Membrane Association Dictates Ligand Specificity for the Innate Immune Receptor NOD2

Abstract

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 Crohn's 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-κB) response, supporting the hypothesis that the native-membrane environment confers ligand specificity to the NOD2 receptor for NF-κB signaling. While N-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 and activating a NOD2-dependent NF-κB 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 receptor's 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.

Figure 1

Biologically relevant muramyl dipeptide fragment MDP-(ld), the 6-amino-derivatives of the naturally occurring MDP-(ld), and its unnatural diastereomer MDP-(ll). The amine functionality installed at the 6-position provides a chemical handle for surface tethering in SPR.

Figure 2

An amine functionalized peptidoglycan library. (a) Synthetic route for the modular synthesis of differentially protected peptidoglycan fragments. (b) Library of amine functionalized peptidoglycan fragments.

Figure 3

Relative NF-κB activation of synthetic peptidoglycan fragments in HEK293T-NOD2myc/Tet-op cells. Relative luciferase activity was measured after 8 h of stimulation with peptidoglycan fragments (*P < 0.03, activates in a NOD2-dependent manner; **P < 0.01). Error bars represent SEM of experiments performed at least six times. Experimental details can be found in the Supporting Information.

Figure 4

Representative BSI measurements. (a) Binding data with NOD2 presented in vesicles derived from the HEK293T-NOD2myc/Tet-op cells in which the protein is expressed. Binding to the peptidoglycan library was referenced against membrane vesicles prepared from HEK293T cells not expressing NOD2 (Figure S6). Curves are fits to a single-site Langmuir binding isotherm. Experimental methods and data analysis are described in the Methods and Supporting Information. (b) Competition binding data. (i) Competition binding experiments in which membrane vesicles were preincubated with excess ligand 2 (10 μM) followed by titration of ligand 20 (red), 22 (green), or 2 (blue). (ii) Membrane vesicles preincubated with excess ligand 22 (10 μM) followed by titration of ligand 2.

Figure 5

Muramyl motif of the MDP ligand proposed to be highly important for NOD2 binding.