Fatty acid binding to CD36 affects oxLDL uptake

Abstract

We investigated the hypothesis that interactions of unesterified fatty acid (FA) with the plasma membrane protein CD36 affect uptake of oxLDL. Because the binding of natural FA to CD36 have not been systematically studied, we first characterized FA binding to CD36 by the biophysical method surface plasmon resonance (SPR). After covalently linking the extracellular domain of CD36 to an SPR chip surface, aqueous FA (solubilized in cyclodextrin) was pulsed across the surface, generating association and dissociation binding curves. We studied structurally different long chain FA: a saturated (palmitic acid); unsaturated (oleic acid); trans‐oleic acid; (elaidic); a polyunsaturated [docosahexaenoic acid (DHA)], and an oxidized FA [9S‐hydroxy‐10E,12Z‐octadecadienoic acid (HODE)]. With the exception of HODE, SPR detected FA binding to CD36, and showed that multiple sites are present with rapid association and dissociation kinetics that were similar to HSA. To study the role in oxLDL uptake of these FA, we used a fluorescent oxLDL (Dii‐oxLDL) live‐cell assay with confocal microscopy imaging. CD36‐mediated uptake in serum‐free media was very low but greatly increased with serum. Addition of FA in serum‐free media increased oxLDL binding and uptake to levels found with serum, and affected CD36 plasma membrane (PM) distribution. Binding/uptake of oxLDL exhibited dose dependency of added FA, with the exception of DHA, which binds avidly to CD36 but did not activate uptake. HODE, which binds poorly to CD36, did not affect oxLDL uptake. The high affinity FA binding to CD36 has important implications that binding at some or all sites could affect its conformation, binding of other ligands, and functional properties.