Direct One-Step Fluorescent Labeling of O-GlcNAc-Modified Proteins in Live Cells Using Metabolic Intermediates

Article abstract of DOI:10.1021/jacs.8b08260

The modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) by the enzyme O-GlcNAc transferase (OGT) has emerged as an important regulator of cellular physiology. Metabolic labeling strategies to monitor O-GlcNAcylation in cells have proven of great value for uncovering the molecular roles of O-GlcNAc. These strategies rely on two-step labeling procedures, which limits the scope of experiments that can be performed. Here, we report on the creation of fluorescent uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) analogues in which the N-acyl group of glucosamine is modified with a suitable linker and fluorophore. Using human OGT, we show these donor sugar substrates permit direct monitoring of OGT activity on protein substrates in vitro. We show that feeding cells with a corresponding fluorescent metabolic precursor for the last step of the hexosamine biosynthetic pathway (HBP) leads to its metabolic assimilation and labeling of O-GlcNAcylated proteins within live cells. This one-step metabolic feeding strategy permits labeling of O-GlcNAcylated proteins with a fluorescent glucosamine-nitrobenzoxadiazole (GlcN-NBD) conjugate that accumulates in a time- and dose-dependent manner. Because no genetic engineering of cells is required, we anticipate this strategy should be generally amenable to studying the roles of O-GlcNAc in cellular physiology as well as to gain an improved understanding of the regulation of OGT within cells. The further expansion of this one-step in-cell labeling strategy should enable performing a range of experiments including two-color pulse chase experiments and monitoring OGT activity on specific protein substrates in live cells.

Full text of DOI:10.1021/jacs.8b08260

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Article
Direct one-step fluorescent labeling of O-GlcNAc modified
proteins in live cells using metabolic intermediates
Hong Yee Tan, Razieh Eskandari, David Shen, Yanping Zhu, Ta-Wei Liu,
Lianne I. Willems, Matthew G. Alteen, Zarina Madden, and David J. Vocadlo
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b08260 • Publication Date (Web): 08 Oct 2018
Downloaded from http://pubs.acs.org on October 8, 2018
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Direct one-step fluorescent labeling of O-GlcNAc modified
proteins in live cells using metabolic intermediates
Hong Yee Tan,† Razieh Eskandari, † David Shen,†,‡ Yanping Zhu, †,‡ Ta-Wei Liu, †,‡ Lianne Willems,†‡
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Matthew G. Alteen,† Zarina Madden†,‡ and David J. Vocadlo*,†,‡
†Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A1S6, Canada.
‡Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A1S6,
Canada.
Supporting Information Placeholder
donor substrate to modify its target proteins.15-16 The glycoside
ABSTRACT: The modification of proteins with O-linked N-
hydrolase N-acetyl--D-glucosaminidase (OGA, GH family 84)
cleaves off O-GlcNAc residues and returns proteins to their
unmodified state.17-18 Given the central regulatory role of OGT in
regulating cellular O-GlcNAcylation, how it distinguishes its
substrates, the basis for its substrate preference, and its regulation
acetylglucosamine (O-GlcNAc) by the enzyme O-GlcNAc
transferase (OGT) has emerged as an important regulator of cellular
physiology. Metabolic labeling strategies to monitor O-
GlcNAcylation in cells has proven of great value for uncovering
the molecular roles of O-GlcNAc. These strategies rely on two-step
labeling procedures, which limits the scope of experiments that can
be performed. Here we report on the creation of fluorescent uridine
5’-diphospho-N-acetylglucosamine (UDP-GlcNAc) analogues in
which the N-acyl group of glucosamine is modified with a suitable
linker and fluorophore. Using human OGT we show these donor
sugar substrates permit direct monitoring of OGT activity on
protein substrates in vitro. We show that feeding cells with a
corresponding fluorescent metabolic precursor for the last step of
the hexosamine biosynthetic pathway (HBP) leads to its metabolic
assimilation and labeling of O-GlcNAcylated proteins within live
cells. This one-step metabolic feeding strategy permits labeling of
within cells are accordingly topics of great interest19-22
.
To address these and other related questions, considerable effort
has been allocated to creating chemical strategies to aid in studying
protein O-GlcNAcylation.23-24 For example, OGT has been found
to be tolerant of subtle structural changes made to the donor
substrate UDP-GlcNAc.25-28 This substrate tolerance has in turn
enabled metabolic feeding of cells with analogues of GlcNAc,
which are assimilated by cellular biosynthetic pathways, leading to
labeling of O-GlcNAcylated proteins with analogues of GlcNAc
bearing small bioorthogonal functional groups. Modification of the
2-acetamido group of GlcNAc, with azide,28 alkyne,29
cyclopropenyl,30 and N-propargyloxy-carbamate,31 as well as
O-GlcNAcylated proteins with
a fluorescent glucosamine-
nitrobenzoxadiazole (GlcN-NBD) conjugate that accumulates in a
time and dose dependent manner. Since no genetic engineering of
cells is required, we anticipate this strategy should be generally
amenable to studying the roles of O-GlcNAc in cellular physiology
as well as to gain an improved understanding of the regulation of
OGT within cells. The further expansion of this one-step in-cell
labeling strategy should enable performing a range of experiments
including two-colour pulse chase experiments and monitoring OGT
activity on specific protein substrate in live cells.
replacement of the 6-hydroxyl group with azide32 or alkyne33
functionalities has enabled subsequent chemoselective tagging of
O-GlcNAcylated proteins with either affinity tags or fluorophores.
These two-step metabolic feeding strategies have proven widely
useful for inventorying O-GlcNAcylated proteins,29, 32-36 mapping
O-GlcNAc to the genome,37 imaging O-GlcNAcylated proteins in
situ,30, 34, 38 and to uncover fundamental biology33, 35, 39. While
these metabolic labeling strategies have demonstrated value, these
approaches, as well as alternative chemoenzymatic labeling
methods,40-41 all rely on a two-step process, which limits their scope
in live cell imaging. Notably, most, though not all,34, 42 GlcNAc
precursor analogues give rise to conjugates that can be
deglycosylated by OGA, which results in observed changes being
an aggregate of both their installation and removal. Additionally,
the potential for incomplete reactions for relatively slow ligations
or non-specific reactions that may occur spontaneously when using
INTRODUCTION
The modification of hundreds of nuclear, cytoplasmic, and
mitochondrial proteins by N-acetylglucosamine (GlcNAc) residues
O-linked to serine and threonine residues (O-GlcNAc)1 is emerging
2-3
as a major regulator of cellular function.
Roles for O-GlcNAc
have recently been uncovered in regulating gene transcription,4
nutrient sensing,5-6 and stress response7-8. Furthermore, various
diseases are associated with changes in O-GlcNAc modification
excess of either metabolic probes or with various functionalities
during some chemoselective ligations,
43-44
may limit potential
applications that can be accessed using these strategies. Single step
labeling methods that permit chemoenzymatic labelling of cell
surface glycosylation have recently emerged.45 Inspired by these
recent reports as well as work from the Kohler group, who showed
metabolic feeding with a suitably protected, diazirine-modified,
GlcNAc-1-phosphate analogue (GlcNDAz) enabled labelling of O-
GlcNAcylated proteins,42 we set out to develop a one-step
metabolic labelling strategy that allows direct labeling of O-
such as cancers,9
cardiovascular disease
and
10
11-12
neurodegenerative disorders.13-14 Remarkably, despite the
abundance of O-GlcNAc on hundreds of structurally diverse
proteins within cells, only a single glycosyltransferase acts to
install this modification. Uridine diphosphate-N-acetyl-D-
glucosamine:polypeptidyl transferase (OGT, GT family 41) uses
uridine-5’-diphospho-N-acetylglucosamine (UDP-GlcNAc) as a
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GlcNAc modified proteins in vitro and within cells. We envisioned
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that this approach may ultimately facilitate studies focused on
monitoring kinetics of protein-specific O-GlcNAc modification,
intracellular trafficking of O-GlcNAc modified proteins within
cells, as well as the cellular stability of O-GlcNAcylated proteins.
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RESULTS AND DISCUSSION
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During the course of ongoing studies, we found the use of the
standard radioactivity-based glycosyltransferase assay for OGT22
to be somewhat cumbersome for use as a frequently used primary
assay. We therefore were interested in developing a more
convenient fluorescent assay to monitor glycosyltransfer catalyzed
by OGT. Analysis of the structure of human OGT, including a
ternary complex with both donor and acceptor substrates bound,46
suggested that this enzyme might tolerate moieties appended to the
2-acetamido group that are significantly larger than the diazirine-
containing sidechain used by Kohler and coworkers42 or
electrophilic derivatives used by Jiang and coworkers25.
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We hypothesized that the small, photochemically robust
fluorophore, 4-nitro-2,1,3-benzoxadiazole (NBD, Figure 1)
appended to GlcNAc might be tolerated by OGT. Looking toward
the longer term goal of metabolic engineering, we reasoned that the
low molecular weight of this fluorochrome, coupled with its
amphiphilic physicochemical properties, should confer adequate
solubility of downstream probes, yet not hinder their diffusion
through the plasma membrane. We also recognized that positioning
of this fluorochrome would be critical if it were to be tolerated by
the enzymes of the hexosamine biosynthetic pathway (HBP,
Figure 1), which also converts salvaged GlcNAc into UDP-
GlcNAc. In particular, previous studies showed that UDP-N-
acetylglucosamine pyrophosphorylase 1(AGX1), which converts
Figure 1. Metabolic labelling strategy for cellular biosynthesis of
UDP-GlcN--Ala-NBD. Following diffusion into cells and
deacetylation of Ac3GlcN--Ala-NBD-1-P(Ac-SATE)2 to form GlcN-
-Ala-NBD-1-P, this active -phosphate sugar readily enters into the
biosynthetic pathway and is converted into UDP-GlcN--Ala-NBD
(1), which can then be transferred by OGT onto a nucleocytoplasmic
proteins.
To qualitatively screen these potential donor substrates (1-4) we
N-acetyl--D-glucosamine-1-phosphate
into
UDP-GlcNAc,
used nucleoporin p62 (Nup62), which is a well-established protein
represents a biosynthetic bottleneck for analogues of GlcNAc.47
Indeed, engineering of cell lines through site-directed mutagenesis
of the active site of AGX1 was required to enable metabolic
labeling using diazirine-containing GlcNAc precursors.42 Our
examination of the AGX1 active site, however, suggested that
linkers positioning the fluorophore away from the amide moiety
might be better tolerated by this enzyme.
acceptor substrate for OGT,
in reactions containing
22, 28, 48
recombinant full length human OGT (hOGT). To confirm
enzymatic transfer and more readily distinguish the best substrates
we included uridine diphosphate (UDP), a competitive inhibitor of
OGT having a low nanomolar Ki value,15 to partially antagonize
OGT (Figure 2B). The resulting reactions were analyzed by SDS-
PAGE followed by laser fluorescence scanning of the acrylamide
gel. Remarkably, all analogues were transferred by OGT onto
Nup62, with probes 1, 3, and 4 appearing to be more efficient than
probe 2. The presence of 200 µM UDP reduced modification of
Nup62, indicating transfer was being catalyzed by OGT. Based on
these results we selected probes 1 and 3 for more detailed kinetic
analysis since these linkers were structurally related and could
therefore be more readily compared in downstream assays.
Bearing these points in mind we designed a series of UDP-
GlcNAc analogues in which the NBD fluorochrome was attached
to the 2-acetamido position of GlcNAc through linkers of
increasing length (Figure 2A) including -alanine (-Ala, 1), 5-
aminovaleric acid (AVA, 2), glycine--alanine (Gly--Ala, 3), and
tetraethyleneglycol (PEG4, 4). We accordingly prepared N-
hydroxysuccinimidyl activated linkers appended to NBD using
facile and general procedures (see supporting information). We
then coupled these activated esters with either uridine 5’-
diphospho-2-amino-2-deoxy--D-glucopyranose (UDP-GlcNH2,
5) or uridine 5’-diphospho-N-aminoacetyl--D-glucosamine
(UDP-GlcNAc-NH2, 6) to obtain the final compounds (Scheme 1).
The reaction of UDP-GlcNH2 (5) with the N-hydroxysuccinimide
esters (OSu) of NBD--alanine (7), NBD-aminovaleric acid (8) and
NBD-PEG4 (9) in the presence of NaHCO3 in a mixture of
MeCN:H2O afforded compounds 1, 2, and 4 accordingly. The
reaction of UDP-GlcNAc-NH2 (6) with NBD--alanine (7) using
the same conditions gave us the analogue containing a glycine--
alanine linker (3). We expected that these various linkers should
enable the NBD group to adopt a range of positions away from the
OGT active site. Moreover, they also have varying solubility,
polarity, and flexibility, variations that might lead to preferences by
OGT and the enzymes of the HBP, including AGX1.
To establish quantitative measures of the kinetic parameters
governing processing of these substrates by OGT, we next used the
UDP-GlcNAc analogues 1 and 3 as sugar donors in OGT-catalyzed
transfer reactions using the known O-GlcNAcylated protein
acceptors: calcium/calmodulin-dependent kinase IV (CaMKIV)49
and Nup62 48. Previously established kinetic parameters for Nup62
(Km(app)1 = 5.8 ± 0.7 M; kcat(app)1 = 0.84 ± 0.04 nmol/min/mg
hOGT) and CaMKIV (Km(app)1 = 2.1 ± 0.5 M; kcat(app)1 = 0.037 ±
0.002 nmol/min/mg hOGT) obtained using radioactively labelled
UDP-GlcNAc, were used as the benchmark of OGT assay.22 To
make quantitative measurements, we constructed a standard curve
using lysozyme covalently modified with NBD as an in-gel
standard (Supplementary Figures S1 and S2) in conjunction with
quantitative in-gel laser fluorescence scanning (Supplementary
Figure S3). We then assessed transfer of the NBD-sugar moiety of
UDP-GlcNAc analogues (1 and 3) by first using a fixed
concentration of UDP-GlcN-Gly--Ala-NBD (3) and varying
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CaMKIV and Nup62 concentrations. For CamKIV we found no
saturation up to
We next varied the concentrations of donor substrates including
UDP-GlcN-Gly--Ala-NBD (3), and UDP-GlcN--Ala-NBD (1),
while keeping fixed the concentration of the acceptor protein
substrates. In this way we determined that the Km(app)2 values for
both unnatural sugars ranged between 40- to 150-fold higher than
the value measured for UDP-GlcNAc (Figure 4), presumably due
to the bulkier 2-N-acyl substituent. The kcat/Km values, which are
the physiologically relevant rate constants since the cellular
reactions are all in competition, are within 40-fold of the natural
substrate UDP-GlcNAc. Interestingly, these differences in second
order rate constants (kcat/Km) depended on the identity of the protein
acceptor. In any event, these data show that hOGT processes both
of these UDP-NBD-sugar conjugates (1 and 3) quite well, indicating
they could potentially be useful substrates to replace the standard
radioassay for directly measuring group transfer and thereby enable
convenient monitoring of hOGT activity in vitro. Finally, we tested
whether human OGA (hOGA) would remove this modification
from proteins by incubating Nup62 that we had enzymatically
glycosylated with GlcN-NBD with hOGA. Even over long
incubation times, no decrease in fluorescence of the GlcN-NBD
glycosylated Nup62 protein was observed (Supplementary Figure
S4) indicating, as we expected,50 that this artificial GlcNAc sugar
cannot be cleaved by OGA. More saliently for our objectives, based
on these collective kinetic results, we expected that both UDP-
GlcN--Ala-NBD (1) and UDP-GlcN-Gly--Ala-NBD (3) would
be competent substrates for OGT within the cellular milieu, with
the fluorophore sugar modification accumulating on O-
GlcNAcylated proteins over time.
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Figure 2. UDP-GlcNAc analogues are accepted as substrates by
OGT. (A) UDP-GlcNAc analogues (1-4), (B) Laser scanning of
acrylamide gel assays show that UDP-GlcNAc-NBD analogues (1-
4) can be used as donor sugars by OGT using Nup62 as an acceptor
protein substrate.
Given these promising results, we accordingly set out to
synthesize membrane permeable biosynthetic precursors of UDP-
GlcN--Ala-NBD (1) and UDP-GlcN-Gly--Ala-NBD (3) that
could diffuse into the cell and be assimilated by cellular
biosynthetic pathways. Given that a diazirine-modified GlcNAc
analogue proved an incompetent substrate for the early steps of the
HBP in which N-acetylglucosamine kinase (NAGK)
phosphorylates the 6-hydroxyl of GlcNAc or N-acetylglucosamine-
phosphate mutase (AGM1) isomerizes GlcNAc-6-P to GlcNAc-1-
the limit of solubility of the protein (Figure 3A). Therefore, the
kcat(app)1 and Km(app)1 values obtained when using CamKIV should
be considered estimates. Nevertheless, on the basis of a linear
regression of the second order region of the Michaelis-Menten plot
we observe that the kcat/Km values for both CamKIV and Nup62
when using donor substrate 3 remained within approximately 3-
fold of the value observed when using UDP-GlcNAc (Figure 3A,
3B). Given that we saw no saturation for CaMKIV, we next used
only Nup62 as an acceptor substrate when examining UDP-GlcN-
-Ala-NBD (1) as a donor substrate. With this substrate we found
by standard non-linear fitting, that the kcat/Km value for Nup62 was
again comparable with that measured for UDP-GlcNAc using the
radioactivity-based assay (Figure 3C). Overall, these data indicate
that these unnatural donor sugars do not greatly perturb binding and
modification of protein substrates as assessed using these two
model proteins.
P,42 we considered using
a suitably protected advanced
intermediate within the HBP that would enable us to bypass these
two enzymatic steps. Previous successes have been observed in
metabolic feeding using per-O-acetylated 1--phospho-sugars in
which the phosphate group is protected with either two O-
acetoxymethyl51 or S-acetyl-2-thioethyl (SATE) groups42. These
protecting groups mask both hydrogen bond donors and charged
groups yet are sufficiently stable to enable passage across the cell
membrane, after which the protecting groups are removed through
the
action
of
intracellular
carboxyesterases
and
phosphodiesterases. Given our examination of the active site of
AGX1 (Supplementary Figure S5), we were optimistic
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Scheme 1. Synthetic route to NBD-conjugates of UDP-GlcNAc analogues (1-4).
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Acceptor
A. CamKIV
Donor
Km(app)1
(M protein substrate)
kcat(app)1
(nmol/min/mg hOGT)
kcat / Km
(nmol/min/mg hOGT/M acceptor)
UDP-GlcN-Gly--Ala-NBD, 3
UDP-GlcN-Gly--Ala-NBD, 3
35  17
0.72  0.19
0.35  0.03
24  20
59  26
B. NUP62
C. NUP62
5.9  1.3
UDP-GlcN--Ala-NBD, 1
0.70  0.15
0.22  0.01
314  16
Figure 3. Kinetic analysis of hOGT catalyzed transfer of UDP-Gly--Ala-NBD (3) and UDP--Ala-NBD (1) onto CamKIV and Nup62 at
various protein concentrations at a fixed substrate concentration.
a large NOE with the vicinal proton H2, confirming that the product
was, as desired, -configured (Spectra in Supplementary
Material). Ac3GlcN-Gly--Ala-NBD--1-P(Ac-SATE)2 (10) was
similarly synthesized (Spectra in Supplementary Material).
that Ac3GlcN--Ala-NBD--1-P(Ac-SATE)2 (10) and Ac3GlcN-
Gly--Ala-NBD--1-P(Ac-SATE)2 (11) would be deacylated
within cells to the corresponding -phosphate sugars GlcN--Ala-
NBD--1-P and GlcN-Gly--Ala-NBD--1-P, which would in
turn, through the action of AGX, be converted within the cell to the
donor sugars UDP-GlcN--Ala-NBD (1) and UDP-GlcN-Gly--
Ala-NBD (3). Given our in vitro results described above, we
reasoned that this strategy would lead to OGT modifying O-
GlcNAcmodified proteins within cells directly with the
corresponding fluorescent sugar.
With the two suitably protected candidate metabolic precursors of
the HBP in hand (10 and 11), we evaluated whether treatment of
wild-type HeLa cells with these probes used at concentrations
ranging from 25 to 200 µM resulted in modification of proteins
with the fluorescent NBD-sugar analogues. Notably, the smaller
diazirine-containing precursor was reported as not being
incorporated by WT HeLa cells, however, we felt that given the
slightly longer linkers we used here in conjunction with analysis of
the AGX1 structure, that these probes might be tolerated. Upon
analysis of the lysates from treated cells using laser scanning
fluorescence imaging of acrylamide gels, we were surprised to
observe little, if any, increase in modification of proteins when
using Ac3GlcN-Gly--Ala-NBD--1-P(Ac-SATE)2 (10), even
when increasing probe concentrations up to 200 µM and incubating
cells for up to 24 h (Supplementary Figure S6). Presumably, this
failure to label proteins stems from limited or no conversion of
precursor (10) into UDP-GlcN-Gly--Ala-NBD (3) by AGX1,
though this idea remains speculative and it is possible this
compound is simply catabolized within cells. However, upon
incubation of cells with Ac3GlcN--Ala-NBD--1-P(Ac-SATE)2
(11), we observed fluorescent labelling of cellular proteins
(Supplementary Figure S7). Cells treated for 24 h with 50 µM of
probe 11 exhibited no visible signs of toxicity and continued to
proliferate at a normal rate. The labelling efficiency of proteins in
both MEF and HeLa cells treated for 8 hours at various doses of
probe 11 showed a dose-dependent increase in protein labeling
(Figure 5B) as assessed by fluorescence intensity. These data
suggest that this labeling approach should prove general for use
with various cell lines.
For the synthesis of Ac3GlcN--Ala-NBD--1-P(Ac-SATE)2
(10), we first attempted to couple per-O-acetylated glucosamine
with NBD--Ala-OSu (7), followed by deprotection of anomeric
acetate and installation of the protected phosphate moiety.
Unfortunately, though the acylation and selective deprotection
proceeded efficiently, we obtained only the undesired -phosphate
that we attributed to steric hindrance by the NBD moiety. We
therefore decided to install the phosphate group on 1,3,4,6-tetra-O-
acetyl-2-azidoacetyl-2-deoxy-D-glucopyranose (15) and 1,3,4,6-
tetra-O-acetyl-2-azido-2-deoxy-D-glucopyranose (16) prior to the
N-acyl group in order to enable formation of the stereochemically
desired -glycosylphosphates (Scheme 2). Starting with
glucosamine hydrochloride (12) we converted this to the
corresponding
1,3,4,6-tetra-O-acetyl-2-azido-2-deoxy-
glucopyranoside (14) using imidazole-1-sulfonyl azide52 followed
by per-O-acetylation. Selective deprotection of the anomeric
hydroxyl
afforded
3,4,6-tri-O-acetyl-2-azido-2-deoxy-D-
glucopyranose (18), which was subsequently converted into the
protected phosphite ester through the use of phosphoramidite S,S'-
((((diisopropylamino)phosphanediyl)bis(oxy))bis(ethane-2,1-
diyl)) diethanethioate in the presence of 1H-tetrazole. Low
temperature oxidation of the phosphine intermediate using m-
chloroperbenzoic acid (mCPBA) afforded the protected 3,4,6-tri-
O-acetyl-2-azido-2-deoxy-glucopyranosyl phosphate derivative
(Ac3GlcN3--1-P(Ac-SATE)2, 20). We next chose to generate an
intermediate iminophosphorane from azide (20) using tributyl
phosphine, which readily reacted in-situ with NBD--Ala-OSu (7)
to form the desired amide Ac3GlcN--Ala-NBD--1-P(Ac-
SATE)2 (11). Spectral characterization of amide 11 to establish the
stereochemistry at the anomeric position showed that H1 displayed
We next sought to establish whether known O-GlcNAc modified
proteins are labeled using this one step labeling strategy. We
therefore immunoprecipitated FG-containing nucleoporins using
monoclonal MAb414, which readily precipitates Nup62, Nup153,
and Nup214.53 Imaging of fluorescence associated with the
resulting immunoprecipitated proteins and simultaneous detection
of Nup62, Nup153, and Nup214 using MAb414 revealed
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colocalization of fluorescence from NBD on three proteins with
UDP-GlcN--Ala-NBD (1), which in turn is used as a substrate by
OGT to modify target proteins with the NBD-sugar analogue. We
then turned
1
2
3
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8
molecular weights corresponding to those of Nup62, Nup153, and
Nup214 (Figure 6A). These data are consistent with Ac3GlcN--
Ala-NBD--1-P(Ac-SATE)2 (11) being converted within cells to
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Acceptor[a]
A. CamKIV
Donor
Km(app)2
(M[b]
kcat(app)2
(nmol/min/mg hOGT)
kcat / Km
)
(nmol/min/mg hOGT/M acceptor)
UDP-GlcN-Gly--Ala-NBD, 3
UDP-GlcN-Gly--Ala-NBD, 3
3860  350
51  14
1.60  0.11
0.43  0.03
1.37  0.14
8.3  2.1
B. NUP62
C. NUP62
UDP-GlcN--Ala-NBD, 1
36  6
0.30  0.01
8.4  1.5
Figure 4. Kinetic analysis of hOGT catalyzed transfer of UDP-GlcN-Gly--Ala-NBD (3) and UDP-GlcN-Gly--Ala-NBD (1) onto CaMKIV
and Nup62 at various substrate concentrations with fixed protein concentration.
to establishing whether we could use this probe in conjunction with
fluorescence microscopy. This is of particular interest since recent
reports have shown that metabolic two-step labeling, in conjunction
with fluorescence lifetime imaging (FLIM) using genetically
encoded fluorescent fusion proteins, can be used to quantify the
extent of O-GlcNAc modification of target proteins of interest
within cells.30, 38 We therefore incubated MEF cells for 20 hours
with 100 µM. Ac3GlcN--Ala-NBD--1-P(Ac-SATE)2 (11). After
fixing and washing the cells, fluorescence microscopy revealed
widespread labeling throughout the cytoplasm and nucleus (Figure
5C). Immunocytochemical imaging of MAb414 immunoreactivity,
superimposed over NBD fluorescence, supports components of the
nuclear pore being modified with GlcN--Ala-NBD. Notably, we
observe significant NBD fluorescence associated with what may be
the nucleolus within cells. The basis for this labeling is unclear but
given that many nuclear proteins are O-GlcNAc modified, this may
point to a specific set of proteins that are preferentially modified
using this probe or, alternatively, some non-specific accumulation
of the probe within this compartment.
To confirm that OGT is responsible for modification of the
proteins in cells we used engineered mouse embryonic fibroblast
(MEF) cells [MEF(OGTF/Y)] in which the OGT gene is flanked by
loxP recombination sites and also harbor a transgene encoding the
Cre recombinase from which expression of Cre can be induced by
treatment of cells with tamoxifen or related analogues.54
Accordingly, these cells enable inducible deletion of the OGT gene.
We incubated these cells with 4-hydroxytamoxifen (4-HT), or
vehicle alone, for 56 hours prior to treatment with 50 µM Ac3GlcN-
-Ala- NBD--1-P(Ac-SATE)2 (11) for 16 hours. We confirmed
that OGT protein levels were greatly decreased by immunoblot and
also observed, consistent with this decrease in OGT levels, that
OGA levels also decreased, as did overall O-GlcNAc levels
(Figure 6B). Notably, control cells having normal levels of OGT
showed labeling of proteins when treated with 11 whereas cells in
which the OGT gene had been deleted showed much reduced
protein labeling. These data indicate that OGT is the principle
enzyme responsible for fluorescent labeling of proteins with
Ac3GlcN--Ala-NBD--1-P(Ac-SATE)2 (11) within cells. As an
additional test whether
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OH
OAc
OH
OAc
1. (ClCH2CO)2O,
NaOMe, Et3N,
MeOH, rt
1
2
O
Ac2O
H2NNH2.HOAc
THF, rt
O
O
O
HO
HO
AcO
AcO
HO
HO
AcO
AcO
pyridine, rt
NaN , MeOH,
3
80oC
2.
OH
OAc
NH2
OH
R
OH
R
R
HCl
3
4
15
16
, R1 = NHCOCH3N3
13
14
17
, R1 = NHCOCH3N3
, R1 = NHCOCH3N3 (67%)
18
, R2 = N3 (93%)
12
(44% over 3 steps)
, R2 = N3
Im-SO2-N3
, R2 = N3 (87% over 2 steps)
5
6
CuSO45H2O,
K2CO3, MeOH, 2h
7
8
9
OAc
H2
O
OAc
O
nOe
H1
phosphoramidite,
1H-tetrazole,
MeCN/THF, rt
1.
AcO
AcO
AcO
NBD--Ala-OSu
AcO
NH
10
11
12
13
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2. mCPBA, 40oC to rt,
PBu3,
0oC to rt, THF
R
O
R
O
O
O
O
P
P
NH
O
O
O
19, R1 = NHCOCH3N3
O
(40% over 2 steps)
SAc
O2N
SAc
N
O
20
, R2 = N3
N
(25% over 2 steps)
SAc
SAc
10
11
, R1 = NHCO(CH2)2 (55%)
, R2 = CH2 (37%)
Scheme 2. Synthetic route to Ac3GlcN-Gly--Ala-NBD--1-P(Ac-SATE)2 (10) and Ac3GlcN--Ala-NBD--1-P(Ac-SATE)2 (11). This
strategy enables late-stage conjugation of NBD fluorophore using the Staudinger-Vilarrasa coupling.
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Figure 6. Cellular proteins are modified by O-GlcN-NBD through the
action of OGT. (A) Immunoprecipitation of nucleoporins. Left lane:
Immunoblotting using mAB414 antibody indicates nucleopore proteins
that are immunoprecipitated; Middle lane: Fluorescent bands indicate
proteins that were glycosylated with the GlcN-NBD residue; Right
lane: Merger of left and middle lane indicating overlapping
immunoreactivity and fluorophore. (B) Left blot: Treatment of MEF
cells with 4-HT leads to decreased OGT. Middle blot: GlcN-NBD
labeled proteins in the nuclear extract of control and OGT KO MEF
cells obtained from incubating cells with compound 11. Right blot:
Fluorescence signal from immunoprecipitated Nup62 is diminished
after knocking out OGT.
Figure 5. (A) Metabolic precursors Ac3GlcN-Gly--Ala-NBD--1-
P(Ac-SATE)2 (10) and Ac3GlcN--Ala-NBD--1-P(Ac-SATE)2 (11)
to donor sugars UDP-GlcN--Ala-NBD (1) and UDP-GlcN-Gly--
Ala-NBD (3). (B) Dose-dependence (0 to 200 µM) of protein labeling
in mouse embryonic fibroblasts (left) and HeLa (right) cells; WCL:
whole cell lysate (C) top left: HeLa cell lines stained with mAB414;
top middle: HeLa cells after incubation with compound (11); top right:
merged image depicting HeLa nucleus after treatment of compound
(11); bottom left: CTD110.6 stained HeLa cells indicating O-
GlcNAcylated proteins; bottom middle: HeLa cells after treatment with
compound (11); bottom right: merged image of depicting HeLa cells
treated with compound (11) and the O-GlcNAcylated proteins in the
cytoplasmic region.
CONCLUSIONS
We show that OGT tolerates unnatural analogues of the donor
substrate UDP-GlcNAc in which the 2-acetamido group is replaced
by relatively large substituents including, as shown here, by
fluorophores such as NBD. This observation should open the door
to convenient new direct transfer assays for OGT that do not rely
on the use of radioactivity, which should prove useful for the wider
community. We build on these observations to show that one step
labeling of O-GlcNAcylated proteins within live cells can be
realized by feeding cells with a suitable metabolic precursor that
can be assimilated at a late stage by the hexosamine biosynthetic
pathway. In particular, we find that feeding cells with suitably
protected 1-phosphosugar analogues, exemplified here by
Ac3GlcN-Gly--Ala-NBD--1-P(Ac-SATE)2 (10), leads to
labeling of known O-GlcNAcylated proteins such as nucleoporins
with a GlcN-NBD residue. We find the structure of the N-acyl
linker moiety plays a key role in governing the utility of such
probes and suggests that further tuning of the structure may enable
more efficient labeling. Notably, genetic engineering of cells to
express mutant AGX1 tolerant of modifications to the N-acyl group
OGT function is required for modification of O-GlcNAcylated
proteins with unnatural metabolic precursor (11), we examined
modification of nuclear pore protein Nup62 using these same cell
lines. Immunoprecipitation of Nup62 from these cells revealed
that only in control cells containing normal levels of OGT did we
observe
significant
GlcN--Ala-NBD-labeled
Nup62.
Collectively, these data provided strong evidence for OGT being
responsible for fluorescent labeling of O-GlcNAcylated proteins by
11 within cells.
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is unnecessary and does not lead to more efficient labeling in cells
1
2
3
4
(Supplementary Figure S8). Accordingly, we expect that this
approach will prove useful in a wide range of cells to explore the
physiological roles and regulation of O-GlcNAc in different
cellular models.
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Though we find that OGT is clearly the principle transferase
mediating the labeling of cells with GlcN-NBD, it is possible that
other transferases may tolerate this unnatural donor sugar, though
given our observations this would likely be at a lower level of
efficiency than OGT-catalyzed transfer. An obvious limitation to
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strategies, is that the kinetics of OGT-catalyzed transfer of these
unnatural GlcNAc analogues do not match the kinetics of transfer
of GlcNAc itself. Nevertheless, such one step metabolic in cell
labeling presents the possibility of performing a range of
experiments.
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One can envision for example, generating additional probes that
incorporate photophysically distinct fluorochromes that could
enable two colour pulse-chase experiments. Additionally, we
expect that engineering these metabolic precursors should permit
delivery of other moieties of interest, such as biotin, which could
enable simple proteomic strategies to studying O-GlcNAcylation.
Perhaps most notably, this one step strategy may be used in
conjunction with approaches described recently in which
fluorescence lifetime imaging (FLIM) to monitor the extent of
modification of specific proteins of interest directly within cells.30,
38
These FLIM studies made use of two-step chemoselective
ligation chemistry, which does not permit continuous imaging of
protein modification in cells. Accordingly, this one-step metabolic
feeding strategy, when used in conjunction with genetically
engineered proteins of interest fused to suitable fluorescent
proteins, could permit monitoring the spatiotemporal action of
OGT activity on specific recombinant target proteins of interest
within live cells in real time using FLIM.
ASSOCIATED-CONTENT
Supporting figures, Experimental methods, NMR characterization,
and kinetic data figures. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: dvocadlo@sfu.ca
ORCID:
David J. Vocadlo: 0000-0001-6897-5558
Hong Yee Tan: 0000-0003-3530-9350
Notes:
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The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
Financial support through a Discovery grant from the Natural
Sciences and Engineering Research (D.V., NSERC, RGPIN/-2015-
05426) and Project grant from the Canadian Institutes of Health
Research (D.V., CIHR PJT-148732) is gratefully acknowledged.
H.Y.T. was supported by the NSERC CREATE program ChemNet.
The authors thank J. Kohler (UT Southwestern) for the AGX
mutant cell line and N. Zachara (Johns Hopkins) for the inducible
OGT KO MEF cells. D.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.
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OAc
R =
HN
O
OH
AcO
AcO
O
P
N
N
O
O
O
HN
HO
HO
O
O
S
S
O
O
P
intracellular
UTP
HN
O
NO2
O
OH
O
R
O
O
Ac3GlcN--Ala-NBD--1-P(Ac-SATE)2
R
OH
GlcN--Ala-NBD--1-P
O
HO
HO
PPi
O
AGX1
O
One-step labeling of
O-GlcNAc
HN
OH
O
NH
O
HO
HO
R
O
P
O
N
O
O
HN
O
O
P
O
O-GlcN--Ala-NBD
Modified Protein
O
O
Na
O
Na
HO
OH
R
OGT
UDP-GlcN--Ala-NBD
UDP
Table of contents (TOC) figure.
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