title: The expression of recombinant human LOX-1 and identifying its mimic ligands by fluorescence polarization-based high throughput screening.
abstract: Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) was identified as a major receptor for oxidized low-density lipoprotein (oxLDL) in endothelial cells. LOX-1 critically mediates the endothelial dysfunction and the progression of atherosclerosis by oxLDL stimulation. It might be an important target for vascular endothelium. In order to obtain human LOX-1 and identify its mimic ligand for facilitating the study of LOX-1 function, a recombinant plasmid pPIC9K-His-hLOX-1 was structured and expressed human LOX-1 in Pichia pastoris GS115. Western blot analysis ensured the expressed recombinant human LOX-1 protein and a receptor-ligand binding assay showed that it had high binding affinity with oxLDL. With this receptor protein, a competitive fluorescence polarization-based high throughput screening method was established in a 384-well microplate to isolate the mimic ligands of human LOX-1. The evaluating parameter Z' value of 0.72 for this method showed that fluorescence polarization-based high throughput screening assay was robust and the results had a high reliability. By the fluorescence polarization-based high throughput screening assay, a total of 20,316 chemicals were screened, and 2 chemicals were identified that they have a high affinity with human LOX-1. Competitive uptake DiI-oxLDL assay by human LOX-1 transfected CHO-K1 cells further confirmed that two chemicals block the uptake of DiI-oxLDL. And the preliminary results indicated that isolated mimic ligands may act as a function of antagonist. The discovery of human LOX-1 mimic ligand would benefit to further study the function of LOX-1 and identify a novel avenue for prevention and treatment atherosclerosis.
authors.affiliation: National Centre for Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
authors.forename: Tiantai
authors.initials: T
authors.lastname: Zhang
authors.forename: Zhentai
authors.initials: Z
authors.lastname: Huang
authors.forename: Ying
authors.initials: Y
authors.lastname: Dai
authors.forename: Xiuping
authors.initials: X
authors.lastname: Chen
authors.forename: Ping
authors.initials: P
authors.lastname: Zhu
authors.forename: Guanhua
authors.initials: G
authors.lastname: Du
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D008078:Cholesterol, LDL / Q000378:metabolism / Q000493:pharmacokinetics; D006224:Cricetinae; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004355:Drug Stability; D005454:Fluorescence Polarization / Q000379:methods*; D015870:Gene Expression; D006801:Humans; D008024:Ligands; D018716:Molecular Mimicry; D011994:Recombinant Proteins / Q000378:metabolism; D051127:Scavenger Receptors, Class E / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D008078:Cholesterol, LDL; D008024:Ligands; C495137:OLR1 protein, human; D011994:Recombinant Proteins; D051127:Scavenger Receptors, Class E; D004121:Dimethyl Sulfoxide