Oligomerization is required for the activity of recombinant soluble LOX-1.
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.
Cao
Wei
W
Department of Biological Technologies, Wyeth Research, Cambridge, MA 02140, USA.
Calabro
Valerie
V
Root
Adam
A
Yan
Grace
G
Lam
Khetemenee
K
Olland
Stephane
S
Sanford
Jocelyn
J
Robak
Angela
A
Zollner
Richard
R
Lu
Zhijian
Z
Ait-Zahra
Mostafa
M
Agostinelli
Rita
R
Tchistiakova
Lioudmila
L
Gill
Davinder
D
Harnish
Douglas
D
Paulsen
Janet
J
Shih
Heather H
HH
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D001665:Binding Sites; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D003432:Cross-Linking Reagents / Q000737:chemistry; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000276:immunology; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D051127:Scavenger Receptors, Class E / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D000906:Antibodies; D003432:Cross-Linking Reagents; D007141:Immunoglobulin Fc Fragments; D008077:Lipoproteins, LDL; C495137:OLR1 protein, human; D011993:Recombinant Fusion Proteins; D051127:Scavenger Receptors, Class E; C099252:oxidized low density lipoprotein