Abstract 163: Functional Analysis of Novel Mutations in Human SR-BI

Abstract

Scavenger receptor class B, type I (SR-BI) is an 82 kDa glycosylated high density lipoprotein (HDL) receptor that is imperative for mediating HDL-cholesterol (HDL-C) uptake into the liver for whole body cholesterol excretion. Recently, two novel point mutations in human SR-BI, S112F or T175A, were discovered in patients with high HDL-C levels. We hypothesize that the high HDL-cholesterol levels are a direct result of each mutant SR-BI’s inability to bind and mediate the selective uptake of cholesteryl esters (CE) from HDL particles. To test this hypothesis, we created SR-BI cDNA encoding the S112F- or T175A point mutations. Cell surface biotinylation and flow cytometry analysis confirmed the plasma membrane localization of both mutant receptors, although T175A-SR-BI expressed at slightly lower levels than wild-type SR-BI. Following transient transfection into COS-7 cells, both S112F- and T175A-SR-BI mutant receptors displayed a greater than 75% decrease in [125I]HDL binding and a greater than 90% decrease in selective uptake of [3H]HDL-COE, as compared to wild-type SR-BI. Both mutant SR-BI receptors also displayed an impaired ability to mediate the release of free cholesterol from cells to HDL. PFO-PAGE revealed that the mutant receptors maintained their ability to form dimers and higher order oligomers, suggesting that the lack of function is not due to a change in the oligomeric status of SR-BI. Interestingly, introduction of alanine at residue 175 disrupts the consensus sequence for N-linked glycosylation at Asn173. Immunoblot analysis confirmed that the T175A-SR-BI migrated slightly faster than wild-type SR-BI, and we hypothesize that receptor function may be compromised due to changes in the glycosylation pattern of this receptor. It is also important to note that S112 and T175 are located in a highly conserved hydrophobic region of the extracellular domain that has previously been shown to be important for receptor function. As such, we speculate that mutations at one or both of these residues may also have a significant impact on HDL-cholesterol transport due to changes in hydrophobicity of this region of SR-BI.