Influence of hypoxanthine and other purines on the lecithin:cholesterol acyltransferase reaction in the plasma of rat and other species.

1. Esterification of radiolabelled cholesterol in the plasma of rat, mouse, pig, ox and, to a lesser extent, guinea pig was partially inhibited by hypoxanthine, xanthine and guanine; esterification in human plasma and in plasma from 12 other vertebrate species was unaffected by purines. 2. Esterification of endogenous cholesterol and the formation of lysolecithin in rat plasma were decreased in the presence of purines indicating that it was the lecithin:cholesterol acyltransferase (LCAT) reaction that was inhibited rather than the isotopic equilibration of labelled cholesterol with the endogenous substrate lipoproteins. 3. Maximum inhibition of the LCAT reaction in rat plasma occurred at 1.4 mM hypoxanthine or xanthine; inhibition was not dependent upon the concentration of LCAT or plasma lipoproteins but increased with the amount of lipoprotein depleted rat plasma (LDRP) present in the incubation mixture. 4. Partial inhibition of the LCAT reaction in rat or mouse plasma by purines had no significant effect on the fatty acyl composition of the cholesteryl esters (CE) formed by LCAT. 5. In the presence of heated rat plasma, LDRP or, to a lesser extent, rat high density lipoproteins (HDL) prepared from heated plasma, the LCAT reaction in human plasma was inhibited by hypoxanthine. 6. Rat HDL and LDRP prepared from plasma pre-incubated at 37 degrees C for 4 hr before heating increased and decreased, respectively, the inhibitory effect of hypoxanthine on human plasma LCAT compared with HDL and LDRP prepared from unincubated rat plasma.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 plasma lecithin:cholesterol acyltransferase in man. III. Role of high density lipoprotein cholesteryl esters in the activating effect of a high-fat test meal.

Plasma lecithin:cholesterol acyltransferase (LCAT) activity is increased during the clearance phase of alimentary lipemia induced by a high-fat test meal in normal subjects. Ultracentrifugal fractionation of high density lipoproteins (HDL) into HDL(2), HDL(3), and very high density (VHD) subfractions followed by analyses of lipid and protein components has been accomplished at intervals during alimentary lipemia to seek associations with enzyme changes. HDL(2) lipids and protein increased substantially, characterized primarily by enrichment with lecithin. HDL(3), which contain the main LCAT substrates, revealed increased triglycerides and generally reduced cholesteryl esters which were reciprocally correlated, demonstrating a phenomenon previously observed in vitro by others. Both changes correlated with LCAT activation, but partial correlation analysis indicated that ester content is primarily related to triglycerides rather than LCAT activity. The VHD cholesteryl esters and lysolecithin were also reduced. Plasma incubation experiments with inactivated LCAT showed that alimentary lipemic very low density lipoproteins (VLDL) could reduce levels of cholesteryl esters in HDL by a nonenzymatic mechanism. In vitro substitution of lipemic VLDL for postabsorptive VLDL resulted in enhanced reduction of cholesteryl esters in HDL(3) and VDH, but not in HDL(2), during incubation. Nevertheless, augmentation of LCAT activity did not result, indicating that cholesteryl ester removal from substrate lipoproteins is an unlikely explanation for activation. Since VHD and HDL(3), which contain the most active LCAT substrates, were also most clearly involved in transfers of esters to VLDL and low density lipoproteins, the suggestion that LCAT product lipoproteins are preferentially involved in nonenzymatic transfer and exchange is made. The main determinant of ester transfer, however, appears to be the level of VLDL, both in vitro and in vivo. Rose, H. G., and J. Juliano. Regulation of plasma lecithin: cholesteryl acyltransferase in man. III. Role of high density lipoprotein cholesteryl esters in the activating effect of a high-fat test meal.     

Cellular pregnenolone esterification by acyl-CoA:cholesterol acyltransferase

Pregnenolone (PREG) can be converted to PREG esters (PE) by the plasma enzyme lecithin: cholesterol acyltransferase (LCAT), and by other enzyme(s) with unknown identity. Acyl-CoA:cholesterol acyltransferase 1 and 2 (ACAT1 and ACAT2) convert various sterols to steryl esters; their activities are activated by cholesterol. PREG is a sterol-like molecule, with 3-β-hydroxy moiety at steroid ring A, but with much shorter side chain at steroid ring D. Here we show that without cholesterol, PREG is a poor ACAT substrate; with cholesterol, the V(max) for PREG esterification increases by 100-fold. The binding affinity of ACAT1 for PREG is 30-50-fold stronger than that for cholesterol; however, PREG is only a substrate but not an activator, while cholesterol is both a substrate and an activator. These results indicate that the sterol substrate site in ACAT1 does not involve significant sterol-phospholipid interaction, while the sterol activator site does. Studies utilizing small molecule ACAT inhibitors show that ACAT plays a key role in PREG esterification in various cell types examined. Mice lacking ACAT1 or ACAT2 do not have decreased PREG ester contents in adrenals, nor do they have altered levels of the three major secreted adrenal steroids in serum. Mice lacking LCAT have decreased levels of PREG esters in the adrenals. These results suggest LCAT along with ACAT1/ACAT2 contribute to control pregnenolone ester content in different cell types and tissues.     

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.     

Lecithin-cholesterol acyltransferase (LCAT) catalyzes transacylation of intact cholesteryl esters. Evidence for the partial reversal of the forward LCAT reaction

Lecithin-cholesterol acyltransferase (LCAT) catalyzes the intravascular synthesis of lipoprotein cholesteryl esters by converting cholesterol and lecithin to cholesteryl ester and lysolecithin. LCAT is unique in that it catalyzes sequential reactions within a single polypeptide sequence, a phospholipase A2 reaction followed by a transacylation reaction. In this report we find that LCAT mediates a partial reverse reaction, the transacylation of lipoprotein cholesteryl oleate, in whole plasma and in a purified, reconstituted system. As a result of the reverse transacylation reaction, a linear accumulation of [3H]cholesterol occurred during incubations of plasma containing high density lipoprotein labeled with [3H]cholesteryl oleate. When high density lipoprotein labeled with cholesteryl [14C]oleate was also included in the incubation the labeled fatty acyl moiety remained in the cholesteryl [14C]oleate pool showing that the formation of labeled cholesterol did not result from hydrolysis of the doubly labeled cholesteryl esters. The rate of release of [3H]cholesterol was only about 10% of the forward rate of esterification of cholesterol using partially purified human LCAT and was approximately 7% in whole monkey plasma. Therefore, net production of cholesterol via the reverse LCAT reaction would not occur. [3H]Cholesterol production from [3H]cholesteryl oleate was almost completely inhibited by a final concentration of 1.4 mM 5,5'-dithiobis(nitrobenzoic acid) during incubation with either purified LCAT or whole plasma. Addition of excess lysolecithin to the incubation system did not result in the formation of [14C]oleate-labeled lecithin, showing that the reverse reaction found here for LCAT was limited to the last step of the reaction. To explain these results we hypothesize that LCAT forms a [14C]oleate enzyme thioester intermediate after its attack on the cholesteryl oleate molecule. Formation of this intermediate allows [3H]cholesterol to be liberated from the enzyme by exchange with unlabeled cholesterol of plasma lipoproteins. The liberated [3H]cholesterol thereby becomes available for reesterification by LCAT as indicated by its appearance as newly synthesized cholesteryl linoleate.     

Metabolism of low-density lipoprotein free cholesterol by human plasma lecithin-cholesterol acyltransferase

The metabolism of cholesterol derived from [3H]cholesterol-labeled low-density lipoprotein (LDL) was determined in human blood plasma. LDL-derived free cholesterol first appeared in large alpha-migrating HDL (HDL2) and was then transferred to small alpha-HDL (HDL3) for esterification. The major part of such esters was retained within HDL of increasing size in the course of lecithin-cholesterol acyltransferase (LCAT) activity; the balance was recovered in LDL. Transfer of preformed cholesteryl esters within HDL contributed little to the labeled cholesteryl ester accumulating in HDL2. When cholesterol for esterification was derived instead from cell membranes, a significantly smaller proportion of this cholesteryl ester was subsequently recovered in LDL. These data suggest compartmentation of cholesteryl esters within plasma that have been formed from cell membrane or LDL free cholesterol, and the role for HDL2 as a relatively unreactive sink for LCAT-derived cholesteryl esters.     

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)     

Nascent high density lipoproteins from liver perfusates of orotic acid-fed rats

Uniformly fatty livers from orotic acid-fed rats secreted almost no very low density lipoproteins (VLDL) but normal amounts of nascent high density lipoproteins (HDL) accumulated in perfusates. When lecithin:cholesterol acyltransferase (LCAT) was inhibited, nascent HDL were uniformly discoidal and lacked cholesteryl esters. Lipid and apoprotein compositions of nascent HDL from normal and fatty livers were similar whether LCAT was inhibited or not. Apolipoprotein B-100 was not detected in perfusates of uniformly fatty livers, but small amounts of apolipoprotein B-48 were present in HDL2 fractions. Nascent lipoproteins were not seen in Golgi compartments, but lipid-rich particles were clearly evident in endoplasmic reticulum cisternae adjacent to the cis face of the Golgi complex, suggesting that orotic acid blocks VLDL secretion by preventing translocation of nascent particles from the endoplasmic reticulum to the cis Golgi compartment. The accumulation of normal amounts of discoidal HDL in liver perfusates despite virtual absence of triglyceride-rich lipoproteins in Golgi secretory compartments, the space of Disse, and the perfusate is inconsistent with the concept that nascent HDL are exclusively a product of surface remnants cast off during lipolysis of chylomicrons and VLDL.     

Lipid transfers between reconstituted high density lipoprotein complexes and low density lipoproteins: effects of plasma protein factors

In this study we examined the transfer of lipids between reconstituted high density lipoprotein discs (r-HDL) and human low density lipoproteins (LDL) in the presence and absence of lecithin:cholesterol acyltransferase (LCAT) or of plasma phospholipid transfer protein (PLTP). We found that spontaneous transfer of phospholipids from r-HDL to LDL occurred by an apparent first order reaction with a half-time of 5.8 to 6.9 hr depending on the phospholipid. During the time of incubation of r-HDL with LDL (from 0 to 25 hr), the phospholipid content of r-HDL decreased more than 30%, the free cholesterol content increased 2.5-fold, and low levels of cholesteryl esters appeared in r-HDL. These compositional changes gave rise to small discoidal particles with a limiting diameter of 77 A and two molecules of apoA-I per particle. When LCAT was included in the reaction mixture, the r-HDL lost even more phospholipid, lost some free cholesterol, and gained cholesteryl esters relative to the apolipoprotein content, due to the enzymatic reaction. The products of the LCAT reaction had a diameter of 93 A and three, rather than two, apoA-I molecules per particle. Inclusion of PLTP into the reaction mixture accelerated the transfer of phospholipids (half-time of 1.7 hr) and the formation of the 77 A product. In addition to these compositional and morphological changes, which may be important in the interconversions of native HDL subspecies, the prolonged incubations revealed some slow reactions, such as the esterification of LDL cholesterol by LCAT, a background formation of cholesteryl esters in r-HDL, and an apparent hydrolysis of cholesteryl esters in LDL in the presence of r-HDL.     

Alteration of plasma HDL cholesteryl ester composition with transgenic expression of a point mutation (E149A) of human LCAT

We have previously identified a single amino acid mutation (hE149A) in human LCAT that increases its in vitro reactivity with phosphatidylcholine species containing sn-2 arachidonate (Wang et al. 1997. J. Biol. Chem. 272: 280-286). The purpose of the present study was to determine whether in vivo overexpression of hE149A compared with human wild-type LCAT (hLCAT-wt) would be sufficient to enrich the steady state composition of plasma HDL cholesteryl esters (CE) with long chain (>18 carbon) polyunsaturated fatty acyl species. Transgenic lines with 20-fold overexpression of hLCAT were created and studied between 12 and 16 weeks of age while consuming a chow diet. Transgenic overexpression of hE149A compared with hLCAT-wt significantly enriched HDL with CE species containing 20:4 (45%) and 22:6 n-3 (108%), at the expense of those containing 18:2, without a significant change in the plasma HDL concentration, particle size, or phospholipid fatty acyl composition. Removing the contribution of endogenous mouse LCAT by crossing the transgenic mice into the mouse LCAT knockout background resulted in even greater changes in HDL CE composition, with a 2.4-, 5-, and 5-fold increase in 20:4, 20:5 n-3, and 22:6 n-3 cholesteryl esters in the hE149A mice compared with hLCAT-wt Tg mice, respectively. Our results demonstrate that in vivo expression of hE149A significantly enriches HDL cholesteryl esters in 20- and 22-carbon fatty acyl species without affecting HDL concentration or size. Furthermore, the data suggest that endogenous mouse LCAT in hLCAT transgenic mice contributes to the plasma HDL CE pool out of proportion to its mass, presumably because the hLCAT transgene is poorly activated by mouse apolipoprotein A-I.     

Familial lecithin:cholesterol acyltransferase deficiency. Biochemistry of the cornea

Opacification of the cornea from lipid accumulation is an early and characteristic feature of familial lecithin:cholesterol acyltransferase (LCAT) deficiency. Visual impairment in a female age 48 years led to keratoplasty and the first detailed analysis of cornea in this disorder. Multilaminar figures were present, and total lipid extracts were enriched with phospholipid and cholesterol; cholesteryl esters were reduced, and accounted for about 12% of the cholesterol. Linoleate C18:2 was the predominant residue in the cholesteryl ester fatty acid fraction, with a C18:1/18:2 ratio of 1:6.5. This ratio differs from that in normal cornea, and from that in plasma and in other tissue deposits in LCAT deficiency. Various disorders of the HDL/LCAT system in plasma can lead to corneal lipid accumulation and opacification. These disorders may share general defects of lipid clearance from the cornea, but this study of LCAT cornea indicates that the character of the accumulating lipid is significantly influenced by active local metabolism, irrespective of the defect in the HDL/LCAT system also present.     

Lecithin:cholesterol acyltransferase deficiency increases atherosclerosis in the low density lipoprotein receptor and apolipoprotein E knockout mice.

The purpose of the present study was to test the hypothesis that lecithin:cholesterol acyltransferase (LCAT) deficiency would accelerate atherosclerosis development in low density lipoprotein (LDL) receptor (LDLr-/-) and apoE (apoE-/-) knockout mice. After 16 weeks of atherogenic diet (0.1% cholesterol, 10% calories from palm oil) consumption, LDLr-/- LCAT-/- double knockout mice, compared with LDLr-/- mice, had similar plasma concentrations of free (FC), esterified (EC), and apoB lipoprotein cholesterol, increased plasma concentrations of phospholipid and triglyceride, decreased HDL cholesterol, and 2-fold more aortic FC (142 +/- 28 versus 61 +/- 20 mg/g protein) and EC (102 +/- 27 versus 61+/- 27 mg/g). ApoE-/- LCAT-/- mice fed the atherogenic diet, compared with apoE-/- mice, had higher concentrations of plasma FC, EC, apoB lipoprotein cholesterol, and phospholipid, and significantly more aortic FC (149 +/- 62 versus 109 +/- 33 mg/g) and EC (101 +/- 23 versus 69 +/- 20 mg/g) than did the apoE-/- mice. LCAT deficiency resulted in a 12-fold increase in the ratio of saturated + monounsaturated to polyunsaturated cholesteryl esters in apoB lipoproteins in LDLr-/- mice and a 3-fold increase in the apoE-/- mice compared with their counterparts with active LCAT. We conclude that LCAT deficiency in LDLr-/- and apoE-/- mice fed an atherogenic diet resulted in increased aortic cholesterol deposition, likely due to a reduction in plasma HDL, an increased saturation of cholesteryl esters in apoB lipoproteins and, in the apoE-/- background, an increased plasma concentration of apoB lipoproteins.     

Recombinant human LCAT normalizes plasma lipoprotein profile in LCAT deficiency

Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for cholesterol esterification in plasma. Mutations in the LCAT gene leads to two rare disorders, familial LCAT deficiency and fish-eye disease, both characterized by severe hypoalphalipoproteinemia associated with several lipoprotein abnormalities. No specific treatment is presently available for genetic LCAT deficiency. In the present study, recombinant human LCAT was expressed and tested for its ability to correct the lipoprotein profile in LCAT deficient plasma. The results show that rhLCAT efficiently reduces the amount of unesterified cholesterol (?30%) and promotes the production of plasma cholesteryl esters (+210%) in LCAT deficient plasma. rhLCAT induces a marked increase in HDL-C levels (+89%) and induces the maturation of small preβ-HDL into alpha-migrating particles. Moreover, the abnormal phospholipid-rich particles migrating in the LDL region were converted in normally sized LDL.     

HDL2b lipoproteins as an acceptor of cholesterol from erythrocyte membrane and the role of lecithin-cholesterol-acyltransferase during this process

The role of lecithin-cholesterol-acyltransferase (LCAT) in net cholesterol transport from erythrocyte membranes to one of high density lipoprotein subclasses--HDL2b--was studied in vitro. It was shown that an addition of partially purified LCAT to the incubation medium containing human HDL2b and human erythrocytes triggers free cholesterol transport from erythrocyte membranes to HDL2b. In this case, the rise in HDL cholesterol occurs mainly due to the formation of cholesteryl esters produced by LCAT. The significance of cholesterol ecceptor properties of HDL2b in vivo is discussed.     

Remodeling of HDL by CETP in vivo and by CETP and hepatic lipase in vitro results in enhanced uptake of HDL CE by cells expressing scavenger receptor B-I.

The transport of HDL cholesteryl esters (CE) from plasma to the liver involves a direct uptake pathway, mediated by hepatic scavenger receptor B-I (SR-BI), and an indirect pathway, involving the exchange of HDL CE for triglycerides (TG) of TG-rich lipoproteins by cholesteryl ester transfer protein (CETP). We carried out HDL CE turnover studies in mice expressing human CETP and/or human lecithin:cholesterol acyltransferase (LCAT) transgenes on a background of human apoA-I expression. The fractional clearance of HDL CE by the liver was delayed by LCAT transgene, while the CETP transgene increased it. However, there was no incremental transfer of HDL CE radioactivity to the TG-rich lipoprotein fraction in mice expressing CETP, suggesting increased direct removal of HDL CE in the liver. To evaluate the possibility that this might be mediated by SR-BI, HDL isolated from plasma of the different groups of transgenic mice was incubated with SR-BI transfected or control CHO cells. HDL isolated from mice expressing CETP showed a 2- to 4-fold increase in SR-BI-mediated HDL CE uptake, compared to HDL from mice lacking CETP. The addition of pure CETP to HDL in cell culture did not lead to increased selective uptake of HDL CE by cells. However, when human HDL was enriched with TG by incubation with TG-rich lipoproteins in the presence of CETP, then treated with hepatic lipase, there was a significant enhancement of HDL CE uptake. Thus, the remodeling of human HDL by CETP, involving CE;-TG interchange, followed by the action of hepatic lipase (HL), leads to the enhanced uptake of HDL CE by cellular SR-BI.These observations suggest that in animals such as humans in which both the selective uptake and CETP pathways are active, the two pathways could operate in a synergistic fashion to enhance reverse cholesterol transport.     

Lecithin:cholesterol acyltransferase-induced transformation of HepG2 lipoproteins

Previous studies with the human hepatoblastoma-derived HepG2 cell line in this laboratory have shown that these cells produce high density lipoproteins (HDL) that are similar to HDL isolated from patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency. Experiments were, therefore, performed to determine whether HepG2 HDL could be transformed into plasma-like particles by incubation with LCAT. Concentrated HepG2 lipoproteins (d less than 1.235 g/ml) were incubated with purified LCAT or lipoprotein-deficient plasma (LPDP) for 4, 12, or 24 h at 37 degrees C. HDL isolated from control samples possessed excess phospholipid and unesterified cholesterol relative to plasma HDL and appeared as a mixed population of small spherical (7.8 +/- 1.3 nm) and larger discoidal particles (17.7 +/- 4.9 nm long axis) by electron microscopy. Nondenaturing gradient gel analysis (GGE) of control HDL showed major peaks banding at 7.4, 10.0, 11.1, 12.2, and 14.7 nm. Following 4-h LCAT and 12-h LPDP incubations, HepG2 HDL were mostly spherical by electron microscopy and showed major peaks at 10.1 and 8.1 nm (LCAT) and 10.0 and 8.4 nm (LPDP) by GGE; the particle size distribution was similar to that of plasma HDL. In addition, the chemical composition of HepG2 HDL at these incubation times approximated that of plasma HDL. Molar increases in HDL cholesteryl ester were accompanied by equimolar decreases in phospholipid and unesterified cholesterol. HepG2 low density lipoproteins (LDL) isolated from control samples showed a prominent protein band at 25.6 nm with GGE. Active LPDP or LCAT incubations resulted in the appearance of additional protein bands at 24.6 and 24.1 nm. No morphological changes were observed with electron microscopy. Chemical analysis indicated that the LDL cholesteryl ester formed was insufficient to account for phospholipid lost, suggesting that LCAT phospholipase activity occurred without concomitant cholesterol esterification.     

A novel lecithin-cholesterol acyltransferase antioxidant activity prevents the formation of oxidized lipids during lipoprotein oxidation.

Recent investigations suggest that high-density lipoprotein (HDL) may play an anti-atherogenic role as an antioxidant and inhibit the oxidative modification of low-density lipoprotein (LDL). The antioxidant activity of HDL has been proposed to be associated with several HDL-bound proteins. We have purified one HDL-associated protein, lecithin:cholesterol acyltransferase (LCAT), to apparent homogeneity and have found that LCAT is not only capable of esterifying cholesterol in the plasma, but can also prevent the accumulation of oxidized lipids in LDL. Addition of pure human LCAT to LDL or palmitoyl-linoleoyl phosphatidylcholine/sodium cholate (PLPC) micelles inhibits the oxidation-dependent accumulation of both conjugated dienes and lipid hydroperoxides. LCAT also inhibits the increase of net negative charge that occurs during oxidation of LDL. LCAT has the ability to prevent spontaneous oxidation and Cu2+ and soybean lipoxygenase-catalyzed oxidation of lipids. The antioxidant activity of LCAT appears to be enzymatic, since the enzyme is active for up to 10 h in the presence of mild free-radical generators. The catalytic serine, residue 181, may mediate this activity and act as a reusable proton donor. Chemical modification of the active serine residue with diisopropylfluorophosphate completely inhibits the ability of LCAT to prevent lipid oxidation. Thus, in addition to its well-characterized phospholipase and acyltransferase activities, LCAT can also act as an antioxidant and prevent the accumulation of oxidized lipid in plasma lipoproteins.     

Affinity of lipid transfer protein for lipid and lipoprotein particles as influenced by lecithin-cholesterol acyltransferase

The effects of lecithin-cholesterol acyltransferase (LCAT) on the transfer of cholesterol esters mediated by lipid transfer protein (LTP) and its affinity for lipid and lipoprotein particles were investigated. When the single bilayer vesicle preparations (containing phosphatidylcholine, cholesterol, cholesteryl ester, and apolipoprotein- (apo) A-I at the molar ratio of 90:30:1.2:0.18) or high density lipoprotein 3 (HDL3) were used as the cholesteryl ester donor and low density lipoproteins (LDL) as the acceptor, the transfer activity of LTP was enhanced by the addition of low concentrations of LCAT. In contrast, no enhancement of cholesteryl ester transfer was observed upon addition of LCAT to either the discoidal bilayer particle preparations (containing phosphatidylcholine, cholesterol, cholesteryl ester, and apo-A-I at the molar ratio of 90:30:1.2:1.0) or high density lipoprotein 2 (HDL2). Although both apo-A-I and apo-A-II promoted the transfer of cholesteryl ester from vesicles to LDL, the additional enhancement of the transfer by LCAT was observed only with the vesicles containing apo-A-I. Gel permeation chromatography of LTP/vesicle and LTP/HDL3 mixtures in the presence and absence of LCAT showed that the affinity of LTP for both the vesicles and HDL3 increased upon addition of LCAT. In contrast, neither HDL2 nor discoidal bilayer particles showed any significant enhancement of LTP binding upon addition of LCAT. By using LCAT covalently bound to Sepharose 4B, a maximal interaction between LTP and bound LCAT was shown to occur at the ionic strength of 0.16. Deviation from this ionic strength reduced the extent of the interaction. At the ionic strength of 0.01 and 0.5, the elution volume of LTP was identical to that of bovine serum albumin.     

Cloning and expression of a novel lysophospholipase which structurally resembles lecithin cholesterol acyltransferase

Lecithin cholesterol acyltransferase (LCAT) is the key enzyme in the esterification of plasma cholesterol and in the reverse cholesterol transport on high-density lipoprotein (HDL). We have found a novel LCAT-related gene among differentially expressed cDNA fragments between two types of foam cells derived from THP-1 cells, which are different in cholesterol efflux ability, using a subtractive PCR technique. The deduced 412-amino-acid sequence has 49% amino acid sequence similarity with human LCAT. In contrast to the liver-specific expression of LCAT, mRNA expression of the gene was observed mainly in peripheral tissues including kidney, placenta, pancreas, testis, spleen, heart, and skeletal muscle. The protein exists in human plasma and is probably associated with HDL. Moreover, we discovered that the recombinant protein hydrolyzed lysophosphatidylcholine (lysoPC), a proatherogenic lipid, to glycerophosphorylcholine and a free fatty acid. We have therefore named this novel enzyme LCAT-like lysophospholipase (LLPL), through which a new catabolic pathway for lysoPC on lipoproteins could be elucidated.