Increases in the particle size of high-density lipoproteins induced by purified lecithin: cholesterol acyltransferase: effect of low-density lipoproteins

Homogeneous subpopulations of human high-density lipoproteins subfraction-3 (HDL3) have been incubated at 37 degrees C with purified lecithin: cholesterol acyltransferase, human serum albumin and varying concentrations of human low-density lipoproteins (LDL). Changes in HDL particle size and composition during these incubations were monitored. Incubation of HDL3a (particle radius 4.3 nm) in the absence of LDL resulted in an esterification of more than 70% of the HDL free cholesterol after 24 h of incubation. This, however, was sufficient to increase the HDL cholesteryl ester by less than 10% and was not accompanied by any change in particle size. When this mixture was incubated in the presence of progressively increasing concentrations of LDL, which donated free cholesterol to the HDL, the molar rate of production of cholesteryl ester was much greater; at the highest LDL concentration HDL cholesteryl ester content was almost doubled after 24 h and there was an increase in the HDL particle size up to the HDL2 range. In the case of HDL3b (radius 3.9 nm), there were again only minimal changes in particle size in incubations not containing LDL. In the presence of the highest concentration of LDL tested, however, the particles were again enlarged into the HDL2 size range after 24 h incubation. These HDL2-like particles were markedly enriched with cholesteryl ester but depleted of phospholipid and free cholesterol when compared with native HDL2. Furthermore, the ratio of apolipoprotein A-I to apolipoprotein A-II resembled that in the parent-HDL3 and was very much lower than that in native HDL2. It has been concluded that purified lecithin: cholesterol acyltransferase is capable of increasing the size of HDL3 towards that of HDL2 but that other factors must operate in vivo to modulate the chemical composition of the enlarged particles.     

Comparison of human plasma low- and high-density lipoproteins as substrates for lecithin: cholesterol acyltransferase

The interstrand crosslinks that appear in stored depurinated DNA interfere with the counting of apurinic sites and strand breaks by sucrose gradient analysis. They could not be cleaved at acid or alkaline pH, or by treatment with methoxyamine.     

Binding affinity and reactivity of lecithin cholesterol acyltransferase with native lipoproteins.

The first step in the reaction of lecithin cholesterol acyltransferase (LCAT) with lipoproteins is the interfacial binding of the enzyme to the lipid surfaces. In this study the equilibrium dissociation constants (Kds) for the interaction of pure human plasma LCAT with LDL, HDL2, HDL3, and a reconstituted discoidal HDL (rHDL) were determined by the activity-inhibition method. In addition, enzyme kinetics were measured with each of the lipoprotein substrates. Based on phospholipid concentrations, the Kd values (0.9 x 10(-5) to 4.6 x 10(-5) M) increased in the order rHDL = HDL3 相似文献   

In vitro transformation of apoA-I-containing lipoprotein subpopulations: role of lecithin:cholesterol acyltransferase and apoB-containing lipoproteins

Two populations of apoA-I-containing lipoproteins are found in plasma: particles with apoA-II [Lp(AI w AII)] and particles without apoA-II [Lp(AI w/o AII)]. Both are heterogeneous in size. However, their size subpopulation distributions differ considerably between healthy subjects and patients with coronary artery diseases. The metabolic basis for such alterations was studied by determining the role of lecithin:cholesterol acyltransferase (LCAT) and apoB-containing lipoproteins (LpB) in the size subpopulation distributions of Lp(AI w AII) and Lp(AI w/o AII). ApoB-free and LCAT-free plasmas, prepared by affinity chromatography, and whole plasma were incubated at 4 degrees C and 37 degrees C for 24 hr. After incubation, Lp(AI w AII) and Lp(AI w/o AII) were isolated by anti-A-II and anti-A-I immunosorbents. Their size subpopulation distributions were studied by nondenaturing gradient polyacrylamide gel electrophoresis. At 4 degrees C most Lp(AI w AII) particles were in the range of 7.0-9.2 nm Stokes diameter. Incubation of plasma at 37 degrees C resulted in an overall enlargement of particles up to 11.2 nm and larger. These particles were enriched with cholesteryl ester and triglyceride and depleted of phospholipids and free cholesterol. Removal of LpB or LCAT from plasma prior to incubation greatly reduced their enlargement. At 4 degrees C, Lp(AI w/o AII) contained mostly particles of 8.5 and 10.1 nm. Incubation at 37 degrees C abolished both subpopulations with the formation of a new subpopulation of 9.2 nm. This transformation was identical in apoB-free plasma but was not seen in LCAT-free plasma. Our study shows that transformation of Lp(AI w AII) requires both LCAT and LpB. However, LpB is not necessary for the transformation of Lp(AI w/o AII) in vitro. The relevance of these in vitro studies to in vivo lipoprotein metabolism was demonstrated in a subject with hepatic triglyceride lipase deficiency.     

Effect of lecithin:cholesterol acyltransferase on distribution of apolipoprotein A-IV among lipoproteins of human plasma

The effect of cholesterol esterification on the distribution of apoA-IV in human plasma was investigated. Human plasma was incubated in the presence or absence of the lecithin:cholesterol acyltransferase (LCAT) inhibitor 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) and immediately fractionated by 6% agarose column chromatography. Fractions were monitored for apoA-IV, apoE, and apoA-I by radioimmunoassay (RIA). Incubation resulted in an elevated plasma concentration of cholesteryl ester and in an altered distribution of apoA-IV. After incubation apoA-IV eluted in the ordinarily apoA-IV-poor fractions of plasma that contain small VLDL particles, LDL, and HDL2. Inclusion of DTNB during the incubation resulted in some enlargement of HDL; however, both cholesterol esterification and lipoprotein binding of apoA-IV were inhibited. Addition of DTNB to plasma after incubation and prior to gel filtration had no effect on the apoA-IV distribution when the lipoproteins were immediately fractionated. Fasting plasma apoE was distributed in two or three peaks; in some plasmas there was a small peak that eluted with the column void volume, and, in all plasmas, there were larger peaks that eluted with the VLDL-LDL region and HDL2. Incubation resulted in displacement of HDL apoE to larger lipoproteins and this effect was observed in the presence or absence of DTNB. ApoA-I was distributed in a single broad peak that eluted in the region of HDL and the gel-filtered distribution was unaffected by incubation either in the presence or absence of DTNB. Incubation of plasma that was previously heated to 56 degrees C to inactivate LCAT resulted in no additional movement of apoA-IV onto lipoproteins, unless purified LCAT was present during incubation. The addition of heat-inactivated LCAT to the incubation, had no effect on movement of apoA-IV. These data suggest that human apoA-IV redistribution from the lipoprotein-free fraction to lipoprotein particles appears to be dependent on LCAT action. The mechanism responsible for the increased binding of apoA-IV to the surface of lipoproteins when LCAT acts may involve the generation of "gaps" in the lipoprotein surface due to the consumption of substrate from the surface and additional enlargement of the core. ApoA-IV may bind to these "gaps," where the packing density of the phospholipid head groups is reduced.     

Surface plasmon resonance biosensor studies of human wild-type and mutant lecithin cholesterol acyltransferase interactions with lipoproteins

Binding of lecithin cholesterol acyltransferase (LCAT) to lipoprotein surfaces is a key step in the reverse cholesterol transport process, as the subsequent cholesterol esterification reaction drives the removal of cholesterol from tissues into plasma. In this study, the surface plasmon resonance method was used to investigate the binding kinetics and affinity of LCAT for lipoproteins. Reconstituted high-density lipoproteins (rHDL) containing apolipoprotein A-I or A-II, (apoA-I or apoA-II), low-density lipoproteins (LDL), and small unilamellar phosphatidylcholine vesicles, with biotin tags, were immobilized on biosensor chips containing streptavidin, and the binding kinetics of pure recombinant LCAT were examined as a function of LCAT concentration. In addition, three mutants of LCAT (T123I, N228K, and (Delta53-71) were examined in their interactions with LDL. For the wild-type LCAT, binding to all lipid surfaces had the same association rate constant, k(a), but different dissociation rate constants, k(d), that depended on the presence of apoA-I (k(d) decreased) and different lipids in LDL. Furthermore, increased ionic strength of the buffer decreased k(a) for the binding of LCAT to apoA-I rHDL. For the LCAT mutants, the Delta53-71 (lid-deletion mutant) exhibited no binding to LDL, while the LCAT-deficiency mutants (T123I and N228K) had nearly normal binding to LDL. In conclusion, the association of LCAT to lipoprotein surfaces is essentially independent of their composition but has a small electrostatic contribution, while dissociation of LCAT from lipoproteins is decreased due to the presence of apoA-I, suggesting protein-protein interactions. Also, the region of LCAT between residues 53 and 71 is essential for interfacial binding.     

Role of plasma lecithin:cholesterol acyltransferase in the metabolism of high density lipoproteins

The role of the plasma lecithin:cholesterol acyltransferase reaction in the esterification of the cholesterol of human and baboon plasma high density lipoproteins has been studied. Human plasma was incubated in vitro, and the initial rate of cholesterol esterification in lipoprotein fractions obtained by chromatography on hydroxylapatite was determined. The rate of esterification was greater in the high density lipoprotein fraction than in the low density lipoprotein fraction. High density lipoproteins from human and baboon plasma were filtered through columns of Sephadex G 200, and the relative concentrations in the effluent of key lipids involved in the acyltransferase reaction were determined. The ratio of esterified to unesterified cholesterol varied across the lipoprotein peak obtained from either type of plasma. The relative concentration of lecithin compared to sphingomyelin also varied across the peaks obtained with human high density lipoproteins. When human or baboon plasma was incubated with cholesterol-(14)C and the high density lipoproteins were filtered through Sephadex, the specific activity of the esterified cholesterol varied across the lipoprotein peak. Similar results were obtained when plasma esterified cholesterol was labeled in vivo by the injection of labeled mevalonate into baboons. The data suggest that the acyltransferase reaction is the major source of the esterified cholesterol of the high density lipoproteins.     

Evidence that lecithin:cholesterol acyltransferase acts on both high-density and low-density lipoproteins

In incubations of plasma containing lipoproteins at physiological concentrations it has been confirmed that high-density lipoproteins (HDL) are the major initial recipients of the esterified cholesterol formed in the reaction catalysed by lecithin:cholesterol acyltransferase. It has also been confirmed, however, that a small proportion of the esterified cholesterol of lecithin:cholesterol acyltransferase origin is incorporated directly into low-density lipoproteins (LDL), via a pathway that bypasses the HDL. This direct incorporation of esterified cholesterol into LDL is compatible with either of two general models. Model A proposes that lecithin:cholesterol acyltransferase does not interact directly with LDL but rather that it acts only on lipoproteins outside the LDL fraction. According to model A, while most of the esterified cholesterol so formed is incorporated into HDL, a small proportion is transferred directly to LDL. Model B, by contrast, proposes that a direct incorporation of esterified cholesterol into LDL is the result of a direct action of lecithin:cholesterol acyltransferase on the free cholesterol associated with LDL. To differentiate between these two models, experiments have been performed in which incubation mixtures containing LDL, HDL and a source of lecithin:cholesterol acyltransferase were supplemented with free [3H]cholesterol which had previously been incorporated into either LDL or HDL. It was found that, of the esterified [3H]cholesterol which was subsequently formed, the proportion recovered in the LDL fraction was much greater in the incubations to which the free [3H]cholesterol had been added as a component of LDL than in those to which it had been added as a component of HDL. This essentially excluded model A but was consistent with model B. It has been concluded that, while most of the lecithin:cholesterol acyltransferase may interact with particles in the HDL fraction, a small proportion of the enzyme interacts directly with LDL.     

Studies on the substrate specificity of human and pig lecithin: cholesterol acyltransferase: role of low-density lipoproteins

The substrate properties of low-density lipoprotein (LDL) fractions from human and pig plasma and of lipoprotein a [Lp(a)] upon incubation with either pig or human lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) were investigated and compared with those of pig high-density lipoproteins (HDL) or human HDL-3. The cholesterol esterification using purified native pig LDL-1, human LDL, or Lp(a) as a substrate was approximately 36-42% that of pig HDL or human HDL-3, while cholesteryl ester formation with pig LDL-2 was 41-47%. No significant difference was found in the substrate activity between pig HDL and human HDL-3, and between human LDL and Lp(a), respectively. After depletion of pig LDL-1, pig LDL-2, and human LDL from apolipoprotein A-I (apoA-I), cholesteryl ester formation decreased to about 22-28% of the value found with pig HDL. Depletion of human LDL from apolipoprotein E (apoE) did not result in significantly different esterification rates in comparison to native LDL. Total removal of non-apoB proteins from human LDL resulted in esterification rates of approximately 10-15% that of HDL. Readdition of apoA-I to all these LDL fractions produced solely in apoA-I-depleted LDL fractions an increase of cholesteryl ester formation, whereas in those LDL fractions that were additionally depleted from apoE and/or from apoC polypeptides, a further decrease in the esterification rate occurred.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of lecithin: cholesterol acyltransferase by human apolipoprotein A-IV

Human plasma apoproteins (apo) A-I and A-IV both activate the enzyme lecithin:cholesterol acyltransferase (EC 2.3.1.43). Lecithin:cholesterol acyltransferase activity was measured by the conversion of [4-14C] cholesterol to [4-14C]cholesteryl ester using artificial phospholipid/cholesterol/[4-14C]cholesterol/apoprotein substrates. The substrate was prepared by the addition of apoprotein to a sonicated aqueous dispersion of phospholipid/cholesterol/[4-14C]cholesterol. The activation of lecithin:cholesterol acyltransferase by apo-A-I and -A-IV differed, depending upon the nature of the hydrocarbon chains of the sn-L-alpha-phosphatidylcholine acyl donor. Apo-A-I was a more potent activator than apo-A-IV with egg yolk lecithin, L-alpha-dioleoylphosphatidylcholine, and L-alpha-phosphatidylcholine substituted with one saturated and one unsaturated fatty acid regardless of the substitution position. When L-alpha-phosphatidylcholine esterified with two saturated fatty acids was used as acyl donor, apo-A-IV was more active than apo-A-I in stimulating the lecithin:cholesterol acyltransferase reaction. Complexes of phosphatidylcholines substituted with two saturated fatty acids served as substrate for lecithin:cholesterol acyltransferase even in the absence of any activator protein. Essentially the same results were obtained when substrate complexes (phospholipid-cholesterol-[4-14C]cholesterol-apoprotein) were prepared by a detergent dialysis procedure. Apo-A-IV-L-alpha-dimyristoylphosphatidylcholine complexes thus prepared were shown to be homogeneous particles by column chromatography and density gradient ultracentrifugation. It is concluded that apo-A-IV is able to facilitate the lecithin:cholesterol acyltransferase reaction in vitro.     

Identification of the active-site serine in human lecithin: cholesterol acyltransferase

Purified human lecithin:cholesterol acyltransferase (LCAT) was covalently labeled by [3H]diisopropylflourophosphate with concomitant loss of enzymatic activity (M. Jauhiainen and P.J. Dolphin (1986) J. Biol. Chem. 261, 7023-7043). Some 60% of the enzyme was labeled in 1 h. Cyanogen bromide (CNBr) cleavage of the labeled, reduced, and carboxymethylated protein, followed by gel permeation chromatography yielded a 5- to 6-kDa peptide (LCAT CNBr-III) containing at least 60-70% of the incorporated label. Comparison of the amino acid composition of LCAT CNBr-III with that of the CNBr peptides predicted from the LCAT sequence (J. McLean et al. (1986) Proc. Natl. Acad. Sci. USA 83, 2335-2339) indicates that LCAT CNBr-III is peptide 168-220. In 22 cycles of automated Edman degradation of CNBr-III a radioactive derivative was only observed at cycle 14, and of the predicted CNBr fragments only peptide 168-220 contains a serine at position 14 from the amino terminus. Tryptic peptides predicted from the sequence should contain Ser181 at positions 22 and 23 from the N-terminus of fragments 160-199 and 159-199, respectively. On the other hand, Ser216 should be in position 15 from the N-terminus in fragment 202-238. Radiolabel sequencing of the tryptic digest of [3H]diisopropylphosphate-LCAT resulted in recovery of radioactivity in cycles 22 and 23, whereas cycle 15 yielded negligible radioactivity. These results establish that Ser181 is the major active site serine in human LCAT.     

The interaction of apolipoproteins with lecithin:cholesterol acyltransferase

The rate of lecithin:cholesterole acyltransferase reaction was measured in a cholesterol-containing single bilayer lecithin vesicle system. ApolipoproteinA-I (apoA-I) activated the enzyme by itself; the other components of apolipoproteins of high density lipoproteins (HDL) (rho = 1.08--1.2 g/cm3), or rabbit serum gamma globulin inhibited the reaction. The reaction which was activated by pure apoA-I was strongly inhibited by anti-apoA-I antibody. Quantitative analysis of the results showed that the lecithin:cholesterol acyltransferase reaction was activated by the binding of apoA-I to the surface of lipid substrates. The rate of the lecithin:cholesterol acyltransferase-catalyzed reaction was strictly proportional to the surface density of apoA-I. The inhibition was due to the decrease of the amount of apoA-I on the lipid surface, either through competitive exclusion by apoA-II or by other proteins, or through specific extraction with antibody. The presence of components of apoHDL, other than apoA-I, prevented the inhibitory action of anti-apoA-I antibody.     

Synthetic substrates of lecithin: cholesterol acyltransferase

Investigation of the substrate specificity of lecithin: cholesterol acyltransferase has been greatly aided by the use of synthetic particles containing the molecular lipid substrates and the apolipoprotein activators of the enzyme. These synthetic particles, in vesicle or disc-like micelle form, are described in some detail noting their preparation, properties, advantages, and limitations as substrates for lecithin:cholesterol acyltransferase. The reactions of the enzyme with the synthetic particles are reviewed in terms of acyl donor and acceptor specificity, activation by apolipoproteins, effects of various inhibitors, and the kinetics of the reaction.     

Purification of human plasma lecithin:cholesterol acyltransferase by covalent chromatography

Human plasma lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) has been purified more than 20,000 fold from plasma in 10% yield. This new procedure is composed of only four steps, including ultracentrifugation of plasma to yield a 1.21-1.25 kg/l density fraction, covalent binding of LCAT in this fraction to thiopropyl-Sepharose followed by adsorption of the enzyme to wheat-germ lectin-Sepharose for elimination of albumin and finally batch-wise treatment of the desorbed LCAT with hydroxyapatite to remove residual impurities. The purified enzyme was free of apolipoprotein A-I, A-II, B, C-I, C-II, C-III and E as checked by double immunodiffusion and SDS-electrophoresis, which latter method also demonstrated the absence of hitherto characterized lipid transfer proteins. Only traces of apolipoprotein D were present in the preparation as detected by immunoblotting. The purified enzyme retained alpha- and beta-LCAT activities. Non-denaturing and denaturing polyacrylamide gel electrophoresis yielded apparent molecular masses of 69 and 66 kDa, respectively, for the enzyme which on isoelectric focusing produced one major and one minor isoform with pI values of 4.20 and 4.25, respectively. Apolipoprotein A-I was required to transform artificial lecithin-cholesterol liposomes into substrates for the purified LCAT.     

Purification and characterization of human plasma lecithin:cholesterol acyltransferase.

A highly purified (approximately 12 000-fold) homogeneous preparation of human plasma lecithin:cholesterol acyltransferase (LCAT) with 16% yield was obtained by a combination of density ultracentrifugation, high density lipoprotein affinity column chromatography, hydroxylapatite chromatography, and finally chromatography on anti-apolipoprotein D immunoglobulin-Sepharose columns to remove apolipoprotein D. This enzyme preparation was homogeneous by the following criteria: a single band by polyacrylamide gel electrophoresis in 8 M urea; a single band on sodium dodecyl sulfate gel electrophoresis with an apparent molecular weight of 68 000 +/- 1600; a single protein peak with a molecular weight of 70 000 on a calibrated Sephadex G-100 column. Its amino acid composition was different from human serum albumin and all other apoproteins isolated from lipoprotein fractions.     

Distribution and localization of lecithin:cholesterol acyltransferase and cholesteryl ester transfer activity in A-I-containing lipoproteins

Two types of A-I-containing lipoproteins are found in human high density lipoproteins (HDL): particles with A-II (Lp(A-I with A-II] and particles without A-II (Lp(A-I without A-II]. We have studied the distribution of lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer (CET) activities in these particles. Lp(A-I with A-II) and Lp(A-I without A-II) particles were isolated from ten normolipidemic subjects by anti-A-I and anti-A-II immunosorbents. Most plasma LCAT mass (70 +/- 15%), LCAT (69 +/- 16%), and CET (81 +/- 15%) activities were detected in Lp(A-I without A-II). Some LCAT (mass: 16 +/- 7%, activity: 17 +/- 8%) and CET activities (7 +/- 8%) were detected in Lp(A-I with A-II). To determine the size subspecies that contain LCAT and CET activities, isolated Lp(A-I with A-II) and Lp(A-I without A-II) particles of six subjects were further fractionated by gel filtration column chromatography. In Lp(A-I without A-II), most LCAT and CET activities were associated with different size particles, with the majority of the LCAT and CET activities located in particles with hydrated Stokes diameters of 11.6 +/- 0.4 nm and 10.0 +/- 0.6 nm, respectively. In Lp(A-I with A-II), most of the LCAT and CET activities were located in particles similar in size: 11.1 +/- 0.4 nm and 10.6 +/- 0.3 nm, respectively. Ultracentrifugation of A-I-containing lipoproteins resulted in dissociation of both LCAT and CET activities from the particles. Furthermore, essentially all CET and LCAT activities were recovered in the non-B-containing plasma obtained by anti-LDL immunoaffinity chromatography. This report, therefore, provides direct evidence for the association of LCAT and CET protein with A-I-containing lipoproteins. Our conclusions pertain to fasting normolipidemic subjects and may not be applicable to hyperlipidemic or nonfasting subjects.