Reverse cholesterol transport (transfer of macrophage-cholesterol in the subendothelial space of the arterial wall to the liver) is terminated by selective high density lipoprotein (HDL)-cholesteryl ester (CE) uptake, mediated by scavenger receptor class B, type 1 (SR-B1). We tested the validity of two models for this process: "gobbling," one-step transfer of all HDL-CE to the cell and "nibbling," multiple successive cycles of SR-B1-HDL association during which a few CEs transfer to the cell. Concurrently, we compared cellular uptake of apoAI with that of apoAII, which is more lipophilic than apoAI, using HDL-[H]CE labeled with [I]apoAI or [I]apoAII. The studies were conducted in CHO-K1 and CHO-ldlA7 cells (LDLR) with (CHO-SR-B1) and without SR-B1 overexpression and in human Huh7 hepatocytes. Relative to CE, both apoAI and apoAII were excluded from uptake by all cells. However, apoAII was more highly excluded from uptake (2-4×) than apoAI. To distinguish gobbling nibbling mechanisms, media from incubations of HDL with CHO-SR-B1 cells were analyzed by non-denaturing PAGE, size-exclusion chromatography, and the distribution of apoAI, apoAII, cholesterol, and phospholipid among HDL species as a function of incubation time. HDL size gradually decreased, nibbling, with the concurrent release of lipid-free apoAI; apoAII was retained in an HDL remnant. Our data support an SR-B1 nibbling mechanism that is similar to that of streptococcal serum opacity factor, which also selectively removes CE and releases apoAI, leaving an apoAII-rich remnant.
Apolipoprotein (apo) A-II, the second most abundant protein after apo A-I of human plasma high-density lipoproteins (HDL), is the most lipophilic of the exchangeable apolipoproteins. The rate of microsolubilization of dimyristoylphosphatidylcholine (DMPC) membranes by apo A-I to give rHDL increases as the level of membrane free cholesterol (FC) increases up to 20 mol % when the level of reaction decreases to nil. Given its greater lipophilicity, we tested the hypothesis that apo A-II and its reduced and carboxymethylated monomer (rcm apo A-II) would form rHDL at a membrane FC content of >20 mol %. According to turbidimetric titrations, the DMPC/apo A-II stoichiometry is 65/1 (moles to moles). At this stoichiometry, apo A-II forms rHDL from DMPC and FC. Contrary to our hypothesis, apo A-II, like apo A-I, reacts poorly with DMPC containing ≥20 mol % FC. The rate of formation of rHDL from rcm apo A-II and DMPC at all FC mole percentages is faster than that of apo A-II but nil at 20 mol % FC. In parallel reactions, monomeric and dimeric apo A-II form large FC-rich rHDL coexisting with smaller FC-poor rHDL; increasing the FC mole percentage increases the number and size of FC-rich rHDL. On the basis of the compositions of coexisting large and small rHDL, the free energy of transfer of FC from the smallest to the largest particle is approximately −1.2 kJ. On the basis of our data, we propose a model in which apo A-I and apo A-II bind to DMPC via surface defects that disappear at 20 mol % FC. These data suggest apo A-II-containing HDL formed intrahepatically are likely cholesterol-rich compared to the smaller intracellular lipid-poor apo A-I HDL.
Reassembled high density lipoproteins (rHDL) of various sizes and compositions containing apo A-I or apo A-II as their sole protein, dimyristoyl phosphatidylcholine (DMPC), and various amounts of free cholesterol (FC) have been isolated and analyzed by differential scanning calorimetry (DSC) and by circular dichroism to determine their stability and the temperature dependence of their helical content. Our data show that the multiple rHDL species obtained at each mol% FC usually do not have the same mole% FC as the starting mixture and that the size of the multiple species increases in a quantized way with their respective mol% FC. DSC studies reveal multiple phases or domains that can be classified as virtual DMPC, which contains a small amount of DMPC that slightly reduces the melting temperature Tm, a boundary phase that is adjacent to the apo A-I or apo A-II that circumscribes the discoidal rHDL, and a mixed FC + DMPC phase that has a Tm that increases with mol% FC. Only the large rHDL contain virtual DMPC whereas all contain boundary phase and various amounts of mixed FC + DMPC according to increasing size and mol% FC. As reported by others, FC stabilizes the rHDL. For rHDL (apo A-II) compared to rHDL (apo A-I), this occurs in spite of the reduced number of helical regions that mediate binding to the DMPC surface. This effect is attributed to the very high lipophilicity of apo A-II and the reduction in the polarity of the interface between DMPC and the aqueous phase with increasing mol% FC, an effect that is expected to increase the hydrophobic associations with the non polar face of the amphipathic helices of apo A-II. These data are relevant to the differential effects of FC and apolipoprotein species on intracellular and plasma membrane nascent HDL assembly and subsequent remodeling by plasma proteins.
Reverse cholesterol transport (RCT), the transfer of cholesterol from peripheral tissues, including the subendothelial space of the arterial wall, to the liver for disposal, is a current model of HDL atheroprotection. The final RCT step, selective hepatic HDL-cholesteryl ester (CE) uptake, is mediated by scavenger receptor class B type I (SR-BI). The net receptor reaction of SR-BI vs. HDL is distinct from that of LDL vs. the LDL receptor. LDL holo particle uptake is succeeded by steps that breakdown apo B-100 and hydrolyze and recycle the CE. In contrast, HDL-CE uptake is selective, occurring without a concomitant net uptake of the major HDL protein, apo A-I and even though apo E and apo A-I bind equally well to SR-BI, apoA-I-containing particles mediate 2-fold more selective CE uptake. The reaction of HDL with SR-BI is similar to the activity of a streptococcal serum opacity factor (SOF) against HDL_both reactions selectively remove CE from HDL leaving remnants. In addition, SOF catalyzes the displacement of apo A-I leaving an apo A-II-rich neo HDL, an effect that was assigned to the greater lipophilicity of apo A-II vs. apo A-I. Thus, we tested the hypothesis that the same occurs during the interaction of HDL with SR-BI, i.e., that apo A-II vs. apo A-I is selectively excluded from cellular uptake via SR-BI. Herein, we compare the selective uptake of HDL-CE vs. HDL-apo A-I and apo A-II. Cellular uptake of HDL-[ 3 H]CE labeled with [ 125 I]apo A-I or [ 125 I]apo A-II was compared in CHO-K1 and CHO-ldlA7 cells (LDL-R -/- ) with and without over expression of mouse SR-BI, and Huh7 human hepatocytes. Cell-associated 125 I and 3 H were determined by γ- and β-counting respectively. Uptake of CE, apo A-I, and apo A-II SR-BI-over expressing CHO cells was 32,800 ± 4800, 9.3 ± 2.7, and 2.5 ± 0.2 nmol/mg cell protein. The corresponding values for Huh7 cells were 9,700 ± 1,800, 15 ± 2.4, and 7.6 ± 0.9 nmol/mg cell protein. Relative to CE, both apo A-I and apo A-II were excluded from uptake by all cells. However, relative to the apo A-I and apo A-II contents of HDL, uptake of apo A-I was twice that of apo A-II, thus supporting the hypothesis that the more lipophilic apo A-II is selectively excluded from cellular uptake via SR-BI and retained in the neo HDL remnant.
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