Recruitment of cell phospholipids and cholesterol by apolipoproteins A-I1 and A-I: formation of nascent apolipoprotein-specific HDL that differ in size, phospholipid composition, and reactivity with LCAT

Studies were carried out to determine whether apolipoprotein (apo) A-11, like apoA-I, can recruit phospholipid and cholesterol from cell membranes, thereby forming nascent apoA-11-specific HDL. ApoA-I1 and apoA-I were purified from plasma and each was incubated with C H O cells at a concentration of 10 pg/ml. Lipid-containing complexes were isolated from the medium in both cases; the composition of the apoA-11and apoA-I-specific complexes were similar where percent protein, phospholipid, and cholesterol were 35 + 3, 38 k 2, and 25 k 1 for apoA-11, respectively, and 40 2 for apoA-I, respectively. O n a per mole of apolipoprotein basis, apoA-I recruited significantly more phospholipid and cholesterol than dimeric apoA-I1 suggesting that apoA-I with its greater number of alpha helices binds more lipid. By electron microscopy, nascent apoA-11and apoA-I-specific particles were predominantly discoidal in morphology. ApoA-I1 complexes were unique in their nondenaturing polyacrylamide gradient gel size distribution as six distinct populations of particles with diameters of 8.1, 9.3, 10.4, 11.8, 13.1, and 14.6 nm were routinely noted, compared with apoA-I which formed only three major populations with diameters of 7.3, 9.2, and 11.0 nm. Nascent apoA-I complexes incubated with purified 1ecithin:cholesterol acyltransferase (LCAT) were transformed into predominantly 8.4 nm particles. The latter is similar in size to plasma HDL?,, LpA-I particles, suggesting that extracellularly assembled apoAI-lipid complexes can directly give rise to a major plasma LpA-I subpopulation upon interaction with LCAT. Unlike apoA-I, apoA-11-lipid complexes could not serve as substrates for LCAT and did not undergo transformation. This study also demonstrates, for the first time, that apoA-I1 and apoA-I show a preference in phospholipid recruitment from membranes. Although phosphatidylcholine is the major phospholipid removed by both apolipoproteins, apoA-I1 preferentially recruits phosphatidylethanolamine (PE) as its second most abundant phospholipid while apoA-I recruits sphingomyelin. As PE is usually associated with the inner leaflet of the membrane, it is likely that dimeric apoA11, compared with apoA-I, can penetrate farther into the membrane and extract PE. This ability of apoA-I1 to insert more deeply into the lipid milieu may explain the known ability of apoA2, 35 + 1, and 24 I1 to resist dissociation from the mature HDL particle.Forte, T. M., J. K. Bielicki, R. Goth-Goldstein, J. Selmek, and M. R. McCall. Recruitment of cell phospholipids and cholesterol by apolipoproteins A-I1 and A-I: formation of nascent apolipoproteinspecific HDL that differ in size, phospholipid composition, and reactivity with LCAT. J Lipid Res. 1995. 36: 148-157. Supplementary key words apolipoprotein A-l apolipoprotein A-I1 membrane interactions phosphatidylcholine phosphatidylethanolamine sphingomyelin * nascent HDL 1ecithin:cholesterol acyltransferase * Chinese hamster ovary cell There is an inverse relationship between the concentration of plasma high-density lipoproteins (HDL) and risk for premature coronary artery disease. Plasma H D L possess two major apolipoproteins (apo), apoA-I and apoA-11, where the former constitutes approximately 70% of the total H D L protein and the latter, 20%. In plasma, H D L are composed of distinct apolipoprotein specific populations including apoA-I with apoA-I1 (LpAI/AII) and apoA-I without apoA-I1 (LpAI) (1). Recent reports also indicate that plasma contains some apoA-I1 without apoA-I (LpAII) H D L but this subpopulation accounts for less than 10% of total apoA-I1 (2, 3 ) . The in vivo site of assembly of nascent, Le., discoidal, apolipoprotein-specific H D L has not been completely resolved. Potential mechanisms for assembly include (1) a Abbreviations: apo, apolipoprotein; HDL, high density lipoprotein; CHO, Chinese hamster ovary; LCAT, 1ecithin:cholesterol acyltransferase; PC, phosphatidylcholine; PE, phosphatidylethanolamine; FBS, fetal bovine serum; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. IT) whom correspondence should be addressed. 148 Journal of Lipid Research Volume 36, 1995 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 1, 2013 w w w .j.org D ow nladed fom direct mechanism whereby cells producing apolipoproteins, e.g., hepatocytes, secrete the pre-assembled particles or (2) an indirect mechanism whereby particles are assembled extracellularly by interaction of free apolipoproteins with pre-existing lipoproteins or cell membranes. Early studies of Stein and Stein (4) indicated that apoHDL was able to stimulate removal of cholesterol and phospholipid from cell membranes. Previous reports on incubations of cholesterol-loaded macrophages (5), cholesterol-enriched fibroblasts (6), and endothelial and smooth muscle cells (7) with apoA-I and apoA-I1 suggested that both proteins can promote cholesterol efflux from cells. Lipidated complexes of apoA-I and apoA-I1 described in these studies contained both phospholipid and unesterified cholesterol; however, the physical-chemical properties of the complexes were not determined. These studies provide little insight into the nature of the nascent particles resulting from interaction of either apoA-I or apoA-I1 with cell membranes. We have recently shown that lipid-free, genetically engineered apoA-I can recruit phospholipid and cholesterol from Chinese hamster ovary (CHO) cell membranes and that, in so doing, is able to generate several discrete, nascent HDL subpopulations (8). The generation of nascent HDL resulting from the interaction of lipid-free apolipoproteins with cell membranes has important implications in reverse cholesterol transport because it provides a mechanism whereby excess cholesterol may be removed from cells unable to catabolize cholesterol. ApoA-I and apoA-I1 are both amphipathic proteins that possess several lipid binding helices. However, the properties of the helical repeats are clearly different for the two proteins where apoA-I1 contains class A2 and apoA-I contains a class A, amphipathic helices (9). In the present investigation, we addressed the question whether apoA-I and apoA-I1 isolated from plasma are equally effective in binding and removing phospholipid and cholesterol from C H O cell membranes and whether apoA-11, like apoA-I, is able to form discrete nascent HDL subpopulations. As there are structural differences' between these two proteins, we also evaluated whether they had the same or different preferences for membrane phospholipids and whether the nascent particles they assembled could function as substrates for the 1ecithin:cholesterol acyltransferase (LCAT) reaction. MATERIALS AND METHODS Isolation of apolipoproteins Fresh human plasma was obtained from the blood bank and 1.0 mg/ml EDTA and 0.05 mg/ml gentamicin sulfate were added; the plasma was maintained at 4°C during processing. The HDL fraction was isolated at d 1.063-1.21 g/ml from approximately 1 1 plasma. Washed HDL were treated with 3 M guanidine hydrochloride at 37'C for 3 h for isolation of apoA-I as previously described (10). ApoA-11 was isolated from the apoA-I-depleted HDL using 6 M guanidine hydrochloride according to Blanche et al. (11). Isolated apoA-I was purified on a Sephacryl S-200 H R column and apoA-I1 on a DEAE-Sepharose C L 6B column. Eluted fractions were assayed for purity of apoAI and apoA-I1 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and dot blotting using monospecific antiapoA-I and anti-apoA-I1 antibodies. In each case, only a single band of purified protein was seen on SDS-PAGE; apoA-I1 was electrophoresed in the absence of reducing agents and was present in the homodimeric form. The purified proteins were dialyzed into a buffer containing 150 mM NaCl, 20 mM Tris, and 0.27 mM EDTA, pH 8.0, and refrigerated. Cell incubations and isolation of apolipoprotein-lipid complexes C H O C1 9 cells were grown in 850 cm2 roller bottles with DMEM/F12 (1:l) and 10% fetal bovine serum (FBS) as previously described (8). Prior to onset of incubations, cells were rinsed three times with Hank's balanced salt solution and incubated overnight with serum-free medium; this medium was discarded before incubations with purified apolipoproteins to insure that apolipoproteins in FBS were not carried over. Cells were then incubated for 24 h with serum-free medium containing either apoA-I (10 pglml) or apoA-I1 (10 pg/ml). Conditioned medium was subsequently collected and placed on ice. Gentamicin sulfate (0.05 mg/ml), ED?;4 (1 mg/ml), and PMSF (0.5 mM) were added and cell debris was removed by filtration. The conditioned medium was concentrated by ultrafiltration (Amicon stirred cell, YMlO membranes) and apoA-1or apoA-II-lipid complexes were isolated at d < 1.235 g/mI as previously described (7). These fractions were extensively dialyzed against saline-Tris-EIYTA prior to further analyses. Electrophoretic analyses The size distribution of particles was determined by nondenaturing polyacrylamide gradient (4-30%) gel electrophoresis according to previously described procedures (12). Peak positions of apolipoprotein-lipid complexes on gradient gels are based on the diameter of globular protein standards electrophoresed on the same gel. Standards used to calibrate the gels were thyroglobulin, apoferritin, lactate dehydrogenase, and albumin with Stokes diameters of 17.0 m, 12.2 nm, 8.16 m, and 7.1 nm, mpctively. SDS-polyacrylamide gel electrophoresis was carried out on 4-20% gels obtained from Schleicher and Schuell (Keene, NH) according to the procedure of Laemmli (13). Agarose gel electrophoresis was carried out on Beckman Paragon gels according to the manufacturer's instructions. Proteins were transferred to nitrocellulose by diffusion and bands were probed with either antibody to apoA-I or apoA-11 as previously described (14). Forte el ai. Phospholipid and cholesterol recruitment by apoA-I1 and apoA-I 149 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 1, 2013 w w w .j.org D ow nladed fom