Mapping and characterization of the binding site for specific oxidized phospholipids and oxidized low density lipoprotein of scavenger receptor CD36.
Recent studies have identified a novel family of oxidized phosphatidylcholines (oxPC(CD36)) that serve as highly specific ligands for scavenger receptor CD36. oxPC(CD36) accumulate in vivo and mediate macrophage foam cell formation as well as promote platelet hyper-reactivity in hyperlipidemia via CD36. The structural basis of oxPC(CD36) binding to CD36 has not been elucidated. We used liquid-phase binding to glutathione S-transferase fusion proteins containing various regions of CD36 to initially identify the region spanning CD36 amino acids 157-171 to contain a major binding site for oxPC(CD36). A bell-shaped pH profile and salt concentration dependence suggest an electrostatic mechanism of the binding. Two conserved, positively charged amino acids in the region 157-171 (lysines at positions 164 and 166) were identified as critical for oxPC(CD36) and oxidized low density lipoprotein (oxLDL) binding to CD36. Lysine neutralization with chemical modifier or site-directed mutagenesis of lysine 164/166 to alanine or glutamate, but not to arginine, abolished binding. Cells expressing full-length CD36 with mutated lysines (164 and 166) failed to recognize oxPC(CD36) and oxLDL. Synthetic peptides mimicking the CD36 binding site, but not mutated or scrambled peptides, effectively prevented: (i) oxLDL binding to CD36, (ii) macrophage foam cell formation induced by oxLDL, and (iii) platelet activation by oxPC(CD36). These data indicate that CD36 (160-168) represents the core of the oxPC(CD36) binding site with lysines 164/166 being indispensable for the binding.
Kar
Niladri S
NS
Department of Molecular Cardiology, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Ashraf
Mohammad Z
MZ
Valiyaveettil
Manojkumar
M
Podrez
Eugene A
EA
D000818:Animals; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism; D018955:CD36 Antigens / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D017353:Gene Deletion; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D051379:Mice; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding
D018955:CD36 Antigens; D008077:Lipoproteins, LDL; D010743:Phospholipids; C099252:oxidized low density lipoprotein