Abstract 676: Novel Human Variants Of SR-B1 Impact HDL-cholesterol Clearance

Abstract

The reverse cholesterol transport (RCT) pathway is the primary avenue by which our bodies lower plasma cholesterol levels. In RCT, peripheral cholesterol is transported by high density lipoproteins (HDL) to the liver for excretion. Cholesterol delivery is facilitated by a high-affinity interaction between HDL and its receptor, scavenger receptor class B member 1 (SR-B1). Recently, novel variants within the gene encoding SR-B1 were identified in a population of Icelandic people. Four missense mutations - V32M-, V111M-, V135I-, and G319V-SR-B1- were associated with elevations in plasma HDL-cholesterol. Based on these clinical phenotypes, we hypothesized that these conserved variants of SR-B1 would disrupt HDL-cholesterol transport. To assess the functions of the variants, we transiently transfected vector, wild-type, or mutant SR-B1 constructs into COS7 cells which express low endogenous SR-B1. All mutants displayed decreased DiI-HDL binding, yet only G319V-SR-B1 displayed a significant decrease in DiI lipid uptake, by 42% compared to wild-type. SR-B1 also mediates cholesterol efflux from cells into HDL. When assayed, only V32M- and G319V-SR-B1 decreased cholesterol efflux to HDL by 25 and 52%, respectively, compared to wild-type. Additionally, we demonstrated a decrease in total and cell surface expression of the G319V-SR-B1 variant compared to wild-type SR-B1, suggesting that impairments in function could be due to decreased expression. SR-B1 forms oligomers to facilitate its functions, and all variants maintained the ability to form higher order oligomers. To further characterize the SR-B1 variants, we utilized a recently-developed purification system to generate full-length SR-B1 protein and performed microscale thermophoresis to monitor changes in affinity for HDL. Paralleling our results from cellular assays, all SR-B1 variants displayed impaired HDL binding. When mapped to a homology model of full-length SR-B1, all four mutations are within the extracellular region, but the functional perturbations caused by each mutation warrants further studies. These mutants, combined with previously characterized SR-B1 variants, can be integrated as important pieces in the puzzle of cholesterol homeostasis and cardiovascular disease.