title: Multivalent Interactions of Human Primary Amine Oxidase with the V and C22 Domains of Sialic Acid-Binding Immunoglobulin-Like Lectin-9 Regulate Its Binding and Amine Oxidase Activity.
abstract: Sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9) on leukocyte surface is a counter-receptor for endothelial cell surface adhesin, human primary amine oxidase (hAOC3), a target protein for anti-inflammatory agents. This interaction can be used to detect inflammation and cancer in vivo, since the labeled peptides derived from the second C2 domain (C22) of Siglec-9 specifically bind to the inflammation-inducible hAOC3. As limited knowledge on the interaction between Siglec-9 and hAOC3 has hampered both hAOC3-targeted drug design and in vivo imaging applications, we have now produced and purified the extracellular region of Siglec-9 (Siglec-9-EC) consisting of the V, C21 and C22 domains, modeled its 3D structure and characterized the hAOC3-Siglec-9 interactions using biophysical methods and activity/inhibition assays. Our results assign individual, previously unknown roles for the V and C22 domains. The V domain is responsible for the unusually tight Siglec-9-hAOC3 interactions whereas the intact C22 domain of Siglec-9 is required for modulating the enzymatic activity of hAOC3, crucial for the hAOC3-mediated leukocyte trafficking. By characterizing the Siglec-9-EC mutants, we could conclude that R120 in the V domain likely interacts with the terminal sialic acids of hAOC3 attached glycans whereas residues R284 and R290 in C22 are involved in the interactions with the active site channel of hAOC3. Furthermore, the C22 domain binding enhances the enzymatic activity of hAOC3 although the sialic acid-binding capacity of the V domain of Siglec-9 is abolished by the R120S mutation. To conclude, our results prove that the V and C22 domains of Siglec-9-EC interact with hAOC3 in a multifaceted and unique way, forming both glycan-mediated and direct protein-protein interactions, respectively. The reported results on the mechanism of the Siglec-9-hAOC3 interaction are valuable for the development of hAOC3-targeted therapeutics and diagnostic tools.
authors.affiliation: Medicity Research Laboratory, University of Turku, Turku, Finland.
authors.forename: Heli
authors.initials: H
authors.lastname: Elovaara
authors.affiliation: Structural Bioinformatics Laboratory, Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland.
authors.forename: Vimal
authors.initials: V
authors.lastname: Parkash
authors.affiliation: Medicity Research Laboratory, University of Turku, Turku, Finland.|Turku Doctoral Program of Molecular Medicine, University of Turku, Turku, Finland.
authors.forename: Ruth
authors.initials: R
authors.lastname: Fair-Mäkelä
authors.affiliation: Structural Bioinformatics Laboratory, Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland.|Pharmaceutical Sciences Laboratory, Pharmacy, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland.
authors.forename: Outi M H
authors.initials: OM
authors.lastname: Salo-Ahen
authors.affiliation: Structural Bioinformatics Laboratory, Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Guédez
authors.affiliation: Structural Bioinformatics Laboratory, Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland.
authors.forename: Eva
authors.initials: E
authors.lastname: Bligt-Lindén
authors.affiliation: Structural Bioinformatics Laboratory, Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland.
authors.forename: Janne
authors.initials: J
authors.lastname: Grönholm
authors.affiliation: Medicity Research Laboratory, University of Turku, Turku, Finland.|Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland.
authors.forename: Sirpa
authors.initials: S
authors.lastname: Jalkanen
authors.affiliation: Structural Bioinformatics Laboratory, Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland.
authors.forename: Tiina A
authors.initials: TA
authors.lastname: Salminen
mesh_terms: D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001120:Arginine; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D000072417:Protein Domains; D055550:Protein Stability; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012663:Semicarbazides / Q000493:pharmacokinetics; D063265:Sialic Acid Binding Immunoglobulin-like Lectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera / Q000235:genetics; D020349:Surface Plasmon Resonance
chemical_list: D015703:Antigens, CD; D015815:Cell Adhesion Molecules; D011994:Recombinant Proteins; C412227:SIGLEC9 protein, human; D012663:Semicarbazides; D063265:Sialic Acid Binding Immunoglobulin-like Lectins; C010059:carbamylhydrazine; D001120:Arginine; C076668:AOC3 protein, human; D006631:Amine Oxidase (Copper-Containing)