Mannan Chain Length Controls Lipoglycans
Signaling via and Binding to TLR2
This information is current as
of June 17, 2017.
Jérôme Nigou, Thierry Vasselon, Aurélie Ray, Patricia
Constant, Martine Gilleron, Gurdyal S. Besra, Iain Sutcliffe,
Gérard Tiraby and Germain Puzo
J Immunol 2008; 180:6696-6702; ;
doi: 10.4049/jimmunol.180.10.6696
http://www.jimmunol.org/content/180/10/6696
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References
The Journal of Immunology
Mannan Chain Length Controls Lipoglycans Signaling via and
Binding to TLR21
Jérôme Nigou,2* Thierry Vasselon,‡ Aurélie Ray,* Patricia Constant,* Martine Gilleron,*
Gurdyal S. Besra,§ Iain Sutcliffe,¶ Gérard Tiraby,† and Germain Puzo*
T
he innate immune system is the first line of host defense
against invading pathogens. It is mediated by phagocytes
including macrophages and dendritic cells that recognize
microorganisms via a limited number of germline-encoded pattern
recognition receptors, among which are the TLRs. TLRs are a
family of at least 12 membrane proteins that trigger innate immune
responses through NF-␬B-dependent and IFN regulatory factordependent signaling pathways (for recent reviews, see Refs. 1–3).
They are type I transmembrane proteins that possess an N-terminal
ectodomain of leucine-reach repeats, which are involved directly
or through accessory molecules in ligand binding, a single transmembrane domain, and a C-terminal cytoplasmic Toll/IL-1 receptor domain that interacts with Toll/IL-1 receptor domain-containing adaptator molecules to induce intracellular signaling. TLRs are
widely expressed in many cell types, and the immune sentinel
cells, such as macrophages, neutrophils, and dendritic cells, express most of them. Some TLRs are expressed at the cell surface,
*Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5089, Department of Molecular Mechanisms of Mycobacterial Infections, Toulouse; †Cayla Invivogen, Research Department, Toulouse; ‡Institut de Génétique Moléculaire de Montpellier, Centre National
de la Recherche Scientifique Unité Mixte de Recherche 5535, Montpellier, France;
and §School of Biosciences, University of Birmingham, Birmingham, and ¶School of
Applied Sciences, Northumbria University, Newcastle Upon Tyne, United Kingdom
whereas others are found almost exclusively in intracellular compartments such as endosomes (1–3).
TLRs recognize conserved microbial-associated molecular
patterns that are essential for the survival of the microorganism
and are therefore difficult for it to alter. However, they are unusual in that some can recognize several structurally unrelated
ligands. TLR2, which plays a major role in detecting Grampositive bacteria, is most probably the TLR that achieves the
highest diversity in ligand recognition. Indeed, its ligands are as
diverse as lipopeptides, lipoteichoic acid, peptidoglycan, porins, zymosan, or glycosyl-phosphatidyl-myo-inositol (GPI)3 anchors. However, TLR2 generally functions as a heterodimer
with either TLR1 or TLR6, which appears to be involved in
discrimination of the acylation state of lipoproteins (4 – 6) and
GPI anchors (7). Indeed, triacylated lipoproteins and GPIs are
preferentially recognized by the TLR2/TLR1 complex, whereas
diacylated lipoproteins and GPIs are recognized by the TLR2/
TLR6 complex. The later heterodimer is also required for activation by zymosan and peptidoglycan (4).
Recently, lipoglycans have been defined as a new class of TLR2
agonists (8 –16). These ligands are recognized in the context of
TLR2 heterodimerization with TLR1 (17–20). Lipoglycans are
found in some genera of the order Actinomycetales (21) but have
Received for publication October 16, 2007. Accepted for publication March 4, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from Centre National de la Recherche Scientifique. G.S.B. was supported by a Personal Research Chair from James Bardrick, a
Royal Society Wolfson Research Merit Award, as a former Lister Institute-Jenner
Research Fellow, the Medical Research Council (G9901077 and G0500590), and The
Wellcome Trust (081569/2/06/2).
2
Address correspondence and reprint requests to Dr. Jérôme Nigou, Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique
Unité Mixte de Recherche 5089, 205 route de Narbonne, 31077 Toulouse Cedex 4,
France. E-mail address: [email protected]
www.jimmunol.org
3
Abbreviations used in this paper: GPI, glycosyl-phosphatidyl-myo-inositol;
A-Pam3CSK4, Alexa Fluor 488-labeled synthetic lipopeptide; AraLAM, uncapped
LAM; BCG, bacillus Calmette-Guérin; HEK-Flag-TLR2, HEK293 cells stably expressing a Flag-TLR2 protein; HSA, human serum albumin; LAM, lipoarabinomannan; LM, lipomannan; ManLAM, mannose-capped LAM; Manp, mannopyranose;
MPI, mannosyl-phosphatidyl-myo-inositol; PILAM, phospho-myo-inositol-capped
LAM; PIM, phosphatidyl-myo-inositol mannosides; ReqLAM, LAM from Rhodococcus equi; RruLAM, LAM from Rhodococcus ruber; RvLM, LM from Mycobacterium
tuberculosis H37Rv; SaeLM, LM from Saccharothrix aerocolonigenes; TotLAM,
LAM from Turicella otitidis; TpaLAM and TpaLM, LAM and LM, respectively, from
Tsukamurella paurometabolum; Araf, arabinofuranose; CCD, charge-coupled device.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
TLR2 is a pattern-recognition receptor that is activated by a large variety of conserved microbial components, including lipoproteins, lipoteichoic acids, and peptidoglycan. Lipoglycans are TLR2 agonists found in some genera of the phylogenetic order
Actinomycetales, including Mycobacterium. They are built from a mannosyl-phosphatidyl-myo-inositol anchor attached to a
(␣136)-linked D-mannopyranosyl chain whose units can be substituted by D-mannopyranosyl and/or D-arabinofuranosyl units. At
this time, little is known about the molecular bases underlying their ability to induce signaling via this receptor. We have recently
shown that the anchor must be at least triacylated, including a diacylglyceryl moiety, whereas the contribution of the glycosidic
moiety is not yet clearly defined. We show herein that lipoglycan activity is directly determined by mannan chain length. Indeed,
activity increases with the number of units constituting the (␣136)-mannopyranosyl backbone but is also critically dependent on
the substitution type of the 2-hydroxyl of these units. We thus provide evidence for the definition of a new pattern that includes
the nonlipidic moiety of the molecules, most probably as a result of the (␣136)-mannopyranosyl backbone being a highly
conserved structural feature among lipoglycans. Moreover, we demonstrate that lipoglycans can bind cell surface-expressed TLR2
and that their ability to induce signaling might be, at least in part, dictated by their avidity for the receptor. Finally, our data
suggest that lipoglycans and lipoproteins have a common binding site. The present results are thus discussed in the light of the
recently published crystal structure of a TLR1-TLR2-lipopeptide complex. The Journal of Immunology, 2008, 180: 6696 – 6702.
The Journal of Immunology
6697
Table I. Structural and functional features of lipoglycans
Structural Features
Activities
Lipoglycana
Size (kDa)
Mannan Chain
Size/Lengthb
% Substitution
by Araf c
Studiesd
TLR2 Activation
EC50 (ng/ml)e
% Inhibition
Pam3CSK4/TLR2f
BCGLM
RvLM
SaeLM
TpaLM
RruLM
ReqLM
TpaLAM
PIM6
fPILAM
sPILAM
PIM2
RvManLAM
BCGManLAM
AraLAM
TotLAM
5.5
5.5
7
3
7
8
13
2.6
ND
17
1.4
17
17
17
9
25/14
25/14
36/14
11/11
28/27
36/23
11/11
5/5
ND
25/16
1/1
30/18
30/18
30/17
28/28
0
0
0
0
42
29
Arabinan chain
0
Bulky arabinan
Bulky arabinan
0
Bulky arabinan
Bulky arabinan
Bulky arabinan
100
(20, 28)
UD
(15)
(16)
(25)
(26)
(16)
(12)
UD
(29–31)
(12)
(32, 33)
(32, 34)
(35)
(27)
1
1
1
5
15
25
50
100
1,000
2,000
2,000
2,000
2,000
4,000
⬎10,000
79
ND
ND
ND
ND
ND
ND
74
ND
ND
65
ND
60
ND
ND
been extensively studied in the genus Mycobacterium (22, 23).
Interestingly, two polymorphisms in the exon part of TLR2 that
attenuate receptor signaling also enhance the risk of developing
tuberculosis and leprosy (24). Mycobacteria contain a family of
lipoglycans whose archetypes are phosphatidyl-myo-inositol mannosides (PIM), lipomannan (LM), and lipoarabinomannan (LAM).
They all share a conserved mannosyl-phosphatidyl-myo-inositol
anchor (MPI), based on a sn-glycero-3-phospho-(1-D-myo-inositol) unit with one ␣-D-mannopyrannose (Manp) unit linked at O-2
of the myo-inositol. This MPI anchor contains four potential sites
of acylation: positions 1 and 2 of the glycerol unit, position 6 of the
Manp unit linked at O-2 of the myo-inositol, and position 3 of the
myo-inositol. O-6 of myo-inositol can be glycosylated by one or
five Manp units, yielding PIM2 and PIM6. LMs correspond to
polymannosylated PIMs and are built from a conserved (␣136)Manp backbone in which some units are substituted, generally at
O-2, by single ␣-Manp units. LAMs correspond to LMs with an
attached D-arabinan domain (Fig. 1). In some mycobacterial species, the nonreducing termini of the arabinosyl side-chains can
be modified by a cap motive consisting of either oligomannosyl
or phospho-myo-inositol units. LM (13, 14) and phospho-myoinositol-capped LAM (PILAM) (8, 10) have been described as
strong TLR2 agonists, whereas PIMs (11, 12, 20) were found to
be weak agonists. Lipoglycans identified in other genera such as
Rhodococcus, Corynebacterium, Tsukamurella, Turicella, and
Saccharothrix also consist of a MPI anchor glycosylated by a
(␣136)-Manp backbone. However, in most cases, they are simpler in structure. Most particularly, the arabinan moiety of the
molecule can be reduced to single arabinosyl susbtituents (15,
16, 25–27) (Fig. 1). These different lipoglycan variants, either
LAM or LM, show variable TLR2-dependent proinflammatory
activities (15, 16).
Thus far the molecular bases underlying the ability of lipoglycans to induce signaling via TLR2 are poorly understood. We have
recently shown, using Mycobacterium bovis bacillus CalmetteGuérin (BCG) LM as a model, that the MPI anchor must be at least
triacylated (20). In the present study, we have used highly purified
lipoglycans with different carbohydrate domains to get further insights into the contribution of the glycosidic moiety.
Materials and Methods
Materials
Nomenclature and structural features of the lipoglycans studied are shown
in Fig. 1 and Table I. Lipoglycans, mycobacterial LAMs (PILAM, ManLAM, AraLAM) (36), LMs (RvLM, BCGLM) (28), PIMs (PIM2, PIM6)
(12), SaeLM (15), TpaLAM and TpaLM (16), RruLAM (25), ReqLAM
(26), and TotLAM (27) were purified as previously described. Their purity
was determined by a set of analyses, including SDS-PAGE, chemical degradations, mass spectrometry, and NMR. The 19-kDa lipoprotein was provided by John T. Belisle (Colorado State University, Fort Collins, CO).
Pam3CSK4, and Pam2CSK4 lipopeptides were from Invivogen. Pam3CSK4
used for binding experiments was from Boehringer Mannheim. The fluoroprobe Alexa Fluor 488 was coupled to Pam3CSK4 (A-Pam3CSK4) using
a labeling kit from Molecular Probes as described previously (37). Recombinant human soluble CD14 (sCD14) was purified from conditioned medium of Schneider-2 insect cells transfected with cDNA encoding human
CD14 as previously described (38).
Complexes between sCD14 and Pam3CSK4, A-Pam3CSK4, PIM6,
PIM2, BCGLM, or BCGLAM were formed by incubating Pam3CSK4 (8
␮g/ml), A-Pam3CSK4 (8 ␮g/ml), PIM2 (5.6 ␮g/ml), PIM6 (10 ␮g/ml),
SaeLM (28 ␮g/ml), BCGLM (22 ␮g/ml), and BCGManLAM (68 ␮g/ml)
with sCD14 (100 ␮g/ml) overnight at 37°C in Dulbecco’s PBS containing
0.05% pyrogen-free human serum albumin (HSA).
THP-1 experiments
The THP-1 monocyte/macrophage human cell line was maintained in continuous culture with RPMI 1640 medium (Invitrogen), 10% FCS (Invitrogen) in an atmosphere of 5% CO2 at 37°C, as nonadherent cells. Lipoglycans were added in duplicate or triplicate, at concentrations of 10 or 20
␮g/ml, to the THP-1 cells (5 ⫻ 105 cells/well) in 24-well culture plates and
then incubated for 20 h at 37°C. Supernatants from THP-1 cells were
assayed for TNF-␣ by sandwich ELISA using commercially available kits
and according to manufacturer’s instructions (R&D Systems).
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
a
BCGLM, BCGManLAM, PIM6, and PIM2 were purified from M. bovis BCG; RvLM and RvManLAM were from M. tuberculosis H37Rv; AraLAM was from M. chelonae;
fPILAM and sPILAM were from M. fortuitum and M. smegmatis, respectively; SaeLM, TpaLAM and TpaLM, RruLAM, ReqLAM, and TotLAM were from Saccharothrix
aerocolonigenes, Tsukamurella paurometabolum, Rhodococcus ruber, Rhodococcus equi, and Turicella otitidis, respectively.
b
Mannan chain size corresponds to the total number of Manp units of the mannan domain, whereas mannan chain length refers to the number of units of the (␣136)-Manp
chain. Except for PIM2 and PIM6, these are average values from a Gaussian distribution.
c
Values correspond to the proportion of the (␣136)-Manp units that are directly substituted by Araf units (see Fig. 1).
d
These references provide structural features of the various lipoglycans; UD indicates unpublished data from our group.
e
Determined by stimulation of HEK-TLR2 cells (see Fig. 3).
f
Inhibition of A-Pam3CSK4 binding to surface-expressed TLR2; values were obtained using a 20-fold molar excess of lipoglycan/sCD14 complexes compared to
A-Pam3CSK4/sCD14 complexes (see Fig. 5).
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A MICROBIAL PATTERN GOVERNED BY ITS GLYCOSIDIC MOIETY
HEK-TLR2 experiments
The HEK-Blue-2 cell line (Invivogen), a derivative of HEK293 cells that
stably expresses the human TLR2 and CD14 genes along with a NF-␬Binducible reporter system (secreted alkaline phosphatase), was used according to the manufacturer’s instructions. Cells were plated at 5 ⫻ 104
cells per well in 96-wells plates and the different lipoglycans were added
at concentrations ranging from 0.1 to 10,000 ng/ml in the HEK-Blue Detection medium (Invivogen) that contains a substrate for alkaline phosphatase. Alkaline phosphatase activity was measured after 18 to 40 h by reading OD at 630 nm. To investigate the CD14 and TLR dependence of
lipoglycan activity, HEK-TLR2 cells were preincubated for 30 min at
37°C, before stimuli addition, with various Abs: 10 ␮g/ml monoclonal
anti-CD14 (clone 134620, R&D Systems), 2 ␮g/ml monoclonal anti-TLR1
(Invivogen), 2 ␮g/ml monoclonal anti-TLR6 (Invivogen), or an IgG1 isotype control (eBioscience).
TLR2 binding assay
Results
Activation of THP-1 cells
We first compared the relative ability of different lipoglycans (Fig.
1) to stimulate the release of TNF-␣ by THP-1 cells. These cells
express at their surface high levels of TLR2 but barely detectable
levels of TLR4 (not shown) (41). We previously established, using
blocking Abs, that the cytokine release induced by the various
lipoglycans studied here was dependent on TLR2 but not TLR4
(15, 16). Each lipoglycan was tested at concentrations of 10 and 20
␮g/ml (Fig. 2). LMs were found to be the most active lipoglycans.
However, activity seemed to depend on the size of the mannan
domain and/or the presence and length of lateral chains and additional domains (Fig. 2). Indeed, SaeLM, BCGLM, and TpaLM, the
most active molecules, have mannan domains built from 36, 25,
and 11 Manp units on average, respectively, and differ by the side
chains substituting the (␣136)-Manp backbone, which are dimannosyl units, monomannosyl units, or no units, respectively (15,
16, 20) (Fig. 1, Table I). In contrast, PIM6 and PIM2, which have
a smaller mannan domain, with 5 and 1 Manp units, respectively,
were virtually inactive in this model (data not shown). As previously established by us and others, LAM with a bulky arabinan
domain (such as ManLAMs or TpaLAM) are inactive or weakly
active (Fig. 2) (13, 16). This is likely due to a masking of the LM
domain of the molecule because removal of the arabinan domain
by chemical treatments restores the proinflammatory activity of the
resulting LM moiety (13, 16), as shown here by the activity of
TpaLM as compared with that of TpaLAM (Fig. 2). More interestingly, a direct substitution of the (␣136)-Manp backbone by
arabinosyl units also inhibits the TLR2-inducing activity. Indeed,
partial substitution (as the in case of RruLAM and ReqLAM) resulted in lipoglycans with a lowered activity compared with that of
FIGURE 1. Structural models of lipoglycans tested in the present study.
M, D-mannopyranoside units; A, D-arabinofuranoside units; Man, mannose; PI, phosphoinositol; MPI, mannosyl-phosphatidyl-myo-inositol.
BCGLM, RvLM, SaeLM, TpaLAM and TpaLM, RruLAM, ReqLAM, and
TotLAM are LM or LAM from M. bovis BCG, Mycobacterium tuberculosis H37Rv, Saccharothrix aerocolonigenes, Tsukamurella paurometabolum, Rhodococcus ruber, Rhodococcus equi, and Turicella otitidis,
respectively.
LMs (Fig. 2) despite the presence in the mannan moiety of these
molecules of 28 and 36 Manp units, respectively (25, 26) (Fig. 1,
Table I). Moreover, complete substitution (as in the case of TotLAM) resulted in a fully abrogated activity. Because in these three
lipoglycans the (␣136)-Manp backbone is substituted at the 2-hydroxyl, these data indicate that this position of the Manp units
plays a critical role in the ability of lipoglycans to induce signaling
via TLR2. However, the presence of Manp, but not arabinofuranose (Araf), units glycosylating these positions can compensate for the loss of the free hydroxyl groups.
Comparing the relative ability of lipoglycans to stimulate the
release of TNF-␣ by THP-1 cells thus allows a better understanding of the structure/function relationship of these molecules. Lipoglycan activity appears to depend on the size of the mannan
chain and is critically dependent on the substitution type of the
2-hydroxyl of the (␣136)-Manp backbone that must be free or
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The HEK293 cells stably expressing a Flag-TLR2 protein (HEK-FlagTLR2) (39) were cultured in complete culture medium on glass coverslips precoated with 0.5% gelatin for 24 – 48 h before experiments. The
cells were washed twice with PBS containing 0.05% HSA and incubated for 15 min at 37°C in PBS-HSA with A-Pam3CSK4/sCD14 complexes (160 ng/ml Pam3CSK4, 2 ␮g/ml sCD14) in the presence or not
of a 10- or 20-fold molar excess of unlabelled lipoglycan/sCD14 or
Pam3CSK4/sCD14 complexes. At the end of incubation, coverslips
were washed several times with PBS-HSA, fixed for 30 min in PBS
containing 4% paraformaldehyde, stained for TLR2 by successive incubation for 30 min at 4°C with a mouse anti-Flag M2 mAb and a
Cy3-labeled anti-mouse Ab from Sigma-Aldrich, and mounted. Images
were obtained using a fluorescence microscope equipped with a CoolSNAP HQ charge-coupled device (CCD) camera (Ropper Scientific),
and cell-surface fluorescence intensity ratios of A-Pam3CSK4 to FlagTLR2 were measured from CCD images either manually using the
Metamorph software (see Fig. 6B) or automatically using a visual
scripting interface for the ImageJ software (see Fig. 6C) (that can be
downloaded from http://www.mri.cnrs.fr) (40). Data are expressed as
arbitrary units.
The Journal of Immunology
FIGURE 2. TNF-␣ production by human THP-1 monocyte/macrophage
cell line in response to various lipoglycans. Lipoglycans were tested at 10
(filled bars) and 20 (open bars) ␮g/ml. ManLAM was from M. bovis BCG
(BCGManLAM).
Activation of HEK-TLR2 cells
To tentatively overcome this limitation, we used HEK293 cells
stably transfected with human TLR2 and CD14 genes (HEK-TLR2
cells) and a NF-␬B-inducible reporter system (secreted alkaline
phosphatase). The 19-kDa lipoprotein (LP19), a well-known mycobacterial TLR2 agonist (42), and also BCGLM and BCGManLAM induced NF-␬B activation in a dose-dependent manner in
HEK-TLR2 cells (Fig. 3) but not in the parent HEK cells (data not
shown), demonstrating that activation was specific for TLR2.
LP19 and BCGLM were strong agonists of the receptor, and an
EC50 of 1 ng/ml was determined for both molecules (Fig. 3). In
sharp contrast, but in agreement with data obtained with THP-1
cells, BCGManLAM was a weak agonist with an EC50 of 2 ␮g/ml,
thus exhibiting an activity reduced by more than three orders of
magnitude as compared with BCGLM. In a similar way, we determined the EC50 value for each lipoglycan (Table I). Data were
in agreement with those obtained with THP-1 cells (Fig. 2). The
most active molecules were the various LMs as well as RruLAM
FIGURE 3. NF-␬B activation in HEK-TLR2 cells by various stimuli.
Cells (5 ⫻ 104) were plated in 96-wells plates and stimulated at 37°C for
24 h by LP19 (E), BCGLM (Œ), or BCGManLAM (f). NF-␬B activity
was determined by reading OD at 630 nm.
FIGURE 4. TLR2-dependent activity of lipoglycans as a function of
their mannan chain length. The mannan chain length corresponds to the
number of mannosyl units building the (␣136)-Manp backbone. For lipoglycans labeled with asterisks, only the numbers of Manp units of the
(␣136)-Manp backbone that are not substituted by Araf units have been
taken into account. Lipoglycan activity corresponds to decadic logarithm of
the EC50 value determined by stimulation of HEK-TLR2 cells (Fig. 3,
Table I).
and ReqLAM, that is, lipoglycans with the highest number of nonsubstituted mannosyl units. We noticed that SaeLM and BCGLM
showed the same EC50 of 1 ng/ml. The mannan domain of SaeLM
is larger when compared with that of BCGLM. However, this
mainly results from longer side chains for the former (i.e., dimannosyl vs single mannosyl units) (15) (Fig. 1, Table I). Nevertheless, the length of the mannan chain (i.e., the number of mannosyl
units building the (␣136)-Manp backbone) is the same in both
molecules: 14 mannosyl units on average (Fig. 1, Table I). We thus
hypothesized that mannan chain length, rather than mannan chain
size, might be a key parameter determining lipoglycan activity. We
therefore plotted the activity of the lipoglycans (log of EC50 value)
as a function of their mannan chain length (number of mannosyl
units constituting the chain backbone) (Fig. 4). Intriguingly, a linear relationship was observed for lipoglycans containing solely
mannosyl units, that is, LMs (BCGLM, RvLM, SaeLM, and
TpaLM) and PIMs (PIM6 and PIM2), demonstrating that LM activity is directly determined by the mannan chain length. However,
lipoglycans substituted with arabinosyl units, that is, LAMs,
showed an activity weaker than that expected from their mannan
chain length. For example, although ManLAMs have a mannan
chain identical with that of LMs from the same species, they only
show an activity equivalent to that of PIM2 (Fig. 4, Table I), suggesting that their bulky arabinan domain masks the mannan chain
in such a way that they behave like molecules with a mannan
restricted to a single mannosyl unit. Similarly, TpaLAM activity is
equivalent to that of a LM with a mannan length of 7 mannosyl
units, whereas its mannan is made from 11 mannosyl units (Figs.
1 and 4, Table I). The arabinan domain of TpaLAM is much simpler and smaller than that of ManLAMs (16) (Fig. 1), and thus its
steric hindrance on the mannan domain is lowered as compared
with that observed with ManLAMs. Concerning LAMs whose
mannan chain is directly substituted by arabinosyl units, that is,
RruLAM and ReqLAM, their activity corresponds to that of a
mannan chain with 9 and 8 units, respectively. This is less than
would be expected if we take into account only the number of
Manp units that are not substituted (i.e., 16 in both cases; see Fig.
4, lipoglycan names labeled with asterisks), suggesting that Araf
substitution has side effects. Finally, the EC50 of TotLAM, which
has no free Manp units, was found to be higher than 10 ␮g/ml, a
value ⬎5-fold higher than that obtained for PIM2. The activity of
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
glycosylated by Manp, but not Araf, units. However further investigations, such as into the role of the mannan chain size, are limited
by the fact that cytokine production by THP-1 cells shows a weak
dynamic of response. Indeed, the dose-dependent activation of the
cells is limited to a sharp range of lipoglycan concentration, and
thus the results only give a “yes” or “no” response concerning
lipoglycan activity.
6699
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A MICROBIAL PATTERN GOVERNED BY ITS GLYCOSIDIC MOIETY
all the lipoglycans was dependent on the expression of CD14 (Fig.
5A) and on the heterodimerization of TLR2 with TLR1 but not
TLR6 (Fig. 5B) as determined by blocking Ab experiments and in
agreement with previous data obtained on mycobacterial LAM and
LM (13, 17–20).
Binding to TLR2
We next asked whether the activity of lipoglycans was determined
by their ability to bind TLR2. Vasselon et al. (37) have recently
developed a binding assay of fluorescently labeled synthetic triacylated lipopeptide Pam3CSK4 (A-Pam3CSK4) to TLR2 expressed on the surface of TLR2-transfected HEK cells (HEK-FlagTLR2). They have shown that TLR2 strongly and specifically
binds A-Pam3CSK4 when presented as a complex with sCD14. We
used this assay to determine whether lipoglycans were able to
compete for Pam3CSK4 binding to TLR2. Lipoglycan/sCD14
complexes were prepared and we found that their addition in a 10or 20-fold molar excess to A-Pam3CSK4/sCD14 complexes resulted in an inhibition of Pam3CSK4 binding to TLR2 (Fig. 6,
Table I). Inhibition was shown to be dose dependent as evidenced
with PIM2 and PIM6 complexes. These results demonstrate that
lipoglycans directly bind TLR2 and that their binding site is at
least partially common with that of bacterial lipopeptides. Inter-
FIGURE 6. Lipoglycans compete with A-Pam3CSK4 for binding to
TLR2. HEK-Flag-TLR2 cells were incubated for 15 min at 37°C with
A-Pam3CSK4/sCD14 complexes and a 10-fold (B) or 20-fold (A and C)
molar excess of the indicated lipoglycan/sCD14 or Pam3CSK4/sCD14
complexes. A, CCD images of Cy3-Flag-TLR2 and A-Pam3CSK4 fluorescence in representative cells (center and right panels, respectively) and the
merged images (left panels) are shown. B and C, Ratio of cell-surface
fluorescence intensities of A-Pam3CSK4 to Flag-TLR2 measured from
CCD images. Results are expressed as arbitrary units and are the mean of
at least 20 independent measures (B) or of 1264 –3584 measures (C). Statistical analysis of the data for C gives p values ⬍10⫺24 as assessed by a
Student’s t test comparison of the different conditions by pairs. Inhibition
percentages are indicated on the graphs.
estingly, BCGLM was a better competitor than PIM6, which was
better than PIM2 and ManLAM (Fig. 6C; p ⬍ 10⫺24). The best
agonists (Table I) were thus the best competitors (Fig. 6), suggesting that lipoglycan ability to induce signaling via TLR2 is at least
partially dictated by their avidity for this receptor.
Discussion
MPI-anchored lipoglycans, which are found in some genera of the
Actinomycetales order, are agonists of TLR2. These are complex
molecules bearing one to four fatty acids on their MPI anchor and
showing a great degree of heterogeneity in their carbohydrate moiety (23). Although recent studies have shown that the lipidic part
of the molecule is necessary for its activity, the contribution of the
glycosidic moiety is still poorly understood. In this study, we have
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 5. Lipoglycan activity is dependent on CD14 expression (A)
and TLR2 heterodimerization with TLR1 (B). HEK-TLR2 cells were preincubated for 30 min at 37°C, before lipoglycan addition, with various Abs:
10 ␮g/ml monoclonal anti-CD14, 2 ␮g/ml monoclonal anti-TLR1, 2 ␮g/ml
monoclonal anti-TLR6, or an IgG1 isotype control (at the corresponding
concentration). BCGManLAM, PIM2, and PIM6 were tested at a concentration of 500 ng/ml and TpaLM, RvLM, BCGLM, and SaeLM at a concentration of 10 ng/ml. Pam3CSK4 (5 ng/ml) and Pam2CSK4 (0.05 ng/ml)
lipopeptides were used as positive controls of TLR2/TLR1 and TLR2/
TLR6 agonists, respectively.
The Journal of Immunology
and for initiating signaling. The crystal structure of the TLR1TLR2 heterodimer in complex with Pam3CSK4 has been recently
reported (45). The triacylated lipopeptide appears to form a bridge
between TLR2 and TLR1 with the two ester-bound fatty acyl
chains inserted deep into a pocket in the hydrophobic core of
TLR2, the third amide-linked acyl chain occupying a hydrophobic
channel at the surface of TLR1 and the conserved polar head located at the region of contact between the two receptors. In contrast, the four lysine residues have only limited interactions with
TLR1 or TLR2, which is consistent with previous studies suggesting strongly that the highly variable polypeptide chain of lipoproteins is not included in the pattern that is recognized by TLR2 and
TLR1 (44).
Herein we have found that lipoglycans PIM2, PIM6, BCGLM,
and BCGManLAM can compete with fluorescently-labeled
Pam3CSK4 for binding to cell surface-expressed TLR2 (Fig. 6).
Our results thus show that the binding site for lipoglycans and
triacylated lipopeptides is overlapping, presumably because the
lipid anchors of both classes of molecules share common structural
similarities, including a diacylglyceryl moiety shown to be critical
for activity of both classes of ligands (20, 44). We also found that
variation in the length and nature of the carbohydrate chain of
lipoglycans does not affect the specificity of recognition by TLR2/
TLR1 or TLR6 heterodimers but rather modulates the sensitivity
of responses mediated by TLR2-TLR1 heterodimers (Fig. 5B). The
observation that the most potent agonists were the best competitors
of Pam3CSK4 suggests that the most active molecules have a
higher affinity for the receptor complex. Thus, in contrast to the
peptidic chain of lipoproteins, the (␣136)-mannopyranosyl backbone, which is a highly conserved structural feature of lipoglycans,
is an integral part of the microbial-associated molecular pattern
and probably establishes close contacts with the receptors. However, further studies will be required to test this hypothesis.
Acknowledgments
We gratefully acknowledge Dr. Daniel Drocourt, Elise Armau, and Sophie
Gauthier (Cayla/Invivogen, Toulouse, France) for helpful discussions and
technical assistance. We thank John T. Belisle (Colorado State University,
Fort Collins, CO) for providing the 19-kDa lipoprotein (through National
Institutes of Health, National Institute of Allergy and Infectious Diseases,
contract NO1 AI-75320 titled, “Tuberculosis Research Materials and Vaccine Testing”).
Disclosures
The authors have no financial conflicts of interest.
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