The Role of Palmitoylation of CD36 in the Uptake of Oxidized Low Density Lipoprotein

CD36 is a scavenger receptor located in a variety of cell types including monocytes, endothelial cells, epithelial cells, and various cultured cell lines. CD36 has been identified as the receptor for thrombospondin; the receptor-ligand pair was shown to mediate interactions between platelets and monocytes, tumor cells and matrix. Since then, the discovered receptor functions of CD36 have broadened to include CD36 acting as a receptor for collagen, oxidized low density lipoprotein (oxLDL), fatty acids, anionic phospholipids, and malaria parasitized erythrocytes. The study of the binding of oxLDL with CD36 is crucial because oxidized LDL is a carrier of cholesterol esters and after CD36 internalizes the ligand, the individual cell becomes lipid-laden and oxidized LDL activates the cell to signal various inflammatory mediators. The net result of these actions is the build-up of atherosclerotic lesions in the arteries by forcing the individual cell to become lipid-laden.

CD36 is comprised of 472 amino acids, arranged with the bulk of the protein being extracellular, two transmembrane domains, and two short cytoplasmic tails located on either end of the receptor. Earlier work in this laboratory had shown that four cysteine residues located on the tails of the receptor were modified by palmitate, a hydrophobic, 16-carbon saturated fatty acid. A non-palmitoylated CD36 mutant (CD36-4S) was constructed by changing each of those cysteine residues to serine.

Having created a non-palmitoylated mutant of CD36, the endocytosis of oxLDL could be analyzed and compared. The experimental procedures consisted of transfecting two sets of CD36, CD36-4S, and pcDNA3 (as a negative control) into Chinese hamster ovary cells as plasmids. One set was fixed with paraformaldehyde before the adding of the FA6/Alexa 488 antibodies, a mouse antibody to CD36 and a fluorescent goat antibody to mouse, so that the dispersion of CD36 versus CD36-4S on the cellular membrane could be viewed under a laser microscope; the other set was fixed with paraformaldehyde after incubating with the FA6/Alexa 488 for one hour to allow the ligand to be internalized along with the CD36. Next, all six samples were mounted on slides and viewed using a confocal laser microscope.

Both CD36 and CD36-4S cells fixed prior to activation had equal distributions of the receptor-ligand pair on the cellular membrane. Yet, upon examining the CD36 and CD36-4S cells fixed after activation, only 22% of the CD36-4S mutant version internalized oxLDL, while, 89% of the CD36 wild type version internalized oxLDL.

In conclusion, the non-palmitoylated version of CD36 is equal in efficiency in the binding of oxLDL with the wild type version of CD36. Yet, the non-palmitoylated version was less efficient in internalizing oxLDL than the wild type version. Additional experiments will be done to confirm these results and to analyze the detailed molecular steps required for binding and internalization of oxLDL through CD36. Since these steps contribute to atherosclerosis and plaque accumulation in the arteries, possible future research could address methods to block the endocytosis of oxLDL with CD36, while not affecting any of the receptor's other biological processes.