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.

Bacterial Derived Carbohydrates Bind Cyr1 and Trigger Hyphal Growth in Candida albicans

“… 27 Recently, we have developed robust solution chemistry to access amino versions of peptidoglycan fragments which utilizes a benzyl protected carbohydrate ( 5 , Scheme 1 ). 26 Here, a tripeptide ( 7 , Scheme 1 ), which contains benzyl protecting groups, was synthesized using solution based chemistry (see the Supporting Information for synthetic details; Supplemental Scheme 1 ). 7 was coupled to 5 using coupling reagents to minimize epimerization, and the resulting product was globally deprotected using catalytic hydrogenation to yield 4 , a novel MTP whose tripeptide contains l -alanine, d -isoglutamine, and l -lysine.…”

Synthesis and Application of Methyl N,O‐Hydroxylamine Muramyl Peptides

“…NF‐κB luciferase assay : An established assay was used . See the Supporting Information for additional details/statistical analysis.…”

Peptidoglycan O-Acetylation as a Virulence Factor: Its Effect on Lysozyme in the Innate Immune System

“…NF- K B is also activated through a signal cascade in phagocytes via the recognition by the intracellular receptors nucleotide-binding oligomerization domain-containing protein 1 and 2 (NOD1 and NOD2). Gram-negative, and some Gram-positive, PG fragments containing γ- d -Glu- m DAP activate NOD1, while it has been demonstrated that for NOD2, the minimal fragment needed for recognition is a MurNAc residue, suggesting that it could function as a universal PG receptor [30,31]. It should be noted, however, that although lysozyme-released fragments adequately stimulate TLR2 and NOD2, released monomers containing 1,6-anhMurNAc products have been shown to have much weaker activation abilities [32].…”