Detection of heterozygotes for familial lecithin: cholesterol acyltransferase (LCAT) deficiency.

"Rocket" immunoelectrophoresis using specific anti-lecithin: cholesterol acyltransferase (LCAT) antiserum showed no immunoreactive protein in two patients with familial LCAT deficiency. Subnormal quantity of plasma LCAT was found in the maternal grandmother, the parents, and in two of four siblings of the patients (3.3-3.4 mg/l vs. 5.4 +/- 0.5 mg/l in 12 controls). The immunochemical quantitation of the enzyme correlated well (r = .93) with LCAT activity in an artificial substrate assay. These two methods allow detection of heterozygotes for LCAT deficiency.     

Detection of lecithin: cholesterol acyltransferase (LCAT) in a human hepatoma cell line

A human hepatoma cell line (HepG-2) was probed for the presence of lecithin: cholesterol acyltransferase (LCAT) using an antiserum to human plasma LCAT. Double immunodiffusion analysis using antiserum to human plasma LCAT revealed a single precipitin line in the sonicated cell homogenate. This precipitin line showed a reaction of identity with highly purified plasma LCAT. The presence of LCAT within the hepatoma cells was also confirmed by an immunofluorescence test. In contrast, the cell culture supernate showed a weak and inconsistent precipitin line. These data suggest that HepG-2 cells synthesize LCAT but secretion of the enzyme by these cells into the culture medium may be partially or totally impaired.     

Biochemical and compositional analyses of recombinant lecithin:cholesterol acyltransferase (LCAT) obtained from a hepatic source

Lecithin:cholesterol acyltransferase (LCAT) is an important plasma glycoprotein which plays a central role in lipid metabolism. This protein is responsible for generation of cholesteryl esters in plasma and it has been proposed to play a pivotal role in the reverse cholesterol transport pathway. Structural and functional studies of LCAT have employed various expression systems for production of recombinant LCAT (rLCAT). However, recent studies have shown some differences in the oligosaccharide structure and composition of rLCAT. In this study, we have generated a new hepatic based expression system using McArdle-RH7777 (Mc-7777) cells to produce a recombinant protein most similar to human plasma LCAT. The expressed glycoprotein was compared to the LCAT expressed in previously characterized baby hamster kidney (BHK) cells. Both proteins were compared on the basis of their carbohydrate structure and composition as well as their functional properties. Although the functional properties of both glycoproteins were similar, the carbohydrate structure was significantly different. While BHK-LCAT contained bi-, tri-, and tetraantennary structures, Mc-7777 LCAT presented only biantennary oligosaccharide structures. The difference in glycosylation pattern of rLCAT from Mc-7777 and BHK cells underlines the importance of appropriate expression system, both in vivo and in vitro.     

Characterization of functional residues in the interfacial recognition domain of lecithin cholesterol acyltransferase (LCAT)

Lecithin cholesterol acyltransferase (LCAT) is an interfacial enzyme active on both high-density (HDL) and low-density lipoproteins (LDL). Threading alignments of LCAT with lipases suggest that residues 50-74 form an interfacial recognition site and this hypothesis was tested by site-directed mutagenesis. The (delta56-68) deletion mutant had no activity on any substrate. Substitution of W61 with F, Y, L or G suggested that an aromatic residue is required for full enzymatic activity. The activity of the W61F and W61Y mutants was retained on HDL but decreased on LDL, possibly owing to impaired accessibility to the LDL lipid substrate. The decreased activity of the single R52A and K53A mutants on HDL and LDL and the severer effect of the double mutation suggested that these conserved residues contribute to the folding of the LCAT lid. The membrane-destabilizing properties of the LCAT 56-68 helical segment were demonstrated using the corresponding synthetic peptide. An M65N-N66M substitution decreased both the fusogenic properties of the peptide and the activity of the mutant enzyme on all substrates. These results suggest that the putative interfacial recognition domain of LCAT plays an important role in regulating the interaction of the enzyme with its organized lipoprotein substrates.     

Phosphatidylcholine fluidity and structure affect lecithin:cholesterol acyltransferase activity

The purpose of this study was to test the hypothesis that lipid fluidity regulates lecithin:cholesterol acyltransferase (LCAT) activity. Phosphatidylcholine (PC) species were synthesized that varied in fluidity by changing the number, type (cis vs. trans), or position of the double bonds in 18 or 20 carbon sn-2 fatty acyl chains and recombined with [(3)H]cholesterol and apolipoprotein A-I to form recombinant high density lipoprotein (rHDL) substrate particles. The activity of purified human plasma LCAT decreased with PC sn-2 fatty acyl chains containing trans versus cis double bonds and as double bonds were moved towards the methyl terminus of the sn-2 fatty acyl chain. The decrease in LCAT activity was significantly correlated with a decrease in rHDL fluidity (measured by diphenylhexatriene fluorescence polarization) for PC species containing 18 carbon (r(2) = 0.61, n = 18) and 20 carbon (r(2) = 0.93, n = 5) sn-2 fatty acyl chains. rHDL were also made containing 10% of the 18 carbon sn-2 fatty acyl chain PC species and 90% of an inert PC ether matrix (sn-1 18:1, sn-2 16:0 PC ether) to normalize rHDL fluidity. Even though fluidity was similar among the PC ether-containing rHDL, the order of PC reactivity with LCAT was significantly correlated (r(2) = 0.71) with that of 100% PC rHDL containing the same 18 carbon sn-2 fatty acyl chain species, suggesting that PC structure in the active site of LCAT determines reactivity in the absence of measurable differences in bilayer fluidity. We conclude that PC fluidity and structure are major regulators of LCAT activity when fatty acyl chain length is constant.     

Structural differences between wild-type and fish eye disease mutant of lecithin:cholesterol acyltransferase

Fluorescence spectroscopy has been used to investigate the conformational changes that occur upon binding of wild type (WT) and mutant (Thr123Ile) lecithin:cholesterol acyltransferase (LCAT) to the potential substrates (dioleoyl-phosphatidyl choline [DOPC] and high density lipoprotein [HDL]). For a detailed analysis of structural differences between WT and mutant LCAT, we performed decompositional analysis of a set of tryptophan fluorescence spectra, measured at increasing concentrations of external quenchers (acrylamide and KI). The data obtained show that Thr123Ile mutation in LCAT leads to a conformation that is likely to be more rigid (less mobile/flexible) than that of the WT protein with a redistribution of charged residues around exposed tryptophan fluorophores. We propose that the redistribution of charged residues in mutant LCAT may be a major factor responsible for the dramatically reduced activity of the enzyme with HDL and reconstituted high density lipoprotein (rHDL).     

An amino acid exchange in exon I of the human lecithin: cholesterol acyltransferase (LCAT) gene is associated with fish eye disease.

The exons of the lecithin:cholesterol acyltransferase (LCAT) gene in DNA samples from two of the original Swedish Fish Eye Disease patients have been amplified by polymerase chain reactions and sequenced by the dideoxy method. The two patients apparently were unrelated. In both patients a mutation in codon 10 of the first exon was found, altering proline10 to leucine. We note that the mutations causing Fish Eye Disease as well as those causing classical LCAT deficiency are spread over most of the translated gene. Why these various mutations in the same gene give rise to two different disease phenotypes remains unexplained.     

Purification of horse (Equus caballus) serum lecithin:cholesterol acyltransferase

1. A method for the purification of horse serum lecithin:cholesterol acyltransferase has been established. 2. The method involves the adsorption of the enzyme from diluted horse serum on DEAE-Sephadex A-50, (NH4)2SO4 fractionation, 1-butanol treatment, and chromatographic techniques of DEAE-Sepharose CL-6B, DEAE-Sephadex A-50, Affi-Gel blue and hydroxylapatite. 3. The resultant enzyme preparation essentially formed a single main band when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. 4. The final purification of the enzyme was 20,000-fold with 7% yield. 5. The apparent mol. wt of the enzyme was 64,000. 6. The activity of the enzyme was stable for 3 days at 0 degree C.     

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.     

Isolation and properties of porcine lecithin:cholesterol acyltransferase

Lecithin: cholesterol acyltransferase (LCAT, phosphatidylcholine: sterol O-acyltransferase, EC 2.3.1.43) was purified approximately 20 000-fold from pig plasma by ultracentrifugation, phenyl-Sepharose and hydroxyapatite chromatography. Purified LCAT had an apparent relative molecular mass of 69 000 +/- 2000. By isoelectrofocusing it separated into five or six bands with pI values ranging from pH 4.9 to 5.2. The amino acid composition was similar to that of the human enzyme. An antibody against pig LCAT was prepared in goat. The antibody reacted against pig LCAT and gave a reaction of partial identity with human LCAT. Incubation of pig plasma or purified enzyme with the antibody virtually inhibited LCAT activity. The same amount of antibody inactivated only 62% of the LCAT activity in human serum. Pig and human LCAT were activated to the same extent by either human or pig apolipoprotein A-I (apo-A-I) using small liposomes as substrate. Human apoA-I, however, caused a higher esterification rate for both enzymes. Using apoA-I and small liposomes as a substrate, the addition of apoC-II up to 4 micrograms/ml had no effect on the LCAT reaction, but above this concentration LCAT was inhibited. Small liposomes with phosphatidylcholine/cholesterol molar ratios of 3:1 up to 8.4:1 did not show any significant differences in the LCAT reaction, when used as substrates in the presence of various amounts of apoA-I and albumin. In contrast, the LCAT activity was significantly reduced by liposomes with phosphatidylcholine/cholesterol molar ratios below 3:1.     

Selective extraction of lecithin:cholesterol acyltransferase (EC 2.3.1.43) from human plasma

A method for the rapid extraction of lecithin:cholesterol acyltransferase (LCAT) from human plasma or serum has been developed. The method is based on direct treatment of acidified plasma of fully conserved enzyme activity, with the strong ion exchanger Q-Sepharose, which under the experimental conditions bound all LCAT but only about 10% of the total protein content of the plasma, no albumin and essentially no lipoproteins. This corresponds to a 10-fold purification. Only traces of apolipoprotein A-I remained in the quantitatively desorbed LCAT preparation which, however, contained a residual fraction of apolipoprotein D and acidic plasma proteins.The present one-step procedure for extraction of LCAT in high yields from human plasma represents a simple and efficient alternative to the first step in previously described methods for preparation of the enzyme to homogeneity.     

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.     

Selectivity and contribution of lecithin: cholesterol acyltransferase to plasma cholesterol ester formation

Selectivity factors (Vm/Km) for human and rat lecithin: cholesterol acyltransferases (LCAT) for the transfer of various acyl groups from the 2-position of phosphatidylcholine were determined. By multiplying these values by the proportions of acyl groups at the 2-position of phosphatidylcholine, one can predict the proportions of molecular species of cholesterol ester which will be synthesized by LCAT. In human subjects fasted overnight, the molecular composition of plasma cholesterol ester was found to reflect the LCAT selectivity relatively accurately. This result supports the concepts that hepatic acyl-CoA:cholesterol acyltransferase (ACAT) does not contribute significantly to the synthesis of plasma cholesterol ester and that removal of cholesterol ester from plasma is not selective with respect to molecular species under these conditions. In contrast to the results with humans, the molecular composition of plasma cholesterol ester formed in spontaneously hypertensive rats fed a high-cholesterol diet and then fasted overnight differs from that which is predicted from LCAT selectivity and the proportion of various fatty acids at the 2-position of phosphatidylcholine: these results suggest that cholesterol ester is formed mainly via the ACAT reaction.     

A dual role for lecithin:cholesterol acyltransferase (EC 2.3.1.43) in lipoprotein oxidation

Lecithin:cholesterol acyltransferase (LCAT) is a key enzyme involved in lipoprotein metabolism. It mediates the transesterification of free cholesterol to cholesteryl ester in an apoprotein A-I-dependent process. We have isolated purified LCAT from human plasma using anion-exchange chromatography and characterized the extracted LCAT in terms of its molecular weight, molar absorption coefficient, and enzymatic activity. The participation of LCAT in the oxidation of very low density lipoproteins (VLDL) and low-density lipoproteins (LDL) was examined by supplementing lipoproteins with exogenous LCAT over a range of protein concentrations. LCAT-depleted lipoproteins were also prepared and their oxidation kinetics examined. Our results provide evidence for a dual role for LCAT in lipoprotein oxidation, whereby it acts in a dose-responsive manner as a potent pro-oxidant during VLDL oxidation, but as an antioxidant during LDL oxidation. We believe this novel pro-oxidant effect may be attributable to the LCAT-mediated formation of oxidized cholesteryl ester in VLDL, whereas the antioxidant effect is similar to that of chain-breaking antioxidants. Thus, we have demonstrated that the high-density lipoprotein-associated enzyme LCAT may have a significant role to play in lipoprotein modification and hence atherogenesis.     

Effects of detergents on the lecithin:cholesterol acyltransferase reaction

This paper describes the effect of an ionic (sodium dodecyl sulfate; SDS) and a nonionic detergent (Triton X-100) on the substrate and enzyme components of the lecithin: cholesterol acyltransferase (LCAT) reaction. When the enzyme sources (purified or partially purified) or the respective substrates [high-density lipoproteins (HDL) or proteoliposomes] were preincubated with detergents, a consistent trend in LCAT activity was only seen when partially purified LCAT was used as the enzyme source. This trend indicated an approximately 25% increase in enzyme activity over the control when 10(-4) M SDS and 2 X 10(-3)% Triton X-100 were present in the preincubation mixtures, respectively. Those observations suggested that, during the preincubations and subsequent assays, the enzyme (in the presence of detergents) was allowed to dissociate from the endogenous substrate and subsequently interact with the exogenous substrate molecules. Additional experiments utilizing molecular-sieve chromatography with whole plasma and partially purified enzyme also showed that dissociation of LCAT/lipoprotein complexes occurred in the presence of detergent. SDS was also shown to enhance the reaction of LCAT in whole plasma with anti-LCAT antibody in an enzyme-linked immunoassay system, indicating that the detergent treatment facilitated the exposure of additional antigenic sites, perhaps via dissociation of the enzyme from plasma lipoproteins.