Glycan-Gold Nanoparticles as Multifunctional Probes for Multivalent Lectin-Carbohydrate Binding: Implications for Blocking Virus Infection and Nanoparticle Assembly

Article abstract of DOI:10.1021/jacs.0c06793

Multivalent lectin-glycan interactions are widespread in biology and are often exploited by pathogens to bind and infect host cells. Glycoconjugates can block such interactions and thereby prevent infection. The inhibition potency strongly depends on matching the spatial arrangement between the multivalent binding partners. However, the structural details of some key lectins remain unknown and different lectins may exhibit overlapping glycan specificity. This makes it difficult to design a glycoconjugate that can potently and specifically target a particular multimeric lectin for therapeutic interventions, especially under the challenging in vivo conditions. Conventional techniques such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) can provide quantitative binding thermodynamics and kinetics. However, they cannot reveal key structural information, e.g., lectin's binding site orientation, binding mode, and interbinding site spacing, which are critical to design specific multivalent inhibitors. Herein we report that gold nanoparticles (GNPs) displaying a dense layer of simple glycans are powerful mechanistic probes for multivalent lectin-glycan interactions. They can not only quantify the GNP-glycan-lectin binding affinities via a new fluorescence quenching method, but also reveal drastically different affinity enhancing mechanisms between two closely related tetrameric lectins, DC-SIGN (simultaneous binding to one GNP) and DC-SIGNR (intercross-linking with multiple GNPs), via a combined hydrodynamic size and electron microscopy analysis. Moreover, a new term, potential of assembly formation (PAF), has been proposed to successfully predict the assembly outcomes based on the binding mode between GNP-glycans and lectins. Finally, the GNP-glycans can potently and completely inhibit DC-SIGN-mediated augmentation of Ebola virus glycoprotein-driven cell entry (with IC50 values down to 95 pM), but only partially block DC-SIGNR-mediated virus infection. Our results suggest that the ability of a glycoconjugate to simultaneously block all binding sites of a target lectin is key to robust inhibition of viral infection.

Full text of DOI:10.1021/jacs.0c06793

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Article
Glycan-Gold Nanoparticles as Multifunctional Probes for
Multivalent Lectin-Carbohydrate Binding: Implications
for Blocking Virus Infection and Nanoparticle Assembly
Darshita Budhadev, Emma Poole, Inga Nehlmeier, Yuanyuan Liu, James Hooper, Elizabeth Kalverda, Uchangi
Satyaprasad Akshath, Nicole Hondow, W. Bruce Turnbull, Stefan Pohlmann, Yuan Guo, and Dejian Zhou
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 16 Sep 2020
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Glycan-Gold Nanoparticles as Multifunctional Probes for Multivalent
Lectin-Carbohydrate Binding: Implications for Blocking Virus Infection
and Nanoparticle Assembly
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Darshita Budhadev†,╬, Emma Poole†,╬, Inga Nehlmeier‡, Yuanyuan Liu†,§, James Hooperɧ, , Elizabeth
Kalverda,† Uchangi Satyaprasad Akshath,† Nicole Hondowɸ, W. Bruce Turnbull†, Stefan Pöhlmann‡,*,
Yuan Guoɧ,*, and Dejian Zhou†,*
† School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United
Kingdom.
‡ Infection Biology Unit, German Primate Center – Leibniz Institute for Primate Research, 37077 Göttingen, and Faculty of
Biology and Psychology, University of Göttingen, 37073 Göttingen, Germany.
§ Permanent address: School of Pharmaceutical and Chemical Engineering, Chengxian College, Southeast University, Nan-
jing 210088, China.
ɧ School of Food Science & Nutrition and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2
9JT, United Kingdom.
ɸ School of Chemical and Process Engineering, University of Leeds, Leeds LS2 9JT, United Kingdom.
╬ These authors contributed equally to this work.
KEYWORDS. Gold nanoparticle, glycoconjugate, multivalent lectin-glycan interaction, fluorescence quenching, binding
affinity, multi-modal readout, virus inhibition.
ABSTRACT: Multivalent lectin-glycan interactions are widespread in biology and are often exploited by pathogens to bind and
infect host cells. Glycoconjugates can block such interactions and thereby prevent infection. The inhibition potency strongly depends
on matching the spatial arrangement between the multivalent binding partners. However, the structural details of some key lectins
remain unknown and different lectins may exhibit overlapping glycan specificity. This makes it difficult to design a glycoconjugate
that can potently and specifically target a particular multimeric lectin for therapeutic interventions, especially under the challenging
in vivo conditions. Conventional techniques such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) can
provide quantitative binding thermodynamics and kinetics. However, they cannot reveal key structural information, e.g. lectin’s bind-
ing site orientation, binding mode, and inter-binding site spacing, which are critical to design specific multivalent inhibitors. Herein
we report that gold nanoparticles (GNPs) displaying a dense layer of simple glycans are powerful mechanistic probes for multivalent
lectin-glycan interactions. They can not only quantify the GNP-glycan-lectin binding affinities via a new fluorescence quenching
method, but also reveal drastically different affinity enhancing mechanisms between two closely-related tetrameric lectins, DC-SIGN
(simultaneous binding to one GNP) and DC-SIGNR (inter-crosslinking with multiple GNPs), via a combined hydrodynamic size and
electron microscopy analysis. Moreover, a new term, potential of assembly formation (PAF), has been proposed to successfully
predict the assembly outcomes based on the binding mode between GNP-glycans and lectins. Finally, the GNP-glycans can potently
and completely inhibit DC-SIGN-mediated augmentation of Ebola virus glycoprotein-driven cell entry (with IC50 values down to 95
pM), but only partially block DC-SIGNR-mediated virus infection. Our results suggest that the ability of a glycoconjugate to simul-
taneously block all binding sites of a target lectin is key to robust inhibition of viral infection.
can bind simultaneously with each other and form a single en-
tity. This gives the highest affinity enhancement and selectivity
due to the most favorable enthalpy and entropy terms.13 While
those without such spatial and orientation match may inter-
crosslink with each other to maximize binding enthalpy and
form large scale assemblies, but this typically gives lower affin-
ity enhancement and binding specificity due to an unfavorable
entropy penalty.15 Therefore, understanding the structural
mechanism underlying the affinity enhancement in multivalent
protein-ligand binding is key to the design of potent, specific
multivalent inhibitors against a target multivalent receptor. No-
tably, the development of potent glycoconjugates to block virus
binding and infection of host cells can prevent virus mutation
Introduction
Multivalent lectin-glycan interactions are widespread and me-
diate many important biological functions which include cell-
cell communication, pathogen-host cell recognition, attachment
and infection, and modulation of immune responses.1-9 As most
monovalent lectin-glycan binding events are often too weak to
be bio-functional, many lectins form multimeric structures to
cluster their carbohydrate-binding-domains (CRDs) for effi-
cient binding with spatially matched multivalent glycans to en-
hance binding affinity and specificity.10-14 The overall multiva-
lent affinity is not only directly linked to the monovalent affin-
ity, but also the glycan valency and the mode of binding. In gen-
eral, a pair of spatially matched multivalent binding partners
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and developing resistance, a unique advantage over other anti-
surface densely glycosylated glycoprotein which is responsible
viral strategies.9
for initiating HIV-DC-SIGN interaction to facilitate HIV infec-
tion. The GNP-glycans are thus good mimics of gp120 for prob-
ing its interaction with DC-SIGN. Furthermore, they also dis-
play excellent colloidal stability and show no signs of aggrega-
tion in biologically relevant media, allowing for unambiguously
detection of changes in GNP aggregation induced by lectin
binding. Additionally, as GNP’s extinction coefficient scales
linearly with its volume (SI, Figure S1), the use of a small 5 nm
GNP allows for access to a wide concentration range required
for affinity quantitation (see later section) of weak binders with-
out introducing significant “inner-filter” effects.
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A number of different scaffolds, including DNAs, proteins,
polymers, dendrimers, C60 derivatives, vesicles, and inorganic
nanoparticles, have been employed to construct glycoconju-
gates14, 16-22 to study multivalent lectin-glycan recognition and
develop effective interventions against certain diseases. Some
of these have exhibited excellent potency in inhibiting pathogen
infection.9, 11, 13-14, 16-20, 23-24 Most of the bindings have been eval-
uated by conventional biophysical techniques such as surface
plasmon resonance (SPR),14, 22 and/or isothermal titration calo-
rimetry (ITC).14, 25-27 Despite their wide use and good capacity
in providing quantitative binding affinity, thermodynamic and
kinetic data, these techniques cannot reveal key structural infor-
mation of the target lectin, e.g. binding site orientation, inter-
binding site distance, and binding mode which are critical to
design potent, specific multivalent glycan inhibitors. Mean-
while, the unique size-dependent physical properties of nano-
materials have been harnessed to study lectin-glycan interac-
tions. A good example here are gold nanoparticles (GNPs)28
whose strong, size- and aggregation-state dependent absorp-
tion,29-30 powerful signal amplification in SPR,31 and surface en-
hanced Raman scattering32-33 have been widely exploited for bi-
osensing and diagnostic applications. Despite these successes,
glycan conjugated GNPs (GNP-glycans) have not been ex-
ploited as new structural and mechanistic probes for multivalent
lectin-glycan interactions.
Recently, we have shown that CdSe/ZnS quantum dots
(QDs) displaying a dense layer of mannose glycans are power-
ful structural probes for multivalent lectin-glycan binding.34-36
The QD probes can not only quantify the binding affinity via a
ratiometric FRET readout but also dissect the different binding
modes between a pair of closely-related, almost identical te-
trameric lectins, DC-SIGN37-38 and DC-SIGNR39 (collectively
abbreviated as DC-SIGN/R hereafter). DC-SIGN/R are im-
portant lectin receptors which play a key role in facilitating the
HIV, Ebola virus and Zika virus infections.34-35, 40-41 Moreover,
DC-SIGN is also key to immune regulation,1, 5, 42-43 making it an
highly attractive target for developing immunotherapies against
important human diseases such as cancer, allergy, and autoim-
mune diseases.1, 42, 44 However, its tetrameric structure remains
unknown, making it difficult to develop novel glycoconjugates
that can potently and specifically target DC-SIGN for therapeu-
tic interventions, especially when the overlapping glycan spec-
ificity of various lectins is considered.1 Using the QD-glycans,
we have found that DC-SIGN’s 4 CRDs bind simultaneously to
one QD and give an impressive affinity enhancement factor ()
of up to 1.5 million fold over the corresponding monovalent
binding. The QD-glycans also potently inhibit DC-SIGN-
mediated augmentation of Ebola virus entry into host cells with
sub-nM IC50 values.35 Despite such success, the cytotoxicity
and long term toxicity of the CdSe QD scaffold has significantly
limited its potential use as DC-SIGN targeting therapeutic
agents, especially under in vivo conditions.
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Using DC-SIGN/R as model lectins, we show that the
GNP’s strong fluorescent quenching property47 can be exploited
as a reliable method to quantify multivalent GNP-glycan-lectin
binding for the 1st time. Moreover, its nanoscale size, and high
contrast under electron microscopy imaging have been inte-
grated into a multimodal readout to reveal the different binding
modes and affinity enhancement mechanisms for DC-SIGN/R
(e.g. simultaneous binding for DC-SIGN, but inter-crosslinking
for DC-SIGNR). Furthermore, the GNP-lectin binding modes
are found to be directly linked to the GNP’s ability to block lec-
tin mediated virus infection of host cells: only a GNP-glycan
which binds simultaneously to all binding sites of the target lec-
tin can potently and completely block virus infection, but not
that showing a cross-linking binding mode.
Results and Discussion
Ligand design and synthesis. The schematic structures of the
GNP-glycan conjugates and the chemical structures of the gly-
can ligands used in this study are shown in Figure 1.
Figure 1. Schematic of the GNP-glycans used in this study. The
GNP is coated with DHLA-based glycan ligand containing either
one (A) or three (B) terminal -1-mannose or -1-mannose--1,2-
mannose to tune its surface glycan valency. The chemical struc-
tures of glycan ligands are depicted underneath.
To address this issue, here we have constructed polyvalent
glycan-nanoparticle probes on a relatively small 5 nm GNP to
take the advantages of GNPs’ excellent biocompatibility, low-
/non-toxicity, and robust gold-thiol surface chemistry for easy
control of the glycan density and surface presentation.28 More-
over, after coating with lipoic acid (LA) based glycan ligands
containing a terminal mannose (Man) or mannose--1,2-
mannose (DiMan), the resulting GNP-glycans are of the similar
size and mannose glycan coating to gp120 trimer,45-46 the HIV
Each ligand contains three unique functional domains, a di-
hydrolipoic acid (DHLA) for strong binding to the GNP surface
via the formation of 2 strong Au-S bonds to impose excellent
stability;48 a short, flexible oligo-(ethylene glycol) linker to en-
hance water solubility, and to resist non-specific adsorption,35,
49
and a terminal an -1-mannose or -1-mannose-α-1,2-man-
nose (abbreviated as DHLA-Man or DHLA-DiMan, respec-
tively, hereafter) for specific binding to DC-SIGN/R.34-35 We
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good yields using the standard BOC protection and amide cou-
have found previously that DC-SIGN binds more efficiently to
QDs capped with higher mannose densities.34 Moreover, gly-
coconjugates with higher glycan valency have often shown to
exhibit better virus inhibition potencies.9, 14, 16, 21, 50 Thus we are
interested to study whether GNP-glycans’ DC-SIGN/R binding
affinity and virus inhibition potency can be further enhanced by
increasing its surface glycan density. Thus, a branched version
of the DHLA-glycan ligand, each containing three terminal gly-
cans, are also synthesized (denoted as DHLA-(Man)3 or DHLA-
(DiMan)3, respectively).50 The branched ligands have the same
DHLA anchoring group for GNP binding as the monomeric
glycans. Thus, a similar number of ligands are expected to coat
each GNP, allowing us to prepare more densely glycosylated
GNPs as shown schematically in Figure 1.
pling chemistries. Details of the synthetic procedures and spec-
troscopic data of the intermediate compounds were provided in
the Supporting Information (SI). Second, -mannose and -
mannose--1,2-mannose appending a hydrophilic di(ethylene
glycol)-azide linker (N3-EG2-Man-/DiMan) to provide some
flexibility to the terminal glycans were synthesized using our
established procedures.34-35 Third, the LA-acetylene linkers
were coupled efficiently to N3-EG2-Man/DiMan via the Cu-cat-
alyzed click chemistry16 to give the desired LA-Man/DiMan or
LA-(Man)3/(DiMan)3 ligands (see Experimental Section for the
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general procedures and spectroscopic data, and SI for their H
and 13C NMR spectra). Finally, the LA-glycan ligands were re-
duced quantitatively to their corresponding DHLA forms by
tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl)51-52
before they were used to prepare the GNP-glycans.
Scheme 1. Synthetic route to LA-Man-/DiMan ligands
GNP-glycan preparation. GNP-glycans (GNP capped with
the DHLA-glycan ligands) were prepared by incubation of a 5
nm citrate stabilized GNP with the above DHLA-glycan ligands
in water at a ligand:GNP molar ratio of 1000:1 for 24 h. Any
free unbound ligands were removed by ultra-filtration using
30K MWCO filter tubes and washing with pure water. GNP-
glycans prepared using LA-glycans or their reduced DHLA-
glycan forms were found to be identical in terms of hydrody-
namic sizes and stability. Thus the air-stable LA-glycan ligands
could be directly used to make the GNP-glycans, eliminating
the need of reduction and handling air-sensitive DHLA-glycan
ligands that were essential in QD-glycan preparation via cap-
exchange.34, 40, 48 This made the GNP-glycan preparation simple
and straightforward. This result was fully consistent with earlier
literature reports that dialkyldisulfides were cleaved upon bind-
ing to gold surfaces, forming identical self-assembled monolay-
ers to their alkylthiol counterparts.53 The GNP-glycans were
highly stable, no changes of physical appearance or precipita-
tion were observed after prolonged storage (>6 months) at 4 oC.
They were uniform and monodisperse in both water and in a
binding buffer (20 mM HEPES, 100 mM NaCl, 10 mM CaCl2,
pH7.8) with hydrodynamic diameters (Dhs) of ~11 nm (see SI,
Figure S2 and Table S1). Such Dh values matched well to the
size of gp120 trimer (~12 nm).45 Moreover, the GNPs were
densely coated with mannose containing glycans similar to
those found on gp120 surface. Using the ligand amount differ-
ence between that used and that remained in the supernatant af-
ter conjugation (measured by a phenol-sulfuric acid assay),35
the numbers of ligands bound on each GNP were estimated as
~490, ~690, 720, and ~650 for LA-Man, LA-DiMan, LA-
(Man)3 and LA-(DiMan)3, respectively. Using the Dh values and
the method reported by the Mirkin group,54 the average inter-
glycan distances were estimated as ~0.97, ~0.83, ~0.46, and
~0.49 nm for GNP-Man, GNP-DiMan, GNP-(Man)3 and GNP-
(DiMan)3, respectively (SI, Table S2). The average inter-gly-
can distances of GNP-Man/DiMan fell well within the majority
of inter-glycan sequon distances (e.g. 0.7-1.3 nm) found on
gp120,46 but those of GNP-(Man/Diman)3 were considerably
smaller. These suggested that GNP-Man/DiMan were good
mimics of gp120 for probing its DC-SIGN interaction.
Scheme 2. Synthetic route to LA-(Man)3-/(DiMan)3 ligands
Quantifying GNP-glycan-DC-SIGN/R binding affinity. Be-
sides strong plasmonic absorption, GNPs are well-known for
their strong and universal fluorescence quenching property.47
Moreover, its quenching has shown to follow the nano-surface
energy transfer (NSET),55-56 rather than Förster resonance en-
ergy transfer (FRET) mechanism. As a result, its quenching is
The synthetic routes to the monomeric LA-glycan and branched
trimeric LA-(glycan)3 ligands are shown in Schemes 1 and 2,
respectively. First, the LA based linker molecules each contain-
ing one or three terminal acetylene groups were synthesized in
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Figure 2. (A) Schematic showing the quenching of DC-SIGN/R
more superior and covers a longer distance range than most or-
ganic quenchers relying on the FRET mechanism.47, 55 Indeed,
the GNP’s superior quenching ability has been widely exploited
for biosensing applications,57-58 but not as a readout method for
lectin-glycan binding quantification. Here the GNP’s outstand-
ing fluorescence quenching was exploited as a new readout for
multivalent lectin-glycan binding quantification for the first
time (Figure 2A). To do this, we first introduced a cysteine site-
specifically on the CRD (outside its glycan binding pocket so
not affecting its glycan binding properties) of DC-SIGN/R ex-
tracellular segments by mutagenesis. The mutant proteins have
shown to retain faithfully the tetrameric structure and glycan
binding properties of wildtype proteins.34-35 We then labeled the
proteins using a maleimide modified Atto-594 and confirmed
the labeling by high resolution mass spectroscopy (SI, Figure
S3). We then recorded the fluorescence spectra of labeled DC-
SIGN/R without and with each GNP-glycan under a fixed pro-
tein:GNP molar ratio of 1:1 over a concentration range of 0-64
nM. All binding studies were carried out in a binding buffer
containing a large excess of bovine serum albumin (BSA, 1
mg/mL final concentration). The inclusion of large excesses of
BSA, a non-target serum protein of high abundance in vivo, in
binding studies made the conditions resemble more closely to
real biological situations. Moreover, this also greatly reduced
non-specific interactions and adsorption of proteins and GNPs
to surfaces (a main source of experimental errors at low con-
centrations, e.g. < 10 nM).59
(dye-labeled) fluorescence upon binding to a GNP-glycan, which
acts as a readout for binding quantification. (B) Typical fluores-
cence spectra of varied concentrations of DC-SIGN without (solid
lines) and with (broken lines) 1 molar equivalent of GNP-DiMan
(EX = 590 nm). (C, D) The quenching efficiency (QE%)-
concentration relationship for DC-SIGN (C) or DC-SIGNR (D)
binding to the various GNP-glycans fitted by the Hill’s equation.
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To analyze the binding data quantitatively, we first calculated
the quenching efficiency (QE) at each concentration (C) via:
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QE% = (IF0 - IF)/IF0 × 100%
(1)
Where IF0 and IF are the integrated fluorescence of the protein
in the absence and presence of each GNP-glycan, respectively.
Given that the fluorescence of DC-SIGN/R increases linearly
with C in the absence of GNP-DiMan (SI, Figure S3), and GNPs
can completely quench various fluorophores at close proximity
(e.g. > 99.9%),47 the QE% thus faithfully represents the percent-
age of proteins bound to the GNP-glycan. Thus, the apparent
binding dissociation constant (Kd) can be derived from the QE-
C relationship by fitting with Hill’s equation:
QE = QEmax × Cn/(Kdn + Cn)
(2)
Where QEmax, Kd, C, and n are the maximum QE (fixed at 100),
apparent binding dissociation constant, protein concentration,
and Hill coefficient, respectively.
Table 1. Summary of the fitting parameters of DC-SIGN/R binding
The typical fluorescence spectra showing the binding be-
tween DC-SIGN and GNP-DiMan were given in Figure 2B. In
the absence of GNP-DiMan, DC-SIGN’s fluorescence intensity
was found to increase linearly with concentration. While in the
presence of GNP-DiMan, its fluorescence was greatly reduced
and increasingly deviated from the linear relationship with the
increasing concentration (SI, Figure S4). This result was fully
consistent with that, with the increasing concentration, an in-
creasing proportion of DC-SIGN were bound to and quenched
by GNP-DiMan in close proximity. The specificity of DC-
SIGN-GNP-DiMan binding was verified by two controls: (1)
mixing DC-SIGN with a GNP capped with a control DHLA-
EG2-OH ligand lacking the terminal glycan produced negligible
quenching; (2) free mannose could effectively compete with
GNP-DiMan binding to DC-SIGN, leading to a significant,
dose-dependent fluorescence recovery (SI, Figure S5).
to various GNP-glycans derived from Figures 2C and 2D.
GNP-glycan + Protein
Kd (nM)
n
R2
GNP-Man + DC-SIGN
33.1 ± 2.1 0.82±0.07 0.997
18.7 ± 0.3 0.77±0.01 0.999
3.9 ± 0.3 0.59±0.03 0.999
3.6 ± 0.1 0.42±0.01 1.00
214 ± 68 0.68±0.10 0.994
133 ± 20 0.63±0.07 0.996
152 ± 37 0.99±0.14 0.954
GNP-(Man)3 + DC-SIGN
GNP-DiMan + DC-SIGN
GNP-(DiMan)3 + DC-SIGN
GNP-Man + DC-SIGNR
GNP-(Man)3 + DC-SIGNR
GNP-DiMan + DC-SIGNR
GNP-(DiMan)3 + DC-SIGNR
42 ± 2
0.48 ± 0.03 0.999
Figures 2C and 2D showed the best fits of the QE-C relation-
ships for DC-SIGN/R binding to each GNP-glycan. The result-
ing fitting parameters were summarized in Table 1. Four key
points could be drawn from the binding data given in Table 1.
(1) DC-SIGN bound more strongly to GNP-glycans than DC-
SIGNR did. This effect was particularly pronounced for GNP-
DiMan where the binding affinity difference was as high as ~40
fold. As GNP-DiMan here presented a good mimic of gp120,
its stronger binding affinity with DC-SIGN over DC-SIGNR
could thus help explain why DC-SIGN was found more effec-
tive than DC-SIGNR in transmitting infections for some HIV
strains.60 This result also agreed well with those reported previ-
ously with QD-glycans where the binding Kds were measured
ratiometrically via FRET.34 Given that the CRDs in DC-
SIGN/R had the same mannose binding motifs,61 the difference
here indicated that DC-SIGN/R might adopt different modes in
binding to GNP-glycans, similar to those observed with QD-
glycans.35 (2) Further increasing the GNP surface glycan den-
sity via capping with the dendritic LA-(glycan)3 ligands signif-
icantly improved the binding affinity with DC-SIGNR (e.g. Kd
~42 v.s. ~152 nM, or 3.6 fold enhancement for GNP-(DiMan)3
v.s. GNP-DiMan), but not much with DC-SIGN, suggesting that
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DiMan before and after binding to wildtype DC-SIGN/R (unla-
DC-SIGNR may prefer binding to GNPs with even higher gly-
beled) by dynamic light scattering (DLS).62 GNP-DiMan dis-
played a single Dh species of ~11 nm with a narrow distribution
(full width at half maximum, FWHM = 3.5 nm, Figure 3A1) in
the binding buffer. After mixing with DC-SIGN (protein: GNP
molar ratio = 20:1), the size of the dominate species was signif-
icantly increased (Dh = ~41 nm) and broadened (FWHM ~28
nm, Figure 3A2). This result was consistent with that expected
for each GNP-glycan being bound by a limited number of DC-
SIGN molecules, forming a protein shell on the GNP. In con-
trast, mixing DC-SIGNR with GNP-DiMan produced a domi-
nant species whose Dh was gradually increased from ca. 300 to
>2000 nm over a period of 4 h (Figure 3A3). Such sizes were
clearly too big to be individual GNP-DC-SIGNR assemblies, a
strong indication of macroscopic assembly, arising presumably
from DC-SIGNR and GNP-DiMan inter-crosslinking.
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can densities. (3) Low nM Kd (e.g. 3.9 nM) for DC-SIGN bind-
ing with GNP-DiMan was obtained, which was ~250,000 fold
tighter than the corresponding monomeric binding between Di-
Man and DC-SIGN CRD (Kd = 0.9 mM),25 suggesting that a
polyvalent display of the glycans on the GNP greatly enhanced
its multivalent binding affinity with DC-SIGN. These results
were fully consistent with those reported for other multivalent
lectin-glycoconjuagte interactions.10, 14, 18, 22 (4) A higher degree
of affinity enhancement was observed for DC-SIGN binding to
GNP-DiMan over GNP-Man, possibility due to the former be-
ing able to exploit more efficiently the CRD’s secondary glycan
bind sites than the later.61 Overall, these results agreed well with
those reported previously with the QD-glycans.35
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Probing DC-SIGN/R-GNP-glycan binding mode and affin-
ity enhancing mechanism. First we monitored the Dhs of GNP-
Figure 3. Typical Dh distribution histograms of GNP-DiMan only (10 nM, A1); GNP-DiMan (10 nM) + wildtype DC-SIGN (200 nM, A2)
or GNP-DiMan (16 nM) + wildtype DC-SIGNR (640 nM, A3). TEM images of cryo-prepared samples of GNP-DiMan only (40 nM, B1),
GNP-DiMan (40 nM) + DC-SIGN (1.5 M, B2) or GNP-DiMan (40 nM) + DC-SIGNR (1.5 M, B3), and their corresponding GNP nearest
neighbor distance (NND) histograms (C1, C2, C3). The red curves show the Guanssian fits of the histograms.
This assumption was verified by transmission electron micros-
copy (TEM) imaging of cryo-prepared GNP-DiMan-protein
samples via rapid plunge freezing followed by vacuum drying
which we had shown previously to preserve the native disper-
sion states of nanoparticle solutions (SI, Figure S6).63 In the
absence of DC-SIGN/R, GNP-DiMan appeared as isolated sin-
gle particles (Figure 3B1) and gave a randomly distributed
nearest neighbor distance (NND) over a range of 10 to 100 nm
(Figure 3C1). Binding of DC-SIGN with GNP-DiMan also
gave isolated single particles (Figure 3B2), although the result-
ing NND distribution histogram could be fitted well (R2 =
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Figure 4. Dh-Cprotein relationships for GNP-glycan (10 nM) binding
0.982) by a single Gaussian distribution to yield an average
NND of ~44 nm (Figure 3C2). Interestingly, this NND value
matched well to the Dh size of GNP-DiMan-DC-SIGN assem-
bly (~41 nm), suggesting that the formation of a layer of DC-
SIGN on the GNP prevented individual GNPs from coming
close to each other. In contrast, binding of DC-SIGNR with
GNP-DiMan produced large scale, closely-packed GNP assem-
blies of hundreds of nm cross (Figure 3B2) with a rather small
NND of ~7.9 nm (Figure 3C3). This result agreed well with the
DLS measurement, confirming that binding of DC-SIGNR led
to macroscopic assemblies of GNPs via DC-SIGNR-GNP-
DiMan intercross linking. These results revealed that the bind-
ing modes of DC-SIGN/R to GNP-DiMan were clearly differ-
ent: DC-SIGN must have bound simultaneously to one GNP-
DiMan via all of its four binding sites and formed a layer of
proteins surrounding each GNP, giving rise to isolated single
particles. In contrast, DC-SIGNR and polyvalent GNP-DiMan
inter-crosslinked each other, and formed large scale but closely-
packed GNP-DC-SIGNR assemblies. Therefore, by harnessing
GNPs’ nanoscale size and high contrast under TEM imaging,
we have developed a new multimodal readout which success-
fully dissected the distinct modes of GNP-DiMan in binding to
DC-SIGN/R. This result agreed fully with those reported previ-
ously with QD-glycans.35
to wildtype DC-SIGN/R at different concentrations. (A) DC-SIGN
binding to GNP-DiMan (black squares, fitted by Hill’s equation: Y
= a + (b-a) × Xn/(kn + Xn); where a =11.1±0 nm; b = 80±99 nm; k
= 200 ± 990 nM and n =0.53±0.36, R2 = 0.997) or GNP-Man (red
dots, fitted by a linear relationship, Y = a + b × X; where a = 11.6
±0.7 nm; b = 0.091±0.010, R2 = 0.928); (B) DC-SIGNR binding to
GNP-(DiMan)3 (blue triangles) or GNP-(Man)3 (green triangles).
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To provide a more quantitative explanation of the data, here we
introduced a new term: the potential of assembly formation
(PAF):
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PAF = C0(GNP) × C0(protein)/(Kd)2
(3)
Where C0(GNP), C0(protein) and Kd are GNP-glycan and protein
starting concentrations and their apparent binding Kd, respec-
tively. As Kd indicates 50% binding at equilibrium, the PAF
thus represents the ratio of a reaction quotient to equilibrium of
a reversible binding reaction. Where PAF =1 would indicate
equilibrium (50%) binding, while PAF >1 indicates binding be-
ing favorable and PAF >10 being strongly favorable (>90%).
Similarly, PAF < 1 indicates binding being unfavorable and
PAF <0.1 being strong unfavorable (<10% binding).
Using this definition and the apparent Kds given in Table 1,
the PAFs for DC-SIGN binding to GNP-Man and GNP-DiMan
were in the range of 0.18-1.8, and 13-131, respectively. While
those for DC-SIGNR binding to GNP-(Man)3 and GNP-
(DiMan)3 ranged from 0.011 to 0.11, and 0.11 to 1.1. Combin-
ing the PAF value and binding mode would allow us to predict
the assembly outcome. For example, with a PAF of >10 and
simultaneous binding, most of the added DC-SIGN molecules
should readily bind to GNP-DiMan to saturate its surface bind-
ing capacity, giving rise to a rapidly increasing and quickly sat-
urated Dh with the increasing protein concentration. This was
exactly what was measured from DLS. In contrast, the PAF of
DC-SIGN-GNP-Man binding span across 1, which would indi-
cate a gradually increased binding, again matching well to a
positive, linear Dh-concentration relationship (Figure 4A). For
inter-crosslinking binding between DC-SIGNR and GNP-
glycans, large-scale assemblies were only observed for GNP-
(DiMan)3 mixed with the highest protein concentration (200
nM) which had a PAF of 1.1 and indicated the binding became
favorable. While all other conditions where PAFs were < 1, no
significant binding (assembly) was observed (Figure 4B). To-
gether, these results established PAF as a useful indicator for
predicting the binding (assembly) outcome of multivalent lec-
tin-glycan binding partners. Furthermore, the excellent agree-
ment between the predicted outcome based on PAFs (derived
from Kds measured via the GNP quenching method) and those
observed from DLS implied that this new fluorescence quench-
ing based binding affinity method was highly credible and reli-
able. Interestingly, the ability of lectins to bind and inter-cross-
link with virus surface glycoproteins had shown to play an im-
portant role in virus neutralization.64-66 Thus, we anticipate that
the PAF criteria proposed here could act as a useful tool to pre-
dict lectin-virus interactions and neutralization.
The distinct binding modes exhibited by DC-SIGN/R could also
help explain their different affinities with GNP-glycans. For
DC-SIGN, its simultaneous binding with one GNP via all 4 of
its CRDs should yield strong affinity enhancement from both
the favorable enthalpy and entropy terms. For DC-SIGNR,
whilst its inter-crosslinking mode of binding with GNP-glycans
could maximize the binding enthalpy, the resulting large scale
assemblies would incur significant entropy penalty. As a result,
DC-SIGN should bind more strongly to GNP-glycans than DC-
SIGNR did, matching well to the apparent Kd data measured by
fluorescence quenching (Table 1).
Correlation between lectin-GNP-glycan affinity and assem-
bly. The credibility of this GNP quenching based affinity meas-
urement method was further verified by the DLS data. For ex-
ample, binding of an increasing amount of DC-SIGN to a fixed
amount of GNP-DiMan (10 nM) gave a more rapid increase of
Dh than that to GNP-Man (Figure 4A), consistent with the for-
mer being a stronger binding partner than the latter (apparent
Kd: 3.9 v.s. 33 nM). Moreover, mixing an increasing amount of
DC-SIGNR with GNP-(Man)3 yielded no noticeable Dh
changes throughout the concentration range studied (20-200
nM), indicating no measurable binding (Figure 4B). While
mixing DC-SIGNR with GNP-(DiMan)3 also produced no ap-
parent Dh changes initially (≤100 nM); as DC-SIGNR concen-
tration was increased further, a dramatic increase of Dh was ob-
served, particularly at 200 nM, a clear indication of the for-
mation of large scale GNP-DC-SIGNR assemblies (see Figure
4B, and SI, Figures. S7-S10).
Blocking DC-SIGN/R-mediated augmentation of Ebola vi-
rus glycoprotein-driven transduction. The distinct binding
modes and affinities of GNP-glycans with two important viral
receptors, DC-SIGN/R, should result in different abilities in
blocking DC-SIGN/R-mediated virus infection of host cells. To
investigate this potential and any possible correlation, human
embryonic kidney 293T cells transfected to express DC-SIGN
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NA = IC50/[IC50 + C]
or DC-SIGNR on their membrane were used as described pre-
(4)
viously.35 Murine leukemia virus (MLV) vector particles bear-
ing the Ebola virus surface glycoprotein (EBOV-GP) and en-
coding the luciferase gene were used to model Ebola virus entry
into cells. The virus particles can bind efficiently to DC-
SIGN/R (via their surface EBOV-GP) and binding results in in-
creased transduction as determined by luciferase expression in
host cells.34-35 Binding of high affinity GNP-glycans to host cell
surface DC-SIGN/R could block these lectin receptors from fur-
ther binding to EBOV-GP, thereby reducing luciferase gene
transduction. Indeed, pretreatment of 293T cells with GNP-
glycans efficiently inhibited DC-SIGN-mediated augmentation
of transduction in a dose-dependent manner (SI, Figure S11).
GNP-DiMan/-(DiMan)3 were found to be more potent in block-
ing the transduction than GNP-Man/-(Man)3. This result was
consistent with the former being stronger DC-SIGN binders
than the latter. The normalized dose-dependent inhibition data
were fitted well by a simple competition model:
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8
Where NA, IC50 and C were normalized luciferase activity, GNP
concentration giving 50% inhibition, and GNP concentration,
respectively. Whilst DC-SIGNR-mediated augmentation of
gene transduction was also inhibited by GNP-glycans, the re-
sulting normalized dose-dependent inhibition data were best fit-
ted by a modified competition model:
NA = EC50n/[EC50n + C n] (5)
Where the inhibition coefficient, n, was found to be in the range
of 0.4 to 0.6 (Table 2), indicating a poor inhibition efficiency.
Moreover, GNP-glycans could not completely inhibit DC-
SIGNR-mediated transduction, even at high concentrations (see
Figure 5A/B, and SI, Figure S12). This result revealed that
GNP-glycans were much less effective in blocking DC-SIGNR-
mediated augmentation of gene transduction than that of DC-
SIGN-dependent transduction.
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Figure 5. Plot of normalized luciferase activities of DC-SIGN- or DC-SIGNR- expressing 293T cells as a function of the concentration of
GNP-DiMan (A) or GNP-(DiMan)3 (B) inhibitors. Data for virus particles bearing the EBOV-GP are shown in dots while those of control
virus particles bearing the VSV-G are shown in triangles. Schematics beneath showing the different inhibition efficiencies of GNP-glycans
against DC-SIGN (C) or DC-SIGNR (D) mediated infections. (C) For DC-SIGN expressing host cells, all of the binding sites of its surface
DC-SIGNs are bound and blocked by GNP-glycans, making them unavailable for further binding to virus surface EBOV-GPs to initiate
infection. (D) For DC-SIGNR expressing host cells, GNP-glycans cross-link with some surface DC-SIGNRs but the bindings are weak and
dynamic at the edges, allowing the virus to access the unblocked DC-SIGNR binding sites to initiate infection.
Table 2. Summary of inhibition data for GNP-glycans against DC-
SIGN/R-mediated EBOV-GP driven infection of 293T host cells.
* shows the EC50 values for DC-SIGNR.
GNP-(DiMan)3 DC-SIGNR
2.6±0.1*
0.53±0.02 0.997
We believe this result is reasonable from both the binding affin-
ity (GNP-glycans bind more weakly to DC-SIGNR than to DC-
SIGN) and binding mode points of view. Each GNP-glycan
could bind simultaneously to all 4 of DC-SIGN’s binding sites,
completely blocking them from further binding to virus surface
EBOV-GPs to initiate cell entry (Figure 5C). In contrast, the
inter-crosslinking mode of binding between GNP-glycans and
DC-SIGNR meant that each GNP could only bind to 2 of the 4
binding sites in DC-SIGNR. It would be very difficult for GNP-
glycans to cross-link all cell surface DC-SIGNRs to fully block
their binding sites. Even if this was possible, the DC-SIGNRs
GNP-glycan
Receptor
IC/EC50
n
R2
(nM)
GNP-Man
DC-SIGN
DC-SIGN
DC-SIGN
0.26±0.08
1.57±0.25
0.095±0.017
0.15±0.03
7.3±1.2*
53±17*
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1
1
1
0.889
0.937
0.921
0.890
GNP-(Man)3
GNP-DiMan
GNP-(DiMan)3 DC-SIGN
GNP-Man
DC-SIGNR
DC-SIGNR
DC-SIGNR
0.42±0.04 0.980
0.61± 0.14 0.902
GNP-(Man)3
GNP-DiMan
0.63±0.09* 0.56±0.02 0.990
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at the cluster edge would still only bind divalently (via 2 out- EBOV-GP-dependent entry was measured as 0.095±0.017 nM.
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ward facing binding sites) to GNP-glycans, where the binding
would be weak and dynamic. These, together with any unbound
binding sites, could act as toe-holds for binding to viral surface
EBOV-GPs to initiate cell uptake and infection (Figure 5D).
Therefore, an inhibitor that inter-crosslinks with target recep-
tors would be much less effective in blocking virus infection
than its simultaneous binding counterpart, making it almost im-
possible to achieve complete inhibition (Figure 5C/D). This ef-
fect was clearly demonstrated from a side-by-side comparison
of virus inhibition data for two GNP-glycan-lectin pairs show-
ing similar affinity but with distinct binding modes, e.g. GNP-
Man-DC-SIGN (Kd: 33±2 nM) v.s. GNP-(DiMan)3-DC-SIGNR
(Kd: 42±2 nM). The simultaneous binding pair (GNP-Man-DC-
SIGN) clearly displayed higher inhibiting potencies over its
cross-linking counterpart (GNP-(DiMan)3-DC-SIGNR) across
the whole range of concentrations studied (SI, Figure S13A),
even on a Kd normalized concentration (C/Kd) term to eliminate
the effect of small Kd differences (SI, Figure S13B). The dif-
ference was even clearer by comparing the relative infection ra-
tio between the cross-linking and simultaneous binding pairs
where a stable ratio of ~1.6 was observed at low GNP concen-
trations (≤ 3 nM, or ≤ 1/10 of Kds). As concentration was in-
creased, comparable to the Kds, the ratio was increased mark-
edly and reached ~27 at 50 nM (SI, Figure S13C). The obser-
vation that the potency difference for GNP inhibitors of differ-
ent binding modes was more pronounced at high concentrations
where most cell surface lectins would be blocked by GNPs,
matched well to what was expected from our proposed mecha-
nism above.
This value was considerably lower (by almost an order of mag-
nitude) than those measured for some of the most potent gly-
coconjugate inhibitors of Ebola virus infection reported previ-
ously (e.g. the giant globular multivalent glycofullerenes, IC50:
0.67 nM21, the virus-like glycodendrinanoparticles, IC50: 0.91
nM,16 and our previous QD-EG3-DiMan, IC50: 0.70±0.2 nM35).
This result demonstrated an outstanding potency of the GNP-
glycans in blocking DC-SIGN-mediated EBOV-GP driven in-
fection of host cells. Importantly, as virus surface glycans are
maintained by host cell’s glycosylation apparatus, they are
mostly conserved and unaffected by virus mutation.67 There-
fore, the potential advantages of developing potent glycoconju-
gate viral inhibitors over other anti-viral strategies include two
folds: (1) it can reduce the chances of virus mutation by block-
ing its entry to host cells, and (2) its treatment potency is un-
likely affected by virus mutation, allowing us to provide a po-
tentially long lasting solution.
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Conclusion
In summary, we have developed polyvalent GNP-glycans as
new powerful structural and mechanistic probes for multivalent
lectin-glycan interactions. By exploiting GNPs’ outstanding
fluorescence quenching property, we have developed a new
method for quantifying multivalent lectin-glycan interaction,
revealing that a polyvalent display of mannose containing gly-
cans on GNPs greatly enhances their binding affinities with two
vitally important viral receptors, DC-SIGN/R. Moreover, by ex-
ploiting GNPs’ nanoscale size and high TEM contrast, we have
revealed the distinct binding mode and affinity enhancing
mechanisms for GNP-glycans binding with DC-SIGN/R. Im-
portantly, the assembly outcomes of the dose-dependent GNP-
lectin binding are in perfect agreement with those predicted
from their respective binding modes and affinities, verifying the
credibility of our new affinity method. Furthermore, we have
found that GNP-glycans can potently block DC-SIGN-
mediated EBOV-GP driven viral infections of host cells with
IC50 values down to 95±17 pM, making it the most potent gly-
coconjugate inhibitor against EBOV-GP driven infection. No-
tably, its inhibition potency will unlikely be affected by virus
mutation, allowing us to provide potentially a long lasting solu-
tion. Finally, we have revealed that only a GNP-glycan inhibitor
showing a simultaneous-, but not a crosslinking-, binding mode
is able to completely block the target lectin mediated virus in-
fection. Together, these data provide a useful guidance in de-
signing polyvalent glycoconjugates for potent, specific inhibi-
tion of virus infection. Given their low-/non- toxicity and excel-
lent biocompatibility, the GNP-glycans are perfectly placed for
a wide range of applications, from probing fundamental struc-
tural mechanisms of glycobiology, developing novel biophysi-
cal and biomedical assays, to developing novel therapeutics
against deadly virus infections and immune dysregulation dis-
eases such as cancer, allergy and autoimmune diseases.
Besides, comparing the GNP-lectin binding affinity (Table 1)
and inhibition potency (Table 2) data revealed that the Kd and
IC50/EC50 values did not always follow the expected positive
correlation (e.g. stronger binders being better inhibitors), even
for those showing the same binding mode. The Kd/IC50 relation-
ship appeared to be affected by GNP surface glycan density. A
positive Kd/IC50 correlation was only observed for GNPs having
similar glycan densities (e.g. GNP-Man v.s. GNP-DiMan, or
GNP-(Man)3 v.s. GNP-(DiMan)3), but not for those having
large glycan density differences (e.g. GNP-Man v.s. GNP-
(Man)3 or GNP-DiMan v.s. GNP-(DiMan)3). The latter gave an
unexpected negative Kd/IC50 correlation (e.g. stronger binders
being worse inhibitors), possibly caused by the different bind-
ing environments used in GNP-lectin binding and virus inhibi-
tion studies. The former was performed in solution, where both
the GNPs and lectins were free to move in all three dimensions,
allowing them to easily adjust their relative positions to maxim-
ize binding. Under such conditions, it appeared that a high gly-
can density on GNP surface was beneficial for lectin binding.
Whereas in virus inhibition studies, all valid bindings (capable
of blocking virus binding) could only take place on two-dimen-
sions with cell membrane bonded lectins. The binding partners,
particularly cell surface lectins, would exhibit greatly reduced
freedom of movement which might have led to a different gly-
can density for optimal binding (and hence virus inhibition).
Experimental Section
Materials. Gold nanoparticle (nominal diameter 5 nm, max ~520
nm) was purchased from Sigma Aldrich as a stabilized suspension
in citrate buffer or synthesized in house. 2-(2-aminoethoxy)-etha-
nol, Di-tert-butyl decarbonate, sodium sulfate, sodium hydride
(60% dispersion in mineral oil), 3-bromo-1-propyne, potassium hy-
droxide, trifluoroacetic acid, triethylamine, sodium bicarbonate, O-
(6-Chlorobenzotriazol-1-yl)-N, N, N’, N’-tetramethyluronium hexa
fluorophosphate (HCTU), lipoic acid (LA), tris(2-carboxyethyl)
phosphine hydrochloride (TCEP.HCl), copper sulfate, sodium
Importantly, luciferase activities of control virus particles bear-
ing the vesicular stomatitis virus glycoprotein (VSV-G), which
cannot use DC-SIGN/R for augmentation of cell entry, were not
affected significantly by GNP-glycan treatment, confirming
that the observed inhibitory effects were specific (SI, Figure
S10). It was noteworthy to point out that the IC50 value of GNP-
DiMan in blocking DC-SIGN-mediated augmentation of
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ascorbate, Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] amine
33.7, 27.5, 24.9. HRMS: calculated m/z for C57H99N10O28S2
(TBTA), tris(hydroxymethyl)aminomethane (Tris base), guanidine
hydrochloride, anhydrous DMF and other chemicals were pur-
chased from Sigma Aldrich, Alfa Aesar, Fluorochem, Thermo Sci-
entific, VWR International or Acros organics with > 99% impurity
and used as received without further purification unless specified
elsewhere. All the solvents were obtained in >99% purity from
Fischer Scientific and used as received. When used as reaction sol-
vents in anhydrous reactions, THF and CH2Cl2 were dried and de-
oxygenated using an Innovative Technology Inc. PureSolv® sol-
vent purification system. Ultra-pure water (resistance >18.2
MΩ.cm) purified by an ELGA Purelab classic UVF system, was
used for all experiments and making buffers.59, 62
(M+H)+ 1435.6066; found 1435.6096.
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LA-(DiMan)3: Yield = (69%, 0.030 mmol). H NMR (500 MHz,
D2O) δ (ppm): 8.03 (s, 3H, triazole-H), 5.11 (d, 3H, J 1.8 Hz, H-1),
5.03 (d, 3H, J = 1.9 Hz, H-1’), 4.64 – 4.62 (m, 6H), 4.60 (d, 6H, J
2.9 Hz), 4.08 (dd, 3H, J 3.4, 1.8 Hz, H-2), 3.97 (td, 9H, J = 4.5, 2.5
Hz), 3.92 – 3.68 (m, 40H), 3.66 – 3.61 (m, 29H), 3.58 – 3.53 (m,
3H), 3.21 – 3.13 (m, 2H), 2.42 (dq, 1H, J 12.3, 6.1 Hz), 2.18 (t, 2H,
J 7.0 Hz), 1.91 (dd, 1H, J 13.1, 6.8 Hz), 1.67 (m, 1H), 1.59 – 1.49
(m, 4H), 1.32 (p, 3H, J 7.7 Hz). ). 13C NMR (125 MHz, D2O) δ:
179.0 (C=O), 146.5, 127.9, 104.8 (C-1), 100.9 (C-2), 81.2, 75.8,
75.3, 74.2, 72.8, 72.7, 72.5, 72.2, 72.0 (2), 71.9, 71.3 (2), 70.1, 69.4
(2), 69.0, 66.1, 63.7, 63.4, 62.9, 62.3, 59.0, 52.6, 42.8, 40.6, 38.4,
36.2, 30.1, 27.4. HRMS: calculated m/z for C75H130N10O43S2
(M+H)2+ 961.3862; found 961.3858.
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Glycan-ligand synthesis by click chemistry. The general proto-
cols employed in preparing the LA-glycan ligands via click chem-
istry were as follow.16 To a 1:1 (v:v) mixed THF:H2O solution (2.0-
5.0 mL) containing the glycan-EG2-N3 (1.1 mole equiv.) and LA-
linker (1 mole equiv.) were added CuSO4.5H2O (0.036 mole
equiv.), TBTA (0.063 mole equiv.) followed by sodium ascorbate
(0.135 mole equiv.) and the resulting solution was stirred at RT.
After 3 h, TLC confirmed the consumption of all starting materials.
The solvent was then evaporated and the crude product was puri-
fied by size exclusion chromatography using Biogel P2 column us-
ing water as eluent to afford the desired pure LA-glycan product.
LA-Man: Yield = (67%, 0.18 mmol). 1H NMR (500 MHz, CD3OD)
δ (ppm): 8.04 (s, 1H, triazole-H), 4.8 (d, 1H, J 1.6 Hz, Man H-1),
4.65 (s, 2H), 4.6 (t, 2H, J 5 Hz), 3.9 (t, 2H, J 5.1 Hz), 3.86-3.81 (m,
3H), 3.8 (dd, 1H, J 1.7 Hz, 3.4 Hz, Man H-2), 3.72 (d, 1H, J 5.5
Hz), 3.69-3.67 (m, 3H), 3.66 (s, 2H), 3.65-3.63 (m, 2H), 3.62-3.61
(m, 4H), 3.60-3.57 (m, 2H), 3.53 (t, 2H, J 5.5 Hz), 3.39-3.34 (m,
3H), 3.20-3.15 (m, 1H), 3.12-3.07 (m, 1H), 2.49-2.42 (m, 1H), 2.20
(t, 2H, J 7.4 Hz), 1.92-1.85 (m, 1H), 1.73-1.59 (m, 4H), 1.50-1.40
(m, 2H). 13C NMR (125 MHz, CD3OD) δ: 176.1 (C=O), 145.7
(C=CH), 126.0 (C=CH), 101.7 (Man C-1), 74.6, 72.6, 72.1 (Man
C-2), 71.6, 71.5, 71.4, 71.3, 70.8, 70.6, 70.4, 68.6, 67.7, 65.0, 63.0
(Man C-6), 57.6, 51.5, 41.3, 40.3, 39.3, 36.8, 35.7, 29.8, 26.7.
HRMS: calculated m/z for C27H49N4O11S2 (M+H)+ 669.2834;
found 669.2838.
LA-DiMan: Yield = (65%, 0.080 mmol). 1H NMR (500 MHz,
CD3OD) δ (ppm): 8.05 (s, 1H, triazole-H), 5.11 (d, J 1.7 Hz, 1H),
4.96 (d, J 1.8 Hz, 1H), 4.66 (s, 2H), 4.60 (t, 2H, J 5.1 Hz), 3.97 (dd,
1H, J 3.3, 1.8 Hz), 3.91 (dd, 2H, J 5.5, 4.7 Hz), 3.88 – 3.78 (m, 6H),
3.72 – 3.65 (m, 8H), 3.62 (dt, 6H, J 5.0, 1.3 Hz), 3.58 (dt, 3H, J
11.2, 1.6 Hz), 3.53 (t, 2H, J 5.5 Hz), 3.43 – 3.32 (m, 3H), 3.17 (ddd,
1H, J 10.9, 7.1, 5.4 Hz), 3.10 (dt, 1H, J 11.0, 6.9 Hz, 1H), 2.46 (dtd,
1H, J 12.1, 6.7, 5.4 Hz), 2.20 (t, 2H, J 7.4 Hz), 1.89 (dq, 1H, J 12.7,
6.9 Hz), 1.77 – 1.57 (m, 4H), 1.45 (qt, 2H, J 9.4, 5.9 Hz). 13C NMR
(125 MHz, CD3OD) δ: 176.1 (C=O), 145.7 (C=CH), 126.0
(C=CH), 104.2 (Man C-1), 100.1 (Man C-1’), 80.6, 75.1, 74.7,
72.4, 72.1, 71.9, 71.6, 71.5, 71.4, 71.3, 70.8, 70.6, 70.5, 69.0, 68.8,
67.9, 65.0, 63.2, 63.1, 57.6, 51.5, 41.3, 40.4, 39.3, 36.8, 35.7, 29.8,
26.7. HRMS: calculated m/z for C33H58N4O16S2 (M+H)+ 831.3233;
found 831.3242.
LA-(Man)3: Yield = (60%, 0.060 mmol). 1H NMR (500 MHz, D2O)
δ (ppm): 8.03 (s, 3H, triazole-H), 4.87 (d, 3H, J 1.8 Hz, H-1), 4.64
– 4.61 (m, 6H), 4.60 (s, 6H), 3.98 – 3.96 (m, 6H), 3.95 (dd, 3H, J
3.5, 1.8 Hz, H-2), 3.89 (d, 1H, J 2.0 Hz), 3.87 (d, 2H, J 1.7 Hz),
3.84 – 3.79 (m, 6H), 3.77 (d, 2H, J 5.6 Hz), 3.74 (d, 7H, J 3.1 Hz),
3.67 (d, 3H, J 9.8 Hz), 3.65 – 3.61 (m, 25H), 3.61 – 3.53 (m, 3H),
3.21 – 3.12 (m, 2H), 2.42 (dq, 1H, J 12.3, 6.1 Hz), 2.18 (t, 2H, J
7.0 Hz), 1.91 (dt, 1H, J 13.6, 6.8 Hz), 1.71 – 1.64 (m, 1H), 1.58 –
1.48 (m, 4H), 1.33 (q, 3H, J 7.7 Hz). 13C NMR (125 MHz, D2O) δ:
176.5 (C=O), 144.0, 125.3, 99.9 (C-1), 72.7, 70.5, 69.9, 69.5, 69.4,
68.8, 67.5, 66.7, 66.3, 63.5, 60.9, 59.7, 56.5, 50.0, 40.2, 38.0, 35.9,
GNP-glycan preparation. 10 mL of 5 nm GNP stock was concen-
trated to 250 L using a 30 KDa MWCO spin column and washed
with H2O (3 × 200 µL) to remove any impurities. Then DHLA-
glycan ligands dissolved in H2O were added to the GNP solution in
a molar ratio of GNP:DHLA-glycan = 1:1000. The resulting solu-
tion was mixed and stirred at RT in dark for overnight. The result-
ing mixture was passed through a 30 KDa MWCO spin column by
centrifugation at 15,000 × g for 2 min and the residues were washed
with H2O (3 × 200 µL) to give the GNP-glycan stock. The filtrate
and washing through liquids were combined and used to evaluate
the glycan loading on GNPs as described previously.34 The concen-
tration of the GNP-glycans was determined from its absorbance at
520 nm using the Beer-Lambert law and a GNP molar extinction
co-efficient of 1.1 × 107 M−1⋅cm−1.
Protein production and labeling. The soluble extracellular seg-
ments of DCSIGN (DC020) and DCSIGNR (DSR034) were ex-
pressed as inclusion bodies in E. coli and purified by Man-Se-
pharose affinity column followed by Superdex size exclusion col-
umn as reported previously.34 The mutant proteins, DC020 Q-274C
and DSR034 R278C were constructed by site directed mutagenesis
and labelled with atto594-maleimide as described previously.35, 40
The labeled proteins were purified by mannose-Sepharose affinity
column. All the proteins obtained were characterized by high reso-
lution mass spectroscopy (HRMS) and their corresponding spectra
were shown in SI, Figure S1. The dye labeling efficiency (per pro-
tein monomer) for DC-SIGN and DC-SIGNR was determined to
be 87 % and 82 %, respectively, based on the relative intensity of
the labeled and unlabeled protein peaks measured from MS.
Fluorescence spectra. All fluorescence spectra were recorded on
a Cary Eclipse Fluorescence Spectrophotometer using a 0.70 mL
quartz cuvette under a fixed λEX of 595 nm over a range of 605-750
nm. All measurements were performed in a binding buffer (20 mM
HEPES, 100 mM NaCl, 10 mM CaCl2, pH 7.8) containing 1 mg/mL
of BSA to minimize any nonspecific absorption of the GNPs and
proteins on surfaces. For apparent Kd measurement, the concentra-
tions of labeled DC-SIGN or DC-SIGNR protein and GNP-glycans
were varied simultaneously in a fixed protein:GNP molar ratio of
1:1. The samples were incubated at RT for 20 min before recording
the fluorescence spectra. The fluorescence spectra of the protein in
the absence of the GNP-glycans, recorded under identical experi-
ment conditions, were used to determine the quenching efficiency.
The instrument PMT voltages were adjusted to compensate the low
fluorescence signals at low concentrations. The quenching effi-
ciency (QE) of DC-SIGN/R binding to each GNP-glycan was cal-
culated via Eq. 1 and the resulting QE-concentration relationship
was fitted by Hill’s equation (Eq. 2) to derive the apparent binding
Kd values.
Dynamic light scattering. The hydrodynamic diameters (Dh) of
wildtype DC-SIGN/R and GNP-glycans in pure water and in a
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Journal of the American Chemical Society
Page 10 of 13
binding buffer (20 mM HEPES, 100 mM NaCl, 10 mM CaCl2, pH
tion, LA-glycan ligands syntheses and their 1H and 13C NMR spec-
7.8) were recorded on a Malvern ZETASizer-Nano using disposa-
ble polystyrene cuvettes as reported in our previous papers.35, 68 For
monitoring GNP-DiMan binding to wildtype DC-SIGN/R, a series
of samples, each containing GNP-DiMan (10 nM) and a varying
amount of wildtype DC-SIGN/R, were mixed in a binding buffer at
RT for 20 mins before DLS measurement was carried out. 10 con-
secutive scans were performed for each sample and the resulting
volume Dh distribution histograms for each sample were combined
and fitted by Gaussian distributions to estimate their Dhs.35
tra, and supporting figures showing fluorescence spectra of GNP-
DiMan binding to DC-SIGN, mannose competition studies, Dh dis-
tribution histograms of the GNP-glycans, DC-SIGN/R, and GNP-
glycan binding to DC-SIGN/R at different concentrations, TEM
images of cryo-prepared GNP-DiMan + DC-SIGN/R samples, and
the original data of GNP-glycans inhibiting DC-SIGN/R-mediated
EBOV-GP driven infections. This material is available free of
charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author(s)
9
TEM imaging. Three samples: (1) GNP-DiMan, (2) GNP-DiMan
+ wildtype DC-SIGN, and (3) GNP-DiMan + wildtype DC-SIGNR
were incubated (final CGNP = 16 nM and Cprotein=640 nM) in a bind-
ing buffer overnight. The samples were prepared by plunge-freez-
ing into liquid ethane followed by warming under vacuum to cap-
ture the GNP dispersions in their native dispersed state as demon-
strated in our previous paper.63 Briefly, 3.5 L of suspension was
placed onto a plasma-cleaned TEM grid with a continuous carbon
support film, blotted, and plunge frozen into liquid ethane. The
TEM grids were then warmed to RT over several minutes by plac-
ing the specimens in the liquid nitrogen cooled storage container in
a rotary pumped vacuum desiccator.
The samples were then analyzed using an FEI Titan3 Themis 300
G2 S/TEM equipped with FEI SuperX energy dispersive X-ray
(EDX) spectrometers and a Gatan OneView CCD. Images were
collected for each sample, with EDX spectroscopy used to confirm
that the small nanoparticles imaged were indeed GNPs as Au spe-
cies were detected together with other peaks (carbon, oxygen, sili-
con, and copper) due to the microscope, TEM grid or support film.
A series of images at the same magnification were recorded for
each sample, allowing easy comparison of the nanoparticle disper-
sion state of the three samples. The TEM images were analyzed
automatically by MATLAB scripts to derive the nearest neighbor
distances (NNDs) as reported previously.35
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* Stefan Pöhlmann- Infection Biology Unit, German Primate Cen-
ter, Kellnerweg 4, 37077 Gottingen, Germany; orcid.org/0000-
0001-6086-9136; Email: spoehlmann@dpz.eu.
* Yuan Guo- School of Food Science&Nutrition and Astbury Cen-
ter for Structural Molecular Biology, University of Leeds, UK; or-
cid.org/0000-0003-4607-7356; Email: y.guo@leeds.ac.uk .
* Dejian Zhou- School of Chemistry and Astbury Center for Struc-
tural Molecular Biology, University of Leeds, UK; orcid.org/ 0000-
0003-3314-9242; Email: d.zhou@leeds.ac.uk.
Author Contributions
╬
D.B., and E.P. contributed equally to this work.
Funding Sources
UK Biotechnology and Biological Sciences Research Council
(grant no: BB/R007829/1). EU Horizon 2020 Marie Sklodowska-
Curie Fellowship (grant no: 797597). UK Engineering and Physical
Sciences Research Council DTP PhD scholarship (grant no:
EP/M50807X/1).
Notes
Virus Inhibition. The effects of GNP-glycans (glycan = Man, Di-
Man, (Man)3 or (DiMan)3) on Ebola virus glyco-protein (EBOV-
GP) driven entry into 293T cells were assessed by using our estab-
lished procedures.34-35 Briefly, 293T cells seeded in 96-well plates
were transfected with plasmids encoding DC-SIGN or DC-SIGNR
or control transfected with empty plasmid (pcDNA). The cells were
washed at 16 h post transfection and further cultivated at 37°C, 5%
CO2 in Dulbecco's modified eagle medium (DMEM) containing
10% fetal bovine serum (FBS). At 48 h post transfection, the cells
were exposed to twice the final concentration of GNP-glycan in-
hibitor in DMEM supplemented with 10% FBS for 30 min in a total
volume of 50 µL. Thereafter, the resulting cells were inoculated
with 50 µL of preparations of MLV vector particles encoding the
luciferase gene and bearing either EBOV-GP (which can use DC-
SIGN/R for augmentation of host cell entry) or the vesicular sto-
matitis virus glycoprotein (VSV-G, which cannot use DC-SIGN/R
for augmentation of host cell entry). Under these conditions, bind-
ing of GNP-glycan nanoparticles to 293T cell surface DC-SIGN/R
receptors can block EBOV-GP interactions with these lectin recep-
tors, resulting in reduced transduction efficiency of the virus parti-
cles and hence reducing the cellular luciferase activity. At 6 h post
inoculation, 100 µL of fresh DMEM culture medium was added
and the cells incubated for another 72 h. Thereafter, luciferase ac-
tivities in cell lysates were determined using a commercially avail-
able kit (PJK), following the manufacturer’s instructions, as de-
scribed in our previous publication.
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We thank the UK Biotechnology and Biological Sciences Research
Council (grant no: BB/R007829/1) and the EU Horizon 2020 via a
Marie Sklodowska-Curie Fellowship (grant no: 797597) for fund-
ing this research. E.P. thanks the University of Leeds and the UK
Engineering and Physical Sciences Research Council for providing
a DTP PhD scholarship (grant no: EP/M50807X/1).
ABBREVIATIONS
GNP, gold nanoparticle; DC-SIGN, Dendritic Cell-Specific Inter-
cellular adhesion molecule-3-Grabbing Non-integrin; DC-SIGNR,
DC-SIGN related lectin found on endothelial cells; TLC, thin layer
chromatography; HPLC: high performance liquid chromatography;
NMR, nuclear magnetic resonance; MS, mass spectrometry.
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