Exploring the Binding Specificity of Siglec-Derived Peptides to Human Primary Amine Oxidase Using an Integrative...

Human primary amine oxidase (AOC3) is a membrane-anchored protein expressed on the luminal surface of the vascular endothelium at sites of inflammation. It binds via protein-protein interactions to the CE-loops of the C2₂ domain in Siglec-9 and Siglec-10, but the exact mechanism of interaction is unknown on an atomic level. The AOC3 binding to a radiolabeled Siglec-9 peptide can be used to image inflammation by positron emission tomography (PET). In the present study, a combination of computational and wet lab experiments is used to map the binding of the original and designed shorter Siglec-derived peptides to AOC3, to determine what features in the CE-loop are important for the interaction. To select the peptides for wet lab studies, docking and molecular dynamics simulations were first conducted to predict the binding mode of the peptides in the AOC3 active site channel, and MMGBSA calculations to predict binding energies. Selected peptides were further analyzed through microscale thermophoresis to affirm binding and saturation transfer difference NMR (STD-NMR) to determine the binding epitope to AOC3. Our results unveil that the shorter cyclic Siglec-9 and Siglec-10 peptides derived from the CE-loop bind to AOC3. Our results summarize that a tryptophan and an arginine in both peptides are important for the AOC3 interactions, but their binding mode differs slightly. Our results suggest that the designed shorter Siglec-9 peptide could be advantageous in PET imaging of inflammation and the binding properties of the Siglec-10 peptides hint a biological role for the CE-loop of the C2 domains in Siglec-10.