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Title: Regulation of ligand recognition and endocytosis by the LOX-1 scavenger receptor
Author: Lacey, Katie
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2012
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Lectin-like oxidised low-density lipoprotein receptor-1 (LOX-1) is a scavenger receptor that binds a wide range of ligands including oxidised low-density lipoprotein (OxLDL). LOX-1-mediated recognition of OxLDL can cause endothelial dysfunction and apoptosis. LOX-1 is implicated in foam cell formation and atherosclerotic plaque initiation and progression. The C-type lectin-like domain of LOX-1 binds OxLDL but the molecular basis for this recognition is unclear. Glycosidase or protease-mediated removal of exposed N-linked carbohydrates or protein epitopes on OxLDL led to the conclusion that LOX-1 recognises carbohydrates but not protein determinants on OxLDL. We hypothesised that the identification of such glycans would enable us to target the LOX-1-0xLDL interaction. Screening of a glycan array using recombinant LOX-1 identified GaINAcα1-3Galβ (carbohydrate 1) and Galα1-3(Galα1-4)Galβ1-4GlcNAcβ (carbohydrate 2) as potential LOX-1 ligands. Carbohydrate 1 was unable to block the interaction but 10 mM carbohydrate 2 almost completely abolished OxLDL binding. A simpler carbohydrate: Galα1-3(Galα1-4)Galβ-pMP (carbohydrate 3) was tested and showed improved blocking capability. Carbohydrate 3 administration to ApoE-/- transgenic mice fed a fat-rich diet showed decreased incidence of arterial atherosclerosis in comparison to controls. LOX-1 mediates endocytosis of OxLDL, in a clathrin- and AP-2-independent but dynamin-2-dependent manner and is reliant upon a novel DOL endocytic motif. Yeast-based genetic screens to identify cytosolic factors that bound wild-type (DOL) but not mutant (DAL) LOX-1 endocytic motifs, identified components of the AP-4 adaptor complex. Knockdown of the AP-4E subunit in epithelial cells expressing LOX-1, resulted in a significant decrease in LOX-1-mediated OxLDL internalisation. LOX-1 endocytosis also appears to be an actin-dependent process involving plasma membrane ruffles possibly regulated by the Rho Kinase, ROCK. Pharmacological inhibition of ROCK caused a significant decrease in LOX-1- mediated OxLDL endocytosis. This work has shed new light into the mechanisms regulating LOX-1-mediated ligand binding and endocytosis and has identified soluble inhibitors that block this interaction and are potential atherosclerosis therapeutics.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available