Biomedical subjects

Y L Marcel

Publications and source records attributed to Y L Marcel.

At least 37 records · Page 2Linked to original sources

PubMed1996

Effect of the apolipoprotein A-I and surface lipid composition of reconstituted discoidal HDL on cholesterol efflux from cultured fibroblasts.

Five series of reconstituted discoidal HDL (LpA-I) particles have been prepared, and their constituents, apolipoprotein A-I (apoA-I), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), unesterified cholesterol (UC), phosphatidylinositol (PI), or sphingomyelin (SM), have been systematically varied to elucidate the relationship between HDL composition and cholesterol efflux from non-cholesterol-loaded human skin fibroblasts. The physical properties, such as hydrodynamic diameters, alpha-helix contents, and surface potentials, of these LpA-I have been measured and related to the ability of the LpA-I to accept cellular cholesterol. The results show that for LpA-I particles containing 2, 3, or 4 apoA-I per particle, Lp4A-I are the best acceptors of cellular cholesterol, followed by Lp3A-I and then Lp2A-I particles. Discoidal Lp2A-I with variations in POPC content, from 121 to 266 mol/particle; show no difference in their abilities to promote cholesterol efflux. Similarly, inclusion of 7 and 15 mol of free cholesterol to Lp2A-I also does not affect their ability to accept cellular cholesterol. However, increasing the content of either PI or SM, up to 20 mol/particle, is associated with significantly increased abilities of the LpA-I to promote cholesterol efflux. The efflux of cellular cholesterol to discoidal LpA-I particles is independent of specific changes in apoA-I conformation and charge, but appears to be positively related to major changes in the size of the lipoprotein particle. The study suggests that in contrast to interlipoprotein cholesterol transfers, the efflux of cholesterol from cultured fibroblasts is less sensitive to factors that affect the frequency of molecular collisions and more dependent on the ability of an HDL particle to absorb and retain cholesterol molecules. Since SM and PI appear to modulate this adsorption/desorption of cholesterol to HDL, variations in the concentration of these lipids within HDL would be expected to affect plasma cholesterol homeostasis.

Apolipoprotein A-I↗

PubMed1996

Specific phospholipid association with apolipoprotein A-I stimulates cholesterol efflux from human fibroblasts. Studies with reconstituted sonicated lipoproteins.

To understand how the lipid composition of high density lipoprotein mediates the efflux of cellular cholesterol, we have characterized the effects of variations in the lipid composition of well defined model sonicated apolipoprotein A-I (apoA-I)-containing lipoprotein (LpA-I) particle on cholesterol efflux from cultured human skin fibroblasts. LpA-I particles with varying content of phosphatidylcholine (POPC), phosphatidylinositol, sphingomyelin, cholesterol ester, and triolein were prepared by co-sonication. Association of as little as 5 mol of phosphatidylcholine with apoA-I is sufficient to transform lipid-free apoA-I into a distinct lipoprotein-like particle that is a significantly better acceptor of cellular cholesterol. Increasing the ratio of POPC/apoA-I from 5/1 to 35.5/1 in the sonicated LpA-I is associated with a significant increase in the release of cellular cholesterol. At low POPC/apoA-I ratios, native gradient gel electrophoresis of the LpA-I shows these lipoproteins to be small complexes (around 5-6 nm), with only 1 molecule of apoA-I (Lp1A-I). At a POPC/apoA-I ratio above 11/1, LpA-I form well defined complexes that contain 2 molecules of apoA-I (Lp2A-I) and range in size from 7.6 to 7.7 nm. Inclusion of sphingomyelin into an Lp1A-I further stimulates cholesterol efflux significantly. In contrast, inclusion of either sphingomyelin or phosphatidylinositol into a sonicated Lp2A-I has no effect on cholesterol efflux. Incorporation of cholesterol ester and/or triolein into an Lp2A-I particle is associated with a small reduction in cholesterol efflux to these lipoproteins. Therefore, cholesterol efflux from human fibroblasts is directly proportional to the amount and type of phospholipid in a sonicated LpA-I particle. Changes in the conformation and charge of apoA-I that result from changes in the lipid composition of a sonicated LpA-I particle appear to directly affect the ability of the lipoprotein to bind and retain cholesterol molecules. These data therefore suggest that the adsorption/desorption of cholesterol molecules to/from a sonicated LpA-I complex may be less sensitive to interfacial lipid-lipid interactions, but may depend on a conformation-dependent ability of apoA-I to bind cholesterol.

Apolipoprotein A-I↗

PubMed1996

Serum albumin is a significant intermediate in cholesterol transfer between cells and lipoproteins.

The function of albumin in the movement of cholesterol into and out of non-cholesterol-loaded fibroblasts has been investigated. Cholesterol efflux from cholesterol labeled normal human skin fibroblasts to fatty acid-free human serum albumin (HSA) is biphasic with a rapid first phase that plateaus at about 15 min followed by a nearly linear phase up to 90 min, the longest incubation in this study. Saturation of efflux is observed at about 10 mg of albumin/mL. Efflux is specific to albumin since other molecules, such as ovalbumin or gelatin, do not induce efflux. The ability of HSA to induce cellular cholesterol efflux is low compared to reconstituted discoidal lipoprotein A-I (LpA-I). HSA at 2 mg/mL produces a rate of cholesterol efflux similar to that of LpA-I at 45 micrograms of protein/mL; however, these concentrations are within the physiological range for both HSA and apolipoprotein A-I (apoA-I). The efflux to the medium containing both LpA-I and HSA is greater than that to each of them alone but does not show complete additivity, indicating a competition between HSA and LpA-I. The HSA-mediated cholesterol movement is bidirectional as demonstrated by the transfer of cholesterol from HSA-(3H)- cholesterol complexes to fibroblasts; moreover, the HSA-mediated transfer is much faster than that from cholesterol-containing LpA-I (0.8 versus 0.2 pmol (micrograms of cell protein)-1 (90 min)-1. However, the presence of either low-density lipoprotein (LDL) or LpA-I in the incubation medium significantly inhibits the transfer of cholesterol from HSA-(3H)-cholesterol complexes to fibroblasts, thus allowing the bidirectional transfer of cholesterol between HSA and cells to possibly operate as a net efflux. In conclusion, albumin plays a significant role in cholesterol transfer between cells and lipoproteins.

Apolipoprotein A-I↗

PubMed1996

Role of Lp A-I and Lp A-I/A-II in cholesteryl ester transfer protein-mediated neutral lipid transfer. Studies in normal subjects and in hypertriglyceridemic patients before and after fenofibrate therapy.

The two major subclasses of HDL contain apo A-I only (Lp A-I) or both apo A-I and apo A-II (Lp A-I/A-II). We have carried out experiments to quantify the participation of Lp A-I and Lp A-I/A-II in the neutral lipid transfer reaction in normal and hypertriglyceridemic subjects. Thirteen hypertriglyceridemic subjects were studied before and after fenofibrate therapy. Fenofibrate treatment resulted in decreases in total cholesterol, triglycerides (TG), and VLDL cholesterol of 19%, 48%, and 70%, respectively, and a 28% increase in HDL cholesterol, with no significant change in the proportion of Lp A-I and Lp A-I/A-II particles. The abundance of cholesteryl ester transfer protein (CETP) mRNA in peripheral adipose tissue decreased with treatment in four of five patients studied; however, no change occurred in plasma CETP mass. Using an isotopic transfer assay, we demonstrated that both Lp A-I and Lp A-I/A-II participated in the CE transfer reaction, with no change after fenofibrate therapy. This finding suggests that the marked increase in HDL cholesterol during fenofibrate therapy is due to normalization of plasma TG and hence decreased opportunity for mass transfer of lipid between HDL and TG-rich proteins in vivo. In this population of hypertriglyceridemic subjects, CETP was distributed in both the Lp A-I and Lp A-I/A-II subfractions of HDL, with preferential association with the smaller Lp A-I poor. In contrast, in nine normal subjects studied, negligible amounts of CETP were associated with Lp A-I/A-II. Nonetheless, the Lp A-I/A-II fraction of HDL contributed significantly to total CE mass transfer in normolipidemic plasma. Lp A-I/A-II is an efficient donor for CE transfer to TG-rich lipoproteins, and its low affinity for CETP may in fact facilitate neutral lipid transfer either by a shuttle mechanism or by formation of a ternary complex.

Adult↗

PubMed1995

Apolipoprotein A-I conformation in reconstituted discoidal lipoproteins varying in phospholipid and cholesterol content.

The effects of the size and cholesterol content on the conformation of apolipoprotein A-I (apoA-I) have been studied in reconstituted discoidal lipoproteins containing two apoA-I per particle (Lp2A-I). The immunoreactivity of a series of 13 epitopes distributed along the apoA-I sequence has been evaluated in Lp2A-I with a phospholipid/apoA-I molar ratio ranging from 31 to 156 and in Lp2A-I with constant phospholipids but varying in cholesterol content from 0 to 22 molecules. The results are compatible with a three domain structure in apoA-I in which the central domain is located between residues 99 and 143 and postulated to be a hinged domain that responds differentially to changes in phospholipid and cholesterol contents. Increasing the phospholipid content results in significant changes of epitope immunoreactivity throughout the N-terminal and central domains of apoA-I with fewer modifications in the C-terminal domain. In contrast, increasing Lp2A-I of two central epitopes, A11 (residues 99-132) and 5F6 (residues 118-148), and an extreme N-terminal epitope, 4H1 (residues 2-8). Interestingly, the effects of increasing cholesterol or phospholipids on these epitopes are opposite. This suggests a specific effect of cholesterol on the central domain tertiary structure between residues 99 and 143. Competition binding assays among pairs of antibodies binding to apoA-I on Lp2A-I are best explained by invoking inter- as well as intramolecular competitions. The specificity of the intermolecular competitions suggests an N to C termini arrangement of the two apoA-I molecules around the disc. Increasing the phospholipid content of Lp2A-I mainly increases the competitions between 3G10 and antibodies binding to most adjacent epitopes. Simultaneously as Lp2A-I enlarges, several of these antibodies also enhance the binding of 3G10. This has been interpreted as evidence of a structural rearrangement of apoA-I as a result of the size increase where the alpha-helix (residues 99-121) that contains the 3G10 epitope is increasingly interacting with lipids resulting in the enhanced expression of this epitope. The increasing interactions of apoA-I helices with lipids in the enlarging disc are compatible with previous reports of a greater apoA-I stability in the large discs. By contrast, cholesterol has limited but specific effects on antibody competitions and decreases the interaction of the N-terminal domain with the domain containing 3G10, either by direct cholesterol protein interaction or by modification of the lipid phase packing.

Antibodies, Monoclonal↗

PubMed1995

Role of apolipoprotein A-I in cholesterol transfer between lipoproteins. Evidence for involvement of specific apoA-I domains.

A series of monoclonal antibodies against epitopes spanning different domains of apoA-I have been tested for their effects on unesterified cholesterol transfer between low density lipoprotein (LDL) and well-defined homogenous lipoproteins reconstituted with phosphatidylcholine, cholesterol, and apoA-I (LpA-I). Antibodies 2G11 (reacting between residues 25 and 110), A05 (residues 25-82), A03 (residues 135-140), A44 and r5G9 (residues 149-186), and 4A12 (residues 173-205) significantly inhibit cholesterol transfer from LDL to Lp2A-I while they enhance transfer in the opposite direction, thus causing an increased net transfer to LDL. Most of these monoclonal antibodies (mAbs) also enhance phospholipid transfer to LDL but in a lesser and variable proportion relative to cholesterol. Their epitopes are mainly contained within domains that are predicted to be amphipathic alpha-helices. In contrast, mAbs 4H1 (residues 2-8), 3G10 (residues 96-121), and 5F6 (residues 116-141) have little or no effect on either cholesterol or phospholipid transfer, and the epitopes for these three mAbs have been shown in earlier studies to be structurally and functionally related. Their immunoreactivity responds similarly to variation in lipoprotein cholesterol content, and the antibodies binding to these sites compete with one another and have similar effects on the cholesterol esterification reaction. Thus, the current results are compatible with the hypothesis that they form an integrated domain with a common function in cholesterol metabolism, possibly as part of a hinge domain. Most mAbs were found to increase significantly the alpha-helicity of apoA-I in the Lp2A-I immunecomplexes, suggesting that they may increase the stability of the lipid-bound apoA-I. However, not unexpectedly, there is no correlation between the effects of mAbs on alpha-helicity and their effects on cholesterol or phospholipid transfer since each mAb has a discrete effect on these transfers. These studies demonstrate the specificity of LpA-I particles in cholesterol transport and document the existence of apoA-I domains with different functions in cholesterol transport.

Antibodies, Monoclonal↗

PubMed1995

Effect of LpA-I composition and structure on cholesterol transfer between lipoproteins.

The effect of high density lipoprotein composition on the rates of unesterified cholesterol exchange between low density lipoproteins (LDL) and well-defined homogeneous discoidal lipoproteins (LpA-I) reconstituted with phosphatidylcholine, cholesterol, and apolipoprotein A-I (apoA-I) has been investigated. LpA-I containing cholesterol and 2, 3, and 4 apoA-I molecules per particle differed in their ability to accept or donate cholesterol. A significant cholesterol exchange occurs between LDL and Lp2A-I (7.8 and 9.6 nm), while there is little or no cholesterol exchange detectable between LDL and Lp3A-I (10.8 and 13.4 nm) and Lp4A-I (17.0 nm) complexes. The cholesterol transfer from LDL to the cholesterol-free Lp2A-I (9.6 nm), Lp3A-I (13.4 nm), and Lp4A-I (17.0 nm) particles also shows significant cholesterol transfer to Lp2A-I, while there is no detectable transfer to Lp3- and 4A-I particles. The rates of cholesterol transfer to cholesterol-free and cholesterol-containing Lp2A-I appear to differ significantly. Cholesterol transfer from LDL to cholesterol-free Lp2A-I is zero order with respect to acceptor concentrations when the Lp2A-I/LDL ratio is above 10. Transfer rates from LDL to cholesterol-free Lp2A-I are faster for the smaller Lp2A-I (8.5 nm) than to the larger Lp2A-I (9.7 nm) and exhibit half-times (t1/2) at 25 degrees C of 4.0 and 5.3 h, respectively. In contrast, cholesterol transfer from LDL to cholesterol-containing Lp2A-I remains dependent upon acceptor concentrations to an acceptor/donor particle ratio of 80. In addition, transfer from LDL to cholesterol-containing Lp2A-I is faster to the 9.6 nm than to 7.8 nm particles, with t1/2 of 1.4 and 2.3 h, respectively. The rates of cholesterol transfer from Lp2A-I to LDL are higher than in the opposite direction, in particular for the small Lp2A-I (7.8 nm), which has a t1/2 of approximately 50 min. The results show that changes in the composition and structure of apoA-I-containing particles have a significant effect on inter-lipoprotein exchange of cholesterol. This suggests that the kinetics of cholesterol transfer to and from reconstituted discoidal LpA-I particles cannot be fully explained by passive aqueous diffusion.

Apolipoproteins A↗

PubMed1995

Apolipoprotein B and E basic amino acid clusters influence low-density lipoprotein association with lipoprotein lipase anchored to the subendothelial matrix.

Lipoprotein accumulation in the subendothelial matrix is an important step in atherogenesis. We have previously shown that addition of lipoprotein lipase (LPL) markedly increased binding of apolipoprotein B (apoB)-containing lipoproteins to an endothelial cell-derived matrix, and this enhanced lipoprotein binding was inhibited by apoE. In the present studies we examined the role of various regions of apoB in the binding of LDL to LPL-containing endothelial cell matrix and the ability of various apoE domains to decrease lipoprotein retention. We studied three apoB epitope-specific monoclonal antibodies for their ability to block the binding of 125I-LDL to LPL-containing matrix. Of these, monoclonal antibody 4G3, which recognizes an arginine-containing epitope in apoB, was the most effective in reducing LDL binding. Chemical modification of LDL apoB lysines or arginines markedly reduced the ability of the lipoprotein to block the binding of 125I-LDL to LPL-containing matrix, suggesting that apoB positively charged amino acids are involved in the interaction. Furthermore, polyarginine or polylysine markedly decreased 125I-LDL binding to LPL-containing matrix, whereas polyleucine was ineffective. These data suggest that apoB positively charged regions are important in LDL binding. To explore the role of charge modifications on apoE by single arginine-cysteine interchanges, we examined the effects of the three major human apoE isoforms (apoE2, apoE3, and apoE4). ApoE3 was the most effective in decreasing 125I-LDL retention, followed by apoE4; apoE2 was the least effective. Similarly, apoE2-containing HDL was much less effective than apoE3-containing HDL in decreasing 125I-LDL retention.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗

PubMed1994

Apoprotein B structure and receptor recognition of triglyceride-rich low density lipoprotein (LDL) is modified in small LDL but not in triglyceride-rich LDL of normal size.

We compared the effect of lipid composition and particle size of triglyceride-rich low density lipoprotein (LDL) upon apoprotein B conformation and binding to the LDL receptor. Three groups of triglyceride-rich LDL were studied: (a) LDL isolated from chronic hypertriglyceridemic individuals (HTG-LDL); (b) normal LDL made triglyceride-rich by in vitro incubation with triglyceride emulsion and the neutral lipid transfer protein (R-LDL); and (c) LDL from normolipidemic individuals made acutely hypertriglyceridemic by intravenous infusion of 10% Intralipid (IV-LDL). HTG-LDL was small and dense, whereas R-LDL and IV-LDL had normal size. HTG-LDL, but not R-LDL or IV-LDL, exhibited decreased binding to the LDL receptor on human skin fibroblasts in studies at 4 degrees C and reduced degradation at 37 degrees C. Apoprotein B conformation was assessed by circular dichroism and by analyzing the immunoreactivity of different monoclonal antibodies. HTG-LDL but not R-LDL or IV-LDL showed a change in the CD spectra and a consistent decrease in the immunoreactivity of monoclonal antibody 3F5 (2.5-fold) which recognizes an epitope adjacent to the receptor binding domain of apoprotein B. These findings suggest that in triglyceride-rich LDL, the relative content of neutral lipid in the core of LDL in the absence of changes in the size of the particle does not significantly affect apoprotein B conformation or its affinity for the LDL receptor.

Antibodies, Monoclonal↗

PubMed1994

Two-site enzyme immunoassay of cholesteryl ester transfer protein with monoclonal and oligoclonal antibodies.

We developed a sandwich-type enzyme immunoassay to measure cholesteryl ester transfer protein (CETP) mass in human plasma. A specific monoclonal antibody (TP-4) that recognizes an epitope located in the C-terminal domain was used for antigen capture and an anti-CETP peptide antibody directed against the 290-306 residue was used for detection. Bound antibodies were revealed with an antibody-peroxidase conjugate specific for rabbit IgG. The presence of 10 mL/L Triton X-100 in the incubation buffer increased antigen exposure of CETP in plasma. The curves for CETP in standard plasma and partially purified CETP were parallel. This technique is rapid (results within 6 h), accurate, precise (mean intra- and interassay CVs 3.6% and 8.4%, respectively), and simple to perform. Assay sensitivity is at microgram concentrations, with a working range of 20-200 micrograms/L. In 40 normolipidemic healthy subjects, the mean CETP concentration in plasma was 1.1 +/- 0.4 mg/L. A strong correlation between CETP concentration and CETP activity (r = 0.91, n = 42) was observed. In plasma, the bulk of CETP was found in high-density lipoprotein fractions. Therefore, this assay may be a useful tool for investigations of CETP and its significance in relevant diseases.

Antibodies↗

PubMed1994

Dietary fish oil-induced decrease in low density lipoprotein binding to fibroblasts is mediated by apolipoprotein E.

In a previous study we demonstrated that isocaloric substitution of fish oil (FO) for lard in the diet of cynomolgus monkeys resulted in low density lipoproteins (LDL) that were poorer competitors for binding of a standard 125I-labeled LDL and led to less cholesteryl ester accumulation in skin fibroblasts (Linga, V., et al. 1993. J. Lipid Res. 34: 769-778). The decreased binding and cholesteryl ester accumulation by FO LDL appeared related to the LDL apolipoprotein E (apoE) content. We hypothesized that FO LDL had reduced binding to skin fibroblasts due to a decrease in receptor active apoE. To test this hypothesis and determine the relative contribution of apoE versus apolipoprotein B (apoB) in binding of LDL to skin fibroblasts, LDL from cynomolgus monkeys fed lard or FO-containing diets were isolated, characterized, radioiodinated, and tested for binding in the absence or presence of a 10-fold molar excess of monoclonal antibody to the receptor binding domain of apoE (1D7) or apoB-100 (MB47). FO LDL were smaller, contained less apoE (E/B molar ratio = 0.48 +/- 0.03 vs. 1.85 +/- 0.22; P < 0.001), and had a weaker binding affinity (Kd = 11.3 +/- 1.6 vs. 3.8 +/- 0.80 microgram/ml; P < 0.01) compared to the lard counterparts. Furthermore, the apoE/B molar ratio of LDL appeared inversely related to the Kd for binding to skin fibroblasts. Incubation of LDL with skin fibroblasts in the presence of a 10-fold molar excess of monoclonal antibody directed at the receptor binding domain of apoB-100 (MB47) eliminated 96 +/- 3% of binding of FO LDL, but eliminated only 43 +/- 18% of binding for lard LDL. Incubation with a 10-fold molar excess of monoclonal antibody to the receptor-binding domain of apoE (1D7) eliminated only 23 +/- 6% of FO LDL binding to fibroblasts relative to a no-antibody control, but for lard LDL 44 +/- 11% of binding to fibroblasts was eliminated. Both antibodies together blocked all binding of LDL from both diet groups. In a fluid phase precipitation assay > 75% of the LDL particles from both diet groups was precipitated with saturating amounts of MB47, indicating that the proportion of LDL particles expressing this epitope was the same for both diet groups. The same assay using 1D7 showed approximately 4-fold greater precipitation of LDL in the lard versus FO group.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Apolipoprotein A-I domains involved in the activation of lecithin:cholesterol acyltransferase. Importance of the central domain.

The reaction of highly purified lecithin:cholesterol acyltransferase (LCAT) with defined reconstituted discoidal apoA-I-containing lipoproteins (LpA-I) with 2, 3, or 4 apoA-I molecules/particle (Lp2, 3, or 4A-I) has been studied in the presence of a number of specific anti apoA-I antibodies. Among nine anti-apoA-I monoclonal antibodies (mAbs) reacting with epitopes distributed over 80% of the sequence, three significantly inhibit the LCAT reaction with all particles. The position of their epitopes located in the middle to COOH-terminal region between residues 96-121 (3G10), 135-148 (A03), and 149-186 (A44) is compatible with an inhibition by steric hindrance over a central domain. Antibody 4H1 binding to the NH2 terminus (residues 2-8) profoundly increases (5-fold) the LCAT reaction with Lp2A-I (7.8 nm), but not with other particles. Other mAbs, A11 and 5F6, binding to epitopes (residues 99-139 and 118-141) enhance LCAT reactivity with the small Lp2A-I (7.8 nm) and Lp3A-I (10.8 nm) but not with their larger counterparts. Most mAbs have similar effects on LCAT reaction with native high density lipoprotein3 as with LpA-I. The inhibitory or enhancing effects of these mAbs are also observed with Fab fragments and not related to their binding affinity for apoA-I containing reconstituted lipoprotein particles. The intercalation of epitopes for mAbs that inhibit or enhance LCAT reaction with small LpA-I is compatible not with steric hindrance but with conformational modifications of apoA-I and indirectly of the lipids in small particles. We propose that enhancing mAbs act by stabilization of an apoA-I conformation which is not favored in small LpA-I, i.e. by increasing binding of amphipathic helices to lipids or by interfering with the mobility of a hinged domain. The epitopes for the inhibitory mAbs can be shown to overlap on several LpA-I models, indicating that steric hindrance over a single site is a possible mechanism of inhibition.

Antibodies, Monoclonal↗

PubMed1993

Apolipoprotein A-I conformation in discoidal particles: evidence for alternate structures.

To define the conformation of apolipoprotein A-I in discoidal particles, the immunoreactivity of a series of epitopes distributed along the apoA-I sequence has been evaluated in lipid-free apoA-I and in lipid-bound form. To this end, reconstituted discoidal lipoproteins, here called LpA-I, and defined by number of apoA-I per particle (e.g., Lp2A-I), have been prepared with palmitoyloleoylphosphatidylcholine, cholesterol, and apoA-I. Four LpA-I have been obtained and studied: two in the Lp2A-I class, 7.8 and 9.6 nm in diameter, and two in the Lp3A-I class, 10.8 and 13.4 nm. The immunoreactivity of all the epitopes tested was significantly different in LpA-I particles compared to lipid-free apoA-I, demonstrating that binding to lipids produces a drastic change in apoA-I conformation. Specific domains in the primary sequence become highly exposed while others are masked. Although the variation in immunoreactivity of the epitopes between various LpA-I was not drastic, significant differences in the calculated ED50 values were observed for a number of antibodies in small versus large particles within each class (Lp2A-I or Lp3A-I), indicating that particle size can modulate apoA-I conformation. In addition, when the competition between pairs of mAbs was analyzed in order to understand the relative position of epitopes, highly significant differences were observed as a function of particle size within each class. In particular, the competition between mAbs recognizing epitopes in the central region of apoA-I was greater in the larger particles than in their small counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

PubMed1993

Cholesterol efflux from fibroblasts to discoidal lipoproteins with apolipoprotein A-I (LpA-I) increases with particle size but cholesterol transfer from LpA-I to lipoproteins decreases with size.

To understand the role of different discoidal lipoproteins in cellular cholesterol efflux, defined discoidal lipoproteins containing 2, 3, or 4 apolipoproteins (apo) A-I per particle (Lp2A-I, Lp3A-I, and Lp4A-I) were prepared from mixtures of apoA-I and phospholipids with or without cholesterol. Each particle had a slow pre beta migration on agarose gel electrophoresis which further decreased as the number of apoA-I increased. Incubation of cholesterol-labeled human fibroblasts with the different LpA-I at an equimolar concentration in apoA-I showed that the best acceptors of cellular cholesterol were Lp4A-I, followed by Lp3A-I and Lp2A-I. Cholesterol efflux to these particles was positively correlated to the number of apoA-I, to the ratio of phospholipids to apoA-I, and to the size of particles, three interrelated parameters. To follow the subsequent movement of cellular cholesterol after it became associated with LpA-I, cholesterol- and apoA-I-labeled LpA-I were incubated with plasma which resulted in parallel modifications of each labels electrophoretic migration with time. However, [3H]cholesterol-labeled LpA-I transferred from pre beta to alpha migration with a precursor-product relationship while 125I-LpA-I progressively shifted from pre beta to alpha migration. The change in electrophoretic migration of 125I-LpA-I is independent of cholesterol and appears related only to a modification of apoA-I charge. Lp2A-I was fastest in changing its electrophoretic migration to alpha, followed by Lp3A-I and then Lp4A-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein A-I↗

PubMed1993

Structure of the human apolipoprotein D gene promoter region.

A human genomic clone of 18.2 kbp encompassing the apolipoprotein D (apoD) exon-1 and 2 and 10 kbp of upstream sequence was isolated and characterized. DNA sequencing and primer extension analysis revealed a transcriptional initiation site located 27 bp downstream of a consensus TATA box sequence. The exon 1 was 66 bp long. Computer analysis of DNA sequence from positions -557 to +129 revealed some putative transcriptional regulatory elements including a stretch of (pyrimidine/purine)26 located from nucleotide -263 to nucleotide -212, which could potentially form Z-DNA. Steroid hormone regulatory elements were identified which may be related to the modulation of apoD gene expression by androgens and estrogens in vitro.

Apolipoproteins↗

PubMed1993

Uptake of high density lipoprotein cholesterol ester by HepG2 cells involves apolipoprotein E localized on the cell surface.

High density lipoprotein (HDL) cholesterol ester (CE) is taken up by many cells without simultaneous uptake of HDL apoprotein. The studies described herein demonstrate that the selective uptake of cholesterol ester by HepG2 cells is reduced by antibody directed against the receptor-binding domain of apoE (monoclonal antibody (mAb) 1D7) but not by antibody directed against the NH2-terminal portion of the molecule. The reduction, by 1D7, of HDL cholesteryl ester uptake is not due to apoE acquisition by the labeled HDL preparation or by the transfer of [3H]CE of HDL to apoE-containing lipoproteins and uptake by the apoB/E or apoE receptors. Rather, it appears that mAb 1D7 recognizes apoE localized at the cell surface of HepG2 cells. This conclusion is supported by the fact that: 1) reduction of HDL-CE uptake by HepG2 cells is observed within 15 min after the addition of the antibody-ligand mixture; 2) 1D7 is similarly effective in reducing the selective uptake of HDL-CE when added to the ligand or to the cells; 3) three different anti-apoE mAbs (1D7, 3B7, and 3H1) bind specifically to the surface of the cells. We have also demonstrated that heparin (5 mg/ml) does not reduce the amount of apoE-immunoreactive material bound at the cell surface when added before or after the binding period. 1D7, but not 3B7 or 3H1, binds less in the presence of heparin. The observations are consistent with a localization of apoE on the cell membrane rather than on lipoproteins bound to apoB/E or apoE receptors.

Antibodies, Monoclonal↗

PubMed1993

Contribution of apoB-48 and apoB-100 triglyceride-rich lipoproteins (TRL) to postprandial increases in the plasma concentration of TRL triglycerides and retinyl esters.

After the ingestion of a fat-rich meal, there is a postprandial increase in the plasma concentration of both apolipoprotein B-48- and apoB-100-containing triglyceride-rich lipoproteins (apoB-48 and apoB-100 TRL). In order to determine the contribution of these lipoproteins to postprandial lipemia, the concentration of triglycerides (TG) and retinyl esters (RE) was measured in apoB-48 and apoB-100 TRL after an oral fat load. Six normolipidemic male subjects were fed heavy cream (1 g fat per kg body weight) containing vitamin A (3000 retinol equivalents). TRL were isolated by ultracentrifugation from plasma samples obtained at regular intervals after the meal, and apoB-100 TRL were separated from apoB-48 TRL by affinity chromatography using monoclonal antibodies. Postprandial increase in plasma TG concentration was due to an increase in TG in the TRL fraction, which in turn was predominantly (82 +/- 4%) due to an increase in TG in apoB-48 TRL. Contribution of apoB-100 TRL to postprandial increase in TRL TG was 3-27% in individual subjects. ApoB-100 TRL remained a significant carrier of total plasma triglyceride in the fed state, as reflected by similar apoB-100 and apoB-48 TRL TG concentrations at 2, 4, and 6 h after the fat meal. Retinyl esters were regularly detected in apoB-100 TRL. Seventy-five (+/- 9) percent of the increase in TRL-RE was due to RE in apoB-48 TRL and 25 +/- 9% was due to RE in apoB-100. These data suggest that RE in plasma are not always associated with apoB-48-containing lipoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1993

Cholesteryl ester transfer protein and high density lipoprotein responses to cholesterol feeding in men: relationship to apolipoprotein E genotype.

The apolipoprotein (apo) E isoform is an important determinant of the plasma lipoprotein distribution of apoE and of the metabolism of apoE-containing lipoproteins. We have determined the effects of apoE genotype on the plasma lipoprotein response to cholesterol feeding in 30 young normal male subjects (5 E3/2, 11 E3/3, 14 E4/3) under rigorously controlled dietary conditions. Two diets, differing only in cholesterol content (low cholesterol (LC): 80 mg cholesterol/1000 kcal and high cholesterol (HC): 320 mg cholesterol/1000 kcal), were compared using a random crossover design. At the end of the HC as compared to the LC period, total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), and HDL2-C increased by an average of 15%, 21%, 7%, and 23%, respectively, for the three genotype groups combined (P < 0.001 for each). The LDL-C response to dietary cholesterol did not differ among the apoE genotypes. However, the increase in HDL-C varied significantly according to the apoE genotype (E3/2: 0 change, E3/3: +4%, E4/3: +12%; P < 0.05). The plasma cholesteryl ester transfer protein (CETP) response to cholesterol feeding also differed amongst the three apoE genotype groups (E3/2: +37%, E3/3: +18%, E4/3: +9%) (P < 0.05). ApoE genotype has significant and opposite effects on plasma CETP and HDL-C responses to dietary cholesterol in men.

Adipose Tissue↗