Substrate For Lecithin:cholesterol Acyltransferase Research Articles

Novel Cocrystallization of Apolipoprotein A‐I with Butyric Acid

Diseases of lipid metabolism, in particular cardiovascular atherosclerosis, remain the number one cause of morbidity and mortality in Western society. The well-documented anti-atherogenic role of high-density lipoprotein (HDL) is related to its involvement in reverse cholesterol transport. When the transporter ABCA1 initiates cholesterol efflux from peripheral and hepatic cells, apolipoprotein A-I (apoA-I) binds the free cholesterol together with phospholipid to form discoidal nascent HDL (“good cholesterol”). The enzyme lecithin:cholesterol acyltransferase (LCAT) catalyzes the esterification of the cholesterol carried by HDL. ApoA-I activates LCAT for reverse cholesterol transport, wherein LCAT esterifies the cholesterol within the nascent HDL2 and facilitates maturation of HDL to a mature spherical form. After esterification, cholesterol can no longer diffuse back to the peripheral and hepatic cells. The esterified cholesterol, or cholesteryl ester, molecules form the hydrophobic core of the mature HDL particle and leave the circulation upon uptake by a hepatic scavenger receptor for excretion by the liver. Research in the Atkinson laboratory has implicated the arginine 123 residue of apoA-I in the function of LCAT. Based on the X-ray crystal structure, the Arg123 residues of each apoA-I monomer enclose a hydrophobic tunnel into which the lipid substrates of LCAT may be sequestered. ApoA-I has a closed conformation without lipid bound that transforms to a “double belt” encircling the nascent HDL disc. When apoA-I activates LCAT, the tunnel should widen in order to present the substrates to LCAT for esterification. However, the mechanism by which apoA-I interfaces with LCAT substrates remains unknown. Cocrystallization of apoA-I with the lipid component, butyric acid, will enable us to assess how the “double belt” of apoA-I opens in three-dimensional space.

A fluorescence method to detect and quantitate sterol esterification by lecithin:cholesterol acyltransferase

We describe a simple but sensitive fluorescence method to accurately detect the esterification activity of lecithin:cholesterol acyltransferase (LCAT). The new assay protocol employs a convenient mix, incubate, and measure scheme. This is possible by using the fluorescent sterol dehydroergosterol (DHE) in place of cholesterol as the LCAT substrate. The assay method is further enhanced by incorporation of an amphiphilic peptide in place of apolipoprotein A-I as the lipid emulsifier and LCAT activator. Specific fluorescence detection of DHE ester synthesis is achieved by employing cholesterol oxidase to selectively render unesterified DHE nonfluorescent. The assay accurately detects LCAT activity in buffer and in plasma that is depleted of apolipoprotein B lipoproteins by selective precipitation. Analysis of LCAT activity in plasmas from control subjects and sickle cell disease (SCD) patients confirms previous reports of reduced LCAT activity in SCD and demonstrates a strong correlation between plasma LCAT activity and LCAT content. The fluorescent assay combines the sensitivity of radiochemical assays with the simplicity of nonradiochemical assays to obtain accurate and robust measurement of LCAT esterification activity.

Effects of cross-link breakers, glycation inhibitors and insulin sensitisers on HDL function and the non-enzymatic glycation of apolipoprotein A-I

Hyperglycaemia, a key feature of diabetes, is associated with non-enzymatic glycation of plasma proteins. We have shown previously that the reactive alpha-oxoaldehyde, methylglyoxal, non-enzymatically glycates apolipoprotein (Apo)A-I, the main apolipoprotein of HDL, and prevents it from activating lecithin:cholesterol acyltransferase (LCAT), the enzyme that generates almost all of the cholesteryl esters in plasma. This study investigates whether the glycation inhibitors aminoguanidine and pyridoxamine, the insulin sensitiser metformin and the cross-link breaker alagebrium can inhibit and/or reverse the methylglyoxal-mediated glycation of ApoA-I and whether these changes can preserve or restore the ability of ApoA-I to activate LCAT. Inhibition of ApoA-I glycation was assessed by incubating aminoguanidine, pyridoxamine, metformin and alagebrium with mixtures of methylglyoxal and discoidal reconstituted HDL (rHDL) containing phosphatidylcholine and ApoA-I, ([A-I]rHDL). Glycation was assessed as the modification of ApoA-I arginine, lysine and tryptophan residues, and by the extent of ApoA-I cross-linking. The reversal of ApoA-I glycation was investigated by pre-incubating discoidal (A-I)rHDL with methylglyoxal, then incubating the modified rHDL with aminoguanidine, pyridoxamine or alagebrium. Aminoguanidine, pyridoxamine, metformin and alagebrium all decreased the methylglyoxal-mediated glycation of the ApoA-I in discoidal rHDL and conserved the ability of the particles to act as substrates for LCAT. However, neither aminoguanidine, pyridoxamine nor alagebrium could reverse the glycation of ApoA-I or restore its ability to activate LCAT. Glycation inhibitors, insulin sensitisers and cross-link breakers are important for preserving normal HDL function in diabetes.

Biogenesis and speciation of nascent apoA-I-containing particles in various cell lines

It is generally thought that the large heterogeneity of human HDL confers antiatherogenic properties; however, the mechanisms governing HDL biogenesis and speciation are complex and poorly understood. Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only alpha-nascent apolipoprotein A-I-containing particles (alpha-LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Interestingly, incubation of exogenous apoA-I with either HepG2 or macrophages generates both alpha-LpA-I and prebeta1-LpA-I. Furthermore, glyburide inhibits almost completely the formation of alpha-LpA-I but not prebeta1-LpA-I. Similarly, endogenously secreted HepG2 apoA-I was found to be associated with both prebeta1-LpA-I and alpha-LpA-I; by contrast, CaCo-2 cells secreted only alpha-LpA-I. To determine whether alpha-LpA-I generated by fibroblasts is a good substrate for LCAT, isolated alpha-LpA-I as well as reconstituted HDL [r(HDL)] was reacted with LCAT. Although both particles had similar V(max) (8.4 vs. 8.2 nmol cholesteryl ester/h/microg LCAT, respectively), the K(m) value was increased 2-fold for alpha-LpA-I compared with r(HDL) (1.2 vs. 0.7 microM apoA-I). These results demonstrate that 1) ABCA1 is required for the formation of alpha-LpA-I but not prebeta1-LpA-I; and 2) alpha-LpA-I interacts efficiently with LCAT. Thus, our study provides direct evidence for a new link between specific cell lines and the speciation of nascent HDL that occurs by both ABCA1-dependent and -independent pathways.

Oxidation of Methionine Residues to Methionine Sulfoxides Does Not Decrease Potential Antiatherogenic Properties of Apolipoprotein A-I

The initial stage of oxidation of high density lipoproteins (HDL) is accompanied by the lipid hydroperoxide-dependent, selective oxidation of two of the three Met residues of apolipoprotein A-I (apoA-I) to Met sulfoxides (Met(O)). Formation of such selectively oxidized apoA-I (i.e. apoA-I(+32)) may affect the antiatherogenic properties of HDL, because it has been suggested that Met(86) and Met(112) are important for cholesterol efflux and Met(148) is involved in the activation of lecithin:cholesterol acyl transferase (LCAT). We therefore determined which Met residues were oxidized in apoA-I(+32) and how such oxidation of apoA-I affects its secondary structure, the affinity for lipids, and its ability to remove lipids from human macrophages. We also assessed the capacity of discoidal reconstituted HDL containing apoA-I(+32) to act as substrate for LCAT, and the dissociation of apoA-I and apoA-I(+32) from reconstituted HDL. Met(86) and Met(112) were present as Met(O), as determined by amino acid sequencing and mass spectrometry of isolated peptides derived from apoA-I(+32). Selective oxidation did not alter the alpha-helicity of lipid-free and lipid-associated apoA-I as assessed by circular dichroism, and the affinity for LCAT was comparable for reconstituted HDL containing apoA-I or apoA-I(+32). Cholesteryl ester transfer protein mediated the dissociation of apoA-I more readily from reconstituted HDL containing apoA-I(+32) than unoxidized apoA-I. Also, compared with native apoA-I, apoA-I(+32) had a 2- to 3-fold greater affinity for lipid (as determined by the rate of clearance of multilamellar phospholipid vesicles) and its ability to cause efflux of [(3)H]cholesterol, [(3)H]phospholipid, and [(14)C]alpha-tocopherol from lipid-laden human monocyte-derived macrophages was significantly enhanced. By contrast, no difference was observed for cholesterol and alpha-tocopherol efflux to lipid-associated apolipoproteins. Together, these results suggest that selective oxidation of Met residues enhances rather than diminishes known antiatherogenic activities of apoA-I, consistent with the overall hypothesis that detoxification of lipid hydroperoxides by HDL is potentially antiatherogenic.

Lipid-free apolipoproteins A-I and A-II promote remodeling of reconstituted high density lipoproteins and alter their reactivity with lecithin:cholesterol acyltransferase

We examined the effect of lipid-free apolipoprotein A-I (apoA-I) and apoA-II on the structure of reconstituted high density lipoproteins (rHDL) and on their reactivity as substrates for lecithin:cholesterol acyltransferase (LCAT). First, homogeneous rHDL were prepared with either apoA-I or apoA-II using palmitoyloleoylphosphatidylcholine (POPC) and cholesterol. Lipid-free apoA-I and apoA-II were labeled with the fluorescent probe dansyl chloride (DNS). The binding kinetics of apoA-I-DNS to A-II-POPCrHDL and of apoA-II-DNS to A-I-POPCrHDL were monitored by fluorescence polarization, adding the lipid-free apolipoproteins to the rHDL particles in a 1:1 molar ratio. For both apolipoproteins, the binding to rHDL was rapid, occurring within 5 min. Next, the effect on rHDL structure and particle size was determined after incubations of lipid-free apolipoproteins with homogeneous rHDL at 37°C from 0.5 to 24 h. The products were analyzed by non-denaturing gradient gel electrophoresis followed by Western blotting. The effect of apoA-I or apoA-II on 103 Å A-II-POPCrHDL was a rearrangement into 78 Å particles containing apoA-I and/or apoA-II, and 90 Å particles containing only apoA-II. The effect of apoA-I or apoA-II on 98 Å A-I-POPCrHDL was a rearrangement into complexes ranging in size from 78 Å to 105 Å containing apoA-I and/or apoA-II, with main particles of 78 Å, 88 Å, and 98 Å. Finally, the effect of lipid-free apoA-I and apoA-II on rHDL as substrates for LCAT was determined. The addition of apoA-I to A-II-POPCrHDL increased its reactivity with LCAT 24-fold, reflected by a 4-fold increase in apparent Vmax and a 6-fold decrease in apparent Km, while the addition of apoA-II to A-II-POPCrHDL had no effect on its minimal reactivity with LCAT. In contrast, the addition of apoA-II to A-I-POPCrHDL decreased the reaction with LCAT by about one-half. The inhibition was due to a 2-fold increase in apparent Km; there was no significant change in apparent Vmax. Likewise, the addition of apoA-I to A-I-POPCrHDL inhibited the reaction with LCAT to about two-thirds that of A-I-POPCrHDL without added apoA-I. ▪ In summary, both lipid-free apoA-I and apoA-II can promote the remodeling of rHDL into hybrid particles of primarily smaller size. Both apoA-I and apoA-II affect the reactivity of rHDL with LCAT, when added to the reaction in lipid-free form. These results have important implications for the roles of lipid-free apoA-I and apoA-II in HDL maturation and metabolism.—Durbin, D. M., and A. Jonas. Lipid-free apolipoprotein A-I and A-II promote remodeling of reconstituted high density lipoproteins and alter their reactivity with lecithin:cholesterol acyltransferase.

Regiospecific esterification of estrogens by lecithin:cholesterol acyltransferase.

Lecithin:cholesterol acyltransferase (LCAT), the enzyme that esterifies cholesterol in blood, also esterifies other steroids at the 3beta-hydroxyl. These steroids, like cholesterol, are delta5-3beta-hydroxysteroids, such as pregnenolone and dehydroepiandrosterone. One unusual LCAT substrate is the estrogen, estradiol, which is esterified at the 17beta-hydroxyl. The esterification of estradiol by LCAT has been reported to produce a powerful antioxidant that protects low density lipoprotein (LDL) from oxidation. We investigated the substrate specificity of LCAT, comparing the esterification of four different steroids (estradiol, estriol, testosterone, and 5-androstene-3beta, 17beta-diol) by human LCAT in blood and by acyl-coenzyme A:acyltransferase in tissue (placenta and fat). Estradiol was esterified only at the D ring 17beta-hydroxyl group in both blood and tissue. In contrast, although testosterone has a D ring structure identical to that of estradiol, and it was esterified at the 17beta-hydroxyl by acyl-coenzyme A:acyltransferase in tissue, it was not esterified by LCAT. When 5-androstenediol was the substrate in the tissues, both the 3beta- and 17beta-esters were synthesized, but the major product was the 17beta-ester. Conversely, although 5-androstenediol was an excellent substrate for LCAT, only the 3beta-hydroxyl was esterified. No 17beta-ester was formed. The comparison of the esterification of estriol by acyl-coenzyme A:acyltransferase and LCAT was also surprising. In the tissues, estriol is esterified at both D ring hydroxyls, and both are esterified about equally. Although estriol is an extremely polar estrogen, it is esterified by LCAT, albeit at a very slow rate. Although again both D ring hydroxyls were esterified, the LCAT esterification site was mainly at the 17beta-hydroxyl. Esterification of estriol at the 17beta-hydroxyl in preference to the 16alpha-hydroxyl is especially striking, because the 17beta-hydroxyl group is sterically shielded by the C-18 methyl group, making esterification at this position energetically much more difficult. Furthermore, these studies demonstrate that esterification of the 17beta-hydroxyl group by LCAT is unique to estrogens. It suggests that this unusual regiospecific esterification of C-17 of the estrogens underlies a distinct stereochemical requirement for the powerful antioxidant action that has reported for the estradiol esters formed by LCAT.

The Hydrophobic Face Orientation of Apolipoprotein A-I Amphipathic Helix Domain 143–164 Regulates Lecithin:Cholesterol Acyltransferase Activation

Apolipoprotein A-I (apoA-I) activates the plasma enzyme lecithin:cholesterol acyltransferase (LCAT), catalyzing the rapid conversion of lipoprotein cholesterol to cholesterol ester. Structural mutants of apoA-I have been used to study the details of apoA-I-LCAT-catalyzed cholesterol ester formation. Several studies have shown that the alpha-helical segments corresponding to amino acids 143-164 and 165-186 (repeats 6 and 7) are essential for LCAT activation. In the present studies, we examined how the orientation of the hydrophobic face, independent of an increase in overall hydrophobicity, affects LCAT activation. We designed, expressed, and characterized a mutant, reverse of 6 apoA-I (RO6 apoA-I), in which the primary amino acid sequence of repeat 6 (amino acids 143-164) was reversed from its normal orientation. This mutation rotates the hydrophobic face of repeat 6 approximately 80 degrees. Lipid-free RO6 apoA-I showed a marked stabilization when denatured by guanidine hydrochloride, but showed significant destabilization to guanidine hydrochloride denaturation in the lipid-bound state compared with wild-type apoA-I. Recombinant high density lipoprotein discs (rHDL) formed from RO6 apoA-I, sn-1-palmitoyl-sn-2-oleoyl phosphati-dylcholine, and cholesterol were approximately 12 A smaller than wild-type apoA-I rHDL. The reduced size suggests that one of the repeats did not effectively participate in phospholipid binding and organization. The sn-1-palmitoyl-sn-2-oleoyl phosphatidylcholine RO6 rHDL were a less effective substrate for LCAT. Mapping the entire lipid-free and lipid-bound RO6 apoA-I with a series of monoclonal antibodies revealed that both the lipid-free and lipid-bound RO6 apoA-I displayed altered or absent epitopes in domains within and adjacent to repeat 6. Together, these results suggest that the proper alignment and orientation of the hydrophobic face of repeat 6 is an important determinant for maintaining and stabilizing helix-bilayer and helix-helix interactions.

Lecithin–cholesterol acyltransferase activity in normocholesterolaemic and hypercholesterolaemic roosters: modulation by lipid apheresis

Lipid apheresis, a recently described procedure for the elimination of lipid but not apolipoproteins from plasma, was applied to normocholesterolaemic and hypercholesterolaemic roosters. Lipid apheresis resulted in an immediate reduction in plasma unesterified cholesterol concentration, which was sustained for 150 min. The reduction in unesterified cholesterol concentration was higher in the normocholesterolaemic animals than in the hypercholesterolaemic animals. Lipid apheresis induced changes in the ratio of plasma unesterified to total cholesterol in normocholesterolamic animals but not in hypercholesterolaemic animals. In hypercholesterolaemic animals, lecithin-cholesterol acyltransferase (LCAT) activity was not affected by lipid apheresis, whereas in normocholesterolaemic animals LCAT activity was acutely reduced for 150 min after lipid apheresis. Saturated LCAT kinetics occurred in the hypercholesterolaemic animals but not in the normocholesterolaemic animals. LCAT obeyed Michaelis-Menten kinetics. After lipid apheresis, there was a pool of unesterified cholesterol that was available as substrate for LCAT to a greater extent in hypercholesterolaemic animals than in normocholesterolaemic animals. These observations may have important implications for lipid apheresis as a treatment for atherosclerosis.

Dietary fatty acid modification of HDL phospholipid molecular species alters lecithin: cholesterol acyltransferase reactivity in cynomolgus monkeys.

In the following report, cynomolgous monkeys, fed atherogenic diets containing either saturated, monounsaturated, polyunsaturated (n-6 Poly) or fish oil (n-3 Poly) fat as 35% of total calories, provide a model for the study of dietary fat effects on plasma lipoproteins and atherosclerosis. We have previously described the ability of polyunsaturated fat diets to lower plasma described the ability of polyunsaturated fat diets to lower plasma high density lipoprotein (HDL) cholesterol levels and alter HDL subpopulation distribution in the primate model. These experiments investigate possible mechanisms responsible for such modifications. Animals fed polyunsaturated fat had significantly lower plasma concentrations of HDL cholesterol, total plasma cholesterol, and apolipoprotein A-I. Such changes were reflected in the distribution of protein among HDL subfractions, with the most remarkable modification in subclass distribution being the preponderance of small HDL particles in the n-3 Poly-fed animals. Striking alterations were also observed in the distribution of phosphatidylcholine (PC) molecular species (diet effect P < 0.0001 for all major molecular species). Phosphatidylcholine isolated from lipoproteins were used to make recombinant HDL (rHDL) particles. The reaction rate of purified lecithin:cholesterol acyltransferase (LCAT) with particles made from n-3 Poly-derived PC was 50% of that determined using rHDL formed with PC from other dietary groups (P < 0.0001). When the distribution of LCAT-derived rHDL cholesteryl esters was analyzed, LCAT demonstrated little selectivity for certain PC molecular species except in n-3 Poly-derived rHDL where 18:2-containing PC was selectively utilized. These data demonstrate that differences in dietary fat intake can significantly alter HDL PC concentration and molecular species distribution. We suggest that diet-induced alterations in HDL PC molecular species modify the type of cholesteryl esters produced during the LCAT reaction thereby affecting the plasma cholesteryl ester pool. We also propose that dietary n-3 Poly affects cholesteryl ester metabolism in part via LCAT by lowering PC (LCAT substrate) availability, altering the rate of the LCAT reaction, and decreasing HDL cholesterol concentrations; however, n-6 Poly dietary fat effects on HDL concentration appear to be through some mechanism other than LCAT.

Recruitment of cell phospholipids and cholesterol by apolipoproteins A-II 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-II, like apoA-I, can recruit phospholipid and cholesterol from cell membranes, thereby forming nascent apoA-II-specific HDL. ApoA-II and apoA-I were purified from plasma and each was incubated with CHO cells at a concentration of 10 micrograms/ml. Lipid-containing complexes were isolated from the medium in both cases; the composition of the apoA-II- and apoA-I-specific complexes were similar where percent protein, phospholipid, and cholesterol were 35 +/- 3, 38 +/- 2, and 25 +/- 1 for apoA-II, respectively, and 40 +/- 2, 35 +/- 1, and 24 +/- 2 for apoA-I, respectively. On a per mole of apolipoprotein basis, apoA-I recruited significantly more phospholipid and cholesterol than dimeric apoA-II suggesting that apoA-I with its greater number of alpha helices binds more lipid. By electron microscopy, nascent apoA-II- and apoA-I-specific particles were predominantly discoidal in morphology. ApoA-II 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 lecithin:cholesterol acyltransferase (LCAT) were transformed into predominantly 8.4 nm particles. The latter is similar in size to plasma HDL3a, LpA-I particles, suggesting that extracellularly assembled apoA-I-lipid complexes can directly give rise to a major plasma LpA-I subpopulation upon interaction with LCAT. Unlike apoA-I, apoA-II-lipid complexes could not serve as substrates for LCAT and did not undergo transformation. This study also demonstrates, for the first time, that apoA-II and apoA-I show a preference in phospholipid recruitment from membranes. Although phosphatidylcholine is the major phospholipid removed by both apolipoproteins, apoA-II 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 apoA-II, compared with apoA-I, can penetrate farther into the membrane and extract PE. This ability of apoA-II to insert more deeply into the lipid milieu may explain the known ability of apoA-II to resist dissociation from the mature HDL particle.

Reactivity of lecithin-cholesterol acyl transferase (LCAT) towards glycated high-density lipoproteins (HDL)

Hyperglycaemia in diabetic patients results in non-enzymatic glycation of plasma proteins, including lipoproteins such as high-density lipoproteins (HDL). We studied the effects of in vitro HDL glycation on the activity of lecithin-cholesterol acyl transferase (LCAT), a key enzyme in HDL plasma metabolism. LCAT was prepared from non-diabetic subjects and HDL by sequential density ultracentrifugation (in the density range of 1.063–1.21 g/ml) from both diabetic and non-diabetic patients. HDL from non-diabetic patients were glycated in vitro by incubating lipoproteins with 100 mmol/l glucose for various times at 37°C with sodium cyanoborohydride as reducing agent. Glycation of HDL protein was quantified by measuring the percentage of derived amino acid residues using the TNBS assay. Kinetic parameters of LCAT were first determined using native HDL from non-diabetic patients and in vitro glycated HDL. With native HDL, K m and V max were 51.1 ± 4.2 μmol/l ( n = 8) and 12.9 ± 2.4 nmol/ml/h ( n = 8), respectively. Enzyme reactivity, calculated as the V max/ K m ratio, was 0.25 ± 0.04 h −1 ( n = 8). In the case of moderate glycation (derived residues < 30%; n = 19) a significant increase in both K m (18.2 ± 3.4%; mean ± S.D.) and V max (9.3 ± 2.4%) was observed. In contrast, with a high level of glycation (derived residues > 30%; n = 8), both parameters fell ( K m, 25 ± 6.3%; V max, 34.1 ± 3.3%). In addition, whatever the level of glycation, enzyme reactivity was lower in the presence of in vitro glycated HDL. This decrease in LCAT reactivity was not due to a peroxidative process nor to an alteration of the protein and lipid composition of in vitro glycated HDL. It could, however, be explained by glycation of lysine residues in apolipoprotein A-I, which is the most potent activator of LCAT. In a second series of experiments, native diabetic HDL preparations were used as LCAT substrate. No alteration in K m values was observed, but there was a significant decrease in both V max (28%) and enzyme reactivity (32%). This difference in K m and V max alterations between native diabetic HDL and in vitro glycated HDL with low levels of glycation might be explained by the impact of physiological modifications, other than glycation, which could differently affect the chemicophysical properties of HDL in diabetic patients. In conclusion, the decrease in LCAT reactivity observed with both native diabetic HDL and in vitro glycated HDL could affect the reverse cholesterol transport of HDL and thereby contribute to the atherosclerotic process in diabetic patients.

Incorporation of dietary n−3 fatty acids into molecular species of phosphatidyl choline and cholesteryl ester in normal human plasma

To understand the differences in the antiatherogenic actions of eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6), we determined their incorporation into molecular species of phosphatidylcholine (PC) and cholesteryl ester (CE) after feeding 12 g marine lipid concentrate/d to six normolipidemic males for 28 d. The time course of incorporation of EPA into plasma PC and CE showed a precursor-product relationship. In contrast, the DHA concentration of CE was markedly lower than that in PC, and the EPA-DHA ratio was 2-6-fold higher in CE than in PC at all time intervals. Three PC species--16:0-20:5, 16:0-22:6, and 18:0-20:5--increased, whereas 18:1-18:2, 18:0-18:2, and 16:0-20:3 decreased. In vitro formation of CE species in plasma by lecithin-cholesterol acyltransferase (LCAT) showed an increased formation of 20:5 CE but not 22:6 CE, indicating that DHA is a poor substrate for LCAT. These results demonstrate a differential incorporation of EPA and DHA into plasma lipids, which may be related to the differences in their biological effects.

Site-directed mutagenesis and structure-function analysis of the human apolipoprotein A-I. Relation between lecithin-cholesterol acyltransferase activation and lipid binding.

We have mutagenized the human apoA-I gene and have generated cell lines which express normal and mutant apoA-I forms. Point mutations were introduced which changed Gln-1, Gln-2 to Arg,Arg, Pro99 to His, and Pro121 to His. In addition, the following amino acid deletions (delta) were generated: delta 113-124, delta 148-186, delta 212-233, and delta 213-243. The apoA-I form isolated from the culture medium of C127 cells was analyzed for its ability to activate lecithin-cholesterol acyltransferase (LCAT) and to bind to phospholipid vesicles and high density lipoprotein (HDL). Compared with the wild type (WT) apoA-I, the relative activation of LCAT achieved by the point mutations Gln-1, Gln-2----Arg,Arg, Pro99----His, and Pro121----His were 106 +/- 7, 92 +/- 6, and 77 +/- 9%, respectively. Kinetic analysis of one mutant apoA-I form showed similar Vmax but a 15-fold increase in the Km of the mutant apoA-I form. Furthermore, the activation achieved by the internal deletion mutants delta 113-124, delta 148-186, delta 212-233, and delta 213-243 was 47 +/- 3, 0.5 +/- 0.4, 28 +/- 4 and 13 +/- 5%, respectively. Mutants deficient in their ability to activate LCAT displayed alterations in liposome and HDL binding, compared with WT as determined by density gradient ultracentrifugation analysis of the culture medium. Thus, the peak recovery (approximately 50%) of apoA-I bound to HDL was at density 1.14 g/ml for the WT apoA-I, at 1.18 g/ml for the mutants delta 113-124 and delta 148-186, and at d greater than 1.21 g/ml for the delta 212-233 and delta 213-243. Electron microscopy of the proteoliposome LCAT substrate generated by WT and mutant apoA-I forms showed that the carboxyl-terminal deletion mutants which displayed aberrant binding to HDL also displayed reduced ability to convert the spherical lecithin-cholesterol vesicles into discs compared with WT. The findings suggest that (a) the importance of the carboxyl terminus of apoA-I for LCAT activation is related to its ability to bind to lipid and/or to form discoidal substrate for LCAT, and (b) the interaction of several domains of apoA-I are required for the activation of LCAT.

Age related changes in the lipoprotein substrates for the esterification of plasma cholesterol in rats

The activity of the enzyme lecithin:cholesterol acyltransferase (LCAT) and the properties of its lipoprotein substrates have been investigated in 6- and 19-month-old Fischer-344 rats. These studies were carried out to determine the nature of the relationship between the observed hypercholesterolemia and the age-related decrease in the fractional rate of lipoprotein cholesterol esterification. The distribution of LCAT activity of plasma fractions was determined following gel chromatography and ultracentrifugation respectively. LCAT activity was found to be associated with the high density lipoprotein (HDL) fraction when rat plasma was passed through a Bio-Gel A-5 M column. Upon density gradient ultracentrifugation for 24 h it was found associated with HDL fraction; d = 1.125–1.21 g/ml. However, following prolonged ultracentrifugation (40 h), the majority of the LCAT activity was displaced into the lipoprotein-free infranatant (d > 1.225 g/ml). The dissociation of LCAT from its complex with HDL occurred to a smaller extent in aged rat plasma than in young rat plasma. Substrate specificity studies indicated that HDL was a considerably better substrate for LCAT than very low density lipoproteins (VLDL) in both young and aged rats. In addition, HDL from young rats was a better substrate for LCAT than the HDL from aged rats. Incubation experiments followed by the isolation of lipoproteins and the subsequent analyses of their cholesterol contents revealed that the age-related hypercholesterolemia was mainly due to an increase in the cholesterol carried by lipoprotein fractions d = 1.025–1.07 g/ml (LDL + HDL 1). These and other low density lipoproteins d < 1.025 g/ml) were poor substrates for LCAT. However, these lipoproteins could provide free cholesterol for esterification by first transferring it to HDL (d = 1.07–1.21). The HDL isolated from the plasma of aged rats was enriched with apolipoprotein (apo) E and these lipoprotein particles were found to be inferior substrates for LCAT. These data suggest that the decreased fractional rate of esterification observed in aged rats is due to the slower utilization of the HDL lipid substrate pool by the enzyme LCAT as a result of the accumulation of unfavorable substrates (compositionally altered HDL particles) for the LCAT reaction.

An investigation of the role of lecithin: cholesterol acyltransferase and triglyceride-rich lipoproteins in the metabolism of pre-beta high density lipoproteins

Small high density lipoproteins (HDL) with pre-beta electrophoretic mobility (pre-beta HDL) have recently been shown to be the primary acceptor of cholesterol from cultured cells. We studied the metabolism of these particles by incubating serum at 37°C in the presence and absence of active lecithin: cholesterol acyltransferase (LCAT). We found that the serum pre-beta HDL concentration decreased in the presence of LCAT, but when LCAT was inhibited the concentration remained constant, or increased, depending on the method of inhibition. This suggests that pre-beta HDL are a substrate for LCAT. We also found a significant negative correlation between levels of LCAT activity and pre-beta HDL in 28 fasting healthy subjects, this provides evidence that the activity of LCAT regulates, at least in part the concentration of these particles in vivo. During the early phase of incubation there was a more rapid decrease in pre-beta HDL concentration which was greater in the post-prandial than fasting state. When we infused a triglyceride emulsion into 6 subjects or added this to serum in vitro we observed an immediate fall in pre-beta HDL concentration. These findings suggest that pre-beta HDL interact with triglyceride rich particles. We investigated the origin of pre-beta HDL from blood lipoproteins during their lipolysis, in vivo and in vitro and found that they were produced from both triglyceride-rich and high-density lipoproteins. Formation from triglyceride-rich lipoproteins was evident by the rise in pre-beta HDL concentration during heparin-induced lipolysis when fasting and post-prandially. The rise was greater post-prandially and particularly marked in 4 hypertriglyceridaemic patients following a fat load. Generation from alpha-HDL was evident when we prolonged the action of the heparin-released lipases by incubation of post-heparin sera at 37°C. Continued formation of pre-beta HDL occurred at an equal rate in the fasting and post-prandial samples suggesting release by lipolysis of alpha-HDL. This was supported by the action of lipases on serum and isolated HDL in vitro, where triglyceride lipase rather than phospholipase activity appeared more effective at releasing pre-beta HDL. These findings suggest binding and release of pre-beta HDL by triglyceride-rich lipoproteins depending on the prandial state and production from alpha-HDL through the action of lipases.

Inhibition of cholesteryl ester transfer protein activity by monoclonal antibody. Effects on cholesteryl ester formation and neutral lipid mass transfer in human plasma.

We have employed a neutralizing monoclonal antibody, prepared against the Mr 74,000 cholesteryl ester transfer protein (CETP), to investigate the regulation of lecithin:cholesterol acyltransferase (LCAT) activity by cholesteryl ester (CE) transfer, and also to determine which lipoproteins are substrates for LCAT in human plasma. The incubation of normolipidemic plasma led to transfer of CE from HDL to VLDL, and of triglycerides from VLDL to LDL and HDL. This net mass transfer of neutral lipids between the lipoproteins was eliminated by the monoclonal antibody. However, CE transfer inhibition had no effect on the rate of plasma cholesterol esterification in plasma incubated from 10 min to 24 h at 37 degrees C. In the absence of CE transfer, HDL and LDL exhibited cholesterol esterification activity, whereas VLDL did not. The rate of CE formation in HDL was three to four times greater than in LDL during the first hour of incubation, but CE formation in HDL decreased after 6-8 h, while that in LDL continued. Thus, (a) the Mr 74,000 CETP is responsible for all neutral lipid mass transfer in incubated human plasma, (b) the rate of CE formation in plasma is not regulated by CE transfer from HDL to other lipoproteins, and (c) HDL is the major initial substrate for LCAT; LDL assumes a more significant role only after prolonged incubation of plasma.

Cholesterol esterification by lecithin-cholesterol acyltransferase in A-I-free plasma

Lecithin-cholesterol acyltransferase (LCAT) mass, activity and endogenous cholesterol esterification rate were measured in plasma and apolipoprotein A-I-free (A-I-free) plasma from two normolipidemic and two hyperlipidemic subjects, and from a patient with Tangier disease. A-I was removed from plasma by an anti-A-I immunosorbent. LCAT activity was measured using an exogenous substrate. The plasma LCAT concentration of the four non-Tangier subjects was 4.63 ± 0.64 μg/ ml (mean ± S.D.); means of 26 ± 7% of total LCAT mass and 22 ± 11% of plasma LCAT activity were found in their A-I-free plasma. The plasma LCAT concentration of the Tangier subject was 1.49 μg/ml. About 95% of LCAT mass and all LCAT activity were found in the A-I-free plasma. Thus, the LCAT mass (1.4 μg/ml) and activity (43.1 nmol/h per ml) in Tangier A-I-free plasma were not significantly different from that found in the four non-Tangier A-I-free plasmas (mass = 1.21 ± 0.44 μg/ ml; activity: 27.3 ± 18.4 nmol/h per ml). Although the LCAT activity per unit mass of the enzyme in plasma and A-I-free plasma were comparable (24.9 ± 2.8 vs. 22.8 ± 7.8 nmol/h per μg LCAT, n = 5), the plasma cholesterol esterification rate of A-I-free plasma from all subjects was lower than that found in plasma (7.5 ±2.7 vs. 13.0 ±3.8 nmol/h per μg LCAT). In conclusion, although A-I-containing lipoproteins are the preferred substrates of LCAT, other LCAT substrates and cofactors are found in A-I-free plasma along with LCAT. Thus, non-A-I-containing particles can serve as physiological substrates for cholesterol esterification mediated by LCAT.

Sphingomyelin liposomes with defined fatty acids: metabolism and effects on reverse cholesterol transport

Small unilamellar liposomes prepared from sphingomyelins with defined 14C-labeled fatty acids were studied after injection into rats. The liposomes contained trace amounts of [ 3H]cholesteryl linoleyl ether (CLE), which served as a nonexchangeable and nonhydrolyzable marker. The liposomes were cleared from the circulation with an initial t 1 2 of about 90 min. [ 14 C]18:0- and [ 14 C]18:1- containing sphingomyelins were cleared at a similar rate, but [ 14 C]18:2- sphingomyelin disappeared much faster. The liver accounted for up to 70% of [ 3H]cholesteryl ether injected with 18:0-sphingomyelin liposomes, and for up to 50% with liposomes prepared from 18:1 or 18:2-sphingomyelin. The initial uptake of the liver appeared to be of the entire particle, and the loss of 14C label with time indicated metabolism of the sphingomyelins. With [ 14 C] 18:0- sphingomyelin liposomes, up to 8% of liver radioactivity was recovered in neutral lipids 6 h after injection, and this value was 17 and 22% with [ 14 C]18:2- and [ 14 C]18:1- sphingomyelins , respectively. The recovery in ‘carcass’ of [ 3H]cholesteryl ether 3 h after injection of [ 14 C] 18:2- sphingomyelin liposomes was 33%, and of 14C label, 21%. Injection of 18:1- or 18:2-sphingomyelin liposomes (5.4 μmol/100 g body weight) resulted in a 2-fold increase of plasma unesterified cholesterol; a 30% increase was seen with 18:0 liposomes (2.63 μmol/100 g body weight). In experiments with cultured cells, the unsaturated sphingomyelin liposomes alone enhanced cholesterol efflux more extensively than the saturated ones, but their efficacies became similar when mixed with apoprotein (apo) A-I. At equimolar concentration, apo C-III 1 or C-III 2 had a smaller effect than apo A-I. It is concluded that 18:1- or 18:2-sphingomyelin tends to form small unilamellar liposomes which may reach also extrahepatic tissues. The liposomes are able to enhance cholesterol release in vitro and in vivo. Since they are not a substrate for lecithin-cholesterol acyltransferase, they should be able to deliver the free cholesterol to the liver, where they are also rapidly metabolized.

Lecithin: cholesterol acyltransferase and its action on different substrates.

Lecithin : cholesterol acyltransferase (LCAT) is synthesized by the liver and is responsible for the production of most of the cholesteryl esters (CE) transported in serum of mahxand of many animals (1). HDL, especially HDL3 are assumed to represent the main substrate for LCAT, and apoA-I was found to be a necessary cofactor for the LCAT reaction (2). In recent publications we could demonstrate that the properties of LCAT purified from either pig or human plasma are similar. Using liposomes consisting of phosphatidylcholine (PC) and unesterified 3 H-cholesterol (FC) as substrate the enzymes of both species esterified FC at the same rate, if the molar PC/FC ratio was more than 3:1. The esterification rate was significantly reduced, if the molar ratio of PC/FC was below 3:1 (3). Since such differences in the molar PC/FC ratios of HDL™ and LDL-fractions may play a role in the substrate properties of these lipoproteins, we further tested the action of LCAT from both species on isolated native HDLor LDL-fractions. Here we could demonstrate that the esterification rate of FC in LDL was 30-40% that in HDL (4). Furthermore, these LDL-fractions became even better substrates, if the molar PC/FC ratio was raised to >3:1 by incubation with liposomes in the presence of cholesteryl ester transfer/exchange protein (CETP).