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Journal of Molecular and Cellular Cardiology 52 (2012) 538–549
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Journal of Molecular and Cellular Cardiology
journal homepage: www.elsevier.com/locate/yjmcc
Review article
Protein O-linked β-N-acetylglucosamine: A novel effector of cardiomyocyte metabolism and function
Victor M. Darley-Usmar a, Lauren E. Ball b, John C. Chatham a,⁎
a
b
Division of Molecular and Cellular Pathology, Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA
Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, SC, USA
a r t i c l e
i n f o
Article history:
Received 5 July 2011
Received in revised form 11 August 2011
Accepted 12 August 2011
Available online 22 August 2011
Keywords:
O-GlcNAc
Glucotoxicity
Cardiac metabolism
Cardioprotection
Reactive oxygen species
a b s t r a c t
The post-translational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the
O-linked attachment of the monosaccharide β-N-acetyl-glucosamine (O-GlcNAc) is emerging as an important
mechanism for the regulation of numerous biological processes critical for normal cell function. Active synthesis
of O-GlcNAc is essential for cell viability and acute activation of pathways resulting in increased protein O-GlcNAc
levels improves the tolerance of cells to a wide range of stress stimuli. Conversely sustained increases in OGlcNAc levels have been implicated in numerous chronic disease states, especially as a pathogenic contributor
to diabetic complications. There has been increasing interest in the role of O-GlcNAc in the heart and vascular
system and acute activation of O-GlcNAc levels have been shown to reduce ischemia/reperfusion injury, attenuate vascular injury responses as well mediate some of the detrimental effects of diabetes and hypertension on
cardiac and vascular function. Here we provide an overview of our current understanding of pathways regulating
protein O-GlcNAcylation, summarize the different methodologies for identifying and characterizing OGlcNAcylated proteins and subsequently focus on two emerging areas: 1) the role of O-GlcNAc as a potential regulator of cardiac metabolism and 2) the cross talk between O-GlcNAc and reactive oxygen species. This article is
part of a Special Section entitled “Post-translational Modification.”
© 2011 Elsevier Ltd. All rights reserved.
Contents
1.
2.
3.
4.
Introduction
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Regulation of O-GlcNAc synthesis and turnover . . . . . . . . . . . . . . . . . . . . . . . . .
Pharmacological and molecular modulation of O-GlcNAc turnover . . . . . . . . . . . . . . . . . .
Identifying and characterizing O-GlcNAcylated proteins . . . . . . . . . . . . . . . . . . . . .
4.1.
Probing for protein O-GlcNAc modification with antibodies, enzymatic labeling, and metabolic
4.2.
Detection of the sites of O-GlcNAc modification by tandem mass spectrometry (MS/MS)
. .
4.3.
Enrichment of O-GlcNAcylated peptides for ETD MS/MS . . . . . . . . . . . . . . . . . .
5.
O-GlcNAcylation as a metabolic sensor and regulator
. . . . . . . . . . . . . . . . . . . . . .
6.
Mitochondria as an integrator of redox and O-GlcNAc signaling
. . . . . . . . . . . . . . . . .
7.
O-GlcNAc and redox cell signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Disclosures
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Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
⁎ Corresponding author at: Division of Molecular and Cellular Pathology, Department of
Pathology, University of Alabama at Birmingham, 1670 University Boulevard, Volker Hall
G001 Birmingham, AL 35294-0019, USA. Tel.: +1 205 934 0240.
E-mail address: [email protected] (J.C. Chatham).
0022-2828/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.yjmcc.2011.08.009
Classical protein glycosylation involves the synthesis of complex
elongated oligosaccharide structures via N-linkage on asparagine
and O-linkage on the hydroxy amino acids serine (Ser) and threonine
(Thr), as well as hydroxyproline, hydroxylysine, and tyrosine residues
V.M. Darley-Usmar et al. / Journal of Molecular and Cellular Cardiology 52 (2012) 538–549
of proteins that become secreted or membrane component glycoproteins [1]. Importantly these glycosylation reactions occur exclusively
in the ER and Golgi; however, in 1984 a novel glycosylation modification was first reported in which a single β-N-acetyl-glucosamine moiety was attached via an O-linkage to serine (Ser) and threonine (Thr)
residues of cytoplasmic and nuclear proteins [2]. This modification,
now commonly known as O-GlcNAc, has been shown to be present
in over 1000 nuclear, cytoplasmic and mitochondrial proteins and
has been found in all metazoans, as well as in some bacteria, protozoa,
viruses and fungi [3–7]. O-GlcNAcylation is distinct from traditional
glycosylation in that it is restricted to the cytoplasm, nucleus and mitochondria and it is not extended into complex elongated structures;
it also exhibits parallels with protein phosphorylation, in that it responds to acute stimuli, alters protein function and enzyme activity
and modifies the same or proximal Ser/Thr residues as phosphorylation [3–7].
It has become increasingly apparent that O-GlcNAc modification
of proteins is an important mechanism for the regulation of numerous biological processes critical for normal cell function such as signal
transduction [8–11], proteasome activity [12,13], apoptosis [14,15],
nuclear transport [16], translation and transcription [17]. Most of
our understanding of the impact of alterations in O-GlcNAc levels
on cell function has come from studies of chronic disease models
such as cancer [18–20], senescence [21–23], neurodegeneration
[11,14,24,25] as well as diabetes and diabetic complications [26,27].
However, a number of studies have demonstrated that O-GlcNAc is
essential for cell viability [28–30] and that acute activation of pathways resulting in increased protein O-GlcNAc levels improves the tolerance of cells to a wide range of stress stimuli [29].
There has been increasing interest in the role of O-GlcNAc in the
heart and vascular system and acute activation of O-GlcNAc levels has
been shown to reduce ischemia/reperfusion injury [31–41], attenuate
vascular injury responses [42] as well mediate some of the detrimental
effects of diabetes [21,43–49] and hypertension [50–53] on cardiac and
vascular function. The growing awareness of the effects of altered OGlcNAc on cardiovascular function is reflected in part by a relatively
large number of reviews on this subject over the past few years
[37,52–58]. Therefore, in light of these and other recent comprehensive
reviews on O-GlcNAc biology [3–7], we provide a brief summary on the
regulation of O-GlcNAc synthesis, followed by a practical overview regarding the 1) modulation of cellular O-GlcNAc levels and 2) the characterization and identification of O-GlcNAcylated proteins. We then
focus on two emerging areas, namely the role of O-GlcNAc as a potential
regulator of cardiac metabolism and the cross talk between O-GlcNAc
and reactive oxygen species, with an emphasis on the role of mitochondria in mediating this interaction.
2. Regulation of O-GlcNAc synthesis and turnover
The primary components involved in regulating O-GlcNAc synthesis and turnover are summarized in Fig. 1. Uridine-diphosphate-Nacetylglucosamine (UDP-GlcNAc) is the sugar nucleotide donor for
the synthesis of O-GlcNAc modified proteins, by O-GlcNAc transferase
(OGT). OGT has a high affinity for UDP-GlcNAc, which provides it with
a competitive advantage over nucleotide transporters in the endoplasmic reticulum and Golgi that compete for cytoplasmic UDPGlcNAc, but also makes it highly sensitive to changes in UDP-GlcNAc
concentrations [59]. Thus, the rate of O-GlcNAc synthesis and overall
levels of O-GlcNAc protein modification are highly dependent on the
flux through hexosamine biosynthesis pathway (HBP). HBP flux is
regulated largely by L-glutamine-D-fructose 6-phosphate amidotransferase (GFAT), which converts fructose-6-phosphate to glucosamine6-phosphate with glutamine as the amine donor [60].
Cell culture studies suggest that ~2–5% of total glucose entering the
cell is metabolized via GFAT [61]; however, the extent to which this
holds true for a highly metabolic organ, such as the heart is unknown.
539
Fig. 1. The hexosamine biosynthesis pathway (HBP) and protein O-GlcNAcylation. Glucose imported into the cells is rapidly phosphorylated to glucose-6-phosphate and converted to fructose-6-phosphate, which is subsequently metabolized to glucosamine-6phosphate by L-glutamine-D-fructose 6-phosphate amidotransferase (GFAT). Synthesis
of glucosamine-6-phosphate is dependent on availability of glutamine, which is formed
by glutamine synthetase (GTS). Glucosamine-6-phosphate is subsequently metabolized
by glucosamine 6-phosphate N-acetyltransferase (Emeg32) to N-acetylglucosamine-6phosphate, which is converted to N-acetylglucosamine-1-phosphate by phosphoacetylglucosamine mutase (Agm1) and the synthesis of UDP-uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) is catalyzed by UDP-N-acetylglucosamine pyrophosphorylase
(Uap1). Flux through the HBP can be increased with glucosamine, which is phosphorylated by hexokinase (HK) to form glucosamine 6-phosphate thereby bypassing GFAT. UDPGlcNAc is the obligatory substrate for OGT (uridine-diphospho-N-acetylglucosamine:
polypeptide β-N-acetylglucosaminyltransferase) leading to the formation of O-linked βN-acetylglucosamine (O-GlcNAc)-modified proteins. β-N-acetylglucosaminidase (OGA)
catalyzes the removal of O-GlcNAc from the proteins.
GFAT is considered to be the primary rate-determining step in the
HBP and exists in two isoforms (GFAT1 and GFAT2), transcribed from
separate genes. In the heart GFAT2 is the most abundant isoform [62]
and in rodent models of aging [23] and pressure overload hypertrophy
[63] there were significant increases in GFAT2 mRNA levels, but little or
no change in GFAT1 mRNA levels. In both studies there were also increased UDP-GlcNAc levels, consistent with an increase in HBP flux,
suggesting that GFAT expression and HBP flux in the heart are subject
to regulation in response to chronic stress. It is also of note that both
GFAT1 and GFAT2 are subject to phosphorylation by cAMP-dependent
protein kinase [64,65]; in addition both AMPK and CaMKII lead to phosphorylation of Ser243 of human GFAT1 resulting in increased enzyme
activity [66]. While our understanding of the regulation of GFAT activity
remains poorly understood, the fact that it appears to be a target for
AMPK, raises the possibility that it is a key link between AMPK, the
HBP and O-GlcNAc signaling.
Following its synthesis by GFAT, glucosamine-6-phosphate is metabolized via glucosamine 6-phosphate N-acetyltransferase (Emeg32),
phosphoacetylglucosamine mutase (Agm1) and finally UDP-Nacetylglucosamine pyrophosphorylase (Uap1) to UDP-GlcNAc (Fig. 1).
The HBP is often referred to as a “nutrient-sensing” pathway, and this
highlighted by the fact that in addition to glucose availability, UDPGlcNAc synthesis is dependent on amino acid metabolism, specifically
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V.M. Darley-Usmar et al. / Journal of Molecular and Cellular Cardiology 52 (2012) 538–549
the availability of glutamine; acetyl-CoA derived from either pyruvate
or fatty acid metabolism as well as high energy phosphates in the
form of ATP and UTP. Thus, the synthesis of UDP-GlcNAc is critically dependent on the coordinated action of several key metabolic pathways in
addition to glucose. The importance of the HBP and UDP-GlcNAc synthesis for cell viability is also emphasized by the fact that gene deletion
of either EMeg32 or Agm1 is embryonically lethal [67,68]; surprisingly
however, remarkably little is known about the regulation of HBP flux
in the heart or indeed in other tissues.
In addition to being a substrate for traditional glycosylation, UDPGlcNAc is also a substrate for OGT, a unique glycosyl transferase,
which catalyzes the attachment of O-GlcNAc to specific Ser/Thr residues of nuclear and cytoplasmic proteins. In humans and mice there
is a single OGT gene located on the X chromosome [69–71], which
through alternative splicing leads to three isoforms: 1) nucleocytoplasmic, ncOGT, (100 kDa); 2) short form, sOGT (78 kDa) and 3) a
mitochondrial form, mOGT (103 kDa); each isoform has the same
C-terminal catalytic domains, but different N-terminal domains contributing to different localization and targeting [69,70,72]. In muscle,
kidney and liver, OGT exists as a heterotrimer consisting of two 110 kDa
and one 78 kDa subunits [73]; in other tissues it is a homotrimer of three
110 kDa subunits [71]. The catalytic C-terminal domain is altered by tyrosine phosphorylation and is also a target for O-GlcNAcylation [71], suggesting a potential feedback regulatory element on OGT activity. Later
we will discuss in more detail the fact that insulin increases both tyrosine
phosphorylation and O-GlcNAcylation of OGT, leading to increased activity and transient translocation from nucleus to cytoplasm [74,75]. The Nterminal domain of OGT contains multiple tetratricopeptide repeats
(TPR), which mediate the protein–protein interactions involved in the
recognition and activation of specific protein targets [59,76].
The steady state level of protein O-GlcNAcylation is dependent on
the rate of synthesis catalyzed by OGT as well as its rate of removal
catalyzed by β-N-acetylglucosaminidase (O-GlcNAcase) [77,78]. As
with OGT, O-GlcNAcase is highly conserved across species and is
expressed in all tissues and unlike lysosomal hexosaminidases has a
pH optimum in the neutral range, pH 4.5–6 [77,78]. Also similar to
OGT, O-GlcNAcase is also encoded by a single gene, with at least
two alternative spliced isoforms [6]. The predominant splice variants
are, a long N100 kDa form, and a short form (~70 kDa) with the OGlcNAcase catalytic regions in the N-terminal domain [6,79]. The Cterminal region of the long isoform has a histone acetyl-transferase
like domain with a caspase cleavage site between the two regions
suggesting potential regulation in response to apoptosis; however,
cleavage per se does not appear to affect the O-GlcNAcase activity,
suggesting that this may have other regulatory roles, such as stabilization, localization or targeting [80–82]. The short form of OGlcNAcase has a smaller C-terminal region and lacks the putative HAT
domain found in the long form [6,79]; little is known regarding the different functions or localization of these two O-GlcNAcase isoforms, although the long isoform appears to exhibit greater catalytic activity
than the short isoform [79]. As with GFAT and OGT, our knowledge of
the regulation of O-GlcNAcase activity remains limited; however, it is
known to be both phosphorylated and modified by O-GlcNAc [78,80].
Changes in cellular O-GlcNAc levels occur rapidly, with a response
time of seconds to minutes in response to extracellular stimuli
[83,84], which not only distinguishes it from traditional forms of glycosylation, but also draws parallels with phosphorylation. Further
similarities to phosphorylation are emphasized by the fact that OGlcNAc also modifies Ser/Thr residues and in some cases phosphorylation and O-GlcNAcylation occur on the same residues. It is increasingly evident that there is a complex and extensive cross talk
between O-GlcNAcylation and phosphorylation; for example, Wang
et al., [85] reported that increasing overall cellular O-GlcNAc levels
by inhibition of O-GlcNAcase decreased phosphorylation at 280 sites
and increased phosphorylation at 148 sites. The potential for rapid cycling between O-GlcNAcylation and phosphorylation is further
emphasized by the observations that OGT and O-GlcNAcase forms
transient complexes with kinases and protein phosphatases [86,87].
3. Pharmacological and molecular modulation of O-GlcNAc turnover
Experimentally O-GlcNAc levels can be increased by activating
synthesis, which is commonly achieved by elevating extracellular glucose concentrations, adding extracellular glucosamine, or by preventing its removal by inhibiting O-GlcNAcase. Hyperglycemia (~ 25 mM
glucose) increases overall O-GlcNAc levels in many cell lines including neonatal and adult cardiomyocytes [34,45,46]. While some of
the effects of hyperglycemia in cardiomyocytes can be reversed by inhibition of GFAT with either 6-diazo-5-oxo-L-norleucine (DON) or Odiazoacetyl-L-serine (azaserine) [34,46], hyperglycemia clearly has
many other effects on cellular function other than increasing metabolism via the HBP. Additionally while azaserine and DON are commonly used as GFAT inhibitors, they are better characterized as
amidotransferase inhibitors [88]; thus, conclusions based on their
use should be made with caution. Glucosamine is readily transported
by the glucose transporter systems, with a Km of 2–4 mM for GLUT1
and GLUT4 [89], the primary glucose transporters in cardiomyocytes
and subsequently phosphorylated by hexokinase to glucosamine 6phosphate. Glucosamine 6-phosphate directly enters the HBP bypassing the key regulatory enzyme GFAT (Fig. 1), resulting in an increase
in UDP-GlcNAc and O-GlcNAc levels. In the perfused heart, 5 mM glucosamine leads to 2 to 3 fold increase in O-GlcNAc levels within
15 min [32] and as little as 100 μM glucosamine has been shown to
significantly augment O-GlcNAc levels in the heart [90]. Such studies
highlight the dynamic nature of the HBP and O-GlcNAc synthesis in
the heart; however, increasing UDP-GlcNAc levels can also impact
the synthesis of other glycoconjugates [91,92] and elevated levels of
other intermediates in the HBP have the potential to alter other metabolic pathways such as glycogen synthesis [93]. Nevertheless, at the
cellular level increasing OGT protein levels appear to have the same
effects on cardiomyocytes as adding glucosamine [33,94], suggesting
that the predominant effect of glucosamine, at least in the short
term, is to increase cellular O-GlcNAc levels. Several studies have
reported that the addition of glucosamine results in depletion of ATP
levels [95]; however, we have not observed any impact of glucosamine
treatment on ATP levels in either cardiomyocytes or the intact perfused
heart [34,36,90]. Furthermore, up to 10 mM glucosamine also had no
adverse effect on cardiac function [90].
PUGNAc [O-(2-acetamido-2-deoxy-D-glucopyranosylidene) aminoN-phenylcarbamate] is a widely used competitive inhibitor of OGlcNAcase [96,97] and by attenuating the removal of O-GlcNAc leads
to significant increases in cellular O-GlcNAc levels in isolated cardiomyocytes, the isolated perfused heart and in vivo [31,34,38,98]. Although a common tool for increasing O-GlcNAc levels PUGNAc is also
equally effective as an inhibitor of lysosomal hexosaminidases [99],
which raises concerns about is specificity. Recently two new classes of
O-GlcNAcase inhibitors have been described; GlcNAcastatin [100] and
NAG-thiazolines [99,101], both of which exhibit several orders of magnitude of greater specificity for O-GlcNAcase over other hexosaminidases and with Ki in vitro in the pico to nanomolar range. We have
successfully used the NAG-thiazoline derivatives 1,2 dideoxy-2′propyl-α-D-glycopyranoso-[2,1-d]-Δ2′-thiazoline (NAG-Bt) and 1,2
dideoxy-2′-ethylamino-α-D-glucopyranoso-[2,1-d]-Δ2′thiazoline
(NAG-Ae) to increase O-GlcNAc levels in the perfused heart and
cardiomyocytes [37]. In the perfused heart NAG-Bt had an EC50 of
~ 30 μM for increasing overall O-GlcNAc levels [37]; in recent studies, in cardiomyocytes we found that NAG-Ae, also known as
thiamet-G, had an EC50 of ~ 100 nM for increasing O-GlcNAc levels
[102]. In addition to effectively increasing O-GlcNAc levels in isolated cardiomyocytes and hearts, preliminary studies in vivo demonstrate that i.p. administration of thiamet-G at a dose of 50 mg/kg
resulted in a 2–3 fold increase in cardiac O-GlcNAc levels within
V.M. Darley-Usmar et al. / Journal of Molecular and Cellular Cardiology 52 (2012) 538–549
2 h of administration (Chatham data not shown). It is also worth
noting that Yuzwa et al., [103] reported that thiamet-G added to
drinking water was effective at increasing tissue O-GlcNAc levels;
however, they did not report on the O-GlcNAc levels in the heart.
We have observed no detrimental effects of O-GlcNAcase inhibitors
on cardiac function in the perfused heart; thus, there appears to be
no acute adverse effects of increasing cardiac O-GlcNAc levels by
inhibiting O-GlcNAcase.
While it is possible to pharmacologically increase O-GlcNAc levels
by either increasing synthesis or inhibiting degradation, there are
limited options for decreasing O-GlcNAc levels. The most common
approach has been to attenuate OGT protein levels at the cellular
level either by using siRNA methods [33], increase O-GlcNAcase expression [40] or to delete OGT completely using Cre-lox technology
[30]. In cell culture, OGT-null mouse embryonic fibroblasts (MEFs)
grow normally up to 48 h, after which growth slows and cell death
occurs around 4–5 days [30]. Watson et al., [104] reported the development of a cardiac specific, inducible OGT KO mouse and found that a reduction in OGT levels had no adverse effects under basal conditions, but
did accelerate the development of contractile dysfunction in response
to pressure overload. The lack of any effects of decreased OGT levels in
the absence of additional stress was somewhat surprising, given that
OGT gene deletion typically leads to cell death [30]. The reason for
this lack of phenotype is unclear and could be linked to transduction
mosaicism or incomplete penetrance, which can occur in such inducible models, or that while OGT levels are markedly lower, that there
has been an adaptive response leading to decreased O-GlcNAcase
protein, so that turnover rates of O-GlcNAc may not be significantly
altered. Certainly in other cell systems deletion of OGT leads to a reduction in O-GlcNAcase protein, in what seems to be an attempt to
maintain O-GlcNAc levels constant [30]. However, it is also possible
that since cardiomyocytes are essentially terminally differentiated,
that they are able to survive with very low levels of OGT until subject
to additional stress.
The lack of high affinity specific inhibitors of OGT has represented
a significant limitation in investigating the role of O-GlcNAc on cardiomyocyte function. Walker and colleagues identified potential OGT
inhibitors, using in vitro high throughput screening approaches
[105]. These compounds were subsequently commercially available
as OGT inhibitors (TimTec, LLC); however, while there have been
some reports in cell-based systems demonstrating the effectiveness
of these compounds in lowering O-GlcNAc levels [39,106], they
have not yet been fully validated as OGT inhibitors and thus should
be used with caution. Indeed our experience with these compounds
in decreasing O-GlcNAc levels cardiomyocytes have been unsuccessful and we found that in an isolated perfused heart model as little of
5 μM of “TT04” resulted in a significant decline in cardiac function
(Chatham, data not shown). Recently, Gloster et al. [107] described
a novel sulphonated UDP-GlcNAc analog, which inhibited OGT in
vitro with Ki of 8 μM and significantly lowered O-GlcNAc levels in
COS-7 cells. Clearly further studies are needed to better characterize
such inhibitors, but if their specificity can be fully validated and any
effects on other glycosylation reactions characterized, the availability
of these or similar compounds could be a useful addition for the pharmacological modulation of cardiomyocyte O-GlcNAc turnover.
4. Identifying and characterizing O-GlcNAcylated proteins
4.1. Probing for protein O-GlcNAc modification with antibodies, enzymatic labeling, and metabolic labeling
While it is increasingly appreciated that O-GlcNAcylation is an
abundant PTM modification it has been very difficult to detect due
to the low stoichiometry of modification with an estimated 5–10% occupancy at a particular site [108]; the fragile nature of the O-linkage
during tandem mass spectrometric (MS/MS) sequencing, and the
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lack of tools available to study this modification. However, recent
breakthroughs in enrichment strategies and mass spectrometric sequencing approaches have greatly facilitated the unambiguous identification of O-GlcNAc modified peptides. These techniques are revealing
the ubiquitous and dynamic nature of this posttranslational modification and provide direct evidence that this previously overlooked modification provides an additional layer of posttranslational regulation of
protein function (Table 1).
Pan-specific anti-O-GlcNAc antibodies (CTD110.6, Pierce; RL2
Covance) have proven invaluable for monitoring changes in global
protein O-GlcNAc modification by immunoblotting or for probing
for the presence of the modification on a specific protein of interest.
However, these antibodies vary greatly in their specificity and sensitivity toward different OGT substrates and thus recognize only a subset of O-GlcNAc modified proteins. To overcome this limitation, Teo et
al. [109] have recently generated a suite of monoclonal anti-O-GlcNAc
antibodies, now available from Millipore, which were combined for
the immunoprecipitation of a more comprehensive pool of O-GlcNAc
modified proteins. Identification of these proteins by mass spectrometry revealed over 200 candidate O-GlcNAc modified proteins [109].
These new antibodies represent much needed new tools that when
coupled to MS/MS should facilitate the simultaneous identification of
both the modified protein and the sites of modification.
Recent developments in chemoenzymatic techniques, where the
O-GlcNAc moiety is enzymatically tagged with a chemically reactive
handle, are enabling large-scale mass spectrometry-based proteomic
analyses of O-GlcNAc modified proteins. The enzymatic transfer of
[ 3H] galactose by β-1,4-galactosyltransferase (GalT), was originally
utilized as a sensitive and definitive alternative to western blotting
with antibodies or HRP-conjugated lectins to confirm that a protein
of interest possesses the O-GlcNAc modification [110]; however, the
catalytic activity of GalT is now being exploited for chemoenzymatic
tagging of GlcNAc using a genetically engineered GalT1 (Y298L) enzyme which accommodates larger chemically reactive analogs of
UDP-galactose, UDP-N-azido-acetylgalactosamine (UDP-GalNAz) or
UDP-keto-galactose [111]. Once the azido-sugar is incorporated it
is reacted by azide-alkyne Huisgen cycloaddition (“click chemistry”) in the presence a copper catalyst with biotinylated or otherwise functionalized tags thereby enabling visualization and affinity
purification [112]; specificity for O-GlcNAc is obtained by cleaving
N-linked glycoproteins with PNGase F. This approach has been utilized to identify 32 putative O-GlcNAc modified proteins [113].
Cell membranes are permeable to per-O-acetylated Nazidoacetylglucosamine (Ac4GlcNAz), which once inside the cell is
deacetylated to GlcNAz by intracellular esterases; this approach has
been developed to incorporate chemically reactive sugars into OGlcNAcylated proteins in intact cells. Initially endogenous enzymes
in the GlcNAc salvage pathway were exploited to convert GlcNAz to
UDP-GlcNAz and GlcNAz is then transferred to serine and threonine
residues of substrate proteins by OGT. However, to overcome the low
level of incorporation of GlcNAz into proteins, Bertozzi and coworkers
have used N-azidoacetylgalactosamine (GalNAz) as a source of GlcNAz
for metabolic labeling [114]; GalNAz can be converted to UDP-GlcNAz
by UDP-galactose 4′-epimerase (GALE); following epimerization, UDPGlcNAz is incorporated into O-GlcNAc modified proteins by OGT. This approach appears to be much more robust for the metabolic labeling of OGlcNAc modified proteins [115]. The resulting azido sugar is then reacted
with phosphine containing functionalized linkers for the enrichment and
detection of GlcNAz containing proteins [116,117]. Pratt and colleagues
recently published a modification of the metabolic labeling strategy,
where the GlcNAc analog contains a reactive alkyne (GlcNAlk) rather
than an azide. The UDP-GlcNAlk is less susceptible to epimerization to
the UDP-galactosamine analog and can be reacted by click chemistry
with azide containing probes [118].
One drawback to chemoenzymatic and metabolic labeling is that
the biotin-tagged O-GlcNAc modified peptide itself is rarely identified
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Table 1
Methods utilized for the characterization of O-GlcNAc modified proteins.
Methodology for the enrichment and
detection of O-GlcNAc modified proteins
O-GlcNAcylated proteins
identified (no.)
Identification of O-GlcNAc
modification site (no.)
Combined with
quantitative methods
References
Tissue or cell type
Anti-O-GlcNAc immunoprecipitation
and detection by LC–MS/MS with CID or ETD.
Yes (215)
Yes (51)
No
No
Yes
Yes (SILAC)
[109]
[162]
HEK293 cells
Cos-7 cells
Lectin Weak Affinity Chromatography (LWAC)
and detection by LC MS/MS with ETD.
Yes
Yes
Yes
Yes (62)
Yes (145)
Yes (58)
Yes
Yes (142)
Yes (iTRAQ)
Yes (SILAC)
[123]
[124]⁎
[126]
[128]
Brain cortex
Brain cortex
Brain
Embryonic stem cells
Yes
Yes
Yes
Yes
Yes
No
No
Yes (ETD)
No
Yes (BEMAD)
Yes (iTRAQ)
[163]
[164]
[165]
[113]
[127]
Brain
Xenopus oocytes
Brain
MCF-7 cells
Erthyrocytes
Chemoenzymatic labeling: enzymatic
incorporation of a reactive sugar
analog followed by click chemistry.
Detection by LC–MSMS with CID or ETD.
(25)
(20)
(pilot study)
(32)
(25)
Chemoenzymatic labeling: enzymatic
incorporation of a reactive sugar analog
followed by click chemistry with a photocleavable
biotin linker. Detection by LC MS/MS with ETD.
Yes (64)
Yes (141)
Yes (SILAC)
[125]⁎
Hela cells
Metabolic labeling in intact cells followed by
gel-based separation and LC–MS/MS
Yes (199)
Yes (pilot study)
Yes (374)
No
No
No
Yes
No
No
[116]
[115]
[118]
Hela cells
Jurkat cells
NIH–3T3 cells
Asterisk indicates studies amenable to simultaneous identification of the sites of phosphorylation.
by tandem mass spectrometry. Therefore, these approaches typically
yield a list of putative candidate proteins for which the presence
and site(s) of O-GlcNAc modification must be confirmed in further
studies; however, adaptations of the chemoenzymatic labeling strategy, which permit mapping the sites of O-GlcNAc modification are
described below. A chemoenzymatic labeling approach has also
been developed to monitor dynamic changes in the stoichiometry of
O-GlcNAc modification on a protein of interest [119]. This involves labeling O-GlcNAc with a large tag that increases the molecular weight
of the protein sufficiently to be resolved by SDS-PAGE. While this approach does not reveal the percent occupancy of O-GlcNAc modification at a particular amino acid, it offers a mechanism to screen for
ligands or physiological conditions that influence the extent of
mono-, di-, or tri-O-GlcNAc modification of a given protein. This approach provides an alternative to using site-specific anti-O-GlcNAc
antibodies, high-resolution mass spectrometry, or radiolabels for the
interrogation of crosstalk between O-GlcNAc and other posttranslational modifications.
4.2. Detection of the sites of O-GlcNAc modification by tandem mass
spectrometry (MS/MS)
The standard approach used to identify sites of posttranslational
modification, is to digest the protein of interest using trypsin or
other proteases. The resulting peptides are fragmented and peptide
sequence and site(s) of modification can then be identified from the
fragmentation pattern of the peptide by MS/MS. The O-GlcNAc modification, however, is not stable during conventional MS/MS approaches; upon collision with gas molecules, the GlcNAc moiety is
lost from the Ser/Thr side chain prior to fragmentation of the peptide
backbone precluding direct detection of the site of modification. Thus,
O-GlcNAc as well as other fragile modifications go undetected during
routine mass spectrometric analysis. The reproducible neutral loss of
the monosaccharide from the peptide and/or generation of the GlcNAc
oxonium ion (204.2 Da) may indicate the presence of an O-GlcNAc
modified peptide and the mass spectrometer can be programmed to acquire additional data on peptides that exhibit this behavior [108]; nevertheless, the specific modification site cannot be identified.
Recent advances in alternative fragmentation approaches, electron capture dissociation (ECD) and electron transfer dissociation
(ETD), permit sequencing of peptides with fragile modifications,
such as O-GlcNAc [120]. The availability of ETD capability on commercially available ion trap and Orbitrap instruments and the continued
development of algorithms for searching ETD MS/MS data against
protein databases are enabling detection of this evasive modification
[121,122]. With a mass spectrometric method now available that allows
direct identification of O-GlcNAc modified peptides and unambiguous
determination of the site of modification, the remaining challenge is selectively enriching O-GlcNAc modified peptides from complex mixtures
to overcome the limited dynamic range of the instrumentation.
4.3. Enrichment of O-GlcNAcylated peptides for ETD MS/MS
To facilitate the identification of the sites of modification, OGlcNAcylated peptides can be immunoprecipitated with pan-O-GlcNAc
antibodies, enriched with wheat germ agglutinin (WGA) or succinylated
WGA lectins, or affinity purified following chemoenzymatic labeling. The
older O-GlcNAc antibodies, RL2 and CTD110.6, were of limited utility
for immunoprecipitation due to either recognition of a limited number of proteins or relatively poor elution efficiency; however the
newer antibodies appear to have improved properties for immunopurification of O-GlcNAcylated peptides and proteins [109].
During lectin weak affinity chromatography, a long WGA agarose column (N3 m in length) is utilized to separate O-GlcNAc modified peptides
from unmodified peptides [123,124]. WGA lectin interacts predominately
with the N-acetylhexosamines (HexNAc), N-acetylglucosamine (GlcNAc)
and N-acetylgalactosamine (GalNAc). Highly glycosylated peptides are
retained on the column while the migration of monosaccharide
containing peptides is weakly retarded. Mass spectrometric analysis of the enriched mono-glycosylated peptides reveals the site of
attachment at Asn or Ser/Thr but does not distinguish GlcNAc
from GalNAc. The advantage of this approach is its simplicity and
coupled with electron transfer dissociation MS/MS, the sites of Olinked HexNAc are directly identified without the need for enzymatic labeling or chemical derivatization. Recent studies have also
coupled this enrichment approach to quantitative proteomic strategies to identify sites of O-GlcNAc modification that are dynamically regulated [125,126].
Adaptations to the chemoenzymatic tagging approach, implemented
specifically to enable mass spectrometric identification of biotin-tagged
O-GlcNAc modified peptides, offer an alternative enrichment strategy
that can be coupled to quantitative proteomic methodology. To
V.M. Darley-Usmar et al. / Journal of Molecular and Cellular Cardiology 52 (2012) 538–549
overcome the difficulties in eluting biotinylated peptides from streptavidin beads and the inefficient backbone fragmentation of biotinylated peptides during MS/MS, peptides can be released from streptavidin beads by
base catalyzed β-elimination [127] or by the use of a photocleavable biotinylated alkyne crosslinker [125]. One caveat of β-elimination is that
this results in the release of peptides that no longer contain an OGlcNAc tag, thus further studies must be performed to validate
the site(s) of modification. In contrast, following photolysis the
photocleavable linker leaves behind a tag that provides a signature fragmentation pattern by MS/MS and enables assignment of the site of
modification. Although the chemoenzymatic tagging approach requires
multiple sample preparation steps, advantages of this workflow include
the ability to couple this approach to established quantitative proteomic
procedures.
The use of isobaric tags (iTRAQ) or stable isotopic labeled amino
acids in cell culture (SILAC) prior to enrichment of O-GlcNAcylated
peptides permits the simultaneous identification of the site and relative quantitation of the extent of O-GlcNAc modification following a
stimulus [126,128,129]. These studies require a high mass accuracy
tandem mass spectrometer with the capability to fragment peptides
by collision induced dissociation and electron transfer dissociation.
The ability to couple quantitative proteomic approaches to the sitespecific identification of O-GlcNAc modification will permit the unbiased discovery of the temporal dynamics and regulation of this modification during signal transduction. While alterations in the patterns
of protein O-GlcNAc modification have been observed under varying
physiological conditions in the heart, to date these proteins have
not been identified. As sites of protein O-GlcNAc modification are
published, they are made publically available in the Cell Signaling
Phosphosite database [130] and dbOGAP [131]. The dbOGAP repository also contains information regarding known sites of phosphorylation
that are in close proximity to the sites of O-GlcNAc modification. Large
scale site-mapping experiments are revealing a loose consensus sequence and this information has been utilized to generate an algorithm
for predicting sites of O-GlcNAc modification [131]. It has been estimated that with the emergence of these new MS and enrichment strategies,
the total number of O-GlcNAc modification sites mapped over the past
2 years is greater than those identified during the past 20 years [3].
543
attenuate glucose entry. This is also consistent with the widely accepted
notion that chronically elevated cellular O-GlcNAc levels is a contributing factor to the etiology of insulin resistance as well as a key mediator
of glucose toxicity associated with the adverse effects of diabetes on
numerous tissues including cardiomyocytes [5,21,27,55]. It is of note
that GFAT which regulates glucose flux into the HBP, is phosphorylated
and activated by AMPK [66] and since O-GlcNAcylation is reported to
increase AMPK activity, this could represent a feed forward mechanism, which if sustained may also contribute to O-GlcNAc mediated
glucotoxicity (Fig. 2).
While the studies highlighted above demonstrate a clear link between
the HBP, O-GlcNAcylation levels and glucose metabolism it is striking that
the focus is primarily on relatively long term regulation with an emphasis
on the pathogenesis of insulin resistance and glucotoxicity. However, as
noted above changes in O-GlcNAcylation can occur rapidly and we have
demonstrated that relatively acute increases in O-GlcNAc levels can modulate the responses of cardiomyocytes and the intact heart to stress and
agonist stimulation [32,47,83,84]. This raises the question as to whether
short term modulation of O-GlcNAc synthesis in the heart, could influence
cardiac metabolism and substrate selection. To address this question, we
recently examined the effects of glucosamine (0–10 mM) on substrate
utilization in the intact heart perfused with physiologically relevant
concentrations of glucose, lactate, pyruvate, palmitate and insulin [90].
Perfusion for 60 min with as little as 50 μM glucosamine significantly increased both UDP-GlcNAc and O-GlcNAc levels. While there are potential pathways for glucosamine metabolism, which could lead to an
increase in glycolysis [141,142], we found that glucosamine had no effect on either glucose oxidation or glycolytic flux [90]. Surprisingly,
however, we found a significant increase in palmitate oxidation with a
concomitant decrease in lactate and pyruvate oxidation reaching a
maximal effect at 100 μM glucosamine. In contrast to Luo et al., who
reported that glucosamine stimulated fatty acid oxidation in cultured
adipocytes was associated with increased AMPK and ACC phosphorylation [140], we observed no effect of glucosamine on either AMPK or ACC
phosphorylation. On the other hand, membrane levels of FAT/CD36,
which plays a key role in fatty acid transport in the heart, were significantly increased; furthermore, preliminary data suggested that FAT/CD36 is
also a potential target for O-GlcNAcylation [90].
5. O-GlcNAcylation as a metabolic sensor and regulator
The HBP has long been recognized as a glucose-sensing pathway
[6,26,132], but as noted earlier since it intersects with several key
metabolic pathways it might be more accurately characterized as nutrient or metabolic sensing pathway. Increased flux through the HBP
was first linked to the development of cellular insulin resistance in
1991 [61] and has been further supported by studies demonstrating
that glucosamine infusion in vivo resulted in skeletal muscle insulin
resistance [93,133,134] and also that GFAT overexpression in mice
leads to insulin resistance [135,136]. Glucosamine induced insulin resistance is also mediated by increased O-GlcNAcylation of IRS-1 and
IRS-2 [134]. Increased cellular O-GlcNAc levels also decreased insulin
stimulated GLUT4 translocation, reduced insulin-stimulated phosphorylation of IRS-1 and Akt as well as increased O-GlcNAc modifications of
GLUT4, IRS-1 and Akt2 [137]. It is worth noting, however, that at the cellular level increased O-GlcNAcylation does not appear to be essential for
the development of glucose/insulin-induced insulin resistance [138].
The syntaxin 4 binding protein, Munc18c is also modified by OGlcNAc [139]; Munc18c contributes to membrane fusion events, involved in insulin-stimulated GLUT4 translocation and its modification
by O-GlcNAc attenuated the response to insulin [139]. Chronic activation of the HBP also resulted in O-GlcNAcylation of AMPK, which was associated with increased activity and increased fatty acid oxidation [140].
Taken together, these data support the concept of O-GlcNAcylation as
glucose sensing mechanism, and that elevated O-GlcNAc levels on several different key regulatory proteins, acts in a feedback manner to
Fig. 2. Acute and transcriptional regulation of cardiac metabolism mediated by the
hexosamine biosynthesis pathway (HBP) and O-GlcNAc. Flux through the HBP is regulated by glutamine-D-fructose 6-phosphate amidotransferase (GFAT) and is dependent
on the availability of glucose and glutamine. O-GlcNAc transferase (OGT) is subject to
both O-GlcNAcylation (green circles) and phosphorylation (red circles) mediated by
insulin, both of which have been reported to increase its activity. AMPK, which is
well recognized as a key regulator of cellular metabolism has been reported to be a target
for O-GlcNAcylation and that this increases its activity; furthermore, GFAT has been shown
to be phosphorylated by AMPK also leading to increased activity. This provides a feed forward
loop leading to a further increase in OGT activity and subsequent increase in O-GlcNAcylation.
Increases in O-GlcNAc levels can potentially acutely regulate cardiac metabolism, not only by
modification of AMPK, but also by O-GlcNAcylation of other key mediators of carbohydrate
and fatty acid metabolism. A number of transcription factors, which play a critical role in
the longer-term regulation of metabolism, are also known to be O-GlcNAcylated.
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The spontaneously hypertensive rat (SHR), is a widely used geneticmodel of hypertension, which expresses a different cd36 mRNA compared to Wistar controls rats and in the SHR heart, CD36 protein levels
at plasma membrane are reduced [143]. Earlier studies by Des Rosiers
and colleagues showed that the contribution of exogenous long-chain
fatty acids to β-oxidation was significantly reduced in hearts from SHR
rats [144] and they recently reported that this could be due to alterations
in post-translational modifications of CD36 protein including changes in
O-GlcNAc modification [145]. Consistent with our observations, they
found that in hearts from control Wistar rats, 0.5 mM glucosamine increased fatty acid oxidation N2 fold, but had no effect on fatty oxidation
in hearts from SHRs, despite the fact that overall O-GlcNAc levels were increased to the same extent. They also reported that based on the predicted sequences for the wild-type and mutant CD36, the mutant CD36
had fewer potential sites for post-translational modification, including
reduced O-GlcNAc modification sites. Further support for the HBP and
by extension O-GlcNAc in regulating cardiac metabolism, was provided
by the observation that glutamine, which is required for GFAT activity
(Fig. 1), also increases long-chain fatty acid metabolism and this can be
reversed by inhibition of the HBP with azaserine [146]. Taken together
these studies suggest a novel mechanism by which the HBP and OGlcNAcylation could play a role in the acute regulation of cardiac metabolism. Specifically, these data have led us to postulate that an increase in
glucose flux through the HBP, resulting in increased O-GlcNAcylation of
CD36, leads to increased fatty acid transport and oxidation, which
would have an indirect feedback effect on decreasing carbohydrate
oxidation.
Young and colleagues have shown that cardiac metabolism exhibits
time of day dependent changes in both fatty acid and glucose metabolism
which was associated with diurnal variations in total protein OGlcNAcylation and abolished in hearts from cardiac clock mutant
(CCM) mice [147]. Specifically they found that during the active phase
there was increased myocardial protein O-GlcNAcylation suggesting increased flux of glucosyl moieties through the hexosamine biosynthetic
pathway. Thus the cardiomyocyte circadian clock regulates nonoxidative
fatty acid (i.e. triglyceride) and glucose (i.e. glycogen and O-GlcNAc) metabolism [148]. They also found that increased protein O-GlcNAcylation
results in altered gene and protein expression of cardiomyocyte circadian
clock components, as well as protein O-GlcNAcylation of CLOCK, raising
the possibility that non-oxidative glucose metabolism is an integral component of the cardiomyocyte circadian clock, potentially mediated via
protein O-GlcNAcylation.
Yang et al., [74] provided further support for a close link between OGlcNAc and metabolic regulation, by showing that insulin treatment
leads to translocation of OGT from the nucleus to the plasma membrane,
where it catalyzes the O-GlcNAcylation of key components of the insulin
signaling pathway thereby attenuating insulin signal transduction. It was
postulated that this represented one of the mechanisms contributing to
O-GlcNAc mediated insulin resistance; however, these data also suggests
a novel feedback loop, which could contribute to the short-term regulation of cellular metabolism. While further studies are clearly needed to
demonstrate that HBP flux and O-GlcNAc turnover play a central and coordinated role in regulating cardiac metabolism it is clear that there is a
close relationship between the HBP, O-GlcNAcylation and both acute regulation of myocardial substrate utilization as well as transcriptionally
mediated regulation as summarized in Fig. 2.
6. Mitochondria as an integrator of redox and O-GlcNAc signaling
It is well established that mitochondria play a central role in integrating cellular energy metabolism, redox signaling and cell survival
pathways; however, the role of O-GlcNAcylation in regulating these
processes is poorly understood. The notion that mitochondrial proteins might be potential targets for O-GlcNAc modification was first
raised by the identification of a specific mitochondrial isoform of
OGT (mOGT), which is localized to the inner membrane of
mitochondria and contains mitochondrial targeting sequence [72].
While initial reports suggested that mOGT had either limited catalytic
activity or that substrates for OGT in the mitochondria were very limited [11,72], more recently, a number of studies have suggested that
O-GlcNAcylation of mitochondria proteins contributes to both to
acute cardioprotection [33,38] as well as mitochondrial dysfunction
and increased programmed cell death [45,110].
Some of the first evidence to suggest that O-GlcNAc had a direct effect
on mitochondrial function was the demonstration that acute increases in
O-GlcNAc levels in the heart or in isolated cardiomyocytes, attenuated
mPTP opening, a critical step in the initiation of programmed cell death
[33,38,39,149]. For example, mitochondria isolated from hearts of
mice treated with the O-GlcNAcase inhibitor, PUGNAc were resistant
to Ca2+-induced mPTP formation as assessed by mitochondrial swelling
assay. Furthermore, overexpression of OGT in neonatal cardiomyocytes
also decreased Ca2+-induced mitochondrial swelling; whereas, OGT inhibition increased mitochondrial swelling [38,39]. VDAC has been identified as a potential O-GlcNAc target and it has been hypothesized that
O-GlcNAcylation of VDAC preserves mitochondrial integrity by interfering with mPTP formation [38]. In addition we have shown that increasing overall O-GlcNAc levels either by increasing O-GlcNAc synthesis with
glucosamine, and increased OGT expression or decreasing O-GlcNAc degradation by inhibiting O-GlcNAcase all attenuated H2O2-induced loss of
mitochondrial membrane potential and cyctochrome c release [33],
which was associated with increased mitochondrial Bcl-2 levels. Since
Bcl-2 inhibits the mPTP opening possibly by interaction with VDAC, one
of the putative components of mPTP [150,151]. However, given that
VDAC is not required for mPTP opening and the continued uncertainty
regarding the molecular composition of the mPTP [152], whether OGlcNAcylation of VDAC directly contributes to the attenuation of mPTP
opening, has yet to be verified.
Despite the apparent protective effects associated with increasing mitochondrial O-GlcNAc levels, it was initially reported that overexpression
of mOGT was toxic to cells [72]. More recently, Shin et al. [110] developed
an inducible expression system enabling selective expression of either
mOGT or ncOGT isoforms in mammalian cells. They reported that increasing mOGT levels induced apoptosis in both a human embryonic kidney fibroblast cell lime (EcR-293) and insulinoma derived INS-1 cell lines. The
extent to which this would be applicable to the heart or cardiomyocytes
is not known; however, given the high energetic demand of the heart
and the importance of tight control of oxidative metabolism, it is likely
that dysregulation of mitochondrial O-GlcNAcylation would also be detrimental to cardiomyocytes. Evidence in support of this was provided by
Hu et al. [45], who reported that hyperglycemia induced mitochondrial
dysfunction in cardiomyocyte was mediated via O-GlcNAcylation of mitochondrial proteins. Importantly they identified several mitochondrial
proteins as targets for O-GlcNAc modification that are members of respiratory complexes, such as NDUFA9 of complex I, and the mitochondrial DNA-encoded, subunit I of Complex IV (COX I). They also found
that hyperglycemia induced increases in mitochondrial O-GlcNAc levels
resulted in impaired activity of Complex I, III and IV and that this could
be reversed by increasing expression of O-GlcNAcase.
The study by Hu et al. [45] was the first to show that in cardiac mitochondria, proteins in the respiratory complexes are modified by OGlcNAc and that this may contribute to impaired function induced by
hyperglycemia. However, a number of key questions remain, including
the regulation of mitochondrial O-GlcNAc synthesis and the mechanism
by which removal of O-GlcNAc from mitochondrial proteins is catalyzed. Earlier reports speculated that the very low levels of mitochondrial O-GlcNAcylation could be due to limited substrate availability,
i.e., UDP-GlcNAc [72]. The fact that even under basal conditions there
was evidence of mitochondrial GlcNAcylation suggests that is not the
case in the heart; however, this leaves open the question whether
UDP-GlcNAc is actively transported into mitochondria, as it is in the
ER and Golgi [153,154] and the extent to which availability of UDPGlcNAc is rate limiting for mitochondrial O-GlcNAc synthesis? Perhaps
V.M. Darley-Usmar et al. / Journal of Molecular and Cellular Cardiology 52 (2012) 538–549
even more important for our understanding of O-GlcNAc cycling in the
mitochondria is the mechanism by which O-GlcNAc is removed from
mitochondrial proteins. Hu et al. [45] reported low levels of OGlcNAcase activity in mitochondria isolated from mouse hearts and
showed that increasing cellular levels of O-GlcNAcase attenuated the
hyperglycemia induced increase in mitochondrial O-GlcNAc levels.
However, as far as we are aware, no mitochondrial protein has been
shown to have the hexosaminidase activity required to catalyze the removal of O-GlcNAc from proteins; in other words to date only one half
of the O-GlcNAc cycle in the mitochondria, namely mOGT has been
identified.
While it is well established that hyperglycemia leads to increased
O-GlcNAc levels in multiple cells and organs including the intact heart
and cardiomyocytes, it is important to note that unlike glucosamine
which is primarily metabolized via the HBP, increasing glucose levels
has multiple effects, including increased oxidative stress. Indeed it
has been reported that hyperglycemia leads to increase generation
of mitochondrial superoxide and it is this increase in reactive oxygen
species (ROS), which leads to activation of the HBP and increased OGlcNAc synthesis [48,49]. It has also been proposed that mitochondrial superoxide inhibits GAPDH, which further contributes to the increased flux of glucose through the HBP, by attenuated glycolysis
[48,49]. However, since relatively little is known regarding the regulation of GFAT, OGT, and O-GlcNAcase, we cannot rule out the possibility
that ROS could directly modulate their activities. Thus, hyperglycemia
has the potential to increase O-GlcNAc levels both by directly increasing
glucose flux through the HBP as well as by ROS mediated increase in
pathways leading to O-GlcNAc synthesis.
While there are clearly many gaps in our knowledge regarding the
regulation of mitochondrial O-GlcNAcylation it is increasingly evident
that mitochondrial proteins are targets for O-GlcNAc modification.
Our current understanding of effects of altered O-GlcNAc levels on
mitochondria function is summarized in Fig. 3. There is no doubt that
in response to an acute stress, including oxidative stress there is an increase in cellular O-GlcNAc levels, and pharmacological approaches to
augment this increase either by activating HBP flux or inhibiting OGlcNAcase activity, increases tolerance of mitochondria to oxidative stress
and thereby improves cell survival. In cardiomyocytes this increased tolerance to oxidative stress as discussed above is associated with increased
O-GlcNAc levels on specific mitochondrial proteins, such as VDAC. In the
case of a chronic stress such as that seen with hyperglycemia, there can
be both a direct increase in O-GlcNAc synthesis due to increase glucose
metabolism via the HBP as well as activation of the HBP mediated by increased mitochondrial ROS. Under sustained conditions, this has the potential to establish a feed forward loop where impaired mitochondrial
function leads to greater ROS production and this could further augment
the increase in O-GlcNAcylation, contributing to further mitochondrial
dysfunction and ultimately cell death. Hu et al. [45] demonstrated that
in cardiomyocytes there is a clear association between hyperglycemia, increased O-GlcNAcylation of key respiratory chain proteins and impaired
mitochondrial function.
The above discussion has focused on direct O-GlcNAc modification
of mitochondrial proteins; however, it is should be recognized that
changes in O-GlcNAc turnover may have indirect effects on mitochondrial function via O-GlcNAcylation of proteins that could influence mitochondrial function, but are not themselves mitochondrial
proteins. One potentially intriguing example is GSK-3β, where increased O-GlcNAcylation attenuates its activity [76]. Furthermore,
the cytoprotection associated with stress-induced increase in OGlcNAc was reported to be due to increased heat shock protein expression as a consequence of O-GlcNAc-mediated inhibition of GSK3β [30]. This is may be particularly relevant in the context of ischemic
cardioprotection where GSK-3β is a major convergence point for multiple cardioprotection strategies as well as a key effector for activation
of downstream targets [155–157], including attenuation of the mitochondrial permeability transition pore (mPTP).
545
Fig. 3. The intersection between O-GlcNAcylation, ROS and mitochondrial function. In
response to an acute stress (upper panel), including that induced by ROS, there is a
rapid increase in cellular and potentially mitochondrial O-GlcNAcylation as a result of
either an increased flux through the hexosamine biosynthesis pathway (HBP) and/or
decreased O-GlcNAcase activity (OGA). This acute increase in O-GlcNAc levels has
been shown to improve tolerance of mitochondria to oxidative stress and attenuate
loss of mitochondrial membrane potential leading to increased cell survival. Strategies
designed to acutely augment this response have been shown to improve cell survival;
conversely decreased O-GlcNAc levels lead to decreased cell survival. Chronic increases
in cellular O-GlcNAc levels are typically associated with metabolic disease such as diabetes and the resulting hyperglycemia. Sustained increases in glucose levels can increase O-GlcNAc synthesis by directly increasing flux through the HBP as well as
indirectly, as a result of hyperglycemia induced ROS production, which has also been
shown to stimulate O-GlcNAc synthesis (lower panel). Hyperglycemia is also known
to lead to impaired mitochondrial function in part due to increased O-GlcNAcylation
of mitochondrial proteins, which could in turn further increase ROS production and
thereby in a feed forward manner continue to increase O-GlcNAc levels with an even
greater decline in mitochondrial function. This O-GlcNAc mediated impairment of mitochondrial function, could then increase the vulnerability to additional stress thus
leading to increased cell death. Indeed, targeted overexpression of mitochondrial
OGT has been shown to lead to increased apoptosis.
7. O-GlcNAc and redox cell signaling
In addition to being increased in response to various stress stimuli
the generation of reactive oxygen species (ROS) is an inevitable
byproduct of normal mitochondrial metabolism. Rather than just
leading to non-specific modifications of lipids and proteins, it is widely recognized that ROS and reactive nitrogen species (RNS) play an integral role in regulating redox cell signaling pathways [158,159]. An
emerging concept in redox biology is the concept of “redox tone”
[158,159], where cell signaling, metabolism, and cell survival outcomes
are linked to the antioxidant and thiol status of cells. Interestingly, several links between O-GlcNAc protein modification and oxidative stress
suggest that O-GlcNAc signaling may represent an important mechanism that integrates both metabolic and redox signaling and thereby
modulate redox tone.
One example of the intersection between redox signaling and
O-GlcNAc is nitric oxide synthase (NOS), which is subject to both
phosphorylation and O-GlcNAcylation and is responsible for the
generation of nitric oxide (NO), one of the most widely recognized reactive species involved in redox signaling. Endothelial NOS (eNOS) is
activated by Akt-mediated phosphorylation of Ser1177, which increases its activity; however this same site it also a target for OGlcNAc modification, leading to lower activity and decreased NO production [49]. The interaction between O-GlcNAc and redox signaling
can also occur at the transcriptional level; for example the transcription factor FoxO1, which is activated by O-GlcNAcylation [160] also
regulates the transcription of oxidative stress responsive enzymes,
such as catalase and MnSOD. Taken together these observations highlight the potential role of O-GlcNAc turnover integrating both acute
and transcriptional responses to oxidative stress thereby playing a
key role in regulating redox signaling.
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8. Conclusions
Our understanding of the potential importance of protein OGlcNAcylation in mediating the function and stress response of the
heart and vascular system has only emerged over the past 5–
10 years. This is highlighted by the fact that a PubMed search combining heart/cardiac/vascular with O-GlcNAc yields a total of only 65 papers, of which 51 or 78% have been published in the last 5 years.
Studies to date have demonstrated that consistent with other tissues
and organs changes in overall levels of O-GlcNAcylation occur rapidly
in response to various stimuli emphasizing the dynamic nature of OGlcNAc synthesis and removal. In agreement with much of the earlier
literature on O-GlcNAcylation in other biological systems, increased
O-GlcNAc levels have been linked to the adverse effects of metabolic
disease, especially diabetes, on both cardiomyocyte and vascular
function. There is also emerging evidence to suggest that dysregulation in O-GlcNAc cycling in vascular smooth muscle may be associated with hypertension; however, further work is needed to establish a
true causal relationship. There is also substantial evidence demonstrating that acute augmentation of cardiac O-GlcNAc levels either
by increasing synthesis or inhibiting degradation affords significant
ischemic cardioprotection and this has been demonstrated at the cellular level, the perfused heart and in vivo. While much of the emphasis on studies of O-GlcNAcylation in the heart have been focused on
its role in mediating the response to various pathophysiological stresses, recent data has emerged demonstrating that acute and relatively
subtle changes in O-GlcNAcylation can directly modulate cardiac metabolism, illustrating that it also contribute to the regulation of normal physiological processes.
Parallels are frequently drawn between phosphorylation and OGlcNAcylation, and while this is not surprising given that both respond rapidly to a wide range of stimuli and modify similar protein
residues via an O-linkage, there are however some notable differences. The most obvious difference is that in contrast to the hundreds
of kinases and phosphatases involved in regulating phosphorylation,
there is a single OGT responsible for adding O-GlcNAc to proteins
and single OGA to remove O-GlcNAc. This raises a key question as to
how the kind of specificity associated with phosphorylation can be
achieved with O-GlcNAcylation? One putative mechanism is the multiple TPRs in the N-terminal domain of OGT, which play a key role in
recognition and activation of specific protein targets [59,76]. In support of this there are clear examples of OGT forming transient complexes with key protein targets in response to specific stimuli
[74,87]; however considerable work remains, to better understand
the activation and regulation of the formation of these protein
complexes.
Given that O-GlcNAcylation appears to be as abundant as phosphorylation, it is perhaps surprising that it is not as widely studied. It is worth
remembering, however, that the recognition of phosphorylation as an
important mechanism for regulating protein function took many decades
to be fully appreciated. As discussed by Krebs in his 1992 Nobel lecture,
while the discovery of serine/threonine phosphorylation occurred as
early as 1933; it was only by 1968 that it was recognized that phosphorylation could be regulated by extracellular stimuli [161] and by 1976,
43 years after it was first described, the number of enzymes known to
undergo phosphorylation/dephosphorylation was over 20 [161]. It is
now a little over 25 years since protein O-GlcNAcylation was first described [2] and currently more than 1000 proteins have been identified
as being targets for this modification (http://cbsb.lombardi.georgetown.
edu/statistic.php). One reason why studies of O-GlcNAcylation have not
emerged more rapidly is due, at least in part, because that it is a low molecular weight modification and unlike phosphorylation is uncharged,
therefore does not lead to gel shifts. In addition it is very labile not only
in traditional MS analyses, but also if tissue samples are processed without O-GlcNAcase inhibitors, it is readily lost from proteins, as would occur
with phosphorylation in the absence of phosphatase inhibitors. Finally,
the lack of available reagents has also been a hindrance; until very recently, there were only 2 commercially available pan O-GlcNAc antibodies
(CTD110.6 and RL2) and antibodies to OGT and OGA have only become
widely available in the past 3–5 years. In addition a critical limitation in
trying to elucidate O-GlcNAc dependent signaling networks is the lack
of site specific O-GlcNAc antibodies. Antibodies generated against synthetic O-GlcNAcylated peptides have provided site-specific (S1011-OGlcNAc IRS-1 [108]) and pan-specific (CTD110.6) antibodies; however,
the synthesis and deprotection of O-GlcNAc peptides is significantly
more challenging than that of phosphopeptides.
It is clear that O-GlcNAcylation is an integral component of the
complex signaling network involved in regulating cellular responses
to physiological and pathophysiological stimuli; however, it is also
equally clear that our understanding of the fundamental mechanisms
involved in regulating O-GlcNAc turnover is surprisingly limited. A
major challenge has been the lack of reliable tools, including antibodies, which are taken for granted in the investigation of many
PTMs; however, as the methods for identifying and characterizing
O-GlcNAcylated proteins are continuing to improve and become
more widely available, it is anticipated that there will be an increasing
number of investigators driven to elucidate more fully the role of OGlcNAc signaling in the cardiovascular system.
Disclosures
None.
Acknowledgments
This work was supported by NIH R01 grants ES10167, AA13395 and
DK075867 to VDU; DE020925 to LEB; and HL101192 and HL079364 to
JCC.
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