Mapping the interaction site and effect of the Siglec-9 inflammatory biomarker on human primary amine oxidase

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.

Siglecs Modulate Activities of Immune Cells Through Positive and Negative Regulation of ROS Generation

“…Such Siglec-binding with oxidase enzyme through protein-protein interactions enhances hAOC3 activity and generates hydrogen peroxide ( 39 ). Siglec-9 also binds to sialoglycoprotein hAOC3 through sialic acid-Siglec binding via its V domain ( 40 ).…”

Vascular adhesion protein-1-targeted PET imaging in autoimmune myocarditis

“… 20 Trp8 was additionally mutated to alanine since its predicted binding site is not conserved in human VAP-1 and rat VAP-1. 23 Thereafter, the residues in the resulting CAALSLSAAGLTLCPSK peptide were scrambled manually by avoiding Siglec-9-derived amino acid repeats and by distributing polar residues throughout the sequence to avoid hydrophobic patches. The constructed [ 68 Ga]Ga-DOTA-control, CSALATGLSALALCPSK, the R3A/R9A, and the [ 68 Ga]Ga-DOTA-Siglec-9 peptides were docked into the homology model for rat VAP-1 (rVAP-1) with Gold 24 using the pyridazinone inhibitor in the crystal structure of hVAP-1 (Protein Data Bank (PDB) ID 4bty) 25 as the center of the binding site.…”

“…The constructed [ 68 Ga]Ga-DOTA-control, CSALATGLSALALCPSK, the R3A/R9A, and the [ 68 Ga]Ga-DOTA-Siglec-9 peptides were docked into the homology model for rat VAP-1 (rVAP-1) with Gold 24 using the pyridazinone inhibitor in the crystal structure of hVAP-1 (Protein Data Bank (PDB) ID 4bty) 25 as the center of the binding site. The rVAP-1 model was created with Modeller 26 using the published sequence alignment 23 and hVAP-1 structure (PDB ID 4bty). Additionally, all peptides were docked into hVAP-1 for comparison.…”

“…Trp8 in the [ 68 Ga]Ga-DOTA-Siglec-9 peptide binds close to the specificity pocket of hVAP-1 (Asp180, Thr210, and Leu177). 23 , 25 This binding site is not conserved in rVAP-1 where the corresponding residues are Gln180, Lys210, and Gln177. 23 Trp8 in the docked [ 68 Ga]Ga-DOTA-Siglec-9 and R3A/R9A peptides binds near Gln180 and Gln177 in rVAP-1, whereas the constructed 68 Ga-DOTA-control with scrambled sequence and without any tryptophanes does not interact with these residues (Supplemental Figure 1).…”