STRUCTURAL FEATURES OF SELECTIVE AND NONSELECTIVE NOD RECEPTOR AGONISTS

Abstract

Muramyl peptides are fragments of bacterial peptidoglycan playing an important role in the activation of innate immune system. Core structure of muramyl peptides is composed of N-acetylmuramic acid residue (MurNAc) covalently bound to a peptide consisting of 2-5 amino acid residues. The first residue in the peptide chain is usually L-alanine, the 2nd is D-isoglutamine or D-isoglutamic acid, the 4th and 5th are D-alanines. The 3rd residue is most variable being represented by L-lysine or L-ornithine in Gram-positive bacteria, or by meso-diaminopimelic acid (meso-DAP) in Gram-negative bacteria. Muramyl peptides are potential immunostimulants and adjuvants. Two receptors, NOD1 and NOD2, are known to play a central role in muramyl peptide recognition. However, the data on NOD receptor specificity for several common muramyl peptides are not available or incomplete. The goal of this study was to extend knowledge on possible relationships between structure of natural, or chemically modified muramyl peptides, and their selectivity towards NOD1 and NOD2 receptors. We investigated ability of natural muramyl peptides and their derivatives to activate NOD1 and NOD2. To this end, we used a novel biological test system, which represents modified HEK293T cells with NF-κB-driven reporter gene (luciferase) expression and knock-outs of endogenous NOD1 and/or NOD2 genes. As a result, we have shown that NOD2 has a broader spectrum of natural agonists than previously thought. In particular, NOD2 recognizes muramyl peptides from Gram-negative bacteria containing a meso-DAP residue, which have been considered inactive towards NOD2. NOD1 recognizes only muramyl peptides containing a mesoDAP residue; however, this molecule should not be exposed as a terminal residue, as thought earlier, but it may also be present within the peptide chain. This data imply that natural muramyl peptides containing mesoDAP have a dual receptor specificity (NOD1 and NOD2), i.e. they are non-selective NOD receptor agonists. Destruction of the MurNAc residue eliminates ability of these muramyl peptides to activate NOD2, but has no effect on their NOD1 agonism. Hence, modified muramyl peptides containing meso-DAP and a destroyed MurNAc residue are selective NOD1 agonists. Natural muramyl peptides featuring an intact MurNAc and no meso-DAP are selective NOD2 agonists. Conclusions: 1) we have characterized key structural features of selective NOD1 and NOD2 agonists as well as non-selective NOD receptors agonists; 2) we show the importance of these new data for understanding innate immune response regulation, and for development of novel muramyl peptide immunostimulants.