Exploring the Binding Proteins of Glycolipids with Bifunctional Chemical Probes

Article abstract of DOI:10.1002/anie.201608827

Glycolipids are important structural components of biological membranes and perform crucial functions in living systems, including signaling transduction and interaction with extracellular environment. However, the mechanistic exploration of glycolipids in vivo is challenging because they are not genetically encoded. Herein, we designed and synthesized a series of bifunctional monogalactosyldiacylglycerol (MGDG) probes as a model by introducing diazirine and terminal alkyne moieties on an aliphatic chain. In combination with proteome profiling and molecular modeling, we have demonstrated that MGDG alleviates inflammation by antagonizing TLR4.

Full text of DOI:10.1002/anie.201608827

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201608827
German Edition: DOI: 10.1002/ange.201608827
Anti-Inflammatory Compounds
Exploring the Binding Proteins of Glycolipids with Bifunctional
Chemical Probes
Xiaohui Liu+, Ting Dong+, Yu Zhou, Niu Huang, and Xiaoguang Lei*
Dedicated to Prof. Samuel J. Danishefsky on the occasion of his 80th birthday
Abstract: Glycolipids are important structural components of
biological membranes and perform crucial functions in living
systems, including signaling transduction and interaction with
extracellular environment. However, the mechanistic explora-
tion of glycolipids in vivo is challenging because they are not
genetically encoded. Herein, we designed and synthesized
a
series of bifunctional monogalactosyldiacylglycerol
(MGDG) probes as a model by introducing diazirine and
terminal alkyne moieties on an aliphatic chain. In combination
with proteome profiling and molecular modeling, we have
demonstrated that MGDG alleviates inflammation by antag-
onizing TLR4.
G
lycolipids are a class of lipids which contain a carbohydrate
component and contribute to many (patho)physiological
processes. Besides exerting structural effects on membranes,
numerous investigations have revealed that glycolipids par-
ticipate in cellular recognition, cell signaling transduction,
and modulation of protein activities.[1] For example, glyco-
sphingolipids are the most common glycolipids in mammalian
cell membranes, playing important roles in cell–cell signaling,
pathogen–cell surface interactions, and in cellular regulatory
functions.[2] Lipopolysaccharide, an endotoxin of Gram-
negative bacteria, can trigger pro-inflammatory response by
its lipid A unit.[3] Eritoran, an analogue of lipid A, was
developed as an antagonist of LPS (Figure 1).[4] Fatty acid
esters of monogalactosyldiacylglycerol (MGDG), categorized
as glycolipids, are essential components of the thylakoid
Figure 1. Representative examples of glycolipids.
membrane in chloroplasts, accounting for 80% of the
membrane lipids found in green plant tissues. MGDG share
a common galactosyl glycerol moiety attached with two fatty
acid chains. Crude MGDG extracts have been shown to
possess anti-viral,[5a] anti-tumor,[5b] anti-proliferative[5c] and
anti-inflammatory activities.[5d] In particular, dilinolenoyl
MGDG has also been reported to show anti-inflammatory
effects in human peripheral blood neutrophils.[6] However,
the exact modes of action remain elusive. Thus, designing
probes to comprehensively dissect the mechanism of glyco-
lipids is of great importance.
[*] X. Liu[+]
School of Pharmaceutical Science and Technology, Tianjin University
Tianjin 300072 (China)
A better understanding of cellular mechanisms and
processes mediated by glycolipids not only contributes to
biochemical studies but also guides therapeutic development.
Glycolipid chemical probe analogues are undoubtedly valu-
able tools for understanding their behaviors in physiologically
relevant contexts. Recently, a variety of chemical probes have
emerged for the study of lipid–protein interactions in their
native environment.[7–11] For example, Bertozzi and co-work-
ers prepared a series of glycophosphatidylinositol (GPI)
fluorescent probes and unraveled the biological function of
the GPI anchor.[12] In pioneering work, the Kohler research
group developed photoreactive probes that can be used to
study transient glycan-mediated interactions.[13,14] In most of
these probesꢀ designs, besides a “recognition” group and
“tag” group, “photoreactive trap” group was additionally
T. Dong,[+] Prof. Dr. X. Lei
Beijing National Laboratory for Molecular Sciences, Key Laboratory
of Bioorganic Chemistry and Molecular Engineering of Ministry of
Education, Department of Chemical Biology, College of Chemistry
and Molecular Engineering, Synthetic and Functional Biomolecules
Center, and Peking-Tsinghua Center for Life Sciences, Peking
University
Beijing 100871 (China)
E-mail: xglei@pku.edu.cn
Homepage: http://www.chem.pku.edu.cn/leigroup/
Y. Zhou, Prof. Dr. N. Huang
National Institute of Biological Sciences (NIBS)
Changping District, Beijing 102206 (China)
[+] These authors contributed equally to this work.
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201608827.
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incorporated to strengthen the interaction between glycoli-
pids and their interacting protein targets.[1a]
Despite diverse methods developed for studying the
behaviors of lipids, care must be taken to ensure that the
chemical probes do not compromise the original activity of
parent compounds.[15] In light of this requirement, the Yao
group developed a series of remarkable minimalist linkers
characterized by incorporation of a diazirine and terminal
alkyne on a single aliphatic chain.[16] Haberkant and co-
workers reported a 15 carbon long bifunctional fatty acid
which can be effectively used for the global profiling of
protein lipid interactions in living cells.[17] Furthermore, the
Hang group demonstrated a bifunctional fatty acid chemical
reporter applied to the analysis of protein–protein interac-
tions of S-palmitoylated membrane receptors.[18] Inspired by
these discoveries, we propose that a bifunctional fatty acid
modified probe equipped with a diazirine and terminal alkyne
may be useful for mechanistic studies of complex glycolipids.
As described in Figure 2, we firstly prepared glycolipid probes
by replacing one fatty acid chain with the bifunctional chain,
which can be crosslinked to its protein binding partner after
UV irradiation. Click chemistry is then used to label the
alkyne group in the bifunctional chain with biotin-N3 or
fluorescein-N3 (Fluo-N3), allowing the respective identifica-
tion or visualization of the crosslinked protein–lipid complex.
Scheme 1. Syntheses of dilinolenoyl MGDG and its minimalist probe.
attention and several synthetic routes have been described.[20]
As shown in Scheme 1b, our synthesis of MGDG commenced
with the acetate deprotection of 2, followed by TIPDSCl
protection and catalytic hydrogenation to give the diol 3.[20e]
Coupling of linolenic acid to the diol 3 afforded the silylated
MGDG in 72% yield, which was then deprotected by
Et3N·3HF to yield the dilinolenoyl MGDG 4 in 97% yield.
Coupling of the minimalist linker to the primary alcohol gave
the minimalist probe 5.
Figure 2. General scheme for the profiling of cellular target(s) of
glycolipids.
Dilinolenoyl MGDG attracted our attention for its
impressive anti-inflammatory activity both in vitro and in
vivo.[6,19] We expected that by incorporating the bifunctional
fatty acid into MGDG, we could explore the mechanism for
its anti-inflammatory activity. We envisioned that the replace-
ment of one of the linolenoyl chains would not significantly
change its size and properties. As a proof of concept, three
chemical probes of MGDG were synthesized by either direct
modification on the galactosyl moiety with the minimalist
linker or by individual replacement of the fatty acids with the
designed bifunctional fatty acid. To our knowledge, this is the
first time this kind of bifunctional fatty acid has been
incorporated into therapeutically important complex glyco-
lipids for target identification.
In order to obtain the above mentioned chemical probes,
we firstly synthesized the bifunctional fatty acid according to
the reported procedure.[17] The bifunctional fatty acid 1 was
synthesized from the acid and the TMS-protected alkyne in
three steps (Scheme 1a). Then we tried to synthesize MGDG.
As an important glycolipid, MGDG has attracted much
Scheme 2. Syntheses of bifunctional MGDG probes.
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We then set out to synthesize MGDG probes modified
We next performed endogenous proteome labeling fol-
with the bifunctional fatty acid chain. The bifunctional fatty
acid 1 was coupled to the sn-1 position of the diol 3, followed
by a second esterification with linolenic acid to yield the
silylated MGDG probe 7. After deprotection of the com-
pound with Et3N·3HF, we could get the probe 8 in 88% yield
(Scheme 2a). In a like manner, another probe with the
bifunctional fatty acid at the sn-2 position (probe 11) was
obtained (Scheme 2b).
After we synthesized the MGDG probes, we initially
estimated the anti-inflammatory activities by using the
commercially available ELISA kit which allows the determi-
nation of phospho-p38, total p38 and cell number in the same
assay. Consistent with a literature result,[5d] MGDG shows
strong inhibition of p38 phosphorylation without influencing
total cell number. Probe 11 proved to be a more potent
inhibitor of p38 phosphorylation than MGDG, while probe 8
showed a decreased inhibitory effect. However, the minimal-
ist probe 5 was biologically inactive, thereby indicating the
galactosyl moiety is essential for the target recognition
(Figure 3a). Furthermore, when we re-measured the p38
inhibition effect of probes 8 and 11 after washing, we found
that probe 8 totally lost its activity, which indicated probe 8
could not photo cross-link to its target protein (Figure S1 in
the Supporting Information). Overall, probe 11 could be
applied as a satisfactory chemical tool of MGDG for target
ID.
lowed by pull-down/LC-MS/MS aiming to identify the func-
tional target of MGDG. We incubated human chondrocytes
with probe 5 (negative control) or 11 for 1 h and then
irradiated with UV light for 20 min. Subsequently the cells
were lysed and subjected to CuI-catalyzed click reaction
conditions with biotinylated azide. The resulting lysates were
then incubated with streptavidin-labeled beads. After wash-
ing the beads, the precipitated proteins were resolved by SDS-
PAGE and stained with silver. As shown in Figure 3b, we cut
down the gel bands preferentially pulled down by probe 11
rather than 5 and then sent them for MS analysis. As shown in
Table S1, we excluded the proteins present in both the 11-
treated group and the 5-treated group in the mass analysis,
and finally focused on two potential candidates: Toll-like
receptor 4 (TLR4) and KTN-1 (Figure 3b). With these two
potential targets in mind, we further blotted the elution with
KTN-1 antibody and TLR4 antibody, respectively. As shown
in Figure 3c, only TLR4 was validated to exist in the
precipitate, thus demonstrating that TLR4 may be the
target of MGDG. Meanwhile, when we incubated human
chondrocytes with 20 mm MGDG, and probe 11 and then
exposed to UV irridation for 0 or 20 min, the immunopreci-
pitated TLR4 protein conjugates were subjected to Western
blot analysis. We found that the interaction between TLR4
and probe 11 is both UV- and CuAAC-dependent. In the
presence of equal concentrations of MGDG and probe 11, the
quantity of recombinant
TLR4 pulled down by 11
was reduced (Figure S2),
confirming that MGDG
directly interacts with
TLR4.
MGDG could antago-
nize LPS-mediated
TLR4 activation in
Furthermore,
a
dose-responsive
by using
manner
Human TLR4 lucifer-
ase-(LUCPorterꢁ)
stable reporter cell line
(Figure 3d). However,
metabolites of MGDG
did not show inhibitory
effect (Figure S3).
Toll-like receptors
(TLRs) are evolutionar-
ily conserved pattern
recognition molecules,
which have been impli-
cated in the pathobiol-
ogy of inflammation.[21]
As the most extensively
characterized Toll-like
receptor, TLR4 is crit-
ical to innate immunity
responses stimulated by
LPS binding, which lead
to the activation of
Figure 3. a) Human chondrocytes were treated with 100 UmLÀ1 IL-1a in the presence of 20 mm indicated
compounds for 60 min and then phospho-p38 was measured by using p38 kit. b) Human chondrocytes cell
lysates were incubated with 20 mm probe 11 or 5 for 1 h and then exposed to UV irradiation, followed by
conjugating with biotin tag through Cu-catalyzed azide/alkyne cycloaddition (CuAAC) reaction, finally the lysates
were used for pull-down with streptavidin-agarose beads, and the precipitates resolved by SDS-PAGE were stained
by silver. The indicated bands were analyzed by mass spectrometry. c) The elution was blotted with KTN-1 and
TLR4 antibody, respectively. d) MGDG inhibited LPS-mediated TLR4 activation in a dose-dependent manner by
using the TLR4/MD-2/CD14/IL-8 Prom/LUCPorterꢀ reporter cell line.
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downstream signaling pathways, including the nuclear factor-
kB (NF-kB) and the p38 MAPK (mitogen activated protein
kinase) pathways, finally resulting in the induction of systemic
inflammation.[22–26] Consequently, the anti-inflammatory
effect of MGDG may be reasonably explained by TLR4
antagonism.
Next, we further explored the binding mode of MGDG to
TLR4. Previous structural research indicated that the hete-
rodimer TLR4/MD-2 is essential for the recognition of its
natural ligands LPS or synthetic antagonists.[27] Eritoran is an
analog of LPS and inhibits its activity by binding to the TLR4/
MD-2 complex.[4] In 2007, Lee and co-workers demonstrated
crystallographically that Eritoran binds to a large internal
pocket in MD-2.[28] Thus, we modeled the binding pose of
MGDG with TLR4/MD-2 complex. As shown in Figure 4a,b,
MGDG shared a similar binding mode with Eritoran. Both
two fatty chains of MGDG inserted into the pocket of MD-2.
To gain experimental evidence to support this modeling, we
found that both Eritoran and MGDG could outcompete the
interaction between 11 and TLR4/MD-2. (Figure 4c). In
contrast to Eritoran, the binding mode between MGDG and
TLR4/MD-2 shows that fatty chain on sn-2 position forms
favorable hydrophobic interactions with hTLR4 residues
including F440, F463, L444, K388, Q436, which can well
explain why probe 11 could form a covalent bond with TLR4
but probe 8 could not (Figure S4). In order to evaluate which
of them is critical for the binding between MGDG and TLR4,
we individually mutated these five residues of TLR4 into
alanine. Among these mutants, both TLR4F440A/MD-2 and
TLR4F463A/MD-2 largely decreased the ability to interact with
MGDG. The other three mutants retained the binding affinity
for MGDG in a manner similar to wild-type hTLR4,
indicating that F440 and F463 are essential for its binding to
MGDG (Figure 4d). Previous functional studies have ideti-
fied that both F440 and F463 are required for cell activation
by LPS.[29,30] Thus, besides binding like Eritoran (the same
binding mode with Eritoran), we could expect that MGDG
would antagonize TLR4 in a more effective way by preclud-
ing LPS induction. Since Eritoran entered a phase III clinical
trial for the treatment of severe sepsis, an excessive inflam-
matory response to infection, MGDG is promising for
inflammation therapy.[31] Taken together, these results estab-
lish that MGDG acts as an antagonist to exert anti-inflam-
matory activity by targeting the receptor complex of TLR4
and MD-2.
Figure 4. a) Modeled binding pose of MGDG with TLR4/MD-2 com-
plex. b) Comparison of the binding poses between MGDG (purple)
and Eritoran (gray) (PDB ID: 2Z65). c) We pre-incubated human
TLR4/MD-2 recombinant with MGDG or Eritoran for 1 h and then
added probe, 1 h later, the protein mixture was exposed to UV
irradiation for 20 min and then subjected to CuI-catalyzed click
reaction condition with Cyanine5 azide. Direct in-gel fluorescence
detection of the modified proteins was performed. d) HEK293 cells
were transiently transfected with the indicated expression plasmids,
and then were pre-incubated with 20 or 40 mm of MGDG or Eritoran
for 1 h. We isolated the TLR4 proteins by immunoprecipitation and
further incubated with probe 11 for 3 h. The protein mixture was
subjected to photo-crosslinking and CuAAC click chemistry. The
resulting lysates were then incubated with streptavidin-labeled beads,
resolved by SDS-PAGE, and analyzed by Western blot.
In conclusion, we have designed and developed a new
type of bifunctional glycolipid probe which could be used
effectively for mechanistic studies, especially for target
identification. This probe design was demonstrated with
MGDG, and among the synthesized probes, probe 11,
modified with bifunctional fatty acid chain in the sn-2 position
retained anti-inflammatory activity. Further biochemical
studies revealed for the first time that the anti-inflammatory
activity of MGDG was achieved by direct antagonism of
TLR4. Thus, we expect the reported strategy of using
bifunctional glycolipid probes to elucidate complex protein–
glycolipid interactions will find more applications in biomed-
ical research.
Acknowledgements
We thank Dr. Jiang Zhou (Peking University) for HRMS
analysis, Dr. Alexander Jones and Jing Zhang for helpful
discussions. Financial support from the National High Tech-
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Received: September 9, 2016
Published online: && &&, &&&&
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Communications
Anti-Inflammatory Compounds
X. Liu, T. Dong, Y. Zhou, N. Huang,
X. Lei*
&&&& — &&&&
Exploring the Binding Proteins of
Glycolipids with Bifunctional Chemical
Probes
Search and find: A bifunctional probe
design based on monogalactosyldiacyl-
glycerol (MGDG) for mechanistic studies
of glycolipids in living system was devel-
oped. A typical MGDG probe enabled the
identification of TLR4 as its cellular
target. In combination with biochemical
studies and molecular modeling, it was
demonstrated that binding of MGDG to
TLR4/MD-2 leads to its anti-inflammatory
activity.
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www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
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