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: gwhart@jhmi.edu (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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