title: 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 β1-6GlcNAc 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.
authors.affiliation: Department of Disease Glycomics,  Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047,  Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sano
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Korekane
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Ohtsubo
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Yamaguchi
authors.forename: Masaki
authors.initials: M
authors.lastname: Kato
authors.forename: Yukinao
authors.initials: Y
authors.lastname: Shibukawa
authors.forename: Michiko
authors.initials: M
authors.lastname: Tajiri
authors.forename: Hideki
authors.initials: H
authors.lastname: Adachi
authors.forename: Yoshinao
authors.initials: Y
authors.lastname: Wada
authors.forename: Michio
authors.initials: M
authors.lastname: Asahi
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Taniguchi
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers; D006801:Humans; D007700:Kinetics; D008024:Ligands; D016133:Polymerase Chain Reaction; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D051128:Scavenger Receptors, Class F / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D017931:DNA Primers; D008024:Ligands; D011134:Polysaccharides; C495126:SCARF1 protein, human; D051128:Scavenger Receptors, Class F