Synthesis and application of peptidoglycan derivatives to study the recognition and activation of the innate immune receptor NOD2
Date
2017
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
The bacterial cell wall fragment, muramyl dipeptide, has been recognized since the 1970s as the minimal fragment necessary to initiate an innate immune response. Within the last 20 years, the innate immune protein NOD2 was implicated to have direct involvement in the muramyl dipeptide-mediated immune response. Mechanistic work posed NOD2 as the receptor for muramyl dipeptide, however explicit biochemical evidence to directly link NOD2 and muramyl dipeptide as interacting partners remained elusive. In an effort to gain a better understanding of the NOD2 receptor we developed: 1) synthetic routes for natural and modified forms of peptidoglycan derivatives including muramyl dipeptide, and 2) an in vitro binding assay to assess the affinity of NOD2 for muramyl dipeptide and other peptidoglycan fragments. This thesis discusses the application of synthetic peptidoglycan tools and our in vitro binding assay in combination with cellular assays to describe the action of the NOD2 innate immune receptor. ☐ The first report of a direct interaction between NOD2 and muramyl dipeptide using the surface plasmon resonance in vitro binding assay was established in this thesis. Follow up work then demonstrated that NOD2 is capable of binding a variety of peptidoglycan ligands. Within the scope of NOD2-ligand binding exists peptidoglycan fragments smaller than muramyl dipeptide that are also capable of activating an innate immune response, thus indicating that muramyl dipeptide is not the smallest bacterial derived fragment necessary to activate such a response. Evidence is then presented to demonstrate that NOD2 membrane-association confers a more stringent ligand specificity to the NOD2 receptor when signaling via the NF-ĸ B pathway. Finally, structure-activity relationships identify the NOD2 binding motif on muramyl dipeptide. ☐ In order to better understand the regions of muramyl dipeptide important for NOD2 binding, a synthetic route allowing for the late stage modification of the 2-position of muramyl dipeptide was developed. The route allowed for the installation of a variety of acyl groups on the amine at the 2-position. The effect of the 2-position of muramyl dipeptide on NOD2 binding and activation was largely unexplored prior to this work. The resultant library of muramyl dipeptides with varying N-acylation was subsequently assayed for its ability to activate and stabilize the NOD2 receptor, and structure activity relationships were established. The studies performed in this thesis will aid in developing a better understanding of NOD2 function and signaling, and provides a better understanding of how our bodies recognize and respond to bacteria.
Description
Keywords
Innate immune receptor, Innate immunity, MDP, Muramyl dipeptide, NOD2