Human Lecithin:cholesterol Acyltransferase Research Articles

Analysis of human lecithin–cholesterol acyltransferase activity by carboxyl-terminal truncation

Lecithin–cholesterol acyltransferase (LCAT) is a key enzyme in reverse cholesterol transport and catalyzes the esterification of cholesterol in human plasma. Human LCAT is a glycosylated protein, containing 416 amino acids and a proline-rich region at the C-terminus. To address the function of the C-terminal region of LCAT as well as that of the proline-rich region, we constructed and expressed LCAT mutants with C-terminal truncations at different positions. The expression of wild-type LCAT in COS-1 cells resulted in an enzymatically active protein that was secreted by the cells. The mutants lacking the proline-rich region at the C-terminus were expressed and secreted at levels comparable to those of wild-type (∼50% of wild-type concentrations in cell media). The proline-deletion mutants were similar to wild-type LCAT in terms of phospholipase or transferase activities with various interfacial substrates, including reconstituted HDL, proteoliposomes, LDL, and micelles of platelet activating factor. Thus, the binding of LCAT to the diverse interfaces is not affected by the removal of its C-terminal region. Also, the activation by apolipoproteins and access of water-insoluble substrates to the active site are not significantly affected by the deletion of the proline-rich region. However, deletions of the proline-rich region, including the five amino acids nearest to the C-terminus, resulted in approximately an 8-fold increase in the specific activity of LCAT towards the water-soluble substrate, p-nitrophenylbutyrate. This suggests that the C-terminal proline-rich region may interfere with the access of this water-soluble substrate to the active site of LCAT, and may form part of a protective covering of the active site of LCAT while in solution. Further deletions at the C-terminus, beyond the proline-rich region, impaired the secretion of the enzyme, implying that this region may play a critical role in either the secretion or folding of LCAT in COS-1 cells.

The gene cluster containing the LCAT gene is conserved between human and pig.

A pooled DNA probe from P1 artificial chromosome clones (PACs) containing the human lecithin:cholesterol acyl transferase (LCAT) gene cluster was used in fluorescence in situ hybridization (FISH) experiments assigning the genes to pig chromosome 6p13. In addition, probes derived from the coding regions in the human gene cluster were used in long range mapping experiments to show that the overall structures of the human and porcine LCAT gene clusters are identical. Both the linear order and the close physical distance of five apparently unrelated genes have been maintained throughout 90 million years of divergent evolution between human and pig. The extremely dense clustering of the genes in the LCAT gene cluster suggests that this gene organization has biological significance. The conservation of the gene cluster between human and pig supports this suggestion.

Potential gene therapy for lecithin-cholesterol acyltransferase (LCAT)-deficient and hypoalphalipoproteinemic patients with adenovirus-mediated transfer of human LCAT gene.

Overexpression of human lecithin-cholesterol acyltransferase (LCAT) in transgenic mice results in an increase of the antiatherogenic HDLs. To investigate the potential use of LCAT for gene therapy, a recombinant adenovirus was constructed in which the human LCAT cDNA was expressed under the control of the human cytomegalovirus immediate/early promoter followed by a chimeric intron (AdCMV human LCAT). Human apolipoprotein (apo) A-I transgenic mice infected with AdCMV human LCAT by intravenous injection accumulated reactive LCAT in the plasma. LCAT activity was increased 201-fold in the plasma of mice infected with 1 x 10(6) pfu AdCMV human LCAT, from 45 +/- 2 to 9068 +/- 812 nmol.mL-1.h-1, in comparison with basal LCAT activity measured in control mice, 5 days after injection. Plasma HDL cholesterol levels rose from 117 +/- 12 to 797 +/- 48 mg/dL, and plasma human apo A-I concentrations increased from 247 +/- 14 to 616 +/- 17 mg/dL, in AdCMV human LCAT infected mice compared with control mice. HDL particles were larger and had a different electrophoretic mobility. Studies of cholesterol efflux by incubation of serum with cholesterol-loaded Fu5AH cells showed that serum from AdCMV human LCAT-infected mice promoted a significantly higher efflux than did that of the controls. These data establish the potential of this approach for treatment of subjects with LCAT gene defects as well as patients with low plasma levels of apo A-I and HDL cholesterol.

Overexpression of lecithin:cholesterol acyltransferase in transgenic rabbits prevents diet-induced atherosclerosis.

Lecithin:cholesterol acyltransferase (LCAT) is a key plasma enzyme in cholesterol and high density lipoprotein (HDL) metabolism. Transgenic rabbits overexpressing human LCAT had 15-fold greater plasma LCAT activity that nontransgenic control rabbits. This degree of overexpression was associated with a 6.7-fold increase in the plasma HDL cholesterol concentration in LCAT transgenic rabbits. On a 0.3% cholesterol diet, the HDL cholesterol concentrations increased from 24 +/- 1 to 39 +/- 3 mg/dl in nontransgenic control rabbits (n = 10; P < 0.05) and increased from 161 +/- 5 to 200 +/- 21 mg/dl (P < 0.001) in the LCAT transgenic rabbits (n = 9). Although the baseline non-HDL concentrations of control (4 +/- 3 mg/dl) and transgenic rabbits (18 +/- 4 mg/dl) were similar, the cholesterol-rich diet raised the non-HDL cholesterol concentrations, reflecting the atherogenic very low density, intermediate density, and low density lipoprotein particles observed by gel filtration chromatography. The non-HDL cholesterol rose to 509 +/- 57 mg/dl in controls compared with only 196 +/- 14 mg/dl in the LCAT transgenic rabbits (P < 0.005). The differences in the plasma lipoprotein response to a cholesterol-rich diet observed in the transgenic rabbits paralleled the susceptibility to developing aortic atherosclerosis. Compared with nontransgenic controls, LCAT transgenic rabbits were protected from diet-induced atherosclerosis with significant reductions determined by both quantitative planimetry (-86%; P < 0.003) and quantitative immunohistochemistry (-93%; P < 0.009). Our results establish the importance of LCAT in the metabolism of both HDL and apolipoprotein B-containing lipoprotein particles with cholesterol feeding and the response to diet-induced atherosclerosis. In addition, these findings identify LCAT as a new target for therapy to prevent atherosclerosis.

Hyperalphalipoproteinemia in human lecithin cholesterol acyltransferase transgenic rabbits. In vivo apolipoprotein A-I catabolism is delayed in a gene dose-dependent manner.

Lecithin cholesterol acyltransferase (LCAT) is an enzyme involved in the intravascular metabolism of high density lipoproteins (HDLs). Overexpression of human LCAT (hLCAT) in transgenic rabbits leads to gene dose-dependent increases of total and HDL cholesterol concentrations. To elucidate the mechanisms responsible for this effect, 131I-HDL apoA-I kinetics were assessed in age- and sex-matched groups of rabbits (n=3 each) with high, low, or no hLCAT expression. Mean total and HDL cholesterol concentrations (mg/dl), respectively, were 162+/-18 and 121+/-12 for high expressors (HE), 55+/-6 and 55+/-10 for low expressors (LE), and 29+/-2 and 28+/-4 for controls. Fast protein liquid chromatography analysis of plasma revealed that the HDL of both HE and LE were cholesteryl ester and phospholipid enriched, as compared with controls, with the greatest differences noted between HE and controls. These compositional changes resulted in an incremental shift in apparent HDL particle size which correlated directly with the level of hLCAT expression, such that HE had the largest HDL particles and controls the smallest. In vivo kinetic experiments demonstrated that the fractional catabolic rate(FCR, d(-1)) of apoA-I was slowest in HE (0.328+/-0.03) followed by LE (0.408+/-0.01) and, lastly, by controls (0.528+/-0.04). ApoA-I FCR was inversely associated with HDL cholesterol level (r=-0.851,P<0.01) and hLCAT activity (r=-0.816, P<0.01). These data indicate that fractional catabolic rate is the predominant mechanism by which hLCAT overexpression differentially modulates HDL concentrations in this animal model. We hypothesize that LCAT-induced changes in HDL composition and size ultimately reduce apoA-I catabolism by altering apoA-I conformation and/or HDL particle regeneration.

Glycosylation structure and enzyme activity of lecithin:cholesterol acyltransferase from human plasma, HepG2 cells, and baculoviral and Chinese hamster ovary cell expression systems

The glycosylation state of lecithin:cholesterol acyltransferase (LCAT) may be important in determining its enzymatic activity. We compared glycosylation structure, enzyme kinetics, and phosphatidylcholine (PC) acyl specificity of human LCAT from four sources: human plasma (pLCAT), media from HepG2 cells (HepG2 LCAT), media from SF21 cells infected with a recombinant baculovirus (bLCAT) and media from stably transfected Chinese hamster ovary (CHO) cells (CHO LCAT). bLCAT was underglycosylated (molecular weight approximately 50 kDa) and resistant to digestion by N-glycanase F, endoglycosidase F, and neuraminidase. CHO and HepG2 LCAT were overglycosylated (approximately 68 kDa and approximately 70-75 kDa) compared to pLCAT (approximately 65 kDa). CHO LCAT, like pLCAT, was sensitive to N-glycanase F and neuraminidase but not to endoglycosidase F. HepG2 LCAT demonstrated resistance to N-glycanase F and endoglycosidase F. Apparent Km values for all four enzymes were similar (1.4-9.2 microM cholesterol) for recombinant high density lipoproteins (rHDL) containing sn-1 16:0, sn-2 18:1 PC (POPC). Apparent Vmax values (nmol cholesteryl ester formed/h per micrograms) were 52.6 for pLCAT, 48.6 for CHO LCAT, 15.3 for bLCAT, and 8.3 for HepG2 LCAT. Changes in PC acyl specificity in the presence and absence of cholesterol were characterized by comparing the ratio of LCAT activity on rHDL containing sn-1 16:0, sn-2 20:4 PC (PAPC) or POPC (PAPC/POPC activity ratio). The ratios for pLCAT, bLCAT, CHO LCAT, and HepG2 LCAT activity were 0.63, 0.49, 0.56, and 0.51 with cholesterol and 0.34, 0.29, 0.36, and 0.99 without cholesterol, respectively. We conclude that LCAT source influences glycosylation structure, which affects the apparent Vmax for cholesteryl ester formation with only minor changes in apparent Km or acyl substrate specificity.

Lecithin:Cholesterol Acyltransferase Overexpression Generates Hyperalpha-lipoproteinemia and a Nonatherogenic Lipoprotein Pattern in Transgenic Rabbits

Cholesterol esterification within plasma lipoprotein particles is catalyzed by lecithin:cholesterol acyltransferase (LCAT). The impact of the overexpression of this enzyme on plasma concentrations of the different plasma lipoproteins in an animal model expressing cholesteryl ester transfer protein was evaluated by generating rabbits expressing human LCAT. A 6.2-kilobase human genomic DNA construct was injected into the pronuclei of rabbit embryos. Of the 1002 embryos that were injected, 3 founder rabbits were characterized that expressed the human LCAT gene. As in mice and humans, the principal sites of mRNA expression in these rabbits is in the liver and brain, indicating that the regulatory elements required for tissue-specific expression among these species are similar. The alpha-LCAT activity correlated with the number of copies of LCAT that integrated into the rabbit DNA. Compared with controls, the high expressor LCAT-transgenic rabbits total and high density lipoprotein (HDL) cholesterol concentrations were increased 1.5-2.5-fold with a 3.1-fold increase in the plasma cholesterol esterification rate. Analysis of the plasma lipoproteins by fast protein liquid chromatography indicates that these changes reflected an increased concentration of apolipoprotein E-enriched, HDL1-sized particles, whereas atherogenic apolipoprotein B particles disappeared from the plasma. The concentrations of plasma HDL cholesterol were highly correlated with both human LCAT mass (r = 0.93; p = 0.001) and the log LCAT activity (r = 0.94; p < 0.001) in the transgenic rabbits. These results indicate that overexpression of LCAT in the presence of cholesteryl ester transfer protein leads to both hyperalpha-lipoproteinemia and reduced concentrations of atherogenic lipoproteins.

Cloning of rabbit LCAT cDNA: increase in LCAT mRNA abundance in the liver of cholesterol-fed rabbits.

Structure of rabbit lecithin:cholesterol acyltransferase (LCAT) and molecular basis for the effects of cholesterol feeding on LCAT expression were investigated by cloning and sequencing LCAT cDNA from rabbit. The rabbit and human sequences are 91% identical at the nucleotide level and 93% identical at the amino acid level. The interfacial substrate active site, asparagine-linked glycosylation sites, and sites at which rare mutations cause human familial LCAT deficiency are all highly conserved in the rabbit protein. The apparent molecular mass of rabbit LCAT, as determined by immunoblot analysis, was approximately equal to that of human LCAT. Rabbits showed 2.6- and 5.5-fold increases in serum LCAT activity 3 and 6 weeks, respectively, after switching to a cholesterol-enriched diet. Northern blot analysis revealed that the abundance of LCAT mRNA in liver increased 1.6- and 2.8-fold after 3 and 6 weeks, respectively, of cholesterol feeding. The marked temporal relation between the increase in serum LCAT activity and the liver LCAT mRNA abundance suggest that the regulation of LCAT activity in vivo may be primarily determined by changes in the amount of LCAT mRNA.

Chicken Lecithin-Cholesterol Acyltransferase: MOLECULAR CHARACTERIZATION REVEALS UNUSUAL STRUCTURE AND EXPRESSION PATTERN

Rapidly growing oocytes in the laying hen are, in addition to the liver, targets of the so-called "reverse cholesterol transport" (RCT) (Vieira, P.M., Vieira, A.V., Sanders, E.J., Steyrer, E., Nimpf, J., and Schneider, W.J. (1995) J. Lipid Res. 36, 601-610), pointing to the importance of this process in nonplacental reproduction. We have begun to delineate the details of this unique transport pathway branch by molecular characterization of the first nonmammalian lecithin-cholesterol acyltransferase (LCAT), the enzyme that catalyzes an early step in RCT. The biological significance of the enzyme is underscored by the high degree of protein sequence identity (73%) maintained from chicken to man. Interestingly, the conservation extends much less to the cysteine residues; in fact, two of the cysteines thought to be important in mammalian enzymes (residues 31 and 184 in man) are absent from the chicken enzyme, providing proof of their dispensability for enzymatic activity. Antibodies prepared against a chicken LCAT fusion protein cross-react with human LCAT and identify a 64-kDa protein present in enzymatically active fractions obtained by hydrophobic chromatography of chicken serum. The developmental and tissue distribution pattern of LCAT in females is striking; during embryogenesis and adolescence, LCAT expression is extremely high in liver but undetectable in brain. Upon onset of laying, however, brain LCAT mRNA increases suddenly and is maintained at levels 5 times higher than in liver, in stark contrast to most mammals. In adult roosters, the levels of LCAT transcripts in brain are lower than in liver. Together with the molecular characterization of chicken LCAT, these newly discovered developmental changes and gender differences in its expression establish the avian oocyte/liver system as a powerful model to delineate in vivo regulatory elements of RCT.

Role of lecithin:cholesterol acyltransferase and apolipoprotein A-I in cholesterol esterification in lipoprotein-X in vitro

Lipoprotein-X (Lp-X) is an abnormal particle present in the plasma of patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency syndromes or cholestatic liver disease. Compared to other lipoproteins, Lp-X contains a high content of unesterified cholesterol (30%, w/w) to phosphatidylcholine (60%, w/w). The objective of this study was to evaluate the role of LCAT and apolipoprotein A-I (apoA-I) in Lp-X metabolism in vitro and to elucidate the regulation of cholesterol esterification in this unique lipoprotein. Lp-X isolated from sera of patients with obstructive jaundice had a high content of unesterified cholesterol and phosphatidylcholine and contained apolipoprotein E, apoCs, and albumin. Although human recombinant LCAT used as an enzyme source did bind to isolated Lp-X, no cholesterol esterification was detected. However, addition of human apoA-I in the presence of albumin resulted in significant cholesterol esterification in Lp-X (Vmax 0.25 +/- 0.04 nmol/h per microgram LCAT protein). Exogenous apoA-I did not change the size of Lp-X particle as determined by quasi-elastic light scattering analysis. A reduction in Lp-X size was observed when both apoA-I and LCAT were included in the reaction mixture (from 47 nm to 42 nm). Furthermore, addition of apoA-I (but not HDL) dramatically changed the electrophoretic mobility of Lp-X from cathodic to anodic migration. Such changes are not due to displacement of apoC or apoE proteins from Lp-X by apoA-I. While increasing apoA-I concentration (up to 35 micrograms/ml) in the reaction mixture stimulated cholesterol esterification in Lp-X, addition of apoA-I at the concentration of 8 micrograms/ml inhibited cholesterol esterification in VLDL, LDL, and HDL by more than 50%. Albumin was required for the LCAT reaction to Lp-X. Our results suggest that while LCAT binds to isolated Lp-X, apoA-I is needed for the LCAT reaction to proceed. The presence of apoA-I does not result in the displacement of apoCs and apoE from Lp-X and addition of apoA-I changes the electrophoretic mobility but not the size of Lp-X.

Expression of human lecithin-cholesterol acyltransferase in transgenic mice. Effect of human apolipoprotein AI and human apolipoprotein all on plasma lipoprotein cholesterol metabolism.

Human (Hu) lecithin-cholesterol acyltransferase (LCAT) is a key enzyme in the plasma metabolism of cholesterol. To assess the effects of increased plasma levels of LCAT, four lines of transgenic mice were created expressing a Hu LCAT gene driven by either its natural or the mouse albumin enhancer promoter. Plasma LCAT activity increased from 1.2- to 1.6-fold higher than that found in control mouse plasma. Lipid profiles, upon comparing Hu LCAT transgenics to control animals, revealed a 20 t0 60% increase in total and cholesteryl esters that were mainly present in HDL. The in vivo substrate specificity of Hu LCAT was assessed by creating animals expressing Hu apo AI + Hu LCAT (HuAI/ LCAT), Hu apo AI + Hu apo AII + Hu LCAT (HuAI/ AII/LCAT), and Hu apo AII + Hu LCAT (HuAII/LCAT). Plasma cholesterol was increased up to 4.2-fold in HuAI/ LCAT transgenic mice and twofold in the HuAI/AII/LCAT transgenic mice, compared with HuAI and HuAI/AII transgenic mice. HDL cholesteryl ester levels were increased more than twofold in both the HuAI/LCAT and HuAI/AII/LCAT mice compared with the HuAI, HuAI/AII, and HuLCAT animals. The HDL particles were predominantly larger in the HuAI/LCAT and the HuAI/AII/LCAT mice compared with those in HuAI, HuAII/LCAT, and HuLCAT animals. The increase in LCAT activity in the HuAI/LCAT and HuAI/AII/LCAT mice was associated with 62 and 27% reductions respectively, in the proportion of Hu apo AI in the pre beta-HDL fraction, when compared with HuAI and HuAI/AII transgenic mice. These data demonstrate that moderate increases in LCAT activity are associated with significant changes in lipoprotein cholesterol levels and that Hu LCAT has a significant preference for HDL containing Hu apo AI.

Human plasma lecithin:cholesterol acyltransferase. On the substrate efficiency of cholest-5-ene-3ß-thiol as a fatty acyl acceptor

Lecithin: cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyses cholesteryl ester formation from lecithin and cholesterol present in the surface of plasma lipoproteins. Sterol fatty acid acceptors have previously been shown to require the presence of a trans conformation of the A/B ring and a 3ß-OH group. Our laboratory has, however, demonstrated that two thiol sites within LCAT can become fatty acylated following lecithin cleavage although this does not appear to be essential for catalysis. In order to assess the ability of LCAT to donate a fatty acid derived from the sn-2 position of lecithin and present as an acyl enzyme intermediate (linked via an oxyester bond to Ser-181) to a sulfhydryl residue, we evaluated the ability of cholest-5-ene-3 ß-thiol to act as a substrate for cholesterol ester formation by LCAT. Thiocholesterol was a good terminal fatty acyl acceptor when incorporated into synthetic proteoliposomes containing lecithin /thiocholesterol/apo A-I in the molar ratios of 250:15:0.8. The K m for thiocholesterol was 203.6 μM with a V max of 5.3 nmol thiocholesteryl ester formed/h per μg. The K m for cholesterol when substituted for thiocholesterol in the proteoliposomes was 29.5 μM with a V max of 8.8 nmol cholesteryl ester formed/h per μg. Thiocholesterol and cholesterol were shown to occupy the same catalytic site in LCAT. Thus, thiocholesterol exhibits approx. 10% of the substrate efficiency of cholesterol when incubated with pure human LCAT. We conclude that LCAT can transacylate a fatty acyl moiety from the sn-2 position of lecithin to the 3ß-OH group of thiocholesterol forming a cholesteryl thioester. Although the 3ß-SH group is not as good a terminal acceptor as the 3ß-SH group of cholesterol, LCAT is clearly capable of transacylating a fatty acid esterified via an oxyester linkage to one containing a thioester.

Secretion of active human lecithin-cholesterol acyltransferase by insect cells infected with a recombinant baculovirus.

Difficulties in purifying the plasma enzyme lecithin-cholesterol acyltransferase (LCAT) have hampered detailed studies of its (patho)physiological role in lipoprotein metabolism and of structure-function relationships. Potentially, baculovirus-driven expression systems offer a powerful means to produce significant amounts of LCAT. Accordingly, full-length LCAT cDNA was cloned into pVL 1392, a high-level expression derivative of Autographa californica nuclear polyhedrosis virus (AcNPV), and the resultant plasmid was co-transfected into Trichoplusia ni insect cells (High 5 line) with a linearized viral DNA using lipofectin. Such viral DNA had a lethal mutation and grew only when recombined with a pVL1392-type rescue plasmid; cells infected with recombinant Autographa californica LCAT virus changed from a fibroblast-like morphology to rounded, but lacked the polyhedrin occlusion bodies characteristic of wild-type AcNPV infections. Enzymically active recombinant LCAT (rLCAT), sensitive to sulphydryl reagents, was secreted in the late phase of infection (36-48 h) but was absent with wild-type infections. The secreted protein had an apparent molecular mass of 53 kDa by SDS/PAGE, lower than that of native plasma LCAT; it was susceptible to endoglycosidase H digestion and was bound by concanavalin A, suggesting that precursor high-mannose N-glycan chains had not undergone full maturation to complex types. Pretreatment of the cells with tunicamycin to inhibit the first step of N-glycosylation led to intracellular accumulation of immature rLCAT (approximately 46-48 kDa) and a marked reduction in enzyme secreted. We conclude that the baculovirus gene-expression system will permit production of biologically active normal and mutant forms of LCAT protein.

Tissue-specific expression of the human gene for lecithin: cholesterol acyltransferase in transgenic mice alters blood lipids, lipoproteins and lipases towards a less atherogenic profile.

Lecithin:cholesterol acyltransferase (LCAT) is a key enzyme in the reverse cholesterol pathway but its role in lipid metabolism is still unclear. We have generated mice transgenic for a 7-kb genomic DNA fragment comprising the 6 exons and 5 introns of the LCAT gene with 1932 bp of 5' flanking and 908 bp of 3' flanking sequences. One line had integrated about 30 copies and expressed about 40-fold increased LCAT activity in a human test system. The expression showed correct tissue specificity of the human LCAT gene. Increased LCAT activity resulted in a decrease of plasma triacylglycerols below 50% of fasting controls. This reduction was seen in all lipoprotein fractions. Lipoprotein lipase activity did not change significantly, whereas hepatic triacylglycerol lipase increased markedly. Plasma total cholesterol was similar in fasting transgenic and control mice, but low-density lipoprotein and very low-density lipoprotein cholesterol were reduced to about 50%. High-density lipoprotein cholesterol increased about 20%, accompanied by a correspondingly increased size and a higher cholesterol efflux-stimulating activity of transgenic LCAT high-density lipoprotein. Both apolipoprotein A-I and A-II plasma concentrations increased in transgenic mice. Plasma triacylglycerol and cholesteryl ester fatty acid distribution showed an increased proportion of palmitic acid, whereas oleic, linoleic and arachidonic acid decreased, thus resembling more closely the human situation. Overexpression of the human LCAT gene provokes major changes in plasma lipoprotein and apolipoprotein concentrations, resulting in a less atherogenic plasma lipoprotein profile through a reduction in atherogenic and an increase in anti-atherogenic lipoproteins.

Overexpression of Human Lecithin Cholesterol Acyltransferase Leads to Hyperalphalipoproteinemia in Transgenic Mice

Lecithin cholesterol acyltransferase (LCAT) is a key enzyme which catalyzes the esterification of free cholesterol present in plasma lipoproteins. In order to evaluate the role of LCAT in HDL metabolism, a 6.2-kilobase (kb) fragment consisting of 0.851 and 1.134 kb of the 5'- and 3'-flanking regions, as well as the entire human LCAT gene, was utilized to develop transgenic mice. Three different transgenic mouse lines overexpressing human LCAT at plasma levels 11-, 14-, and 109-fold higher than non-transgenic mice were established. Northern blot hybridization analysis demonstrated that the injected 6.2-kb fragment contained the necessary DNA sequences to direct tissue specific expression of the human LCAT gene in mouse liver. Compared to age- and sex-matched controls, total cholesterol and HDL cholesterol levels were increased in all 3 transgenic mice lines by 124-218 and 123-194%, respectively, while plasma triglyceride concentrations remained similar to that of control animals. Fast protein liquid chromatography analysis of transgenic mouse plasma revealed marked increases in high density liposportin (HDL)-cholesteryl ester and phospholipid as well as the formation of larger size HDL. Thus, the majority of the increase in transgenic plasma cholesterol concentrations was due to accumulation of cholesteryl ester in HDL consistent with enhanced esterification of free cholesterol in mouse HDL by human LCAT. Plasma concentrations of apoA-I, apoA-II, and apoE were increased in high expressor homozygote mice who also demonstrated an accumulation of an apoE-rich HDL1. Like the mouse enzyme, human LCAT was found to be primarily associated with mouse HDL. Our studies demonstrate a high correlation between plasma LCAT activity and total as well as HDL cholesterol levels establishing that in mice LCAT modulates plasma HDL concentrations. Overexpression of LCAT in mice leads to HDL elevation as well as increased heterogeneity of the HDL lipoprotein particles, indicating that high levels of plasma LCAT activity may be associated with hyperalphalipoproteinemia and enhanced reverse cholesterol transport.

Site-specific detection and structural characterization of the glycosylation of human plasma proteins lecithin:cholesterol acyltransferase and apolipoprotein D using HPLC/electrospray mass spectrometry and sequential glycosidase digestion.

Site-specific structural characterization of the glycosylation of human lecithin:cholesterol acyltransferase (LCAT) was carried out using microbore reversed-phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESIMS). A recently described mass spectrometric technique involving monitoring of carbohydrate-specific fragment ions during HPLC/ESIMS was employed to locate eight different groups of glycopeptides in a digest of a human LCAT protein preparation. In addition to the four expected N-linked glycopeptides of LCAT, a di-O-linked glycopeptide was detected, as well as three additional glycopeptides. Structural information on the oligosaccharides from all eight glycopeptides was obtained by sequential glycosidase digestion of the glycopeptides followed by HPLC/ESIMS. All four potential N-linked glycosylation sites (Asn20, Asn84, Asn272, and Asn384) of LCAT were determined to contain sialylated triantennary and/or biantennary complex structures. Two unanticipated O-linked glycosylation sites were identified at Thr407 and Ser409 of the LCAT O-linked glycopeptide, each of which contain sialylated galactose beta 1-->3N-acetylgalactosamine structures. The three additional glycopeptides were determined to be from a copurifying protein, apolipoprotein D, which contains potential N-linked glycosylation sites at Asn45 and Asn78. These glycopeptides were determined to bear sialylated triantennary oligosaccharides or fucosylated sialylated biantennary oligosaccharides. Previous studies of LCAT indicated that removal of the glycosylation site at Asn272 converts this protein to a phospholipase (Francone OL, Evangelista L, Fielding CJ, 1993, Biochim Biophys Acta 1166:301-304). Our results indicate that the carbohydrate structures themselves are not the source of this functional discrimination; rather, it must be mediated by the structural environment around Asn272.

Effects of site-directed mutagenesis on the serine residues of human lecithin:cholesterol acyltransferase.

Lecithin:cholesterol acyltransferase (LCAT) is a serine protease-type enzyme that esterifies cholesterol in human plasma and is activated by apolipoprotein A-I in high-density lipoproteins. LCAT contains 22 serine residues, including Ser181, which is thought to be part of the catalytic site. In order to determine the importance of these serine residues in LCAT, we prepared six LCAT mutants: LCAT (Ser19-->Ala), LCAT (Ser181-->Gly), LCAT (Ser208-->Ala), LCAT (SEr216-->Ala), LCAT (Ser225-->Ala) and LCAT (Ser383-->Ala). We also replaced the adjacent asparagine residues in two additional mutants, LCAT (Ser19-->Ala, Asn20-->Thr) and LCAT (Ser383-->Ala, Asn384-->Thr), in order to ascertain the effect of the serines on N-glycosylation. The mutant complementary DNA (cDNA) were subcloned into a eukaryotic expression vector (pSG5) and expressed in COS-6 cells. By polymerase chain reaction analysis, LCAT-specific messenger RNA (mRNA) was found in all mutant and wild-type transfectants. Western blot analysis revealed LCAT-specific bands in media and lysates of the transfected cells. With two exceptions, the amounts of LCAT mass secreted by the transfectants were similar to that of the wild type (mean, 90% mass of wild type; range, 34-138%). Except for LCAT (Ser181-->Gly), which was inactive, the specific activities of the remainder of the mutant enzymes were also similar (mean 95% activity of wild type; range, 65-169%). These results indicate that Ser181 is part of the catalytic site and that stereoconservative substitutions for serines have minor effects on the synthesis, secretion and specific activities of human LCAT.

Role of sn-2 acyl group of phosphatidylcholine in determining the positional specificity of lecithin-cholesterol acyltransferase.

Although human plasma lecithin-cholesterol acyltransferase (LCAT) is believed to be specific for the sn-2 position of phosphatidylcholine (PC), our recent studies showed that it derives a significant percent of acyl groups from the sn-1 position of certain PC species. To understand the physicochemical basis for this altered positional specificity, we determined the effect of sn-2 acyl group of PC on the enzyme activity and utilization of 16:0 from the sn-1 position by purified human and rat LCATs. Positional isomers of PC containing 16:0 at sn-2 were better substrates for human LCAT than the corresponding sn-1-16:0 isomers, whereas the reverse was true for rat LCAT. The positional specificity of human LCAT varied greatly depending on the nature of the acyl group at sn-2. The sn-1 contribution from various sn-1-16:0-2-acyl PCs for cholesteryl ester (CE) synthesis was 1.0% from 16:0-16:0, 1.4% from 16:0-20:5, 7.3% from 16:0-18:1, 47.0% from 16:0-20:3, 49.9% from 16:0-20:4, 54.9% from 16:0-22:6, and 72.3% from 16:0-18:0. There was a linear relationship between the percentage of 16:0 CE formed (from sn-1 position) and the acyl chain length at sn-2 position (r = 0.94). Rat LCAT also transferred some 16:0 from sn-1 position of 16:0-22:6, 16:0-20:3, and 16:0-18:0 PCs, but not from the other natural PCs tested. The phospholipase A activity of both LCATs in the presence of 16:0-20:4 PC showed the same positional specificity as CE synthesis, indicating that the specificity is determined at the formation of acyl-enzyme intermediate. These results show that the positional specificity of LCAT is influenced by the structure of PC, especially the chain length of the sn-2 acyl group.

Substrate and positional specificities of human and mouse lecithin-cholesterol acyltransferases. Studies with wild type recombinant and chimeric enzymes expressed in vitro

Human lecithin-cholesterol acyltransferase (LCAT) preferentially attacks sn-1 position of 16:0–20:4 phosphatidylcholine (PC), producing more 16:0 cholesteryl ester (CE) than 20:4 CE. In contrast, rat and mouse LCATs produce mostly 20:4 CE from the same PC. To understand the structural basis for this difference in positional specificity, we studied the specificities of recombinant mouse and human LCATs and several chimeric constructs of the two. The rLCATs retained the substrate and positional specificities of the plasma enzymes when expressed in COS-1 cells. Human and mouse LCAT cDNAs were each cleaved into three fragments, recombined in various combinations, and the chimeric products were analyzed for their specificities. When the N-terminal, or (and) C-terminal segments of human LCAT were replaced by the corresponding mouse LCAT segments, the chimeric products exhibited the specificity of intact human enzyme. However, when the middle segment, containing the residues 130–306 was replaced by the corresponding mouse LCAT segment, the enzyme exhibited the specificity of mouse LCAT. Similarly, the mouse rLCAT exhibited the specificity of human enzyme when its central segment, but not its N-terminal or C-terminal segment was replaced by the corresponding segment from human LCAT. These results show that the substrate and positional specificities of LCAT are controlled by the central domain of LCAT protein, corresponding to the amino acid residues 130–306.

Overexpression of human lecithin-cholesterol acyl transferase in transgenic rabbits leads to hyperalphalipoproteinemia

Overexpression of human lecithin-cholesterol acyl transferase in transgenic rabbits leads to hyperalphalipoproteinemia