Influence of lipid environment on insulin binding in cultured hepatoma cells

Three mouse monoclonal antibodies (Mabs) to human apo A-I were produced using apolipoprotein A-I or HDL3 as immunogens. These monoclonal antibodies, 2G11, 4A12 and 4B11, were characterized for their reactivity with isolated apolipoprotein A-I and HDL in solution. The immunoblotting patterns of the HDL3 two-dimensional electrophoresis show that these three monoclonal antibodies reacted with all the polymorphic forms of apolipoprotein A-I. Cotitration experiments indicated that they correspond to three distinct epitopes. In order to locate these three antigenic determinants on the isolated apolipoprotein A-I, the reactivity of the three monoclonal antibodies has been studied on CNBr-cleaved apolipoprotein A-I. The monoclonal antibodies 2G11 and 4A12 addressed to the amino (CNBr 1) and carboxy (CNBr 4) terminal segments, respectively. In comparison with the monoclonal antibodies characterized by Weech et al. ((1985) Biochim. Biophys. Acta 835, 390-401), monoclonal antibody 4A12 is the only one described in the literature which is specific of the carboxy terminal segment of apolipoprotein A-I. Monoclonal antibody 4B11 does not react with any CNBr fragment, its binding is temperature dependent, it could be directed to a conformational epitope. Relative differences were demonstrated in the expression of the three epitopes in HDL subfractions isolated by density gradient ultracentrifugation. According to Curtiss and Edgington ((1985) J. Biol. Chem. 260, 2982-2993) our results indicate the existence of an immunochemical heterogeneity in the organization of apolipoprotein A-I at the surface of HDL particles as well as in the soluble form of apolipoprotein A-I.     

Immunochemical characterization of six monoclonal antibodies to human apolipoprotein A-I: epitope mapping and expression.

We have produced and characterized six murine monoclonal antibodies to human apolipoprotein A-I named A-I-9, A-I-12, A-I-15, A-I-16, A-I-19, and A-I-57. All monoclonal antibodies were specific for apolipoprotein A-I and bound between 55% and 100% of 125I-labeled high density lipoproteins (HDL) in a fluid phase radioimmunoassay. All antibodies possessed a higher affinity to apoA-I in HDL than to free, delipidated apoA-I. Two of them, particularly A-I-12 and A-I-15, which were directed to the same or very close epitopes on the molecule, recognized very poorly the delipidated protein. Binding of apoA-I to phospholipid restored the immunoreactivity of the monoclonal antibodies to the protein suggesting that lipids play an important role in determining the immunochemical structure of apoA-I. Using CNBr fragments and synthetic peptides, the epitopes for the antibodies were mapped as follows: A-I-19, CNBr fragment 1; A-I-12 and 15, CNBr fragment 2; A-I-9 and A-I-16, CNBr fragment 3; A-I-57, CNBr fragment 4. Antibody A-I-57 failed to recognized a mutant form of apoA-I, A-IMilano (Arg173----Cys) by immunoblotting and by competitive radioimmunoassay demonstrating that substitution of a single amino acid in human apoA-I may cause the loss of an antigenic determinant.     

Monoclonal antibodies specific for different regions of human apolipoprotein A-I. Characterization of an antibody that does not bind to a genetic variant of apoA-I (Glu----136 Lys)

Three monoclonal mouse hybridoma antibodies, designated 2AI, 4AI, and 5AI, specific for human plasma apolipoprotein A-I (apoA-I) were characterized. In an enzyme-linked immunosorbent assay (ELISA) each of the antibodies reacted with purified apoA-I and with A-I in normal human serum. Immunoblotting of apoA-I subjected to isoelectric focusing revealed that the three antibodies reacted with all the charge isomorphs of apoA-I and with proapoA-I. Using a solid phase competitive displacement assay, the antigenic determinant for antibody 5AI could be localized to cyanogen bromide fragment 3 of apoA-I (residues 113-148), while the epitope for antibody 4AI resided in cyanogen bromide fragment 4. Dot blot experiments and data obtained by the competitive displacement assay revealed that antibody 2AI reacts with high affinity with CNBr fragment 2 but that it also reacts with lower affinity with fragments 1 and 4. The antibody 5AI did not bind to a genetic variant of apoA-I (Glu----136 Lys), demonstrating that the substitution of a single amino acid in human apoA-I can cause the loss of an antigenic determinant.     

Apolipoprotein A-II/A-I ratio is a key determinant in vivo of HDL concentration and formation of pre-beta HDL containing apolipoprotein A-II

Overexpression of human apolipoprotein A-II (apo A-II) in mice induced postprandial hypertriglyceridemia and marked reduction in plasma HDL concentration and particle size [Boisfer et al. (1999) J. Biol. Chem. 274, 11564-11572]. We presently compared lipoprotein metabolism in three transgenic lines displaying plasma concentrations of human apo A-II ranging from normal to 4 times higher, under ad libitum feeding and after an overnight fast. Fasting dramatically decreased VLDL and lowered circulating human apo A-II in transgenic mice; conversely, plasma HDL levels increased in all genotypes. The apo A-I content of HDL was inversely related to the expression of human apo A-II, probably reflecting displacement of apo A-I by an excess of apo A-II. Thus, the molar ratios of apo A-II/A-I in HDL were significantly higher in fed as compared with fasted animals of the same transgenic line, while endogenous LCAT activity concomitantly decreased. The number and size of HDL particles decreased in direct proportion to the level of human apo A-II expression. Apo A-II was abundantly present in all HDL particles, in contrast to apo A-I mainly present in large ones. Two novel findings were the presence of pre-beta migrating HDL transporting only human apo A-II in the higher-expressing mice and the increase of plasma HDL concentrations by fasting in control and transgenic mice. These findings highlight the reciprocal modifications of VLDL and HDL induced by the feeding-fasting transition and the key role of the molar ratio of apo A-II/A-I as a determinant of HDL particle metabolism and pre-beta HDL formation.     

Apolipoproteins A-I,A-II and E are independently distributed among intracellular and newly secreted HDL of human hepatoma cells

Whereas hepatocytes secrete the major human plasma high density lipoproteins (HDL)-protein, apo A-I, as lipid-free and lipidated species, the biogenic itineraries of apo A-II and apo E are unknown. Human plasma and HepG2 cell-derived apo A-II and apo E occur as monomers, homodimers and heterodimers. Dimerization of apo A-II, which is more lipophilic than apo A-I, is catalyzed by lipid surfaces. Thus, we hypothesized that lipidation of intracellular and secreted apo A-II exceeds that of apo A-I, and once lipidated, apo A-II dimerizes. Fractionation of HepG2 cell lysate and media by size exclusion chromatography showed that intracellular apo A-II and apo E are fully lipidated and occur on nascent HDL and VLDL respectively, while only 45% of intracellular apo A-I is lipidated. Secreted apo A-II and apo E occur on small HDL and on LDL and large HDL respectively. HDL particles containing both apo A-II and apo A-I form only after secretion from both HepG2 and Huh7 hepatoma cells. Apo A-II dimerizes intracellularly while intracellular apo E is monomeric but after secretion associates with HDL and subsequently dimerizes. Thus, HDL apolipoproteins A-I, A-II and E have distinct intracellular and post-secretory pathways of hepatic lipidation and dimerization in the process of HDL formation. These early forms of HDL are expected to follow different apolipoprotein-specific pathways through plasma remodeling and reverse cholesterol transport.     

Studies on the proteins involved in the interaction of high-density lipoprotein with isolated human small intestine epithelial cells.

Treatment of 125I-labelled high-density lipoprotein ([125I]HDL3) with monospecific polyclonal antibodies against apolipoproteins A-I and A-II resulted in a dose-dependent inhibition of the [125I]HDL3 binding to isolated human small intestine epithelial cells by 25% and 50%, respectively. Both antibodies also inhibited intracellular degradation of [125I]HDL3 by 80%. Treatment of enterocytes with polyclonal antibody against apolipoprotein A-I binding protein, a putative HDL receptor, inhibited both binding and degradation of [125I]HDL3 by these cells by 50%. Antibodies to apolipoprotein A-I, A-II and apo A-I-binding protein also inhibited [125I]HDL3 binding to cholesterol-loaded cells.     

Monoclonal antibodies as probes of high-density lipoprotein structure: identification and localization of a lipid-dependent epitope

Eight stable murine monoclonal antibodies (mabs) were raised against human high-density lipoproteins (HDL). Three different antibody reactivities were demonstrated by immunoblotting. A group of five antibodies were specific for apolipoprotein A-I (apoA-I) and bound to similar or overlapping epitopes. The second type of reactivity, shown by mab-32, was specific for apoA-II. In the third group, two antibodies showed high reactivity with apoA-II and slight cross-reactivity with apoA-I. The properties of two antibodies, mab M-30 specific for apoA-I and mab M-32 specific for apoAII, were characterized in detail as probes of HDL structure. The association of 125I-labeled HDL or synthetic complexes of apoA-I and phosphatidylcholine with mab M-30 was lipid dependent. Mab M-32 binding to apoA-II was independent of lipid. The lipid-dependent epitope bound by mab M-30 has been localized to an 18 amino acid synthetic apoA-I peptide. Moreover, studies with HDL2, HDL3, and immunoadsorbed HDL subfractions indicate that binding of mab M-30 to HDL is influenced by some component within the microenvironment individual HDL particles. These lines of evidence suggest that the molar ratio of apoA-I to apoA-II is the critical determinant. Binding of mab M-32 to HDL increased the reactivity of HDL to mab M-30 in a dose-dependent manner, indicating an unusual form of cooperativity between two mabs that recognize different proteins in HDL. These monoclonal antibodies will be valuable in studies of the metabolic significance of protein-protein and lipid-protein interactions in HDL.     

Characterization of human high-density lipoprotein subclasses LP A-I and LP A-I/A-II and binding to HepG2 cells

Plasma HDL can be classified according to their apolipoprotein content into at least two types of lipoprotein particles: lipoproteins containing both apo A-I and apo A-II (LP A-I/A-II) and lipoproteins with apo A-I but without apo A-II (LP A-I). LP A-I and LP A-I/A-II were isolated by immuno-affinity chromatography. LP A-I has a higher cholesterol content and less protein compared to LP A-I/A-II. The average particle mass of LP A-I is higher (379 kDa) than the average particle weight of LP A-I/A-II (269 kDa). The binding of 125I-LP A-I to HepG2 cells at 4 degrees C, as well as the uptake of [3H]cholesteryl ether-labelled LP A-I by HepG2 cells at 37 degrees C, was significantly higher than the binding and uptake of LP A-I/A-II. It is likely that both binding and uptake are mediated by apo A-I. Our results do not provide evidence in favor of a specific role for apo A-II in the binding and uptake of HDL by HepG2 cells.     

High-density lipoprotein and its apolipoproteins inhibit cytolytic activity of complement. Studies on the nature of inhibitory moiety

Human high-density lipoprotein (HDL) and its apolipoproteins A-I and A-II inhibit complement-mediated lysis of human and sheep erythrocytes. This inhibitory activity under study is exerted after C9 is bound to membrane-associated C5b-8 complexes but prior to completed assembly and insertion of the C5b-9 complex. In this paper, we define some structure-activity relationships of the inhibitory moiety. With the exception of weak lytic inhibitory activity found in LDL/VLDL pools and in some unconcentrated minor fractions of plasma obtained by hydrophobic chromatography, all inhibitor activity was found in fractions which contained either apolipoprotein A-I, apolipoprotein A-II, or both. Intact HDL has a high level of inhibitor activity but delipidation by chloroform-methanol extraction was associated with an increase in activity on a protein-weight basis. Purified apolipoprotein A-I and apolipoprotein A-II exhibited equal inhibitory activity, greater than that exhibited by intact HDL. Nevertheless, ultracentrifugal fractions in which no free apolipoproteins could be demonstrated still possessed inhibitory activity. These experiments suggest that delipidation of HDL is not necessary for expression of inhibitor activity, although we could not rule out the possibility that apolipoproteins in dynamic equilibrium with HDL are responsible for the inhibitor activity observed in whole serum and plasma and in HDL preparations. Limited proteinase digestion completely abolished the inhibitory activity of partially delipidated HDL. Phospholipase C had little or no effect on the inhibitory activity of delipidated HDL, apolipoprotein A-I or apolipoprotein A-II, but reduced the inhibitory activity of intact HDL. These data suggest that the phospholipid polar headgroups are not necessary for inhibitory activity. However, the loss of these headgroups is associated with decreased activity, possibly due to increased hydrophobicity of HDL, or increased association among HDL micelles, and subsequent decrease in effective molar concentration of the inhibitory moiety.     

Characterization and measurement of human apolipoprotein A-II by radioimmunoassay

The development of a radioimmunoassay for apolipoprotein A-II (apo A-II) is described. Initial studies revealed a lack of immunological identity between purified apo A-II used as the standard and serum or HDL. Extensive testing of different buffers, standards, antisera, tracers, utilization of a detergent, and heating of sera failed to resolve the problem. Gel filtration of iodinated and non-iodinated apo A-II on Sephadex G-100 columns showed that apo A-II, in dilute solution, elutes in a higher molecular zone than expected with a broad, assymetrical profile. The use of a subfraction of the tracer in the assay resulted in parallelism in the serum and standard dilution curves. The apo A-II assay was sensitive, specific, and reproducible. Apo A-II added to sera was fully recovered and delipidation did not affect the immunoreactivity of either serum or HDL. Apo A-II contributed approximately 20% to the protein mass of HDL. Comparison of these results with those obtained by radial immunodiffusion, and with previously reported data, indicates that the reactivity of apo A-II in its native and delipidated forms may be markedly influenced by different immunologic methodologies and their specific reagents. Caution should thus be shown at present in assigning absolute concentrations to apo A-II in serum or HDL.     

Expression of a monoclonal antibody-defined aminoterminal epitope of human apoC-I on native and reconstituted lipoproteins

Nine distinct mouse monoclonal antibodies were produced in two fusions using holo-human very low density lipoprotein (VLDL) as antigen. On immunoblotting first with human VLDL and then with isolated human apoC-I, seven of the antibodies, representing three isotypes, manifested specificity for apoC-I. Two antibodies were directed against apoB. To assess whether the seven anti-apoC-I antibodies were directed against the same or distinctively different epitopes, cross-competition assays were performed wherein 125I-labeled monoclonal antibodies were made to compete with unlabeled antibodies for occupancy on immobilized VLDL-associated apoC-I. All antibodies cross-competed to varying extents implying that they were directed against closely spaced epitopes, but based on these experiments three different epitopes were defined. On immunoblotting with CNBr fragments, all of the epitopes were assigned to the CNBr I fragment of human apoC-I (amino acids 1-38) suggesting that the NH2-terminal region of apoC-I is more immunogenic in mice than other parts of the molecule when apoC-I is associated with VLDL. A competitive solid-phase radioimmunoassay (RIA) was developed employing one of the anti-apoC-I antibodies (A3-4). VLDL was adsorbed to plastic microtiter wells, and a limiting amount of the antibody was reacted with the adsorbed VLDL. The amount of monoclonal antibody that bound to the immobilized VLDL-apoC-I was determined with a 125I-labeled goat anti-mouse IgG antibody. The addition of competitor apoC-I complexed with lipids resulted in reduced binding of the anti-apoC-I antibody to the immobilized VLDL-apoC-I. Competitor complexes consisted of an artificial lipid emulsion (Intralipid) incubated with apoC-I at phospholipid/apoC-I ratios of 1:1 to 60:1 (w/w). As the lipid/protein ratios were increased, the competitive displacement curves produced by the complexes become progressively steeper, while isolated lipid-free apoC-I produced curves with very shallow slopes, suggesting that a conformation-dependent epitope was being probed. Other apoproteins (C-II, C-III, A-I, A-II, and E) whether lipid-free or complexed with lipids did not compete. Fractionation of the 30:1 apoC-I-Intralipid complex by gel permeation chromatography suggested that apoC-I bound to phospholipids was the most effective competitor. This was confirmed by testing of apoC-I-DMPC complexes, which yielded curves that paralleled those produced by apoC-I-Intralipid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Measurement of human high density lipoprotein apolipoprotein A-1 in serum by radioimmunoassay.

A sensitive and specific double antibody radioimmunoassay for the major apolipoprotein (apo A-I) of human serum high density lipoprotein (HDL) was developed. Initial studies indicated that direct measurements of apo A-I concentration in whole untreated sera or isolated high density lipoprotein fractions yielded variable results, which were lower than those obtained in the corresponding samples which had been subjected to delipidation. Subsequently, it was observed that heating diluted sera or HDL for 3 hr at 52 degrees C prior to assay resulted in maximal increases in apo A-I immunoreactivity to levels comparable to those found in the delipidated specimens. This simple procedure permitted multiple sera to be assayed efficiently with full recovery of apo A-I.     

Interaction of apolipoprotein A-II with recombinant HDL containing egg phosphatidylcholine, unesterified cholesterol and apolipoprotein A-I

The preparation of discoidal, recombinant HDL (r-HDL) containing various phospholipids, apolipoproteins and a range of concentrations of unesterified cholesterol has been reported by several investigators. The present study describes the preparation of r-HDL containing both apolipoprotein (apo) A-I and apo A-II. r-HDL with 100:1 (mol:mol) egg PC.apo A-I and 0 (Series I), 5 (Series II) or 10 (Series III) mol% unesterified cholesterol were prepared by the cholate dialysis method. The resulting complexes had a Stokes' radius of 4.7 nm and contained two molecules of apo A-I per particle. When the r-HDL (2.0 mg apo A-I) were supplemented with 1.0 mg of apo A-II, one of the apo A-I molecules was replaced by two molecules of apo A-II. This modification was not accompanied by a loss of phospholipid, nor by major change in particle size. The addition of 2.5 or 4.0 mg of apo A-II resulted in the displacement of both apo A-I molecules from a proportion of the r-HDL and the formation of smaller particles (Stokes' radius 3.9 nm), which contained half the original number of egg PC molecules and three molecules of apo A-II. The amount of apo A-I displaced was dependent on the concentration of unesterified cholesterol in the r-HDL: when 2.5 mg of apo A-II was added to the Series I, II and III r-HDL, 44, 60 and 70%, respectively, of the apo A-I was displaced. Addition of 4.0 mg of apo A-II did not promote further displacement of apo A-I from any of the r-HDL. By contrast, the association of apo A-II with r-HDL was independent of the concentration of unesterified cholesterol and was a linear function of the amount of apo A-II which had been added. It is concluded that (1), the structural integrity of egg PC.unesterified cholesterol.apo A-I r-HDL, which contain two molecules of apo A-I, is not affected when one of the apo A-I molecules is replaced by two molecules of apo A-II; (2), when both apo A-I molecules are replaced by apo A-II, small particles which contain three molecules of apo A-II are formed; and (3), the displacement of apo A-I from r-HDL is facilitated by the presence of unesterified cholesterol in the particles.     

Regulation of high density lipoprotein receptors in cultured macrophages: role of acyl-CoA:cholesterol acyltransferase

The interaction of human serum high density lipoproteins (HDL) with mouse peritoneal macrophages and human blood monocytes was studied. Saturation curves for binding of apolipoprotein E-free [125I]HDL3 showed at least two components: non-specific binding and specific binding that saturated at approximately 40 micrograms HDL protein/ml. Scatchard analysis of specific binding of apo E-free [125I]-HDL3 to cultured macrophages yielded linear plots indicative of a single class of specific binding sites. Pretreatment of [125I]HDL3 with various apolipoprotein antibodies (anti apo A-I, anti apo A-II, anti apo C-II, anti apo C-III and anti apo E) and preincubation of the cells with anti-idiotype antibodies against apo A-I and apo A-II prior to the HDL binding studies revealed apolipoprotein A-I as the ligand involved in specific binding of HDL. Cellular cholesterol accumulation via incubation with acetylated LDL led to an increase in HDL binding sites as well as an increase in the activity of the cytoplasmic cholesterol esterifying enzyme acyl-CoA:cholesterol acyltransferase (ACAT). Incubation of the cholesterol-loaded cells in the presence of various ACAT inhibitors (Sandoz 58.035, Octimibate-Nattermann, progesterone) revealed a time- and dose-dependent amplification in HDL binding and HDL-mediated cholesterol efflux. It is concluded that the homeostasis of cellular cholesterol in macrophages is regulated in part by the number of HDL binding sites and that ACAT inhibitors enhance HDL-mediated cholesterol efflux from peripheral cells.     

Apolipoprotein A-I from normal human plasma: definition of three distinct antigenic determinants

We have prepared, selected and cloned four mouse hybridomas that secreted monoclonal antibodies against human plasma apolipoprotein A-I. These antibodies are all of the IgG-I subclass, and were named anti-A-I 6B8, 5G6, 3D4 and 5A6. We characterized the specificity of the antibodies, finding that all four of them reacted similarly, and with only the major proteins having the molecular weight and isoelectric focusing characteristics of apolipoprotein A-I. The antibodies reacted with all known charge-polymorphs of apolipoprotein A-I and pro apolipoprotein A-I. Thus, the polymorphs of apolipoprotein A-I are alike in that they all contain the antigenic sites of these four antibodies. In a solid-phase, antibody competition radioimmunoassay we found inhibition or enhancement of antibody binding to apolipoprotein A-I, according to the pair of antibodies tested. Antibodies 6B8, 5G6 and 3D4 were different from one another and reacted with different antigenic determinants, but 5A6 was similar to 3D4 and reacted at the same site. We compared the reactions of the four antibodies with CNBr-cleaved fragments of apolipoprotein A-I separated by polyacrylamide gel electrophoresis. We found three different patterns of reaction with the apolipoprotein A-I fragments; 6B8, 5G6 and 3D4 were different, but 5A6 resembled 3D4. Thus, the four antibodies reacted with at least three different antigenic sites in apolipoprotein A-I, which were present in different CNBr fragments of apolipoprotein A-I, but not on fragment 4 which forms the carboxy-terminal segment.     

Studies on the interaction between apolipoprotein A-II-enriched HDL3 and cultured bovine aortic endothelial (BAE) cells

The specific binding of high-density lipoproteins (HDL) to a number of cell and membrane types has been reported. The aim of this study was to investigate the ligand specificity of HDL binding sites on bovine aortic endothelial (BAE) cells and in particular to investigate the role of apo A-II in the interaction. In order to do this we prepared AII-HDL3 particles by incubating HDL3 with apo HDL. These particles were enriched in apo A-II, contained virtually no apo A-I, and were similar to HDL3 in terms of size and lipid composition. As these particles resemble the native HDL3 structure we believe they are probably a more suitable model for investigation of ligand specificity than artificial recombinants. AII-HDL3 particles were shown to bind to cells with similar affinity and capacity as HDL3. Further experiments indicated that HDL3 and AII-HDL3 competed with each other for binding and displayed similar affinities for a common binding site(s). The results suggest that apo A-II, as well as apo A-I, play an important role in the process of HDL recognition by putative HDL receptors on endothelial cells.     

Isolation of apolipoproteins A-I, A-II and E and their estimation by rocket immunoelectrophoresis in blood plasma of dis-alpha-lipoproteinemic patients

The methods for isolation of pure apolipoproteins A-I, A-II and E from the blood plasma of donors for preparation of monospecific rabbit antisera against these apolipoproteins and their estimation in human blood plasma using immunoelectrophoresis are described. It was found that the average content of apolipoprotein A-I (apo A-I) in the blood plasma of healthy males is 126.6 mg%, that of apolipoprotein A-II (apo A-II) is 56.8 mg%, that of apolipoprotein E (apo E) is 10.2 mg%. The apo A-I content in blood plasma is increased in hyper-alpha-lipoproteinemic patients and is decreased in hypo-alpha-lipoproteinemic ones, i. e. there is a direct relationship between the changes in concentration of high density lipoproteins (HDL) and apo A-I. The concentration of apo A-II in dis-alpha-lipoproteinemias varies within a narrow range. A considerable increase of the alpha-cholesterol/apo A-I ratio suggesting an increased capacity of HDL to transport cholesterol in hyper-alpha-lipoproteinemic patients is observed. There exists an indirect correlation between the changes in the contents of apo A-I and apo E in dis-alpha-lipoproteinemic patients.     

Apolipoprotein A-II inhibits high density lipoprotein remodeling and lipid-poor apolipoprotein A-I formation

The high density lipoproteins (HDL) in human plasma are classified on the basis of apolipoprotein composition into those containing apolipoprotein (apo) A-I but not apoA-II, (A-I)HDL, and those containing both apoA-I and apoA-II, (A-I/A-II)HDL. Cholesteryl ester transfer protein (CETP) transfers core lipids between HDL and other lipoproteins. It also remodels (A-I)HDL into large and small particles in a process that generates lipid-poor, pre-beta-migrating apoA-I. Lipid-poor apoA-I is the initial acceptor of cellular cholesterol and phospholipids in reverse cholesterol transport. The aim of this study is to determine whether lipid-poor apoA-I is also formed when (A-I/A-II)rHDL are remodeled by CETP. Spherical reconstituted HDL that were identical in size had comparable lipid/apolipoprotein ratios and either contained apoA-I only, (A-I)rHDL, or (A-I/A-II)rHDL were incubated for 0-24 h with CETP and Intralipid(R). At 6 h, the apoA-I content of the (A-I)rHDL had decreased by 25% and there was a concomitant formation of lipid-poor apoA-I. By 24 h, all of the (A-I)rHDL were remodeled into large and small particles. CETP remodeled approximately 32% (A-I/A-II)rHDL into small but not large particles. Lipid-poor apoA-I did not dissociate from the (A-I/A-II)rHDL. The reasons for these differences were investigated. The binding of monoclonal antibodies to three epitopes in the C-terminal domain of apoA-I was decreased in (A-I/A-II)rHDL compared with (A-I)rHDL. When the (A-I/A-II)rHDL were incubated with Gdn-HCl at pH 8.0, the apoA-I unfolded by 15% compared with 100% for the apoA-I in (A-I)rHDL. When these incubations were repeated at pH 4.0 and 2.0, the apoA-I in the (A-I)rHDL and the (A-I/A-II)rHDL unfolded completely. These results are consistent with salt bridges between apoA-II and the C-terminal domain of apoA-I, enhancing the stability of apoA-I in (A-I/A-II)rHDL and possibly contributing to the reduced remodeling and absence of lipid poor apoA-I in the (A-I/A-II)rHDL incubations.     

Differential effect of ultracentrifugation on apolipoprotein A-I-containing lipoprotein subpopulations

Two populations of apolipoprotein (apo) A-I-containing lipoprotein particles are found in high density lipoproteins (HDL): those that also contain apo A-II[Lp(A-I w A-II)] and those that do not [Lp(A-I w/o A-II)]. Lp(A-I w/o A-II) comprised two distinct particle sizes with mean hydrates Stokes diameter of 10.5 nm for Lp(A-I w/o A-II)1 and 8.5 nm for Lp(A-I w/o A-II)2. To study the effect of ultracentrifugation on these particles, Lp(A-I w/o A-II) and Lp(A-I w A-II) were isolated from the plasma and the ultracentrifugal HDL (d 1.063-1.21 g/ml fractions) of five normolipidemic and three hyperlipidemic subjects. The size subpopulations of these particles were studied by gradient polyacrylamide gel electrophoresis. Several consistent differences were detected between plasma Lp(A-I w/o A-II) and HDL Lp(A-I w/o A-II). First, in all subjects, the relative proportion of Lp(A-I w/o A-II)1 to Lp(A-I w/o A-II)2 isolated from HDL was reduced. Second, particles larger than Lp(A-I w/o A-II)1 and smaller than Lp(A-I w/o A-II)2 were considerably reduced in HDL. Third, a distinct population of particles with approximate Stokes diameter of 7.1 nm usually absent in plasma was detected in HDL Lp(A-I w/o A-II). Little difference in subpopulation distribution was detected between Lp(A-I w A-II) isolated from the plasma and HDL of the same subject. When plasma Lp(A-I w/o A-II) and Lp(A-I w A-II) were centrifuged, 14% and 4% of A-I were, respectively, recovered in the D greater than 1.21 g/ml fraction. Only 2% A-II was found in this density fraction. These studies show that the Lp(A-I w/o A-II) particles are less stable than Lp(A-I w A-II) particles upon ultracentrifugation. Among the various Lp(A-I w/o A-II) subpopulations, particles larger than Lp(A-I w/o A-II)1 and smaller than Lp(A-I w/o A-II)2 are most labile.     

Apo A-II participates in HDL–liposome interaction by the formation of new pre-β mobility particles and the modification of liposomes

Interaction between high density lipoproteins (HDL) and liposomes results in both a structural modification of HDL and the generation of new pre-β HDL-like particles. Here, phosphatidylcholine liposomes and human HDL were incubated at liposomal phospholipid/HDL phospholipid (L-PL/HDL-PL) ratios of 1:1, 3:1 and 5:1 with a subsequent assessment of the distribution of apolipoprotein (apo) A-I, apo A-II, free cholesterol (FC) and PL between newly generated pre-β mobility lipoproteins and non-disrupted liposomes. Both at L-PL/HDL-PL ratios of 3:1 and 5:1 the fraction of liposomal-derived PL associated with pre-β fraction was significantly higher than those accepted by α-HDL. We found that 78% of apo A-I released from HDL was incorporated into pre-β mobility fraction. The relative contents of PL and apo A-I in pre-β fraction were constant irrespective of the initial L-PL/HDL-PL ratio in the incubation mixture and accounted for approximately 83 and 11%, respectively. Apo A-II was detached from HDL to a similar extent as apo A-I and distributed evenly between pre-β fraction and non-disrupted liposomes. Apo A-II constituted approximately 1%, by weight, in these fractions at all L-PL/HDL-PL ratios investigated. It corresponded approximately to 10% of pre-β fraction protein mass. Both liposomes and pre-β fraction accepted comparable amounts of FC released from HDL. This data indicated that during the interaction between human HDL and phosphatidylcholine liposome apo A-II participates both in structural modification of liposomes and in the generation of pre-β mobility fraction of constant content of PL, apo A-I and apo A-II. Involvement of apo A-II in HDL–liposome interaction may influence the anti-atherogenic properties of liposomes.