Assessment of direct binding interaction between CD36 and its potential lipid ligands using a peptide mimic of the receptor labeled with a fluorophore.

Abstract

Cluster of differentiation 36 (CD36) is a cell-surface receptor that recognizes diverse substances. We have presented indirect evidence that a short segment of the receptor comprising amino acids 149-168 contains a site for binding of its lipid ligands (e.g., distinct fatty acids and aldehydes). However, experimental support for their direct interactions is yet to be achieved. For this, we devised a fluorescence intensity assay, where a synthetic peptide consisting of CD36 amino acids 149-168 labeled with fluorescein isothiocyanate (FITC-CD36149-168) and its variant peptides were used as positive and negative probes, respectively. First, we obtained results indicating that 1-palmitoyl-2-(5-keto-6-octenedioyl)phosphatidylcholine (an established CD36 ligand) but not 1-palmitoyl-2-arachidonyl-phosphatidylcholine (a non-ligand of the receptor) bound in a saturable and specific manner to FITC-CD36149-168. Strikingly, the assay allowed us to provide the first evidence supporting direct and specific binding between the CD36 segment and fatty aldehydes (e.g., Z-11-hexadecenal). However, this method failed to illustrate specific interactions of the segment with fatty acids, such as oleic acid. Nonetheless, our findings offer further insight into the biologically relevant ligands and the role of CD36. In addition, we suggest that this fluorescence-based technique provides a convenient means to evaluate protein (peptide)-lipid interactions.