Mono- and Di-Fucosylated Glycans of the Parasitic Worm S. mansoni are Recognized Differently by the Innate Immune Receptor DC-SIGN

Article abstract of DOI:10.1002/chem.202002619

The parasitic worm, Schistosoma mansoni, expresses unusual fucosylated glycans in a stage-dependent manner that can be recognized by the human innate immune receptor DC-SIGN, thereby shaping host immune responses. We have developed a synthetic approach fo

Full text of DOI:10.1002/chem.202002619

Accepted Article
Accepted Article
Title: Mono- and Di-Fucosylated Glycans of the Parasitic Worm S.
mansoni are Recognized Differently by the Innate Immune
Receptor DC-SIGN
Authors: Apoorva D Srivastava, Luca Unione, Margreet A Wolfert,
Pablo Valverde, Ana Arda, Jesús Jiménez-Barbero, and
Geert-Jan Boons
This manuscript has been accepted after peer review and appears as an
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To be cited as: Chem. Eur. J. 10.1002/chem.202002619
Link to VoR: https://doi.org/10.1002/chem.202002619
01/2020

10.1002/chem.202002619
Chemistry - A European Journal
FULL PAPER
Mono- and Di-Fucosylated Glycans of the Parasitic Worm S.
mansoni are Recognized Differently by the Innate Immune
Receptor DC-SIGN
Apoorva D. Srivastava,+[a] Luca Unione,+[a] Margreet A. Wolfert,[a, b] Pablo Valverde,[c] Ana Ardá,[c] Jesús
Jiménez-Barbero,[c, d, e] and Geert-Jan Boons*[a, b, f]
[a]
A. D. Srivastava, Dr. L. Unione, Dr. M. A. Wolfert, Prof. Dr. G. -J. Boons
Department of Chemical Biology and Drug Discovery
Utrecht Institute for Pharmaceutical Sciences, and Bijvoet Center for Biomolecular Research, Utrecht University
Universiteitsweg 99, 3584 CG Utrecht (Netherlands)
E-mail: g.j.p.h.boons@uu.nl
[b]
[c]
Dr. M. A. Wolfert, Prof. Dr. G. -J. Boons
Complex Carbohydrate Research Center, University of Georgia
315 Riverbend Road, Athens, GA 30602 (USA)
Dr. P. Valverde, Dr. A. Ardá, Prof. Dr. J. Jiménez-Barbero
Molecular Recognition and Host-Pathogen Interactions
CIC bioGUNE
Bizkaia Technology Park, Building 800, 48162 Derio, Bizkaia (Spain)
Dr. J. Jiménez-Barbero
Ikerbasque, Basque Foundation for Science
48013 Bilbao, Bizkaia (Spain)
[d]
[e]
Dr. J. Jiménez-Barbero
Department of Organic Chemistry II
University of the Basque Country
UPV/EHU, 48940 Leioa, Bizkaia (Spain)
Prof. Dr. G. -J. Boons
[f]
Department of Chemistry
University of Georgia
Athens, GA 30602 (USA)
[+]
A. D. S. and L. U. contributed equally
Supporting information for this article is available.
Abstract: The parasitic worm, Schistosoma mansoni, expresses
unusual fucosylated glycans in a stage-dependent manner that can
be recognized by the human innate immune receptor DC-SIGN,
thereby shaping host immune responses. We have developed a
synthetic approach for mono- and bis-fucosylated LacdiNAc (LDN-F
and LDN-DF, respectively), which are epitopes expressed on
glycolipids and glycoproteins of S. mansoni. It is based on the use of
monosaccharide building blocks having carefully selected amino-
protecting groups, facilitating high yielding and stereoselective
glycosylations. The molecular interaction between the synthetic
glycans and DC-SIGN was studied by NMR and molecular modeling,
which demonstrated that the α1,3-fucoside of LDN-F can coordinate
with the Ca2+-ion of the canonical binding site of DC-SIGN allowing for
additional interactions with the underlying LDN backbone. The 1,2-
fucoside of LDN-DF can be complexed in a similar manner, however,
in this binding mode GlcNAc and GalNAc of the LDN backbone are
placed away from the protein surface resulting in a substantially lower
binding affinity. Glycan microarray binding studies showed that the
avidity and selectivity of binding is greatly enhanced when the glycans
are presented multivalently, and in this format Lex and LDN-F gave
strong responsiveness whereas no binding was detected for LDN-DF.
The data indicates that S. mansoni has developed a strategy to avoid
detection by DC-SIGN in a stage-dependent manner by the addition
of a fucoside to a number of its ligands.
Introduction
Schistosomes are parasitic helminths that infect over 250 million
people worldwide and are responsible for 280,000 deaths
annually.[1] They can manipulate the host’s immune system to
establish chronic infections. Although the molecular basis of these
immune-modulatory mechanisms remain poorly understood, it
has been established that glycans of schistosomes can induce
specific innate immune responses in the infected host, which in
turn affects adaptive immunity. This occurs through an interplay
between Toll like receptors (TLRs) and C-Type lectin receptors
(CLRs) of dendritic cells (DCs) thereby tuning immune responses
toward an immune activation or tolerant state.[2] Schistosomes
can biosynthesize a vast array of glycoconjugates, many of which
are expressed at specific stages of their complex life cycle. The
glycoproteins and glycolipids of schistosomes lack sialic acid and
contain
a variety of terminal glycan epitopes, including
fucosylated antigens such as, Lewisx (Lex), pseudo-Lewisy
(pseudo
Ley),
GalNAcb1,4(Fuca1,3)GlcNAc
(LDN-F),
(F-LDN-F),
Fuca1,3GalNAcb1,4(Fuca1,3)GlcNAc
GalNAcb1,4(Fuca1,2Fuca1,3)GlcNAc
(LDN-DF)
and
(DF-
Fuca1,2Fuca1,3GalNAcb1,4(Fuca1,2Fuca1,3)GlcNAc
LDN-DF) (Table 1).[3] These glycan motifs are rarely found in
mammalian glycoconjugates but are typical signatures of
schistosomes.[4]
Dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-
SIGN), which is a C-type lectin expressed by immature dendritic
cells, has been implicated in schistosomiasis.[5] DC-SIGN can
recognize fucosylated glycan moieties presented by S. mansoni,
such as Lex,[3b,6] LDN-F,[3a,5a] and pseudo_Ley.[8] Further
1
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10.1002/chem.202002619
Chemistry - A European Journal
FULL PAPER
fucosylation of these motifs to give LDN-DF or DF-LDN-DF
appears to abolish detection by DC-SIGN.[3a]
of amino protecting groups to construct an LDN derivative that can
be glycosylated with a fucosyl donor having a temporary
protecting group at C-2 to allow installation of the subsequent 1,2-
linked fucoside. The amino protecting groups need to be selected
in such a way that they promote the introduction of b-glycosides,
but do not sterically block the introduction of the 1,3-fucoside.
Furthermore, the target compounds need to be modified by an
aminopentyl linker, which is required for glycan microarray
printing.
Table 1. Major glycan structures of Schistosoma mansoni egg proteins.
Glycan Structure
Abbreviation Symbol
"4
GalNAcβ1-4GlcNAc-
Galβ1-4(Fucα1-3)GlcNAc-
LDN
"4
Lex
!3
We have found that the monosaccharide building blocks 1-4
are appropriate for the assembly of aminopentyl modified LDN-F
(12) and LDN-DF (20) (Scheme 1). In this respect, the NHTroc
protecting group of 1 assures that a b-glycoside is formed when
glycosylated with acceptor 2, which has its C-2 amino group
masked as azide. The presence of the latter functional group is
important because after oxidative removal of the Nap ether, an
acceptor is formed (6) that sterically is sufficiently unencumbered
for glycosylation with fucosyl donor 3. The latter compound also
has a removable PMB ether allowing the installation of an 1,2-
fucoside using donor 4. After assembly of a tetrasaccharide, the
azide can be converted into Troc and a glycosylation with properly
protected aminopentanol will give linker modified compound LDN-
DF (19) as only the b-anomer.
"4
GalNAcβ1-4(Fucα1-3)GlcNAc-
LDN-F
!3
"4
GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc- LDN-DF
!3
!2
Key:
: N-Acetyl-D-glucosamine
: L-Fucose
: N-Acetyl-D-galactosamine
: D-Galactose
The carbohydrate binding site of DC-SIGN is composed of a
wide solvent exposed surface flanked by flexible loop,
conferring broad glycan binding selectivity.[9] As a matter of fact,
in addition to fucosylated glycans, high-mannose structures can
also be recognized by DC-SIGN. Most structural studies dealing
with DC-SIGN-glycan binding have focused on high mannose-
a
containing oligosaccharides.[10] Conversely,
a
structural
AcO
OAc
O
OBn
STol
OR
O
O
HO
NapO
OC(NPh)CF3
AcO
OTDS
understanding of binding of fucosyl containing glycans derived
from S. mansoni is limited to Lex.[6b] A structural model, based on
docking, indicates that the binding of pseudo-Ley occurs through
rearrangement of the protein to accommodate the additional
fucoside at galactoside, and supports substantial plasticity of DC-
SIGN.[8] Thus, it is surprising that bis-fucosylated glycans such as
LDN-DF are not recognized by DC-SIGN calling for further studies.
Here, we report the first chemical synthesis of LDN-DF and
OBn
BnO
N3
TrocHN
1
2
3, R = Bn
4, R = PMB
AcO
OAc
O
AcO
AcO
OBn
OAc
OBn
O
O
O
AcO
O
OTDS
iii
O
RO
O
i
OTDS
TrocHN
1 + 2
N3
TrocHN
N3
O
OR
OBn
5, R = Nap
6, R = H
7, R = Bn
13, R = PMB
BnO
OH
ii
AcO
LDN-F by
a
convergent block synthetic approach. The
OAc
HO
HO
OBn
OH
O
O(CH2)5NH2
NHAc
O
O
O
compounds were obtained in ample quantities making it possible
to examine their molecular interactions with DC-SIGN using STD-
NMR, chemical shift perturbation, trNOESY and molecular
modeling studies. It was found that DC-SIGN can interact with the
terminal fucoside of LDN-DF, however, no further interactions are
possible with the underlying glycan resulting in a low affinity
binding. On the other hand, the binding of LDN-F leads to
interactions of the fucoside, the GalNAc moiety and the acetyl
group of GlcNAc of the underlying LDN motif, leading to a
substantial higher affinity. The binding of DC-SIGN with LDN-DF,
LDN-F, Lex and Lex-Lex and SLex-Lex was also examined in a
glycan microarray format, which showed strong responsiveness
of Lex and LDN-F but no binding to LDN-DF was observed,
indicating that through multivalent interactions avidity and
selectivity of binding is substantially enhanced. The data indicates
that S. mansoni has developed a strategy to avoid detection by
DC-SIGN by the addition of a fucoside to its ligands.
AcO
O
O
O
OR
O
TrocHN
iv
AcHN
viii
NHTroc
7
O
O
OBn
OH
OBn
OH
8, R = TDS
9, R = H
12
BnO
v
HO
vi
10, R = C(NPh)CF3
vii
11, R = (CH2)5N(Bn)Cbz
AcO
OAc
OBn
AcO
AcO
O
OAc
O
OBn
AcO
O
O
O
OTDS
O
TrocHN
O
N3
OTDS
O
TrocHN
O
N3
xi
x
ix
O
13
O
OBn
OH
BnO
OBn
O
OBn
BnO
OBn
BnO
HO
14
15
OH
AcO
OAc
OH
OBn
O
O
O
HO
O
O
AcO
O
O
O(CH2)5NH2
NHAc
O
OR
AcHN
xv
TrocHN
NHTroc
O
O
O
O
OH
OBn
HO
BnO
O
O
OH
20
OBn
OH
OBn
16, R = TDS
17, R = H
HO
BnO
xii
xiii
xiv
18, R = C(NPh)CF3
19, R = (CH2)5N(Bn)Cbz
Results and Discussion
Chemical synthesis
Scheme 1. Synthesis of LDN-F and LDN-DF epitopes. Reagents and
conditions: i) TMSTOf, DCM, -30 °C, 91%; ii) DDQ, DCM, DCM/H2O, 84%; iii)
DPS/Tf2O, DCM, -60 °C to -40 °C, 68% for 7 and 73% for 13; iv) (a) PPh3,
THF/H2O (b) trocCl, NEt3, DCM, 63% (over two steps); v) HF/py in pyridine; vi)
The preparation of Schistosoma-derived glycans has received
relatively little attention,[11] and the chemical synthesis of LDN-DF
has not been reported. The latter compound represents a
challenging synthetic target because it requires careful selection
2,2,2-Trifluoro-N-phenylacetimidoyl
chloride,
DBU,
DCM;
vii)
HO(CH2)5N(Bn)Cbz, TMSTOf, DCM, -30 °C, 66% (over 3 steps); viii) (a) Zn dust,
2
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AcOH, Ac2O, THF (b) NaOMe, MeOH (c) Pd/C, H2, H2O/ MeOH, 45% (over
three steps); ix) DDQ, DCM/H2O, 74%; x) TMSTOf, DCM/ DMF, -30 °C to +5 °C,
83%; xi) (a) PPh3, THF/H2O (b) TrocCl, NEt3, DCM; xii) HF/py in pyridine; xiii)
perturbation analysis of the protein backbone, Nuclear
Overhauser Effect (NOE) evaluation, and molecular modeling.
The NMR-based strategy combines molecular recognition
analysis from the perspective of the protein and ligand thereby
providing a detailed atomic view of the interactions. 1H-STD-NMR
experiments were performed to establish which parts of LDN-F
2,2,2-Trifluoro-N-phenylacetimidoyl
chloride,
DBU,
DCM;
xiv)
HO(CH2)5N(Bn)Cbz, TMSTOf, DCM, -30 °C, 31% (over five steps); xv) (a) Zn
dust, AcOH, Ac2O, THF (b) NaOMe, MeOH (c) Pd/C, H2, H2O/MeOH, 37% (over
three steps).
1
and LDN-DF make direct contacts with the lectin. H-15N HSQC
experiments on the 15N-labeled carbohydrate recognition domain
(CRD) of DC-SIGN were conducted to identify protein residues
involved in ligand binding and to establish whether differences
exist between the two epitopes. Furthermore, 1H-15N HSQC
titration experiments provided binding affinity estimations. Finally,
trNOESY experiments characterized the ligand conformation in
the bound state and additionally provided short intermolecular
distances between the ligand and the protein, thus defining the
precise binding pose.
A
TMSOTf-mediated glycosylation of N-phenyl trifluo-
racetimidate donor 1 with acceptor 2 afforded disaccharide 5 in a
yield of 91%. The Nap ether of 5 was removed using DDQ in a
mixture of DCM and H2O to give acceptor 6 in a yield of 84%. Next,
fucosyl donors 3 and 4, having Bn and PMB ether at C-2,
respectively, were preactivated with DPS/ Tf2O in the presence of
TTBP at -60 °C,[12] which was followed by the addition of acceptor
6. The resulting reaction mixture was allowed to slowly warm to -
40ºC resulting in formation of trisaccharides 7 and 13, which were
isolated as mainly the a-anomers in good yield (7, 68%; J1,2 = 3.5
1H Saturation Transfer Difference (STD) NMR. 1H-STD NMR
spectra resulting from the interaction of LDN-F and LDN-DF with
DC-SIGN extracellular domain (ECD) which tetramerizes in
solution, along with the corresponding reference spectra are
shown in Figures 1a and 1b, respectively. It demonstrates that
DC-SIGN can bind both glycans but with substantial differences
in binding mode. Both ligands bind through the terminal fucoside.
The STD data for LDN-F are similar to those previously reported
for the structurally related Lex tri-saccharide,[6b] indicating a similar
mode of binding. As for Lex, LDN-F is recognized by DC-SIGN
through the fucoside and additional contacts are made with the
GalNAc pyranosyl ring and the N-acetyl group of the GlcNAc
moiety. The N-acetyl moiety of GalNAc exhibited only a weak STD
effect, indicating that it does not contribute substantially to binding
(Figure 1a). Detectable STD effects of the interaction of DC-SIGN
with LDN-DF were restricted to H2, H4 and Me of the terminal 1,2-
fucoside with a remarkable absence of STD signals from the
internal 1,3-fucoside and the GalNAc and GlcNAc moieties
(Figure 1b). The much lower intensities of the STD signals of the
LDN-DF compared to those acquired for the LDN-F suggests
weaker binding of the former. The blank 1H-STD NMR
experiments of LDN-F and LDN-DF ligands alone are shown in SI
(Figures S1 and S2).
Chemical shift perturbation analysis. The primary
carbohydrate binding site of DC-SIGN is composed of an
extended loop (from W343 to D355) and residues in b-strand-4
(from N363 to D367), which surround the principal Ca+2 ion.[10b]
Additionally, residues in b-strands-3 and -2 (from E356 to G361
and F313, respectively) shape a secondary binding region.
Chemical shift perturbation analysis using the 15N-labeled protein
was performed to examine which residues of the CRD of DC-
SIGN interact with the glycans. Thus, the 15N-labeled CRD DC-
SIGN was titrated with LDN-F and LND-DF and 1H-15N-HSQC
spectra were acquired at every titration point (Figure S3). Both
ligands provided similar chemical shift perturbation (CSP) profiles
involving amino acids of the primary and secondary binding site.
However, upon addition of the same number of equivalents, the
CSP of the lectin backbone NH resonances were much larger for
LDN-F than for LDN-DF (Figure 1c), indicating weaker binding of
the latter compound. In-deed, fitting of the CSP to the
corresponding binding isotherms for LDN-F yielded a KD of 1.5 ±
0.4 mM (Figure S3). Protein saturation was not possible with LDN-
DF providing an imprecise KD estimation, but substantially larger
than 6 mM.
1
1
Hz; JC,H = 174.9 Hz) and (13, 73%; J1,2 = 3.8 Hz; JC,H = 175.4
Hz). Alternative glycosylation conditions resulted in poor
anomeric selectivity (Table S1). Trisaccharide 7 was further
modified with an anomeric aminopentyl linker and to attain b-
anomeric selectivity, the azide was converted into NHTroc (®8)
by a two-step procedure entailing reduction using triphenyl
phosphine followed by reaction of the resulting amine with TrocCl.
Next, glycosyl donor 10 was prepared by removal of the anomeric
TDS ether of 8 with HF/pyridine followed by reaction with N-
phenyl trifluoroacetimidate in the presence of DBU. As antici-
pated,
a TMSOTf mediated glycosylation of 10 with N-
benzyloxycarbonyl-N-benzyl-5 aminopentanol gave 11 as only
the b-anomer in 87% yield. The latter trisaccharide was globally
deprotected by a three-step procedure in which the NHTroc was
converted to NHAc using zinc dust, followed by global
deacetylation and hydrogenation to provide spacer modified LDN-
F 12. A number of alternative strategies were explored to prepare
LND-F, and it was found that judicious selection of amino
protecting groups was critical, and the use of bulky protecting
groups such as N-phthalimido resulted in a donor-acceptor
reactivity mismatch, resulting in almost immediate hydrolysis or
degradation of donor (Table S1). In addition to donor 4 having a
temporary PMB ether, several alternatives were examined but
these gave disappointing results (Table S1). Next, attention was
focused on the preparation of spacer modified LDN-DF 20. Thus,
treatment of trisaccharide 13 with DDQ in DCM/H2O afforded
acceptor 14. Several reaction conditions were explored (Table
S2) to install a fucoside, and tetrasaccharide 15 was obtained with
high a-anomeric selectivity in good yield (83%) when the
glycosylation was performed in DCM in the presence of DMF[13]
and promoted by 1 equivalent of TMSTOf at -20 °C followed by
1
slow warming to +5 °C (J1,2 = 3.9 Hz; JC,H = 173.3 Hz). The
anomeric TDS group of 15 was removed and the resulting lactol
(17) converted into a N-phenyl trifluoroacetimidate 18, which was
coupled with N-benzyloxycarbonyl-N-benzyl-5-aminopentanol to
give 19. Global deprotection of the latter compounds gave target
tetrasaccharide 20 in an overall good yield (37% over three steps).
Molecular basis of binding of LDN-F and LDN-DF to DC-SIGN
The molecular basis of the interactions of DC-SIGN with LDN-F
(12) and LDN-DF (20) in solution was examined by combining
Saturation Transference Difference (STD-NMR), chemical shift
3
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Figure 1. 1H STD-NMR spectra for the interaction of full-length DC-SIGN with (a) LDN-F and (b) LDN-DF. Relative STD and epitope mapping are shown on the left
side for both ligands and refer to double difference between the STD spectrum in the presence of the protein and that in the absence of it. STD spectra were
acquired in the same conditions, however, drastic difference in absolute STD intensities between the two ligands exist. Specifically, the maximum STD signal
detected for LDN-DF ligand corresponds to just the 25% of the strongest STD signal detected for the LDN-F. (c) Average chemical shift perturbation upon the
addition of 100 equivalents of LDN-F (red) and LDN-DF (blue) respect to the protein. The cut-offs values were determined as “mean+stdev” and are indicated with
dotted lines. Structural models for the complexes of CRD DC-SIGN with the LDN-F ligand (d) and the LDN-DF ligand (e) obtained from MD simulations.
4
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Transferred NOESY. Recently,[14] the molecular complexes of
DC-SIGN with A and B histo blood group antigens have been
studied by trNOESY experiments. Key intermolecular NOEs
between the Hg protons of V351 with H1 and H2 of the fucoside
moiety were observed. The importance of the van der Waals
stabilizing contacts involving the Me groups of V351 for the
recognition of branched fucosylated epitopes has also been
shown by X-ray crystallography,[6a] and is in agreement with
mutagenesis studies, demonstrating that substitution of this
residue has important implications for the binding of Lewis type
epitopes.[15] Therefore, a trNOESY experiment was carried out for
LDN-F in the presence of 0.2 equivalents of the CRD of DC-SIGN.
After addition of the protein to the NMR tube, strong negative NOE
were observed for the ligand protons, which contain information
on the bound ligand conformation. Inter-molecular NOE
correlations between the methyl groups of V351 and H1-Fuc, H2-
Fuc, and the methyl group of GlcNAc were observed (Figure 2).
Consistent with the STD analysis, Fuc H1 and H2 are in close
contact with the lectin. Moreover, the strong STD described above
for the methyl moiety of the GlcNAc residue is supported by the
tr-NOE correlation between this group and V351. As control, the
NOESY spectrum of the free ligand was measured which
displayed very weak negative NOE effects. Unfortunately, a good
tr-NOESY spectrum could not be recorded for the LDN-DF
complex, probably due to its rather low binding affinity.
corresponding monosaccharide in the deposited 1SL5 structure.
The resulting binding pose was minimization by MD simulation
that resulted in a structure that is in excellent agreement with the
experimental data, including HSQC chemical shift perturbation
and epitope mapping through STD. For LDN-DF, a similar
approach was used by superimposing the terminal 1,2-linked
fucoside in the primary Ca2+ binding site. This starting binding
pose placed H2 of the terminal fucoside in close proximity to the
protein backbone, which is in agreement with the STD data. The
alternative binding pose through the inner fucoside was discarded
due to steric clashes. Analysis of the MD trajectory showed that
for the complex of LDN-F with DC-SIGN, the ligand conformation
remained fairly well defined as revealed by the low dispersion of
the φ and ψ angles (Figure S4). This arrangement favors
hydrophobic contacts between V351 and the Fuc and GlcNAc
moieties, which were maintained throughout the entire MD run
(Figure 1d). Additionally, the bound geometries were validated by
simulating the STD spectrum with CORCEMA-ST.[17] The match
between the expected and the experimental STD intensities for
LDN-F was excellent, further supporting the proposed binding
model (Figure S5).
The derived bound structure for LDN-DF was very different
(Figure 2e). In this case, the LDN backbone was far from the
protein with only the terminal fucoside making interactions. In this
case, the contacts between the Ac of GlcNAc and Hγ protons of
V351 were only transient and there was not a preferential spatial
arrangement of both groups to make substantial van der Waals
contacts. Although the LDN-F moiety of LDN-DF preserved
conformational rigidity, the α1,2-fucoside linkage was rather
flexible (Figure S1) providing
a loosely defined epitope
presentation around the primary binding site. In this case, the
fitting between the CORCEMA-ST simulations with the
experimental STD was less accurate, which is probably due to the
weak STD signals and the inability of the MD simulation to
reproduce the flexibility of the complex.
Glycan microarray binding studies
The CRDs of DC-SIGN are clustered in tetramers, and such an
arrangement can greatly amplify the avidity and specificity when
interacting with glycans epitopes that are present in a multivalent
arrangement.[9b-18] To examine the importance of multivalency,
LDN-F (12) and LDN-DF (20) and a number of control glycans
including Lex (21), Lex-Lex (22), SLex (23) and SLex-Lex (24),
which all are equipped with an anomeric aminopentyl moiety,
were immobilized on N-hydroxysuccinimide (NHS)-activated
glass slides in replicates of 6 by piezoelectric printing. After
incubation overnight in a saturated NaCl chamber, unreacted
esters were quenched with ethanolamine. First, the glycan
microarray was probed with biotinylated Aleuria aurantia lectin,
which recognizes a1,2- a1,3- and a1,6-fucosides, and
Streptavidin-AlexaFluor635. As anticipated all compounds
showed strong responsiveness (Figure S3) confirming proper
spot morphology and printing. Next, sub-arrays were incubated
with various concentrations of recombinant human DC-SIGN-Fc
chimera premixed with anti-IgG Fc-biotin and Streptavidin-
AlexaFluor635 in TSM binding buffer containing Ca2+. After
incubation for 1 h, the slide was washed, dried by centrifugation
and scanned for fluorescence intensity. LDN-F (12), Lex (21) and
Lex-Lex (22) exhibited strong responsiveness whereas no binding
was detected for LDN-DF (20) (Figure 3). This observation
indicates that the avidity and selectivity of binding is greatly
Figure 2. (a) NOESY spectrum of the complex of DC-SIGN with LDN-F. (b, c
and d) The quest of protein/ligand intermolecular NOEs. (e) Three-dimensional
model from the MD simulation showing the key intermolecular NOEs between
the ligand and the V351 residue of the lectin.
Molecular modeling. The NMR data were employed to derive
three-dimensional models for the complexes of DC-SIGN with
LDN-F and LDN-DF. For LDN-F, the experimental STD and
intermolecular NOE data showed close contacts of H1-Fuc, H2-
Fuc and Ac-GlcNAc with the methyl group of V351 of the protein.
For the histo blood A and B antigens,[16] it has been demonstrated
that DC-SIGN binds the fucosyl ring exclusively through
coordination of the C-3 and C-4 hydroxyls with the Ca2+ ion. The
crystallographic structure of DC-SIGN complexed with Lex (pdb
code 1SL5) also fulfills this requirement.[6a] Therefore, the
pyranosyl ring of LDN-F was superimposed onto the
5
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10.1002/chem.202002619
Chemistry - A European Journal
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enhanced when the binding is probed on a multivalent surface. At
a higher concentration of DC-SIGN, SLex-Lex (24) also exhibited
responsiveness whereas this was not the case for SLex, indicating
that the internal Lex moiety can be recognized by DC-SIGN. When
the microarray binding studies were performed in the absence of
Ca2+, no binding was observed confirming specificity of binding.
additional contacts, and as a result the binding affinity is
approximately an order of magnitude lower.
Previous studies have shown that antibodies directed against
Lex and LDN-F can block the binding of DC-SIGN to soluble egg
antigen of S. mansoni whereas an antibody against LDN-DF had
no effect on binding.[3a,22] These observations lead to the
conclusion that DC-SIGN can recognize Lex and LDN-F but not
LDN-DF. Our studies have shown that LDN-DF can interact with
DC-SIGN albeit with a substantial lower affinity than for Lex and
LDN-F. The glycan microarray studies demonstrated that the
avidity and selectivity of binding is greatly enhanced when the
glycans are presented in a multivalent manner, and in this format
Lex and LDN-F gave strong responsiveness whereas no binding
was detected for LDN-DF. The extracellular domain of DC-SIGN
occurs as a tetramer. Furthermore, the glycans of S. mansoni are
presented on its cell surface as glycoproteins and glycolipids, and
thus it is anticipated that such assemblies can make multivalent
interactions with DC-SIGN, resulting in enhancement in avidity[9b]
and magnify selectivities.[23]
Recently, it was shown that schistosomula extracellular
vesicles (EVs) carry surface glycoproteins and glycolipids with a
specific subset of fucosylated structures, such as Lex, pseudo_Ley
and LDN-F motifs that mediate internalization by moDCs in a DC-
SIGN dependent manner.[24] It is also known known that DC-SIGN
signaling via fucosides decreases pro-inflammatory responses,[25]
indicating that S. mansoni may exploit these structures to dampen
host immune response. Fucosylated glycans such as LDN, Lex,
LDN-F and LDN-DF are expressed in a stage-dependent manner
during the life cycle of S. mansoni.[26] Interestingly, an increase in
di-fucosylated N-glycans and glycolipids occurs during the
transition from immature to mature eggs and in the miracidia
stage.[21,27] During these stages, ligands, for DC-SIGN such as Lex,
are expressed at low levels. Thus, it is like that these changes
result in a lack of detection by DC-SIGN, thereby modulating the
host’s immune system.
Further studies are required to determine the importance of
the density of specific glycans during the different life stages of
Schistosomes and their influence on DC-SIGN detection and
subsequent skewing of host immune responses. Structures such
as LDN-DF and LDN-F are part of complex oligosaccharides in
which multiple of these epitopes can be presented. Potentially,
such structures can make multivalent interactions leading to high
avidity of binding. These features of molecular recognition require
further investigation. In addition to DC-SIGN, other C-type lectins
have been implicated in sensing helminth glycans by human DCs.
Among those, the macrophage galactose-type lectin MGL
exhibits high specificity for S. mansoni glycans terminating in Gal-
NAc.[28] It is conceivable that these lectins act in concert to detect
patterns of glycans thereby shaping immune responses. Finally,
the synthetic approaches of LDN-F and LDN-DF will promote
further biological studies to address the role of uniquely
fucosylated glycans in S. mansoni infectivity and may lead to the
development of immune-modulatory compounds.
β4
R
α3
4×106
12
3 µg/mL
β4
R
α3
10 µg/mL
20
21
3×106
2×106
1×106
0
α
2
β4
R
R
R
R
α3
β4
β3 β4
α3
α3
α3
α3
22
α3 β4
23
12 20 21 22 23 24
α3 β4
β3 β4
α3
24
Figure 3. Microarray results of the glycan library printed at 100 µM for binding
to DC-SIGN (3 and 10 µg/mL). Bars represent the mean ± SD.
Conclusions
The recognition of fucosylated structures such as Lex, LDN-F and
pseudo_Ley, by DC-SIGN has been implicated in
schistosomiasism,[5a-19] resulting in modulation of innate and
adaptive immune responses.[20] Further fucosylation of these
epitopes to give structures such LDN-DF or DF-LDN-DF
abolishes binding.[3a] During the life cycle of S. mansoni,
fucosylated glycans such as LDN, Lex, LDN-F and LDN-DF are
expressed in a stage-dependent manner, thereby shaping host
immune responses.[3c-21] We have investigated, at a molecular
level, in which way the terminal 1,2-fucoside of LDN-DF
influences recognition by DC-SIGN. Such a study required well-
defined glycans, which were obtained by a chemical approach in
which amino protecting groups were carefully selected to facilitate
high yielding and stereoselective chemical glycosylations. The
molecular recognition of LDN-F and LDN-DF by DC-SIGN was
studied by NMR assisted by molecular modeling, which revealed
that in solution it can recognize both glycans but with substantial
differences in affinity and binding mode. The HSQC titration
experiments provided a dissociation constant for LDN-F of 1.5 ±
0.4 mM at the monovalent level, whereas the kD for LDN-DF could
only be estimated but is substantially larger than 6 mM. In the
case of the LDN-F, the α1,3-fucoside coordinates with the Ca2+-
ion of the CRD of DC-SIGN, placing the GlcNAc residue in close
proximity to the protein surface thereby allowing for additional
interactions. The affinity and structural model for LDN-F and Lex
are very similar,[6b] indicating that the presence of a β4GalNAc vs.
a β4Gal moiety does not substantially alter binding. The terminal
α1,2-linked fucoside of LDN-DF can also bind into the canonical
binding site of DC-SIGN but in this case, the GlcNAc and GalNAc
residues are placed away from the protein surface preventing
Experimental Section
General procedure for glycosylations for the synthesis of 7 and 13
Thioglycoside donor (4 eq), diphenyl sulfoxide (4 eq) and 2,4,6-tri-tert-
butylpyrimidine (4 eq) were dissolved in DCM and stirred in the presence
of pre-activated molecular sieves (4 Å) for 30 min. Next, the temperature
6
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10.1002/chem.202002619
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was lowered (-60 ºC), followed by the addition of trifluromethanesulfonic
anhydride (4 eq). A solution of acceptor (1 eq) in anhydrous DCM was
added dropwise along the wall of the flask and the reaction was left stirring
while the temperature was slowly raised to -40 ºC. The reaction was
quenched with triethyl amine, the molecular sieves were filtered off, and
DCM was removed in vacuo. The residue was purified by silica gel column
chromatography.
equipped with a cryoprobe in the presence of 0.2 equivalents of DC-SIGN
(180 µM of protein), with a mixing time of 400 ms, at 298 K.
Molecular modelling
Initial geometries of ligands 12 and 20 were built in the Glycam web
(http://glycam.org). Proton-proton distances derived from NOESY spectra
(using the isolated spin-pair ap-proximation) were used to check the
goodness of the minimized structures. The initial pdb coordinates for CRD
of DC-SIGN were derived from the crystal structure Protein Database
(PDB) 1SL5. The magnesium ion was replaced by calcium, and the fucose
pyranose ring of glycans 12 and 20 was superimposed onto the
corresponding sugar in the deposited 1SL5 structure. The resulting binding
poses were used as starting points for molecular dynamics (MD)
simulations. The MD simulations were performed using the Amber16
program4 with the ff99SB force field parameters for protein and
GLYCAM_06h for the saccharides. Thereafter, the starting 3D geometries
were placed into a 12 Å octahedral box of explicit TIP3P waters, and
counterions were added to maintain electroneutrality. Two consecutive
minimization stages were performed involving (1) only the water molecules
and ions and (2) the whole system with a higher number of cycles, using
the steepest de-scent algorithm. The system was subjected to two rapid
molecular dynamic simulations (heating and equilibration) before starting
the real dynamic simulation. The equilibrated structures were the starting
points for the final MD simulations at constant temperature (300 K) and
pressure (1 atm). 500 ns Molecular dynamics simulations without
constraints were recorded, using an NPT ensemble with periodic boundary
conditions, a cut-off of 10 Å, and the particle mesh Ewald method. A total
of 250 000 000 molecular dynamics steps were run with a time step of 1 fs
per step. Coordinates and energy values were recorded every 10000 steps
(10 ps) for a total of 25 000 MD models. A detailed analysis of the
glycosydic linkages for glycans 12 and 20 was performed along the MD
trajectory using the cpptraj module included in Amber-Tools 16 package
and are represented in Figure S4.
General procedure for conversion of azide (N3) into NHTroc
Compounds 7 and 13 (1 eq) were dissolved in THF and water was added.
Next trimethylphosphine (5 eq) was added. The reaction mixture was
stirred under an atmosphere of Ar for 2 h, after which the solvent was
evaporated in vacuo and the residue was co-evaporated with toluene twice.
The residue was dissolved in DCM, followed by the addition of 2,2,2-
trichloroethyl chloroformate (2 eq) and triethylamine (2 eq). The reaction
mixture was stirred for 1 h after which it was diluted by DCM and washed
with water. The organic layer was dried (MgSO4), filtered, and the filtrate
concentrated in vacuo. The residue was purified by silica gel column
chromatography.
General
procedure
for
the
glycosylation
of
5-(N-
benzyloxycarbonyl,N-benzyl)aminopentyl linker
The donors 10 and 18 (1 eq) and 5-(N-benzyloxycarbonyl,N-
benzyl)aminopentyl linker (5 eq) were dissolved in DCM, and stirred in the
presence of pre-activated molecular sieves (4 Å) under an admosphere of
Ar for 30 min. The reaction mixture was cooled (-50 °C), followed by the
addition of trifluoromethanesulfonic acid (0.2 eq). The temperature was
slowly warmed up to -30 °C, after which TLC showed complete
consumption of donor and the formation of a new product. The reaction
mixture was quenched with triethyl amine, the molecular sieves were
filtered off, and the solvent was evaporated. The residue was purified by
silica gel column chromatography.
Protein expression
Microarray
The extracellular domain of DC-SIGN was obtained as previously de-
scribed.[16] The carbohydrate recognition domain of DC-SIGN in its 15N
labelled form was obtained as previously described.[14]
The synthetic compounds were printed at 100 µM on activated glass slides
by piezoelectric non-contact printing (sciFLEXARRAYER S3, Scienion Inc).
Printing was validated by assaying the binding to biotinylated Aleuria
aurantia lectin (AAL) and detection by Streptavidin-AlexaFluor635 (see
Figure S6). Recombinant human DC-SIGN-Fc Chimera was assayed
premixed with anti-IgG Fc-biotin and Streptavidin-AlexaFluor635. The
fluorescence was measured using a GenePix 4000 B microarray scanner
(Molecular Devices) and data were processed with GenePix Pro 7
software and further analysed using our home written Microsoft Excel
macro. Data were fitted using Prism software (GraphPad Software, Inc).
Further details are given in the Supporting Information.
1H Saturation transfer difference (STD) NMR
The samples for saturation-transfer difference (STD) NMR experiments
were prepared using the extracellular domain of DC-SIGN at 10 µM
concentration in 25 mM Tris-d11, 150 mM NaCl, 4 mM CaCl2 in D2O (pD
8) using lectin/ligand ratios of 1:60. The temperature was set to 298 K.
STD experiments were performed at 600 MHz Bruker spectrometer, using
standard Bruker pulse sequences without water suppression nor protein
spin-lock filter. Protein saturation was achieved with a Gaussian-shaped
pulse of 49 ms. The on-resonance frequency was set at aliphatic regions
(0.76 ppm) and the off-resonance frequency at 100 ppm. Blank STD
experiments of the ligands alone were acquired in the same conditions.
The results of blank 1H-STD NMR experiments for ligands 12 and 20 are
shown in Figures S1 and S2, respectively.
Acknowledgements
This research was supported by the Netherlands Organization for
Scientific Research (NWO; TOP-PUNT grant 718.015.003 to G.-
J.B.), the Human Frontier Science Program Organization (HFSP;
grant LT000747/2018-C to L.U.), the European Research Council
(ERC-2017-AdG, project number 788143-RECGLYC-ANMR to
J.J.-B.), the Agen-cia Estatal Investigación of Spain (AEI; grant
RTI2018-094751-B-C21 to J.J.-B.) and the Severo Ochoa
Excellence Accreditation (SEV-2016-0644 to J.J.-B.).
Chemical shift perturbation analysis
1H-15N-HSQC-based experiments were performed using 15N-labeled CRD
DC-SIGN at 50 µM, with 2 mM DTT-d10, at 800 MHz Bruker spectrometer
equipped with a cryoprobe, at 310 K. Eight and ten titration points were
acquired for ligands 12 and 20 respectively, with ligand concentrations
varying from 0 to 0.5 mM for the former and from 0 to 1.5 mM for latter.
Averaged chemical shift perturbation (CSP) and dissociation constants
(kD) were calculated using the CcpNmr Analysis 2.4.2.3 The chemical shift
perturbation analysis was performed based on the protein backbone
assignment deposited in the BMRB database with the code 27854. The
results from this analysis are shown in Figure S3.Transferred NOESY
spectrum for glycan 12, was acquired at 800 MHz Bruker spectrometer
Conflict of interest
The authors declare no conflict of interest.
7
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[25] a) S.I. Gringhuis, T.M. Kaptein, B.A. Wevers, A.W. Mesman, T.B.
Geijtenbeek, Nat Commun. 2014, 5, 3898.
Keywords: glycans • chemical synthesis • molecular recognition
• NMR • immune modulation
[26] a) R.D. Cummings, A.K. Nyame, Biochim Biophys Acta, 1999, 1455,
363–374; b) K.H. Khoo, A. Dell, Adv Exp Med Biol. 2001, 491, 185-205;
c) C.H. Hokke, A.M. Deelder, Glycoconj. J. 2001, 18, 573-587.
[27] a) J. Jang-Lee, R.S. Curwen, P.D. Ashton, B. Tissot, W. Mathieson, M.
Panico, A. Dell, R.A. Wilson, S.M. Haslam, Mol Cell Proteomics. 2007, 6,
1485-1499.
[1]
[2]
[3]
a) P. Kalantari, S.C. Bunnell, M.J. Stadecker, Front. Immunol. 2019, 10,
26; b) P.T. LoVerde, Adv Exp Med Biol. 2019, 1154, 45-70.
a) T.B.H. Geijtenbeek, S.J. van Vliet, A. Engering, B. A 't Hart, Y. van
Kooyk, Annu. Rev. Immunol. 2004, 22, 33-54.
a) I. van Die, S.J. van Vliet, A.K. Nyame, R.D. Cummings, C.M.C. Bank,
B. Appelmelk, T.B.H. Geijtenbeek, Y. van Kooyk, Glycobiology 2003, 13,
471-478; b) C.H. Hokke, A.M. Deelder, K.F. Hoffmann, M. Wuhrer, Exp.
Parasitol. 2007, 117, 275-283; c) C.H. Hokke, A. van Diepen, Mol
Biochem Parasitol. 2017, 215, 47-57.
[28] a) S.J. van Vliet, E. van Liempt, E. Saeland, C.A. Aarnoudse, B.
Appelmelk, T. Irimura, T.B. Geijtenbeek, O. Blixt, R. Alvarez, I. van Die,
Y. van Kooyk, Int. Immunol. 2005, 17, 661-669.
[4]
[5]
a) M.L. Mickum, N.S. Prasanphanich, J. Heimburg-Molinaro, K.E. Leon,
R.D. Cummings, Front. Genet. 2014, 5, 262.
a) T.B.H. Geijtenbeek, J. den Dunnen, S.I. Gringhuis, Future Microbiol.
2009, 4, 879-890; b) Y. van Kooyk, T.B.H. Geijtenbeek, Nat Rev Immunol.
2003, 3, 697-709.
[6]
a) Y. Guo, H. Feinberg, E. Conroy, D.A. Mitchell, R. Alvarez, O. Blixt, M.
E. Taylor, W.I. Weis, K. Drickamer, Nat. Struct. Mol. Biol. 2004, 11, 591-
598; b) K. Pederson, D.A. Mitchell, J.H. Prestegard, Biochemistry. 2014,
53, 5700-5709.
[8]
[9]
a) S. Meyer, E. van Liempt, A. Imberty, Y. van Kooyk, H. Geyer, R. Geyer,
I. van Die, J Biol Chem. 2005, 280, 37349-59.
a) E. van Liempt, C.M.C. Bank, P. Mehta, J.J. García-Vallejo, Z.S. Kawar,
R. Geyer, R.A. Alvarez, R.D. Cummings, Y. van Kooyk, I. van Die, FEBS
Lett. 2006, 580, 6123-6131; b) D.A. Mitchell, A.J. Fadden, K. Drickamer,
J Biol Chem. 2001, 276, 28939-28945.
[10] a) H. Feinberg, R. Castelli, K. Drickamer, P.H. Seeberger, W.I. Weis, J
Biol Chem. 2007, 282, 4202-4209; b) H. Feinberg, D.A. Mitchell, K.
Drickamer, W.I. Weis, Science. 2001, 294, 2163-2166; c) J. Angulo, I.
Díaz, J.J. Reina, G. Tabarani, F. Fieschi, J. Rojo, P.M. Nieto, Chem. Bio.
Chem. 2008, 9, 2225-2227.
[11] a) K. Brzezicka, B. Echeverria, S. Serna, A. van Diepen, C.H. Hokke,
N.C. Reichardt, ACS Chem. Biol. 2015, 10, 1290-1302; b) K. Agoston, J.
Kerékgyártó, J. Hajkó, G. Batta, D.J. Lefeber, J.P. Kamerling, J.F.G.
Vliegenthart, Chemistry. 2002, 8, 151-61.
[12] a) I.A. Gagarinov, T. Fang, L. Liu, A.D. Srivastava, G.-J. Boons, Org. Lett.
2015, 17, 928-931.
[13] a) S.R. Lu, Y.H. Lai, J.H. Chen, C.Y. Liu, K.K. Mong, Angew. Chem. Int.
Ed. 2011, 50, 7315-7320.
[14] a) P. Valverde, S. Delgado, J.D. Martínez, J.B. Vendeville, J. Malassis,
B. Linclau, N.C. Reichardt, F.J. Cañada, J. Jiménez-Barbero, A. Arda,
ACS Chem. Biol. 2019, 14, 1660-1671.
[15] a) T.B.H. Geijtenbeek,G.C.F. van Duijnhoven, S.J. van Vliet, E. Krieger,
G. Vriend, C.G. Figdor, Y. van Kooyk, J. Biol. Chem. 2002, 277, 11314-
11320.
[16] a) J.D. Martínez, P. Valverde, S. Delgado, C. Romanò, B. Linclau, N.C.
Reichardt, S. Oscarson, A. Ardá, J. Jiménez-Barbero, F.J. Cañada,
Molecules, 2019, 24, E2337.
[17] a) N.R. Krishna, V. Jayalakshmi, Top. Curr. Chem. 2008, 273, 15-54.
[18] a) L.L. Kiessling, R.A. Splain, Annu. Rev. Biochem. 2010, 79, 619-653.
[19] a) E. RodrIguez, S.T.T. Schetters, Y. van Kooyk, Nat. Rev. Immunol.
2018, 18, 204-211.
[20] a) R.M. Anthony, L.I. Rutitzky, J.F. Jr. Urban, M.J. Stadecker, W.C.
Gause, Nat Rev Immunol. 2007, 7, 975-87.
[21] a) C.H. Smit, A. van Diepen, D.L. Nguyen, M. Wuhrer, K.F. Hoffmann,
A.M. Deelder, C.H. Hokke, Mol Cell Proteomics. 2015, 14, 1750-69.
[22] a) J. den Dunnen, S.I. Gringhuis, T.B.H. Geijtenbeek, Cancer Immunol.
Immun. 2009, 58, 1149-1157.
[23] a) D.A. Mann, M. Kanai, D.J. Maly, L.L. Kiessling, J. Am. Chem. Soc.
1998, 120, 10575-10582; b) R.T. Lee, Y.C. Lee, Glycoconj. J. 2000, 17,
543-551; c) T.K. Dam, T.A. Gerken, C.F. Brewer, Biochemistry. 2009, 48,
3822-3827.
[24] a) M.E. Kuipers, E.N.M. Nolte-‘t Hoen, A.J. van der Ham, A. Ozir-
Fazalalikhan, D.L. Nguyen, C.M. de Korne, R.I. Koning, J.J. Tomes, K.
F. Hoffmann, H.H. Smits, C.H. Hokke, Journal of Extracellular Vesicles,
2020, 9:1, DOI: 10.1080/20013078.2020.1753420.
8
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Entry for the Table of Contents
Chemically synthesised oligosaccharides derived from the parasite worm S. mansoni made it possible to investigate by a combination of
analytical techniques molecular interactions with the innate immune receptor DC-SIGN. It showed that by further fucosylation of antigens such
as LDN-NF, detection by DC-SIGN is prevented, which in turn may be important for shaping immune responses during different phases of the
life cycle of the parasite.
9
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