Efflux of Cellular Cholesterol and Phospholipid to Apolipoprotein A-I Mutants

Abstract

Human plasma apolipoprotein A-I (apoA-I) and recombinant full-length proapoA-I (apoA-I-(؊6 -243)) as well as four truncated forms of proapoA-I were used as acceptors to study cholesterol and phospholipid efflux from HepG2 cells. Efflux of both cholesterol and phospholipid to the lipid-free plasma apoA-I was twice that of apoA-I-(؊6 -243). When apoA-I was incorporated into reconstituted high density lipoprotein, cholesterol efflux increased, phospholipid efflux decreased and the difference between plasma apoA-I and apoA-I-(؊6 -243) disappeared. Truncation of recombinant apoA-I to residues 222 (apoA-I-(؊6 -222)) and 210 (apoA-I-(؊6 -210)) resulted in a 70 -95% decrease in their ability to promote the efflux of both intracellular and plasma membrane cholesterol. Further truncation to residues 150 (apoA-I-(؊6 -150)) and 135 (apoA-I-(؊6 -135)) fully restored the ability of apoA-I to promote cholesterol efflux. Phospholipid efflux closely paralleled the efflux of cholesterol. Interaction of 125 I-labeled apoA-I with the cells was similar for apoA-I-(؊6 -243), apoA-I-(؊6 -222), and apoA-I-(؊6 -210), but slightly higher for apoA-I-(؊6 -150) and apoA-I-(؊6 -135).When complexed with phospholipid, all forms except apoA-I-(؊6 -210) formed discoidal reconstituted high density lipoprotein particles. When the same amounts of free or lipid-associated apoA-I were compared, association of apoA-I with phospholipid increased cholesterol efflux and decreased phospholipid efflux, and the difference in the ability of different mutants to promote cholesterol and phospholipid efflux disappeared. We conclude that the capacity of lipid-free apoA-I to promote cholesterol efflux is related to its ability to mobilize cellular phospholipid, which apparently involves a region around residues 222-243. A second lipid-binding region is exposed when the carboxylterminal half of apoA-I is absent.

Advanced glycation of apolipoprotein A-I impairs its anti-atherogenic properties

“…ApoA-I was isolated as described previously [19]. Reconstituted HDL (rHDL) (apoA-I/POPC 1:80 mol/mol) was prepared as described previously [20].…”

Apolipoprotein A-I, Phospholipid Vesicles, and Cyclodextrins as Potential Anti-Atherosclerotic Drugs: Delivery, Pharmacokinetics, and Efficacy

“…However, events leading to particle assembly and their relationship to cholesterol ef ux have not been fully elucidated (Mendez 1997). Cholesterol ef ux has been suggested to vary as a result of differences in the capacity of apoA-I to form complexes with PLs responsible for removal of cellular cholesterol (Sviridov et al 1996b). Presumably, the PL layer on the LP surface serves as a region that can solubilize cholesterol, and HDL plays an important role in ef ux, being a small PL-rich LP (Jian et al 1997).…”

“…On the other hand, speci c sequences of apoA-I have been implicated in its ability to ef ux cholesterol (Banka et al 1994;Sviridov et al 1996aSviridov et al , 1996b, denoting a more speci c, possibly receptor-mediated action. The 223-243 region of apoA-I is most likely involved in the formation of apoA-I-lipid complexes (Sviridov et al 1996a(Sviridov et al , 1996b.…”

“…Apolipoprotein-mediated cholesterol removal from microsomal membranes (Nunez and Swaney 1984), from CHO cells (Forte et al 1993), broblasts (Bielicki et al 1992;Asztalos et al 1997), endothelial cells (Savion and Kotev-Emeth 1993), Fu5AH rat hepatoma cells (Fournier et al 1996), and macrophages (Hara and Yokoyama 1991;Yancey et al 1995) requires PL association with the protein (Davidson et al 1995;Forte et al 1995;Sviridov et al 1996b). ApoA-I, in the absence of serum, facilitates cholesterol ef ux from certain types of cells including human monocyte-derived macrophages (HMDMs: Dass et al 1999a) and mouse peritoneal macrophages (MPMs: Gelissen et al 1996), probably by ef uxing and associating with PLs from the PM (Yancey et al 1995).…”

“…Furthermore, monoclonal antibodies recognizing the epitopic region apoA-I-(140-150) speci cally inhibited the ability of apoA-I-containing lipoproteins to promote ef ux of intracellular (but not PM) cholesterol (Sviridov et al 1996b). The carboxy (-COOH) terminus of apoA-I is important for lipid binding (Gillotte et al 1996;Laccotripe et al 1997), while the terminal amphipathic helices 44-65 and 220-241 are the most important for the initial binding of apoA-I to lipids, and hence in ef uxing lipids from PMs (Palgunachari et al 1996).…”