription (see Fig. 1) but the details of the mechanism and the
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impact on various promoters, transcription factors, and the regulation of this system remain to be investigated. There is also evidence to suggest that glycosylation plays a role in regulating translation via glycosylation of the eIF-2 associated p67 (eIF-2A) protein. O-GlcNAc modification of eIF-2A appears to protect the protein from degradation and promotes translation by binding eIF-2 and impeding the inhibitory phosphorylation of eIF-2 [30,31]. Interestingly, this system appears to be sensitive to the nutrient state of the cell and starvation results in the loss of glycosylated eIF-2A that culminates in the inhibition of translation [30,31].
Neurodegenration There is considerable indirect evidence that O-GlcNAc may play a role in neurodegenerative disorders. It is well established that glucose metabolism is reduced in the aging neurons. A reduction in glucose flux results in lower UDP-GlcNAc levels and presumably lower levels of O-GlcNAc modified proteins. There are a variety of O-GlcNAc modified proteins that are enriched in brain neurons including tau, b-amyloid precursor protein, neurofilaments, microtubule-associated proteins, clathrin assembly proteins (AP3 and AP180), synapsin I, collapsin response mediator protein-2 (CRMP-2), ubiquitin carboxyl hydrolase-L1 (UCH-L1), and bsynuclein [32–39]. Interestingly, OGT maps to the Xlinked Parkinson dystonia locus [18] and O-GlcNAcase maps to the AlzheimerÕs disease locus on chromosome 10 [13,40]. Also, several groups have demonstrated both a global as well as protein specific yin-yang relationship between phosphorylation and O-GlcNAc modification (that is when phosphorylation is elevated O-GlcNAc is reduced and vice versa, reviewed in [4,41]). The microtubule-associated protein tau is hyperphosphorylated when in neurofibrillary tangles [42], suggesting that the O-GlcNAc modification may protect tau from aggregation. Understanding the impact of O-GlcNAc on tau and the b-amyloid precursor protein as well as establishing any possible links between disease states and the enzymes OGT and O-GlcNAcase are questions that are currently being pursued by several laboratories.
Signal transduction While the attractive model of O-GlcNAc participating in signal transduction events has been proposed for more than a decade [41], only recently have data emerged implicating O-GlcNAc in specific signal transduction cascades. The hexosamine biosynthetic pathway (HSP), which converts fructose-6-phosphate to UDPGlcNAc (see Fig. 1), the donor sugar nucleotide for OGT, has been implicated in type II diabetes, specifi-
cally in insulin resistance and glucose toxicity [43,44]. Very compelling evidence for the HSP being involved in insulin resistance was generated in 1991 when Marshall et al. [43] showed that inhibition of this pathway prevented hyperglycemia-induced insulin resistance in peripheral tissue. Further work in 1998 by Yki-Jarvinen et al. [45] demonstrated that mice made insulin resistant had elevated O-GlcNAc levels in skeletal muscle. Thus, a clear correlation was established between elevated O-GlcNAc levels and insulin resistance. Using the 3T3L1 adipocyte cell line, we demonstrated that elevation of O-GlcNAc levels via inhibition of O-GlcNAcase with PUGNAc [46] resulted in a defect in insulin-stimulated glucose uptake [47]. Further, we were able to demonstrate that elevated O-GlcNAc levels inhibited the insulin-stimulated phosphorylation of AKT. This work was further supported a few months later when McClain et al. [29] demonstrated that transgenic mice overexpressing OGT in skeletal muscle and adipose tissue were mildly diabetic. Thus, there are both pharmacological and genetic evidence to suggest that elevation of O-GlcNAc levels results in insulin resistance associated with a defect in AKT activation. Lauro and colleagues also demonstrated a defect in insulin signaling and endothelial nitric oxide synthase (eNOS) activation attributed to elevated O-GlcNAc [48,49], further supporting a role for O-GlcNAc in the insulin signaling cascade. There have also been tantalizing hints that O-GlcNAc may be involved in other cascades such as the E-cadherin, b-catenin, PKA, and NF-jB pathways [47,50–52]. There is compelling evidence for O-GlcNAc being involved in glucose toxicity and apoptosis pathways. For example, O-GlcNAcase is cleaved by caspase3 [13], elevation/reduction of O-GlcNAc levels inhibits/ enhances activation of the anti-apoptotic AKT [47,53], and the HSP has been implicated in b-cell death and retinal neuron degeneration [54,55]. Future work will be aimed at not only determining what pathways O-GlcNAc is involved in but also in elucidating the molecular consequences of site-specific O-GlcNAc modification.
Working model and future directions The current nutritional sensor model of O-GlcNAc has been reviewed elsewhere [56]. Briefly, the model proposes that cells are not blindly responding to extracellular stimuli but instead are taking into account their own energy stores. O-GlcNAc, which appears to be highly responsive to nutrient state, modifies signaling components, cytoskeletal components, and the transcriptional and translational machinery. Thus, O-GlcNAc modification could be modulating the proteins that are present and their post-translational state and localization, so that they respond in an appropriate way to extracellular cues.
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Recent advancements in the field, including the development of general [57] and site-specific [58] O-GlcNAc antibodies, the cloning of OGT [8,9] and O-GlcNAcase [12], and the development of mass spectrometry-based and other techniques for protein identification and site-mapping [59–63], should accelerate the field. Future work will take advantage of genetic, biochemical, and pharmacological approaches to elucidate the impact of O-GlcNAc modification on specific proteins and pathways. The emerging picture is that O-GlcNAc, as well as other PTMs in addition to phosphorylation, are playing important dynamic roles in the function of proteins and the general biology of the cell.
Acknowledgments We thank members of the Hart laboratory and Karen M. Wells for critical reading of the manuscript. The ‘‘O-GlcNAc field’’ is rapidly expanding and thus it is likely that we failed to acknowledge the contributions of some of our colleagues (for this we apologize). This work was supported by NIH Grants CA42486, DK38418, and HD13563 to GWH.
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