Investigation of the extent and role of N-linked glycosylation in the human scavenger receptor CD36

Abstract

Human CD36 is a class B scavenger receptor expressed in a variety of cell types such as macrophage and adipocytes. This plasma membrane glycoprotein has a wide range of ligands including oxidised low density lipoprotein (oxLDL) and long chain fatty acids which involves the receptor in diseases such as atherosclerosis and insulin resistance. CD36 is heavily modified post-translationally by N-linked glycosylation and ten putative N-linked glycosylation sites situated in the large extracellular loop of the protein have been identified, however their utilisation and role in the folding and function of the protein have not been characterised. Using mass spectrometry on purified and PNGaseF-deglycosylated CD36, and also by comparing the electrophoretic mobility of different glycosylation-site mutants, this study determined that nine of the ten sites can be modified by glycosylation. Flow cytometric analysis of the different glycosylation mutants expressed in mammalian cells, established that glycosylation is necessary for trafficking to the plasma membrane. Minimally-glycosylated mutants that supported trafficking were identified and indicated the importance of carboxy-terminal sites N247, N321 and N417 and amino-terminal sites N102 and N205. However, unlike the related mouse scavenger receptor SR-BI, no individual site was found to be essential for proper trafficking of CD36. Surprisingly, these minimally-glycosylated mutants appear to be predominantly core glycosylated indicating that mature glycosylation is not necessary for surface expression in mammalian cells. The data also show that neither the nature nor the pattern of glycosylation is relevant to binding of modified LDL.