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The Innate Immune Protein Nod2 Binds Directly to MDP, a Bacterial Cell Wall Fragment
Mammalian Nod2 is an intracellular protein that is implicated in the innate immune response to the bacterial cell wall and is associated with the development of Crohnâs disease, Blau syndrome, and gastrointestinal cancers. Nod2 is required for an immune response to muramyl dipeptide (MDP), an immunostimulatory fragment of bacterial cell wall, but it is not known whether MDP binds directly to Nod2. We report the expression and purification of human Nod2 from insect cells. Using novel MDP self-assembled monolayers (SAMs), we provide the first biochemical evidence for a direct, high-affinity interaction between Nod2 and MDP.Molecular and Cellular Biolog
The Innate Immune Protein Nod2 Binds Directly to MDP, a Bacterial Cell Wall Fragment
Mammalian Nod2 is an intracellular protein that is implicated
in
the innate immune response to the bacterial cell wall and is associated
with the development of Crohnâs disease, Blau syndrome, and
gastrointestinal cancers. Nod2 is required for an immune response
to muramyl dipeptide (MDP), an immunostimulatory fragment of bacterial
cell wall, but it is not known whether MDP binds directly to Nod2.
We report the expression and purification of human Nod2 from insect
cells. Using novel MDP self-assembled monolayers (SAMs), we provide
the first biochemical evidence for a direct, high-affinity interaction
between Nod2 and MDP
Peptidoglycan Modifications Tune the Stability and Function of the Innate Immune Receptor Nod2
Natural
modifications of peptidoglycan modulate the innate immune
response. Peptidoglycan derivatives activate this response via the
intracellular innate immune receptor, Nod2. To probe how these modifications
alter the response, a novel and efficient carbohydrate synthesis was
developed to allow for late-stage modification of the amine at the
2-position. Modification of the carbohydrate was found to be important
for stabilizing Nod2 and generating the proper response. The native
Nod2 ligands demonstrate a significant increase in the cellular stability
of Nod2. Moreover, changing the identity of the natural ligands at
the carbohydrate 2-position allows for the Nod2-dependent immune response
to be either up-regulated or down-regulated. The ligand structure
can be adjusted to tune the Nod2 response, suggesting that other innate
immune receptors and their ligands could use a similar strategy
Membrane Association Dictates Ligand Specificity for the Innate Immune Receptor NOD2
The
human gut must regulate its immune response to resident and
pathogenic bacteria, numbering in the trillions. The peptidoglycan
component of the bacterial cell wall is a dense and rigid structure
that consists of polymeric carbohydrates and highly cross-linked peptides
which offers protection from the host and surrounding environment.
Nucleotide-binding oligomerization domain-containing protein 2 (NOD2),
a human membrane-associated innate immune receptor found in the gut
epithelium and mutated in an estimated 30% of Crohnâs disease
patients, binds to peptidoglycan fragments and initiates an immune
response. Using a combination of chemical synthesis, advanced analytical
assays, and protein biochemistry, we tested the binding of a variety
of synthetic peptidoglycan fragments to wild-type (WT)-NOD2. Only
when the protein was presented in the native membrane did binding
measurements correlate with a NOD2-dependent nuclear factor kappa-light-chain-enhancer
of activated B cells (NF-ÎșB) response, supporting the hypothesis
that the native-membrane environment confers ligand specificity to
the NOD2 receptor for NF-ÎșB signaling. While <i>N</i>-acetyl-muramyl dipeptide (MDP) has been thought to be the minimal
peptidoglycan fragment necessary to activate a NOD2-dependent immune
response, we found that fragments with and without the dipeptide moiety
are capable of binding <i>and</i> activating a NOD2-dependent
NF-ÎșB response, suggesting that the carbohydrate moiety of the
peptidoglycan fragments is the minimal functional epitope. This work
highlights the necessity of studying NOD2-ligand binding in systems
that resemble the receptorâs natural environment, as the cellular
membrane and/or NOD2 interacting partners appear to play a crucial
role in ligand binding and in triggering an innate immune response