Studies of CD36 interacting with fatty acids, oxidized low-density lipoprotein, and the cellular plasma membrane
The glycoprotein CD36 is expressed in the plasma membrane (PM) of many cell types that surround or contact arteries, including macrophages, myocytes, and endothelial cells. CD36 binds oxidized low density lipoprotein (oxLDL), which promotes atherosclerosis, and fatty acids (FA), which promotes their cellular uptake. To gain insights into the molecular mechanisms of uptake, HEK293 cells expressing CD36 were studied by cell biological and fluorescence methods. To test our hypothesis that the PM is not an impermeable barrier to FA and that FA move into cells by diffusion via their uncharged form, we first applied biophysical fluorescence spectroscopy to directly measure transmembrane FA movement and membrane fluidity. Expression of CD36 in HEK293 cells did not increase either transport across the PM or the fluidity of the PM compared to HEK293 cells without CD36; however, CD36 enhanced intracellular FA esterification. Furthermore, the widely used “inhibitors” of FA transport did not alter either the rapid FA transmembrane diffusion in HEK293 cells or diffusion in control experiments with protein-free phospholipid bilayers. To gain new insights into the physiological relevance of FA binding to CD36, we applied surface plasmon resonance (SPR) to quantify FA and oxLDL binding to the ectodomain of CD36. Structurally distinct FA [saturated, monounsaturated (cis and trans), polyunsaturated, ω-3, ω-6, and oxidized FA] were pulsed in a solubilized form (bound to methyl-β-cyclodextrin) across SPR channels, generating real-time association and dissociation binding curves. With the exception of the oxidized FA hydroxyoctadecadienoic acid (HODE), all FA tested bound to CD36 with rapid association and dissociation kinetics similar to human serum albumin. In addition, FA increased oxLDL binding to CD36. To investigate whether FA affect CD36-mediated oxLDL uptake in live cells, we monitored fluorescent oxLDL (Dii-oxLDL) uptake using confocal microscopy. Addition of exogenous FA to serum-free media enhanced dose-dependent oxLDL uptake. Exceptions were ω-3 FA, which bound to CD36, and HODE, which did not bind to CD36, demonstrating FA structure-specific effects on a major function of CD36 and a new mechanistic link between atherosclerosis and high levels of FA in obese and Type-II diabetic individuals.