From the Department of Pathology and Comprehensive
Cancer Center, The University of Michigan Medical School, Ann Arbor,
Michigan 48109, the § Unidad de Genetica Molecular and
¶ Instituto de Patologia Digestiva, Hospital Universitario Marques
de Valdecilla, 39008 Santander, Spain, the
Department of
Chemistry, Graduate School of Science, Osaka University, Machikaneyama
1-1, Toyonaka, Osaka 560-0043, Japan, the ** Department
of Infectious Diseases and Tropical Medicine, Research Institute,
International Medical Center of Japan, Tokyo 162-8655, Japan, the
Department of Molecular Biology and
Biotechnology, University of Sheffield, Sheffield S10 2TN, United
Kingdom, and the §§ Department of Microbiology,
National University of Ireland, Galway, Ireland
Received for publication, December 4, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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NOD2, a protein associated with susceptibility to
Crohn's disease, confers responsiveness to bacterial preparations of
lipopolysaccharide and peptidoglycan, but the precise moiety recognized
remains elusive. Biochemical and functional analyses identified muramyl
dipeptide (MurNAc-L-Ala-D-isoGln) derived
from peptidoglycan as the essential structure in bacteria recognized by
NOD2. Replacement of L-Ala for D-Ala or
D-isoGln for L-isoGln eliminated the ability of
muramyl dipeptide to stimulate NOD2, indicating stereoselective
recognition. Muramyl dipeptide was recognized by NOD2 but not by TLR2
or co-expression of TLR2 with TLR1 or TLR6. NOD2 mutants associated
with susceptibility to Crohn's disease were deficient in their
recognition of muramyl dipeptide. Notably, peripheral blood mononuclear
cells from individuals homozygous for the major disease-associated
L1007fsinsC NOD2 mutation responded to lipopolysaccharide but not to
synthetic muramyl dipeptide. Thus, NOD2 mediates the host response to
bacterial muropeptides derived from peptidoglycan, an activity that is
important for protection against Crohn's disease. Because muramyl
dipeptide is the essential structure of peptidoglycan required for
adjuvant activity, these results also have implications for
understanding adjuvant function and effective vaccine development.
Innate immunity recognizes invading microbes and triggers a
defense response in the host aimed at clearing the invading pathogen. Toll-like receptors (TLRs)1
expressed on the surface of myelomonocytic cells play an important role
in the recognition of microbial components and activation of innate
immunity (1). Each membrane-associated TLR recognizes pathogen-associated molecular patterns that are expressed on
infectious agents (1). Nods, including NOD1 and NOD2, are members of
another family of proteins that have been recently implicated in
intracellular recognition of bacterial components (2, 3). NOD2 is
composed of two NH2-terminal caspase-recruitment domains, a
centrally located nucleotide-binding domain and multiple COOH-terminal
leucine-rich repeats (LRRs), and is expressed in myelomonocytic and
dendritic cells (4, 5). Three genetic variants within the coding region of NOD2, L1007fsinsC, G908R, and R702W, have been
genetically associated with susceptibility to Crohn's disease in
European and American populations (6-9). NOD2 has been shown to
recognize preparations of lipopolysaccharides (LPS) and peptidoglycan
(PGN) through its COOH-terminal LRRs (10), and this activity is
deficient in the disease-associated variants (7). However, the precise bacterial structure recognized by NOD2 remains unknown. In this report
we identified muramyl dipeptide (MDP) derived from peptidoglycan as the
structure in bacteria recognized by NOD2.
Reagents--
LPS from Escherichia coli O55:B5
prepared by the phenol extraction method, further purified by
gel-filtration chromatography, detoxified LPS, and purified lipid A
were obtained from Sigma. LPS from Salmonella
typhimurium was obtained from Sigma. PGN from Staphylococcus
aureus was from Fluka-Chemie (Buchs, Germany). PGN from
Bacillus subtilis was purified as reported (11).
Pam3CysSerLys4, a synthetic bacterial
lipoprotein analog (sBLP), was a gift of A. Zychlinsky (Max Planck
Institute for Infection Biology, Berlin, Germany). MDP and their
analogs MurNAc-L-alanyl-L-isoglutamine and
MurNAc-D-alanyl-D-isoglutamine were obtained
from Bachem (Torrance, CA). Molecules with two and four copies of
GlcNAc-MurNAc attached to L-Ala-D-isoGln, and
their counterparts lacking dipeptide were synthesized as reported (12).
Briefly, the disaccharide glucosaminyl- Transfection and NF- Gel Filtration--
PGN from B. subtilus (0.4 mg) was
digested with mutanolysin (Sigma) for 24 h. After
centrifugation at 100,000 × g for 10 min and
filtration with a 0.22-µm nitrocellulose filter, digested PGN was
fractionated by Superose 12 gel-filtration column chromatography with
10 mM HEPES, 100 mM NaCl, pH 7.4. Purified
bovine serum albumine, chicken egg lysozyme, and bovine cytochrome
c were used as molecular size standards.
Genotype Analysis--
Peripheral blood was obtained from normal
donors and Crohn's disease patients after informed consent according
to Guidelines from the Committee for the Protection of Human Subjects
at the Hospital Universitario Marques de Valdecilla. DNA was tested for the 3020insC NOD2 mutation using the SNaPshot method
(Applied Biosystems, Foster City, CA) based on the dideoxy single-base extension of an unlabeled oligonucleotide primer
(5'-GCCCTCCTGCAGGCCC-3').
Electrophoretic Mobility Shift Assay--
PBMNC were cultured
for 1 h with 1 µg/ml LPS from S. typhimurium
or 10 ng/ml MDP. Then cells were lysed and nuclear extracts were analyzed for the presence of NF- Real-time PCR Analysis--
Total RNA was prepared using TRIZOL
reagent (Invitrogen). To assess mRNA expression, a quantitative PCR
method was used as described previously (5). The generated cDNA was
amplified by using primers for human IL-1 NOD2 was shown to mediate responsiveness to preparations of LPS
and PGN (10). To further characterize the bacterial moiety recognized
by NOD2, we used human embryonic kidney (HEK293T) cells and a
NF-
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
(1-4)-muramic acid was
prepared by stereoselective glycosylation of an N-Troc muramic acid
acceptor with N-Troc-glucosaminyl trichloroacetimidate. The
disaccharide was converted to both disaccharide acceptor and donor,
which was then coupled together by the same glycosylation method to
give a tetrasaccharide. Octasaccharide was obtained in a good yield in
a similar manner. Introduction of the dipeptide moiety of
L-alanyl-D-isoglutamine to
3-O-lactyl groups was performed by deprotection afforded by
the peptidoglycan tetrasaccharide and octasaccharide fragments.
B Activation Assay--
The plasmids
pcDNA3- NOD2, pcDNA3-TLR4, and pDNA3-MD2 have been described
(10). The plasmids expressing TLR1 and TLR6 have been reported (13) and
were generously provided by Dr. A. Hajjar (University of Washington,
Seattle). Expression plasmids producing NH2-terminal
HA-tagged NOD2 variants P268S, P268S/R702W, and P268S/G908R were
generated by the QuikChange XL site-directed mutagenesis kit
(Stratagene, La Jolla, CA). P268S/L1007fsinsC was generated by a PCR
method using P268S DNA as a template. The generated PCR products were
cloned into the pMX-puro expression plasmid (14). The authenticity of
the constructs were confirmed by DNA sequencing. Expression of NOD2
proteins in transfected cells was determined by immunoblotting using
monoclonal anti-HA antibody (Babco, La Jolla, CA) as described (5).
NF-
B activation assays were performed as described (10). LPS, PGN,
and MDP derivaties were added to the cultures in the presence of
calcium phosphate to allow their entry into the cells as reported (10).
Results were normalized for transfection efficiency with values
obtained with pEF-BOS-
gal. Expression of NOD2 proteins in
transfected cells was determined by immunoblotting using monoclonal
anti-HA antibody (Babco, La Jolla, CA) as described (4).
B binding activity as described previously (5).
,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5), and A1
(5'-CGGATGTGGATACCTATAAGG-3' and 5'-GTCATCCAGCCAGATTTAGG-3'). Quantitative real-time PCR was performed in a 7000 sequence
detection system (Applied Biosystems). The ratio of the abundance of
IL-1
and A1 transcripts to that of GAPDH transcripts was calculated as described (5). Specificity of the PCR products was determined by
melting curve analysis.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
B-dependent luciferase reporter to compare the
ability of NOD2 and TLR4 to recognize LPS and PGN. Because NOD2 is an intracellular protein, we assessed NOD2 activity under culture conditions that allow the internalization of the bacterial components into the cells (10). Expression of NOD2 and TLR4 together with its
co-factor MD2 conferred responsiveness to purified LPS prepared by
phenol extraction as reported (7, 10), whereas only NOD2 induced the
response to PGN (Fig. 1A). The
lipid A moiety of LPS mediates TLR4/MD2 activation (15). Consistent
with the latter, NOD2, but not TLR4, did respond to lipid A-depleted
detoxified LPS prepared by alkaline treatment (Fig. 1A),
whereas TLR4, but not NOD2, responded to purified lipid A (Fig.
1A). Notably, TLR4, but not NOD2, was stimulated by highly
purified LPS prepared by gel-filtration chromatography (Fig.
1A). These results indicate that TLR4 and NOD2 recognize
different bacterial components and suggest that PGN present in LPS
preparations may contain the moiety recognized by NOD2. To further
characterize the bacterial structure recognized by NOD2, we digested
purified PGN with the muramidases mutanolysin or Cellosyl, which
degrade the glycan chains and result in the generation of muropeptides
which are composed of N-acetylglucosamine (GlcNAc) and
N-acetylmuramic acid (MurNAc) linked to short peptides (16).
Digested PGN was fractionated by gel-filtration chromatography. Analysis of each fraction revealed a major peak of NOD2-stimulating activity induced by mutanolysin digestion with a relative
molecular mass of less than 12 kDa, consistent with that expected for
muropeptides (Fig. 1B). Similar results were observed when
PGN was digested with Cellosyl, which revealed a single peak of
NOD2 stimulatory activity of less than 12 kDa.2
View larger version (30K):
[in a new window]
Fig. 1.
Recognition of peptidoglycan by NOD2.
A, NOD2 and TLR4 recognize different bacterial components.
HEK293T cells were transfected with 0.1 ng of pcDNA3-NOD2
(NOD2) or 3 ng of pcDNA3-TLR4-FLAG plus 3 ng of
pcDNA3-MD2 (TLR4+MD2) or left untreated ( ) in the
presence of reporter pBxIV-luc and pEF-BOS-
-gal as described (10).
Eight hours post-transfection, 1 µg/ml LPS from E. coli O55:B5 prepared by the phenol extraction method (phenol), LPS
further purified by gel-filtration chromatography (gel
filt.), LPS prepared by the phenol extraction and
alkaline-detoxified (aLPS), purified lipid A from E. coli O55:B5 LPS, or 5 µg/ml PGN from S. aureus
were added with fresh medium. Twenty-four hours post-transfection
B-dependent transcription was determined as described
(10). Values represent mean of normalized data ± S.D. of
triplicate cultures. B, cells were stimulated with
gel-filtration fractions of PGN from B. subtilis digested or
undigested with Mutanolysin for 24 h and fractionated by gel
filtration as described under "Materials and Methods." Mutanolysin
alone did not stimulate NOD2 activity.2 The ability of
2-µl aliquot from each fraction to induce NOD2-dependent
NF-
B activation is shown. Molecular weight markers corresponding to
bovine serum albumin (68K), chicken egg lysozyme
(20K), and bovine cytochrome c (12K)
are indicated.
PGN-derived muropeptides derived from most bacterial species are
composed of GlcNAc-MurNAc linked to short peptides, which include the
conserved dipeptide L-Ala and D-isoGln or
D-Glu (Fig. 2A).
To determine more directly if muropeptides are recognized by NOD2, we
used a panel of synthetic molecules with the structure of GlcNAc-MurNAc
linked to L-Ala-D-isoGln, as well as muramyl dipeptide MurNAc-L-Ala-D-isoGln (MDP), which
lacks GlcNAc (Fig. 2A).
GlcNAc-MurNAc-L-Ala-D-isoGln stimulated NF-B
in a NOD2-dependent manner, whereas disaccharide
GlcNAc-MurNAc in dimeric or tetrameric forms did not (Fig.
2B), indicating that amino acid residues are required for
stimulation of NOD2. However, we cannot formally rule out that the lack
of NOD2 response to GlcNAc-MurNAc in dimeric or tetrameric forms is due
to poor internalization of the synthetic molecules into the cells. MDP
induced potent stimulation of NOD2 (Fig. 2B), indicating
that GlcNAc is not essential for stimulatory activity. To determine
further the specificity of the recognition of MDP by NOD2, we tested
the MDP analogs MurNAc-L-Ala-L-isoGln and
MurNAc-D-Ala-D-isoGln. Notably, replacement of
L-Ala for D-Ala or D-isoGln for
L-isoGln eliminated the ability of MDP to stimulate NOD2,
indicating stereoselective recognition (Fig. 2C). Thus, the
core structure required for recognition of NOD2 is MurNAc attached to
L-Ala and D-isoGln. TLR2 has been proposed to
act as a surface receptor for PGN and certain bacterial lipoproteins including synthetic bacterial lipopeptide (sBLP) (17, 18). Therefore,
we tested the ability of TLR2 and NOD2 to respond to MDP and sBLP. TLR2
mediated a cellular response to sBLP but not to MDP (Fig.
2D). Conversely, MDP, but not sBLP, stimulated NOD2 activity
(Fig. 2D). In addition, we tested whether co-expression of
TLR2 with TLR1 and/or TLR6 could mediate recognition of MDP. TLR2 in
combination with TLR1, TLR6, or TLR1/TLR6 did not activate NF-
B in
response to MDP (Fig. 2D). Thus, NOD2 and TLR2 recognize different bacterial structures.
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The NOD2 variants associated with Crohn's disease, R702W, G908R, and
L1007fsinsC, occur on the same haplotype background, which includes the
common P268S polymorphism (6, 7). To compare the ability of normal and
mutant NOD2 proteins to induce NF-B activity in response to MDP, we
expressed the NOD2 proteins in cells and evaluated their activity in a
functional assay. Both normal and P268S NOD2 induced similar levels of
NF-
B activation in response to MDP (Fig.
3A), which is consistent with
the observation that P268S is not genetically associated with disease
(6, 7). In contrast, the P268S/R702W, P268S/G908R, and
P268S/L1007fsinsC variants induced greatly reduced levels of NF-
B
activation in response to MDP when compared with normal NOD2 (Fig.
3A). Notably, whereas the P268S/R702W and P268S/R702W point
mutants retained some ability to respond to MDP, the truncated
P268/L1007fsinsC protein did not respond at all concentrations of NOD2
and MDP tested (Fig. 3A). Immunoblotting analysis revealed
that the normal and NOD2 mutants were appropriately expressed (Fig.
3B), indicating that the observed differences in MDP
responses could not be explained by differential expression of the
NOD2 constructs.
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We next determined the role of NOD2 in the recognition of MDP by
primary cells from normal and Crohn's disease patients. A panel of
healthy and Crohn's disease individuals were genotyped for the
disease-associated NOD2 alleles, and two individuals (one without
clinical evidence of disease and one individual with Crohn's disease)
who were homozygous for the loss-of-function L1007fsinsC mutation were
identified. The identification of apparently healthy individuals
homozygous for L1007fsinsC is expected in that the penetrance of the
L1007fsinsC mutation is not complete (6-9). PBMNC that are known to
express NOD2 were isolated, incubated with MDP or LPS, and NF-B
activation was assessed in nuclear extracts by an electrophoretic
mobility shift assay (EMSA). Stimulation of PBMNC from individuals
carrying normal or heterozygous NOD2 alleles with either LPS or MDP
resulted in induction of the NF-
B-DNA complex (Fig. 3C).
In contrast, PBMNC from individuals homozygous for L1007fsinsC did
respond to LPS, but not to MDP (Fig. 3C). The DNA binding
complex induced by MDP was shifted by incubation of the nuclear
extracts with an antibody specific for the p50 subunit of NF-
B (Fig.
3C), indicating that the detected protein-DNA complex
contained NF-
B. To further assess NF-
B activation, the mRNA
levels of IL-1
and A1, two NF-
B target genes expressed in PBMNC
(19, 20), were evaluated by quantitative real-time PCR analysis. Both
IL-1
and A1 mRNA levels were induced by incubation with LPS and
MDP in cells from individuals carrying normal or heterozygous NOD2
alleles (Fig. 3, D and E). In contrast, LPS but
not MDP, increased the levels of both IL-1
and A1 mRNA in PBMNC
from individuals homozygous for L1007fsinsC (Fig. 3, D and E). Thus, PBMNC require the expression of normal NOD2 for
their response to MDP but not to LPS. The lack of response to MDP in apparently normal individuals suggest that the presence of certain bacteria in the intestine and/or additional genetic factors may be
required for clinical disease.
MDP is the minimal essential structure of bacterial peptidoglycan
required for biological effects, including activity in Freund's complete adjuvant (21). MDP has been shown to signal through TLR2- and
TLR4-independent mechanisms (22, 23), but the host recognition system
for MDP has not been identified. We present evidence that NOD2 mediates
the recognition of MDP in mammalian cells. Macrophages contain
intracellular hydrolases that digest bacterial PGN and release PGN
fragments including GlcNAc-MurNAc-dipeptide (24, 25). These
muropeptides derived from intracellular and/or phagocytosed bacteria as
well as PGN fragments released during bacterial growth would be
available for recognition by NOD2. The synthetic MDP and
GlcNAc-MurNAc-dipeptides mimicking the natural muropeptides induced
NOD2-dependent activation of NF-B. Thus, NOD2 is likely
to be activated by muropeptides derived from bacteria in
vivo. Although NOD2 can mediate the recognition of muropeptides, the mechanism involved is unclear and remains to be determined. Because
the LRRs are required for recognition, muropeptides could interact
directly with NOD2 through its LRRs or via as yet to be identified
cellular factor(s). Crohn's disease-associated NOD2 variants and PBMNC
from individuals homozygous for L1007fsinsC are defective in their
response to muramyl dipeptide. This result is consistent with the
observation that homozygocity for L1007fsinsC is needed for
susceptibility to Crohn's disease (6, 7). Because activation of
NF-
B in response to bacterial components mediates protection of the
host against pathogens, NOD2-mediated susceptibility to disease may be
caused by a failure to trigger a protective NF-
B pathway in response
to muropeptides. Such a defective response against certain bacterial
products may result secondarily in the diffuse activation of NF-
B
found in intestinal tissue by NOD2-independent mechanisms. The results
presented here suggest that restoring the activity against bacterial
muropeptides may be beneficial to Crohn's disease patients harboring
NOD2 mutations.
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ACKNOWLEDGEMENTS |
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We are grateful to T. Kirikae for stimulating discussions, C. Kirschning for HEK293-TLR2 cells, A. Zychlinsky for sBLP, and A. Hajjar for plasmids. We thank P. Lucas for critical review of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK-61707 (to G. N.) and GM-60421 (to N. I.), a grant from Fundacion Marcelino Botin (to J. L. F.-L.), and by a postdoctoral fellowship from the Crohn's and Colitis Foundation of America (to Y. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶¶ These two authors share senior authorship.
To whom correspondence should be addressed: University
of Michigan Medical School, 1500 E. Medical Center Dr., 4219 CCGC, Ann
Arbor, MI 48109. Tel.: 734-764-8514; Fax: 734-647-9654; E-mail: bclx@umich.edu.
Published, JBC Papers in Press, January 4, 2003, DOI 10.1074/jbc.C200673200
2 N. Inohara, Y. Ogura, and G. Nuñez, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
TLR, Toll-like receptor;
LPS, lipopolysaccharide;
MDP, muramyl dipeptide
MurNAc-L-Ala-D-isoGln;
NF-B, nuclear
factor-
B, PBMNC, peripheral blood mononuclear cells;
PGN, peptidoglycan;
sBLP, synthetic bacterial lipoprotein;
HEK, human
embryonic kidney;
LRR, leucine-rich repeat;
HA, hemagglutinin;
IL, interleukin;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
EMSA, electrophoretic mobility shift assay;
Mur, muramic acid.
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