Chemoenzymatic Synthesis of Complex N-Glycans of the Parasite S. mansoni to Examine the Importance of Epitope Presentation on DC-SIGN recognition

Article abstract of DOI:10.1002/anie.202105647

The importance of multivalency for N-glycan-protein interactions has primarily been studied by attachment of minimal epitopes to artificial multivalent scaffold and not in the context of multi-antennary glycans. N-glycans can be modified by bisecting GlcNAc, core xylosides and fucosides, and extended N-acetyl lactosamine moieties. The impact of such modifications on glycan recognition are also not well understood. We describe here a chemoenzymatic methodology that can provide N-glycans expressed by the parasitic worm S. mansoni having unique epitopes at each antenna and containing core xyloside. NMR, computational and electron microscopy were employed to investigate recognition of the glycans by the human lectin DC-SIGN. It revealed that core xyloside does not influence terminal epitope recognition. The multi-antennary glycans bound with higher affinity to DC-SIGN compared to mono-valent counterparts, which was attributed to proximity-induced effective concentration. The multi-antennary glycans cross-linked DC-SIGN into a dense network, which likely is relevant for antigen uptake and intracellular routing.

Full text of DOI:10.1002/anie.202105647

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Chemoenzymatic synthesis of complex N-glycans of the
parasite S.mansoni to examine the importance of epitope
presentation on DC-SIGN recognition
Authors: Apoorva D. Srivastava, Luca Unione, Mehman Bunyatov, Ivan
A. Gagarinov, Nicola G. A. Abrescia, Sandra Delgado, Ana
Arda, and Geert-Jan Boons
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202105647
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10.1002/anie.202105647
Angewandte Chemie International Edition
RESEARCH ARTICLE
Chemoenzymatic synthesis of complex N-glycans of the parasite
S. mansoni to examine the importance of epitope presentation on
DC-SIGN recognition
Apoorva D. Srivastava,†[a] Luca Unione,†[a] Mehman Bunyatov,[a] Ivan A. Gagarinov,[a] Nicola G. A.
Abrescia,[b,c] Sandra Delgado,[b] Ana Ardá,[b,c] and Geert-Jan Boons*[a,d,e]
[a]
[b]
A. D. Srivastava,† Dr. L. Unione,† M. Bunyatov, Dr. I.A. Gagarinov, Prof. Dr. G.J. Boons
Department of Chemical Biology and Drug Discovery
Utrecht Institute for Pharmaceutical Sciences
Bijvoet Center for Biomolecular Research
Utrecht University
Universiteitsweg 99, 3584 CG Utrecht (Netherlands)
E-mail: g.j.p.h.boons@uu.nl
Dr. N. Abrescia, S. Delgado, Dr. A. Ardá
Molecular Recognition and Host-Pathogen Interactions
CIC bioGUNE, Basque Research and Technology Alliance, BRTA
Bizkaia Technology Park, Building 800, 48162 Derio, Bizkaia (Spain)
Dr. N.G.A. Abrescia, Dr. A. Ardá
Ikerbasque, Basque Foundation for Science, 48013 Bilbao, Bizkaia (Spain)
Prof. Dr. G.J. Boons
[c]
[d]
Complex Carbohydrate Research Center, University of Georgia
315 Riverbend Road, Athens, GA 30602 (USA)
Prof. Dr. G.J. Boons
Department of Chemistry, University of Georgia
Athens, GA, 30602 (USA)
[e]
[†]
These authors contributed equally to this work
Supporting information for this article is given via a link at the end of the document.
Abstract: The importance of multivalency for N-glycan-protein
interactions has primarily been studied by attachment of minimal
epitopes to artificial multivalent scaffold and not in the context of multi-
antennary glycans. N-glycans can be modified by bisecting GlcNAc,
core xylosides and fucosides, and extended N-acetyl lactosamine
moieties. The impact of such modifications on glycan recognition are
also not well understood. We describe here a chemoenzymatic
methodology that can provide N-glycans expressed by the parasitic
worm S. mansoni having unique epitopes at each antenna and
contain core xyloside. NMR, computational and electron microscopy
were employed to investigate recognition of the glycans by the human
lectin DC-SIGN. It revealed that core xyloside does not influence
terminal epitope recognition. The multi-antennary glycans bound with
higher affinity to DC-SIGN compared to mono-valent counterparts,
which was attributed to proximity-induced effective concentration. The
multi-antennary glycans cross-linked DC-SIGN into a dense network,
which likely is relevant for antigen uptake and intracellular routing.
Introduction
Glycan binding proteins play key roles in the battle between host
and pathogens. Pathogens often express glyco-epitopes that can
be detected by glycan binding proteins of the host such as the
mannose-binding lectin, macrophage mannose receptor, or C-
type lectins, resulting in a diverse range of immune responses.
The host also expresses glycan binding proteins such as the
Siglec’s and complement factor H that can detect self-glycan
signatures to maintain immune-homeostasis.[1] A number of
microbes have developed ways to achieve molecular mimicry of
host glycans to avoid immune detection and establish infections.[2]
Binding and structural studies have indicated that glycan binding
proteins recognize relatively small oligosaccharide motifs often
found at termini of complex glycans.[3] There are, however,
indications that the complex architecture of glycans can modulate
recognition of minimal epitopes.[4] For example, N-glycans which
have branched structures can potentially present multiple minimal
epitopes that can engage with multiple glycan binding proteins
resulting in increased binding avidities. It may also facilitate
glycoconjugate clustering, which in turn may influence several
downstream processes. Multivalency has primarily been studied
by attachment of minimal epitopes to artificial multivalent
scaffold,[5] and not in the context of natural multi-antennary
glycans. In addition, N-glycans can be modified by bisecting N-
acetyl glucosamine (GlcNAc), core xylosides and fucosides, and
1
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10.1002/anie.202105647
Angewandte Chemie International Edition
RESEARCH ARTICLE
extended N-acetyl lactosamine (LacNAc) moieties. The impact of
such modification on glycan recognition are also not well
understood. These deficiencies are mainly due to inaccessibility
of structurally defined complex glycans. Here, we report a
synthetic methodology that can provide N-glycans expressed by
the parasitic worm S. mansoni. It includes compounds that have
asymmetrical architectures and contain core xylose and terminal
epitopes such as GalNAcb1,4GlcNAc (Lac-di-NAc) and mono-
and di-Lewis X (Lex). NMR, computational and transmission
electron microscopy were employed to investigate the importance
of glycan complexity for recognition by the human lectin Dendritic
cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN). It revealed
that the core xyloside does not influence terminal epitope
presentation and recognition. Furthermore, it was found that the
multi-antennary glycans bind with higher affinity to DC-SIGN
compared to mono-valent minimal epitopes, which was attributed
to proximity-induced effective concentration. Finally, the studies
uncovered that the multi-antennary glycan can cross-link DC-
SIGN into a dense network, which is likely relevant for antigen
uptake and intracellular routing.
responses.[14] The molecular details by which these complexes
are formed are poorly understood. Moreover, little is known about
the preference of DC-SIGN for specific S. mansoni derived
glycans, and if there are restrictions related to glycan valency or
size. The latter is due to the difficulties of synthesizing highly
complex glycans that are modified by core xylose and have
unusual epitopes such as Lac-di-NAc and LDN-F.
Results and Discussion
Chemoenzymatic synthesis. We set out to develop
a
chemoenzymatic methodology that can provide asymmetric
complex glycans expressed by S. mansoni such as compounds
1, 2 and 3 (Fig. 1). The compounds are modified by core xyloside,
a Lac-di-NAc moiety and various patterns of fucosylation.
In parallel, reference compounds LeXt (C1, Galb1,4-
(Fuca1,3)GlcNAcb1,3Gala-O-aminopropyl; LeX tetraose, Elicityl),
di-LeX
(C2,
Galb1,4-(Fuca1,3)GlcNAcb1,3Galb1,4-
(Fuca1,3)GlcNAcb-O-azidopentyl) and LDN-F (C3, GalNAcb1,4-
(Fuca1,3)GlcNAcb-O-azidopentyl) were prepared or purchased
for NMR studies described below. Challenging aspects of the
preparation of compounds such as 1 are the installation of a core
xyloside and the decoration of each antennae by unique
appendages. The β-1,2-xylosyltransferase from Arabidopsis
thaliana (XYLT), which potentially can install a core xyloside,
operates early in the biosynthesis of N-glycans and has narrow
substrate specify. In particular, it cannot transfer xylose when an
antenna is modified by a galactoside, which greatly complicates
the construction of asymmetrical glycans.[15] Thus, we opted for a
synthetic strategy in which the xyloside was introduced by
chemical glycosylation to give precursors that can be elaborated
by glycosyl transferases into asymmetrical multi-antennary
glycans.
b4
b4
a3
b4 b3 b4
b6
b6
a6
b6
a3
a3
b4 b2
a3
a3
b4
a3
b4
a6
a6
b4 b4
b2
b2
a3
a6
b4
a6
a6
a3
b4 b4
a3
b4
b4 b4
(3)
b4
a3
b2 b2
(1)
b2
b2
(2)
a3
a3
b4 b3 b4
b
b4
a3
b
b4 b3
R
R
a/b
R: -O(CH2)5N3
a3
a3
a3
LeXt (C1)
di-LeX (C2)
LDN-F (C3)
GlcNAc
Man
Xyl
Fuc
Gal
GalNAc
Figure 1. Schematic representation of the different glycans (1-3) and
corresponding fragments (C1-C3).
Thus, heptasaccharide 4a was prepared, which is modified by a
core xyloside, and at potential branching points is modified by the
orthogonal
protecting
groups
levulinoyl
(Lev),
Schistosomes are parasitic helminths that cause chronic
infections in humans associated with high morbidity.[6] N-glycans
of S. mansoni exhibit structural heterogeneity due variations in
core modifications, the number of antennae, and their extensions
into various epitopes. The expression is regulated in stage-
specific manner,[7] and for example during the egg and cercaria
stage, Schistosomes abundantly decorate the core of N-glycans
by xylose.[8]
fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), and
t-butyl-dimethylsilyl (TBS).[16] Sequential removal of these
protecting groups will give acceptors that can be extended by
glycosyl donors such as 5, 6 and 7 to provide, after deprotecting,
a compound such as 8 and 9 (Fig. 2). It was anticipated that the
Lac-di-NAc moiety of 8 and 9 can selectively be modified by
glycosyl transferases. In the next stage of synthesis, the terminal
Gal can be converted into LacNAc, and then enzymatically
extended into a complex structure.[16a] It was expected that
compound 4b would give entry into complex glycans lacking core
xylose. The synthesis of the donors 5, 6 and 7 is presented in the
SI (Scheme S4).
The termini of Schistosome glycans are often fucosylated to
present
epitopes
such
as
Lewis
X
(Lex),
GalNAc b1,4(Fuc a1,3)GlcNAc (LDN-F) and di-Lewis X (di-Lex).[7]
These epitopes can be recognized by DC-SIGN, which is
expressed on dendritic cells,[9] and interacts with conserved
molecular patterns shared by a large group of microorganisms. It
facilitates internalization of pathogens for processing and antigen
presentation.[10] Pathogens can also exploit DC-SIGN for infection
and dissemination making it an important therapeutic target.[11]
DC-SIGN is a homo-tetrameric Ca2+ dependent lectin in which
each monomer presents a carbohydrate recognition domain
(CRD).[12] Potentially, it can engage in multivalent interactions
resulting to high avidity of binding.[13] In vivo, multivalent glycans
on the pathogen surface promote DC-SIGN clustering, thus
facilitating antigen uptake. There are indications that the density
of surface exposed glycans influences cellular signaling, which in
turn leads to enhancement or suppression of proinflammatory
2
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10.1002/anie.202105647
Angewandte Chemie International Edition
RESEARCH ARTICLE
orthogonally protected precursor
building blocks
OBn
OBn
AcO
OAc
OAc
O
OBn
O
OAlloc
O
LevO
BnO
BnO
O
OBn
O
AcO
AcO
O
OC(NPh)CF3
O
OC(NPh)CF3
AcO
BnO
NPhth
NPhth
NPhth
OBn
O
OR
O
O
5
6
O
BnO
O
BnO
O
O
OBn
BnO
NPhth
OBn
O
OBn
O
AcO
AcO
BnO
NPhth
BnO
TBSO
BnO
O
O
BnO
OC(NPh)CF3
4a. R = Ac
4b. R = xylose
OFmoc
NPhth
7
chemical modifications
using 5, 6 and 7
substrate for
OH
OH
O
glycosyl transferases
after mannoside treatment
and galactosylation
HO
HO
HO
O
O
O
HO
NHAc
OH
OH
OH
substrate for
glycosyl transferases
after galactosylation
O
OH
O
O
HO
HO
O
O
HO
HO
NHAc
OH
enzymatic
modifications
O
OR
O
O
O
HO
O
HO
O
O
OH
HO
NHAc
NHAc
HO
HO
HO
O
8. R = Ac
9. R = xylose
OH
OH
O
OH
substrate for
glycosyl transferases
O
O
HO
HO
O
NHAc
NHAc
Figure 2. Overview of the synthetic strategy for the preparation of asymmetrical glycans with and without core xyloside. The coupling of the common precursors,
4a and 4b, which display orthogonal protecting groups at key branching points, with glycosyl donors 5-7 followed by global deprotection afforded asymmetric tri-
antennary glycans, 8 and 9, respectively. The latter intermediates were used as substrates for enzymatic extensions of each antenna to yield the asymmetrical
glycans 1, 2 and 3, which differ for in presence or absence of core-xylose, fucosylation of the arms and for the presence of a di-LeX moiety. Antenna selective arm
extension was possible because the unnatural b-mannoside at the C-2’ arm temporarily blocks it from enzymatic modification. It can, however, be unmasked by a
b-mannosidase and after enzymatic galactosylation, a precursor is obtained that can be elaborated by many glycosyl transferases into a complex structure. The
strategy also exploits that many glycosyl transferases can modify LacNAc but not GlcNAc. The latter can, however, be converted into LacNAc by galactosylation
with GalT1. Key structural features are highlighted by red circles.
exert a conformational control. Compound 16 was treated with HF
in pyridine to remove the anomeric dimethylthexylsilyl group
(TDS), and the resulting anomeric lactol was reacted with 2,2,2-
trifluoro-N-phenylacetimidoyl chloride in presence of DBU to form
Compound 10 (Scheme 1) is an appropriate starting material for
the synthesis of core xylosylated N-glycan 4a. Its mannosyl
moiety is protected at C-2 by an acetyl ester, which can selectively
N-phenyl trifluroacetimidate[18] donor 17 in a yield of 87% over two
steps. The later compound was glycosylated with disaccharide
acceptor 12[19] in presence of TMSOTf as the catalyst to furnish
18 (85%). Next, the Nap ether of 18 was oxidatively removed by
DDQ in the presence of b-pinene as acid scavenger[20] to afford
acceptor 19 (64%). A NIS/TMSOTf mediated glycosylation of
thio-manosyl donor 13, modified by the orthogonal protecting
groups Fmoc and TBS at C-2 at C-4, respectively (see SI,
Scheme S5) with acceptor 19 gave hexasaccharide 20 in a yield
of 77% as only the a-anomer. The benzylidene acetal of 20 was
regioselectively opened by treatment with triethylsilane (Et3SiH)
and dichlorophenylborane (PhBCl2) in DCM at -78°C to furnish
be cleaved to generate an acceptor that can be xylosylated with
donor 11 to provide glycan 16. The anomeric thexyl dimethylsilyl
(TDS) can be cleaved by HF pyridine to give a lactol that can be
converted into donor 17 for glycosylation with acceptor 12 to give
pentasaccharide 18. The naphthylmethyl (NAP) ether and the
benzylidene acetal of the resulting compound can selectively be
cleaved or reductively opened, respectively to give acceptors that
can be glycosylated with thiophenyl mannoside donor 13 and N-
phenyl trifluoroacetimidate donor 14 to provide fully protected
heptasaccharide 4a, which is an appropriate precursor for
antennae selective extension. Alternatively, the acetyl ester of
compound 10 can be left intact to give access to non-xylosylated
N-glycan precursor 4b (see SI, Scheme S2 and S3). Thus,
disaccharide 10 was treated with sodium methoxide in methanol
to give acceptor 15, which was coupled with trichloroacetimidate
11 to provide xyloside bearing trisaccharide 16 as only the b-
anomer. The xylosyl moiety adopted a 1C4 conformation as
acceptor 21 having a hydroxyl at the C-6 position of the central
21]
mannoside. As previously observed,[17a,
upon removal of a
benzylidene acetal, the xyloside moiety adopted
a
4C1
3
conformation, which was reflected by an increase in the J1,2
coupling constant from a di-equatorial (3.7 Hz) to a di-axial
orientation (7 Hz). Finally, a TMSOTf-mediated glycosylation of
mannosyl trichoroacetimidate 14, modified by the orthogonal
protecting groups Alloc and Lev (see SI, Scheme S5), with
acceptor 21 gave heptasaccharide 4a in a yield of 75%. The
corresponding non-xylosylated hexasaccharide core 4b was
synthesized following a similar glycosylation strategy (See SI,
Scheme S2).
evident from the coupling constant between H-1 and H-2 (J1,2
=
3.7 Hz) indicating a di-equatorial orientation. Others have
observed that xylosides protected by acetyl esters in apolar
1
solvent can adopt a C4 conformation.[17] It has been suggested
that the resulting anomeric effect overcomes unfavorable steric
interactions of axial substituents. It appears that protecting groups
at neighboring glycosyl moieties can influence the conformation
of the xyloside, indicating that steric hindrance of these entities
3
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10.1002/anie.202105647
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RESEARCH ARTICLE
Scheme 1. Synthesis of N-glycan precursor 8 starting from key building blocks 10-14.
Next, attention was focused on the installation of the various
branching points to give tri-antennary precursor glycan 8 by
subsequent removal of the orthogonal protecting groups,
glycosylations with donors 5, 6 and 7 followed by deprotection.
Treatment of heptasaccharide 4a with Et3N resulted in the
selective removal of the Fmoc protecting group without affecting
other functionalities to give glycosyl acceptor 22 in a good yield of
81%. The latter acceptor was glycosylated with N-phenyl
TfOH as promoter at -40 °C to afford 25 in a yield of 65%. Next,
the Alloc protecting group was removed by treatment with
tetrakis(triphenylphosphine)-palladium-0
(Pd[PPh3]4)
and
morpholine to afford acceptor 26, which was then glycosylated
with donor 7 using the standard procedure to afford tri-antennary
glycan 27 in 75% yield. The latter compound was subjected to
global deprotection over five steps entailing treatment with
HF/pyridine to remove TBS ether, followed by heating under reflux
with ethylenediamine in n-butanol to cleave the phthalimide-
protecting groups. The exposed free amines and hydroxyls were
acetylated by acetic anhydride in pyridine followed by cleavage of
the esters by sodium methoxide. Finally, the benzyl ethers were
removed by catalytic hydrogenation in the presence of palladium
trifluoroacetimidate
donor
5
in
the
presence
of
trifluoromethanesulfonic acid (TfOH) at -40 °C to afford 23 (80%).
The Lev ester of 23 was selectively removed by treatment with
hydrazine acetate to provide acceptor 24 (66%), which was
coupled with N-phenyl trifluoroacetimidate glycosyl donor 6 using
4
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10.1002/anie.202105647
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RESEARCH ARTICLE
b4
b4
b4
b6
b6
b6
a3
b2
UDP-Gal
B4GalT1
GDP-Fuc
FUT5
b6
a3
b4 b2
b-Man-ase
a6
a6
a6
b2
b2
a6
b4
b4
b2
a3
a6
a6
a6
a6
a3
a3
b4
b2
29
a3
b4
b4b4
b4 b4
b2
28
b4
b4 b4
b2
b4
b4
b2
b4
b2
b2
b2
8
(1)
a3
b4
a3
b6
b6
b6
b6
UDP-Gal
B4GalT1
GDP-Fuc
FUT5
b-Man-ase
a6
a6
a6
a6
b2
b2
b2
a3
b4b2
b4
b4
a6
b4
a6
a6
a6
a3
a3
a3
a3
b4
b4
b4 b4
(2)
b4 b4
b4
b4 b4
b4
b4
b2
b2
b2
b2
9
30
31
a3
i) UDP-GlcNAc,
B3GNT2
ii) UDP-Gal,
B4GalT1
b3b4
b4
b4
b6
b2
b6
b6
UDP-Gal
B4GalT1
GDP-Fuc
FUT5
a6
a6
b4
b4
a6
b4 b2
b2
a3
b2
a3
a6
a6
a6
a3
b4
b4b4
b4b4
b4
b4
a3
b4
a3
b4
a3
b2
b2
32
33
34
b4
b4b3
b3b4
b4
b4 b3 b4
a3 a3
b6
b6
UDP-Gal
B4GalT1
b6
a3 a3
a3 a3
b-Man-ase
a6
b2
a3
b2 a6
a3
a6
b4
b4
a3
b4
a6
b2
a3
a6
a6
b4
b4
b4
b4
b4 b4
b4
a3
b4
b2
b2
b2
35
36
a3
(3)
Scheme 2. Enzymatic extension of precursor glycans 8 and 9 to give asymmetric complex glycan 3.
hydroxide (Pd[OH]2) which afforded the required tri-antennary
glycans containing core xylose. The non-xylosylated
hexasaccharide core 4b was extended by a similar glycosylation
sequence to generate glycan 9 (See SI, Scheme S3).
Compounds 8 and 9 were treated with Helix pomatia β-
mannosidase and the inhibitor 1-deoxyfuconojirimycin,[22] to avoid
the cleavage of core a-fucoside, to yield 28 and 30, respectively
(Scheme 2). The two GlcNAc termini of the a6-arm were
simultaneously galactosylated by B4GalT1 and UDP-galactose to
give derivatives 29 and 31 having terminal LacNAc moieties. The
latter glycans were fucosylated at all three arms by using FUT5
which modified the LacNAc and Lac-di-NAc substrates, affording
compounds 1 and 2. The synthesis of asymmetric glycan 3 started
by treatment of 9 with FUT5 to transform the LDN moiety into
LDN-F epitope providing 32. A key point in our chemoenzymatic
strategy was the capping of the a6b2-arm by a mannosyl moiety,
and thus exposure of 32 to B4GalT1 resulted in the selective
galactosylation of the a6b6-arm. Exposure of the resulting
compounds to B3GNT2 and then B4GalT1 resulted in the
installation of a di-LacNAc moiety to give compound 34. The di-
LacNAc moiety was transformed into LexLex epitope by treatment
with FUT5 to give 35. The GlcNAc residue of the a6b2-arm of 35
was unmasked by treatment with b-mannosidase to give 36 which
was galactosylated by B4GalT1 to provide target compound 3.
8
NMR studies. The interaction of DC-SIGN with the xylosylated
(1) and non-xylosylated (2) glycans was examined by NMR.
Saturation Transference Difference (1H-STD-NMR) experiments
were performed using recombinant extracellular domain (ECD) of
DC-SIGN as receptor, which is organized as a tetramer in solution.
5
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10.1002/anie.202105647
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RESEARCH ARTICLE
The resulting 1H-STD NMR profiles for 1 and 2 were very similar
(Fig. 3a and b). No signals originating from the xyloside were
observed in the 1H-STD spectrum of compound 1, indicating that
this residue does not participate in lectin binding. Comparing the
1H-STD profiles of the complex glycans 1 and 2 with those of Lex
(C1) showed that the contact between the lectin and glycans 1
and 2 takes place exclusively at the terminal epitopes (Fig. 3c).
Due to severe 1H NMR signal overlap, untangling a possible
preference of DC-SIGN for the two Lex moieties and the LDN-F
epitope (Fig. 1) at the different branches of the glycans was
challenging. However, for compound 1, the NMR signals of the
acetyl moieties at the GlcNAc residues of the three arms appear
at different NMR chemical shifts, allowing unequivocal
assignment by NOE spectroscopy (Fig. 3a). The results from the
1H-STD NMR experiment showed that all GlcNAc residues
contribute equally to the binding. A model of the two glycans in
solution further supported that the three branches are equally
accessible for lectin binding (Fig. 3d, 3e).
a
b
(1)
(2)
Off-Resonance
Ac-GlcNAc/GalNAc
H4Fuc
H4Fuc
H4Fuc
H4Fuc
H2Fuc
H4Ga
H1Fuc
H2Fuc
H4Gal
Ac-GlcNAc/GalNAc
1H-STD NMR
H1Fuc
5
4
3
2
5
4
3
2
ppm
Ac-GlcNAc/GalNAc
H1-Fuc
* * *
Ac-GlcNAc
Ac-GalNAc
*
*
*
Man3
NOE
NOE
H1-Fuc/Ac-GlcNAc
* * *
2×
H1-Fuc / Ac - GlcNAc
GlcNAc1a
1×
GlcNAc2
2.1
2.2
2.1
2.0
2.2
2.1
2.0
5.3
5.2
5.1
2.2
2.1
2.0
c
d
e
Lext (C1)
arms
xylose
H2Fuc
H4Fuc
Ac-GlcNAc
H4Gal
TRIS
core
H1Fuc
MeFuc
5
4
3
2
ppm
Figure 3. 1H STD-NMR spectra obtained for the complexes of DC-SIGN (ECD) with (a) glycan (1), (b) glycan (2) and (c) the minimal epitope: the Lex trisaccharide
(C1). The specific and unique 1H-STD NMR signals are highlighted, along with the NOE cross peaks employed for their assignment. In particular the 2D sections
correspond to the NOE correlations between the anomeric protons of the fucose residues and the methyl protons of the corresponding acetamide moieties for each
of the three arms. The 1H-STD NMR profiles are derived from the double difference between the STD spectrum in the presence and in absence of the protein.
Irradiation frequency was set at 0.4 ppm. Protein saturation was achieved with a Gaussian-shaped pulse of 49 ms (Gauss 1.1000, with a power of 1e-05 W. Water
suppression was applied by using the excitation sculpting. (d) and (e). Representative 3D models for glycan (1) and (2) respectively, obtained from MD simulations
analysis. The models support the accessibility of the three arms for providing interactions with the lectin. Alternative views are provided in the SI Fig. S8.
6
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S. mansoni often presents extended N-acetyl lactosamine
(Galβ1,4-GlcNAcβ1,3-Galβ1,4-GlcNAc; poly-LN) chains which
can be modified by fucosylation to form poly-Lewisx moieties
(poly-Lex).[23] The asymmetric glycan 3 presents an extended arm
with two repeating Lex motifs, an LDN-F epitope and a non-
antennary glycans 1, 2 and 3 showed similar CSPs, which did not
extend beyond the canonical carbohydrate binding site,
supporting that despite the complex structures, there are no
additional contacts between the protein and glycans. However,
the induced CSPs were significantly stronger than those observed
1
[25]
fucosylated LacNAc epitope (Fig. 1). The H-STD NMR analysis
for LDN-F (C3)[24] and Lex (C1)
for the same number of
of 3 with DC-SIGN (ECD) (Fig. 4a) showed a similar profile as
observed for glycans 1 and 2 indicating that the terminal Lex and
LDN-F epitopes are equally well recognized. In contrast, the STD
signals from the internal Lex motif were barely visible, indicating
this moiety is less available for lectin recognition. This notion was
further assessed by analyzing the interaction of the
hexasaccharide C2 (di-Lex, Fig. 1) to DC-SIGN (Fig. 4b). The
simpler NMR spectra for this compound allowed a clear
differentiation of the contributions of the internal and terminal Lex
moieties. In particular, the 1H-STD intensities from the internal Lex
moiety only accounted for ~15% of those from terminal Lex.
Next, the interactions of DC-SIGN with the complex glycans 1, 2
and 3 were examined from the protein perspective using receptor-
based NMR methods. To this end, the chemical shift perturbation
(CSP) analysis of the 15N labeled DC-SIGN carbohydrate
recognition domain (CRD) was performed (SI section 5.5). The tri-
equivalents.
To confirm that the multi-antennary glycans bind stronger than
their monovalent counterparts, we used 2-fluoro-fucose (2-F-Fuc)
as probe in competition NMR experiments to determine relative
binding affinities. The addition of the lectin to the fluorinated
probe causes a dramatic decrease in 19F transverse relaxation
time (T2) with respect to the free form, which is indicative of 2-F-
Fuc binding. Next 0.5 equivalent with respect to the probe of
compounds 1, 2 and 3 were added. The addition of a competitor
molecule causes a recovery in T2 for the fluorinated probe, which
is proportional to the relative affinities of the tested compounds
(Fig. 5). Although subtle differences were observed, the results
indicate that the three glycans bind DC-SIGN with a similar affinity.
a
b
A
B
Off-Resonance
1H-STD NMR
(3)
diLeX (C2)
H4Fuc
H4Fuc + H4Fuc
H4Fuc
Ac-GlcNAc/GalNAc
Ac-GlcNAc
H1Fuc
A
H4Gal
H2Fuc
H2Fuc
H4Gal
H1Fuc
Me-Fuc
Me-Fuc
B
5
4
3
2
5
4
3
2
ppm
H1-Fuc
Man3a
Ac-GlcNAc/GalNAc
H4-Fuc
Ac-GlcNAc
*
*
A
B
B
A
*
*
*
*
*
*
*
*
NOE
H1-Fuc / Ac - GlcNAc
GlcN2
GlcN1a
2.11
2.07
ppm
5.20
2.10
3.85
2.10
Figure 4. 1H STD-NMR spectra recorded for the interaction of DC-SIGN (ECD) with (a) glycan (3) and (b) diLex hexasaccharide (C2). The specific and unique 1H-
STD NMR signals are highlighted, along with the NOE cross peaks employed for their assignments. Irradiation frequency was set at 0.4 ppm. Protein saturation
was achieved with a Gaussian-shaped pulse of 49 ms (Gauss 1.1000, with a power of 1e-05 W. Water suppression was applied by using the excitation sculpting.
Next, we compared the relative affinity of the synthetized multi-
antennary N-glycans with respect to the monovalent counterpart.
Thus, in a new sample tube containing 2-F-Fuc and DC-SIGN
(ECD), we added 0.5 equivalents of LDN-F (C3), again with
respect to the fluorinated probe. In this case, the competition with
the monovalent compound caused only a marginal recovery in
transverse relaxation time (T2), which indicates a much lower
affinity of LDN-F (C3) for the lectin. Because the multivalent
molecules contain three putative epitopes for lectin binding, we
further added the compound LDN-F (C3) to reach 1.5 equivalents
with respect to the fluorinated probe, equalizing in this way the
concentration of binder epitopes with respect to the multivalent N-
7
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glycans. Nevertheless, the recovery in T2 transverse relaxation
time of the fluorinated probe is lower than that observed using the
multivalent ligands, which demonstrate that
presentation enhances glycan-lectin binding.
a multivalent
Multivalency is often attributed to a favorable spatial organization
of a multivalent ligand that that can make multiple interactions with
a multivalent protein. DC-SIGN is a tetramer with their CRD
binding sites separated by ∼40 Å.[26] The spatial distance between
two terminal epitopes at the same glycan was estimated as
approx. 20 Å (Fig. 3d and 3e). Thus, the possibility that one glycan
can simultaneously bind to two different CRDs within the same
tetramer through a chelating effect is unlikely. It has been
proposed that “rebinding” can also contribute to cooperativity,[27]
and in this model, as soon as a single ligand-receptor complex
dissociates, the presence of another ligand will increase the
probability of another binding event. It is likely that the trimeric
glycans exhibit higher affinities due to rebinding.
Figure 5. 19F-NMR relaxation filter experiments performed for the fluorine-
containing monosaccharide 2-fluoro-fucose (2F-Fuc) in absence and in
presence of the tetrameric lectin DC-SIGN. The addition of competitor
molecules, 1-3 and C3 causes a recovery in the transverse relaxation time (T2)
for the fluorinated probe, which is proportional to the relative affinities of the
tested compounds.
Multivalent presentation and lattice formation. It is possible
that the antenna of compounds 1-3 engage with more than one
DC-SIGN tetramer to form a higher ordered complexes.[28] To
tetramers by the multivalent glycans. In fact, we observed protein
aggregation in the sample containing glycans 1, 2 and 3 but not
in those containing the monovalent ligands LeX (C1) and LDN-F
(C3), (Fig. S15). These observations further support our
hypothesis that multi-antennary glycans may be able to cross-link
DC-SIGN (EDC) tetramers. Molecular modelling studies support
that the glycans have appropriate geometries to engage with two
DC-SIGN tetramers (Fig. 6g-h). The 3D model of DC-SIGN ECD
assembled into tetramers was generated as previously described
[13] (see SI, Section 6.3).
examine such
a mode of binding, transmission electron
microscopy (TEM) was employed to provide support for the
existence of glycan-lectin complexes. The apo DC-SIGN (ECD)
was used as control. In this format, lectin alone gave a soluble
monodisperse organization (Fig. 6a-c). Conversely, incubation of
the multivalent glycans 1 and 2 with DC-SIGN (ECD) generated a
tangled network (Fig. 6d-e). It is likely, the aggregates arise from
the cross-linking of two or more DC-SIGN CRDs at different ECD
8
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Figure 6. Electron microscopy of DC-SIGN interacting with multivalent glycans. Cryo-TEM images at 50,000x magnification of apo DC-SIGN (a) and in the presence
of compound 1 (d) and 2 (e). (b) apo DC-SIGN visualised by negative-stain; the white circles mark the individual molecules. Scale bar is 100 nm. (c) three class-
averages resulting from 2D classification of extracted particles from negative-stain 2D images and indicating the tetrameric oligomerization of DC-SIGN (see SI). (f)
3D model of DC-SIGN ECD assembled into tetramers. (g) 3D model of DC-SIGN CRD showing the interaction with compound 1 as a single molecule. (h) 3D models
of DC-SIGN ECD (assembled into tetramers) showing the putative interaction with compound 1 and forming an inter-connected network.
epitopes. Xylosylated glycans are highly expressed from the egg
to cercaria stage of development, but substantially decreases
during the schistosomula and adult worm stages.[8] The stage-
Conclusion
S. Mansoni expresses fucosylated glycans that can be
specific expression of core xylosylated glycans implies a role in
snail–schistosome interactions but may be less relevant during
intra-mammalian stages of development. The NMR studies also
uncovered that the multi-antennary glycans bind with higher
affinity to DC-SIGN compared to mono-valent minimal epitopes.
The STD experiments indicated that only the terminal epitopes of
compounds 1-3 engage with DC-SIGN and no further interactions
were observed between protein and glycan. Modeling studies
showed that the distance between the terminal epitopes is too
short to engage with two different CRDs within the same tetramer.
Thus, it is unlikely that avidity enhancement occurs through a
classical chelate effect.[30] Although enhancement of affinity
through proximity-induced effective concentrations has received
relatively little attention, it is likely that the higher affinities of 1-3
can be accounted to such an effect.
Multiple biological properties of DC-SIGN, such as antigen uptake
and processing, can be attributed to receptor clustering. Recently,
it was shown that DC-SIGN mediated uptake of glycoconjugates
by immature moDCs does not directly correlate with their
affinity,[14] and instead it appeared that the size of the cluster is
critical for antigen uptake and routing.[31] Based on this study, we
propose that multi-antennary glycans may be able to cross-link
DC-SIGN (EDC) tetramers into a dense network, where the nodes
are tetramers of DC-SIGN connected through the multivalent
glycans. Physiologically, DC-SIGN is embedded in the cellular
membrane and, recently, the relevance of proper protein
recognized by DC-SIGN on the cell surface of DCs. The
structures of these antigenic glycans and their density on
pathogen surface probably represents a first barrier for host
detection. The molecular basis of complex formation between
DC-SIGN and glycans is not well understood. Moreover, little is
known about a possible preference of DC-SIGN for S. mansoni
derived glycans, and it is unclear whether glycan complexity can
modulate binding. The latter is due to difficulties of synthesizing
highly complex glycans expressed by S. Mansoni required for
structural and functional studies. Current approaches are limited
to relatively simple epitopes or symmetrical structures.[23-24],[28] To
address these limitations, we have developed a chemoenzymatic
approach that can provide N-glycans derived from S. mansoni
including compounds having core xylose and antenna with
unusual structures such as Lac-di-NAc and Lex-Lex. The
compounds made it possible to examine the influence of glycan
complexity on DC-SIGN recognition. It revealed that the core
xyloside does not influence terminal epitope presentation and
lectin binding. Recently, the interaction of DC-SIGN with
symmetric bi-antennary glycans with and without core-xylose was
examined by microarray technology.[29] It showed core xylose
abolished lectin binding for the inner-core mannosyl residues.
Together with the NMR data presented here, it supports a model
in which the xyloside masks or distort the conformation of the
glycan-core but does not impact the presentation of the Lex
9
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RESEARCH ARTICLE
presentation was demonstrated.[28] Although our in-solution
studies do not represent N-glycan presentation on the pathogen
surface, it is conceivable that lectins and glycans presented at a
surface can form similar latices and that the branched nature of
N-glycans contribute to cluster size, which in turn will impact
update and intracellular transport. Future studies will focus on
investigating DC-SIGN clustering and internalization using nano-
or micro-structures, decorated with the types of complex glycans
presented herein. The complex glycans will also be important to
unravel structural elements required for triggering different
signaling pathways during S. mansoni infection, as well as for the
further developing of glycan-based vaccines.
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.), Ramón y Cajal (RYC)
Contract (to A.A), the Agencia Estatal Investigacion of Spain (AEI;
grants RTI2018-095700-B-I00 (to NGAA) and RTI2018-094751-
B-C21) and the Severo Ochoa Excellence Accreditation (SEV-
2016-0644). The staff of the Electron Microscopy Platform at the
CIC bioGUNE and David Gil-Carton are acknowledged for
valuable technical assistance. Dr. Sean Connell is acknowledged
for the support in the 2D classification of DC-SGN ECD tetramers
(EM). Dr. Prof. Jesús Jiménez-Barbero is acknowledged for
insightful discussions, suggestions and comments.
Keywords: Glycan • Chemoenzymatic • Glycosyl transferase •
NMR • Cryo-EM.
10
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Entry for the Table of Contents
The molecular recognition of chemoenzymatically synthesized glycans derived from the
parasitic worm S. mansoni by the human lectin DC-SIGN was investigated by a combination
of NMR, computational and electron microscopy. It uncovered the importance of glycan
complexity for lectin recognition including the influence of glycan valency, core xylosylation
and length of oligo-LacNAc residues.
12
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