Abstract
We have studied the effects of mutations in apoA-I on reconstituted high density lipoprotein (HDL) particle (rHDL(apoA-I)) binding to and cholesterol efflux from wild-type (WT) and mutant forms of the HDL receptor SR-BI expressed by ldlA-7 cells. Mutations in helix 4 or helix 6 of the apoA-I reduced efflux by 79 and 51%, respectively, without substantially altering receptor binding (apparent K(d) values of 1.1-4.4 microg of protein/ml). SR-BI with an M158R mutation bound poorly to rHDL with WT and helix 4 mutant apoA-I; the helix 6 mutant restored tight binding to SR-BI(M158R) (K(d) values of 48, 60, and 7 microg of protein/ml, respectively). SR-BI(M158R)-mediated cholesterol efflux rates, normalized for binding, were high for all three rHDLs (71-111% of control). In contrast, absolute (12-19%) and binding-corrected (24-47%) efflux rates for all three rHDLs mediated by SR-BI with Q402R/Q418R mutations were very low. We propose that formation of a productive complex between apoA-I in rHDL and SR-BI, in which the lipoprotein and the receptor must either be precisely aligned or have the capacity to undergo appropriate conformational changes, is required for efficient SR-BI-mediated cholesterol efflux. Some mutations in apoA-I and/or SR-BI can result in high affinity, but non-productive, binding that does not permit efficient cholesterol efflux.
Highlights
Apolipoprotein A-I1 is the major protein constituent of high density lipoprotein (HDL) and plays crucial roles in the synthesis, structure, and functions of HDL [1]
We have studied the effects of mutations in Apolipoprotein A-I (apoA-I) on reconstituted high density lipoprotein (HDL) particle (rHDL(apoA-I)) binding to and cholesterol efflux from wild-type (WT) and mutant forms of the HDL receptor scavenger receptor class B type I (SR-BI) expressed by ldlA-7 cells
Effects of ApoA-I Mutations on Binding and Cholesterol Efflux Mediated by the Q402R/Q418R SR-BI Double Mutant—To determine whether the properties exhibited by the M158R mutant receptor, relaxed structural requirements for productive binding and restoration of high affinity Reconstituted HDL Particles (rHDLs) binding by the helix 6 mutant (Fig. 2), were common to another SR-BI mutant with reduced plasma HDL binding, we examined cholesterol efflux and ligand binding by ldlA[Q402R/Q418R] cells
Summary
Apolipoprotein A-I (apoA-I) is the major protein constituent of high density lipoprotein (HDL) and plays crucial roles in the synthesis, structure, and functions of HDL [1]. ApoA-I promotes high affinity binding of HDL and rHDL particles to cells (14 –17), and analysis of apoA-I-deficient mice highlighted the importance of apoA-I for the delivery of HDL cholesterol to steroidogenic tissues via selective lipid uptake [18]. A number of the functions of apoA-I appear to be mediated by a cell surface HDL receptor called scavenger receptor class B type I (SR-BI) (14, 15, 19, reviewed in Ref. 20). SR-BI-mediated cholesterol, cholesteryl ester, and triglyceride movement between lipoproteins and cells and the affinity of ligand binding to SR-BI can be influenced by variations in the size and apolipoprotein and lipid compositions of the HDL particles (14, 15, 29, 30, 36 – 40). A The restriction enzyme recognition sites are marked in boldface type. b nt, nucleotide number of the published apoA–I cDNA sequence [53] oligonucleotide position (ϩ) relative to the transcription initiation site (i.e. ϩ1 from ATG). c Amino acid position (ϩ) relative to the mature apoA–I (i.e. ϩ1 from mature apoA–I protein). d Mutagenized residues are marked in boldface type and are underlined
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