Cholesteryl glucosides signal through the carbohydrate recognition domain of the macrophage inducible C-type lectin (mincle)

Article abstract of DOI:10.1039/d0ob02342f

Cholesteryl α-d-glucosides (αGCs) are unique metabolic products of the cancer-causing human pathogenHelicobacter pylori.Viasignalling through the Macrophage inducible C-type lectin (Mincle) and the induction of a pro-inflammatory response, they are thought to play a role in the development of gastric atrophy. Herein, we prepared the first library of steryld-glucosides and determined that they preferentially signal through the carbohydrate recognition domain of human Mincle, rather than the amino acid consensus motif. Lipidated steryld-glucosides exhibited enhanced Mincle agonist activity, with C18 cholesteryl 6-O-acyl-α-d-glucoside (2c) being the most potent activator of human monocytes. Despite exhibiting strong Mincle signalling, sito- (5b) and stigmasterol glycosides (6b) led to a poor inflammatory response in primary cells, suggesting that Mincle is a potential therapeutic target for preventingH. pylori-mediated inflammation and cancer.

Full text of DOI:10.1039/d0ob02342f

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Cholesteryl glucosides signal through the
carbohydrate recognition domain of the
macrophage inducible C-type lectin (mincle)†
Cite this: Org. Biomol. Chem., 2021,
19, 2198
Received 25th November 2020,
Accepted 21st January 2021
Mattie S. M. Timmer, *a Thomas J. Teunissen,a Kristel Kodar,a Amy J. Foster,a
Sho Yamasakib,c,d,e and Bridget L. Stocker*a
DOI: 10.1039/d0ob02342f
rsc.li/obc
Cholesteryl α-D-glucosides (αGCs) are unique metabolic products phatidyl-α-D-glucosides (αCPGs, 3), and monoacylated deriva-
of the cancer-causing human pathogen Helicobacter pylori. Via tives of αCPGs (4) (Fig. 1), which constitute ca. 25% of the
signalling through the Macrophage inducible C-type lectin lipid content of the bacteria.4–6 Collectively, αCG (1) and the
(Mincle) and the induction of a pro-inflammatory response, they αCAGs 2 account for over 80% of the cholesteryl-α-D-glucosides
are thought to play a role in the development of gastric atrophy. found in H. pylori.4 Notwithstanding, how H. pylori cholesteryl
Herein, we prepared the first library of steryl D-glucosides and glucosides influence the cancer-promoting capacity of the bac-
determined that they preferentially signal through the carbo- teria is a topic of much debate.7–14
hydrate recognition domain of human Mincle, rather than the
To provide further insight into the mechanisms by which
amino acid consensus motif. Lipidated steryl D-glucosides exhibi- glucosylated cholesterol metabolites influence the immune
ted enhanced Mincle agonist activity, with C18 cholesteryl 6-O- response, Yamasaki and co-workers isolated metabolites from
acyl-α-D-glucoside (2c) being the most potent activator of human H. pylori and determined that αCAGs 2a and 2b signalled
monocytes. Despite exhibiting strong Mincle signalling, sito- (5b) through the Macrophage inducible C-type lectin (Mincle),
and stigmasterol glycosides (6b) led to
a
poor inflammatory thereby promoting an inflammatory immune response.9 In
response in primary cells, suggesting that Mincle is a potential these studies, a decrease in the inflammatory response to
therapeutic target for preventing H. pylori-mediated inflammation H. pylori was observed in murine strains lacking αCgT.
and cancer.
Further-more, while the antibody-mediated neutralisation of
Mincle did not lead to an increase in bacterial burden in
murine models, inhibition of Mincle led to a reduction in
symptoms associated with gastritis.9 Insomuch, inflammation
mediated by glucosylated cholesterol metabolites may play a
role in the development of gastric cancer, an observation that
is in agreement with clinical studies where the activity of
αCgTs from H. pylori isolates was positively correlated with the
degree of gastric atrophy in patients.8 However, in the clinical
studies, increased αCgT activity resulted in fewer surviving bac-
teria.8 In contrast, the phosphorylated cholesteryl α-D-gluco-
sides (e.g. 3) did not signal through Mincle, but rather the den-
dritic cell immunoactivating receptor (DCAR).9
Helicobacter pylori is a bacterial pathogen that colonises the
stomach and is the leading cause of gastric ulcers, carcinoma,
and lymphoma.1,2 It is estimated that over 50% of the world’s
population is infected with H. pylori,3 and as such, there has
been much interest in deciphering how H. pylori promotes the
growth and development of gastric cancers. H. pylori senses
cholesterol rich regions in host epithelial tissue and, via the
agency of cholesterol-α-glucosyltransferases (αCgT), converts
cholesterol into cholesteryl α-D-glucoside (αCG, 1), cholesteryl
6-O-acyl-α-D-glucosides (αCAGs, e.g. 2a), cholesteryl 6-O-phos-
aSchool of Chemical and Physical Sciences, Victoria University of Wellington, PO Box
600, Wellington, New Zealand. E-mail: mattie.timmer@vuw.ac.nz
bDepartment of Molecular Immunology, Research Institute for Microbial Diseases,
Osaka University, SuitaOsaka 565-0871, Japan
cLaboratory of Molecular Immunology, Immunology Frontier Research Center
(IFReC), Osaka University, SuitaOsaka 565-0871, Japan
dDivision of Molecular Design, Medical Institute of Bioregulation, Kyushu University,
Fukuoka, Fukuoka 812-8582, Japan
eDivision of Molecular Immunology, Medical Mycology Research Center, Chiba
University, Chiba 260-8673, Japan
†Electronic supplementary information (ESI) available: Experimental procedures
and characterization data for all compounds. See DOI: 10.1039/d0ob02342f
Fig. 1 α-Linked cholesteryl glucosides.
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In view of the recent finding that αCAGs 2a and 2b are by the in situ generation of the glucosyl iodide and subsequent
Mincle agonists,9 we became interested in determining how coupling to the steroid of choice. The in situ generated TMS-
modifications to the cholesteryl glucoside scaffold influence protected cholesteryl α-D-glucoside was then subject to Dowex-
Mincle signalling and moreover, where such motifs bind H+ in MeOH to give αCG (1). Rather than precipitation and fil-
human Mincle. Mincle contains a carbohydrate recognition tration to remove TBAI from the reaction mixture,23 we
domain (CRD),15,16 which recognises pathogen-associated obtained higher product yields and purity by employing silica
molecular patterns,17 as well as a cholesterol recognition gel flash column chromatography (95 : 5, CH2Cl2/MeOH, v/v)
amino acid consensus (CRAC) motif18 that is found in hMincle followed by size exclusion chromatography (Sephadex LH-20,
but not murine Mincle,19 and which binds cholesterol metab- CH2Cl2/MeOH, 1 : 1). In this way, αCG (1) was prepared in 80%
olites.18 Thus, we sought to synthesise the two major choles- yield (over three-steps). The same methodology was then
teryl α-D-glucosides of H. pylori, αCG (1) and αCAG (2a),8,10 applied to the synthesis of the sterol analogues, with the gluco-
along with derivatives that contain different steroid backbones syl iodide being coupled to β-sitosterol (10b) or stigmasterol
or acyl chains (2c, 5a,b and 6a,b, Fig. 2). As H. pylori contain- (10c) to yield the TMS-protected steryl glucosides. Global de-
ing cholesteryl β-D-glucoside (βCG, 7) induced macrophage protection then gave β-sitosteryl α-D-glucoside (αSiG, 5a) in
phagocytosis yet bacteria containing αCG (1) did not,7 we were 78%, and stigmasteryl α-D-glucoside (αStG, 6a) in 85% overall
also interested in assessing the immune stimulating properties yields. In all instances, the α-selectively of the glycosylation
1
of βCG (7). The ability of the derivatives to signal through reaction was determined via H and 2D NMR (J1,2 = 3.8 Hz for
Mincle and CRAC- or CRD-mutants thereof would be deter- H-1α), with generally excellent α-selectivity being observed (ca.
mined using the nuclear factor of activated T cells (NFAT)- 9 : 1, α : β).
green fluorescent protein (GFP) reporter cells coupled to
FcRγ,20 while human-derived monocytes would provide an 435 and the appropriate vinyl ester.23,24 To this end, αCG (1)
indication of the compound’s functional immune response.21 was treated with Novozym 435 and vinyl myristate25 to provide
To prepare the steroid-based α-glucosides, an elegant pro- C14-αCAG (2a) in excellent (90%) yield following purification
cedure employing the use of TMS-protected glucosyl by silica gel flash column chromatography and size exclusion
Each steroid-glucoside was then acylated using Novozym
a
iodide,22 as developed by Gervay-Hague,23,24 was employed chromatography (Scheme 1A). Similarly, C18-αCAG (2c) was
(Scheme 1A). To this end, D-glucose (8) was treated with TMSCl prepared via the enzyme-catalysed acylation of αCG (1) using
in DMF to give per silylated glucose 9 in 70% yield, followed vinyl stearate,25 while the acylated β-sitosterol and stigmasterol
glycosides, αSiAG (5b) and αStAG (6b), respectively, were pre-
pared via enzyme-mediated coupling with vinyl myristate.
Next, βCG 7 was prepared via a TMSOTf-mediated glycosylation
reaction between cholesterol (10a) and imidate 1126 to provide
the β-product exclusively, with methanolysis furnishing gluco-
side 7 in 70% yield in 2 steps (Scheme 1B).
The ability of the steryl glucosides to signal through
mMincle or hMincle was then determined using 2B4
NFAT-GFP reporter cells expressing mMincle + FcRγ, hMincle
+ FcRγ, FcRγ (negative control), with trehalose dibehenate
(TDB) serving as the positive control (Fig. 3).27 The 6-O-acyl
steroid derivatives [αCAG (2a and 2c), αSiAG (5b), and αStAG
(6b)] showed good mMincle agonism at both 0.1 and 1 nmol
per well. Of the non-acylated derivatives, αCG (1) exhibited
Fig. 2 Target steryl D-glucosides.
good mMincle agonist activity at 1 nmol per well, αStG (6a) led
to modest mMincle activity, while αSiG (5a), βCG (7) and chole-
sterol (10a) did not signal through mMincle (Fig. 3A).
With the exception of cholesterol (10a), all derivatives acti-
vated hMincle reporter cells at both ligand concentrations
(Fig. 3B), with the non-acylated derivatives αCG (1), αSiG (5a)
and αStG (6a) eliciting slightly less activity than their acylated
counterparts [αCAG (2a), αSiAG (5b) and αStAG (6b), respect-
ively]. However, this difference was not as pronounced as that
observed when using mMincle reporter cells, which might be
attributed to the presence of both the CRD and the CRAC
motif in hMincle. Changes to the steroid backbone appeared
to have little effect on the ability of the steryl glucosides to
signal through hMincle. Cholesterol (10a) only led to a signifi-
Scheme 1 Synthesis of steryl D-glucosides.
cant increase in GFP production by hMincle reporter cells at a
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In contrast, more is known about the CRD in hMincle. This
contains a Ca2+ binding site that includes an EPN motif
involved in the binding of the 3- and 4-hydroxyls of one
glucose moiety of α,α′-trehalose.15 Mutation of this site into
glutamine-proline-aspartic acid (QPD), a putative galactose-
recognition sequence, abrogates the binding of α,α′-trehalose
containing glycolipids (i.e. trehalose dimycolate, TDM) to
Mincle.15
For our steryl glucosides, signalling through the EPN motif
appeared to dominate over the CRAC motif, although for some
steryl glucosides, this preference was less pronounced. For
example, βCG (7) could not signal through either mutated
Mincle receptor, which might reflect the lower binding prefer-
ence of this ligand for hMincle, while stigmasterol glycoside
(αStAG, 6b) signalled through both Mincle domains at higher
ligand concentrations. As has been previously reported, chole-
sterol (10a) was unable to activate hMincle with a mutated
CRAC motif18 but retained activity when using hMincle EPN →
QPD, while TDB signalled through the carbohydrate-binding
EPN motif27 but not to the CRAC motif.
Fig. 3 Ability of steryl glucosides to signal through mMincle and
hMincle. (A) mMincle + FcRγ, (B) hMincle + FcRγ, and FcRγ only expres-
sing 2B4 reporter cells were incubated in plates coated with the pre-
pared ligands (0.1 or 1 nmol per well), TDB (0.1 or 1 nmol per well), or
iPrOH only. After 20 hours, the cells were harvested and analysed for
GFP expression by flow cytometry. Data represents the mean ( SEM) of
three independent experiments performed in duplicate. Statistical sig-
nificance was calculated in comparison to iPrOH only using 2-way
Next, functional immune responses elicited by the steryl
glucosides were investigated. Human monocytes, which have
previously been shown to express Mincle,21 were purified from
whole blood and stimulated with 40 or 80 μM of the steryl
α-glucosides or TDB (positive control), or untreated (negative
control). The secretion of IL-8, a chemokine involved in neu-
trophil chemotaxis, as well as proinflammatory cytokines inter-
leukin (IL)-1β and tumor necrosis factor (TNF)-α, were then
determined by ELISA (Fig. 5). All ligands, except for the non-
lipidated αStG (5a) and cholesterol (10a), led to IL-8 pro-
duction by human monocytes (Fig. 5A), with C18-αCAG (2c) eli-
citing the highest levels of IL-8 at both concentrations of
ligand tested. The strongest pro-inflammatory response was
elicited by monocytes stimulated with αCAG (2c), which con-
tained a stearic acid ester at the 6-O-position of glucose, with
this ligand leading to the production of IL-1β and TNF-α at
both ligand concentrations (Fig. 5B and C). The non-acylated
glucoside αCG (1), and to a lesser extent the stigmasterol
derivative αStG (6a), also led to TNF-α production by human
monocytes when higher concentrations of ligand were used,
however neither ligand resulted in the production of statisti-
cally significant levels of IL-1β. While we cannot conclusively
say that cytokine production by human monocytes in response
to the steryl glucosides is solely dependent on Mincle, the
Mincle signalling observed in the reporter assays indicates that
monocyte activation is, at least in part, due to the engagement
of the steryl glucosides with hMincle.
ANOVA (Sidak’s multiple comparisons test), ***P
0.0001.
≤ 0.001; ****P ≤
concentration of 1 nmol per well.18 Taken together, hMincle
appears to better accommodate changes to the steryl
α-glucoside scaffold than mMincle, though for both Mincle
orthologues incorporation of a lipid chain at the 6-position of
glucose enhanced Mincle signalling. Moreover, βCG (7) only
signalled through human but not murine Mincle.
Given the relevance of H. pylori as a human pathogen, we
then sought to determine how the steryl glucosides signalled
through hMincle. To this end, NFAT-GFP reporter cells with
mutations to the CRAC motif (hMincle R135 → L135)18
(Fig. 4A) or the carbohydrate-binding EPN motif [hMincle EPN
→ QPD]15 (Fig. 4B) were treated with the steryl glucosides. It is
known that a mutation in the hMincle CRAC motif from
Arg135 to Leu prevents cholesterol binding,18 however, further
insight into the exact binding mode remains to be determined.
Taken as a whole, it appears that hMincle is better able to
tolerate a wider variety of steryl glucosides than mMincle. This
may be due to the presence of two ligand-binding sites in
Fig. 4 Binding site for steryl α-glycoside. 2B4 reporter cells expressing hMincle, and indeed, some species-specific differences were
hMincle with mutation in (A) CRAC motif, hMincle(R135L) + FcRγ, and in
(B) CRD, hMincle(EPN-QPD) + FcRγ, were incubated with plate coated
ligand; reporter cells expressing WT hMincle + FcRγ and FcRγ only were
used as controls. After 20 hours, the cells were harvested and analysed
observed, most notably with regard to βCG (7), which was a
ligand for hMincle but not mMincle. This highlights the care
that needs to be taken when translating observations from one
species to another.17 Notwithstanding, the incorporation of a
for GFP expression by Flow cytometry. Data represents the mean ( SEM)
of three independent experiments performed in duplicate.
lipid chain at the 6-position of glucose generally enhanced
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studies using hMincle expressing RAW-Blue cells, or human
monocyte-derived dendritic cells.18 The most marked differ-
ence in functional response to steryl α-glucosides was the
ability of the ligands to stimulate human monocytes to
produce IL-1β and TNF-α. Here, αCAG (2c), containing the C18
lipid, led to a significant production of IL-1β and TNF-α by
human monocytes, while αCG (1) induced a less notable, but
nonetheless, pro-inflammatory response. This result parallels
our earlier observation that long lipids enhance the agonist
activities of Mincle ligands.29 Our SAR data also revealed that
acylated sitosteryl and stigmasteryl glucosides 5b and 6b
exhibited strong Mincle signalling but led to a poor inflamma-
tory response in primary cells.
Given the complex interaction of H. pylori with a number of
different human antigen presenting cell (APC) subsets,30
understanding how to best mitigate the detrimental effects of
H. pylori is a complex task. Notwithstanding, we have, for the
first time, revealed that the CRD is the preferred Mincle
binding site of steryl α-D-glucosides, thereby providing a frame-
work for the future design of Mincle agonists or antagonists.
These findings support the earlier observation that cholesteryl
α-D-glucosides signal through Mincle and lead to an inflamma-
tory response which has been implicated in H. pylori-mediated
gastric cancer progression.9 Moreover, we identified α-D-gluco-
sides (e.g., αSiAG 5b and αStAG 6b) that signal through Mincle
but do not lead to the production of key proinflammatory cyto-
kines by primary human cells. Taken together, it is thus poss-
ible that the synthesis and application of Mincle antagonists
that counter the pro-inflammatory effects of H. pylori metab-
olites might assist in the development of better therapies for
the treatment of gastric cancer.
Fig. 5 Activation of human monocytes by steryl α-glucosides.
Monocytes were purified from whole blood by negative selection,
seeded onto 96-well plate (2 × 105 cell per well) and stimulated with 40
or 80 μM of ligand, TDB was used as a positive control. The expression
of IL-8 (A), TNF-α (B), and IL-1β (C) was measured from supernatants at
24 h using ELISA. Data represents the mean ( SEM) of three indepen-
dent experiments performed in triplicates. Statistical significance was
calculated in comparison to untreated control using 2-way ANOVA
(Sidak’s multiple comparisons test); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001,
****P ≤ 0.0001.
Conflicts of interest
There are no conflicts to declare.
both mMincle and hMincle signalling. Using hMincle mutants
with modifications to the carbohydrate binding EPN motif or
the CRAC motif the steryl glucosides were found to preferen-
tially bind at the EPN motif. However, subtle changes to ligand
structure can influence Mincle-ligand interactions, as illus-
trated by stigmasterol glycoside 6b, which can signal through
both the CRAC motif and the CRD.
Acknowledgements
The authors would like to thank the NZ Health Research
Council for financial support (Hercus Fellowship, BLS). The
use of human leukocyte from healthy donors with written
informed consent was approved by NZ Northern A Health and
Disability Ethics Committee (approval no. 15/NTA/178).
The ability of the steryl α-glucosides to elicit a functional
immune response by human monocytes typically paralleled
studies using the NFAT-GFP reporter cells. All steryl
α-glucosides, except for the non-lipidated sitosterol derivative
5a, led to the production of IL-8 by human monocytes, with 5a
also eliciting only modest hMincle activation in the reporter
cell assay. Similarly, cholesterol-coated plates did not lead to a
significant increase in cytokine production by human mono-
cytes and exhibited only weak hMincle agonist activity in the
reporter cell assay. This may be due to the presentation of the
ligand28 and indeed, cholesterol crystals at concentrations of
100–300 μg mL−1 were used to elicit an immune response in
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