A Tale of Two Scavenger Receptors: Structure‐Function Relationships of Purified SR‐B1 and CD36

Both CD36 and SR-B1 are glycosylated integral membrane proteins belonging to the class B scavenger receptor family and are known to play roles in cardiovascular and atherosclerotic disease. CD36 serves as the receptor for oxidized low density lipoprotein (oxLDL) and facilitates cholesterol accumulation in macrophages and development of atherosclerotic plaques. SR-B1 (scavenger receptor class B type 1) is the primary receptor for high density lipoprotein (HDL), which removes cholesterol from the peripheral atherosclerotic plaques and delivers it to the liver for excretion. Current structural information about class B scavenger receptors is limited to only purified extracellular regions, transmembrane segments, or transient transfection studies in cultured cells, and remains a roadblock to studying the isolated functions of these lipid and cholesterol transporters. Recently, we have successfully expressed and purified full-length human SR-B1 and CD36 using an Sf9 insect cell/baculoviral infection system, which allows for the expression of glycosylated proteins. Purified full-length receptors are a valuable addition to our scientific toolbox and allow us to ask novel questions about the structure-function relationships of scavenger receptors. To verify function within Sf9 cell membranes, we expressed human full-length constructs in plated Sf9 cells and observed significantly increased binding of DiI-HDL and DiI-oxLDL, as well as increased DiI-lipid uptake when comparing SR-B1 and CD36 to empty vector infected cells, respectively. When purified and solubilized in detergent micelles, both receptors remain stable over time as shown by Prometheus thermal shift assays. We have also demonstrated that purified proteins are able to bind to their native ligands (HDL to SR-B1 and oxLDL to CD36) with high affinity by microscale thermophoresis (MST). However, as SR-B1 and CD36 are both scavenger receptors, they are able to bind to a variety of lipoprotein ligands and we have demonstrated these interactions with varying binding affinities by MST. Glycosylation of both CD36 and SR-B1 has been shown to be important for cell surface expression, but the role of glycosylation in ligand binding has remained uncharacterized. We are the first to demonstrate that the glycosylation status of CD36 and SR-B1 does not alter binding affinities or protein stability. These purified receptors and functional assays provide promise for understanding the ways in which class B scavenger receptors perform their critical functions and lay the groundwork for future structure-function studies.