Oligomerization is required for the activity of recombinant soluble LOX-1

• Published in: The FEBS journal 2009-09, Vol.276 (17), p.4909-4920
• Main Authors: Cao, Wei, Calabro, Valerie, Root, Adam, Yan, Grace, Lam, Khetemenee, Olland, Stephane, Sanford, Jocelyn, Robak, Angela, Zollner, Richard, Lu, Zhijian, Ait-Zahra, Mostafa, Agostinelli, Rita, Tchistiakova, Lioudmila, Gill, Davinder, Harnish, Douglas, Paulsen, Janet, Shih, Heather H
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; antagonists; Antibodies - chemistry; atherosclerosis; Binding Sites; Biochemistry; Cell Membrane - metabolism; CHO Cells; Cricetinae; Cricetulus; Cross-Linking Reagents - chemistry; crosslinking; endothelial cells; Fc fusion protein; Humans; Immunoglobulin Fc Fragments - genetics; Immunoglobulin Fc Fragments - immunology; ligands; Lipoproteins, LDL - metabolism; low density lipoprotein; Low density lipoprotein receptors; LOX‐1; Models, Molecular; Molecular biology; Molecular Sequence Data; Oxidation; polyclonal antibodies; Protein Multimerization; Protein Structure, Tertiary; Proteins; receptor oligomerization; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - pharmacology; recombinant protein; scavenger receptor; Scavenger Receptors, Class E - antagonists & inhibitors; Scavenger Receptors, Class E - genetics; Scavenger Receptors, Class E - metabolism; therapeutics
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2009.07190.x

LOX-1 is a scavenger receptor that functions as the primary receptor for oxidized low-density lipoprotein (OxLDL) in endothelial cells. The binding of OxLDL to LOX-1 is believed to lead to endothelial activation, dysfunction, and injury, which constitute early atherogenic events. Because of its potential pathological role in atherosclerosis, LOX-1 has been proposed as a therapeutic target for the treatment of this disease. In order to antagonize the ligand-binding function of cell surface LOX-1, we generated a series of recombinant human LOX-1-crystallizable fragment (Fc) fusion proteins and subsequently characterized their biochemical properties and ligand-binding activities in vitro. Consistent with the notion that oligomerization of cell surface LOX-1 is required for high-avidity binding of ligands, we found that LOX-1-Fc fusion protein containing four ligand-binding domains per Fc dimer, but not the one containing two ligand-binding domains, exhibited ligand-binding activity. Optimal ligand-binding activity could be achieved via crosslinking of LOX-1-Fc fusion proteins with a polyclonal antibody against Fc. The crosslinked LOX-1-Fc protein also effectively inhibited the binding and internalization of OxLDL by cell surface LOX-1. These findings demonstrate that functional oligomerization is required for recombinant LOX-1-Fc to function as an effective antagonist.