Monitoring Innate Immune Receptor Stability via Post‐Translational Modification by OGT

Abstract

The human body's first line of defense against infectious disease is the innate immune system, which is tasked with distinguishing harmful pathogens from commensal and beneficial microbes. On a molecular level, this system contains different pattern recognition receptors (PRRs) that detect microbe associated molecular patterns (MAMPs) specific to particular microorganisms, and the interaction between a PRR and its respective MAMP triggers an immune response. The Nod‐like receptors (NLRs) are one major family of innate immune receptors that contain the proteins Nod1 and Nod2, which use different bacterial cell wall peptidoglycan (PGN) fragments as their designated MAMPs. Binding between the NLR and PGN fragment is mediated by the leucine rich repeat domain (LRR), which exists at the C‐termini of Nod1 and Nod2. Upon LRR‐MAMP stimulation, NF‐kB and other signaling cascades are initiated, ending with the production of proinflammatory cytokines and chemokines. Misregulation of signaling by NLRs can lead to disease, and mutations in Nod1 and Nod2 are associated with gastric cancer and Crohn's disease, respectively. These NLRs are critically important for maintaining the relationship between the human body and the trillions of microorganisms that it hosts, yet so much about their functions and regulation in the body remain unclear. Recently, Nod2 was identified as being O‐‐GlcNAcylated, which is a post‐translational modification mediated by two enzymes that affect thousands of proteins in diverse ways. An emerging cellular thermal shift assay (CETSA) that is used for identifying protein‐ligand binding was repurposed to measure the stabilizing effect of O‐GlcNAc modification on Nod1 and Nod2. When protein O‐GlcNAc levels are increased, melting temperatures of both NLRs increase as well. These data are supported by cycloheximide half‐life experiments and NF‐kB luciferase assays, which demonstrated that the modification also led to increased NLR half‐lives and NF‐kB activity. Additionally, two Nod2 Crohn's associated variants were studied, and both G908R‐Nod2 and 1007fs‐Nod2 are O‐GlcNAcylated in the cell. While the modification led to similar increases in melting temperature and NF‐kB activity for 1007fs‐Nod2, its effects on G908R‐Nod2 are less clear. These results show that CETSA is a useful tool for measuring stability changes caused by O‐GlcnAc modification and that NLRs may be commonly post‐translationally modified to regulate stability.Support or Funding InformationMITZUTANI GLYCOSCIENCE FOUNDATION, NATIONAL SCIENCE FOUNDATION (CAREER CHE 1554967), NIH NATIONAL HEART, LUNG, AND BLOOD INSTITUTE (P01HL107153)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.