Peptidoglycan Probes for Fluorescence Polarization Displacement Assays to Investigate Ligand Recognition by Human Peptidoglycan Recognition Protein 1

Bacterial peptidoglycan fragments (PGNs) are key signaling molecules in mammalian hosts. A central aspect of understanding their biological functions is the biochemical characterization of PGN recognition by host receptors. Herein, we employed two fluorescent PGN probes, 940-NADA and 940-C1-NBD, to demonstrate their binding to human peptidoglycan recognition protein 1 (hPGRP1) using in vitro fluorescence polarization (FP) assay. Additionally, we used a diverse panel of chemically synthetized or isolated PGNs with varying stem peptide lengths, compositions, and amidation status, which reflect the structural diversity of bacterial peptidoglycan, to investigate their ability to displace 940-NADA from hPGRP1 in an FP displacement assay. Lastly, sequestration of PGNs by hPGRP1 attenuated NOD1 signaling in reporter cells and reduced the production of proinflammatory cytokines in THP-1 cells. Together, these results establish fluorescent PGN probes as a versatile platform for detecting hPGRP1-PGN interactions and provide functional evidence supporting the anti-inflammatory role of hPGRP1 in host innate immunity.