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German Edition:DOI:10.1002/ange.201706535
Glycan Editing International Edition:DOI:10.1002/anie.201706535
Modulating Cell-Surface Receptor Signaling and Ion Channel
Functions by In Situ Glycan Editing
Hao Jiang,* Aim8Llpez-Aguilar,LuMeng,Zhongwei Gao,Yani Liu, Xiao Tian, Guangli Yu,
Ben Ovryn, Kelley W. Moremen, and Peng Wu*
Abstract: Glycans anchored on cell-surface receptors are
active modulators of receptor signaling.Astrategy is presented
that enforces transient changes to cell-surface glycosylation
patterns to tune receptor signaling.This approach, termed
in situ glycan editing, exploits recombinant glycosyltransfer-
ases to incorporate monosaccharides with linkage specificity
onto receptors in situ. a2,3-linked sialic acid or a1,3-linked
fucose added in situ suppresses signaling through epidermal
growth factor receptor and fibroblast growth factor receptor.
We also applied the same strategy to regulate the electrical
signaling of apotassium ion channel–human ether-/-go-go-
related gene channel. Compared to gene editing, no long-term
perturbations are introduced to the treated cells.Insitu glycan
editing therefore offers apromising approach for studying the
dynamic role of specific glycans in membrane receptor signal-
ing and ion channel functions.
Upon ligand binding,receptors embedded in the plasma
membrane transduce messages into intracellular signaling
molecules in acascade of events,leading to changes in cell
behavior. By combining the specificity of genetic manipula-
tion and the spatiotemporal resolution of light- or small
molecule-based approaches,itispossible to control receptor
signaling in cultured cells or in living organisms.Elegant
examples include photoswitchable ion channels controlled by
azobenzene[1] and “on-switch” chimeric antigen receptor T
cells tuned by arapamycin analogue.[2]
Essentially all membrane receptors are glycosylated and
the anchored glycans actively mediate receptor signaling by
modulating ligand–receptor binding,receptor dimerization,
endocytosis,and degradation.[3] In many instances the role of
specific glycans in these processes remains obscure despite
the new advances in gene editing[4] and metabolic oligosac-
charide engineering (MOE).[5] MOE remodels glycan struc-
tures by supplementing the growth media with carbohydrate
analogues that get incorporated into glycans by the endog-
enous biosynthetic machinery.[6] To our knowledge there has
been only one report from our previous study where MOE
was employed to control aspecific signaling pathway,inwhich
we discovered that enhanced fucosylation on N-glycans
inhibits Wnt signaling.[7]
Nevertheless,modification of cell-surface glycans using
genetic or MOE approaches are not free of limitations:due to
the functional redundancyofmany glycosyltransferases,
typically more than one gene needs to be knocked out in
order to produce aphenotype,[8] and gene editing of
glycosyltransferases may also introduce unexpected side
effects,for example,chaperone functions.[9] Likewise,MOE
often leads to changes on diverse array of glycans.Therefore,
there is acritical need to explore alternative approaches for
the modification of cell-surface glycans.
Herein, we report amethod, termed in situ glycan editing,
to control receptor signaling and ion channel functions and
demonstrate that it serves as astraightforward and fast
approach to modify glycocalyx (Figure 1). In situ glycan
editing finds its roots in chemoenzymatic glycan labeling
which has been exploited by several laboratories,including
Figure 1. In situ glycan editing of cell-surface glycans. Specific labeling
of glycans terminated with galactose is achieved by in situ Sia editing
with CMP-SiaNAl followed by reaction with an azide probe via CuAAC.
Modulation of cell signaling is achieved by remodeling cell-surface
glycans via in situ Sia editing with CMP-Sia or in situ Fucediting with
GDP-Fuc.
[*] Dr.H
.J
iang, X. Tian, Prof. G. Yu
Key Laboratory of Marine Drugs, Ministry of Education and Qingdao
National Laboratory for Marine Science &Technology and Shandong
Provincial Key Lab of Glycoscience &Glycoengineering, School of
Medicine and Pharmacy,Ocean University of China
5Yushan Road, Qingdao,266003 (China)
E-mail:haojiang@ouc.edu.cn
Dr.A.Lkpez-Aguilar,Dr. B. Ovryn, Prof. P. Wu
Department of Molecular Medicine, The Scripps Research Institute
10550 N. Torrey Pines Road, La Jolla, CA 92037 (USA)
E-mail:pengwu@scripps.edu
Dr.L.Meng, Dr.Z.Gao, Prof. K. W. Moremen
Complex CarbohydrateResearch Center and Department of
Biochemistryand Molecular Biology,University of Georgia
315 Riverbend Road, Athens, GA 30602 (USA)
Dr.Y.Liu
Department of Pharmacology,School of Pharmacy
Qingdao University
38 Dengzhou Road, Qingdao,266021 (China)
Supportinginformation and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201706535.
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our own, to visualize cell-surface higher order glycans.In
chemoenzymatic glycan labeling,which has been successfully
used to visualize N-acetyllactosamine (LacNAc), fucose a1,2-
galactose,TFantigen, among afew others,[10] arecombinant
glycosyltransferase is used to transfer amonosaccharide
bearing achemical tag from the corresponding nucleotide
sugar donor to acell-surface acceptor glycans in situ. Thetag
can be derivatized using bioorthogonal click chemistry to
install abiophysical probe for imaging.
In this study (Figure 1), we demonstrate that in situ glycan
editing using human a2,3-sialyltransferease ST3Gal-IV when
combined with bioorthogonal click chemistry,provides
afacile method to validate the identity of membrane
receptors modified by the in situ added monosaccharides.
We discover that the newly added sialic acid or fucose (using
H. pylori a1,3-fucosyltransferase–a1,3 FucT)[11] without fur-
ther derivatization actively tune the strength of membrane
receptor signaling and ion channel functions.
ST3Gal-IV,one of the 20 Golgi-resident sialyltransferases
annotated in humans,controls sialyl Lewis Xbiosynthesis on
N- and O-glycans in cells of myeloid lineage.[12] In vivo
ST3Gal-IV is known to add sialic acids onto the terminal
galactose of N- and O-glycans,[13] however, the in vitro
specificity and substrate scope of recombinant human
ST3Gal-IV for cell-surface glycan editing has not been
characterized. Kinetic analysis using type II LacNAc as the
acceptor substrate revealed that this enzyme has excellent
activity toward the natural donor substrate CMP-Neu5Ac
(CMP-Sia) with Kmof 0.0734 mmand Vmax of 0.0478 nmol
min@1.The enzyme can also accept an alkyne modified CMP-
Sia analogue (CMP-SiaNAl) as the donor with aslightly lower
activity (Kmof 0.211 mmand Vmax of 0.0404 nmolmin@1;
Supporting Information, Figure S1 a,b).
To assess the ability of the recombinant ST3Gal-IV to
modify glycocalyx, we treated Lec2 Chinese hamster ovary
(CHO) cell mutants that express abundant peripheral
LacNAc disaccharides[14] with CMP-SiaNAl and ST3Gal-IV.
At various time points we subjected the ST3Gal-IV treated
cells to the ligand accelerated copper(I)-catalyzed azide–
alkyne cycloaddition (CuAAC)[15] to install biotin tags to the
newly added SiaNAl (Figure 1). Thebiotinylated cells were
then probed with Alexa Fluor 488-strepavidin enabling flow
cytometry and confocal microscopy analysis.Insitu Sia
editing produced robust fluorescent signals in Lec2 cells
whereas control Lec8 cells lacking terminal galactose only
showed background labeling (Supporting Information, Fig-
ure S1c), and the endogenous sialyltransferase had no con-
tribution to in situ Sia editing on Lec2 cell surface (Support-
ing Information, Figure S2a). After a40min in situ Sia editing
reaction (600 mmof CMP-SiaNAl and 50 mgmL@1of ST3Gal-
IV), the cell-associated fluorescence reached aplateau,
suggesting the saturation of cell-surface accessible sialylation
sites (Supporting Information, Figure S1 c–e). Thehalf-life of
the newly added sialic acid on the cell surface was approx-
imately two hours (Supporting Information, Figure S1f).
To determine ex vivo cell-surface behavior of the
recombinant enzyme,weperformed in situ Sia editing using
three CHO cell lines,Lec2, Lec1, and Lec8;these mutants are
known to have distinct glycosylation patterns:Lec2 expresses
galactose-terminated N-glycans and the unsialylated core
1O-glycan Gal-b-(1-3)-GalNAc;Lec1 does not synthesize
complex or hybrid N-glycans,but its O-glycans synthesis is not
affected;and Lec8 expresses complex type N-glycans and
core 1O-glycans lacking the terminal galactose.[14] In situ Sia
editing was performed by treating all three cell lines with
CMP-SiaNAl and ST3Gal-IV,then newly added SiaNAl was
labeled by biotin via CuAAC. Thelabeled cells were either
lyzed for western blot analysis or stained with Alexa Fluor
488-streptavidin for flow cytometry or confocal microscopic
imaging.Lysates from Lec2 cells treated with CMP-SiaNAl
exhibited robust signal that was abolished upon PNGase-F
mediated N-glycan removal. By contrast, signals were not
detected in lysates from Lec1 and Lec8 cells (Figure 2a). This
result was consistent with flow cytometry analysis (Figure 2b)
and confocal imaging (Figure 2c). Together, these observa-
tions suggest that ex vivo ST3Gal-IV preferentially modifies
N-linked glycans,leaving O-glycans unperturbed in CHO
cells.
After identifying the substrate scope and specificity of
in situ Sia editing,wenext sought to apply this method to
functional study of cell surface proteins.Weselected three
model systems that have been shown to have their function
influenced by their glycosylation state.Epidermal growth
factor receptor (EGFR) possesses eleven N-linked glycosy-
lation sites with potential sialylation or fucosylation,[16] and
performs critical roles in essential cellular processes which
control cell proliferation and migration. Mutations in the
Figure 2. Determinethe labeling specificity of recombinant ST3Gal-IV
in CHO cell mutants via western blot, flow cytometry,and imaging
analysis. a) CHO cell mutants were treated with CMP-SiaNAl or CMP-
Sia (500 mm)and ST3Gal-IV(50 mgmL@1)for 30 min. The labeled cells
were then reacted with biotin-Azvia CuAAC, lyzed, and probed with
anti-biotin antibody (top). The Commassie blue stain proved equal
loading amount (bottom).b)CHO cell mutants were biotinylatedas
described above. The labeled cells were probed with streptavidin-Alexa
Fluor 488 for flow cytometry analysis (n=3). c) Imaging of cell-surface
glycans terminated with galactose. Lec2 cells stained with chloro-
methyl fluorescein diacetate (CMFDA, red) and unstained Lec8 cells
were mixed at a1:4 ratio and cultured for 3days. The cells were
treated with ST3Gal-IVand CMP-Sia or CMP-SiaNAl, then stained with
Alexa Fluor 647-Az (green) and Hoechst 33342 (blue). Scale bars:
20 mm.
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EGFR gene are associated with pathogenesis and progression
of different carcinoma types,inparticular those of pulmonary
origin.[17] Wong et al. found that overexpression of a2,3-
sialyltransferases and a1,3-fucosyltransferases in lung cancer
cells suppress EGF-induced receptor dimerization and phos-
phorylation.[3b] However,enzyme overexpression usually
causes permanent perturbation to the treated cells.To
overcome this drawback we sought to determine if adding
sialic acid or fucose directly on the cell surface via the in situ
glycan editing strategy may convey similar effects.Through
this approach only transient perturbation is introduced
(Supporting Information, Figure S1 f) ;cell viability assay
indicated that the in situ glycan editing is non-toxic to the
treated cells (Supporting Information, Figure S3). Toward
this end, we incubated the adenocarcinoma human alveolar
basal epithelial cell A549, acell line expressing high levels of
EGFR, with ST3Gal-IV and CMP-SiaNAl to determine if
cell-surface EGFR could be modified. Treated cell lysates
were biotinylated and immunoprecipitated using anti-EGFR
or anti-biotin. Western blot analysis indicated that immuno-
precipitated EGFR was modified by biotin and the enriched
biotinylated protein pools contained EGFR, while the control
sample which was subjected to ST3Gal-IV in the presence of
CMP-Sia showed no detectable signal (Figure 3a). These
results confirmed that EGFR were indeed modified by in situ
Sia editing.
Next, we evaluated if in situ Sia or Fuc editing could alter
EGFR dimerization, activation or degradation. A549 cells
were subjected to in situ Sia editing using CMP-Sia as the
glycan donor or in situ Fuc editing using GDP-fucose (GDP-
Fuc) as the glycan donor, followed by EGF stimulation.
Western blotting analysis of the cell lysates indicated that the
formation of the EGFR dimer decreased upon in situ Sia or
Fuc editing by 37%and 25%, respectively (Figure 3b).
Similarly,Tyr 1068 phosphorylation of EGFR was reduced by
36%and 63%ineach case (Figure 3b). In the control
experiment where cells were only treated with ST3Gal-IV
without the CMP-Sia donor, no suppression of EGFR
dimerization was observed (data not shown). Interestingly,
using CMP-SiaNAl as the in situ donor resulted in more
pronounced inhibition of EGFR dimerization (Supporting
Information, Figure S4). It is known that the EGF-stimulated
mitogen-activated protein kinase (MAPK) signaling occurs
primarily in the plasma membrane and that signaling through
EGFR induces the activation of MAPK by phosphorylation
at Tyr1068.[18] Consistent with the suppressive role of in situ
Sia or Fuc editing on EGFR activation, we also observed the
decreased phosphorylation of MAPK by 35%and 29 %,
respectively (Figure 3b). To confirm that these observations
were indeed caused by in situ glycan editing of EGFR, the
above experiments were repeated via in situ Fuc editing in the
presence of the EGFR inhibitor gefitinib.Anadditive effect
was detected, verifying EGFR-suppression was indeed cause
by in situ Fuc editing (Supporting Information, Figure S5).
Surprisingly,despite the pronounced impact of in situ Sia
editing on EGF-induced EGFR dimerization and activation,
little influence of this treatment on EGF-induced EGFR
degradation was observed (Supporting Information, Fig-
ure S6).
Previous studies showed that signaling through EGFR
enhances the migration of cancer cells.[19] To test if in situ Sia
editing has any impact on EGFR dependent migration,
awound healing assay was performed on the EGF treated
A549 cells.Asshown in Figure 3c,insitu Sia editing of EGF
stimulated cancer cells showed significantly suppressed cell
migration, but did not impact non-stimulated cancer cells.To
further validate this result, we treated the cells with aknown
metabolic inhibitor of sialyltransferases,2,4,7,8,9-pentaacetyl-
3Fax-Neu5Ac-CO2Me to block the biosynthesis of sialylated
glycans,[20] and measured the EGF-mediated cell migration.
As aresult, asignificant increase of cell migration was
observed in the inhibitor treated cells as compared with
untreated or in situ Sia edited cells (Figure 3c). Similar effects
were also observed in in situ Fuc edited and defucosylated
A549 cells,and the impact of fucosidase-mediated defucosy-
lation could be rescued by in situ Fuc editing (Supporting
Information, Figure S7).
To evaluate if in situ glycan editing can serve as ageneral
approach to control receptor signaling,weexamined its
impact on fibroblast growth factor receptor (FGFR)-medi-
ated signaling.FGF-FGFR complex comprises two receptor
molecules,two FGFs and aco-receptor heparin.[21] Aberrant
FGF signaling can promote tumor development by directly
Figure 3. Cell-surface in situ glycan editing suppresses EGFR signaling.
a) A549 cells were labeled with CMP-SiaNAl or CMP-Sia and ST3Gal-IV,
then reacted with biotin-Azvia CuAAC. EGFR pull-downed from cell
lysate were resolved and probed with anti-biotin antibody (left);
biotinylatedproteins pull-downed from cell lysate were resolved and
probed with anti-EGFR antibody (right). b) Starved A549 cells were
treated with or without in situ Sia or Fucediting and stimulated with
EGF. Lysates were prepared and analyzed by Western blotting (n=3,
*p<0.05). c) Wound-healing assay for the effects of EGF, in situ Sia
editing and sialylation inhibitor on cells migration. A549 cells were
treated and wounded, the summarized migration area were measured
using ImageJ (n=6, **p<0.01).
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driving cancer cell proliferation and supporting tumor angio-
genesis.[22] It is reported that FGFR signaling is regulated by
its O- and N-glycosylation.[23] Here we chose the human
breast adenocarcinoma cell line MCF-7 that expresses FGFR
to evaluate the efficacy of our strategy.MCF-7 cells were
subjected to in situ Sia editing followed by stimulation with
FGF and heparin. FGFR dimer formation and the down-
stream phosphorylation of MAPK were significantly sup-
pressed upon in situ Sia editing (by 26 %and 58 %, respec-
tively;Supporting Information, Figure S8a,b) compared to
the control unsialylated cells.Similarly,FGFR-mediated
activation of MAPK was also impaired by 47 %upon in situ
Fuc editing (Supporting Information, Figure S8 b).
As the initial evaluation of the in situ glycan editing
strategy was based on growth factor receptors,wenext sought
to assess the use of this technique to modulate the function of
ion channels.Human ether-/-go-go-related gene (hERG)
channel is apotassium channel that belongs to voltage gated
ion channels,[24] which plays important roles in electrical
signaling by repolarizing the cell membrane in the heart.[25]
HERG has only one extracellular N-linked glycosylation
site[26] and its gating properties is known to be related to
protein glycosylation.[27]
To determine if the in situ added monosaccharides could
modulate hERG channel gating properties,HEK-293 cells
stably expressing hERG channels were subjected to in situ Sia
or Fuc editing followed by voltage clamp experiment to
record whole-cell current. Astandard depolarizing pulse
protocol for the hERG channel was employed (Figure 4a).
Thecurrent traces of control untreated cells and in situ glycan
edited cells were recorded (Supporting Information, Fig-
ure S9a, S10a). From the I–Vcurve measured at the end of
the depolarizing clamp steps,nosignificant changes of current
were observed in cells treated with in situ Sia or Fuc editing
(Supporting Information, Figures S9 b, S10 b). Then we com-
pared the steady-state activation (SSA) curve of hERG
channels between the control and the in situ glycan editing
group to study the voltage dependence of activation. SSA
curves based on the tail currents were measured at the initial
point of the repolarizing clamp steps.Asshown in Figure 4b,
there was asignificant depolarizing shift in sialylation group,
with the mean V1/2 shifting from @4.3 mV to 1.6 mV (n=6–8,
p<0.01). By contrast, no significant difference of SSA
relationships between the control group and fucosylated
group was observed (Supporting Information, Figure S10 c).
These data suggested that in situ Sia editing modulates the
hERG channel gating by depolarizing its activation voltage–
more depolarized potential is required to activate hERG
channel after in situ Sia editing.
Although in situ Fuc editing had no apparent impact on
the voltage dependent-activation of hERG channel, it was
detected to have amajor influence on the hERG maximum
current by whole-cell voltage-clamp experiments.The patch
was initially maintained in the whole-cell mode and then
excised into the normal buffer. GDP-Fuc and a1,3 FucT were
freshly added in the buffer and continuously perfused to the
cell for 40 min. During this procedure,maximal tail currents
of hERG channels evoked with Vh=40 mV were recorded
every 15 s. As soon as in situ Fuc editing was applied,
normalized hERG maximal current was suppressed (Fig-
ure 4c). Compared with the control group,the fucosylated
group exhibited accelerated decreasing rate of normalized
hERG maximal tail current during the 40-min perfusion. As
aresult, the normalized hERG maximal tail current of
fucosylated group is approximately 25%lower than that of
control group at the end of the 40-min perfusion (Figure 4c,
bottom), indicating that in situ Fuc editing suppressed hERG
channel activity by inhibiting its current.
In 1979, Paulson and co-workers demonstrated that
sialyltransferases with distinct specificities could be used to
restore Siaa2-6Gal, Siaa2-6GalNAc,orSiaa2-3Gal epitopes
onto the surface of previously de-sialylated erythrocytes.[28]
However,this method has not received wide attention
because of the limited availability of recombinant glycosyl-
transferases that can catalyze glycan transfer on the cell
surface.One exception to this observation is the human a1,3-
fucosyltransferase VI. Demonstrated by McEver,Sackstein,
and others,this enzyme actively transfers fucose from GDP-
Fuc to create sialyl Lewis Xepitopes on the cell-surface to
direct cord blood cell engraftment and homing of mesenchy-
mal stem cells to bone.[29] By contrast, using glycosidases for
global glycan editing is aroutine practice owing to the readily
availability of these enzymes from commercial sources.[30]
In the current study,wedemonstrated that in situ glycan
editing is apowerful approach for transiently modulating cell-
surface receptor signaling and ion channel properties.Via this
approach, sialic acid, fucose,and their analogues can be added
in situ onto the cell surface in alinkage specific manner.Sialic
acid and fucose added in situ exhibited similar effects in
suppressing EGFR and FGFR dimerization and downstream
signaling.Nevertheless,they were found to have distinct
impact on hERG channel gating and activity.Insitu Sia
editing modulates hERG channel activation by depolarizing
Figure 4. Cell-surfaceinsitu glycan editing modulate the hERG chan-
nel functions. a) The pulse protocol for whole-cell voltage clamp
experiment. Cells were held at @80 mV and depolarizedtopre-pulse
potential ranging from @60 to +40 mV with 10-mV increments for 4s,
followed by a @40 mV repolarizing step. Arrows indicate points at
which currents were measured (solid:for I–Vcurves, dash:for SSA
curves). b) SSA curves for hERG channels in untreated and in situ Sia
edited cells (n=6–8). Lines are fits of the data to single Boltzmann
distributions. c) The normalized maximal tail current for hERG chan-
nels in untreatedand in situ Fucedited cells (n=6, **p<0.01).
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SSA relationships whereas in situ Fuc editing inhibits the
maximal hERG tail current. It is possible that the different
modulating patterns observed for in situ Fuc and Sia editing
are caused by different locations and charges of the newly
added a1,3-fucosides and a2,3-sialosides.Itisworth noting
that the fraction of receptors modified by in situ glycosylation
has not been determined in this work. Currently in our
laboratory we are developing amethod for the quantitative
measurement of the modified receptors and evaluating the
feasibility of applying this method to modulate receptor
signaling pathways in more complex systems such as cultured
organoids.
Acknowledgements
This work was supported by the NIH (R01GM113046 and
R01GM111938 to P. W. ,P01GM107012 to K.W.M.), NSFC-
SD Joint Fund (U1606403 to G.Y.), the Fundamental
Research Funds for the Central Universities (201762002 to
H.J.), Taishan Scholar Project Special Funds (to G.Y.)and
China Postdoctoral Science Foundation (2017M612356 to
H.J.). P.W. conceived this project by insightful discussions
with Prof.Rachel Hazan.
Conflict of interest
Theauthors declare no conflict of interest.
Keywords: click chemistry ·fucosylation ·insitu glycan editing ·
sialylation
Howtocite: Angew.Chem. Int. Ed. 2018,57,967–971
Angew.Chem. 2018,130,979–983
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Manuscript received:July 3, 2017
Revised manuscript received: November24, 2017
Version of record online: January 2, 2018
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