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 en-
zymes 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 cell�sresponse 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.
� 2003 Published by Elsevier Science (USA).
Keywords: O-GlcNAc; Post-translational modification; Transcription; Signal transduction; Apoptosis; Neurodegeneration; Nutrient sensing;
Diabetes; Alzheimer�s disease; Glycosylation
In 1984, Torres and Hart described the presence of
O-glycosidically linked GlcNAc monosaccharides oncell-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 labo-
ratories described the existence of O-GlcNAc modified
nucleocytoplasmic proteins [2,3]. Further work demon-
strated 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 mem-
branes was disproved [7]. In recent years, the nucleocy-
toplasmic enzymes for the addition (O-GlcNAc
transferase, OGT) and removal (neutral b-N-acetylglu-cosaminidase, 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
impact of O-GlcNAc modification on mammalian
protein properties and functions.
The enzymes
A soluble O-GlcNAc transferase (OGT) was first pu-
rified and characterized in 1990 [14] but it would be an-
other seven years before the enzyme was cloned [8,9]. The
110 kDa polypeptide has two domains: an N-terminuswith 11.5 tetratricopeptide repeats (TPRs) and a putative
catalytic C-terminus. TPRs are known protein–protein
association domains [15]. The enzyme functions as a tri-
mer 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 ofconcentrations, including the known physiological range,
of UDP-GlcNAc [10]. One possible explanation for these
nearly unsaturatable UDP-GlcNAc kinetics is a Ping-
Pong mechanism for the enzyme, even though this hy-
pothesis has yet to be tested. It was also established that
the enzyme is tyrosine phosphorylated as well as being
Biochemical and Biophysical Research Communications 302 (2003) 435–441
www.elsevier.com/locate/ybbrc
BBRC
* Corresponding author. Fax: 1-410-614-8804.
E-mail address: [email protected] (G.W. Hart).
0006-291X/03/$ - see front matter � 2003 Published by Elsevier Science (USA).
doi:10.1016/S0006-291X(03)00175-X
modified byO-GlcNAc itself [8]. TheTPRs suggested that
the enzymemight be interactingwith other proteins and it
was recently shown that OGT has several binding part-
ners, includingmSin3A,GRIF-1, andOIP106 whichmaybe involved in targeting the enzyme to discrete intracel-
lular 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]. Thiswork 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 ofmodification,we postulate thatOGT
is regulated by localization, binding partners, and PTMs.Future work will focus on establishing the identity of
other binding partners and PTMs and how theymodulate
OGT activity, stability, and localization.
A neutral, nucleocytoplasmic hexosaminidase activity
was described in 1976 [19]. A nucleocytoplasmic, neu-
tral, b-N-acetylglucosaminidase (O-GlcNAcase) that
appears to be responsible for the previously describedactivity 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-GlcNA-
case 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 cleavedduring the induction of apoptosis in cells by treatment
with cytotoxic lymphocyte granules. Surprisingly,
cleavage of full-length 130 kDa O-GlcNAcase into 2
Table 1
Identified mammalian O-GlcNAc modified proteins
Protien Reference Proof Protein Reference Proof
OGT [8] b,c,e,h,k RNA Pol II [64] a–i
Sp-1 [65] a–h SRF [66] a, b
HNF-1 [67] e c-myc [25] a–i,k,l
HCF [63] k Oct1 [63] k
EWS [63] k ERa&b [23] a–i
NF-jB [52] f hhSEC23 [63] k
p53 [68] b,e,g EF-2D [63] k
EIF2aAp67 [30] b–e,g,l EIF4A1 [63] k
EF1 [63] k 40SrpS24 [63] k
Nup62 [3] a–k Nup155 [63] a,c,f
Nup54 [63] k Ran [63] k
K8/18/13 [69] a–e Synapsin I [38] a–h
Annexin I [63] k E-cadherin [50] e
Piccolo [63] k Dynein LC1 [63] k
b-Synuclein [39] b–e,i CRMP-2 [39] b–e,i
APP [33] b,e,f Tau [32] a–h
Talin [70] b,c Clathrin AP-3 [36] b,c,e
Neurofilament H,M,L [34] a–e MAPS [35] b,c
Band 4.1 [71] a–e Cofillin [63] k
AnkyrinG [73] b–f a-Tubulin [72] f
Nucleophosmin [63] k hnRNP p43 [74] b,e,f
Pyruvate Kinase [63] k GAPDH [63] k
Enolase [63] k PGK [63] k
UDPGP [63] k UGP-1 [63] k
aB-Crystallin [60] a–e HSC70 [63] k
HSP90 [63] k PPI [63] k
ProteasomeC2 [63] k UCH-L1 [39] b–e,i
UCH homologue [63] k GSK3b [11] h
PYPp65 [75] c,e CKII [10] a–h,k
i2pp2a [63] k RHO-GDI-a [63] k
b-Catenin [50] e,f,i IRSs [76] f,k,i
eNOS [48] f,i GRIF-1 [17] f
OIP-106 [17] f p85PI3K [49] f
Glut-1 [77] b,f GS [78] 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, Ewing�s 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, glyceraldyde-
3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; Glut, glucose transporter; GS, glycogen synthase.
436 L. Wells et al. / Biochemical and Biophysical Research Communications 302 (2003) 435–441
approximately 65 kDa fragments has no effect on in vi-tro 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 in-
duces 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 proposedthat O-GlcNAc may modify RNA polymerase II that is
in the preinitiation complex or a storage form. On es-
trogen 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 specificmanner. OGT was found to associate with the mSin3A
corepressor complex and OGT activity is necessary for
maximal gene silencing [16]. OGT expression and ele-
vated 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
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 ag-
gregating 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.
L. Wells et al. / Biochemical and Biophysical Research Communications 302 (2003) 435–441 437
impact on various promoters, transcription factors, andthe 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 besensitive 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-Glc-NAc 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, clath-rin assembly proteins (AP3 and AP180), synapsin I,
collapsin response mediator protein-2 (CRMP-2),
ubiquitin carboxyl hydrolase-L1 (UCH-L1), and b-synuclein [32–39]. Interestingly, OGT maps to the X-
linked Parkinson dystonia locus [18] and O-GlcNAcase
maps to the Alzheimer�s disease locus on chromosome
10 [13,40]. Also, several groups have demonstrated both
a global as well as protein specific yin-yang relationshipbetween phosphorylation and O-GlcNAc modification
(that is when phosphorylation is elevated O-GlcNAc is
reduced and vice versa, reviewed in [4,41]). The micro-
tubule-associated protein tau is hyperphosphorylated
when in neurofibrillary tangles [42], suggesting that the
O-GlcNAc modification may protect tau from aggre-
gation. Understanding the impact of O-GlcNAc on tau
and the b-amyloid precursor protein as well as estab-lishing 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 participat-ing in signal transduction events has been proposed for
more than a decade [41], only recently have data
emerged implicating O-GlcNAc in specific signal trans-
duction cascades. The hexosamine biosynthetic pathway
(HSP), which converts fructose-6-phosphate to UDP-
GlcNAc (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 pre-
vented hyperglycemia-induced insulin resistance in pe-
ripheral 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 elevatedO-GlcNAc levels and insulin resistance. Using the 3T3-
L1 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 demon-
strate that elevated O-GlcNAc levels inhibited the
insulin-stimulated phosphorylation of AKT. This work
was further supported a few months later when McClainet al. [29] demonstrated that transgenic mice overex-
pressing 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 at-tributed to elevated O-GlcNAc [48,49], further sup-
porting 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 path-
ways. For example, O-GlcNAcase is cleaved by caspase-3 [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-Glc-
NAc 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 extra-
cellular stimuli but instead are taking into account their
own energy stores. O-GlcNAc, which appears to behighly responsive to nutrient state, modifies signaling
components, cytoskeletal components, and the tran-
scriptional and translational machinery. Thus, O-Glc-
NAc modification could be modulating the proteins that
are present and their post-translational state and local-
ization, so that they respond in an appropriate way to
extracellular cues.
438 L. Wells et al. / Biochemical and Biophysical Research Communications 302 (2003) 435–441
Recent advancements in the field, including the de-velopment 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 path-ways. 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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