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 oxidized low density lipoprotein 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 10 putative glycosylation sites situated in the large extracellular loop of the protein have been identified; however, their utilization and role in the folding and function of the protein have not been characterized. Using mass spectrometry on purified and peptide N-glycosidase F-deglycosylated CD36 and also by comparing the electrophoretic mobility of different glycosylation site mutants, we have determined that 9 of the 10 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 carboxyl-terminal sites Asn-247, Asn-321, and Asn-417. However, unlike SRBI, 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 low density lipoprotein.
Highlights
Human CD36, originally identified in platelets as glycoprotein IV (1), is a class B scavenger receptor localized to the plasma membrane
The sequence of 471 amino acids has two short hydrophobic regions at the carboxyl and amino termini separated by a large hydrophilic region (21); the topology of the protein is unclear with both ditopic (22) and type I (23) topological models proposed
Mutagenesis of either of these two residues resulted in very little cell surface expression of the protein (35); neither site is conserved in human CD36
Summary
Human CD36, originally identified in platelets as glycoprotein IV (1), is a class B scavenger receptor localized to the plasma membrane. FACS analysis of two mutants with an overlapping pattern of intact glycosylation sites N1–7 (CD36N1–7) or N7–10 (CD36N7–10) (Fig. 6A) showed that both reached the plasma membrane but were expressed to very different levels (1.6 and 11% of wild type CD36 (wtCD36), respectively; the top section of Table 3)
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