Asymmetric Total Synthesis of Mycobacterial Diacyl Trehaloses Demonstrates a Role for Lipid Structure in Immunogenicity

Article abstract of DOI:10.1021/acschembio.0c00030

The first asymmetric total synthesis of three structures proposed for mycobacterial diacyl trehaloses, DAT1, DAT2, and DAT3 is reported. The presence of two of these glycolipids, DAT1 and DAT3, within different strains of pathogenic M. tuberculosis was co

Full text of DOI:10.1021/acschembio.0c00030

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Article
Asymmetric Total Synthesis of Mycobacterial Diacyl Trehaloses
Demonstrates a Role for Lipid Structure in Immunogenicity
Mira Holzheimer, Josephine F. Reijneveld, Alexandrea Ramnarine, Georgios Misiakos, David C. Young,
Eri Ishikawa, Tan-Yun Cheng, Sho Yamasaki, D. Branch Moody, Ildiko Van Rhijn, and Adriaan J. Minnaard
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.0c00030 • Publication Date (Web): 15 Apr 2020
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Asymmetric Total Synthesis of Mycobacterial Diacyl Trehaloses
Demonstrates a Role for Lipid Structure in Immunogenicity
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Mira Holzheimer,a Josephine F. Reijneveld,a,b,c, ‡ Alexandrea Ramnarine,b, ‡ Georgios Misiakos,a David
C. Young,b Eri Ishikawa,d,e Tan-Yun Cheng,b Sho Yamasaki,d,e D. Branch Moody,b Ildiko Van Rhijnb,c,*
and Adriaan J. Minnaarda,*
a Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
b Brigham and Women’s Hospital Division of Rheumatology, Immunology and Allergy and Harvard Medical School,
Boston, MA, 02115, USA.
c Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584
CL, Utrecht, The Netherlands.
d Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-
0871, Japan.
e Laboratory of Molecular Immunology, Immunology Frontier Research Center, Osaka University, Suita, Osaka, 565-0871,
Japan.
ABSTRACT: The first asymmetric total synthesis of three structures proposed for mycobacterial diacyl trehaloses, DAT1, DAT2 and
DAT3 is reported. The presence of two of these glycolipids, DAT1 and DAT3, within different strains of pathogenic M. tuberculosis
was confirmed, and it was shown that their abundance varies significantly. In mass spectrometry, synthetic DAT2 possessed nearly
identical fragmentation patterns to presumptive DAT2 from M. tuberculosis H37Rv, but did not co-elute by HPLC, raising questions
as the precise relationship of the synthetic and natural materials. The synthetic DATs were examined as agonists for signaling by the
C-type lectin, Mincle. The small differences in the chemical structure of the lipidic parts of DAT1, DAT2 and DAT3 led to drastic
differences of Mincle binding and activation, with DAT3 showing similar potency as the known Mincle agonist trehalose dimycolate
(TDM). In the future, DAT3 could serve as basis for the design of vaccine adjuvants with simplified chemical structure.
dimycolate,13,14 diacyl trehaloses (DAT)15-17 and pentaacyl
INTRODUCTION
trehaloses (PAT).15,18 Because of the chemical diversity of DAT
Mycobacterium tuberculosis (Mtb), the causative agent of the
and the potential for contamination of even small amounts of
disease tuberculosis (Tb), is responsible for the largest number
bioactive molecular variants, testing natural DAT compounds
of deaths worldwide by a single pathogen, killing an estimated
on cells for immune response is not reliable. To establish the
1.3 million people annually. The ability of Mtb to survive and
molecular structure of these compounds and enable further
persist in the host is estimated to result in billions of latently
biological studies, several of the compounds have been the
infected individuals worldwide, with a high incidence of
target of total synthesis. In DAT and PAT, both of which have
undiagnosed cases.1 After infection of macrophages, Mtb is able
escaped total synthesis until now, the trehalose core is acylated
to survive and replicate in host phagosomes, while withstanding
with the methyl-branched fatty acids mycosanoic,
the hostile acidic environment. The mycobacterial cell envelope
mycolipanolic and mycolipenic acid (Fig. 1).17,18
is one factor that contributes to the resilience of Mtb within host
cells.2 It is a multi-layered barrier, composed of many complex
lipids, glycolipids and glycoproteins, many of which are unique
to Mtb.3-5 It has been shown in the last decades that many of
these cell wall components have antigenic properties and/or
possess immunomodulatory functions. One class of these
mycobacterial cell wall components, comprising of di- and
polyacylated trehaloses, is suggested to be located on the outer
part of the mycobacterial cell wall.6 These trehalose-based
glycolipids are esterified with palmitic or stearic acid at the 2-
and 2’-position as well as with the Mtb-specific multimethyl-
branched acyl residues phthioceranic, hydroxyphthioceranic,
mycosanoic, mycolipanolic and mycolipenic acid. Important
examples are Ac2SGL,7-9 Sulfolipid-1,10-12 trehalose mono- and
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mycolic acid layer of the mycobacterial cell envelope.24,29-31
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However, in aerosol infection mouse models using DAT/PAT
deficient mutants, there were no observed differences in growth
compared to wild-type suggesting that DAT/PAT itself is not
necessary for Mtb survival.29
Recently, Mtb cell wall components – such as trehalose
dimycolate (TDM, also known as cord factor) – have been
identified as high affinity ligands for macrophage-inducible C-
type lectin (Mincle).32-34 Activation of Mincle results in
downstream expression of cytokines, chemokines and growth
factors and leads to recruitment of inflammatory cells to the site
of activation as a central part of the innate immune response to
Mtb.33 Several other Mtb cell wall glycolipids have been
identified as Mincle activators,34,35 and there is growing interest
in using these Mincle ligands for the development of novel
vaccine adjuvants.36
In 2017, it was demonstrated that a DAT-containing extract
from Mtb activated Mincle, too.35 We realized that, apart from
minute amounts of contaminants in natural isolates that can
influence the results, the activation of Mincle could very well
depend on the precise structure of the DAT. Therefore, we
sought to synthesize three different DATs with precisely
defined molecular structure and stereochemistry to study their
Mincle activating properties and to assess the influence of the
acyl substituents on Mincle binding. Furthermore, we aimed to
confirm the presence of these three DATs in different strains of
Mtb including clinical isolates.
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Figure 1 (A) Chemical structures of the mycobacterial diacyl
trehaloses DAT1-3. (B) Retrosynthetic analysis.
DAT was first isolated in 1989 by Daffé et al. and was
initially named SL-IV (Sulfolipid-IV) as the structure was
assigned first as 2,3-diacyl-trehalose-2’-sulfate.15,16 The
structure of this family of acyl trehaloses was eventually
revisited and corrected to be 2,3-diacyl-trehalose, and
depending on the nature of the 3-O-acyl group were termed
DAT1, DAT2 or DAT3 (Fig. 1A). In following reports these
compounds were often referred to as just DAT, presenting a
family of mycobacterial glycolipids rather than defined
molecular structures.17 Many studies have asserted the antigenic
properties of DAT glycolipids by ELISA but these were tested
mainly as mixtures rather than pure compounds. It was
demonstrated multiple times that anti-DAT antibodies are
present in blood sera of Tb patients but not of healthy
controls.17,19-22 This raised great interest in using DAT for the
detection and diagnosis of Tb in patients. The reports utilizing
ELISA for the detection of anti-DAT antibodies, however,
showed a huge variation in sensitivity and specificity depending
on assay design.23
RESULTS AND DISCUSSION
Synthesis. DAT1, DAT2 and DAT3 differ in their chiral acyl
group esterified with the 3-OH of the trehalose core. Therefore,
our synthesis plan involved the preparation of suitably protected
2-palmitoyl trehalose 1 and the three mycobacterial lipids 2, 3,
and 4 as key intermediates necessary to construct the target
diacyl trehaloses. Trehalose 1 could be obtained starting from
,-trehalose by a desymmetrization approach previously
applied in the synthesis of trehalose-based sulfoglycolipids.37,38
The mycobacterial lipids, on the other hand, can be traced back
to the common precursor 5 (Fig. 1B). The synthesis of
mycolipanolic and mycolipenic acid was previously reported by
us and involves copper-catalyzed asymmetric conjugate
addition (Cu-cat. ACA) and an Evans’ aldol reaction to
introduce the stereocenters.39 We sought to improve the current
synthetic procedures to arrive at an efficient, high-yielding total
synthesis.
In recent years, research has focused on elucidating the
biosynthesis of DAT and unravelling its effect on the immune
system.24-27 It was shown that DAT partially inhibits the
proliferation of murine T-cells, suggesting
a role in
immunosuppression and T-cell hyporesponsiveness associated
with Tb.28 Mtb mutants incapable of synthesizing mycolipenic
acid, and therefore deficient in DAT and PAT, show
aggregation in liquid culture due to defects in capsule
attachment, indicating that one of the functions of DAT and
PAT is anchoring the hydrophilic capsule to the hydrophobic
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(R,SFe)-Josiphos-CuBr (1 mol%)
(R,SFe)-Josiphos-CuBr (3 mol%)
1. DIBAL-H (1.2 eq)
O
MeMgBr (1.2 eq)
O
O
MeMgBr (1.2 eq)
O
CH2Cl2, –65 ¬C
1
2
3
4
OTBDPS
OTBDPS
OTBDPS
OTBDPS
EtS
EtS
EtS
EtS
MTBE, –78 ¬C
81%
MTBE, –78 ¬C
92%
dr > 20:1
2. HWE (1.6 eq)
n-BuLi (1.3 eq)
THF, 0 ¬C to rt
94% over 2 steps
5
6
7
8
98% ee
1. DIBAL-H (1.3 eq)
CH2Cl2, –65 ¬C
(2 times)
5
6
7
8
2. TsCl (2 eq)
pyridine (2 eq)
CH2Cl2, 0 °C to rt
88% over 3 steps
NaH2PO4 (4 eq)
2-methyl-2-butene (10 eq)
NaClO2 (10 eq)
OH
1. Mg (6.5 eq)
C16H33Br (6 eq)
THF, rt
1. TBAF (1.6 eq)
THF, rt
OTBDPS
OTBDPS
H
O
C16H33
C16H33
TsO
C16H33
9
2. DMP (1.3 eq)
CH2Cl2, rt
97% over 2 steps
t-BuOH/H2O, rt
O
2. CuBr•SMe2 (0.5 eq)
0 °C to rt
96%
11
10
9
mycosanoic acid 2
92%
10
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O
O
P
OEt
PCy2
PPh2
Wittig reagent (1.5 eq)
n-Bu4NOH (2 eq)
OEt
Fe
EtS
OEt
OH
O
C16H33
C16H33
PhCH3, reflux
THF, 0 ºC to rt
O
(R,SFe)-Josiphos
HWE
12
85%
94%
mycolipenic acid 4
Bn
Aux (1 eq)
Bu2BOTf (1.2 eq)
Et3N (1.3 eq)
PPh3
EtO
Bn
O
LiOH (1.5 eq)
H2O2 (14 eq)
O
N
O
N
OH
O
O
O
Aux
C16H33
C16H33
O
Wittig reagent
OH
13
THF/H2O, 0 °C to rt
OH
O
CH2Cl2
0 to –78 to 0 ¬C
82%
quant.
mycolipanolic acid 3
dr > 20:1
Scheme 1 Asymmetric synthesis of mycosanic (2), mycolipanolic (3) and mycolipenic acid (4).
of 97% over two steps. From 11 all three mycobacterial lipids
could be synthesized in a limited number of steps.
Mycosanoic acid 2 was obtained in 92% yield after Pinnick
oxidation of aldehyde 11. Mycolipenic acid 4 was prepared by
first subjecting 11 to a Wittig reaction followed by alkaline
ester hydrolysis. To install the two remaining stereocenters
present in 3, an Evans’ aldol reaction was performed giving
13 in good yield and excellent dr. The aldol product 13 was
finally hydrolyzed to give mycolipanolic acid 3. Compared to
the previous syntheses of 3 and 4 the yields could be
significantly improved by careful optimization of the
reactions. For mycosanoic, mycolipanolic and mycolipenic
acid excellent overall yields were obtained with 53% over 10
steps, 47% (previously 2%) and 46% (previously 5%) over 11
steps, respectively, making the synthesis of these chiral lipids
highly efficient. In the synthesis of mycolipenic acid,
oxidation, Wittig reaction and ester hydrolysis were
significantly improved whereas in the case of mycolipanolic
acid the Evans’ aldol reaction and the removal of the chiral
auxiliary were optimized to give high yields.39
RCOOH
Cl3C6H2COCl
Et3N
O
O
Ph
Ph
O
O
O
O
O
O
DMAP
HO
O
R
O
O
O
PhCH3 or THF, rt
14a: R1, 70%
14b: R2, 66%
14c: R3, 75%
O
O
C14H29
O
C14H29
O
O
O
Ph
Ph
O
O
O
O
O
O
Si
Si
Si
Si
O
O
1
TBAF/AcOH 1/1
THF, rt,
15a: R1, 96%
15b: R2, 78%
15c: R3, 96%
aq. H2SO4
HO
HO
O
O
Ph
O
O
O
CHCl3/MeOH, rt
O
O
O
O
R
R
O
or Pd/C, Pd(OH)2
H2 (1 atm)THF, rt
DAT1a: R1, 45%
DAT2a: R2, 66%
DAT3a: R3, 78%
O
O
O
O
O
C14H29
OH
C14H29
O
O
Ph
OH
O
HO
HO
OH
OH
R1
=
R2
=
R3
=
C18H37
C18H37
C18H37
OH
With the enantiopure acids 2-4 in hand, the esterification of
palmitoylated trehalose 1 was achieved following the
Yamaguchi procedure. In the case of mycosanoic and
mycolipenic acid the corresponding diacylated products were
obtained in good yields but in order to reach that result for
mycolipanolic acid, the esterification procedure needed to be
carefully optimized to avoid acyl migration and elimination of
the -hydroxyl of 3. By limiting the number of equivalents of
base and keeping the time for acid activation at a
Scheme 2 Completion of the total synthesis of the mycobacterial
glycolipids DAT1-3
.
The synthesis of the chiral enantiopure lipids 2, 3 and 4
(Scheme 1) commenced with Cu-cat. ACA of
methylmagnesium bromide to ,-unsaturated thioester 5
giving 6 in 81% yield and 98% ee. Reduction to the
corresponding aldehyde followed by Horner-Wadsworth-
Emmons reaction produced another ,-unsaturated thioester
7. The second methyl stereocenter was again introduced by
Cu-cat. ACA in excellent yield and dr. Double DIBAL-H
reduction followed by tosylation gave 9 in 88% yield over
three steps. Tosylate 9 was subjected to a Grignard cross-
coupling in the presence copper(I) to install the linear alkyl
tail of 10. Removal of the silyl protecting group followed by
Dess-Martin oxidation gave aldehyde 11 in an excellent yield
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natural compounds (supplemental data) yielded fragmentation
patterns diagnostic for the known structures.
A
948.734
H37Rv
1
2
3
4
5
6
7
8
8
6
4
2
0
2
1
0
A
B
mouse Mincle + FcRγ
100
80
60
40
20
0
950 952
solvent
m/z
front
j011
j117
j257
TDM
DAT
0
0
0
1
DAT
2
DAT
3
6
7
time (min)
1006.774
0
200
400
600
H37Rv
lipids (ng/well)
2
1
0
9
human Mincle + FcRγ
100
80
60
40
20
0
C
1
10
11
12
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1008 1010
0
0
TDM
DAT
m/z
j011
1
DAT
2
j117
j257
DAT
0
0
3
988.767
988.767
1
0
origin
7
8
time (min)
1
0
200
400
600
2
3
T
H37Rv
M
T
T
A
M
G
A
A
lipids (ng/well)
2
2
1
0
D
D
D
1
988.762
988.766
D
0.9
hIg
0
0
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
2
0
mMincle-hIg
j011
j117
0
0
2
0
j257
989 991
m/z
7
8
time (min)
B
DAT-1
DAT-2
DAT-3
m/z 988.766
m/z 988.766
m/z 1006.776
ng/well
H37Rv lipids+
syntheticstandards
1
1
0
no lipid
DAT
DAT
DAT
3
TDM
1
2
1
0
H37Rv lipids
0
Figure 3 Synthetic DAT3 is recognized by human and mouse
Mincle. (A) Before functional assays, DAT1, DAT2, and DAT3
were analyzed by thin-layer chromatography for relative
quantification and the presence of major breakdown products. (B
and C) NFAT-GFP reporter cells expressing mouse Mincle +
FcR or human Mincle +FcR were stimulated with the indicated
amount of DAT1, DAT2, DAT3 or TDM. After 24 hours,
induction of NFAT-GFP was analyzed by flow cytometry. (D)
ELISA-based detection of DAT1, DAT2, DAT3, or TDM by
mouse Mincle-human Ig Fc (mMincle-hIg) fusion proteins.
Bound protein was detected with anti-human Ig-horse radish
peroxidase (HRP) followed by addition of colorimetric substrate
and measurement.
6.6
7.0
7.2 7.6
6
7
time (min)
C
minimum, synthesis of 14b could achieved in good yield.
Notably, in the case of 14a and 14c, no acyl migration was
observed, indicating that the -hydroxyl in 3 might play a role
in acyl migration. Removal of the silyl protecting group of
14a-c under buffered conditions gave the corresponding diols
15a-c in good to excellent yields. The final deprotection – the
removal of the benzylidene protecting group – was achieved
applying a procedure reported by Guiard et al.37 using
aqueous sulfuric acid (DAT1 and DAT3) or by palladium
hydrogenolysis (DAT2, to prevent -hydroxyl elimination)
and provided the three di-O-acyl trehaloses DAT1-3 in
moderate to good yields. The spectral data of DAT1 matched
the reported NMR data of isolated DAT1a.17 Besra’s report
Figure 2 Detection of DAT variants in M. tuberculosis strains.
Lipid extracts from four different M. tuberculosis strains were
analyzed by HPLC-MS: laboratory strain H37Rv, and three
clinical isolates named j257, j011, and j117. Extracted ion
chromatograms of ions corresponding with the ammonium
adduct of DAT1 (calculated m/z = 948.733), DAT2 (m/z =
1006.775), and DAT3 (m/z = 988.764) showed m/z values
consistent with those expected from DATs. (B) Comparison with
synthetic standards showed chromatographic coelution for DAT1
and DAT3 but not for DAT2 indicating that synthetic DAT2 is not
identical to natural DAT2. (C) CID analysis of the standards and
1
describes the H-NMR signals of the anomeric protons of
DAT1 at 5.25 and 5.05 ppm for the acylated and non-acylated
glucose unit, respectively. The spectrum of synthetic DAT1
shows these two anomeric signals at 5.24 and 5.06 ppm,
which is in good agreement. Furthermore, H-2 and H-3 (at the
positions bearing the acyl moieties) in natural DAT1 appear at
4.83 and 5.40 ppm, respectively, and in synthetic DAT1 at
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4.82 and 5.39 ppm. The 13C signals of the anomeric carbons
remarkably, DAT1 only weakly activated murine Mincle.
When using human Mincle expressing cells, DAT2 showed
moderate activation whereas DAT1 again barely induced
Mincle stimulation. In an independent experiment, an ELISA-
based technique was applied that was not dependent on
cellular activation but only on detection of physical
interaction between DAT and soluble Mincle proteins (Fig.
3D). Strong binding of murine Mincle to DAT3 was observed,
but only minimal binding to DAT1 and DAT2, thereby
confirming the results obtained in the cellular activation
assay. These results provide evidence that the chemical
structure of the 3-O-acyl substituent (either mycosanoic,
mycolipanolic, or mycolipenic acid) strongly influences
Mincle binding and activation.
1
2
3
4
5
6
7
8
in natural DAT1 are reported at 95.0 ppm and 92.0 ppm. In the
synthetic material these signals can be found 94.6 ppm and
91.7 ppm, again in good agreement. Additionally, the
carbonyl carbon signals in synthetic DAT1 resonate at 173.5
and 177.6 ppm and the corresponding signals in natural DAT1
can be found at 173.8 and 177.8 ppm. All in all these data
leaves us confident that the structure of synthetic DAT1 is
identical to natural DAT1 as described by Besra (for more
detailed NMR signal comparison, see supporting
information). As for synthetic DAT2 and DAT3, the structural
identity is beyond reasonable doubt because the structures of
the lipid components have been previously established39 and
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1
the H, 13C NMR and mass spectra showed patterns very
similar to synthetic and natural DAT1.
CONCLUSIONS
Detection of DAT1-3 in Mtb strains. Having completed the
total synthesis of DAT1, DAT2 and DAT3 with structures as
described in the literature,17 we sought to determine if the
synthesized glycolipids match the structures of natural
products present in virulent strains of Mtb. Mycobacterial
lipid extracts of the reference strain H37Rv and three clinical
isolates j257, j011 and j117 were analyzed by means of LC-
MS. The extracted-ion chromatograms (Fig. 2A) suggest that
all three DATs are produced by the laboratory strain H37Rv.
Ions consistent with DAT1 and DAT2 were only reliably
detected in the H37Rv strain whereas DAT3 could be detected
in all four strains. The corresponding mass spectra of the
detected natural DATs matched the calculated m/z of each
compound within the expected experimental error of 3-4 ppm.
Collision-induced fragmentation (supplemental data) of the
natural and synthetic DATs yielded interpretable cleavages
(Fig. 2C, H-transfers not shown) that supported the general
In this study, we have accomplished the first total synthesis
of three structurally related mycobacterial DATs. These
synthetic DATs were used as a reference in the detection of
natural DATs in Mtb by LC-MS. This showed that the
presence and abundance of DAT1 and DAT3 differs strongly,
dependent on the Mtb strain. This has important consequences
for the potential use of DATs as markers for Tb infection. In
addition, it might explain a posteriori the irreproducibility
observed in the many attempts to reliably detect DAT by
ELISA. It also showed that the proposed structure of DAT2
does not occur in the studied strains, including the H37Rv
strain. An alternative explanation is that the structure of DAT2
has been assigned incorrectly, as due to the lack of literature
NMR data, a comparison with our synthetic material was not
possible. This will be further investigated.
We found that small changes in the structure of the
branched acyl chain in DAT lead to large differences in
recognition by Mincle. It has been shown previously by
Decout et al. that one of the molecular requirements for
Mincle recognition, besides the trehalose or glucose scaffold,
is the presence of two alkyl chains, either as two separate
esters or as one fatty acid ester with an alkyl chain branched
structures and connectivity. Co-injection (Fig. 2B) of
synthetic standards and natural lipid mixtures showed a
chromatographic match for DAT1 and DAT3. However,
synthetic DAT2 eluted more than a minute earlier than the
natural compound. Thus, whereas the identity of DAT1 and
DAT3 can be considered to have been established beyond
reasonable doubt, we conclude that material with the
structure of synthetic DAT2 does not occur in the H37Rv
strain. This may mean that an isomer of the proposed
structure of DAT2 is present in this strain, or that the
structure of natural DAT2 has been incorrectly assigned.15

to the carbonyl. Additionally, it was previously
demonstrated that the lipid chains can be significantly shorter
than the C80 lipids present in TDM. For instance the synthetic
Mincle ligand GlcC14C18, a glucose esterified at the 6-
position with a C18 alkyl tail with a C14 alkyl branch on the
-position shows even higher potency than TDM.35
Additionally, in a previous report Mincle activation by β-
glucosylceramide was demonstrated, which also contains an
unsaturation in the lipid chain.40 Here, we show that the
presence of the ,-unsaturation in DAT3 enhances Mincle
activation drastically compared to the saturated counterpart
DAT1. This leads us to speculate that the double bond serves
either as a point of interaction (such as --stacking) with
parts of the Mincle binding pocket, or induces a specific
conformation beneficial for binding. All in all, one might
conclude that DAT3 could be an alternative starting point for
adjuvant design for TDM given the higher complexity and
lipophilicity of the latter. For future development of Mtb
vaccine adjuvants even simpler DAT-analogues could be
designed without chiral methyl branches or based on glycose
rather than trehalose.
Mincle activation by DAT1-3. We decided to assess the
Mincle activating properties of the synthetic DATs (Fig. 3) as
well, keeping in mind that our synthetic DAT2 was not present
in the studied Mtb strains. Mincle activation was compared to
the known Mincle-agonist TDM, which is highly potent.
Previous studies have identified various lipid-like trehaloses
that activate Mincle, so we expected that all three forms of
DAT, which differ in small ways in their alkyl chains, were
good candidate activators. Prior to the functional assays, TLC
analysis of synthetic DAT1, DAT2 and DAT3 was performed
to exclude the presence of glycolipid degradation products
and quantification errors (Fig. 3A). Mincle activation requires
the adaptor protein FcR. Therefore, functional Mincle-
activation assays were performed by treatment of NFAT-GFP
reporter cells expressing murine Mincle and FcR (Fig. 3B)
or human Mincle and FcR (Fig. 3C) with the synthetic DAT
variants and TDM. In both assays, DAT3 was able to activate
Mincle. In the case of human Mincle, DAT3 showed similar
potency to the highly potent agonist TDM. DAT2 and,
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Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
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AUTHOR INFORMATION
Corresponding Author
* (A.J.M.) E-mail: a.j.minnaard@rug.nl
* (I.V.R.) E-mail: i.vanrhijn@uu.nl
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Author Contributions
‡These authors contributed equally.
Funding Sources
This work was supported by the Dutch Science Foundation
(NWO), the Bill and Melinda Gates Foundation and AMED
(JP19gm0910010 and JP19ak0101070).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank T. Tiemersma, R. Sneep, and P. van der Meulen
(University of Groningen) for assistance on analyses.
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