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Characterizing the cellular requirements involved in the stability of a human innate immune receptor
Date
2022
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
The innate immune system is the body’s first line of defense against invading pathogen-associated molecular patterns (PAMPs). It relies on innate immune receptors such as Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) to initiate an immune response. NOD2 plays a pivotal role in bacterial peptidoglycan (PG) recognition, leading to the release of anti-inflammatory molecules via the NF-κB pathway. Three single nucleotide polymorphisms of NOD2 are implicated in the pathogenesis of Crohn’s disease (CD), a type of Inflammatory Bowel Disease (IBD) that affects the gastrointestinal tract. NOD2 CD variants are unstable and consequently it leads to their inability to respond to PG ligands. Previous studies have shown that some level of activation and stability is restored upon overexpressing the transcriptionally regulated Heat Shock Protein, HSP70. Additionally, an inducible increase of O-GlcNAcylation, a type of protein glycosylation that is tightly controlled by the enzymes O-linked β-N-acetylglucosamine transferase (OGT) and O-GlcNAcase (OGA), regulates NOD2 activity. However, the molecular mechanism and requirements of these actions have not been elucidated. NOD2 is glycosylated by OGT, but the specific O-GlcNAc residues that orchestrate NOD2 function are unknown. My thesis focused on probing critical protein interactions and modification sites that are crucial for NOD2 function as well as understanding the role of OGT and HSP70 on downstream NOD2-signaling. In working towards this goal, I showed that site-directed mutagenesis of a predicted O-GlcNAc modified serine residue into cysteine affects glycosylation, signaling, binding, and stability of NOD2. Furthermore, a novel interaction between NOD2 and OGT in the presence of HSP70 was identified, forming a NOD2:OGT: HSP70 complex. The complex formation was validated further by a web-based server, ZDOCK, that predicts protein-protein interactions based on a scoring system. Additionally, our findings suggest that O-GlcNAcylation modification of NOD2 is important for HSP70 binding in the cell, and both OGT and HSP70 work in concert to stabilize NOD2. In this work, I also developed chemical biology tools and mass spectrometric strategies for studying NOD2 glycosylation. UDP-sugar donors, which are ultimately used by OGT in the glycosylation reaction, were chemoenzymatically synthesized by OGT. Proteomic workflows were developed in an effort to identify the O-GlcNAcylated residues of NOD2. Two potential O-GlcNAcylation modification sites were identified by LC-MS/MS analysis using two search algorithms, SEQUEST and Byonic. Ultimately, these findings illuminate molecular mechanisms for modifying and potentially stabilizing NOD2 and CD-associated variants.
Description
Keywords
Post-translational modification, Pattern recognition receptors, O-GlcNAcylation, NOD2, Innate Immunity, Glycosylation