The New Chemical Reporter 6-Alkynyl-6-deoxy-GlcNAc Reveals O-GlcNAc Modification of the Apoptotic Caspases That Can Block the Cleavage/Activation of Caspase-8

Article abstract of DOI:10.1021/jacs.7b02213

O-GlcNAc modification (O-GlcNAcylation) is required for survival in mammalian cells. Genetic and biochemical experiments have found that increased modification inhibits apoptosis in tissues and cell culture and that lowering O-GlcNAcylation induces cell death. However, the molecular mechanisms by which O-GlcNAcylation might inhibit apoptosis are still being elucidated. Here, we first synthesize a new metabolic chemical reporter, 6-Alkynyl-6-deoxy-GlcNAc (6AlkGlcNAc), for the identification of O-GlcNAc-modified proteins. Subsequent characterization of 6AlkGlcNAc shows that this probe is selectively incorporated into O-GlcNAcylated proteins over cell-surface glycoproteins. Using this probe, we discover that the apoptotic caspases are O-GlcNAcylated, which we confirmed using other techniques, raising the possibility that the modification affects their biochemistry. We then demonstrate that changes in the global levels of O-GlcNAcylation result in a converse change in the kinetics of caspase-8 activation during apoptosis. Finally, we show that caspase-8 is modified at residues that can block its cleavage/activation. Our results provide the first evidence that the caspases may be directly affected by O-GlcNAcylation as a potential antiapoptotic mechanism.

Full text of DOI:10.1021/jacs.7b02213

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Article
The new chemical reporter 6-alkynyl-6-deoxy-GlcNAc
6AlkGlcNAc) reveals O-GlcNAc modification of the apoptotic
(
caspases that can block the cleavage/activation of caspase-8.
Kelly N Chuh, Anna R Batt, Balyn W Zaro, Narek Darabedian, Nicholas
P. Marotta, Caroline K Brennan, Arya Amirhekmat, and Matthew R. Pratt
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 22 May 2017
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Journal of the American Chemical Society
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The new chemical reporter 6-alkynyl-6-deoxy-GlcNAc (6AlkGlcNAc)
reveals O-GlcNAc modification of the apoptotic caspases that can
block the cleavage/activation of caspase-8
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Kelly N. Chuh,* Anna R. Batt, Balyn W. Zaro, Narek Darabedian, Nicholas P. Marotta, Caro-
†
†
,†,§
line K. Brennan, Arya Amirhekmat, and Matthew R. Pratt*
†
§
Department of Chemistry and Department of Molecular and Computational Biology, University of Southern Cali-
fornia, Los Angeles, CA 90089-0744
ABSTRACT: O-GlcNAc modification (O-GlcNAcylation) is required for survival in mammalian cells. Genetic and bio-
chemical experiments have found that increased modification inhibits apoptosis in tissues and cell culture and that lower-
ing O-GlcNAcylation induces cell death. However, the molecular mechanisms by which O-GlcNAcylation might inhibit
apoptosis are still being elucidated. Here, we first synthesize a new metabolic chemical reporter, 6AlkGlcNAc, for the
identification of O-GlcNAc modified proteins. Subsequent characterization of 6AlkGlcNAc shows that this probe is selec-
tively incorporated into O-GlcNAcylated proteins over cell-surface glycoproteins. Using this probe, we discover that the
apoptotic caspases are O-GlcNAcylated, which we confirmed using other techniques, raising the possibility that the modi-
fication affects their biochemistry. We then demonstrate that changes in the global levels of O-GlcNAcylation result in a
converse change in the kinetics of caspase-8 activation during apoptosis. Finally, we show that caspase-8 is modified at
residues that can block its cleavage/activation. Our results provide the first evidence that the caspases may be directly
affected by O-GlcNAcylation as a potential anti-apoptotic mechanism.
INTRODUCTION
cal experiments have implicated O-GlcNAcylation as an
inhibitor of apoptosis. For example, specific genetic dele-
tion of OGT in T-cells caused a large amount of apoptosis
O-GlcNAc modification is found on serine and threonine
side-chains of proteins throughout the cytosol, nucleus,
and mitochondria of animal and plant cells (Figure 1A).
Unlike many forms of cell surface glycosylation, it only
consists of the addition of the single monosaccharide N-
acetyl-glucosamine that is not elaborated by any addi-
tional carbohydrates. O-GlcNAcylation is also dynamic
through action of the enzyme O-GlcNAc transferase
5
1
,2
in both CD4+ and CD8+ cells, and treatment of pancreat-
ic cancer cell-lines with a small molecule inhibitor of
16
OGT resulted in the induction of apoptosis. However,
the previously identified roles for O-GlcNAcylation in
apoptosis that could explain these results are somewhat
indirect: O-GlcNAcylation can drive the expression of
17
heat-shock proteins, glycosylated phosphofructokinase
and glucose-6-phosphate dehydrogenase have altered
(
OGT) that adds the modification and subsequent remov-
3
al by the enzyme O-GlcNAcase (OGA). Genetic experi-
ments have demonstrated that O-GlcNAcylation is re-
quired for development in mice and Drosophila and for
survival of mammalian cells in culture. Additionally,
changes in the overall cellular levels of this modification
directly contribute to
GlcNAcylation levels are consistently elevated in all types
of cancer when compared to healthy tissue. In contrast,
genetic loss of OGT in mouse neurons results in hyper-
18,19
activities, resulting in the production of NADPH, and
O-GlcNAcylated NFκB has increased transcriptional activ-
4-6
7
16
ity.
While these pathways certainly contribute to cell survival,
we were interested in determining if any of the direct
components of the apoptotic machinery are affected by
O-GlcNAcylation as a more immediate mechanism to
inhibit cell death. Apoptosis in mammalian cells is regu-
lated by the activation of a family of cysteine proteases
a
variety of diseases. O-
8
5
phosphorylated tau, and a combination of biochemical
and pharmacological experiments has demonstrated that
O-GlcNAcylation blocks the aggregation of the neuro-
20-23
termed caspases. These enzymes are translated as inac-
tive zymogens (pro-caspases), preventing the uncon-
trolled activation of cell death. Upon certain stimuli the
caspases are activated by proteolysis in two different
pathways (Figure 1B). The intrinsic pathway begins with
release of mitochondrial proteins, including cytochrome
c, into the cytosol. This results in the formation of a pro-
tein complex that contains multiple copies of pro-
caspase-9. The proteolytic activity of caspase-9 is in-
creased in this complex, resulting in its self-cleavage and
9
,10
degenerative disease associated proteins tau and α-
11,12
synuclein. The general role for O-GlcNAcylation in the
modulation of cell survival during stress potentially links
these two disease observations. The overall levels of O-
GlcNAcylation are significantly elevated by a variety of
cellular stressors in both culture and in vivo models, and
these increased modification levels promote the survival
13-15
of both cells and tissue. Notably, genetic and biochemi-
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activation. Caspase-9 then cleaves and activates the effec-
tor caspases, including caspase-3, which then cleave hun-
cells. This MCR builds upon our previously published
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reporter Ac 6AzGlcNAc, but displays improved detection
3
24-26
dreds of specific substrates resulting in cell death.
The
capabilities because of a superior a signal-to-noise ratio
due to the reverse orientation of the copper-catalyzed
second pathway, called the extrinsic pathway, is typically
initiated from outside the cell through the engagement of
death receptors by appropriate ligands. This leads to a
similar activation and cleavage of caspase-8 that will then
activate the effector caspases. The caspase family mem-
bers have different substrate preferences but all require
an aspartic acid (D) residue immediately N-terminal to
the cleavage site and prefer small amino acids immediate-
30
azide-alkyne cycloaddition (CuAAC) chemistry. When
combined with CuAAC, 6AlkGlcNAc enables the visuali-
zation of labeled proteins in as little as one hour and the
proteomic identification of 469 potentially O-
GlcNAcylated proteins in embryonic mouse fibroblast
cells, including caspases-3 and -8. We subsequently used
biochemical methods to confirm O-GlcNAcylation of all
three of the major apoptotic caspases in both mouse and
human cell lines. We then explored the consequences of
modification on caspase-8. Using small-molecule inhibi-
tors to raise and lower global O-GlcNAcylation levels, we
find that they are inversely related to the kinetics of
caspase-8 activation during apoptosis. Finally, we demon-
strate that caspase-8, a key initiator of the extrinsic apop-
totic pathway, is modified at its cleavage/activation sites
and show that these modifications inhibit cleavage in
vitro and potentially in living cells. Collectively, these
results provide an advanced MCR for the discovery of
potentially O-GlcNAcylated proteins and provide, for the
first time, a potential role for O-GlcNAc in the direct in-
hibition of caspase activation. We predict that this caspa-
se modification may be a function of O-GlcNAcylation in
both development and human disease.
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ly C-terminal.
In addition to the caspases, there are
other proteins that play key roles in apoptosis, including
numerous receptors, scaffolding proteins, and inhibitors
and activators of the pathway.
RESULTS AND DISCUSSION
Development of a selective and robust metabolic
chemical reporter for the identification of potential-
ly O-GlcNAcylated proteins. We and others have
demonstrated that the majority of O-GlcNAcylation
MCRs are not selective for O-GlcNAc and label other
types of glycoproteins. While this does not preclude their
use for the characterization of proteins, it does limit the
number of potentially O-GlcNAcylated proteins that can
be identified in a proteomics experiment, as the enrich-
ment of abundant cell-surface glycoproteins can mask the
29
presence of O-GlcNAcylated proteins. We serendipitous-
ly overcame this selectivity issue with our previously pub-
3
lished MCR (Ac 6AzGlcNAc (1), Figure 2A), which was
excluded from cell-surface glycoproteins but labeled O-
29
GlcNAcylation substrates. We chose to explore a 6-
modified MCR because UDP-6AzGlcNAc is a substrate for
Figure 1. O-GlcNAcylation and the major apoptotic caspases.
(
A) O-GlcNAcylation is the reversible addition of the mono-
31
saccharide N-acetyl-glucosamine to serine and threonine
side-chains of proteins in the cytosol, nucleus and mito-
chondria. (B) Apoptosis is carried out by several caspase pro-
teases, including 3, 8 & 9. The caspases are translated as inac-
tive zymogens (pro-caspases) that are activated by cleavage
at specific sites, directly after certain aspartic acid, D, resi-
dues. Caspases-8 & -9 activate themselves and subsequently
activate caspase-3.
OGT in vitro and because a small-molecule glycoside of
32
6AzGlcNAc can be hydrolyzed by OGA. Notably, we also
demonstrated in vitro that Ac 6AzGlcNAc (1) can bypass
3
the canonical metabolic salvage pathway by transfor-
mation to the 6AzGlcNAc-1-phosphate by the enzyme
29
phosphoacetylglucosamine mutase (AGM1). As men-
tioned above, azido-probes in combination with alkyne-
tags under CuAAC have increased background signal due
30
to non-specific reactivity of alkyne-tags with proteins,
Here, we describe a chemical-proteomics approach that
enabled the discovery of O-GlcNAcylation on all three of
the major apoptotic caspases (3, 8, and 9). To make this
discovery, we developed a new, metabolic chemical re-
which reduces the number of proteins that can be identi-
fied with confidence. Therefore, we synthesized here the
analogous alkyne-bearing MCR Ac
A) in 8 synthetic steps from commercially-available ma-
terial (Figure S1 (SI)). With this potential MCR in hand,
we first compared it to Ac 6AzGlcNAc (1), by treating
3
6AlkGlcNAc (2, Figure
2
3
porter (MCR), termed Ac 6AlkGlcNAc, that shows selec-
tivity for labeling O-GlcNAcylated proteins in mammalian
3
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Journal of the American Chemical Society
surface labeling (Figure 3B), consistent with its selectivity
NIH3T3 cells with either reporter or DMSO vehicle for 16
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h. Analysis by in-gel fluorescence, following lysis and Cu-
AAC with the appropriate fluorescent tag, demonstrated
towards FoxO1 labeling.
Finally, we set out to determine if treatment with MCRs
reduces cellular proliferation or induces apoptosis. To test
these possibilities, NIH3T3 cells were treated in triplicate
that Ac
trum of proteins as Ac
3
6AlkGlcNAc (2) labels a qualitatively similar spec-
6AzGlcNAc (1) (Figure 2B) but
3
with notably improved signal-to-noise (Figure 2B, High
fluorescence “gain”).
3
with either Ac 6AlkGlcNAc (2), Ac
3
6AzGlcNAc (1), or
Ac GlcNAlk before analysis using two different, commer-
4
We next determined whether Ac
3
6AlkGlcNAc (2) is able
cially-available assays: an MTS assay to measure general
cellular-proliferation and an apoptosis assay that
measures the catalytic activity of caspase-3 and -7 (Figure
S5A (SI)). Although there appeared to be a small decrease
in the proliferation of the cells, none of the differences
were statistically significant and no induction of apoptosis
was observed. We next repeated this experiment in HeLa
to label proteins in different mammalian cell-lines. Ac-
cordingly, a small panel of cells was treated with either
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Ac 6AlkGlcNAc (2, 200 μM) or DMSO vehicle for 16 h.
3
The cells were then lysed and subjected to CuAAC condi-
tions with azido-rhodamine. In-gel fluorescence scanning
showed a diversity of modified proteins in all the cell-
lines tested (Figure S2 (SI)), demonstrating the general
cells under Ac 6AlkGlcNAc (2) treatment and observed
3
applicability of Ac
an cell-lines. We then characterized the dose-dependence
and kinetics of Ac 6AlkGlcNAc (2) labeling and compared
it directly to our two previously published MCRs,
3
6AlkGlcNAc (2) to different mammali-
similar results, with no significant change in cellular pro-
liferation and no induction of apoptosis (Figure S5B (SI)).
Together these data demonstrate that Ac 6AlkGlcNAc (2)
has the potential to be a selective probe for the identifica-
tion of O-GlcNAcylated proteins that is relatively benign
to cells.
3
3
29
33
Ac
SI)). These data show that Ac
teins with faster kinetics than Ac
3
6AzGlcNAc (1) and Ac
4
GlcNAlk (Figures S3 and S4
6AlkGlcNAc (2) labels pro-
6AzGlcNAc (1) (Figure
(
3
3
Identification of potentially O-GlcNAcylated pro-
S3A (SI)), which combined with the low background sig-
nal enables proteins to be robustly labeled in as little as 1
teins using Ac
Ac 6AlkGlcNAc (2) to identify potentially modified pro-
teins and simultaneously compared it to Ac GlcNAlk in
3
6AlkGlcNAc. Next, we applied
3
h (Figure S3B (SI)). Additionally, Ac
incorporated more efficiently than Ac
Figure S3C (SI)). Ac 6AlkGlcNAc (2) labeled cells with
slightly slower kinetics compared to Ac GlcNAlk (Figure
S4A (SI)) and at lower overall levels (Figure S4B (SI)).
Finally, we looked at the turnover of Ac 6AlkGlcNAc-
3
6AlkGlcNAc (2) is
4
3
6AzGlcNAc (1)
order to uncover any obvious benefits of the increased
selectivity. Immortalized mouse embryonic fibroblasts
(
3
4
(
MEFs) were treated in triplicate with either
Ac 6AlkGlcNAc (2), Ac GlcNAlk, or Ac GlcNAc as a con-
3
4
4
3
trol. The cells were lysed and subjected to CuAAC condi-
tions with an azide-bearing biotin tag. The proteomes
were then reduced, alkylated, and subjected to biotin-
enrichment using streptavidin-conjugated beads. After
extensive washing to remove unlabeled proteins, on-bead
trypsinolysis afforded peptides that were analyzed using
LC-MS/MS. Labeled proteins were identified as those that
met threshold criteria for enrichment (see Tables S1 & S2
dependent labeling in a pulse-chase experiment (Figure
S4C (SI)). The associated fluorescent signal was steadily
lost over the course of 48 h at a comparable rate to
Ac GlcNAlk-labeling. While some of this signal loss is
4
undoubtedly due to protein degradation, the similarity in
the kinetics suggests that 6AlkGlcNAc, like GlcNAlk and
6
AzGlcNAc, is also a substrate for OGA. We then exam-
ined the selectivity of Ac 6AlkGlcNAc (2) labeling of dif-
3
(
SI) for details) based on spectral counting, resulting in
69 proteins identified as potential O-GlcNAcylated pro-
teins that were labeled by Ac 6AlkGlcNAc (2) (Figure 4A
and Table S1 (SI)) and 433 proteins labeled by Ac GlcNAlk
Figure 4A and Table S2 (SI)), with many of the proteins
ferent classes of glycoproteins by first using two reporter
proteins: GlyCAM-IgG, which is a secreted fusion protein
that contains both N-linked and mucin O-linked cell-
surface glycans, and FoxO1 that is an O-GlcNAcylated
transcription factor. As expected, treatment with
4
3
4
(
showing overlap in the two data sets. Additionally, a large
fraction of the identified proteins has been characterized
as being potentially O-GlcNAcylated in past proteomic
experiments. Although spectral counting is inherently a
semi-quantitative measurement of protein enrichment, as
larger proteins will produce more peptides, we used it
because it allowed us to directly compare the identified
proteins in this study to our previously published data on
Ac GlcNAlk as a control resulted in labeling of both pro-
4
teins (Figure 3A), as this MCR is not excluded from cell-
surface glycoproteins. However, when these cells were
treated with Ac
of GlyCAM-IgG but robust labeling of FoxO1 (Figure 3A),
indicating that Ac 6AlkGlcNAc (2) may be selectively-
3
6AlkGlcNAc (2), we observed no labeling
3
labeling potential O-GlcNAcylation substrates. To con-
firm these results, we also performed an analysis of the
entire repertoire of cell surface glycans using flow cytom-
etry by taking advantage of a copper-chelating azido bio-
29
azide-bearing MCRs.
3
4
tin tag that enables CuAAC on the surface of living cells.
More specifically, NIH3T3 cells were treated with either
Ac
3
6AlkGlcNAc (2) or the non-selective MCRs
GlcNAlk or Ac ManNAlk. Analysis of cell-surface label-
6AlkGlcNAc
(2) treatment resulted in dramatically less global cell-
Ac
4
4
ing by flow cytometry demonstrated that Ac
3
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Figure 3. Ac
surface glycoproteins. (A) Ac
an O-GlcNAcylated reporter protein. NIH3T3 cells expressing
either secreted GlyCAM-IgG or nucleocytoplasmic FoxO1
were treated with DMSO vehicle, Ac
3
6AlkGlcNAc (2) is largely excluded from cell-
6AlkGlcNAc (2) is selective for
3
3
6AlkGlcNAc (2, 200
μM), or Ac GlcNAlk (200 μM) for 24 h. At this time, the pro-
4
teins were enriched from the media or soluble cell lysate,
respectively, subjected to CuAAC with azido-rhodamine and
analyzed by in-gel fluorescence scanning. The data is repre-
sentative of two biological replicates. (B) Ac
is largely excluded from cell surface glycosylation. NIH3T3
cells were treated with Ac GlcNAlk, Ac ManNAlk, or
Ac 6AlkGlcNAc (2) (all at 200 μM) for 16 h. The cells were
3
6AlkGlcNAc (2)
4
4
3
then harvested and subjected to CuAAC with picolyl azido-
biotin. After incubation with FITC-streptavidin, cell-surface
labeling was measured by flow cytometry. Error bars repre-
sent ±s.e.m. from the mean of biological replicates (n = 3),
and statistical significance was calculated using a two-tailed
Student’s t-test.
We first compared the total number of proteins that
could be identified at a statistically-significant level be-
tween the different MCRs (Figure 4B). Not surprisingly
the alkyne-MCRs outperformed the azide counterparts
because of their improved signal-to-noise ratios, and
Ac
potentially O-GlcNAcylated proteins compared to
Ac GlcNAlk. We then collated the overlap between the
proteins identified by Ac 6AzGlcNAc (1), Ac 6AlkGlcNAc
2), and Ac GlcNAlk and found significant overlap be-
3
6AlkGlcNAc (2) enabled the identification of more
Figure 2. Characterization of the metabolic chemical report-
er Ac 6AlkGlcNAc (2). (A) Metabolic chemical reporters
(MCRs) of O-GlcNAcylation. Treatment of cells with
Ac 6AzGlcNAc (1) results in the incorporation of an azide
functionality onto O-GlcNAcylated proteins, which can be
subsequently reacted with alkyne tags using bioorthogonal
3
4
3
3
3
(
4
tween the datasets (Figure 4C). We also annotated the
data based on the sub-cellular localization of the identi-
fied proteins as either intracellular or transmembrane,
and therefore potential O-GlcNAc substrates, or extracel-
lular/lumenal. Proteins without defined localizations
chemistry. Ac
in the same way but with improved signal-to-noise. (B)
Ac 6AlkGlcNAc (2) has improved signal-to-noise compared
to Ac 6AzGlcNAc (1). NIH3T3 cells were treated with either
DMSO vehicle, Ac 6AzGlcNAc (1, 200 μM), or
Ac 6AlkGlcNAc (2, 200 μM) for 16 h, followed by CuAAC with
3
6AlkGlcNAc (2) was hypothesized to function
3
3
were omitted. In our experiments, Ac 6AlkGlcNAc (2)
3
3
enabled the identification of the most intracellular pro-
teins (Figure 4D), which have the opportunity to be O-
GlcNAcylated. Importantly, the previous proteomic iden-
tification of proteins labeled with azide-containing MCRs
was performed in the same cell line, at identical concen-
trations and treatment times. Notably, the apoptotic
caspases-3 and -8 were identified in our proteomics ex-
periment as potential O-GlcNAc substrates from the
3
the appropriate fluorescent tag and analysis by in-gel fluo-
rescence scanning. The data is representative of two biologi-
cal replicates.
3
Ac 6AlkGlcNAc-treated but not Ac GlcNAlk-treated cells
4
(
Figure 4A). To visualize the enrichment of these caspases
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and three known O-GlcNAcylated proteins, the same cells
were treated with Ac 6AlkGlcNAc (2) as above, and the
GlcNAcylated proteins and identified caspases are annotated.
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(B) Number of proteins identified as being enriched to a sta-
tistically significant amount by MCRs in this study (in green)
and a previously published study (in black). (C) Ven diagram
showing the overlap in the proteins identified using
3
labeled proteins were enriched and selectively eluted us-
ing a cleavable azido-azo-biotin tag. Western blotting
confirmed the enrichment of both caspases and the
known O-GlcNAcylated substrates NEDD4-1, pyruvate
kinase (PK), and nucleoporin 62 (nup62) (Figure 5A). Im-
portantly, the abundant, non-O-GlcNAcylated protein β-
actin was not enriched, demonstrating the potential O-
GlcNAc-selective nature of our enrichment. To further
3 3 4
Ac 6AzGlcNAc (1), Ac 6AlkGlcNAc (2), and Ac GlcNAlk. (D)
Graphical representation of the enriched proteins from this
study (in green) and a previously published experiment (in
black) based on their annotated localization. In all experi-
ments, identical cell lines, MCR concentrations, and treat-
ment times were used. The number of proteins in D is less
than the number in B due to proteins with unannotated lo-
calization.
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compare the utility of Ac
3
6AlkGlcNAc (2) to
Ac 6AzGlcNAc (1), we repeated this experiment with both
3
MCRs and compared the efficiency of nup62 enrichment
and found that the alkyne-probe is indeed superior (Fig-
ure 5B). To confirm that caspases-3 and -8 are legitimately
O-GlcNAcylated, we first used chemoenzymatic labeling
35
(Figure S6 (SI)). This method detects endogenous O-
GlcNAcylated proteins by using an engineered galactosyl-
transferase, GalT(Y289L), to modify O-GlcNAc modifica-
tions with the azide-bearing monosaccharide N-
azidoacetyl-galactosamine. The azide of the resulting di-
saccharide can then be detected using CuAAC and alkyne
visualization- or affinity-tags. Lysates from MEFs were
subjected to chemoenzymatic labeling followed by Cu-
AAC with an alkyne-azo-biotin tag (Click Chemistry Tools
#1042) to enable enrichment of O-GlcNAcylated proteins.
Western blotting after elution from streptavidin beads
confirmed that caspase-3 and -8 are indeed endogenously
O-GlcNAcylated (Figure 5B).
Figure 5. Caspases-3 and -8 are genuine O-GlcNAcylated
proteins in mouse cells. (A) Confirmation of Ac
3
6AlkGlcNAc
(
2) labeling of known O-GlcNAcylated proteins and caspase-
3
and -8. Mouse embryonic fibroblasts were treated with
6AlkGlcNAc (2, 200 μM) or DMSO for 16 h, fol-
either Ac
3
lowed by CuAAC with a cleavable azido-biotin tag. After en-
richment on streptavidin beads, the labeled proteins were
eluted and visualized by Western blotting. The non-
glycosylated protein β-actin is a negative control. (B) Nup62
is enriched more efficiently by Ac
when compared to Ac 6AzGlcNAc (1). NIH3T3 cells were
treated with either Ac 6AlkGlcNAc (2, 200 μM),
Ac 6AzGlcNAc (1, 200 μM), or DMSO for 16 h, followed by
3
6AlkGlcNAc (2) labeling
3
3
3
CuAAC with a cleavable azido-biotin tag. After enrichment
on streptavidin beads, the labeled proteins were eluted and
visualized by Western blotting. (C) Chemoenzymatic label-
ing identifies caspases-3 and -8 as O-GlcNAcylated proteins.
MEF cell-lysates were subjected to chemoenzymatic modifi-
cation of endogenous O-GlcNAcylation sites and then Cu-
AAC with alkyne-azo-biotin. Western blotting after enrich-
ment and elution confirmed O-GlcNAcylation of caspases-3,
8, and the positive control Nup62. (D) The WGA lectin en-
Figure 4. Identification of O-GlcNAcylated proteins, includ-
riches caspase-3 and -8. Cell lysates from NIH3T3 cells that
stably express FLAG-tagged caspase-3 or -8 were subjected to
two rounds of affinity enrichment, anti-FLAG followed by
WGA lectin, before analysis by Western blotting. (E) Reverse
ing caspase-3 and -8, using Ac
embryonic fibroblasts were treated with Ac
Ac GlcNAlk, or Ac GlcNAc (all at 200 μM) for 16 h. At this
3
6AlkGlcNAc (2). (A) Mouse
3
6AlkGlcNAc (2),
4
4
time, the corresponding cell-lysates were subjected to Cu-
AAC with azido-biotin, incubation with streptavidin-coated
beads, and trypsinolysis. Proteins identified by LC-MS/MS
are graphically presented as total number of positive minus
total number of control spectral counts. Three known O-
confirmation of Ac 6AlkGlcNAc labeling of caspase-3 and -8.
3
NIH3T3 cells that stably express FLAG-tagged caspase-3 or -8
were treated with either Ac 6AlkGlcNAc (2, 200 μM) or
3
DMSO for 16 h, followed by immunoprecipitation with an
anti-FLAG antibody, CuAAC with an azido-biotin tag and
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streptavidin and Western blotting. The data in all panels is
The major apoptotic caspases are O-GlcNAcylated in
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8
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representative of two biological replicates.
human cancer cell-lines. To confirm caspase O-
GlcNAcylation in human cells, we first treated three can-
cer cell-lines - H1299 (lung), HeLa (cervical), and MCF7
We then stably transformed NIH3T3 cells with FLAG-
tagged caspase-3 or caspase-8. Notably, these proteins
contained mutations at their active sites to prevent the
autoinduction of caspase activity. Immunoprecipitation of
these proteins with anti-FLAG beads was performed, fol-
lowed by a second round of enrichment with the lectin
wheat germ agglutinin (WGA) that binds to O-GlcNAc
modifications. Western blotting against the caspases
showed WGA enrichment of a fraction of the protein,
further indicating that these proteins are endogenously
O-GlcNAcylated (Figure 5D). The stable cells were then
(
breast) - with Ac 6AlkGlcNAc (2) and enriched the la-
3
beled proteins using the cleavable azido-azo-biotin tag.
Western blotting after elution demonstrated MCR label-
ing of caspase-3 and -8, as well as caspase-9, which we did
not detect in our proteomics experiment (Figure 6A). No-
tably, the non-O-GlcNAcylated protein β-actin was once
again largely unenriched compared to inputs in any of the
cell lines and caspase-3 was absent from MCF7 cells as
this cell line is caspase-3 null. We then used chemoen-
zymatic labeling followed by biotin-enrichment and
found that all three caspases are endogenously O-
GlcNAcylated in H1299 cells (Figure 6B). We then turned
our focus to caspase-8, the initiator caspase of the extrin-
sic apoptosis pathway, and used chemoenzymatic en-
richment to show that caspase-8 is O-GlcNAcylated in all
three cancer cell lines (Figure S7 (SI)).
0
1
2
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0
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9
0
treated with Ac 6AlkGlcNAc (2) or DMSO vehicle, fol-
3
lowed by immunoprecipitation of the individual caspases
using an anti-FLAG antibody. Subsequent CuAAC with
azido-biotin, followed by analysis by streptavidin blotting
confirmed the MCR labeling of both caspase -3 and -8
(Figure 5E). Together these data confirm that caspases-3
and -8 are bona fide O-GlcNAcylated proteins in mouse
cells.
Changes in O-GlcNAcylation levels directly affect the
kinetics of caspase-8 cleavage/activation. We next
investigated the consequences of O-GlcNAcylation on
caspase-8 biochemistry by first determining whether the
levels of its modification change during apoptosis. During
the initiation of extrinsic apoptosis, monomers of pro-
caspase-8 are brought into close proximity where they are
able to cleave themselves to generate the fully active pro-
tease, which then goes on to activate the executioner
caspases, such as caspase-3 (Figure 7A). Typically, this
happens through first cleavage after aspartic acid residues
(
D374 and D394) between the large and small subunits of
the catalytic domain, removing the “activation loop,” fol-
lowed by cleavage after at least one other aspartic acid
residue (D210, D216, and/or D223) which removes the
36
pro-domain. Accordingly, HeLa cells were treated with
tumor necrosis factor alpha (TNFα) to induce caspase-8-
mediated apoptosis, which also caused a very slight in-
crease in the global levels of O-GlcNAcylation (Figure S8
(
SI)). Western blotting with two different caspase-8 anti-
bodies clearly showed the time dependent cleav-
age/activation of caspase-8 based on the appearance of a
lower molecular-weight, activated species (p43/41, Figure
Figure 6. The apoptotic caspases are O-GlcNAcylated in
human cancer cell-lines. (A) Metabolic chemical reporter
enrichment. H1299, HeLa, and MCF7 cells were treated with
7B). Chemoenzymatic labeling was used to enrich the
either DMSO vehicle or Ac 6AlkGlcNAc (2, 200 μM) for 16 h.
3
endogenously O-GlcNAcylated fraction of caspase-8 in
this experiment. This modified protein showed less loss
(i.e., cleavage) of pro-caspase-8 and very little of the acti-
vated p43/41 species (Figure 7B), indicating that O-
GlcNAcylation of caspase-8 inhibits the cleavage and acti-
vation of caspase-8. Given this result, we next treated
HeLa cells with either DSMO, 5SGlcNAc or Thiamet-G to
alter O-GlcNAcylation levels (see Figure S9 (SI) for effects
of the inhibitors on O-GlcNAcylation levels). These cells
were then treated with TNFα and the cleavage and activa-
tion of caspase-8 and caspase-3 was visualized by Western
blotting (Figure 7C and Figure S10 (SI)). In all the biologi-
cal replicates, treatment with 5SGlcNAc resulted in faster
kinetics of caspase activation, while Thiamet-G gave
slower rates of activation, when compared to DMSO
Cell lysates were then subjected to CuAAC with the appro-
priate cleavable biotin-tag, followed by enrichment on strep-
tavidin-coated beads. After extensive washing and elution
(
2 2 4
Na S O ), Western blotting revealed the modification of
caspases-3, -8, and -9. (B) Chemoenzymatic modification and
enrichment. H1299 cell-lysates were subjected to chemoen-
zymatic modification of endogenous O-GlcNAcylation sites
and then CuAAC with alkyne-azo-biotin. Western blotting
after enrichment and elution confirmed O-GlcNAcylation of
caspases-3, 8, and -9. In A and B, enrichment of the known
O-GlcNAcylated protein nucleoporin 62 (Nup62) served as a
positive control, and the non-glycosylated protein β-actin
was a negative control. In both experiments, the data is rep-
resentative of two biological replicates.
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effects must occur upstream of NFκB transcriptional ac-
treated cells. Notably, quantitation of the percentage of
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cleaved caspase-8 (p43/41 plus p18) from these replicates
confirms a statistically significant difference in the rate of
caspase-8 activation that inversely depends on O-
GlcNAcylation levels (Figure 7D). Importantly, we did not
observe a change in the overall levels of caspase-8 upon
inhibitor treatment (Figure S11 (SI)). Additionally, the
activation of caspase-8 is through direct engagement of
death receptors by TNFα, indicating that the effects of O-
GlcNAcylation are in this pathway, and these observed
tivity, as the cells were also treated with cycloheximide to
prevent new protein synthesis. This is in contrast to acti-
vation of apoptosis by a stimulus like cellular stress that
could be affected by the established roles for O-GlcNAc in
cellular metabolism and heat shock response.
0
1
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0
Figure 7. O-GlcNAcylation levels affect caspase-8 cleavage/activation. (A) The extrinsic apoptosis pathway. Upon certain signals,
inactive, full-length caspase-8 can be recruited to cell-death receptors where it is oligomerized. Dimers of inactive caspase-8
have increased activity and self-cleave after aspartic acid residues 374 and 384, removing the activation loop and yielding activat-
ed enzyme (p43/p41 and p10 fragments). The p43/41 fragment can then be further cleaved to release the death effector domain
(DED), resulting in the formation of a p18 fragment. Active caspase-8 will then go on to cleave the executionerr caspases, like
caspase-3. (B) The endogenously O-GlcNAcylated fraction of pro-caspase-8 has less cleavage during apoptosis. HeLa cells were
-1
-1
treated with either vehicle or TNFα (10 ng mL ) and cycloheximide (1 μg mL ) to induce apoptosis. After 6 h, the O-
GlcNAcylated proteins were enriched using chemoenzymatic modification and caspase-8 was visualized by Western blotting. (C)
Altering O-GlcNAcylation levels changes the kinetics of caspase-8-mediated apoptosis. HeLa cells were treated with TNFα (10 ng
-1
-1
mL ) and cycloheximide (CHX, 1 μg mL ) to induce apoptosis in the presence or absence of either 5SGlcNAc (OGT inhibitor, 100
μM, 16 h pretreatment) or Thiamet-G (OGA inhibitor, 5 μM, 20 h pretreatment). The kinetics of caspase activation were then
visualized after the indicated times by Western blotting. Active caspases were distinguished from their corresponding zymogen
form by molecular-weight shift. The different amounts of each caspase species was quantitated and normalized to the total
caspase signal in each lane. See Figure S10 for the other biological replicates. (D) Quantitation of the percentage of active caspa-
se-8 (p43/41 plus p18) from the experiments in (C) and Figure S10 (SI). Error bars represent ±s.e.m. from the mean of biological
replicates (n = 2 for 5SGlcNAc and Thiamet-G, n = 3 for DMSO), and statistical significance was calculated using a two-tailed
Student’s t-test.
3
7-39
374
376
Caspase-8 is O-GlcNAcylated near its cleav-
age/activation sites. As mentioned above, the caspases
are activated upon self- or cross-cleavage after particular
aspartic acid residues. Notably, at or near the cleavage
sites required for activation of all three of these caspases
are serine (S) or threonine (T) residues that could be O-
GlcNAcylated, which might affect the efficiency of cleav-
age/activation (Figure S12 (SI)). Importantly, these cleav-
age sites lie in flexible activation loops, and OGT has a
strong preference for unstructured regions of its substrate
proteins.
Here, we focused on these sites ( DSE and
DLSS ; cleavage after D during activation) in caspase-8
384
387
36,40,41
(schematized in Figure 8).
Specifically, H1299 cells
were co-transfected in duplicate with constructs encoding
either HA-tagged wild-type or a FLAG-tagged mutant,
either caspase-8(S375A), caspase-8(S386,387A), or caspa-
374
376
384
387
se-8(AAA) ( DAE and DLAA ) (Figure S13 (SI)). All
of these proteins also contained an active-site mutation
(C to A) to prevent the autoinduction of apoptosis ob-
served in some caspase-8 overexpression experiments.
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The entire pool of endogenously O-GlcNAcylated pro-
teins from these cells were enriched using chemoen-
zymatic labeling (Figure S6), followed by CuAAC with the
cleavable biotin tag, enrichment, and elution as above.
Western blotting showed that all three mutants were en-
riched at significantly lower amounts (normalized to in-
puts) compared to the wild-type protein (Figure 8 and
Figure S13 (SI)). Strikingly, this effect was additive and
caspase-8(AAA) was only enriched at ~10% compared to
the wild-type protein, indicating that essentially all of the
O-GlcNAcylation occurs at the cleavage sites.
DWE ) inhibited the cleavage of this peptide, although
1
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the inhibition by StoW was more pronounced (Figures S15
and S17 (SI)). To our knowledge the effects of mutations
at the P2’ and P3’ positions on caspase-8-mediated cleav-
age have never been systematically tested. Native peptide
2, containing DLSS , again showed notable cleavage,
while the alanine peptide (7, DLAA
cleavage and the tryptophan peptide (8, DLWW ) dis-
played less (Figures S15 and S17 (SI)). We then quantitated
this data to again determine the efficiency of removal of
the activation loop (Figure 9C). We found that the ala-
nine mutations had an overall small inhibitory effect on
caspase-8 activation loop cleavage by 1.2-times compared
to the corresponding serine residues, indicating that the
effect of the mutation alone would be to slow caspase-8
activation slightly. The tryptophan mutations inhibited
cleavage of the loop by 3-times, making these mutations a
reasonable, but less-effective, analog of O-GlcNAcylation
for the inhibition of caspase-8 cleavage.
384
387
384
387
) showed increased
384
387
0
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2
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9
0
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9
0
O-GlcNAcylation
blocks
caspase-8
cleav-
age/activation in vitro and potentially in living cells.
To determine if O-GlcNAcylation can directly affect
caspase-8 self-cleavage/activation, we first performed an
in vitro cleavage assay. As mentioned above, pro-caspase-
8
cleaves itself to generate the fully active protease.
Therefore, active caspase-8 is able to cleave peptides that
correspond to these cleavage sites in vitro. We synthe-
sized peptides surrounding the cleavage site that were
either unmodified (1 and 3) or O-GlcNAcylated (2 and 4,
Figure 9A). These peptides were then incubated in tripli-
cate with recombinant, active human caspase-8 before
analysis using reverse-phase HPLC (RP-HPLC) which sep-
arated any cleavage product from the starting peptide
(
Figure S14 (SI)), and the absolute identity of the peptides
was confirmed by ESI-MS. Caspase-8 was able to readily
cleave both of the unmodified peptides. In contrast, O-
GlcNAcylation almost completely prevented the for-
mation of the cleavage product, with the unmodified pep-
tides 1 and 3 cleaving approximately 28- and 16-times
more efficiently than the O-GlcNAcylated peptides 2 and
4
, respectively (Figure 9A). This leads to a 20-times reduc-
tion in the relative removal of the activation loop (Figure
B), indicating that O-GlcNAcylation at these sites would
9
inhibit the self-activation of caspase-8 during cell death.
We next wanted to test if O-GlcNAcylation at these sites
could affect caspase-8 cleavage in living cells. During
apoptosis in cells both dimerization and caspase-8 cleav-
age have been shown to be required for the generation of
Figure 8. Caspase-8 is O-GlcNAcylated near its cleav-
age/activation sites. Schematic of wild-type caspase-8 con-
3
74
376
384
387
39
taining two cleavage sites ( DSE
and
DLSS ). O-
the fully active caspase-8 enzyme. To determine the con-
GlcNAcylated proteins from H1299 cells transfected with
both HA-tagged wild-type or FLAG-tagged versions of the
indicated mutant caspase-8 proteins were enriched using
chemoenzymatic transfer and visualized by Western blot-
ting. The HA and FLAG blots were visualized simultaneously
to enable a quantitative comparison between the two blots.
Results are the average of two separate biological experi-
ments that were imaged simultaneously (see Figure S13 (SI)).
Error bars represent ± SEM, and statistical significance was
calculated using a two-tailed Student’s t-test.
sequences of O-GlcNAcylation, we planned to test this
cleavage using both a mutant that cannot be O-
GlcNAcylated (i.e., serine to alanine) and a mutant that
could approximate the steric bulk of O-GlcNAc (serine to
tryptophan). We chose tryptophan as a surrogate because
there is no way to site-specifically incorporate O-GlcNAc
using artificial amino acids. To test if these mutations
affected caspase-8 cleavage, we first synthesized the cor-
responding serine to alanine (StoA) and serine to trypto-
phan (StoW) peptides (5-8) and repeated the in vitro
cleavage assay described above. In this experiment, we
With the peptide mutations characterized in vitro, we set
out to test their consequences on caspase-8 activation in
living cells. It has been observed that transient transfec-
tion of caspase-8 can lead to spontaneous dimerization
and cleavage/activation of the protein, presumably due to
high cellular concentrations (Figure 9D). This feature can
be used to directly test any differences in self-activation
caspase-8, without complications from upstream recep-
374
376
found the native peptide 1, containing DSE
, to be fully
cleaved by caspase-8. This increased cleavage compared
to the previous assay could be due to the addition of
DMSO, which was required to solubilize the mutant pep-
tides. Consistent with previous experiments, we found
that substitution of the serine at position P1’ in this pep-
28
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tide by either alanine (5, DAE ) or tryptophan (6,
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more pronounced inhibitory effect on cleavage than mu-
tor-mediated activation steps. Unfortunately, the endog-
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9
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enous caspase-8 in mammalian cells can participate in
this process, so we first used the CRISPR genetic engi-
neering system to generate several clones of a caspase-8
null HeLa cell-line (Figure S18 (SI)). With these cells in-
hand, we next confirmed that overexpression of caspase-8
would cause its auto-dimerization. To accomplish this
goal, caspase-8 null cells were co-transfected with wild-
type HA- and FLAG-tagged caspase-8 constructs bearing
active-site mutations to prevent auto-activation. Im-
portantly, we first confirmed that the antibodies to these
tags do not cross-react (Figure S19A (SI)). Immunoprecip-
itation with an anti-FLAG antibody resulted in co-
enrichment of the HA-tagged caspase-8 and vice versa,
demonstrating that we were indeed inducing dimeriza-
tion of caspase-8 upon overexpression (Figure S19A (SI)).
To determine if global changes in O-GlcNAcylation could
affect this process, we performed the same immunopre-
cipitation in triplicate in cells that were treated with ei-
ther 5SGlcNAc, DMSO, or Thiamet-G to change the over-
all O-GlcNAcylation levels (Figure 9E and Figure S19B
(SI)). Notably, the changes in O-GlcNAcylation had no
significant effect on the co-immunoprecipitation, indicat-
ing it does not alter the ability of caspase-8 to dimerize
during its activation. With these control experiments
completed, we next tested the effects of mutating the ac-
tivation loop from serine to alanine (caspase-8(AAA)) to
prevent O-GlcNAcylation or from serine to tryptophan
(caspase-8(WWW)) to mimic O-GlcNAc. Accordingly,
caspase-8 null cells were transiently transfected with
wild-type caspase-8, caspase-8(AAA), or caspase-
8(WWW). In contrast to previous experiments, these
caspase-8 enzymes do not contain active site mutations.
Cells were collected after different lengths of time post-
transfection, and analysis by Western blotting showed
that at relatively short times (< 36 h) this results in a
“pulse” of cleaved/activated caspase-8 that is then re-
moved, potentially by death of the transfected cell popu-
lation (Figure 9F). Using these conditions, side-by-side
comparison of wild-type caspase-8, caspase-8(AAA), and
caspase-8(WWW) by Western blotting showed that the
caspase-8(AAA) mutant self-cleaved/activated with faster
kinetics than wild-type protein (Figure 9F). Specifically,
cleavage of caspase-8(AAA) could be detected in as little
as 4 h post-transfection, essentially as soon as significant
amounts of protein were detectable. In contrast, wild-type
caspase-8 also self-activated but did not reach peak acti-
vation until 12 h post-transfection. This cellular result is
notable because our in vitro peptide data suggested that
caspase(AAA) should self-cleave/activate with similar, if
not slightly slower, kinetics compared to wild-type pro-
tein. Finally, caspase-8(WWW) self-activated with the
slowest kinetics with a peak at 16 h post-transfection,
consistent with our peptide data and tryptophan acting as
an O-GlcNAc surrogate. These data indicate that O-
GlcNAcylation of caspase-8 can change the kinetics of its
activation living cells by serving as a steric hindrance to
caspase-8 self-cleavage. Importantly, based on our pep-
tide data, bona fide O-GlcNAcylation should have an even
tation to tryptophan.
CONCLUSION
Despite the observations that increased O-GlcNAcylation
inhibits apoptosis and promotes survival in cell culture
and tissues undergoing stress, there have been relatively
few mechanistic explanations for this phenotypic associa-
tion. Here, we demonstrate that the proteins that directly
carry out apoptosis, the caspases, are themselves O-
GlcNAc modified. In order to make this discovery, we
created a new metabolic chemical reporter (MCR),
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Ac 6AlkGlcNAc (2), which we show has significantly im-
proved signal-to-noise compared to our previous probe
29
Ac 6AzGlcNAc (1) while maintaining its selectivity for
3
intracellular proteins over cell surface glycoproteins (Fig-
ures 2 and 3). This is most likely due to the improved ori-
entation of the CuAAC reaction that reduces background
labeling but could also be a result of improved metabolic
stability of the alkyne- versus azide-MCR. Notably, treat-
ment of cells with Ac 6AlkGlcNAc (2) allowed for the vis-
3
ualization of modified proteins in as little as 15 min and a
large amount of labeling in only 1 h (Figure S3B), which
could enable the specific identification of newly O-
GlcNAcylated proteins in response to cellular stimuli
(
e.g., signaling or stress). Like Ac 6AzGlcNAc (1), our new
3
MCR cannot be metabolized to the UDP-6AlkGlcNAc by
the canonical GlcNAc salvage pathway in mammalian
cells, and we believe that it can be converted to
6
AlkGlcNAc-1-phosphate by the action of the enzyme
phosphoacetylglucosamine mutase (AGM1), which we
previously demonstrated can phosphorylate 6AzGlcNAc.
It is also possible that there are uncharacterized small-
molecule kinases that can generate these 1-phosphate
sugars. In either case, a wide range of cell-lines are
strongly labeled by Ac
ing that this MCR will be broadly applicable. By applying
Ac 6AlkGlcNAc (2) to MEF cells in an unbiased prote-
omics experiment, we were able to identify caspase-3 and
8 as being potentially O-GlcNAcylated (Figure 4A). A
parallel proteomics experiment using our non-selective
3
6AlkGlcNAc (2) treatment, indicat-
3
-
33
MCR, Ac GlcNAlk, did not identify these caspases,
4
demonstrating the utility of our new compound in dis-
covery-based experiments. We then confirmed the MCR-
labeling of caspase-3 and -8, as well as caspase-9, in three
different cancer cell types (Figure 6A). Notably, caspase-9
was not identified in our proteomics experiment, suggest-
ing that applying Ac 6AlkGlcNAc (2) in a quantitative
3
proteomics experiment (e.g., SILAC labeling) may uncov-
er additional interesting O-GlcNAcylated proteins. Criti-
cally, we were able to use chemoenzymatic labeling
and/or WGA enrichment of O-GlcNAcylated proteins to
independently confirm the endogenous O-GlcNAc modi-
fication of the caspases (Figures 5 and 6). This type of
independent confirmation is a key first step after MCR-
based identification of any O-GlcNAcylated protein, as
treatment with metabolic reporters may alter the normal
modification state of the cell.
We next chose to focus on caspase-8 given its key roles in
development, immunology, and human disease. By tak-
43
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Page 10 of 18
ing advantage of chemoenzymatic labeling of endogenous bodies to recognize caspase-8; however, we observe a dif-
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O-GlcNAc modifications, we then showed that the O-
GlcNAcylated fraction of caspase-8 is resistant to cleav-
age/activation upon induction of apoptosis by TNFα. It is
possible that O-GlcNAcylation affects the ability of anti-
ference in the amount of cleaved caspase compared to full
length and not an overall reduction in signal in the West-
ern blot, so we think this is unlikely.
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Figure 9. O-GlcNAcylation of the caspase-8 cleavage sites affects its cleavage/activation. (A) O-GlcNAcylation inhibits the self-
cleavage of caspase-8 in vitro. The indicated unmodified or O-GlcNAcylated peptides (2 mM) were incubated with recombinant,
active caspase-8 for 24 h and the percent cleavage was determined using reverse-phase high-performance liquid chromatography
(
RP-HPLC). Results are the mean ±s.e.m of three separate biological experiments (see Figure S14 (SI)). (B) Comparison of the
relative efficiency of caspase-8 activation loop cleavage between unmodified and O-GlcNAcylated peptides. The individual ef-
fects of O-GlcNAcylation shown in (A) were averaged to obtain the overall loop cleavage efficiency. (C) Mutation of the O-
GlcNAcylation sites in caspase-8 to alanine or tryptophan inhibits overall caspase-8 activation loop cleavage in vitro, with tryp-
tophan having a larger inhibitory effect. The individual effects of the alanine and tryptophan mutations (shown in Figure S17
(SI)) were averaged to obtain overall loop cleavage efficiency. (D) Activation of caspase-8 by transient overexpression. Upon
overexpression, caspase-8 will self-dimerize and activate itself without a requirement for cell-death signaling. (E) Changing O-
GlcNAcylation levels does not affect the dimerization of caspase-8. Caspase-8 null HeLa cells were co-transfected with plasmids
encoding HA- and FLAG-tagged, inactive caspase-8, before being treated with 5SGlcNAc (200 μM), DMSO vehicle, or Thiamet-G
(
20 μM) to alter O-GlcNAcylation levels. An anti-FLAG immunoprecipitation was then performed before visualization with an
anti-HA antibody, stripping of the blot, and visualization with an anti-FLAG antibody. The amount of enriched HA-tagged pro-
tein relative to enriched FLAG-tagged protein was then quantified. Results are the average of three separate biological experi-
ments that were imaged simultaneously (Figure S19B (SI)). Error bars represent ±s.e.m. of three biological experiments, and sta-
tistical significance was calculated using a two-tailed Student’s t-test. (F) Caspase-8 with its O-GlcNAc sites mutated to alanine
(
caspase-8(AAA)) activates faster than wild-type protein, and mutation to tryptophan (caspase-8(WWW)) slows the rate of
cleavage. Caspase-8 null HeLa cells were transiently transfected with plasmids encoding either wild-type caspase-8, caspase-
(AAA), or caspase(WWW) for 6 h. After the indicated lengths of time, cells were collected. Analysis by Western blotting shows
8
auto-activation of all proteins. The different amounts of each caspase species were quantitated and normalized to the total
caspase signal in each lane. The data is representative of two biological experiments.
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Page 11 of 18
Journal of the American Chemical Society
age/activation, which may play a role in the regulation of
We also demonstrated that changing the levels of O-
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GlcNAcylation can affect the activation/cleavage of caspa-
se-8 during the initiation of apoptotic signaling by TNFα.
Using site-directed mutagenesis and chemoenzymatic
detection, we found that the vast majority of O-
GlcNAcylation on caspase-8 occurs at serine residues near
the two cleavage sites required for full caspase-8 activa-
tion during apoptosis. We also demonstrated that O-
GlcNAcylation of these sites inhibits their cleavage in
vitro, which has been shown to be required for the full
activation of caspase-8 during apoptosis, and we used
caspase-8 mutants to indicate that O-GlcNAcylation of
these sites can change the kinetics of caspase-8 cleav-
age/activation in living cells. However, we cannot com-
pletely rule out some differences in the cellular experi-
ment, such as the activation of executioner caspases that
then feed-back to activate our overexpressed caspase-8.
We believe that this is certainly not the only O-GlcNAc
modification that causes the phenotypic association be-
tween elevated O-GlcNAcylation levels and cell survival,
but our results suggest that caspase-8 O-GlcNAcylation
could contribute to the anti-apoptotic function of O-
GlcNAc in cells.
programmed cell death in development and disease.
EXPERIMENTAL PROCEDURES
Synthesis of known small molecules. Known com-
pounds, alkyne- and azide-rhodamine (alk-rho and az-
4
2
33
9
43
rho),
UDP-GalNAz,
Thiamet-G
,
Ac
4
5SGlcNAc,
34
44
29
Ac GlcNAlk, Ac
4
4
ManNAlk, and Ac
3
6AzGlcNAc (1) were
synthesized according to literature procedures.
FLAG- and WGA-enrichment of Caspases. NIH3T3
cells stably expressing either FLAG-tagged caspase-3 or -8
were grown to 80% confluency before being treated with
20 μM Thiamet-G for 16 hours. Cells were then harvested
by trypsinization and washed 2X with PBS (2 min, 2,000 x
g, 4 °C). Cell pellets were resuspended in 100 μl of 1% NP-
40 lysis buffer [1% NP-40, 150 mM NaCl, 50 mM triethano-
lamine (TEA) pH 7.4] with Complete Mini, EDTA-free
Protease Inhibitor Cocktail Tablets (Thermo Scientific)
for 20 min and then centrifuged at 4 °C for 10 min at
10,000 x g. The supernatant was collected and the protein
concentration was determined by BCA assay (Pierce,
ThermoScientific). Total cell lysate (4 mg) was diluted to
a final concentration of 1 mg/mL with 1% NP-40 buffer
with Complete Mini, EDTA-free Protease Inhibitor Cock-
tail Tablets (Thermo Scientific). Diluted lysate was then
pre-cleared with Protein-G beads (30 μL, Pierce) pre-
washed 3X with 1 mL of NP-40 buffer, for 1h at 4 °C with
full rotation. Beads were then pelleted by centrifugation
42
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The exact stoichiometry and specific biological contexts
where caspase-8 O-GlcNAcylation has a significant effect
on overall apoptosis remain to be discovered, a line of
investigation we are currently undertaking. Unfortunate-
ly, we have found that the generation of cell-lines that
stably express caspase-8 mutants, like caspase-8(AAA), is
difficult due to variable expression levels that result in
auto-activation of apoptosis. Additionally, transient trans-
fection of caspase-8 (as in Figure 9F) can be effected by
the presence of OGT and OGA inhibitors in the media. To
overcome these issues, we are currently exploring the use
of CRISPR-Cas9 to introduce mutants into the caspase-8
gene at the chromosome level. Intriguingly, all of the
apoptotic caspases in humans and mice, two out of the
three caspases in Drosophila, and ced-3 in C. elegans have
serine or threonine residues at or near their cleavage sites.
However, the O-GlcNAcylation sites we identified here on
caspase-8 are only conserved in mammals. Additionally,
analysis of the known caspase cleavage-sites in substrate
(
2 min, 2,000 x g, 4 °C) and cleared supernatant was
transferred to tubes containing EZview Red ANTI-FLAG
M2 affinity beads (30 μL, Sigma), pre-washed with cold
NP-40 buffer 2X followed by cold PBS 2X. The samples
were placed on a rotator for 2 h at 4 °C. Beads were col-
lected by centrifugation at 2,000 x g for 2 min at 4 °C, and
the supernatant was carefully removed. Beads were sub-
sequently washed 3X by addition of 1 mL cold PBS with
rotation for 5 mins before centrifuging 2 mins at 2,000 x g.
Protein was eluted by addition of 30 μL of 1% SDS buffer
(
5
4% SDS, 150 mM NaCl, 50 mM TEA, pH 7.4) and boiling
min at 98 °C. Supernatant was let cool to room tempera-
ture before being transferred to a new tube in which SDS
concentration was diluted to 0.5% by addition of 1% NP-
4
0 buffer lysis buffer. Resulting diluted FLAG enriched
44
proteins shows that serine or threonine is the second
lysate was then transferred to new tubes containing pre-
washed (PBS 1X, cold NP-40 2X) sWGA agarose resin (60
μL, Vector Labs) and was incubated with rotation for 16 h
at 4 °C. Following enrichment, resin was washed 3X with
PBS before proteins were eluted with 4% SDS buffer and
boiling for 5 min at 98 °C. Samples were then analyzed by
SDS-PAGE (Any Kd, Criterion Gel, Bio-Rad) followed by
analysis by Western blotting.
most favored amino-acid at all of the surrounding eight
residues. Many known caspase substrates have also been
identified as being O-GlcNAcylated in a variety of prote-
omics experiments, although the specific sites of modifi-
cation are largely unknown. Additionally, phosphoryla-
tion of serine/threonine residues near caspase cleavage
45-47
sites has been shown to alter proteolysis rates.
This
raises the possibilities that O-GlcNAcylation of caspases is
an evolutionarily conserved mechanism for modulating
caspase activation and that this modification may also
protect caspase substrates from cleavage. We are current-
ly exploring both of these avenues of research. In conclu-
FLAG-IP of Caspases. NIH3T3 cells stably expressing
either FLAG-tagged Caspase-3 or -8 were grown to 80%
confluency before being treated with 200 μM Ac
4
GlcNAc
or Ac 6AlkGlcNAc (2) for 16 hours. Cells were then har-
3
vested by trypsinization and washed 2X with PBS (2 min,
2,000 x g, 4 °C). Cell pellets were resuspended in 100 μl of
1% NP-40 lysis buffer [1% NP-40, 150 mM NaCl, 50 mM
triethanolamine (TEA) pH 7.4] with Complete Mini,
sion, our new MCR (Ac 6AlkGlcNAc, 2) enabled the dis-
3
covery of caspase O-GlcNAcylation, and our data show
that this modification can inhibit caspase-8 cleav-
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Journal of the American Chemical Society
Page 12 of 18
EDTA-free Protease Inhibitor Cocktail Tablets (Thermo
Scientific) for 20 min and then centrifuged at 4 °C for 10
min at 10,000 x g. The supernatant was collected and the
protein concentration was determined by BCA assay
cold H
9,900 x g, 4 °C). Protein pellet was washed 3X by resus-
pension in cold H O and centrifugation. Protein pellet
was finally resuspended in 14 mL cold H O and poured
over a solid guanidine HCl and vortexted and then diluted
to 25 mL with H O (resulting in 5M final concentration).
The protein solution was diluted to a final concentration
of 1 mg mL with a 5M GuHCl solution (O.D. (A275) ~2.0).
This solution was diluted 10-fold into cold refolding buffer
(5 mM EDTA, 4 mM cysteamine, 2 mM cystamine, 100
mM Tris Base, pH 8.0) with gentle stirring. Protein was
allowed to refold for 48 h at 4 °C without agitation. Re-
2
O (250 mL) and centrifuged immediately (30 min,
1
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5
5
5
5
5
5
5
5
6
2
2
(
Pierce, ThermoScientific). Total cell lysate (2 mg) was
diluted to a final concentration of 0.5 mg/mL with 1% NP-
0 buffer with Complete Mini, EDTA-free Protease Inhibi-
2
4
-1
tor Cocktail Tablets (Thermo Scientific). Diluted lysate
was then pre-cleared with Protein-G beads (30 μL, Pierce)
pre-washed 3X with 1 mL of NP-40 buffer, for 1h at 4 °C
with full rotation. Beads were then pelleted by centrifuga-
tion (2 min, 2,000 x g, 4 °C) and cleared supernatant was
transferred to tubes containing EZview Red ANTI-FLAG
M2 affinity beads (30 μL, Sigma), pre-washed with cold
NP-40 buffer 2X followed by cold PBS 2X. The samples
were placed on a rotator for 2 h at 4 °C. Beads were col-
lected by centrifugation at 2,000 x g for 2 min at 4 °C, and
the supernatant was carefully removed. Beads were sub-
sequently washed 3X by addition of 1 mL cold PBS with
rotation for 5 mins before centrifuging 2 mins at 2,000 x g.
Protein was eluted by addition of 30 μL of 4% SDS buffer
(4% SDS, 150 mM NaCl, 50 mM TEA, pH 7.4) and boiling
5 min at 98 °C. Click chemistry with alkyne-biotin was
performed in the presence of the FLAG-beads assuming
volume of 60 μL. Click chemistry reaction was quenched
by addition of 60 μL of 2X loading buffer and boil 5 min at
98 °C. Samples were then analyzed by SDS-PAGE (Any
Kd, Criterion Gel, Bio-Rad) followed by analysis by strep-
tavidin or Western blotting.
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folding protein was dialyzed 2X into cold H O for 24 h
2
and concentrated using centrifugal filters (30 KDa cutoff,
Amicon Ultra, Millipore) to 3 mL. Buffer was exchanged
1X with 10 mL reaction buffer (10 mM Tris Base, pH 8.0)
and final protein concentration was determined (O.D.
(A275) ~1.5). Purified protein was stored at 4 °C.
Chemoenzymatic labeling. Cells were collected by tryp-
sinization and washed 2X with PBS (2 min, 2,000 x g, 4 °C
)
. Cells were subsequently lysed by adding 13 μL H
2
O, 25
μL 0.05% SDS buffer (0.05% SDS, 5 mM MgCl , 10 mM
2
TEA, pH 7.4) and 1 μL Benzonase (Sigma Ultra Pure) and
incubated on ice for 30 min. Then, 100 μL of 4% SDS buff-
er was added (4% SDS, 150 mM NaCl, 50 mM TEA, pH
7
.4), and the resuspended cell lysate was sonicated briefly
in a bath sonicator and cell debris was pelleted (10 min,
0,000 x g, 15 °C). Protein concentration was determined
1
by BCA Assay (Pierce, ThermoScientific), and the lysate
-1
Expression and purification of GalT(Y289L). pET23a
GalT Y289L plasmid was provided by P. Qasba, National
Cancer Institute, and was subsequently transformed into
was diluted to 1 μg μL in 1% SDS chemoenzymatic buffer
(1% SDS, 20 mM HEPES, pH 7.9). Proteins were precipi-
tated by adding 3X volume of methanol, 0.75X volume
BL21 E. coli (Novagen). A 1 L culture containing ampicillin
chloroform and 2X volume H O followed by vortexing and
2
-1
(
100 μg mL ) was inoculated with 10 mL of 50 mL starter
centrifugation (5 min, 13,000 x g, rt). The aqueous phase
was discarded without disturbing the interface layer be-
fore adding 2.5X volume of methanol, vortexing and pel-
leting protein (5 min, 13,000 x g, rt). The resulting protein
pellet was allowed to air dry for no longer than 20 mins
before being resuspended in 1% SDS chemoenzymatic
buffer (1% SDS, 20 mM HEPES, pH 7.9). Protein concen-
tration was normalized using BCA Assay (Pierce, Ther-
culture grown overnight at 37 °C. The 1 L culture was
grown at 37 °C until an O.D. (A600) of 0.60 was obtained,
at which time expression was induced with isopropyl β-D-
1
-thiogalactopyranoside (ITPG) (1 mM final concentra-
tion, 1,000X stock in water) for 4 h at 37 °C. Cells were
harvested by centrifugation (10 min, 4,000 x g, 4 °C) and
resuspended in 10 mL suspension buffer (25% sucrose w/v
in 1X PBS). Cells were lysed using sonication (30 s pulse,
-1
moScientific) and diluted to 2.5 μg μL in 1% SDS che-
3
0 s rest, 12 min at 4 °C). The resulting lysate was diluted
moenzymatic buffer. To start the chemoenzymatic trans-
fer reaction, these reagents were added in the following
to 80 mL in cold suspension buffer and inclusion bodies
were harvested by centrifugation (30 min, 15,000 x g, 4 °C)
and washed 8-10X or until inclusion bodies turn white in
color, by resuspension in 80 mL suspension buffer fol-
lowed by centrifugation. Inclusion bodies were then
washed 1X with cold wash buffer (10 mM phosphate, pH
order (for 100 μg of total protein): 49 μL of H
labeling buffer (2.5X; 5% NP40, 125 mM NaCl, 50 mM
HEPES, pH 7.9), 55 μL of MnCl (100 mM in H O) and 50
2
O, 80 μL of
2
2
μL of UDP-GalNAz (0.5 mM in 10 mM HEPES, pH 7.9).
Mix by pipetting. Finally, 7.5 μL of purified GalT Y289L
(in 10 mM Tris pH 8.0) was added and reaction mixture
was incubated for 16 h at 4 °C without agitation. Unreact-
ed UDP-GalNAz was then removed by methanol-
chloroform precipitation as described previously. Air
dried protein pellets were resuspended in 1% SDS CuAAC
buffer (1% SDS, 150 mM NaCl, 50 mM TEA, pH 7.4) and
subjected to CuAAC as detailed below.
7
.0) and harvested by centrifugation. Inclusion bodies
were then resuspended in 14 mL cold H O and poured
over solid guanidine HCl and Na SO (resulting in 5M
GuHCl and 300 mM Na SO final concentration), vor-
texted vigorously, and the volume was adjusted to 25 mL
with cold H O. Freshly made NTSB solution (50 mM
DTNB, 1 M Na SO in water pH 8.0) was then added with
2
2
3
2
3
2
2
3
vigorous vortexting to sulfenate free thiols. Completion of
the reaction is indicated by a color change from dark or-
ange to pale yellow. Protein was then precipitated with
Enrichment and selective elution of O-GlcNAcylated
-1
proteins. Cell lysates (~ 1 mg at 1 mg mL ) that had been
labeled either chemoenzymatically or with a metabolic
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Journal of the American Chemical Society
idin enrichment, SDS-PAGE and Western blotting as pre-
chemical reporter were subjected to CuAAC conditions
with freshly made cocktail [azide- or alkyne-azo-biotin
100 μM, 5 mM stock solution in DMSO) (Click Chemistry
Tools); tris(2-carboxyethyl)phosphine hydrochloride
TCEP) (1 mM, 50 mM freshly prepared stock solution in
water); tris[(1-benzyl-1-H-1,2,3-triazol-4-yl)methyl]amine
TBTA) (100 μM, 10 mM stock solution in DMSO);
CuSO •5H O (1 mM, 50 mM freshly prepared stock solu-
1
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viously described.
(
Caspase activation kinetics. HeLa cells were treated at
70-75% confluency with either 100 μM 5SGlcNAc, 5 μM
(
Thiamet-G (1,000X stocks in DMSO), or DMSO vehicle
for 16 h. Apoptosis was then induced by treating with 10
(
-1
-1
ng mL TNFα and 1 μg mL cycloheximide (CHX) for ei-
ther 0, 1, 2, 3, 4, or 6 h. Cells in the media were combined
with cells that were collected by trypsinization. The cells
were then pelleted by centrifugation (6 min, 2,000 x g, 4
4
2
tion in water)]. After 1 h, the proteins were precipitated by
addition of 4 volumes of ice-cold methanol and incuba-
tion at -20 °Cfor 2 h. Precipitated proteins were collected
by centrifugation (30 min, 5,000 x g, 4 °C) and washed 3X
with ice-cold methanol, with resuspension of pellet each
time. The pellet was then air dried for 15 min and then
resuspended in 800 μL of resuspension buffer (6 M urea, 2
M thiourea, 10 mM HEPES pH 8.0) by bath sonication.
Samples were then transferred to 2 mL dolphin-nosed
tubes containing streptavidin beads (25 μL of a 50% slurry
per sample, Thermo) were washed 2 X with 1 mL PBS and
1 X with 1 mL resuspension buffer and resuspended in
resuspension buffer (200 μL). Samples were then incubat-
ed on a rotator for 2 h. Beads were washed 2X with resus-
pension buffer, 2X in PBS (1 ml) and 2X with 1% SDS in
PBS buffer. Beads were then incubated in 25 μL of sodium
dithionite solution (1% SDS, 25 mM sodium dithionite)
for 30 min at room temperature to elute captured pro-
teins. The beads were centrifuged for 2 min at 2,000 x g
and the eluent collected. The elution step was repeated,
and the eluents combined. Proteins were then precipitat-
ed in ice cold methanol (1 mL) overnight in -20 °C. Pro-
tein was collected by centrifugation (10 min, 10,000 x g, 4
°C), and the pellet was allowed to air dry for 5 min, and
then 30 μL 4% SDS buffer (4% SDS, 150 mM NaCl, 50 mM
TEA pH 7.4) was added to each sample. The mixture was
sonicated in a bath sonicator to ensure complete dissolu-
tion, and 30 μL of 2x loading buffer (20% glycerol, 0.2%
bromophenol blue, 1.4% β-mercaptoethanol) was then
added. Samples were then boiled for 5 mins at 98
°Cbefore being separated by SDS-PAGE (4-20% Tris-
Glycine Gel, Bio-Rad).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
°
C) and washed with PBS (1 mL) three times. Cell pellets
were then resuspended in 50 μl of 1% NP-40 lysis buffer
1% NP-40, 150 mM NaCl, 50 mM triethanolamine (TEA)
pH 7.4] with Complete Mini protease inhibitor cocktail
Roche Biosciences) and Z-VAD-FMK (Enzo Life Scienc-
[
(
es) for 15 minutes and then centrifuged (10 min, 10,000 x
g, 4 °C). The supernatant (soluble cell lysate) was collect-
ed and the protein concentration was determined by BCA
assay (Pierce, ThermoScientific). Samples were prepared
-1
at a protein concentration of 2 mg mL with 4X loading
buffer (8% SDS, 40% glycerol, 0.4% bromophenol blue,
2
.8% B-mercaptoethanol, 200 mM Tris-HCl pH 6.8) and
boiled at 5 min at 98 °C. Forty μg of protein was then
loaded per lane for SDS-PAGE separation (Criterion TGX
-20%, Bio-Rad).
™
4
Caspase wild-type vs. Mutant chemoenzymat-
ic/Biotin IP. H1299 cells at 90% confluency were co-
transfected with pCDNA Caspase-8 catalytically dead,
wild-type (HA-tagged, 20 μg) and either (AAA)-mutant
(
2
FLAG-tagged, 20 μg), (S386,387A)-mutant (FLAG-tagged,
0 μg), or (S375A)-mutant (FLAG-tagged, 20 μg) DNA
using Liptofectamine 2000 (Invitrogen), according to
manufacturers protocol. Cells were harvested 48 h post-
transfection and subsequently lysed using SDS-lysis be-
fore being subjected to chemoenzymatic transfer accord-
ing to protocols described above. Following chemoen-
zymatic transfer, protein was precipitated by
MeOH/CHCl and resuspended in 1% SDS buffer. CuAAC
3
was preformed as described previously with alkyne-azo-
biotin. Following CuAAC, enrichment was conducted
according to protocols previously described. Following
the final elution step, both eluents were combine and
proteins were then precipitated in ice cold methanol (1
mL) overnight in -20 °C. Protein was collected by centrif-
ugation (10 min, 10,000 x g, 4 °C), and the pellet was al-
lowed to air dry for 5 min, at which time 20 μL 4% SDS
buffer (4% SDS, 150 mM NaCl, 50 mM TEA pH 7.4) was
added to each sample. The mixture was sonicated in a
bath sonicator to ensure complete dissolution, and 20 μL
of 2X loading buffer (20% glycerol, 0.2% bromophenol
blue, 1.4% β-mercaptoethanol) was then added. Samples
were boiled for 5 min at 98 °C before being subjected to
SDS-PAGE and Western blotting as described above.
Plasmids available upon request.
Endogenous caspase-8 chemoenzymatic/Biotin IP
following induction of apoptosis. HeLa cells were plat-
ed at 2 x 10 cells in a 150 mm dish 48 h prior to activation.
Apoptosis was then induced by treating with 10 ng mL
TNFα and 1 μg mL cycloheximide (CHX) for 6 h. Cells in
the media were combined with cells that were collected
by trypsinization before they were pelleted by centrifuga-
tion (6 min, 2,000 x g, 4 °C) and washed with PBS (1 mL)
three times. Cells were subsequently lysed by adding 26
6
-
1
-1
μL H
MgCl
2
O, 50 μL 0.05% SDS buffer (0.05% SDS, 5 mM
, 10 mM TEA, pH 7.4) containing Z-VAD-FMK (En-
2
zo Life Sciences) and 1 μL Benzonase (Sigma Ultra Pure)
and incubated on ice for 30 min. Then, 200 μL of 4% SDS
buffer was added (4% SDS, 150 mM NaCl, 50 mM TEA, pH
7
.4), and the resuspended cell lysate was sonicated briefly
in a bath sonicator and cell debris was pelleted (10 min,
0,000 x g, 15 °C). Precipitated protein was subjected to
Peptide synthesis. All solid-phase peptide syntheses
were conducted manually on unprotected Rink amide
1
®
ChemMatrix resin, (PCAS BioMatrix) with an estimated
chemoenzymatic transfer followed by click chemistry
with alkyne-azo-biotin before being subjected to streptav-
-1
loading of 0.6 mmol g . Protected O-GlcNAcylated serine
11,45
was prepared as previously described.
Commercially
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 14 of 18
+
+
available N-Fmoc and side-chain protected amino acids
10 eq, Advanced ChemTech) were activated for 20 min
with HBTU (10 eq, Novabiochem) and DIEA (20 eq, Sig-
ma) and then coupled to the resin for 1 h, bubbling with
Da (M + H ); 3 uncleaved, calculated = 835.4 Da (M + H ),
+
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
observed = 835.2 Da (M + H ); 3 cleaved, calculated =
549.2 Da (M + H ), observed = 549.2 Da (M + H ); 4 un-
+
+
+
cleaved, calculated = 1241.5 Da (M + H ), observed = 1241.5
+
+
N
2
to mix. Reaction completion was checked using the
Da (M - H ); 4 cleaved, calculated = 549.2 Da (M + H ),
+
Kaiser test. Briefly, a small amount of resin was incubated
with equal volumes of 5% w/v ninhydrin in EtOH, 80%
w/v phenol in EtOH, and 20 μM KCN in pyridine and
heated to 99 °Cfor 5 min in a sealed tube. If necessary, a
second coupling was conducted with 10 eq amino acid, 10
eq HOBt (Novabiochem) and 15 eq DCC (Sigma) for 2 h,
observed = 549.2 Da (M + H ); 5 uncleaved, calculated =
704.3 Da (M + H ), observed = 704.2 (M + H ); 5 cleaved,
calculated = 505.2 Da (M + H ), observed = 505.2 Da (M +
H ); 6 uncleaved, calculated = 819.3 Da (M + H ), ob-
+
+
+
+
+
+
+
served = 819.2 Da (M + H ); 6 cleaved, calculated = 505.2
+
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Da (M + H ), observed = 505.2 Da (M + H ) 7 uncleaved,
+
with N
Fmoc group was removed with 20% v/v piperidine in
DMF for 5 min with N mixing, and then for an addition 15
2
mixing. After successful coupling, the terminal
calculated = 803.4 Da (M + H ), observed = 803.4 (M +
H ); 7 uncleaved, calculated = 803.4 Da (M + H ), ob-
+
+
+
2
served = 803.3 Da (M + H ); 7 cleaved, calculated = 548.2
+
+
min with fresh 20% piperidine in DMF. When peptides
were completed, the final Fmoc group was removed as
described above and the N-terminal amine was acetylated
with 5 eq each of pyridine and acetic anhydride in DMF.
Peptides were then cleaved from the resin by incubating
Da (M + H ), observed = 548.2 Da (M + H ); 8 uncleaved,
+
calculated = 1033.5 Da (M + H ), observed = 1033.2 Da (M
+ H ); 8 cleaved, calculated = 549.2 Da (M + H ), observed
= 549.2 Da (M + H )
+
+
+
Calcium Phosphate transfection. One day before trans-
fection, 5 x 10 target cells were plated. One hour before
transfection, media was exchanged for fresh media. To an
Eppendorf tube was added water (398 μL, autoclaved), 2M
62 μL (filter sterilized), and 40 μL DNA (500 ng/μL,
0 μg total, phenol/chloroform extracted, EtOH precipi-
tated). Solution was mixed by pipetting, then DNA solu-
tion is added dropwise to a falcon tube containing 500 μL
X HBS (500 mM HEPES, 1.5 mM Na
in
cleavage
cocktail
(95:2.5:2.5
5
2
TFA/H O/Triisopropylsilane) for 3.5 h at room tempera-
ture. The peptides were then diluted ~1/10 in cold diethyl
ether and precipitated overnight (-80 °C). The resulting
suspensions were centrifuged (30 min, 5,000 x g, 4 °C)
CaCl
2
2
and the pellets were resuspended in fresh E
fuged again (30 min, 5,000 x g, 4 °C). The pellets were
then resuspended in H O, flash frozen, and lyophillized.
2
t O and centri-
2
2
2
HPO , 280 mM
4
At this point, glycopeptides 2 and 4 were deprotected
with hydrazine hydrate (80% v/v in MeOH) for 2 h, fol-
lowed by concentration under vacuum. Crude lyophil-
lized material was purified by RP-HPLC (0-50% B gradi-
NaCl, 10 mM KCl, 12 mM dextrose, pH 7.05, filter steri-
lized) while pumping bubbles. The resulting solution was
added dropwise to target cells while swirling slowly. A
fine black precipitate was observed after 30 min and the
media was opaque in color. Seven hours after transfec-
tion, media was exchanged for fresh media.
ent over 60 min; A = 0.1% trifluoroacetic acid in H
2
O; B =
0.1% trifluoroacetic acid, 10% H O, 90% acetonitrile) over
2
a C18 semi-preparative column (Vydac). Purified peptides
were then characterized by ESI-MS (Agilent 6100 series
Caspase-8 knockout with CRISPR. Expression vector
pSpCas9(BB)-2A-Puro (PX459) (Addgene no. 48139) was
digested with BbsI, and a pair of annealed oligonucleo-
tides corresponding the first exon of human caspase-8
+
Quadrupole LC-MS): 1, calculated = 720.3 Da (M + H ),
+
observed = 720.2 Da (M + H ); 2, calculated = 923.4 Da (M
+
+
+ H ), observed = 923.3 Da (M + H ); 3, calculated = 835.4
(
CACCGCTCTTCCGAATTAATAGAC
and
+
+
Da (M + H ), observed = 835.2 Da (M + H ); 4, calculated =
1241.5 Da (M + H ), observed = 1241.2 Da (M + H ); 5, cal-
culated = 704.3 Da (M + H ), observed = 704.2 Da (M +
H ); 6, calculated = 819.3 Da (M + H ), observed = 819.2 Da
(M + H ); 7, calculated = 803.4 Da (M + H ), observed =
803.4 Da (M + H ); 8, calculated = 1033.5 Da (M + H ),
observed = 1033.2 Da (M + H ).
46
AAACGTCTATTAATTCGGAAGAGC), was ligated into
the guide RNA to generate SpCas9-Casp8, which was con-
firmed by sequencing (Laragen, Culver City, CA). HeLa
cells were then transiently transfected with SpCas9-Casp8
using calcium phosphate transfection methods as de-
scribed above. Forty-eight hours post-transfection, 1 ug
+
+
+
+
+
+
+
+
+
+
-1
mL puromycin was added to the media for the next 72 h.
Caspase-8 knockout cells were then further selected for
In vitro caspase-8 cleavage assay. Peptides 1-8 (1000X
stock in DMSO) were added to 4 units of recombinant
caspase-8 (Enzo Life Sciences) in 100 μl of general caspase
activity buffer (20 mM HEPES, 100 mM NaCl, 0.1%
CHAPS, 10% sucrose, 1 mM EDTA, 10 mM DTT, pH 7.2) to
give a final concentration of 2 mM. The mixtures were
incubated overnight at 37 °C and subjected to HPLC anal-
ysis by RP-HPLC. Purified peptides were then character-
-1
by inducing apoptosis with 10 ng mL TNF-alpha and 1 ug
-1
ml cycloheximide for 24 h. Following clonal selection
using cloning cylinders, Western blot analysis showed five
out of five selected colonies had no detectable caspase-8.
Colony number one was selected for sequencing and fur-
ther experiments. A 2 kb fragment of genomic caspase-8
DNA was amplified using PCR with primers
ized by ESI-MS (Agilent 6200 series Quadrupole LC-MS):
CTTTGTGACATGGTACCTGGC
and
+
1
7
uncleaved, calculated = 720.3 Da (M + H ), observed =
GTGTCCGATAATAGGAGATGTT. And additional round
of PCR was then used to introduce attB recombination
sites enabling Gateway cloning (ThermoFisher) into do-
nor vector pDONR201 according to the manufacturers
protocol. E. Coli (DH5α, ThermoFisher) were then trans-
+
20.2 Da (M + H ); 1 cleaved, calculated = 505.2 Da (M +
H ), observed = 505.3 Da (M + H ); 2 uncleaved, calculated
= 923.4 Da (M + H ), observed = 923.2 Da (M + H ); 2
cleaved, calculated = 505.2 Da (M + H ), observed = 505.2
+
+
+
+
+
ACS Paragon Plus Environment

Page 15 of 18
Journal of the American Chemical Society
8(WWW). Media was exchanged after 6 h of transfection,
formed with these plasmids and grown on antibiotic-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
containing agar plates. Twenty colonies were then select-
ed for sequencing (Laragen, Culver City, CA).
and this time was defined as 0 h after transfection. After
the indicated lengths of time, cells in the media were
combined with cells that were collected by trypsinization.
The cells were then pelleted by centrifugation (6 min,
Caspase-8 Overexpression Co-IP. HeLa Caspsae-8
CRISPR cells at 90% confluency were co-transfected with
pCDNA Caspase-8 catalytically dead, wild-type (HA-
tagged, 20 μg) and wild-type (FLAG-tagged, 20 μg) DNA
using Liptofectamine 2000 (Invitrogen), according to
manufacturer’s protocol). Forty-eight hours post transfec-
tion, cells were harvested by trypsinization and then lysed
by resuspension in 150 μL of lysis buffer with 1 mM phe-
nylmethanesulfonyl fluoride (PMSF, 200 mM stock in
isopropyl alcohol) (10X Lysis buffer, Cell Signaling) fol-
lowed by sonication (5 sec on, 5 sec rest, 3X). Protein con-
centration was determined using BCA Assay (Pierce,
ThermoScientific), and 200 μg of lysate was diluted to 1 μg
2,000 x g, 4 °C) and washed with PBS (1 mL) three times.
Cell pellets were then resuspended in 50 μL of 1% NP-40
lysis buffer [1% NP-40, 150 mM NaCl, 50 mM triethanola-
mine (TEA) pH 7.4] with Complete Mini protease inhibi-
tor cocktail (Roche Biosciences) and Z-VAD-FMK (Enzo
Life Sciences) for 15 min and then centrifuged (10 min,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0,000 x g, 4 °C). The supernatant (soluble cell lysate) was
collected and the protein concentration was determined
by BCA assay (Pierce, ThermoScientific). Samples were
-1
prepared at a protein concentration of 3 mg mL with 4X
loading buffer (8% SDS, 40% glycerol, 0.4% bromophenol
blue, 2.8% B-mercaptoethanol, 200 mM Tris-HCl pH 6.8)
and boiled at 5 min at 98 °C. Forty μg of protein was then
loaded per lane for SDS-PAGE separation (Criterion™
TGX 4-20%, Bio-Rad).
-1
μL in lysis buffer. Lysate was then pre-cleared with 30 μL
of magnetic protein-G beads (Cell Signaling, #8740) for 1
h at 4 °C with end-over-end rotation. Inputs were gener-
-1
ated at 2 μg μL using 4X loading buffer (8% SDS, 40%
glycerol,
0.4%
bromophenol
blue,
2.8%
B-
ASSOCIATED CONTENT
mercaptoethanol, 200 mM Tris-HCl pH 6.8) before boil-
ing for 5 min at 98 °C. Magnetic beads were captured us-
ing a magnetic rack and pre-cleared lysate was transferred
to a new, pre-cooled tube. Either FLAG primary antibody
Supporting figures, Experimental methods, NMR characteri-
zation, and proteomic data tables. This material is available
free of charge via the Internet at http://pubs.acs.org.
(
1:50 dilution, Cell Signaling, #2368) or HA primary anti-
AUTHOR INFORMATION
Corresponding Author
body (1:50 dilution, Cell Signaling, #3724) was added to
the pre-cleared lysate. The appropriate isotype control
was used at the same protein concentration as the prima-
ry antibody (Rabbit isotype control, Cell Signaling,
*knchuh@gmail.com and matthew.pratt@usc.edu
#
3900). Thirty μL of magnetic protein-G beads were then
Notes
added and let incubate over night with end-over-end ro-
tation at 4 °C. Protein-G beads were then washed 5X with
The authors have no competing financial interests.
ACKNOWLEDGMENT
5
00 μL of lysis buffer containing 1 mM PMSF. Beads were
the pelleted gently be centrifugation (3 min, 2,000 x g, 4
C) before 3X loading buffer (Cell Signaling, #7722) was
We thank Mary O’Reilly (O’Reilly Science Art) for prepara-
tion of the table of contents figure and Henrik Molina (Rock-
efeller University) for assistance with the proteomics analy-
sis. K.N.C. is a fellow of the National Science Foundation
Graduate Research Fellowship Program (DGE-0937362). This
research was supported by the National Science Foundation
(CHE-1506503 to M.R.P.), the Damon Runyon Cancer Re-
search Foundation (DDR-19-12 to M.R.P.), Susan G. Komen
for the Cure (CCR14299333 to M.R.P.), the American Cancer
Society (RSG-14-225-01-CCG to M.R.P.), and in part by the
National Cancer Institute of the US National Institutes of
Health (CCSG P30CA014089). LC-MS/MS proteomic analysis
was performed at the Rockefeller University Proteomics Re-
source Center.
°
added and samples were boiled for 5 min at 98 °C. After
removal of magnetic beads, samples were analyzed by
SDS-PAGE (4-20% Tris-Glycine Gel, Bio-Rad) and ana-
lyzed by Western blotting.
Caspase-8 Overexpression Co-IP with inhibitor
treatment. HeLa Caspsae-8 CRISPR cells at 90% conflu-
ency were co-transfected with pCDNA Caspase-8 catalyti-
cally dead, wild-type (HA-tagged, 10 μg) and wild-type
(
(
FLAG-tagged, 10 μg) DNA using Liptofectamine 2000
Invitrogen), according to manufacturer’s protocol). Sev-
en hours following transfection cells were replated at 7.5 x
5
1
0 per 10 cm dish for treatment. The following day, cells
were treated with either 5SGlcNAc for 16 h (200 μM,
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