Abstract
Human primary amine oxidase (hAOC3), also known as vascular adhesion protein 1, mediates leukocyte rolling and trafficking to sites of inflammation by a multistep adhesion cascade. hAOC3 is absent on the endothelium of normal tissues and is kept upregulated during inflammatory conditions, which is an applicable advantage for imaging inflammatory diseases. Sialic acid binding immunoglobulin like-lectin 9 (Siglec-9) is a leukocyte ligand for hAOC3. The peptide (CARLSLSWRGLTLCPSK) based on the region of Siglec-9 that interacts with hAOC3, can be used as a specific tracer for hAOC3-targeted imaging of inflammation using Positron Emission Tomography (PET). In the present study, we show that the Siglec-9 peptide binds to hAOC3 and triggers its amine oxidase activity towards benzylamine. Furthermore, the hAOC3 inhibitors semicarbazide and imidazole reduce the binding of wild type and Arg/Ala mutated Siglec-9 peptides to hAOC3. Molecular docking of the Siglec-9 peptide is in accordance with the experimental results and predicts that the R3 residue in the peptide interacts in the catalytic site of hAOC3 when the topaquinone cofactor is in the non-catalytic on-copper conformation. The predicted binding mode of Siglec-9 peptide to hAOC3 is supported by the PET studies using rodent, rabbit and pig AOC3 proteins.
Highlights
Inflammatory cascade entails migration of cells such as leukocytes from the circulation to the site of infection through a complex series of events
Earlier we have shown that the cyclic Siglec-9 peptide (CARLSLSWRGLTLCPSK) binds to immobilized hAOC3 under flow conditions and that the two arginines R3 and R9 of the peptide are critical for the binding[8] (Fig. 1)
Human AOC3 plays a role in the recruitment of leukocytes to sites of inflammation
Summary
Inflammatory cascade entails migration of cells such as leukocytes from the circulation to the site of infection through a complex series of events. The adhesive function involves the interaction with leukocytes by the action of sialylated carbohydrates found on its surface[2,3], while the enzymatic function is responsible for the deamination of primary amines such as, aminoacetone and methylamine, to their corresponding aldehyde products via an oxidative reaction producing hydrogen peroxide and ammonia[4]. We show novel experimental data together with molecular docking simulation that illustrates the binding mode of the Siglec-9 peptide in the active site cavity of hAOC3. These results provide valuable knowledge to aid in the improvement of hAOC3-targeted diagnostic tools and therapeutics
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