While there are a multitude of kinases and phosphatases involved in
the addition and removal of phosphate, only one OGT catalytic subunit
and one O-GlcNAcase have been found. The primary amino acid sequence
of OGT has diverged little from C. elegans to humans11. A polypeptide
related to OGT at the primary sequence level has not been found in yeast
or E. coli. However, the possibility exists that these
organisms may have an OGT with different primary structure, or alternatively,
they may either not have this type of intracellular glycosylation or
they may use a different saccharide besides GlcNAc to accomplish the
In many tissues, the enzyme is found as a homotrimer consisting of
a 110 kDa subunit. However, in some tissues, such as the liver, the
predominant form of OGT is a heterotrimer containing two 110 kDa subunits
and a 78 kDa subunit12.
The 78 kDa subunit appears to result either from proteolysis or from
alternate splicing. Additionally, there seems to be a mitochondrially
arising as an alternate splice variant14.
OGT has a bimodal structure; the N-terminal portion of the protein consists
of 11.5 to 13 tetratricopeptide repeats (TPR), while the C-terminus
contains the catalytic portion. TPR repeats are found in a variety of
proteins and they have been shown to be important in protein-protein
interactions10. The TPRs in OGT are presumably important not only for
substrate recognition, but also for multimerization of the enzyme. The
catalytic portion of OGT shows weak homology to glycogen phosphorylase/glycosyl
Fine mapping of the gene localizes OGT to the X chromosome in mice
and humans. Of particular note, OGT maps to Xq13.1 in human16,
which is also the dystonia Parkinsonism locus. Gene ablation studies
in mice indicate the absolute requirement for O-GlcNAc, as the
embryonic stem (ES) cells are not viable17.
Although an exact substrate sequence motif has not been found, proline
near the site of modification seems to occur frequently10. Interestingly,
this observation alludes to a reciprocal relationship between proline-directed
phosphorylation and O-GlcNAcylation. In addition, OGT is also
modified with both O-GlcNAc and phospho-tyrosine18.
The enzyme that catalyzes the hydrolysis of O-GlcNAc from proteins,
O-GlcNAcase, was purified based on its specificity for O-GlcNAcylated
peptides and its activity at neutral pH, whereas other glycosidases
have an acidic pH optima indicative of their activity residing in lysosomal
organelles19. O-GlcNAcase is ubiquitously expressed with an apparent
molecular weight of 130 kDa. Interestingly, there is a splice variant
of 75 kDa, which is a result of an alternate stop codon. This splice
variant lacks the C-terminal portion of the enzyme and lacks catalytic
activity20. Although the monomer has catalytic activity, purification
of the enzyme from tissue shows that it migrates as a large 340 kDa
complex21 indicating associations with a number of proteins in vivo.
O-GlcNAcase is also a bimodal enzyme. The N-terminal portion
has loose homology to bacterial hyaluronidases21 while the C-terminus
has weak homology to the GCN5-related family of acetyltransferases22.
The N-terminus and C-terminus are joined by a linker domain. The gene
maps to 10q24, which is also associated with late-onset Alzheimer’s
O-GlcNAcase is a good substrate for cleavage by Caspase-323,
an executioner caspase involved in apoptosis. Although the site of cleavage
has not been mapped, cleavage by Caspase-3 does not inhibit O-GlcNAcase
activity in vitro.
It is now widely accepted that phosphorylated residues are important
structural determinants in protein-protein interactions 24.
For example, SH2 domains and 14-3-3 domains bind phosphotyrosine and
phosphoserine/threonine residues, respectively. A potential role for
O-GlcNAc could be to mediate protein-protein interactions as
many proteins that are O-GlcNAcylated are multimeric proteins.
For example, YY1 is a zinc finger DNA-binding transcription factor that
can be modified by O-GlcNAc. The modification causes dissociation
of YY1 from retinoblastoma protein (pRb) allowing it to bind DNA25.
O-GlcNAc could also induce binding as it is estimated that approximately
0.1% of the peptides presented by MHC Class I are O-GlcNAcylated.
This observation indicates that this post-translational modification
is important for recognition by certain T cell receptors26.
It is also apparent that O-GlcNAc modification of RNA polymerase
II and most transcription factors plays a role in assembly of the pre-initiation
complex during the transcription cycle (see below).
Another possible regulatory role for O-GlcNAc could be controlling
the degradation of proteins. Sequences enriched with Pro, Glu, Ser,
and Thr, or PEST sequences, have been proposed to target proteins for
Interestingly, several O-GlcNAc sites have been mapped to PEST
sequences, and it is presumed to protect the protein from degradation.
is reciprocally modified by phosphorylation or O-GlcNAcylation
at Ser 16. When this site is phosphorylated, the protein is rapidly
targeted for degradation, while the O-GlcNAcylated form is degraded
much more slowly28.
Of note, many of both the regulatory and catalytic subunits comprising
the proteasome are modified by O-GlcNAc. O-GlcNAcylation
of the proteasome inhibits is ability to degrade certain proteins29.
By regulating protein half-life, O-GlcNAcylation can exert a
temporal effect on many cellular processes.
Virtually every RNA pol II transcription factor studied to date is modified
with O-GlcNAc. There is no overlying theme in the specific role
of the modification in transcription. In the case of Stat5, the O-GlcNAcylated
form binds the coactivator of transcription CBP, activating Stat5 mediated
However, when Sp1, a ubiquitous transcription factor involved in regulating
housekeeping genes, is modified with O-GlcNAc, it is inhibited
from interaction with the transcriptional machinery31
at some promoters; however, O-GlcNAcylation of Sp1 increases
its activity on other promoters32.
In addition, the C-terminal domain (CTD) of RNA pol II, which is important
for interacting with transcription machinery and is indispensable for
in vivo function, is also extensively O-GlcNAcylated33.
The CTD consists of heptamer repeats that can be multiply modified with
O-GlcNAc. It is known that the CTD is also phosphorylated, and
that this phosphorylation is absolutely necessary for promoter clearance
and elongation. These two modifications are mutually exclusive on RNA
polymerase II CTD34.
Although the exact role of O-GlcNAc has not been delineated,
it may be important for recruitment of RNA pol II to active promoters
or formation of the pre-initiation complex. The O-GlcNAcylated
form could also act as a readily activatable storage form of RNA pol
Transduction of signals from the extracellular surface to the interior,
and the adaptations the cell makes in response to these signals dictates
both survival and differentiation of the cell. Often these signals are
transduced by dynamic post-translational modifications, such as phosphorylation,
and proteolysis. It has been shown that mitogenic activation of lymphocytes
and cerebellar neurons causes rapid and transient changes in O-GlcNAc
levels1,5. These observations indicate that the transitory alterations
in O-GlcNAc are needed to effect a concomitant modification in
activity of target proteins or gene transcription. It is also clear
that O-GlcNAc modulates the insulin signaling pathway (see below).
These studies confirm that O-GlcNAc is a dynamic post-translational
modification that is involved in signaling.
A number of proteins involved in the progression of cells into a cancerous
state have been identified as O-GlcNAc modified proteins. Among
them are -catenin,
p53, pRb family members, and c-Myc4.
In particular, the major O-GlcNAc modification site on c-Myc,
Thr58, is a known hot-spot for mutation in Burkitt`s lymphoma
and is a major GSK3
In addition, O-GlcNAcase activity is increased in certain cancerous
tissues. A recent study comparing different breast cancers with its
corresponding normal tissue showed that there was increased O-GlcNAcase
activity in the tumor with a corresponding decrease in O-GlcNAc
observations raise the notion that O-GlcNAc is important in regulation
of key phosphorylation events that regulate cellular growth.
Both OGT and O-GlcNAcase map to loci linked to neurodegenerative
diseases; the locus for OGT is associated with Parkinson’s disease,
while the locus for O-GlcNAcase is linked with late onset Alzheimer’s
disease. These observations imply that O-GlcNAc may play a role
in neurodegenerative diseases. In fact, tau, a microtubule binding protein
associated in the pathology of Alzheimer’s disease, is both phosphorylated
and O-GlcNAcylated. Tau in normal brains is extensively O-GlcNAc
modified. Hyperphosphorylated tau is found in the aggregates of neurofibrillary
tangles linked with Alzheimer’s. It has been postulated that decreasing
O-GlcNAc levels in the brain leads to the abnormal phosphorylation
of tau37; hence, O-GlcNAc has a protective effect. Beta amyloid
precursor protein (APP), neurofilaments, and many synaptic vesicle proteins
are also extensively O-GlcNAc modified38,39,40. These recent studies
have presented evidence for alterations in O-GlcNAc in neurodegenerative
disease in humans.
It is estimated that approximately 177 million people are affected with
diabetes worldwide41. Approximately 5-10% of these individuals are Type
I diabetic or insulin-dependent, which is an autoimmune disorder that
destroys the body’s ability to synthesize
insulin. Type II diabetes constitutes 90-95% of the population with
diabetes. This condition is characterized by the desensitization of
peripheral tissues to the action of insulin. Studies implicate that
increased flux through the HBP is directly linked to insulin resistance
42. This increased flux would correspondingly increase UDP-GlcNAc levels
and, hence, O-GlcNAcylation, perhaps linking O-GlcNAc
to the induction of insulin resistance. Studies in cultured adipocytes
and transgenic mice have shown that increased O-GlcNAc levels
directly cause insulin resistance. In these model systems, chemically
or genetically increasing O-GlcNAc levels resulted in a Type
II diabetic phenotype in which insulin-stimulated glucose uptake was
impaired43,44. In a cultured adipocyte system, chemically increasing O-GlcNAc
levels inhibited the insulin signaling pathway at the level of PKB/Akt
providing a mechanism by which hyperglycemia can induce insulin resistance.
Additionally, other studies demonstrating increased O-GlcNAcylation
of proteins involved in metabolism and glucose transport, such as glycogen
synthase and GLUT445,46, may further implicate O-GlcNAc in the
etiology of Type II diabetes both in terms of blockage of insulin signaling
and with respect to glucose toxicity.
|| Kearse KP, Hart GW: Lymphocyte activation
induces rapid changes in nuclear and cytoplasmic glycoproteins.
Proc. Natl. Acad. Sci. U.S.A. 88, 1701-1705, 1991
||Fang B, Miller MW: Use
of galactosyltransferase to assess the biological function of O-linked
N-acetyl-d-glucosamine: a potential role for O-GlcNAc during cell
division. Exp. Cell Res. 263, 243-253, 2001
||Zachara NE, ODonnell
N, Cheung WD, Mercer JJ, Marth JD, Hart GW: Dynamic O-GlcNAc modification
of nucleocytoplasmic poteins in response to stress. A survival response
of mammalian cells. J. Biol. Chem. , 30133-30142,
||Zachara NE, Cheung WD, Hart GW: Curr.
Org. Chem. 8, 369-383, 2004
||Griffith LS, Schmitz B: O-linked N-acetylglucosamine
levels in cerebellar neurons respond reciprocally to pertubations
of phosphorylation. Eur. J. Biochem. 262, 824-831,
||Chou TY, Hart GW, Dang CV: c-Myc is glycosylated
at threonine 58, a known phosphorylation site and a mutational hot
spot in lymphomas. J. Biol. Chem. 270, 18961-18965,1995
||Hart GW, Greis KD, Dong LYD, Blomberg MA, Chou
TY, Jiang MS, Roquemore EP, Snow DM, Kreppel LK, Cole RN, Comer
FI, Arnold CS, Hayes BK: “"O-linked N-acetylglucosamine: The
‘yin-yang’ of Ser/Thr phosphorylation?” Glycoimmunology.
Alavi, A., Axford, J.S., eds 1, 115-123, 1995
||Iyer SP, Hart GW: Dynamic nuclear and cytoplasmic:
enzymes of O-GlcNAc cycling. Biochemistry, 42, 2493-2499,
||McClain DA, Crook ED: Hexosamines and insulin resistance.
Diabetes, 45, 1003-1009, 1996
||Kreppel LK, Hart GW: Regulation of a cytosolic
and nuclear O-GlcNAc transferase. Role of the tetratricopeptide
repeats. J. Biol. Chem. 274, 32015-32022, 1999
||Lubas WA, Frank DW, Krause M, Hanover JA: O-linked
GlcNAc transferase is a conserved nucleocytoplasmic protein containing
tetratricopeptide repeats. J. Biol. Chem. 272, 9316-9324,
|| Haltiwanger RS, Blomberg MA, Hart GW: Glycosylation
of nuclear and cytoplasmic proteins. Purification and characterization
of a uridine diphospho-N- acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase.
J. Biol. Chem. 267, 9005-9013, 1992
||Hanover JA, Yu S, Lubas WB, Shin SH, Ragano-Caracciola
M, Kochran J, Love DC: Mitochondrial and nucleocytoplasmic isoforms
of O-linked GlcNAc transferase encoded by a single mammalian gene.
Arch. Biochem. Biophys. 409, 287-297, 2003
||Love DC, Kochan J, Cathey RL, Shin SH Hanover JA,
Kochran J: Mitochondrial and nucleocytoplasmic targeting of O-linked
GlcNAc transferase. J. Cell Sci. 116, 647-654, 2003
||Wrabl JO, Grishin NV: Homology between O-linked
GlcNAc transferases and proteins of the glycogen phosphorylase superfamily.
J. Mol. Biol. 314, 365-374, 2001
||Nolte D, Müller U: Human O-GlcNAc transferase (OGT):
genomic structure, analysis of splice variants, fine mapping in
Xq13.1. Mamm. Genome. 13, 62-64, 2002
||Shafi R, Iyer SP, Ellies LG, O’Donnell N, Marek
KW, Chui D, Hart GW, Marth JD: The O-GlcNAc transferase gene resides
on the X chromosome and is essential for embryonic stem cell viability
and mouse ontogeny. Proc. Natl. Acad. Sci. U.S.A. 97,
||Kreppel LK, Blomberg MA, Hart GW: Dynamic Glycosylation
of nuclear and cytosolic proteins. Cloning and characterization
of a unique O-GlcNAc transferase with multiple tetratricopeptide
repeats. J. Biol. Chem. 272, 9308-9315, 1997
||Dong DL, Hart GW: Purification and characterization
of an O-GlcNAc selective N-acetyl-beta-D-glucosaminidase from rat
spleen cytosol. J. Biol. Chem. 269, 19321-19330, 1994
||Comtesse N, Maldener E, Meese E: Identification
of a nuclear variant of MGEA5, a cytoplasmic hyaluronidase and a
beta-N-acetylglucosaminidase. Biochem. Biophys. Res. Commun.
283, 634-640, 2001
||Gao Y, Wells L, Comer FI, Parker GJ, Hart GW: Dynamic
O-glycosylation of nuclear and cytosolic proteins: cloning and characterization
of a neutral, cytosolic beta-N-acetylglucosaminidase from human
brain. J. Biol. Chem. 276, 9838-9845, 2001
|| Schultz J, Pils B: Prediction of structure and
functional residues for O-GlcNAcase, a divergent homologue of acetyltransferases.
FEBS Lett. 529, 179-182, 2002
||Wells L, Gao Y, Mahoney JA, Vosseller K, Chen C,
Rosen A, Hart GW: Dynamic O-glycosylation of nuclear and cytosolic
proteins: further characterization of the nucleocytoplasmic beta-N-acetylglucosaminidase,
O-GlcNacase. J. Biol. Chem. 277, 1755-1761, 2002
||Pawson T, Nash P: Assembly of cell regulatory systems
through protein interaction domains. Science. 300,
||Hiromura M, Choi CH, Sabourin NA, Jones H, Bachvarov
D, Usheva A: YY1 is regulated by O-linked N-acetylglucosaminylation
(O-glcNAcylation). J. Biol. Chem. 278, 14046-14052,
||Glithero A, Tormo J, Haurum JS, Arsequell G, Valencia
G, Edwards J, Springer S, Townsend A, Pao YL, Wormald M, Dwek RA,
Jones EY, Elliott T: Crystal structures of two H-2Db/glycopeptide
complexes suggest a molecular basis for CTL cross-reactivity. Immunity,
10, 63-74, 1999
||Rechsteiner M, Rogers SW: PEST sequences and regulation
by proteolysis. Trends Biochem. Sci. 21, 267-271,
||Cheng X, Hart GW: Alternative O-glycosylation/O-phosphorylation
of serine-16 in murine estrogen receptor beta: post-translational
regulation of turnover and transactivation activity. J. Biol.
Chem. 276, 10570-10575, 2001
||Zhang F, Su K, Yang X, Bowe DB, Paterson AJ, Kudlow
JE: O-GlcNAc modification is an endogenous inhibitor of the proteosome.
Cell. 115, 715-725, 2003
||Gewinner C, Hart GW, Zachara NE, Cole R, Beisenherz-Huss
C, Groner B:The coactivator of transcription CREB-binding protein
interacts preferentially with the glycosylated form of Stat5. J.
Biol. Chem. 279, 3563-3572, 2004
||Roos MD, Su K, Baker JR, Kudlow JE: O glycosylation
of an Sp1-derived peptide blocks known Sp1 protein interactions.
Mol. Cell Biol. 17, 6472-6480, 1997
|| Majumdar G, Harmon A, Candelaria R, Martinez-Hernandez
A, Raghow R, Solomon SS: O-glycosylation of Sp1 and transcriptional
regulation of the calmodulin gene by insulin and glucagon. Am.
J. Physiol. Endocrinol. Metab. 285, E584-E591, 2003
||Kelly WG, Dahmus ME, Hart GW: RNA polymerase II
is a glycoprotein. Modification of the COOH-terminal domain by O-GlcNAc.
J. Biol. Chem. 268, 10416-10424, 1993
||Kelly WG, Dahmus ME, Hart GW: RNA polymerase II
is a glycoprotein. Modification of the COOH-terminal domain by O-GlcNAc.
J. Biol. Chem. 268, 10416-10424, 1993
||Kamemura K, Hayes BK, Comer FI, Hart GW: Dynamic
interplay between O-glycosylation and O-phosphorylation of nucleocytoplasmic
proteins: alternative glycosylation/phosphorylation of THR-58, a
known mutational hot spot of c-Myc in lymphomas, is regulated by
mitogens. J. Biol. Chem. 277, 19229-19235, 2002
||Slawson C, Pidala J, Potter R: Increased N-acetyl-beta-glucosaminidase
activity in primary breast carcinomas corresponds to a decrease
in N-acetylglucosaminine containing proteins. Bichim. Biophys.
Acta. 1537, 147-157, 2001
|| Lefebvre T, Ferreira S, Dupont-Wallois L, Bussiere
T, Dupire MJ, Delacourte A, Michalski JC, Caillet-Boudin ML: Evidence
of a balance between phosphorylation and O-GlcNAc glycosylation
of Tau proteins—a role in nuclear localization. Bichim. Biophys.
Acta. 1619, 167-176, 2003
||Griffith LS, Mathes M, Schmitz B: Beta-amyloid
precursor protein is modified with O-linked N-acetylglucosamine.
J. Neurosci. Res. 41, 270-278, 1995
||Cole RN, Hart GW: Cytosolic O-glycosylation is
abundant in nerve terminals. J. Neurochem. 79, 1080-1089,
|| Yao PJ, Coleman PD: Reduction of O-linked N-acetylglucosamine-modified
assembly protein-3 in Alzheimer’s disease. J. Neurosci. 18,
|| Retrieved from http://www.who.int/mediacentre/factsheets/fs236/en/
|| Marshall S, Bacote V, Traxinger RR: Discovery
of a metabolic pathway mediating glucose-induced desensitization
of the glucose transport systems. Role of hexosamine biosynthesis
in the induction of insulin resistance. J. Biol. Chem. 266,
||Vosseller K, Wells L, Lane MD, Hart GW: Elevated
nucleocytoplasmic glycosylation by O-GlcNAc results on insulin resistance
associated with defects in Akt activation in 3T3-L1 adipocytes.
Proc. Natl. Acad. Sci. U.S.A. 99, 5313-5318, 2002
|| McClain DA, Lubas WA, Cooksey RC, Hazel M, Parker
GJ, Love DC, Hanover JA: Altered glycan-dependent signaling induces
insulin resistance and hyperleptinemia. Proc. Natl. Acad. Sci.
U.S.A. 99, 10695-10699, 2002
||Parker GJ, Lund KC, Taylor RP, McClain DA: Insulin
resistance of glycogen synthase mediated by O-linked N-acetylglucosamine.
J. Biol. Chem. 278, 10022-10027, 2003
|| Buse MG, Robinson KA, Marshall BA, Hresko RC,
Mueckler MM: Enhanced O-GlcNAc protein modification is associated
with insulin resistance in GLUT1-overexpressing muscles. Am.
J. Physiol. Endocrinol. Metab. 283, E241-E250, 2002