Lecithin:cholesterol acyltransferase reduces the adverse effects of oxidized low-density lipoprotein while incurring damage itself.

In this study, we tried to determine (a) whether there is any permanent effect of oxLDL on the LCAT molecule and its activator lipoprotein apoA-I, and (b) the fate of oxLDL after its exposure to LCAT and apoA-I. Purified LCAT and LDL/oxLDL was incubated at 37 degrees C for 1 h, and separated by gel-permeation chromatography. Its activity was significantly less (30% less) after separating from oxLDL compared to that of LDL. Cofactor activity of isolated apoA-I (incubated with LDL/oxLDL) was found affected by oxLDL. These results indicate modification of the LCAT and apoA-I molecule. But LCAT was found more affected compared to apoA-I in terms of LCAT activity. We are also reporting a novel function of LCAT. It was found to reduce the adverse effects of oxLDL, for example, ability to affect the LCAT activity and TBARS value. This ability of LCAT to repair oxLDL was dose-dependent. But there was no change in its REM values or fluorescence intensity.     

Detoxification of oxidized LDL by transferring its oxidation product(s) to lecithin:cholesterol acyltransferase

In the present study, we isolated modified LCAT (m-LCAT) by hydroxyapatite column chromatography after incubation of crude LCAT (after DEAE SephadexA-50 column chromatography, penultimate step of LCAT purification) with oxidized LDL (oxLDL) at 37 degrees C for 1 h. The activity was found to be about 30% lower than that of native LCAT (n-LCAT). When activity was determined in the presence of oxLDL, m-LCAT was less inhibited than n-LCAT by oxLDL. Treatments of purified LCAT either at 56 degrees C for 30 min, at 100 degrees C for 10 min, or with 6 mM 5-5' -dithiobis-2-nitrobenzoic acid or 9 mM diisopropyl fluorophosphates (each at 37 degrees C for 30 min) resulted in the loss of its cholesterol-esterifying activity. When examined for their ability to detoxify oxLDL, native LCAT and LCAT treated at 56 degrees C for 30 min were found to detoxify oxLDL. These results indicate that oxidation product(s) of LDL is transferred and bound to LCAT in a way that does not depend on its cholesterol-esterifying activity, but rather on the availability of the sulfhydryl group of cysteine residue and the hydroxyl group of serine residue.     

Apolipoprotein E is the major physiological activator of lecithin-cholesterol acyltransferase (LCAT) on apolipoprotein B lipoproteins

Our previous studies have indicated that lecithin-cholesterol acyltransferase (LCAT) contributes significantly to the apoB lipoprotein cholesteryl ester (CE) pool. Cholesterol esterification rate (CER) in apoA-I(-)(/)(-) apoE(-)(/)(-) mouse plasma was <7% that of C57Bl/6 (B6) mouse plasma, even though apoA-I(-)(/)(-) apoE(-)(/)(-) plasma retained (1)/(3) the amount of B6 LCAT activity. This suggested that lack of LCAT enzyme did not explain the low CER in apoA-I(-)(/)(-) apoE(-)(/)(-) mice and indicated that apoE and apoA-I are the only major activators of LCAT in mouse plasma. Deleting apoE on low-density lipoprotein (LDL) reduced CER (1% free cholesterol (FC) esterified/h) compared to B6 (6% FC esterified/h) and apoA-I(-)(/)(-) (11% FC esterified/h) LDL. Similar sized LDL particles from all four genotypes were isolated by fast protein liquid chromatography (FPLC) after radiolabeling with [(3)H]-free cholesterol (FC). LDLs (1 microg FC) from each genotype were incubated with purified recombinant mouse LCAT; LDL particles from B6 and apoA-I(-)(/)(-) plasma were much better substrates for CE formation (5.7% and 6.3% CE formed/30 min, respectively) than those from apoE(-)(/)(-) and apoE(-)(/)(-) apoA-I(-)(/)(-) plasma (1.2% and 1.1% CE formed/30 min). Western blot analysis showed that the amount of apoA-I on apoE(-)(/)(-) LDLs was higher compared to B6 LDL. Adding apoE to incubations of apoA-I(-)(/)(-) apoE(-)(/)(-) very low density lipoprotein (VLDL) resulted in a 3-fold increase in LCAT CER, whereas addition of apoA-I resulted in a more modest 80% increase. We conclude that apoE is a more significant activator of LCAT than apoA-I on mouse apoB lipoproteins.     

Action of lecithin-cholesterol acyltransferase on low-density lipoproteins in native pig plasma

The action of lecithin-cholesterol acyltransferase (LCAT, EC 2.3.1.43) on the different pig lipoprotein classes was investigated with emphasis on low-density lipoproteins (LDL). It was demonstrated previously that LDL can serve as substrate for LCAT, probably because they contain sufficient amounts of apoA-I and other non-apoB proteins, known as LCAT activators. Upon a 24-h incubation of pig plasma in vitro in the presence of active LCAT, both pig LDL subclasses, LDL-1 and LDL-2, fused together, forming one fraction, as revealed by analytical ultracentrifugation. This fusion was time dependent, becoming visible after 3 h and complete after 18 h of incubation. Concomitantly, free cholesterol and phospholipids decreased and cholesteryl esters increased. When isolated LDL-1 and LDL-2 were incubated with purified pig LCAT for 24 h, LDL-1 floated toward higher densities and LDL-2 toward lower densities, although this effect was not as pronounced as in incubations of whole serum. In further experiments, pig serum was incubated for various periods of time in the presence and absence of the LCAT inhibitor sodium iodoacetate. The individual lipoproteins then were separated by density gradient ultracentrifugation or by specific immunoprecipitation and chemically analyzed. Both methods revealed that in the absence of active LCAT there was a transfer of free cholesterol from LDL to high-density lipoproteins (HDL) and a small transfer of cholesteryl esters in the opposite direction. In the presence of LCAT the loss of free cholesterol started immediately in all three lipoprotein classes, was most prominent in LDL, and was proportional to the newly synthesized cholesteryl esters incorporated in each fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of reconstituted high density lipoprotein structure and remodeling by apolipoprotein E

Apolipoprotein E (apoE) enters the plasma as a component of discoidal HDL and is subsequently incorporated into spherical HDL, most of which contain apoE as the sole apolipoprotein. This study investigates the regulation, origins, and structure of spherical, apoE-containing HDLs and their remodeling by cholesteryl ester transfer protein (CETP). When the ability of discoidal reconstituted high density lipoprotein (rHDL) containing apoE2 [(E2)rHDL], apoE3 [(E3)rHDL], or apoE4 [(E4)rHDL] as the sole apolipoprotein to act as substrates for LCAT were compared with that of discoidal rHDL containing apoA-I [(A-I)rHDL], the rate of cholesterol esterification was (A-I)rHDL > (E2)rHDL approximately (E3)rHDL > (E4)rHDL. LCAT also had a higher affinity for discoidal (A-I)rHDL than for the apoE-containing rHDL. When the discoidal rHDLs were incubated with LCAT and LDL, the resulting spherical (E2)rHDL, (E3)rHDL, and (E4)rHDL were larger than, and structurally distinct from, spherical (A-I)rHDL. Incubation of the apoE-containing spherical rHDL with CETP and Intralipid(R) generated large fusion products without the dissociation of apoE, whereas the spherical (A-I)rHDLs were remodeled into small particles with the formation of lipid-poor apoA-I. In conclusion, i) apoE activates LCAT less efficiently than apoA-I; ii) apoE-containing spherical rHDLs are structurally distinct from spherical (A-I)rHDL; and iii) the CETP-mediated remodeling of apoE-containing spherical rHDL differs from that of spherical (A-I)rHDL.     

Localization of an apolipoprotein A-I epitope critical for activation of lecithin-cholesterol acyltransferase.

Apolipoprotein (apo) A-I, the major apoprotein of human high density lipoprotein, is a vital cofactor for lecithin-cholesterol acyltransferase (LCAT), the plasma enzyme responsible for esterification of free cholesterol associated with high density lipoprotein. This esterification is an important component of the reverse cholesterol transport process. An immunochemical approach was used to test the hypothesis that a discrete region of apoA-I was important for LCAT activation. Three human apoA-I-specific monoclonal antibodies were found to inhibit LCAT activation in vitro in a manner directly proportional to their ability to bind to apoA-I-proteoliposomes in fluid phase immunoassays. This relationship was not observed with another four apoA-I-specific antibodies that also were able to bind to the apoA-I proteoliposomes. The use of synthetic peptides representing short amino acid sequences of the apoA-I molecule facilitated the identification of discrete but overlapping apoA-I epitopes for those antibodies that interfered with LCAT-mediated cholesterol esterification. These epitopes spanned amino acid residues 95-121 of mature apoA-I. Therefore, this region is most likely involved in the activation of LCAT by apoA-I.     

Apolipophorin III from Hyphantria cunea shows different anti-oxidant ability against LDL oxidation in the lipid-free and lipid-bound state

Insect apolipophorin III (apoLp-III) and human apolipoprotein A-I (apoA-I) are major protein constituents of the lipoprotein system that share various properties. In order to compare the anti-oxidant ability of apoLp-III and apoA-I in the lipid-free and lipid-bound state, both proteins were purified and synthesized individually as a palmitoyloleoyl phosphatidylcholine (POPC)-reconstituted high-density lipoprotein (rHDL) using the same molar ratio. In the lipid-bound state, apoLp-III and apoA-I showed good anti-oxidant activities against copper-mediated LDL oxidation. Furthermore, apoLp-III and apoA-I, in the lipid-bound state, exhibited potent activities in ferric ion-reducing ability (FRA). However, lipid-free apoLp-III lost the anti-oxidant activity and FRA ability in contrast to lipid-free apoA-I. Lipid-free apoA-I treatment prevented the cellular uptake of oxLDL in macrophages, as visualized by oil-red O staining and detection assays for malondialdehyde (MDA) and lipid hydroperoxide (LPO) in the culture media. However, lipid-free apoLp-III did not prevent the uptake of oxLDL. These results indicate that the putative conformational change of apoLp-III during lipid association is critical for the maintenance of anti-oxidant activity and that the physiologic role of apoLp-III may differ when it is in the lipid-free state and the lipid-bound state.     

Coincubation of PON1, APO A1, and LCAT increases the time HDL is able to prevent LDL oxidation

The inhibition of low-density lipoprotein (LDL) oxidation by high-density lipoprotein (HDL) is a major antiatherogenic property of this lipoprotein. This activity is due, in part, to HDL associated proteins. However, whether these proteins interact in the antioxidant activity of HDL is unknown. LDL was incubated with apolipoprotein A1 (apo A1), lecithin:cholesterol acyltransferase (LCAT), and paraoxonase-1 (PON1) alone or in combination, in the presence or absence of HDL under oxidizing conditions. LDL lipid peroxide concentrations were determined. Apo A1, LCAT, and PON1 all inhibit LDL oxidation in the absence of HDL and enhance the ability of HDL to inhibit LDL oxidation. Their effect was additive rather than synergistic; the combination of these proteins significantly enhanced the length of time LDL was protected from oxidation. This seemed to be due to the ability of PON1 to prevent the oxidative inactivation of LCAT. Apo A1, LCAT, and PON1 can all contribute to the antioxidant activity of HDL in vitro. The combination of apo A1, LCAT, and PON1 prolongs the time that HDL can prevent LDL oxidation, due, at least in part, to the prevention LCAT inactivation.     

Effect of the human plasma apolipoproteins and phosphatidylcholine acyl donor on the activity of lecithin: cholesterol acyltransferase.

The human plasma apoproteins apoA-I and apoC-I enhanced the activity of partially purified lecithin: cholesterol acyltransferase five to tenfold with chemically defined phosphatidylcholine:cholesterol single bilayer vesicles as substrates. By contrast, apoproteins apoA-II, apoC-II, and apoC-III did not give any enhancement of enzyme activity. The activation by apoA-I and apoC-I differed, depending upon the nature of the hydrocarbon chains of phosphatidylcholine acyl donor. ApoA-I was most effective with a phosphatidylcholine containing an unsaturated fatty acyl chain. ApoC-I activated LCAT to the same extent with both saturated and unsaturated phosphatidylcholine substrates. Two of the four peptides obtained by cyanogen bromide cleavage of apoA-I retained some ability to activate LCAT. The efficacy of each of these peptides was approximately 25% that of the whole protein. Cyanogen bromide fragments of apoC-I were inactive. The apoproteins from HDL, HDL2, and HDL3, at low protein concentrations, were equally effective as activators of LCATand less effective than apoA-I. Higher concentrations of apoHDL, apoHDL2, and apoHDL3 inhibited LCAT activity. ApoC and apoA-II were both found to inhibit the activation of LCAT by apoA-I. The inhibition of LCAT by higher concentrations of apoHDL was not correlated with the aopA-II and apoC content.     

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.     

Different Functional and Structural Characteristics between ApoA-I and ApoA-4 in Lipid-Free and Reconstituted HDL State: ApoA-4 Showed Less Anti-Atherogenic Activity

Apolipoprotein A-I and A-IV are protein constituents of high-density lipoproteins although their functional difference in lipoprotein metabolism is still unclear. To compare anti-atherogenic properties between apoA-I and apoA-4, we characterized both proteins in lipid-free and lipid-bound state. In lipid-free state, apoA4 showed two distinct bands, around 78 and 67 Å on native gel electrophoresis, while apoA-I showed scattered band pattern less than 71 Å. In reconstituted HDL (rHDL) state, apoA-4 showed three major bands around 101 Å and 113 Å, while apoA-I-rHDL showed almost single band around 98 Å size. Lipid-free apoA-I showed 2.9-fold higher phospholipid binding ability than apoA-4. In lipid-free state, BS₃-crosslinking revealed that apoA-4 showed less multimerization tendency upto dimer, while apoA-I showed pentamerization. In rHDL state (95:1), apoA-4 was existed as dimer as like as apoA-I. With higher phospholipid content (255:1), five apoA-I and three apoA-4 were required to the bigger rHDL formation. Regardless of particle size, apoA-I-rHDL showed superior LCAT activation ability than apoA-4-rHDL. Uptake of acetylated LDL was inhibited by apoA-I in both lipid-free and lipid-bound state, while apoA-4 inhibited it only lipid-free state. ApoA-4 showed less anti-atherogenic activity with more sensitivity to glycation. In conclusion, apoA-4 showed inferior physiological functions in lipid-bound state, compared with those of apoA-I, to induce more pro-atherosclerotic properties.     

Interconversion between apolipoprotein A-I-containing lipoproteins of pre-beta and alpha electrophoretic mobilities.

Apolipoprotein (apo) A-I-containing lipoproteins can be separated into two subfractions, pre-beta HDL and alpha HDL (high density lipoproteins), based on differences in their electrophoretic mobility. In this report we present results indicating that these two subfractions are metabolically linked. When plasma was incubated for 2 h at 37 degrees C, apoA-I mass with pre-beta electrophoretic mobility disappeared. This shift in apoA-I mass to alpha electrophoretic mobility was blocked by the addition of either 1.4 mM DTNB or 10 mM menthol to the plasma prior to incubation, suggesting that lecithin:cholesterol acyltransferase (LCAT) activity was involved. There was no change in the electrophoretic mobility of either pre-beta HDL or alpha HDL when they were incubated with cholesterol-loaded fibroblasts. However, after exposure to the fibroblasts, the cholesterol content of the pre-beta HDL did increase approximately sixfold, suggesting that pre-beta HDL can associate with appreciable amounts of cellular cholesterol. Pre-beta HDL-like particles appear to be generated by the incubation of alpha HDL with cholesteryl ester transfer protein (CETP) and either very low density lipoproteins (VLDL) or low density lipoproteins (LDL). This generation of pre-beta HDL-like particles was documented both by immunoelectrophoresis and by molecular sieve chromatography. Based on these findings, we propose a cyclical model in which 1) apoA-I mass moves from pre-beta HDL to alpha HDL in connection with the action of LCAT and the generation of cholesteryl esters within the HDL, and 2) apoA-I moves from alpha HDL to pre-beta HDL in connection with the action of CETP and the movement of cholesteryl esters out of the HDL. Additionally, we propose that the relative plasma concentrations of pre-beta HDL and alpha HDL reflect the movement of cholesteryl esters through the HDL. Conditions that result in the accumulation of HDL cholesteryl esters will be associated with low concentrations of pre-beta HDL, whereas conditions that result in the depletion of HDL cholesteryl esters will be associated with elevated concentrations of pre-beta HDL. This postulate is consistent with published findings in patients with hypertriglyceridemia and LCAT deficiency.     

Oxidized low density lipoprotein inhibits macrophage apoptosis through activation of the PI 3-kinase/PKB pathway

Oxidized LDL (oxLDL) is known to induce endothelial adhesion molecule and monocyte chemoattractant protein 1 expression and this is thought to be involved in monocyte recruitment into atherosclerotic lesions. oxLDL has also been found to induce macrophage proliferation. The purpose of the present study was to determine whether oxLDL might also have the ability to increase macrophage populations by inhibiting apoptosis. We found that oxLDL caused a dose-dependent inhibition of the apoptosis that occurs in cultured bone marrow-derived macrophages after macrophage colony-stimulating factor (M-CSF) withdrawal without inducing proliferation. Incubation of macrophages with either native LDL or acetylated LDL had no effect on apoptosis. The prosurvival effect of oxLDL was not inhibited by neutralizing antibodies to granulocyte-macrophage colony-stimulating factor, was maintained in mice homozygous for a mutation in the M-CSF gene, and was not due to other secreted cytokines or growth factors. oxLDL caused activation of the mitogen-activated protein kinases ERK1/2 (extracellular signal-regulated kinases 1 and 2) as well as protein kinase B (PKB), a target of phosphatidylinositol 3-kinase (PI 3-kinase). Furthermore, there was phosphorylation of two important prosurvival PKB targets, I-kappaBalpha(Ser-32) and Bad(Ser-136). The MEK inhibitors PD 98059 and U0126 blocked ERK1/2 activation but did not diminish survival. Conversely, the PI 3-kinase inhibitors LY 294002 and wortmannin blocked PKB activation, and the ability of oxidized LDL to promote macrophage survival.Taken together, these results indicate that oxLDL can directly activate a PI 3-kinase/PKB-dependent pathway that permits macrophage survival in the absence of growth factors.     

Novel N-terminal mutation of human apolipoprotein A-I reduces self-association and impairs LCAT activation

We have identified a novel mutation in apoA-I (serine 36 to alanine; S36A) in a human subject with severe hypoalphalipoproteinemia. The mutation is located in the N-terminal region of the protein, which has been implicated in several functions, including lipid binding and lecithin:cholesterol acyltransferase (LCAT) activity. In the present study, the S36A protein was produced recombinantly and characterized both structurally and functionally. While the helical content of the mutant protein was lower compared with wild-type (WT) apoA-I, it retained its helical character. The protein stability, measured as the resistance to guanidine-induced denaturation, decreased significantly. Interestingly, native gel electrophoresis, cross-linking, and sedimentation equilibrium analysis showed that the S36A mutant was primarily present as a monomer, notably different from the WT protein, which showed considerable oligomeric forms. Although the ability of S36A apoA-I to solubilize phosphatidylcholine vesicles and bind to lipoprotein surfaces was not altered, a significantly impaired LCAT activation compared with the WT protein was observed. These results implicate a region around S36 in apoA-I self-association, independent of the intact C terminus. Furthermore, the region around S36 in the N-terminus of human apoA-I is necessary for LCAT activation.     

Synthesis of reconstituted high density lipoprotein (rHDL) containing apoA-I and apoC-III: the functional role of apoC-III in rHDL

Apolipoprotein (apo) C-III is a marker protein of triacylglycerol (TG)-rich lipoproteins and high-density lipoproteins (HDL), and has been proposed as a risk factor of coronary heart disease. To compare the physiologic role of reconstituted HDL (rHDL) with or without apoC-III, we synthesized rHDL with molar ratios of apoA-I:apoC-III of 1:0, 1:0.5, 1:1, and 1:2. Increasing the apoC-III content in rHDL produced smaller rHDL particles with a lower number of apoA-I molecules. Furthermore, increasing the molar ratio of apoC-III in rHDL enhanced the surfactant-like properties and the ability to lyse dimyristoyl phosphatidylcholine. Furthermore, rHDL containing apoC-III was found to be more resistant to particle rearrangement in the presence of low-density lipoprotein (LDL) than rHDL that contained apoA-I alone. In addition, the lecithin:cholesterol acyltransferase (LCAT) activation ability was reduced as the apoC-III content of the rHDL increased; however, the CE transfer ability was not decreased by the increase of apoC-III. Finally, rHDL containing apoC-III aggravated the production of MDA in cell culture media, which led to increased cellular uptake of LDL. Thus, the addition of apoC-III to rHDL induced changes in the structural and functional properties of the rHDL, especially in particle size and rearrangement and LCAT activation. These alterations may lead to beneficial functions of HDL, which is involved in anti-atherogenic properties in the circulation.     

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.     

Structural and functional properties of reconstituted high density lipoprotein discs prepared with six apolipoprotein A-I variants

Six apolipoprotein A-I (apoA-I) variants containing the following amino acid changes: Pro3----Arg, Pro4----Arg, Lys107----0 (Lys deletion) Lys107----Met, Pro165----Arg, and Glu198----Lys, and the corresponding normal allele products, were isolated by preparative isoelectric focusing from heterozygous individuals. The apoA-I samples were reconstituted with palmitoyloleoyl phosphatidylcholine (POPC) or dipalmitoyl phosphatidylcholine (DPPC), and small amounts of cholesterol, into discoidal high density lipoprotein (HDL) complexes in order to examine their lipid binding and structural properties as well as their ability to activate lecithin:cholesterol acyltransferase (LCAT). Starting with initial molar ratios around 100:5:1 for phosphatidylcholine-cholesterol-apolipoprotein, all the normal and variant apoA-Is were completely incorporated into reconstituted HDL (rHDL). The rHDL particle sizes and their distributions were examined by nondenaturing gradient gel electrophoresis, before and after incubation with LDL, to assess the folding of apoA-I in the complexes. Intrinsic Trp fluorescence properties of the rHDL were measured, as a function of temperature and guanidine hydrochloride concentration, to detect conformational differences in the apoA-I variants. In addition, the LCAT reaction kinetics were measured with all the rHDL, and the apparent kinetic constants were compared. In terms of the structure of the rHDL particles, all the normal variant apoA-Is had similar sizes (94, 96 A) and size distributions, and indistinguishable fluorescence properties, with the exception of the Lys107----0 mutant. This variant formed slightly larger particles that were resistant to rearrangements in the presence of LDL, and had an altered apoA-I conformation in the vicinity of the Trp residues. The kinetic experiments with LCAT indicated that the apoA-I variants, Lys107----0 and Pro165----Arg, in rHDL particles had statistically different (30 to 90%) kinetic constants from the corresponding normal allele products; however, the variability in the kinetic constants among the normal apoA-I products was even greater (40 to 430%). Therefore, we conclude that the effects of these six mutations in apoA-I on the activation of LCAT are minor, and that the structural effects on rHDL, and possibly native HDL, are insignificant with the exception of the Lys107----0 mutation.     

Single repeat deletion in ApoA-I blocks cholesterol esterification and results in rapid catabolism of delta6 and wild-type ApoA-I in transgenic mice

The deletion mutation Delta6 apolipoprotein A-I lacks residues 143-164 or repeat 6 in the mature apoA-I protein. In vitro studies show this mutation dramatically reduces the rate of lecithin:cholesterol acyltransferase (LCAT) catalyzed cholesterol esterification. The present study was initiated to investigate the effect of this mutation on in vivo high density lipoprotein (HDL) cholesterol esterification and metabolism. Transgenic mice expressing human Delta6 apoA-I (TgDelta6 +/+) were created and then crossed with apoA-I knockout mice (-/-) to generate mice expressing only human Delta6 apoA-I (TgDelta6 -/-). Human Delta6 apoA-I was associated with homogeneous sized alpha-HDL, when wild-type mouse apoA-I was present (in TgDelta6 +/+ and +/- mice). However, in the absence of endogenous mouse apoA-I, Delta6 apoA-I was found exclusively in cholesterol ester-poor HDL, and lipid-free HDL fractions. This observation coincides with the 6-fold lower cholesterol ester mass in TgDelta6 -/- mouse plasma compared with control. Structural studies show that despite the structural perturbation of a domain extending from repeat 5 to repeat 8 (137-178), Delta6 apoA-I binds to spherical unilamellar vesicles with only 2-fold less binding affinity. In summary, these data show a domain corresponding to apoA-I repeat 6 is responsible for providing an essential conformation for LCAT catalyzed generation of cholesterol esters. Deletion of apoA-I repeat 6 not only blocks normal levels of cholesterol esterification but also exerts a dominant inhibition on the ability of wild-type apoA-I to activate LCAT in vivo.     

A zebrafish model for the rapid evaluation of pro-oxidative and inflammatory death by lipopolysaccharide, oxidized low-density lipoproteins, and glycated high-density lipoproteins

Oxidation and inflammation are leading causes of nearly all chronic metabolic disorders, and play major roles in cardiovascular disease, cancer, and chronic age-dependent disease. High-density lipoprotein (HDL) and apolipoprotein (apo) A-I have strong antioxidant and anti-inflammatory properties in the plasma. Fructose-induced non-enzymatic glycation of apoA-I can lead to the production of dysfunctional apoA-I and HDL. To compare the physiologic effects of dysfunctional apoA-I and HDL, reconstituted HDL containing native apoA-I (nA-I) or glycated apoA-I (gA-I) was injected into zebrafish embryos in the presence of inflammatory molecules. Co-injection of reconstituted HDL containing VLDL and LDL gA-I (gA-I-rHDL) and lipopolysaccaride (LPS) resulted in acute embryo deaths, while rHDL containing nA-I (nA-I-rHDL) and LPS resulted in significantly enhanced survival. Co-injection of oxidized LDL (oxLDL) and nA-I-rHDL improved embryo survival, while co-injection of oxLDL and gA-I-rHDL aggravated inflammatory deaths. Furthermore, co-injection of oxLDL and HDL(2) (5 ng of protein) or HDL(3) (15 ng of protein) from the young group (22 ± 2 years old) showed significantly increased embryo survival compared with the same co-injection of HDL from the elderly group (71 ± 4 years old). In conclusion, our assay system provides a rapid and economic method to screen antioxidant and anti-inflammatory agents using zebrafish embryos.     

Molecular species of cholesteryl esters formed in abetalipoproteinemia: effect of apoprotein B-containing lipoproteins

In order to study the effects of very low density (VLDL) and low density (LDL) lipoproteins on the activity and specificity of lecithin:cholesterol acyltransferase (LCAT), we determined the molecular species of cholesteryl esters (CE) synthesized in the plasma from three abetalipoproteinemic (ABL) patients, before and after supplementation with normal VLDL or LDL. The patients' plasma had significantly lower concentration of 18:2 CE and higher concentrations of 16:0 CE and 18:1 CE compared to normal plasma. Incubation of ABL plasma with [4-14C]cholesterol at 37 degrees C and the subsequent analysis of labeled CE formed by high performance liquid chromatography revealed that the major species formed was 16:0 CE (34% of total label), whereas similar incubation of the d greater than 1.063 g/ml fraction of normal plasma resulted in the formation of predominantly 18:2 CE (45% of total label). Addition of normal VLDL or LDL to ABL plasma stimulated the total LCAT activity by 30-80% and normalized the CE species synthesized. The LCAT activity of a normal d greater than 1.063 g/ml fraction also was stimulated by the normal VLDL or LDL, but there was no alteration in the species of CE formed. Most of the CE synthesized was found in the added VLDL or LDL with both ABL and normal plasma, indicating that the CE transfer (CET) activity was not affected in ABL plasma. These results suggest that while the VLDL and LDL are required for the maximal activity of LCAT, the species of CE formed are primarily determined by the molecular species composition of phosphatidylcholine in the plasma.