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article
published online: 16 march 2015 | doi: 10.1038/nchembio.1774
O-GlcNAc occurs cotranslationally to stabilize
nascent polypeptide chains
Yanping Zhu1,2, Ta-Wei Liu1,2, Samy Cecioni2, Razieh Eskandari2, Wesley F Zandberg2 & David J Vocadlo1,2*
npg
© 2015 Nature America, Inc. All rights reserved.
Nucleocytoplasmic glycosylation of proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) is recognized as a
conserved post-translational modification found in all metazoans. O-GlcNAc has been proposed to regulate diverse cellular
processes. Impaired cellular O-GlcNAcylation has been found to lead to decreases in the levels of various proteins, which is one
mechanism by which O-GlcNAc seems to exert its varied physiological effects. Here we show that O-GlcNAcylation also occurs
cotranslationally. This process protects nascent polypeptide chains from premature degradation by decreasing cotranslational
ubiquitylation. Given that hundreds of proteins are O-GlcNAcylated within cells, our findings suggest that cotranslational
O-GlcNAcylation may be a phenomenon regulating proteostasis of an array of nucleocytoplasmic proteins. These findings set
the stage to assess whether O-GlcNAcylation has a role in protein quality control in a manner that bears similarity with the role
played by N-glycosylation within the secretory pathway.
H
undreds of nuclear and cytoplasmic proteins are posttranslationally modified with N-acetylglucosamine mono­
saccharide units O-linked to serine or threonine residues
of proteins. This modification, known as O-GlcNAc1, is conserved
among multicellular eukaryotes, where it regulates diverse cellular
processes including, for example, epigenetic regulation of gene
expression2–4, stress response5,6 and circadian rhythm7,8. Remarkably,
O-GlcNAc executes its physiological functions in part by regulating
the levels of various key proteins8–12. In some cases it has emerged
that O-GlcNAc influences protein ubiquitylation8–12, but knowledge
of the various molecular processes by which O-GlcNAc regulates
protein levels remains limited.
Protein O-GlcNAcylation is regulated by only two enzymes:
O-GlcNAc transferase (OGT) installs O-GlcNAc using uridine
5′-diphosphate-N-acetylglucosamine (UDP-GlcNAc) as a donor
substrate13,14, and O-GlcNAcase (OGA) removes O-GlcNAc15. These
two enzymes act together to dynamically modulate the levels of
O-GlcNAc on proteins within cells. Notably, no consensus sequence
governing which residues are O-GlcNAcylated has been found, and
structures of human OGT reveal a long binding cleft, suggesting
that only extended polypeptide substrates can be O-GlcNAcylated16.
Given these data, as well as the observation that OGT stably interacts with actively translating ribosomes17, we considered whether
nascent chains emerging from the ribosome might be cotranslationally O-GlcNAcylated. Although O-GlcNAc is accepted
as a post-translational modification, we found this hypothesis
intriguing given the pleiotropic effects associated with decreased
O-GlcNAcylation. Further, considering recent reports that have
shown a large fraction of nascent polypeptides are cotranslationally degraded, a process that contributes both to protein quality
control and proteostasis18–20, we speculated that if cotranslational
O-GlcNAcylation occurs it could have an important role in governing the fate of nascent polypeptides of a subset of nucleocytoplasmic proteins. Using the known O-GlcNAcylated model proteins
specificity protein (Sp1) and nuclear pore protein p62 (Nup62), we
report that O-GlcNAcylation occurs cotranslationally within both a
cell-free expression system and cells. We further find that cotranslational O-GlcNAcylation protects nascent polypeptide chains of
Sp1 and Nup62 from premature proteasomal degradation by
decreasing cotranslational ubiquitylation. Considering that
hundreds of proteins are O-GlcNAcylated within cells, our data
indicate that cotranslational O-GlcNAcylation may be a common
phenomenon regulating proteostasis of a subset of O-GlcNAcylated
nucleocytoplasmic proteins.
RESULTS
Sp1 is cotranslationally O-GlcNAcylated in vitro
To first test whether OGT can cotranslationally O-GlcNAcylate
proteins, we selected proteins known to be constitutively
O-GlcNAcylated as extensive glycosylation reasonably suggests a
functionally important role. We therefore chose Sp1 as it is highly
O-GlcNAcylated, and its glycosylation status regulates its cellular
levels21,22. Six sites of O-GlcNAcylation are known on Sp123,24 that
are mostly in regions of intrinsic disorder. We used rabbit recticulocyte (RR) lysate as a cell-free expression system that contains all
the cellular machinery for protein production and processing and
is also capable of producing O-GlcNAcylated proteins25. To sensitively monitor O-GlcNAcylation on nascent polypeptides, we used
uridine 5′-diphosphate-N-azidoacetylglucosamine (UDP-GlcNAz),
a close analog of UDP-GlcNAc that has a small pendent azide
functionality. OGT accepts UDP-GlcNAz as a substrate, leading to
formation of O-GlcNAz–modified proteins26. Using the Staudinger
ligation, a highly chemoselective reaction27, the azide group can be
exclusively tagged within RR lysates by using a ­biotin-modified triarylphosphine substituted at one aryl ring with an ortho-­substituted
methyl ester. For these studies, we synthesized a phosphine probe
containing a cleavable linker (biotin-diazo-phosphine probe 1;
Fig. 1a; see Supplementary Results, Supplementary Note for
details of the synthesis and characterization). Using these tools, we
supplemented lysates with polymerase, UDP-GlcNAz and a plasmid
encoding N-terminally Flag-tagged Sp1 and then incubated these
samples for various times. By using cetrimonium bromide (CTAB)
fractionation, we separated peptidyl-tRNAs from mature proteins
released from the ribosome (Fig. 1b). This method permitted us to
detect nascent Sp1 peptidyl-tRNAs in the precipitates, as expected
(Fig. 1c), whereas mature full-length Sp1 protein was only detected
in the supernatants (Supplementary Fig. 1a), indicating that the
fractionation method was effective. By tagging glycosylated proteins obtained from CTAB precipitates using the Staudinger ligation, we were able to observe time-dependent changes in the mass of
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada. 2Department of Chemistry, Simon Fraser
University, Burnaby, British Columbia, Canada. *e-mail: [email protected]
1
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other proteins that are not O-GlcNAcylated,
we assessed the expression in RR lysates of the
O
N
PPh
cytoplasmic protein FBXO22 and Clusterin,
HO
O
O
HN
NH
a protein expressed in the secretory pathN
N
H
H
H
H
way, neither of which are O-GlcNAcylated.
N
N
O
O
O
S
We found that the levels of these proteins
O
O
1
produced were unchanged by OGT inhibib
tion (Supplementary Fig. 1c), indicating that
5′
5′
40S
40S
OGT inhibition does not influence the general
tRNA
60S
60S
G
Staudinger
transcriptional or translational machinery
3′
3′
G
OGT
ligation
G
CTAB-PPT
within RR lysates responsible for the producG
G
Biotin
Nascent
UDP-GlcNAz
Phosphine
tion of these proteins. To determine whether
G O-GlcNAz
polypeptide
biotin probe
(1)
these observations stemmed from indirect
effects of OGT inhibition leading to decreased
O-GlcNAc on components of the ubiquitinCTAB-nascent Flag-Sp1 chain precipitates
proteasome system30, we blocked O-GlcNAc
c
cycling in both directions by simultaneously
Time (min):
0
15
30 60
90
0
15
30 60 90
0
15
30 60 90
adding high concentrations of both OGA and
170
130
OGT inhibitors before initiating translation.
*
*
100
*
Upon inhibiting both OGA and OGT within
70
lysates, we found no change in the amount
55
of mature Sp1 produced relative to that pro40
duced with OGT inhibition alone, indicating
35
that only OGT-catalyzed O-GlcNAcylation of
25
newly synthesized polypeptides influences Sp1
stability (Supplementary Fig. 1d). Together,
15
these results indicate that Sp1 is either rapidly
MW (kDa)
Flag
Strvn
Merge
degraded upon release from the ribosome
or that Sp1 nascent polypeptide chains are
Figure 1 | Sp1 is cotranslationally O-GlcNAcylated in a cell-free expression system. (a) Structure
cotranslationally degraded. On examination
of the biotin-diazo-phosphine probe 1. (b) Schematic of the experimental design. (c) Immunoblot
of immunoblots, we noted that a range of
analysis of CTAB precipitates using anti-Flag antibody and fluorescent streptavidin (Strvn). Sp1
lower-abundance Sp1 polypeptides of smaller
expression in RR lysates was performed by incubating plasmid, T7 polymerase and UDP-GlcNAz
mass (<100 kDa) were absent upon OGT
at 30 °C for the indicated times. Asterisks represent full-length Sp1. MW, molecular weight;
inhibition (Fig. 2a) but were observed when
PPT, precipitation. See Supplementary Figure 9 for uncut gel images.
both OGT and the proteasome were inhibited
­immunoreactive nascent Sp1 polypeptides that bear both O-GlcNAz (Fig. 2b). We speculated these shorter Sp1 polypeptides might be
and the Flag epitope (Fig. 1c). The levels of Sp1 nascent chains nascent chains that, without O-GlcNAcylation, are cotranslationally
increased during early time points but gradually decreased after degraded. To test this hypothesis, we stopped translation at an early
30 min, whereas mature full-length Sp1 proteins within the super- time point when nascent chains are abundant (15 min; Fig. 2c) and
natant increased over time (Fig. 1c and Supplementary Fig. 1a). found, by immunoprecipitation, an array of truncated Sp1 polypepThese data indicate that O-GlcNAz is cotranslationally installed and tides except in the reaction containing OGT inhibitor. Inhibition of
that incorporation of O-GlcNAz does not impair the production of both OGT and the proteasome resulted, however, in levels of these
shorter Sp1 polypeptides that are comparable to those seen in the
full-length Sp1 proteins.
control reaction (Fig. 2c). Similarly, by using CTAB precipitation,
we observed that OGT inhibitor alone led to an absence of Sp1
O-GlcNAc protects nascent Sp1 from degradation
Given that O-GlcNAc can influence protein stability8–12 and that peptidyl-tRNAs (Fig. 2d). We also found that OGT inhibition increased
cotranslational O-GlcNAcylation can occur as shown above, we the extent of Sp1 polypeptide poly­ubiquitination (Supplementary
speculated that cotranslational O-GlcNAcylation might regulate Fig. 1e). Collectively, these data show that O-GlcNAcylation prothe production of stable natively O-GlcNAcylated proteins. To tects nascent Sp1 chains from proteasomal degradation.
probe this hypothesis, we used inhibitors to manipulate the activities of OGT and OGA in RR lysates. To inhibit OGT, we used uri- Cotranslational O-GlcNAcylation occurs within cells
dine 5′-diphosphate (UDP) as well as the substrate analog uridine We next set out to evaluate whether cotranslational O-GlcNAcylation
5′-diphosphate 2-acetamido-2-deoxy-5-thio-D-glucopyranose (UDP- occurs within cells. We therefore performed transfection of
5SGlcNAc, referred to throughout as UDP-5SInh)28. To inhibit HEK293 cells with plasmids encoding either N-terminally 3×
OGA-catalyzed removal of O-GlcNAc, we used Thiamet-G29. We Flag-tagged Sp1 or Nup62. Nup62 was selected as a second model
found that OGT inhibition resulted in near absence of mature protein because it is an excellent substrate for OGT in vitro and is
full-length Sp1 (Fig. 2a), whereas OGA inhibition had no effect both heavily and rapidly modified with O-GlcNAc31. This protein
(Supplementary Fig. 1b). To test whether the observed decrease in is constitutively O-GlcNAcylated and has been proposed to have
mature Sp1 might be due to a failure in transcription or translation over ten O-GlcNAc sites32,33, two of which are mapped to residues
rather than proteasomal degradation, we concomitantly inhibited the T373 and S468 (Uniprot). We treated these transfected cells overproteasome and OGT. This treatment resulted in Sp1 levels that were night with acetylated N-azidoacetylgalactosamine (Ac4GalNAz),
equivalent to those seen in the absence of OGT inhibitor (Fig. 2b). which is biosynthetically converted into UDP-GlcNAz, leading to
OGT-catalyzed O-GlcNAcylation is therefore essential for efficient the formation of O-GlcNAz on proteins34. The following day, after
production of Sp1 in vitro in a cell-free system, and, in the absence transfection and Ac4GalNAz treatment, ribosome-bound nascent
of O-GlcNAc, Sp1 is rapidly degraded by the ubiquitin-proteasome Sp1 polypeptides were purified by isolation of polysomes followed
system. To evaluate whether OGT inhibition might have effects on by immunoprecipitation of Flag-tagged peptides. After tagging of
a
O
Br
N
N
O
H
N
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© 2015 Nature America, Inc. All rights reserved.
320
Flag
Flag
Flag
Flag
Biot
in
2
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Nature chemical biology doi: 10.1038/nchembio.1774
a
Time (min):
b
5
10
30
Time (min):
90
UDP-5SInh – + – – + – – + – – + –
UDP – – + – – + – – + – – + MW (kDa)
170
130
100
5
10
© 2015 Nature America, Inc. All rights reserved.
90
UDP-5SInh – + – – + – – + – – + –
MG132 – + – – + – – + – – + –
UDP – – + – – + – – + – – + MW (kDa)
170
130
100
70
70
55
55
O-GlcNAc is removed following completion of
translation and release of the mature protein.
This observation further suggests that cotranslational O-GlcNAcylation may have a functional role on nascent chains.
O-GlcNAc moderates cotranslational
ubiquitination in cells
Given that both O-GlcNAc and ubiquitin
are found on nascent chains within cells and
that O-GlcNAcylation regulates their sta–
–
+
+
–
– UDP-5SInh
–
–
+
+
–
– UDP-5SInh
+
+
–
–
+ MG132
MW (kDa) –
MW (kDa) –
+
+
–
–
+ MG132
bility in vitro on Sp1, we were interested in
100
170
whether cotranslational O-GlcNAcylation
70
130
contributes to the stability of nascent chains
100
55
70
in cells. To block OGT activity in cells, we
40
55
used acetylated 5-thio-N-acetylglucosamine
40
35
(Ac45SGlcNAc, referred to throughout as
35
25
5SInh), which is transformed by the cellular
15
machinery to generate the OGT inhibitor
25
UDP-5SGlcNAc28. Using this tool compound,
CTAB-PPT/Flag-IB
Flag-IP/Flag-IB
we found that OGT inhibition resulted in
decreased O-GlcNAcylation of nascent chains
Figure 2 | Nascent Sp1 polypeptides are stabilized by cotranslational O-GlcNAcylation in
of Sp1 and Nup62 as well as lower overall levels
a cell-free system. (a) Effect of OGT inhibitors UDP-5SGlcNAc (UDP-5SInh) or UDP on the
of Flag-tagged nascent chains of these proteins
time-dependent production of Sp1. MW, molecular weight. (b) Effect of proteasome inhibition
(Fig. 3a and Supplementary Fig. 2a). Levels
(MG132) on the levels of Sp1 produced under different conditions. (c) Immunoblot (IB) analysis of
of Sp1 nascent chains were decreased by
immunoprecipitated (IP) Flag-tagged Sp1 from RR lysates in which translation was stopped by the
approximately 50% in cells yet were not comaddition of cycloheximide 15 min after the initiation of the expression reaction. (d) Immunoblot
pletely absent, as observed in the cell-free
analysis of Flag-tagged Sp1 nascent chains within CTAB precipitates of nascent Sp1 peptidylsystem. This difference most likely stems
tRNAs that were obtained from RR lysates in which translation was stopped after 15 min by the
from incomplete inhibition of OGT within
addition of cycloheximide. PPT, precipitatation.
cells as compared to lysates, which is apparent when examining the residual level of
O-GlcNAz–modified nascent chains with the biotin-diazo-phosphine O-GlcNAcylation of Sp1 in cells treated with OGT inhibitor
probe, we observed that anti-Flag– and streptavidin–reactive (Fig. 3a). Regardless, these data clearly showed that OGT inhibition
bands were largely coincident, indicating that ribosome-bound Sp1 decreased levels of nascent chains of both Sp1 and Nup62 within
and Nup62 can be cotranslationally O-GlcNAcylated within cells cells. To test whether the decreased levels of these nascent chains
having lower O-GlcNAcylation stemmed from their proteasomal
(Fig. 3a and Supplementary Fig. 2a).
Notably, we found within polysome fractions a substantial degradation, as we hypothesized, we simultaneously inhibited both
portion of the proteins having a molecular weight close to that of the proteasome and OGT within cells. This concomitant inhibition
full-length Sp1 or Nup62 (Fig. 3a and Supplementary Fig. 2a). To resulted in levels of Sp1 and Nup62 nascent chains that were equivclarify whether these long polypeptides were also ribosome-bound alent to those seen in control cells (Fig. 4a and Supplementary
nascent chains or represented contamination of the polysome frac- Fig. 2b), which is in alignment with data from the cell-free
tion with mature full-length proteins, we performed an additional system. These data indicate that cotranslational O-GlcNAcylation
fraction step. The isolated polysome preparation was fractionated stabilizes nascent chains against cotranslational proteasomal
using CTAB to obtain peptidyl-tRNAs, after which we performed degradation both in vitro and in cells.
immunoprecipitation of Flag-Nup62. Using this approach, we
Given our findings regarding stabilization of nascent chains
again observed these long polypeptide chains, indicating that these by cotranslational O-GlcNAcylation, we were intrigued by recent
species were nascent chains linked to the ribosome and not reports showing that cotranslational ubiquitylation labels a large
contaminating mature proteins (Supplementary Fig. 3).
fraction of nascent polypeptides for degradation19,20. We hypothesized that the enhanced cotranslational degradation of nascent
Nascent Sp1 is more heavily O-GlcNAcylated than mature Sp1 chains of Sp1 and Nup62 observed when OGT was inhibited might
Given that O-GlcNAc is installed cotranslationally but is a reversible stem from their increased cotranslational ubiquitylation. To test this
modification, we were curious about the abundance of O-GlcNAc hypothesis, we analyzed the ubiquitin levels of ribosome-bound Sp1
on nascent chains as compared to mature proteins as it is conceiv- and Nup62 nascent polypeptides from cells treated with proteasome
able that glycosylation may either increase or decrease over time. inhibitor alone and cells treated with both proteasome and OGT
We therefore analyzed the extent of O-GlcNAz present on nascent inhibitors. We again noted that proteasome inhibition led to equivachains and mature proteins by quantifying the immunoreactivity of lent levels of these proteins being produced and that concomitant
these species following isolation of Flag-tagged species. Remarkably, inhibition of OGT resulted in increased ubiquitylation of these proby comparing the level of O-GlcNAz present on different species of teins as compared to control cells that were not treated with OGT
Sp1 polypeptide chains (O-GlcNAz/Flag-Sp1), we found that Sp1 inhibitor (Fig. 4a and Supplementary Fig. 2b). As an alternative
nascent chains were more extensively modified with O-GlcNAz approach to address this question, we assessed the effect of reduced
compared to mature proteins isolated from polysome-free fractions. O-GlcNAcylation on Sp1 nascent chains in mouse embryonic fibroShort nascent chains of Sp1 in particular had robustly higher levels blasts (MEFs) in which the ogt gene can be inducibly excised35. Here
of O-GlcNAz than full-length Sp1 nascent chains (Fig. 3b). This we found that loss of OGT resulted in decreased levels of Sp1 nascent
line of data indicates that O-GlcNAc is added cotranslationally to chains. When the OGT gene was excised in the presence of proteaSp1 and that a considerable fraction of the cotranslationally added some inhibitor, we observed an increase in the extent of nascent Sp1
Flag
Flag
c
npg
30
With plasmid
No plasmid
d
With plasmid
No plasmid
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a
+
–
+
+
–
–
+
–
+
+
–
–
+
–
+
+
npg
© 2015 Nature America, Inc. All rights reserved.
Relative O-GlcNAz levels
(O-GlcNAz/Sp1)
_
NC S
_F
L
NC
NC
M
P
Relative O-GlcNAz levels
(O-GlcNAz/Sp1)
Relative O-GlcNAz levels
(O-GlcNAz/Sp1)
3 22 Co
nt
ro
l
5S
In
h
–
–
NC
_F
M L
P_
FL
+
+
Co
nt
ro
l
5S
In
h
+
–
Relative nascent Sp1 levels
(Nascent Sp1/RPS6)
–
–
Relative O-GlcNAz levels on
nascent Sp1 (O-GlcNAz/Sp1)
involved in regulating cellular ubiquitination are known to be O-GlcNAcylated, we
MW (kDa)
1.5
1.5
considered that these might be influenced by
100
O-GlcNAcylation12,36 and therefore performed
70
*
*
*
1.0
1.0
further control experiments. To assess whether
55
*
*
*
*
*
*
*
**
40
reduced O-GlcNAcylation upon OGT inhibi35
0.5
0.5
*
*
*
25
tion alters the activity of ubiquitin systems
*
*
*
Flag
Flag-LE
Strvn
Merge
within cells, we analyzed global protein ubiq0
0
uitylation levels in cells and found that these
were unaffected (Supplementary Fig. 5),
RPS6-Input
suggesting that the effects observed on Sp1
and Nup62 ubiquitylation did not stem from
b
NC MP NC MP NC MP NC MP MW (kDa)
alterations in the general activity of the cellular
NC_FL
1.5
100
1.5
1.5
ubiquitylation machinery. Finally, we further
**
**
70
**
tested whether it is direct O-GlcNAcylation
1.0
1.0
55
1.0
that affects the stability of Sp1 and Nup62
*
*
*
40
**
*
nascent chains by generating mutants of these
NC_S
*
*
0.5
0.5
0.5
35
*
*
*
two proteins in which known O-GlcNAc sites
***
**
*
*
25
*
0
0
0
were eliminated by site-directed mutagenesis.
15
Known O-GlcNAcylation sites of Sp1 include
Flag
Flag-LE
Strvn
Merge
residues S491, S612, T640, S641, S698 and
Figure 3 | Cotranslational O-GlcNAcylation of Sp1 stabilizes nascent polypeptides within cells.
S702, of which S491, S612, S698 and S702
(a) Sp1 is O-GlcNAcylated cotranslationally, and levels of Sp1 nascent chains are reduced
are found in regions of intrinsic disorder. For
upon inhibition of OGT in cells. 24 h after transfection with 3× Flag-tagged Sp1, HEK293 cells
Nup62, the known sites of glycosylation include
were treated with Ac4GalNAz with or without Ac45SGlcNAc (5SInh) overnight. Nascent
T373 and S468, of which S468 is found in a
Flag-tagged Sp1 immunoprecipitated from equal amounts of isolated polysomes were reacted
disordered region. These mutant proteins all
with the biotin-diazo-phosphine probe 1 overnight. Samples were then probed using anti-Flag
showed reductions in their O-GlcNAcylation
antibody and streptavidin (Strvn). Immunoblots of RPS6 were used to control for sample input.
levels, either as a trend or at a significant level
The asterisks indicate coincident anti-Flag– and streptavidin-reactive bands. Values of O-GlcNAz
(Supplementary Fig. 6). We also found that
signals were normalized to corresponding Flag immunoblot signals, whereas nascent Flag-Sp1
loss of these O-GlcNAc sites increased the levsignals were normalized to corresponding RPS6 immunoblot signals. All of the samples were
els of cotranslational ubiquitylation of nascent
normalized to the corresponding control, arbitrarily set as 1. Error bars represent ±s.d. *P < 0.05,
Sp1 and Nup62 chains compared to that of
**P < 0.01 by paired Student’s t-test (n = 3). MW, molecular weight. (b) Analysis of the levels
wild-type proteins (Fig. 4b and Supplementary
of O-GlcNAz on Sp1 nascent chains as compared to mature Sp1 proteins. Cells were treated
Fig. 2c). Furthermore, we were interested in
as in a, except that 5SInh was not added. Sp1 nascent chains were immunoprecipitated from
whether increased cotranslational ubiquitylaisolated polysomes, and mature Sp1 proteins were immunoprecipitated from the polysome-free
tion on nascent chains of these Sp1 mutants
supernatant fractions. Values of O-GlcNAz signals were normalized to corresponding Flag
would increase their susceptibility to proteaimmunoblot signals and then normalized again to the corresponding control, arbitrarily set as 1.
somal degradation, as seen when OGT inhibiError bars represent ±s.d. **P < 0.01, ***P < 0.001 by paired Student’s t-test (n = 3). NC, nascent
tor was used to decrease O-GlcNAcylation of
chain; NC_S, nascent short chain; NC_FL, nascent chain full length; MP, mature protein;
Sp1. To this end, we treated cells expressing
MP_FL, mature protein, full length; LE, long exposure; RPS6, ribosomal protein S6.
Sp1 and these Sp1 mutant proteins with proteasome inhibitor or vehicle. We anticipated
that if the proteosomal susceptibility of nascent
ubiquitination (Supplementary Fig. 4a). Notably, we observed by chains of Sp1 mutants were increased as compared to wild-type Sp1,
immunoblotting that some OGT remained in the knockout cell line proteasome inhibition would lead to greater increases in the levels of
(Supplementary Fig. 4a), perhaps because OGT has a long half-life. mutant Sp1 chains compared to chains of wild-type Sp1. Consistent
This remaining OGT may be part of the reason why Sp1 nascent with our hypothesis, we found that proteasome inhibition resulted
chains were not completely absent, as observed in the cell-free in restoration of the levels of both long and short nascent chains
system. Regardless, the data obtained using this genetic approach of Sp1 mutants, with a much smaller effect for wild-type Sp1
(Fig. 4c and Supplementary Fig. 7). These data indicate that
are similar to those seen when inhibiting OGT in cells.
We next performed a series of control experiments to exclude nascent chains of Sp1 O-GlcNAc–deficient mutants were more
possible off-target effects of the inhibitors used as well as to test extensively degraded by the proteasome as compared to nascent
whether the effects observed were mediated by direct action of chains of wild-type Sp1, which is in keeping with their increased
OGT on Sp1 and Nup62 nascent chains. First, to evaluate whether ubiquitylation. Collectively, these data reveal that the direct
MG132 might have off-target effects that could confound our activity of OGT to cotranslationally O-GlcNAcylate Sp1 and Nup62
results, we repeated several experiments using the structurally dis- protects nascent chains of these proteins against cotranslational
tinct proteasome inhibitor lactacystin. Using this tool compound, ubiquitylation and downstream proteasomal degradation.
we observed results consistent with the findings made using MG132
(Supplementary Fig. 4b), indicating that our observations did not O-GlcNAcylation stabilizes mature Sp1
stem from potential off-target effects of MG132. Next, consistent Given that O-GlcNAc modification has been proposed to modulate
with the direct action of OGT on Sp1 regulating Sp1 levels rather the levels of Sp1 in cells21, we wanted to address whether, in addithan indirect action on the general transcriptional or translational tion to protection at the cotranslational level, there was also regulamachinery in cells, we found that OGT inhibition had no effect on tion at the post-translational level. To this end, we isolated mature
the levels of nascent chains of the cytoplasmic non–O-GlcNAcylated Sp1, which had been released from ribosomes, from polysome-free
protein FBXO22 or on the extent of FBXO22 ubiquitination fractions. We found that ubiquitinylation of mature Sp1 in cells
(Supplementary Fig. 4c,d). Nevertheless, because various proteins treated with OGT inhibitor were clearly elevated compared to levels
Flag-Sp1
5SInh
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+
+
–
5SInh +
–
–
+
–
–
+
–
–
Mr (kDa)
170
130
100
70
Flag
Flag-LE
HA-Ub
55
RPS6-Input
b
1.5
NS
1.0
0.5
0
*
4
3
2
1
0
S612A
T640A
S641A
S698A
S702A
2.5
3
2
*
1
0
more rapidly than degradation of the wild-type
proteins. We therefore tracked the degradation
rates of these mutants by cycloheximide chase
and found, as predicted, that these Sp1 mutants
were degraded more quickly than the corresponding wild-type proteins (Supplementary
Fig. 8c,d). These data therefore provide good
evidence that O-GlcNAcylation can regulate
both the co- and post-translational stability of Sp1, suggesting that O-GlcNAc may
function throughout the lifetime of Sp1 to
protect it from degradation via the ubiquitinproteasome pathway.
DISCUSSION
2.0
*
NS
NS NS
**
* ** **
npg
A
40
A
S6
41
A
A
12
02
S6
S7
A
12
S6
S6
12
A
T6
40
A
S6
41
A
S6
T6
98
A
W
T
Increase in nascent Sp1 level
(Sp1-MG132/Sp1-No MG132)
T6
40
T6
W
S4 T
91
A
S6 A
4
S6 S 0A 12
S6
41 69 S A
12
S6 A S 8A 64
A
12 69 S7 1A
T6
40 S6 A T 8A 02
A 12A 64 S7 A
S6 S 0A 02
41 69 S A
A 8 64
S6 A 1A
98 S70
A 2A
S7
02
A
T640A S698A
WT S491A S612A S641A S702A
Relative HA-Ub levels
(HA-Ub/Sp1)
Using model proteins, we have found that
O-GlcNAcylation occurs not only post1.5
translationally, as previously thought, but also
170
1.0
cotranslationally. Notably, levels of cotrans130
HA-Ub
0.5
lational O-glycosylation by O-GlcNAc on
100
0
nascent chains are higher than those found on
70
the corresponding mature proteins. This obser100
vation on its own suggests that cotranslational
Flag
O-GlcNAcylation most likely has a functional
70
role. Indeed, here we found that cotranslational O-GlcNAcylation regulated the stability
c
of nascent chains by decreasing their cotranslaMG132
–
+
–
+
–
+
–
+
tional ubiquitylation (Fig. 5) as well as by regS612A S612A
4
T640A T640A
ulating Sp1 levels at the post-translational level
** *
S641A S641A S612A S612A
3
through ubiquitylation. Previous research has
S698A S698A T640A T640A
MW (kDa) WT WT S612A S612A S702A S702A S641A S641A
suggested that O-GlcNAc functions to post2
130
NS
translationally regulate the susceptibility of
100
1
proteins including Snail-1 (ref. 11), β-catenin9,
70
Sp1 (ref. 21), clock8 and Δ-lactoferrin37 toward
Flag
0
55
proteasomal degradation. Glucose starvation
coupled with stimulation using cyclic AMP
40
results in rapid proteasomal degradation of
35
Sp1, suggesting that O-GlcNAc protects Sp1
from proteosomal degradation through an
RPS6
undefined mechanism21. Our results suggest
that these previous findings regarding Sp1
Figure 4 | Cotranslational O-GlcNAcylation of Sp1 stabilizes nascent polypeptides from
may in part be accounted for by cotranslapremature proteasomal degradation within cells. (a) Immunoblot analysis of ubiquitinylation
tional O-GlcNAcylation acting to protect both
levels of nascent Sp1 in proteasome inhibited cells in the presence and absence of OGT inhibitor.
nascent chains and mature Sp1 from premaCells were treated as in Figure 3a except that the plasmid pRK5-HA-ubiquitin, which encodes
ture proteosomal degradation by attenuating
ubiquitin with an N-terminal hemagglutinin tag (HA-Ub), was cotransfected into cells.
their ubiquitination.
Nascent Flag-Sp1 polypeptides immunoprecipitated were probed using anti-HA antibody
Accordingly, defining precisely how
to detect ubiquitinylation (Ub). RPS6 was used as a measure for control of sample input.
O-GlcNAc decreases cotranslational ubiquiError bars represent ±s.d. *P < 0.05 from paired Student’s t-test (n = 3). LE, long exposure.
tylation of specific proteins will be of interest.
(b) Analysis of the level of ubiquitinylation of nascent polypeptides of Flag-Sp1 in which
O-GlcNAc may, for example, block cotransdifferent known O-GlcNAc sites were deleted by site-directed mutagenesis. Error bars
lational ubiquitylation, by recruiting proteins
represent ±s.d. *P < 0.05, **P < 0.01, as calculated with one-way analysis of variance (n = 3).
that protect nascent chains by directly blockMW, molecular weight. (c) Analysis of the levels of nascent Sp1 polypeptides obtained from
ing substrate recognition by ubiquitin ligases
various mutants of Sp1 lacking known O-GlcNAc sites. Relative levels of Sp1 nascent chains
or by influencing the folding pathway of
were quantified by dividing total Flag immunoreactivity by the corresponding RPS6
natively O-GlcNAcylated proteins such that
immunoreactivity. Increases in the relative levels of Sp1 nascent chains upon proteasome
nascent chains misfold and are themselves
inhibition were calculated by dividing the relative Flag-Sp1 signal (Flag-Sp1/RPS6) from
recognized by the ubiquitinylation machinMG132-treated cells by the signal from cells not treated with MG132. Error bars represent ±s.d.
ery. Accordingly, future research directed
*P < 0.05, **P < 0.01, as calculated with one-way analysis of variance (n = 3). NS, not significant.
at identifying the set of cotranslationally
O-GlcNAcylated proteins will be of interest,
observed in control cells (Supplementary Fig. 8a,b). We next evalu- as will identifying those proteins whose stability is regulated by
ated the effects of site-directed mutation of known O-GlcNAcylation cotranslational O-GlcNAcylation. Given the presence of O-GlcNAc
sites, and we found that all of the mutants tested showed increased on several hundred cellular proteins38 and the fact that OGT has
ubiquitin modification compared with the wild-type proteins been reported to interact stably with ribosomes17, it is probable
(Supplementary Fig. 8a,b). On the basis of the data above, we that cotranslational O-GlcNAcylation is common to many proteins.
expected that degradation of O-GlcNAc site mutants should occur Seeing as cotranslational O-GlcNAcylation is abundant on these
MW (kDa)
© 2015 Nature America, Inc. All rights reserved.
T640A
S641A S612A S612A
S698A T640A S698A
WT S702A S641A S702A
5
2.0
nt
ro
l
5S
In
h
+
+
Co
+
–
Relative polyubiquitinated Sp1 levels
(polyubiquitinated Sp1/total Sp1)
+
+
nt
ro
l
5S
In
h
+
+
Relative HA-Ub levels
(HA-Ub/Sp1)
+
Co
+
–
nt
ro
l
5S
In
h
+
+
Co
MG132 +
Flag-Sp1 +
Polyubiquitinated
Sp1
a
Relative nascent Sp1 levels
(nascent Sp1/RPS6)
Nature chemical biology doi: 10.1038/nchembio.1774
nature CHEMICAL BIOLOGY | VOL 11 | MAY 2015 | www.nature.com/naturechemicalbiology
323
npg
© 2015 Nature America, Inc. All rights reserved.
article
Nature chemical biology doi: 10.1038/nchembio.1774
model proteins, it is interesting to speculate
5′
5′
40S
40S
whether some sites of O-GlcNAc that are
Loss of OGT
60S
60S
mapped onto proteins stem from cotranslation3′
3′
function
ally installed O-GlcNAc and are residues that
O-GlcNAc
OGT
OGT
OGT Inhibitors
G
or OGT knockout
are not removed as proteins mature. Answering
G
such questions will depend on time-resolved
G
mapping of O-GlcNAcylation sites on both coNascent
Ub
polypeptide
and post-translationally O-GlcNAcylated proteins. On a more functional level, O-GlcNAc is
a cellular stress response6,35 that is also nutrient responsive39–41, and so it seems likely that
cotranslational O-GlcNAcylation is an adapProteasome
tive response enabling protection of nascent
chains during times of stress or high metabolic
G
flux, a view that is consistent with the protecG
G
tive effects of increased O-GlcNAc in various
G
G
models42 and the effects of altered O-GlcNAc
G
levels on the levels of some proteins7–9,37.
Worth noting is that protein N-glycosylation
Full-length protein
within the secretory pathway has been clearly
defined over the past two decades as a major
Figure 5 | Proposed model for regulation of nascent polypeptide chain stability by
factor in protein quality control that helps to
cotranslational O-GlcNAcylation. Nascent polypeptides of Sp1 or Nup62 are modified with
ensure proper folding of various proteins.
O-GlcNAc cotranslationally during their elongation, which can moderate the ubiquitination
The loss of N-glycosylation leads to proand prevent subsequent proteasome degradation leading to efficient production of full-length
teasomal degradation of many proteins43–46.
proteins. In conditions where cotranslational O-GlcNAcylation is blocked by loss of OGT or OGT
N-glycosylation has generally been defined as
inhibition, nascent polypeptides are more heavily polyubiquitinated and are prematurely degraded
occurring cotranslationally47,48. Recent work
by the proteasome.
has shown, however, that N-glycosylation also
occurs post-translationally49 and is essential to
ensure functionally important modification of certain glycosylation 8. Li, M.D. et al. O-GlcNAc signaling entrains the circadian clock by inhibiting
BMAL1/CLOCK ubiquitination. Cell Metab. 17, 303–310 (2013).
sites50. Here we have shown that O-GlcNAcylation can also occur
cotranslationally to protect nascent chains from cotranslational 9. Olivier-Van Stichelen, S. et al. O-GlcNAcylation stabilizes β-catenin
through direct competition with phosphorylation at threonine 41.
degradation, which enables efficient production of the correspondFASEB J. 28, 3325–3338 (2014).
ing full-length proteins. The potential parallels between O-GlcNAc 10.Yang, W.H. et al. Modification of p53 with O-linked N-acetylglucosamine
and N-glycosylation are notable, suggesting that increased insight
regulates p53 activity and stability. Nat. Cell Biol. 8, 1074–1083 (2006).
into cotranslational O-GlcNAcylation may uncover that its role in 11.Park, S.Y. et al. Snail1 is stabilized by O-GlcNAc modification in
hyperglycaemic condition. EMBO J. 29, 3787–3796 (2010).
protein quality control resembles that of N-glycosylation within the
12.Ruan, H.B., Nie, Y. & Yang, X. Regulation of protein degradation by
secretory pathway.
O-GlcNAcylation: crosstalk with ubiquitination. Mol. Cell. Proteomics 12,
Received 21 December 2014; accepted 13 February 2015;
published online 16 March 2015
Methods
Methods and any associated references are available in the online
version of the paper.
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Acknowledgments
Financial support through a Discovery Grant (grant number: RGPIN/298406-2010) the
Natural Sciences and Engineering Research (NSERC) and the Canadian Institutes of
Health Research (CIHR; grant number: MOP-123341) is gratefully acknowledged.
Y.Z. thanks the CIHR for support through a postdoctoral fellowship. D.J.V. acknowledges
the kind support of the Canada Research Chairs program for a Tier I Canada Research
Chair in Chemical Glycobiology and NSERC for support as an E.W.R. Steacie Memorial
Fellow. S.C. acknowledges the Government of Canada and the CIHR for postdoctoral
fellowship support. R.E. acknowledges the Alzheimer Society of Canada and the Michael
Smith Foundation for Health Research for postdoctoral fellowship support.
Author contributions
Y.Z. and D.J.V. designed research; Y.Z. and T.-W.L. performed experiments; R.E. synthesized
UDP-GlcNAz; W.F.Z. synthesized UDP-5SGlcNAc; S.C. synthesized the biotin-diazophosphine probe; Y.Z. and D.J.V. wrote the paper; all authors provided input into the
manuscript.
Competing financial interests
The authors declare competing financial interests: details accompany the online version
of the paper.
Additional information
Supplementary information and chemical compound information is available in
the online version of the paper. Reprints and permissions information is available online
at http://www.nature.com/reprints/index.html. Correspondence and requests
for materials should be addressed to D.J.V.
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325
ONLINE METHODS
Plasmids. Human Sp1, Nup62, FBXO22 and Clu were amplified using synthetic primers to bear N-terminal 3× Flag tags by PCR and then were cloned
into pET28a (Novagen) or pCMV-Tag 2A (Agilent) vectors. Plasmid pRK5HA-Ub was obtained from Addgene.
npg
© 2015 Nature America, Inc. All rights reserved.
Western blotting. Protein samples were run in 12% SDS-PAGE gels and subsequently transferred onto nitrocellulose membranes. For more facile detection of
polyubiquitinated protein species, samples were run using 4–20% SDS-PAGE
gels. Primary antibodies, anti-Flag (Santa Cruz, sc-166355 and sc-807), anti-HA
(Santa Cruz, sc-805), anti-actin (Santa Cruz, sc-47778), anti-ubiquitin (Santa
Cruz, sc-8017) and anti-RPS6 (Santa Cruz, sc-74459) were all used at between
1:1,000 and 1:2,000 dilution. Appropriate fluorescent secondary antibodies
were purchased from Li-Cor and used in 1:20,000 dilution. Images for blots
were obtained using an Odyssey Infrared Imaging System (Li-Cor). To better
illustrate quantitative differences between several bands having both low and
high intensities on one immunoblot for the Flag immunoblots, the immuno­
blots were scanned at two different intensity settings (3.5 and long exposure 6).
In vitro expression. In vitro expression was carried out in TNT-coupled RR
lysate (Promega) according to the user’s manual. Reactions without plasmid
were prepared as the control. UDP-GlcNAz, UDP-5SGlcNAc (Ki = 8 μM),
UDP, ThiametG (Ki = 21 nM) and MG132 were used at a final concentration
of 75 μM, 200 μM, 200 μM, 50 μM and 25 μM, respectively. Before adding
the plasmid to start the reaction, OGT OGA or proteasome inhibitors were
incubated with RRL at RT for 10 min.
Precipitation of peptidyl-tRNA by CTAB. Precipitation of peptidyl-tRNA was
performed essentially as described51. Cell-free translation products (50 μl) were
mixed with 500 μl of 2% (w/v) cetyltrimethylammonium bromide (CTAB) and
vortexed. Then, 500 μl of 0.5 M sodium acetate (pH 5.4) and 100 μg of yeast
tRNA (as carrier) were added to induce the precipitation of peptidyl-tRNA.
After incubation at 30 °C for 10 min, the CTAB precipitates were collected
by centrifugation at 17,000g for 10 min and then washed twice with 1 ml of
acetone/HCl (19:1) to remove the CTAB. The precipitates were air dried and
dissolved in 1× SDS-PAGE loading buffer.
Expression of Sp1 or Nup62 in cells. HEK cells were maintained in highglucose DMEM (Gibco) supplemented with 10% FBS (Gibco), 100 IU/ml
penicillin and 100 μg/ml streptomycin (Gibco). Transfection was carried out
using the calcium phosphate precipitation method as described52. Plasmids
(0.1 ml) were mixed with 0.5 ml of 0.25 M CaCl2, and then 0.5 ml of 2× BBS
(50 mM BES, pH 6.95, 280 mM NaCl and 1.5 mM Na2HPO4) was added and
mixed by pipetting immediately. After incubation for 15 min at room temperature, calcium phosphate-DNA mixture solution (1 ml) was added dropwise to
cell culture in the 10-cm plate and swirled gently and placed in a tissue culture
incubator. For transfections, 12 μg of plasmid encoding Sp1 or its mutants were
used for each 10-cm plate, whereas 6 μg of plasmids encoding Nup62 or mutant
Nup62 were used. For co-expression of HA-tagged ubiquitin, 6 μg of plasmid
pRK5-HA-Ub was cotransfected at the same time as plasmids encoding Sp1
or Nup62 per 10-cm plate. 24 h after the transfection, cells were rinsed with
growth medium, and new growth medium was provided. 8 h after replacing the
medium, Ac4GalNAz or Ac45SGlcNAc in DMSO was added into cell cultures
to yield 200-μM final concentrations of compounds, and equal amounts of
DMSO alone were added to control cultures. Cells were cultured for a further
24 h before cells were harvested. MG132 was added 5 h before harvest to inhibit
treated cultures as well as control cultures.
OGT inducible knockout. The mouse embryonic fibroblast (MEF) cell
line containing lentivirus encoding mutated estrogen receptor (mER)Cre-2A-GFP construct was a kind gift from N. Zachara (Johns Hopkins)35.
Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; 1 g/l glucose) with 10% (v/v) FBS (FBS) and 1% (v/v) penicillin/streptomycin at 37 °C
in a water-jacketed, humidified CO2 (5%) incubator. Typically, cells were plated
nature chemical biology
at 10–25% confluency. Unless otherwise noted, Cre-recombinase was activated
to knock out OGT through incubation with 0.7 μM 4-hydroxytamoxifen (4HT,
Bioshop) 1 d after plating. 4HT was removed 24 h later. Cotransfection of
Sp1 and HA-Ubiquitin was carried out 48 h after inducing OGT knockout
using Turbofect transfection reagent (ThermoScientific), according to the
manufacturer’s manual. MG132 was added 5 h before harvesting cells.
Polysome extraction. To isolate polysomes from HEK cells, ~1.5 × 107 of
cultured cells were rinsed with ice-cold PBS and then directly lysed in 2 ml
ice-cold polysome lysis buffer containing 100 mM Tris (pH 7.4), 50 mM
KCl, 25 mM MgCl2, 100 μg/ml cycloheximide, 1 mM DTT, 100 μM PMSF,
200 μg/ml heparin, 50 μM N-ethylmaleimide (NEM), 40 U/ml RNase inhibitor (Clontech), 1% Triton X-100 (Sigma-Aldrich) and cOmplete mini protease
inhibitor cocktail mix (Roche). Lysates were clarified by centrifugation at
17,000g for 12 min at 4 °C, and the resulting supernatants were then layered
on top of a 3-ml prechilled 35% sucrose cushion in buffer (10 mM Tris pH 7.4,
85 mM KCl and 5 mM MgCl2). After ultracentrifugation of the samples for
2 h at 60,000 r.p.m. in a Beckman Type 90 Ti Rotor at 4 °C, the polysomecontaining pellets were resuspended in 200 μl, pH 7.4, resuspension buffer
containing 50 mM Tris, 100 mM NaCl, 2 mM EDTA, 1% SDS and cOmplete
mini protease inhibitor cocktail mix (Roche). Total proteins in the polysomefree supernatant fractions were precipitated in 5 volumes of methanol at
−80 °C overnight. After 16 h, the proteins were pelleted by centrifugation and
then washed twice using 5 ml cold methanol. After air drying at RT, protein
pellets were resuspended in resuspension buffer.
Immunoprecipitation of ribosome-bound nascent polypeptides and mature
proteins. Polysome extracts or the polysome-free fractions in resuspension
buffer (200 μl) were mixed with an equal volume of cold lysis buffer (10 mM
Tris, pH 8.04, 1 mM PMSF and 0.1% NP-40) and 3 volumes of cold nonionic
detergent solution (50 mM Tris, pH 7.4, 100 mM NaCl, 2 mM EDTA, 1 mM
PMSF, 1.66% Triton X-100, 3.3% BSA and protease inhibitor cocktail). The
solution mixtures were then incubated with 30 μl of anti-Flag immunoprecipitation resin (Genscript) prewashed with PBS at 4 °C overnight. The resins
were pelleted by centrifugation (5,000g, 2 min) and washed three times with
buffer (pH 7.4, 10 mM Tris, 300 mM NaCl and 0.1% Triton X-100) and twice
with PBS. Captured proteins were eluted in PBS using 100 μg/ml Flag peptide
(75 μl) or by boiling directly in 1× SDS loading buffer (75 μl) for 10 min.
Proteins immunoprecipitated were reacted overnight (16 h) with the
bio­tin-diazo-phosphine probe 1 at room temperature.
Cycloheximide chase. 48 h after transfection, HEK cells were incubated with
100 μg/ml of cycloheximide. Cells were harvested at the indicated time points
as shown in Supplementary Figure 8, followed by cell lysis, SDS-PAGE and
western blotting to visualize protein levels.
Synthesis. Ac45SGlcNAc and UDP-5SGlcNAc were synthesized as described
previously28. The cleavable biotin-diazo-phosphine probe 1 was synthesized
as described in the Supplementary Note. UDP-GlcNAz was prepared as
described26.
Statistical analysis of the data. Immunoblot signals were quantified using
Odyssey software (Li-Cor). Values of Flag, streptavidin or HA-Ub signals were
normalized to immunoblot signals of the corresponding loading controls. All
samples were normalized to the corresponding control, which was arbitrarily
set at a value of one. Statistical analyses were carried out using Graphpad Prism
5.03. Data were analyzed using the paired Student’s t-test or one-way analysis
of variance (ANOVA) when comparing more than two values.
51.Hobden, A.N. & Cundliffe, E. The mode of action of α-sarcin and a
novel assay of the puromycin reaction. Biochem. J. 170, 57–61 (1978).
52.Chen, C. & Okayama, H. High-efficiency transformation of mammalian
cells by plasmid DNA. Mol. Cell. Biol. 7, 2745–2752 (1987).
doi:10.1038/nchembio.1774