Biochimie 83 (2001) 575−581

© 2001 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
S0300908401012950/REV

Nucleocytoplasmic O-glycosylation: O-GlcNAc and functional proteomics

Keith Vosseller*, Lance Wells*, Gerald W. Hart**

Department of Biological Chemistry, Johns Hopkins School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA
(Received 20 March 2001; accepted 5 April 2001)

Abstract — The molecular complexity that defines different cell types and their biological responses occurs at the level of the cell’s
proteome. The recent increase in availability of genomic sequence information is a valuable tool for the field of proteomics. While
most proteomic studies focus on differential expression levels, post-translational modifications such as phosphorylation, glycosylation,
and acetylation, provide additional levels of functional complexity to the cell’s proteome. The reversible post-translational
modification O-linked β-N-acetylglucosamine (O-GlcNAc) is found on serines and threonines of nuclear and cytoplasmic proteins. It
appears to be as widespread as phosphorylation. While phosphorylation is recognized as a fundamental mechanism for controlling
protein function, less is known about the specific roles of O-GlcNAc modification. However, evidence is building that O-GlcNAc may
compete with phosphate at some sites of attachment. Aberrant O-GlcNAc modification has been linked to several disease states,
including diabetes and Alzheimer’s disease. Regulated enzymes catalyzing the addition (O-GlcNAc transferase, OGT) and removal
(O-GlcNAcase) of the modification have been cloned and OGT is required for life at the single cell level. Here we review the
properties of O-GlcNAc that suggest it is a regulatory modification analogous to phosphorylation. We also discuss the use of
comparative functional proteomics to elucidate functions for this ubiquitous intracellular carbohydrate modification. © 2001 Société
française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
O-GlcNAc / signal transduction / proteomics / post-translational modification

1. Proteomics and post-translational modifications
The sequencing of genomes from several organisms,
including man, is having a profound impact on the speed
and quality of biological research [1, 2]. However, biology
is carried out mainly by proteins and other macromol-
ecules, whose functional complexity is not reflected in
genomic sequence. The simple model of one gene giving
rise to one protein has been shown to be incorrect for more
than 20 years [3]. There are many ways a cell can increase
the complexity of its proteome when moving from gene to
functional gene products. In eukaryotes, the process of
going from DNA to functional protein usually consists, at
the most basic level, of at least six discreet steps:
transcription, splicing, polyadenylation, transport, transla-
tion, and post-translational modification. At the level of
RNA, alternative transcriptional start sites and alternative
mRNA splicing may increase complexity. mRNA levels
do not always correspond to protein levels [4] due to
translational regulation and differential protein stability.
Cell type specific expression, and different expression
patterns in response to specific external stimuli also
contribute to the uniqueness of a cell’s proteome. Finally,
post-translational modifications have the potential to ex-
* Both authors contributed equally to this article.
**Correspondence and reprints.
E-mail address: gwhart@jhmi.edu (G.W. Hart).
ponentially increase the variety of protein molecular
species in a cell. Virtually all proteins are known to
contain some form of post-translational modification [5].
While we often tend to think in binary modes about
proteins (e.g., an enzyme being ‘on’ or ‘off’), we should
consider that any given modification may have unique
functional influences. While only 20 amino acids are used
in protein synthesis, as many as 140 different amino acids
are found in proteins due to covalent post-translational
modification [6]. In the context of multiple post-
translational modifications of the same protein, the com-
binatorial possibilities become very complex (figure 1).
Recently, the convergence of available coding sequence
databases, computing power, and mass spectrometry tech-
nology has made identification of unknown proteins facile
and rapid enough to design proteomic experiments. Most
proteomic projects to date have targeted protein expres-
sion level differences related to cellular responses [7].
Given that signaling within the cell involves switching
protein function rapidly through post-translational modi-
fications such as phosphorylation, proteomics directed at
function must account for protein changes occurring
post-translationally. Several functional proteomic efforts
have targeted phosphorylation changes in response to
specific cell stimuli such as changes in phosphorylation
due to EGF, PDGF, and Fas receptor activation [8–10]. A
distinct advantage of phosphorylation is that it changes the
pI of the protein which leads to a shift of the spot patterns
576
Figure 1. A single gene can give rise to multiple functional gene products. The image represents the increasing complexity as you
go from genomic DNA to modified proteins, listing three main transitions that are regulated and the four types of analysis that can
be performed in genomic/proteomic experiments.
on a classical two-dimensional gel. This enables one to see
the appearance or disappearance of spots that can then be
identified by mass spectrometry or Edman-degadation
approaches. Similar studies aimed at identifying proteins
displaying dynamic O-GlcNAc modification in response
to changing cellular conditions may reveal points of
regulation by O-GlcNAc in cell signaling. Like phospho-
proteins, O-GlcNAc modified proteins can be specifically
selected for and identified. But first, why should
O-GlcNAc deserve the kind of attention that phosphory-
lation gets?
2. What is O-GlcNAc?
The monosaccharide N-acetylglucosamine is attached
in a β-linkage to serine and threonine hydroxyl groups of
many nuclear and cytoplasmic proteins [11, 12]. It appears
Vosseller et al.
to be as widespread as phosphorylation [13, 14] and has
been found in all multicellular eukaryotes examined. No
consensus motif for O-GlcNAc attachment has been
identified, but many sites are identical to those used by
serine/threonine kinases. The characteristics of O-GlcNAc
set it apart from other forms of complex glycosylation in
several important ways. Enzymes responsible for deter-
mining structures of complex carbohydrates are localized
exclusively to the Golgi and ER. Proteins modified by
complex glycosylation are localized to the lumenal com-
partments of exocytic and endocytic organelles. Thus, the
potential for dynamic responsiveness of complex carbo-
hydrates to signals is limited. Enzymes catalyzing
O-GlcNAc addition (OGT) and removal (O-GlcNAcase)
are found in the cytosol and nucleus and have been cloned
and characterized [15–17]. The proximity of these en-
zymes to their substrates allows for rapid regulation
through post-translational modification of existing pro-
teins. Indeed, protein specific changes in O-GlcNAc levels
O-GlcNAc proteomics
Figure 2. A wide variety of protein classes are modified by O-GlcNAc.
are observed in response to T cell activation [18], and
glucose metabolism and insulin signaling [19]. Addition-
ally, O-GlcNAc turns over more rapidly than the polypep-
tide backbone [20, 21]. Thus, while complex N- and
O-linked glycosylation are not thought to participate in
dynamic signaling responses, O-GlcNAc is poised to do so.
The localization of O-GlcNAc on cytosolic and nuclear
proteins [22] combined with its dynamic characteristics
suggest it may play an important regulatory role in
signaling. O-GlcNAc occurs on functionally diverse
classes of proteins (figure 2). Given the diverse nature of
these proteins, it is unlikely that one unifying function will
be assigned to O-GlcNAc. Just as with phosphate,
O-GlcNAc is likely to have protein and even site-specific
influences on multiple molecular processes. In this regard,
proteomics is a suitable strategy to reveal O-GlcNAc
function in response to varying cell context. Before
discussing proteomic strategies for seeking functions of
O-GlcNAc, we review current evidence that implicates
O-GlcNAc as a regulatory modification.
577
3. Reciprocity with phosphate
Several studies have found a ‘yin-yang’ relationship
between O-GlcNAc modification and phosphorylation.
Globally, it has been shown that phosphatase inhibitors
and kinase activators decrease overall levels of O-GlcNAc
and conversely that kinase inhibitors increase levels of
O-GlcNAc [23]. Furthermore, on a select few proteins the
mapped site for glycosylation and phosphorylation are
identical, such as c-Myc [24], the estrogen receptor [25,
26], and SV-40 large T antigen [27]. This apparent
reciprocity again emphasizes that proteins exist in a
variety of forms through combinatorial post-translational
modification.
4. Protein-protein interactions
Specificity of protein function often depends on induc-
ible protein-protein interactions.
O-GlcNAc may determine or modulate binding part-
ners either directly or indirectly. It remains to be deter
578
mined, if like phosphorylation in the cases of SH2 and
WD domains for instance, if O-GlcNAc is creating a
specific binding domain on modified proteins. However,
nucleocytoplasmic GlcNAc-binding proteins have been
identified [28] and there is evidence that O-GlcNAc is
affecting protein-protein interactions. For instance, a gly-
cosylated Sp1 peptide can prevent Sp1 protein from
binding TAF110, while the unmodified peptide cannot
[29]. Proteomic methodologies can be used to address
many of the questions in regard to protein-protein inter-
actions. One such approach is to use O-GlcNAc modified
and unmodified proteins coupled to a solid support to fish
out interacting proteins. These proteins can be identified
either directly by HPLC-tandem mass spectrometry or
depending on the complexity of the bound proteins by first
separating the proteins on two-dimensional gels.
5. O-GlcNAc and diabetes
There is growing evidence of a link between anomalous
O-GlcNAc modification and diabetes. Hyperglycemia is
associated with insulin resistance or ‘glucose toxicity’ in
type II diabetes , although the underlying molecular
mechanisms for these effects are still largely unknown.
O-GlcNAc transferase (OGT) uses the donor sugar UDP-
GlcNAc in catalyzing the formation of O-GlcNAc. Con-
centrations of UDP-GlcNAc (the end product of the
hexosamine pathway) in the cell are highly sensitive to
ambient glucose levels [30, 31]. Increased flux through the
hexosamine pathway under hyperglycemic conditions
leads to elevated levels of O-GlcNAc modified proteins in
skeletal muscle [32] and in pancreatic beta cells [33]. In
the case of muscle cells, reduced insulin receptor substrate
(IRS) –1 and –2 signaling are associated with their in-
creased O-GlcNAc modification and decreased phospho-
rylation [19]. High concentrations of extracellular glu-
cose, which leads to an increase in O-GlcNAc modified
proteins, causes attenuated insulin stimulated signaling.
For example, the ability of the p85 subunit of PI 3-kinase
and Grb2 to bind IRS-1 are attenuated [34]. Also, hyper-
glycemic conditions increase O-GlcNAc levels on the
transcription factor Sp1 and this correlates with increased
Sp1 dependent TGFβ1 expression [35]. Thus, altered
O-GlcNAc levels under hyperglycemic conditions appear
to perturb normal signaling events required for insulin-
mediated homeostasis. As yet, the specific molecular
targets of hyperglycemia that mediate insulin resistance
are not known. A proteomic screen for proteins whose
state of O-GlcNAc is highly sensitive to extracellular
glucose concentrations and/or insulin signaling may help
target proteins susceptible to improper regulation in the
diabetic disease state.
Understanding the relationship between aberrant
O-GlcNAc modification and the clinical manifestation of
diabetes may reveal a more general mechanism in which
Vosseller et al.
O-GlcNAc could play a role as a nutritional ‘sensor’ in the
cell. Given the relationship between ambient glucose
levels, intracellular UDP-GlcNAc concentrations, and el-
evated O-GlcNAc, this modification is positioned to relay
information about extracellular glucose concentrations to
transcription factors and other components of intracellular
signaling pathways. Lowering of UDP-GlcNAc levels
through a knockout of Emeg32, an enzyme required in the
metabolic pathway leading to the production of UDP-
GlcNAc, lowered cytosolic O-GlcNAc levels while not
affecting other forms of glycosylation appreciably. This
reduced cytosolic glycosylation was associated with cell
cycle and cytoskeletal defects and resistance to apoptosis
[36], further suggesting important links between UDP-
GlcNAc levels and signaling properties of O-GlcNAc.
Thus, the state of O-GlcNAc may represent a ‘set-point’
reflecting the nutritional state of the cell, and operate by
enhancing or dampening a cell’s response to a given
extracellular stimuli. These effects could be mediated by
any number of O-GlcNAc dependent mechanisms.
6. O-GlcNAc enrichment for proteomics
While β-O-GlcNAc was described over 17 years ago
[11], and is critical to cellular processes [37], specific
functions for this modification are not well characterized.
In the case of phosphate being a switch mechanism, its
importance is defined in the dynamic way it regulates
protein function. Thus, in response to some biological
stimulus, capturing proteins in differential states of phos-
phorylation provides insights into the functions of this
post-translational modification [38]. A similar proteomic
approach with O-GlcNAc should also direct us toward
protein specific functions of this modification. Proteomics
usually involves separation of proteins by two-
dimensional polyacrylamide gel electrophoresis (2D-
PAGE), isolation of proteins of interest, digestion with a
protease (usually trypsin) to generate peptides for mass
spectrometry (MS) or tandem mass spectrometry
(MS/MS). The masses of peptides and/or peptide frag-
ments generated by collision induced dissociation in the
mass spectrometer are then compared against theoretical
patterns in sequence databases to identify proteins.
Proteomics is an emerging field and methodologies for
its application are evolving fast. A major challenge to
proteomics is the enormous dynamic range of protein
expression in a cell. For very complex mixtures of
proteins separated by current 2D-PAGE techniques, there
is an overwhelming bias towards detecting high abun-
dance proteins. Regulatory proteins, usually of more
interest in proteomic studies, often tend to be present in
low copy number. Several approaches can be taken to
reduce sample complexity and increase coverage of low
abundance proteins, including pre-fractionation of
samples before analysis by 2D gels, or by using very
O-GlcNAc proteomics
Figure 3. Enrichment for proteins modified by O-GlcNAc. Nucleocytoplasmic extracts of HeLa S3 cells were prepared and subjected
to WGA chromatography. 80 µg of the flow through as well as the 1 M GlcNAc eluted protein were separated by two-dimensional
electrophoresis and the resulting gels were visualized by silver staining.
narrow pI ranges for separation in the first dimension [39].
Affinity tags which selectively isolate subpopulations of
peptides to send to the mass spectrometer [40], may, on
occasion, eliminate the need for gel electrophoresis. In the
case of post-translational modifications, the problem of
low relative abundance may be particularly problematic.
For example, the stoichiometry of phosphate or
O-GlcNAc on a given protein is usually low, but func-
tionally significant. Some proteomic studies which tar-
geted phosphorylation changes, employed techniques that
affinity enrich for either phosphorylated proteins prior to
gel separation [38] or phosphorylated peptides specifically
[41] after protein digestion. These approaches effectively
reduce the complexity of sample and increase the mass of
proteins of interest.
Several tools available for O-GlcNAc detection may be
exploited for enrichment techniques similar to those used
on phosphoproteins. For example, the lectin wheat germ
agglutinin recognizes clustered terminal GlcNAc. WGA
coupled to a solid support such as agarose, can be used to
affinity enrich for O-GlcNAc modified proteins. As an
example, we combined cytosolic and nuclear fractions of
HeLa S3 cells and bound this lysate to a WGA-agarose
column. After extensive washing, we eluted enriched
O-GlcNAc modified proteins specifically by competition
with free GlcNAc. Equal amounts of protein from the
unbound and bound fractions were separated by two-
dimensional polyacrylamide gel electrophoresis and visu-
alized by silver staining (figure 3). There are striking
differences between the stained protein patterns of these
two gels. It is apparent that many spots observed on the
2D gel representing O-GlcNAc enriched proteins are not
579
detectable in the crude unbound material. Thus, we have
effectively revealed low abundance proteins of interest
using this O-GlcNAc affinity enrichment technique. Many
of these proteins would not be detected in crude complex
mixtures.
Some O-GlcNAc specific antibodies have been de-
scribed. For example, RL2 recognizes a subset of
O-GlcNAc sites mainly on nuclear pore proteins [42].
Recently, an antibody recognizing a broader spectrum of
O-GlcNAc sites has been generated [43]. O-GlcNAc
specific antibodies coupled to solid supports such as
agarose could also be used to specifically enrich popula-
tions of proteins modified by O-GlcNAc.
In addition to reducing complexity of sample, the
enrichment strategies described above offer advantages
for mapping sites of O-GlcNAc modification. Since
samples are obtained based on affinity enrichment, sto-
ichiometry of O-GlcNAc is likely to be very high on gel
isolated proteins. A single O-GlcNAc adds 203 Da to a
given peptide otherwise unmodified. In tandem with
identification of proteins, peptides with observed masses
of 203 Da greater than the expected mass may indicate
O-GlcNAc modification. Among other methods described
for O-GlcNAc site mapping [44], further modification of
O-GlcNAc in vitro by enzymatic addition of galactose,
allows for affinity enrichment through binding to the lectin
ricin, which is specific for galactose [45]. Additionally,
base catalyzed β-elimination of O-GlcNAc modified pep-
tides converts glycosylserine to 2-amino-propenoic acid,
and the resulting change from 87 to 69 Da can be used to
identify the site of O-GlcNAc attachment [46]. Recently,
low energy removal of the O-GlcNAc group in the mass
580
spectrometer and monitoring for a reporter ion of 203 Da
was used to target fragmentation and identification of
O-GlcNAc modified peptides [47]. Mapping of
O-GlcNAc attachment sites is a logical and essential
extension of the proteomic strategies suggested here.
Mutagenesis of sites of phosphorylation have been critical
in implicating that modification in protein specific roles
and the same will be true for O-GlcNAc. Proteomic
studies of dynamic O-GlcNAc modification and the map-
ping of attachment sites will help direct us toward the
functions of O-GlcNAc in the cell.
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