N-Glycans of SREC-I (scavenger receptor expressed by endothelial cells): Essential role for ligand binding, trafficking and stability

Abstract

Scavenger receptor expressed by endothelial cells (SREC-I) mediates the endocytosis of chemically modified lipoproteins such as acetylated low-density lipoprotein (Ac-LDL) and oxidized LDL and is implicated in atherogenesis. We produced recombinant SREC-I in Chinese hamster ovary-K1 cells and identified three potential glycosylation sites, Asn 289, Asn 382 and Asn 393, which were all glycosylated. To determine the function of N-glycans in SREC-I, we characterized SREC-I mutant proteins by intracellular distribution and the cellular incorporation rate of Ac-LDL. N382Q/N393Q and N289Q/N382Q/N393Q were sequestered in the endoplasmic reticulum, resulting in a severe reduction in the cellular incorporation of Ac-LDL. N382Q showed a normal cell surface residency and an enhanced affinity for Ac-LDL, resulting in an elevated Ac-LDL cellular incorporation. These results indicate that the N-glycan of Asn 393 regulates the intracellular sorting of SREC-I and that the N-glycan of Asn 382 controls ligand-binding affinity. Furthermore, we detected an enhanced trypsin sensitivity of the N289Q. Glycan structure analyses revealed that the core-fucosylated bi-antennary is the common major structure at all glycosylation sites. In addition, tri- and tetra-antennary were detected as minor constituents at Asn 289. A bisecting GlcNAc was also detected at Asn 382 and Asn 393. Structural analyses and homology modeling of SREC-I suggest that the N-glycan bearing a 1-6GlcNAc branch at Asn 289 protects from proteinase attack and thus confers a higher stability on SREC-I. These data indicate that Asn 289-, Asn 382- and Asn 393-linked N-glycans of SREC-I have distinct functions in regulating proteolytic resistance, ligand-binding affinity and subcellular localization, all of which might be involved in the development of atherogenesis. © The Author 2012.