Lecithin cholesterol acyltransferase

Cholesterol transport in circulation and its removal from tissues depends on the activity of lecithin cholesterol acyltransferase (LCAT). LCAT is a soluble enzyme that converts cholesterol and phosphatidylcholines (lecithins) to cholesteryl esters and lyso-phosphatidylcholines on the surface of high-density lipoproteins. This review presents key background information and recent research advances on the structure of human LCAT, its reactions and substrates, and the expression of the LCAT gene. While the three-dimensional structure of LCAT is not yet known, a partial model now exists that facilitates the study of structure-function relationships of the native enzyme, and of natural and engineered mutants. The LCAT reaction on lipoproteins consists of several steps, starting with enzyme binding to the lipoprotein/lipid surface, followed by activation of LCAT by apolipoproteins, binding of lipid substrates and the catalytic steps giving rise to the lipid products. Quantitative data are presented on the kinetic and equilibrium constants of some of the LCAT reaction steps. Finally, overexpression of the human LCAT gene in mice and rabbits has been used to examine the physiologic role of LCAT in vivo and its protective effect against diet induced atherosclerosis.     

Lecithin:cholesterol acyltransferase activation by synthetic amphipathic peptides

The amphipathic helical theory of Segrest and colleagues (FEBS Lett.:38:247-253, 1974) proposes that the lipid-binding segments of serum apolipoproteins are in an alpha helical conformation. Furthermore the helices have a hydrophobic face and a hydrophilic face with a specific distribution of positively and negatively charged residues. The importance of the pattern of the charged residues in the lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation by the segments is still debated. We designed a 30-residue peptide, GALA, which in the alpha helical conformation has a hydrophilic face composed of glutamic acid residues (Sabbarao et al.: Biochemistry 26:2964-2972, 1987). GALA behaves like the serum apolipoproteins in its interaction with dimyristoylphosphatidylcholine (DMPC) at neutral pH; the amino terminal tryptophan of GALA undergoes a blue shift in its fluorescence emission spectrum, and the circular dichroism (CD) spectrum indicates that GALA acquires alpha helical structure in the presence of DMPC. A DMPC-GALA:19/1 (molar ratio) complex can be isolated by gel-permeation chromatography. This complex has a discoidal structure with the approximate dimensions of 44-A edge thickness and a 170- to 350-A diameter. GALA activates LCAT with DMPC but not with unsaturated phospholipids as the substrate. The apparent partition coefficient of GALA into DMPC vesicles is 100-fold larger than into egg phosphatidylcholine vesicles. The interaction of GALA with unsaturated lipids at neutral pH is so weak that no detectable change in the spectroscopic properties of GALA or the structure of the liposomes can be detected under the conditions used here. The sequence of GALA differs from previously studied model Apo A1 peptides by the absence of positively charged residues on the hydrophilic face. This indicates that positive charges in Apo A1-like peptides are not required in order to form discoidal structures with saturated phospholipids or to activate LCAT with such lipid substrates.     

Lecithin:cholesterol acyltransferase activity and HDL composition in serum of patients with kidney transplants

In comparison with a control group, lecithin:cholesterol acyltransferase activities and HDL-cholesterol concentrations in serum of patients with kidney transplants were unchanged, while apolipoprotein A and HDL-triglyceride concentrations were increased. This constellation shows that the cholesterol metabolism in patients with kidney transplants differs from that of patients undergoing chronic haemodialysis.     

Lecithin/cholesterol acyltransferase modulates diet-induced hepatic deposition of triglycerides in mice

Lecithin/cholesterol acyltransferase (LCAT) is responsible for the esterification of the free cholesterol of plasma lipoproteins. Here, we investigated the involvement of LCAT in mechanisms associated with diet-induced hepatic triglyceride accumulation in mice. LCAT-deficient (LCAT^?/?) and control C57BL/6 mice were placed on a Western-type diet (17.3% protein, 48.5% carbohydrate, 21.2% fat, 0.2% cholesterol, 4.5 kcal/g) for 24 weeks, then histopathological and biochemical analyses were performed. We report that, in our experimental setup, male LCAT^?/? mice are characterized by increased diet-induced hepatic triglyceride deposition and impaired hepatic histology and architecture. Mechanistic analyses indicated that LCAT deficiency was associated with enhanced intestinal absorption of dietary triglycerides (3.6±0.5 mg/dl per minute for LCAT^?/? vs. 2.0±0.7 mg/dl per minute for C57BL/6 mice; P<.05), accelerated clearance of postprandial triglycerides and a reduced rate of hepatic very low density lipoprotein triglyceride secretion (9.8±1.1 mg/dl per minute for LCAT^?/? vs. 12.5±1.3 mg/dl per minute for C57BL/6 mice, P<.05). No statistical difference in the average daily food consumption between mouse strains was observed. Adenovirus-mediated gene transfer of LCAT in LCAT^?/? mice that were fed a Western-type diet for 12 weeks resulted in a significant reduction in hepatic triglyceride content (121.2±5.9 mg/g for control infected mice vs. 95.1±5.8 mg/g for mice infected with Ad-LCAT, P<.05) and a great improvement of hepatic histology and architecture. Our data extend the current knowledge on the functions of LCAT, indicating that LCAT activity is an important modulator of processes associated with diet-induced hepatic lipid deposition.     

Lecithin:cholesterol acyltransferase regulation. Effect of fluidity of dimyristoylphosphatidylcholine vesicles

The regulation of lecithin:cholesterol acyltransferase by changes in phospholipid bilayer fluidity was investigated using pyrene excimer fluorescence to measure fluidity. Fluidity of dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles was decreased by the addition of up to 20% (mol/mol) cholesterol and increased by the addition of up to 10% (mol/mol) lysoDMPC. When both cholesterol and lysoDMPC are present in the bilayer, their individual effects on fluidity are altered. These changes can be explained by complex formation between cholesterol and phospholipid as in the model of Presti et al. (Presti, F.C., Pace, R.J. and Chan, S.I. (1982) Biochemistry 21, 3831-3335). Lecithin:cholesterol acyltransferase activity with these vesicles as substrates was measured to determine whether activity can be modulated by the fluidity changes of the bilayer on which the enzyme acts. When 10% lysoDMPC, a known lecithin:cholesterol acyltransferase inhibitor, is added to the vesicles, inhibition of activity is observed. When 7.5% lysoDMPC is added to vesicles which contain either 5 or 10% cholesterol, lecithin:cholesterol acyltransferase activity increases. This increase in lecithin:cholesterol acyltransferase activity due to vesicle-fluidity increase is sufficient to overcome the decrease in activity due to lecithin:cholesterol acyltransferase inhibition. This is the first report of the ability of lysoDMPC to increase lecithin:cholesterol acyltransferase activity.     

Lecithin:cholesterol acyltransferase deficiency protects against cholesterol-induced hepatic endoplasmic reticulum stress in mice

We recently reported that lecithin:cholesterol acyltransferase (LCAT) knock-out mice, particularly in the LDL receptor knock-out background, are hypersensitive to insulin and resistant to high fat diet-induced insulin resistance (IR) and obesity. We demonstrated that chow-fed Ldlr-/-xLcat+/+ mice have elevated hepatic endoplasmic reticulum (ER) stress, which promotes IR, compared with wild-type controls, and this effect is normalized in Ldlr-/-xLcat-/- mice. In the present study, we tested the hypothesis that hepatic ER cholesterol metabolism differentially regulates ER stress using these models. We observed that the Ldlr-/-xLcat+/+ mice accumulate excess hepatic total and ER cholesterol primarily attributed to increased reuptake of biliary cholesterol as we observed reduced biliary cholesterol in conjunction with decreased hepatic Abcg5/g8 mRNA, increased Npc1l1 mRNA, and decreased Hmgr mRNA and nuclear SREBP2 protein. Intestinal NPC1L1 protein was induced. Expression of these genes was reversed in the Ldlr-/-xLcat-/- mice, accounting for the normalization of total and ER cholesterol and ER stress. Upon feeding a 2% high cholesterol diet (HCD), Ldlr-/-xLcat-/- mice accumulated a similar amount of total hepatic cholesterol compared with the Ldlr-/-xLcat+/+ mice, but the hepatic ER cholesterol levels remained low in conjunction with being protected from HCD-induced ER stress and IR. Hepatic ER stress correlates strongly with hepatic ER free cholesterol but poorly with hepatic tissue free cholesterol. The unexpectedly low ER cholesterol seen in HCD-fed Ldlr-/-xLcat-/- mice was attributable to a coordinated marked up-regulation of ACAT2 and suppressed SREBP2 processing. Thus, factors influencing the accumulation of ER cholesterol may be important for the development of hepatic insulin resistance.     

Lecithin: cholesterol acyltransferase is insufficient to prevent oxidative modification of low-density lipoprotein.

We tried to confirm the antioxidative capability of lecithin:cholesterol acyltransferase (LCAT) reported by Vohl et al. [Biochemistry (1999) 38, 5976-5981]. The enzyme solution protected LDL against oxidation. However, this protection was not due to LCAT enzyme, but to some unknown low-molecular-weight substance(s) in the solution; LCAT itself exerted little protective effect against LDL oxidation.     

Lecithin:cholesterol acyltransferase. Functional regions and a structural model of the enzyme

The amino acid sequence of human lecithin:cholesterol acyltransferase has been determined by degradation and alignment of peptides obtained from tryptic and staphylococcal digestions and the cleavage with cyanogen bromide and consisted of 416 amino acid residues. All of the tryptic peptides of lecithin:cholesterol acyltransferase were isolated and sequenced. Peptides resulting from digestion by staphylococcal protease, cyanogen bromide cleavage, or the combination of the two methods were employed to find overlapping segments. The N terminus of human lecithin:cholesterol acyltransferase was determined to be phenylalanine by sequencing the whole protein up to 40 residues while the C terminus was identified as glutamic acid through carboxypeptidase Y cleavage. Cys50 and Cys74 and Cys313 and Cys356 were identified as the two disulfide bridges while the free sulfhydryl groups were located at positions 31 and 184. The N-glycosylated sites of the protein were assigned to asparagines at positions 20, 84, 272, and 384. The active site of lecithin:cholesterol acyltransferase was identified as serine on position 181 according to its homology with other serine-type esterases which have a common structure of glycine-variable amino acid-active serine-variable amino acid-glycine (Gly-X-Ser-X-Gly) with the variable amino acids disrupting the homology. No long internal repeats or homologies with apolipoproteins were found. The secondary structure is consistent with the results of predictive algorithms. A simple model of the enzyme is proposed on the basis of available chemical data and predictive methods.     

Lecithin retinol acyltransferase forms functional homodimers

Membrane-bound lecithin retinol acyltransferase (LRAT), an essential enzyme in vitamin A processing, catalyzes the formation of retinyl esters from vitamin A and lecithin. Cloned and expressed LRAT has a molecular mass of 25.3 kDa. The enzyme is not homologous to known enzymes and is, therefore, of substantial interest mechanistically. Along these lines, the functional protomeric state of LRAT is of importance. Gel electrophoretic studies on LRAT in the presence of SDS and disulfide reducing agents show the expected 25 kDa monomer. However, gel electrophoresis in the absence of a reducing agent and/or strong denaturing conditions reveals substantial dimer formation. LRAT monomers can be efficiently and irreversibly cross-linked by thiol reactive bismaleimides in retinal pigment epithelial (RPE) membranes generating LRAT homodimers. Cross-linked LRAT homodimers are fully active catalytically. The experiments suggest that LRAT monomers interact in membranes and form functional homodimers through protein-protein interactions and disulfide bond formation.     

Lecithin:cholesterol acyltransferase regulation. II. Effect of fluidity of egg phosphatidylcholine vesicles

The regulation of human plasma lecithin:cholesterol acyltransferase (LCAT) by changes in bilayer fluidity of substrate egg phosphatidylcholine (egg PC) unilamellar vesicles was investigated using pyrene excimer fluorescence to measure fluidity. Fluidity was decreased by adding up to 20% cholesterol or increased by adding up to 10% egg 2-lysophosphatidylcholine (lysoPC). The fluidizing effect of lysoPC was suppressed by the addition of cholesterol. LCAT activity with 10% cholesterol vesicles was decreased by adding 5% lysoPC, yet activity with 5% cholesterol vesicles was unaffected by adding 5% lysoPC. This difference may be explained by a balance between the known LCAT inhibitory effect of lysoPC and its ability to increase bilayer fluidity and thereby increase LCAT activity. LCAT esterification of up to 37% of vesicle cholesterol failed to alter the lysoPC/cholesterol balance sufficiently to influence activity in this system. The findings of our studies are in keeping with modulation of LCAT activity by bilayer fluidity, but fluidity changes caused by enzyme action are not sufficient to regulate that activity.     

Upregulation of acyl-CoA: cholesterol acyltransferase in chronic renal failure

Chronic renal failure (CRF) is associated with profound abnormalities of lipid metabolism and accelerated arteriosclerotic cardiovascular disease. In a recent study, we found marked downregulation of hepatic lecithin-cholesterol acyltransferase, or LCAT, expression, which can account for impaired HDL maturation and depressed HDL cholesterol concentration in CRF. Here, we report on the effect of CRF on acyl-CoA:cholesterol acyltransferase (ACAT) expression. ACAT is an intracellular enzyme that catalyzes esterification of free cholesterol to cholesterol ester for storage or secretion. ACAT plays a major role in hepatic production and release of VLDL, intestinal absorption of cholesterol, foam cell formation, and atherogenesis. We examined hepatic expression of ACAT-1 and ACAT-2 mRNA (Northern blot) and protein (Western blot) abundance and total ACAT activity in male CRF rats (6 wk after 5/6 nephrectomy) and sham-operated controls. The CRF animals showed a significant reduction in creatinine clearance, marked hypertriglyceridemia, modest hypercholesterolemia, and significant upregulation of hepatic tissue ACAT-2 protein and mRNA abundance. In contrast, hepatic ACAT-1 mRNA and protein abundance were unaffected by CRF. Upregulation of ACAT-2 expression was accompanied by a significant increase in hepatic ACAT activity and a significant decrease in hepatic microsomal and whole liver free cholesterol concentration. Thus CRF results in significant upregulation of hepatic ACAT-2 (but not ACAT-1) expression and ACAT activity, which may, in part, contribute to the associated lipid disorders.     

Decreased activity of lecithin:cholesterol acyltransferase and hepatic lipase in chronic hypothyroid rats: Implications for reverse cholesterol transport

Chronic hypothyroidism is frequently associated with atherosclerosis due to increased cholesterol plasma levels; nevertheless, the contribution of impaired reverse cholesterol transport (RCT) in this process has not been completely elucidated. The aim of this study was to evaluate the effect of thyroidectomy (Htx) upon the main stages of RCT in rats. Plasma lipid alterations induced by thyroidectomy showed a slight, but significant, reduction of total plasma triglycerides, a 300% increase of LDL-cholesterol and a 25% decrease in HDL-cholesterol compared to control rats. We evaluated the first stage of RCT determining ₃H-cholesterol efflux in Fu5AH cells. The capacity of HDL obtained from Htx rats to promote cholesterol efflux was similar to that of controls. Lecithin:cholesterol acyltransferase (LCAT) activity, the second stage and the driving force of RCT was 30% lower in Htx animals compared to controls, as determined by reconstituted HDL used as an external substrate. Lipoproteins are remodeled by hepatic lipase; the mean activity of this enzyme in postheparin plasma of Htx animals was reduced by 30% compared to controls, thus suggesting an impaired HDL remodeling by this enzyme in the hypothyroid status. In contrast, lipoprotein lipase activity in the Htx group was unchanged. In summary, this study demonstrates that chronic hypothyroidism in the rat induced an impaired RCT mainly at the cholesterol esterification, and HDL remodeling mediated by hepatic lipase. The latter probably results in an abnormal HDL structure, i.e. phospholipid enrichment, which contributes to decrease HDL-apo AI fractional catabolic rates.