Juan Domingo Toledo

Conformation of apolipoprotein AI in reconstituted lipoprotein particles and particle–membrane interaction: Effect of cholesterol

Discoidal recombinant high density lipoproteins (rHDL) of apolipoprotein AI (apoAI) and palmitoyloleoylphosphatidylcholine (POPC), with or without cholesterol, were prepared by cholate dialysis. By gel filtration, rHDL containing 2-4 (Lp2, Lp3 and Lp4) apoAI molecules/particle were obtained. The ApoAI conformation in these rHDL was investigated by tryptophan fluorescence, denaturation with guanidine HCl, and immunoreactivity with two monoclonal antibodies recognizing epitopes in the N-terminal and central domains. Data show that apoAI conformation is highly dependent on particle size as well as on cholesterol. The ability of rHDL to interact with lipid bilayer was studied by measuring leakage induction on POPC and POPC/cholesterol vesicles loaded with terbium/dipicolinic acid. Among the cholesterol-free rHDL, the most efficient ones were the smallest Lp2. Leakage induction on POPC vesicles is dramatically decreased by the presence of cholesterol in Lp2 and Lp3. All the rHDL, but specially those containing cholesterol, induced more leakage on the POPC/cholesterol than on the POPC vesicles. These results suggest that in small cholesterol-poor particles, apoAI could have a conformation determining a high affinity for membranes, which could facilitate cholesterol efflux. After cholesterol enrichment, a conformational change in apoAI could decrease the affinity for membranes allowing the lipoprotein release.

The central type Y amphipathic α-helices of apolipoprotein AI are involved in the mobilization of intracellular cholesterol depots

We studied the role of a central domain of human apolipoprotein AI (apoAI) in cholesterol mobilization and removal from cells. In order to check different protein conformations, we tested different sized and cholesterol-content reconstituted apoAI particles (rHDL). Meanwhile cholesterol-free discs were active to induce mobilization, only small cholesterol-containing rHDL were active. To test the influence of a central domain in such events, we used two apoAI variants: one, with its central Y helix pair replaced by the C-terminal domain, and the other having a lysine deleted in central region. The helix-swapping variant decrease the cholesterol pool available to acyl-CoA cholesterol acyl transferase and increase mobilization of newly synthesized cholesterol. Instead, the deletion mutant had no effect on both events. We conclude that the central domain of apoAI is involved in cholesterol cell traffic and solubilization, and that a Y-type charge distribution in polar face may be required, as well as a correct helices-polar face orientation.

Effect of reconstituted discoidal high‐density lipoproteins on lipid mobilization in RAW 264.7 and CHOK1 cells

Reconstituted discoidal high‐density lipoproteins (rHDL) resemble nascent HDL, which are formed at the early reverse cholesterol transport steps, and constitute the initial cholesterol (Chol) acceptors from cell membranes. We have used different sized rHDL containing or not Chol, to test their abilities to promote cholesterol and phospholipid efflux from two different cell lines: Raw 264.7 macrophages and CHOK1 cells. All rHDL and lipid‐free apolipoprotein A‐I (apoA‐I) were found to be bound to CHO and RAW cells. In RAW cells, a positive correlation between cellular binding and Chol removal was found for 78 and 96 Å rHDL. Chol‐free rHDL were more effective than Chol‐containing ones in binding to RAW cells and promoting Chol removal. These results were more evident in the 96 Å rHDL. On the other hand, rHDL binding to CHO cells was relatively independent of disc size and Chol content. In spite of the fact that apoA‐I and rHDL promoted Chol efflux from both cellular lines, only in CHOK1 cells this result was also associated to decrease Chol esterification. Among choline‐containing phospholipids, only phosphatidylcholine (PC) (but not sphingomyelin) was detected to be effuxed from both cellular lines. With the only exception of Chol‐free 96 Å discs, the other rHDL as well as apoA‐I promoted PC efflux from RAW cells. Chol‐containing rHDL were more active than Chol‐free ones of comparable size to promote PC efflux from RAW macrophages. Regarding CHO cells, only apoA‐I and Chol‐free 78 Å rHDL were active enough to remove PC. J. Cell. Biochem. 113: 1208–1216, 2012.