Mutant Lecithin:cholesterol Acyltransferase Research Articles

P-Methoxycinnamic Acid Diesters Lower Dyslipidemia, Liver Oxidative Stress and Toxicity in High-Fat Diet Fed Mice and Human Peripheral Blood Lymphocytes.

The pursuit of cholesterol lowering natural products with less side effects is needed for controlling dyslipidemia and reducing the increasing toll of cardiovascular diseases that are associated with morbidity and mortality worldwide. The present study aimed at the examining effects of p-methoxycinnamic acid diesters (PCO-C) from carnauba (Copernicia prunifera)-derived wax on cytotoxic, genotoxic responses in vitro and on dyslipidemia and liver oxidative stress in vivo, utilizing high-fat diet (HFD) chronically fed Swiss mice. In addition, we evaluated the effect of PCO-C on the expression of key cholesterol metabolism-related genes, as well as the structural interactions between PCO-C and lecithin-cholesterol acyl transferase (LCAT) in silico. Oral treatment with PCO-C was able to reduce total serum cholesterol and low-density lipoprotein (LDL) levels following HFD. In addition, PCO-C reduced excessive weight gain and lipid peroxidation, and increased the gene expression of LCAT following HFD. Furthermore, the high affinity of the studied compound (ΔG: −8.78 Kcal/mol) towards the active sites of mutant LCAT owing to hydrophobic and van der Waals interactions was confirmed using bioinformatics. PCO-C showed no evidence of renal and hepatic toxicity, unlike simvastatin, that elevated aspartate aminotransferase (AST) levels, a marker of liver dysfunction. Finally, PCO-C showed no cytotoxicity or genotoxicity towards human peripheral blood lymphocytes in vitro. Our results suggest that PCO-C exerts hypocholesterolemic effects. The safety of PCO-C in the toxicological tests performed and the reports of its beneficial biological effects render this a promising compound for the development of new cholesterol-lowering therapeutics to control dyslipidemia. More work is needed for further elucidating PCO-C role on lipid metabolism to support future clinical studies.

Abstract 226: Mechanism of LCAT Activation by Compound A

Background: Lecithin:cholesterol acyltransferase (LCAT) catalyzes cholesteryl ester (CE) production from free cholesterol (FC) and phosphatidylcholine (lecithin), promoting HDL formation. Objective: To investigate activation of LCAT by Compound A (Amgen), a previously described small-molecule activator of LCAT, with the ultimate goal of developing novel LCAT activators for therapeutic use. Methods: LCAT activity in plasma from Familial LCAT Deficiency (FLD) patients with different mutations was quantitated by TLC before and after addition of Compound A. HEK293 cells were transiently transfected with plasmids containing wild-type (WT) or mutant LCAT cDNA. Media was then incubated with either vehicle or Compound A and LCAT activity was quantitated using a novel plate assay utilizing Methylumbelliferyl Palmitate as a substrate. Results: Compound A increased LCAT activity for a subset of FLD mutations to a level above which renal disease may occur. Mutations of Cys31 in vitro strongly affected basal LCAT activity as well as activation by Compound A. Charged residues at position 31 profoundly decreased activity whereas bulky hydrophobic groups increased LCAT activity up to 3-fold (p < 0.005, all). Mass spectrometry of WT LCAT incubated with Compound A revealed a +103.017 m/z adduct to the tryptic peptide containing Cys31, indicative of a cyanopyrazine adduct to LCAT Cys31. Molecular modeling identified potential binding sites of Compound A to LCAT. Conclusions: Our findings yield important mechanistic insight into LCAT activation that can be used to design novel LCAT activators for therapeutic use.

Plasma lecithin:cholesterol acyltransferase and carotid intima-media thickness in European individuals at high cardiovascular risk

Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for cholesterol esterification in plasma. LCAT is a major factor in HDL remodeling and metabolism, and it has long been believed to play a critical role in macrophage reverse cholesterol transport (RCT). The effect of LCAT on human atherogenesis is still controversial. In the present study, the plasma LCAT concentration was measured in all subjects (n = 540) not on drug treatment at the time of enrollment in the multicenter, longitudinal, observational IMPROVE study. Mean and maximum intima-media thickness (IMT) of the whole carotid tree was measured by B-mode ultrasonography in all subjects. In the entire cohort, LCAT quartiles were not associated with carotid mean and maximum IMT (P for trend 0.95 and 0.18, respectively), also after adjustment for age, gender, HDL-cholesterol (HDL-C), and triglycerides. No association between carotid IMT and LCAT quartiles was observed in men (P=0.30 and P=0.99 for mean and maximum IMT, respectively), whereas carotid IMT increased with LCAT quartiles in women (P for trend 0.14 and 0.019 for mean and maximum IMT, respectively). The present findings support the concept that LCAT is not required for an efficient reverse cholesterol transport and that a low plasma LCAT concentration and activity is not associated with increased atherosclerosis.

Proteinuria in early childhood due to familial LCAT deficiency caused by loss of a disulfide bond in lecithin:cholesterol acyl transferase

IntroductionFamilial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare recessive disorder of cholesterol metabolism characterized by the absence of high density lipoprotein (HDL) and the triad of corneal opacification, hemolytic anemia and glomerulopathy. PatientsWe here report on FLD in three siblings of a kindred of Moroccan descent with HDL deficiency. In all cases (17, 12 and 3 years of age) corneal opacification and proteinuria were observed. In the 17-year-old female proband, anemia with target cells was observed. ResultsHomozygosity for a mutation in LCAT resulted in the exchange of cysteine to tyrosine at position 337, disrupting the second disulfide bond in LCAT. LCAT protein and activity were undetectable in the patients’ plasma and in media of COS7 cells transfected with an expression vector with mutant LCAT cDNA. Upon treatment with an ACE inhibitor and a thiazide diuretic, proteinuria in the proband decreased from 6g to 2g/24h. ConclusionThis is the first report that FLD can cause nephropathy at a very early age.

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).

Two novel point mutations in the lecithin:cholesterol acyltransferase (LCAT) gene resulting in LCAT deficiency: LCAT (G873 deletion) and LCAT (Gly344–>Ser)

We investigated the genetic defects in two patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency. Their clinical manifestations including corneal opacities, anemia, proteinuria, and hypoalphalipoproteinemia were identical for familial LCAT deficiency. Their LCAT activities and the cholesterol esterification rate (CER) were nearly zero, and their LCAT masses were below 10% of normal control values. Sequence analysis of the amplified DNA of case 1 revealed one base deletion of G at base 873 (first position of Val264) in exon 6, leading to a premature termination by frameshift. Sequence analysis of amplified DNA of case 2 revealed a single G to A converting Gly (GGT) to Ser (AGT) substitution at residue 344. When COS-1 cells were transfected with these mutants, LCAT activity in the medium was nearly zero, and the LCAT mass was undetectable (< 0.01 microgram/ml). In contrast, LCAT activity in the medium of COS-1 cells, transfected with wild-type LCAT, was 1.7 nmol/h per ml and the LCAT mass was 0.09 micrograms/ml. The LCAT mass in the cell lysates of the mutants was less than 12% of control for case 1 and 18% of control for case 2. Northern blot analysis of the mRNA of COS-1 cells transfected with the mutants showed the same amounts of LCAT mRNA as compared with wild-type LCAT. Biosynthesis of mutant LCATs was analyzed by pulse-chase and immunocytochemistry in transfected baby hamster kidney cells. SDS-PAGE/fluorography demonstrated that wild-type LCAT was synthesized as a high-mannose type of 56 kDa, which was very slowly converted to a mature form of 67 kDa and was secreted into the media. In contrast to the wild-type LCAT, the mutant precursors were not processed into the mature form but slowly degraded along with chase times. On steady and continuous labeling in the case of wild-type LCAT, the mature 67 kDa form was observed in both the cell lysate and media, whereas no mature form was detected in the cell lysates and media which were transfected mutant LCATs. These data suggest that the mutant LCATs are actually synthesized in an amount comparable to that of wild-type, but they are slowly degraded without being processed into the mature form. The immunocytochemistry revealed that mutant LCATs were mainly retained in the endoplasmic reticulum. These data suggest that these two mutations may disrupt the mutant LCATs' transport from the endoplasmic reticulum into Golgi apparatus, resulting in LCAT deficiency.

A single G to A nucleotide transition in exon IV of the lecithin: cholesterol acyltransferase (LCAT) gene results in an Arg140 to His substitution and causes LCAT-deficiency.

We have characterized the molecular defect causing lecithin:cholesterol acyltransferase (LCAT)-deficiency (LCAT-D) in the LCAT gene in three siblings of Austrian descent. The patients presented with typical symptoms including corneal opacity, hemolytic anemia, and kidney dysfunction. LCAT activities in the plasma of these three patients were undetectable. DNA sequence analysis of polymerase chain reaction (PCR)-amplified DNA of all six LCAT exons revealed a new point mutation in exon IV of the LCAT gene, i.e., a G to A substitution in codon 140 converting Arg to His. This mutation caused the loss of a cutting site for the restriction endonuclease HhaI within exon IV: Upon digestion of a 629-bp exon IV PCR product with HhaI, the patients were found to be homozygous for the mutation. Eight of 11 family members were identified as heterozygotes. Transfection studies of COS-7 cells with plasmids containing a wild-type or a mutant LCAT cDNA revealed that, in contrast to the cell medium containing wild-type enzyme, no enzyme activity was detectable upon expression of the mutant protein. This represents strong evidence for the causative nature of the observed mutation for LCAT deficiency in affected individuals and supports the conclusion that Arg140 is crucial for the structure of an enzymatically active LCAT protein.

Lecithin: cholesterol acyltransferase deficiency: identification of two defective alleles in fibroblast cDNA.

Previous mutations associated with lecithin:cholesterol acyltransferase (LCAT) deficiency have been identified using genomic DNA. To facilitate mutation analysis, we used cDNA from cultured fibroblasts which were shown to express LCAT mRNA. Using reverse-transcriptase PCR, LCAT cDNA was obtained from a 13-year-old boy with complete LCAT deficiency, characterized by low HDL-C (3 mg/dl), nondetectable initial cholesterol esterification rate, LCAT activity, and minimal LCAT mass (0.16 vs. 5-7.5 micrograms/ml). Sequencing of LCAT cDNA clones identified two mutations. A novel frameshift mutations caused by deletion of cytosine at the third nucleotide position of amino acid 168 (exon 5) predicts a disrupted protein catalytic site by converting Ser181-->Ala and creates a Pvu-II restriction site prior to premature truncation at amino acid 238. A C-->T transition results in a substitution of methionine for threonine at amino acid position 321 and creates an Nla-III restriction site on the maternal allele. Expression studies of mutant LCAT cDNA confirmed the virtual absence of LCAT activity in transfected COS-1 cells. The molecular defect in a young male with complete LCAT deficiency has been identified using fibroblast cDNA.

In Vitro Expression of Structural Defects in the Lecithin-Cholesterol Acyltransferase Gene

Classic LCAT deficiency (CLD) and fish eye disease (FED) are two clinically distinct syndromes, associated with defects in the lecithin-cholesterol acyltransferase (LCAT) gene resulting in total (CLD) or partial (FED) enzyme deficiency. In order to investigate the underlying molecular mechanisms that lead to different phenotypic expression in CLD and FED, LCAT mutants associated with either CLD (LCAT147, LCAT156, and LCAT228) or FED (LCAT10, LCAT123, LCAT158, LCAT293, LCAT300, and LCAT347) were expressed in vitro in human embryonic kidney 293 cells and characterized with respect to LCAT expression and enzyme activity. Evaluation of mutant LCAT gene transcription by Northern blot analysis demonstrated LCAT mRNA of normal size and concentration. Although all constructs gave rise to similar intracellular LCAT mass, the amount of enzyme present in the media for LCAT147, LCAT156, and LCAT300 was reduced to less than 10% of normal, suggesting that these mutations disrupted LCAT secretion. Western blot analysis of cell culture media containing wild type or mutant LCAT demonstrated the presence of a single normal-sized band of 67 kDa. The ability of the different enzymes to esterify free cholesterol in high density lipoprotein-like proteoliposomes (alpha-LCAT-specific activity) was reduced to less than 5% of normal for CLD mutants LCAT147 and LCAT228 and FED mutants LCAT10, LCAT123, LCAT293, and LCAT347, whereas that of LCAT156, LCAT158, and LCAT300 ranged from 45 to 110% of control. Although most FED mutant LCAT enzymes retained the ability to esterify free cholesterol present in alpha- and beta-lipoproteins of heat-inactivated plasma, esterification was undetectable in all CLD mutants (LCAT147, LCAT156, and LCAT228). In contrast, all mutant enzymes retained the ability to hydrolyze the water soluble, short-chained fatty acid substrate p-nitrophenolbutyrate. In summary, our studies establish the functional significance of nine LCAT gene defects associated with either FED or CLD. Characterization of the expressed LCAT mutants identified multiple, overlapping functional abnormalities that include defects in secretion and/or disruption of enzymic activity. All nine LCAT mutants retained the ability to hydrolyze the water-soluble PNPB substrate, indicating intact hydrolytic function. Based on these studies we propose that mutations in LCAT residues 147, 156, 228 (CLD) and 10, 123, 158, 293, 300, and 347 (FED) do not disrupt the functional domain mediating LCAT phospholipase activity, but alter structural domains involved in lipid binding or transesterification.

Two different allelic mutations in a Finnish family with lecithin:cholesterol acyltransferase deficiency.

Lecithin:cholesterol acyltransferase (LCAT) deficiency is a genetic disorder associated with low levels of serum HDL cholesterol. The proband of the Finnish LCAT-deficient family had corneal opacities, proteinuria, anemia with stomatocytosis, low serum HDL cholesterol (0.27 mmol/L), and low LCAT activity. Sequence analysis of his LCAT gene revealed compound heterozygosity for two different mutations: a C insertion in exon 1 between nucleotides 932 and 937 and a C-to-T point mutation in exon 6 at position 4976. The C insertion in exon 1 is predicted to result in premature termination and a truncated polypeptide containing only 16 amino acids. The C-to-T point mutation in exon 6 substitutes cysteine for arginine at residue 399. The functional significance of the Arg399-->Cys mutation was examined by expressing the mutated and wild-type LCAT cDNAs in COS cells. COS cells transfected with mutated and wild-type cDNAs showed comparable levels of mature LCAT mRNA. However, LCAT activity in the cell media of COS cells transfected with the mutant LCAT cDNA was significantly lower than that of COS cells transfected with the wild-type cDNA (1.4% versus 12.0% cholesterol esterified, respectively). A polymerase chain reaction-based duplex assay, in which both mutations can be detected simultaneously, was used for preliminary screening of Finnish subjects with serum HDL levels below 0.9 mmol/L; two additional individuals heterozygous for the Arg399-->Cys mutation were identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of N-linked glycosylation of lecithin: cholesterol acyltransferase in lipoprotein substrate specificity

Lecithin: cholesterol acyltransferase (LCAT) is responsible for the formation of cholesteryl ester in plasma. LCAT is a glycoprotein which has a carbohydrate content estimated to be approx. 25% of its total mass. Previous studies of recombinant LCAT have characterized the function of the four N-linked glycosylation sites of LCAT with respect to reconstituted HDL analogue substrates. In order to investigate the relationship between N-linked glycosylation and the ability of LCAT to esterify cholesterol in native plasma lipoproteins, we have expressed a series of mutant LCAT cDNAs in which each of the four glycosylation consensus sequences was eliminated individually. All mutant LCAT proteins were secreted by stably transfected baby hamster kidney cells. The ability of mutant LCATs to esterify cholesterol in purified native lipoproteins indicated that the elimination of the carbohydrate chain at position 20 of recombinant LCAT was associated with a lower activity than the wild type enzyme when HDL was used as a substrate, but no inhibitory effect was observed when LDL was used as a substrate. A mutant enzyme with a substitution of Asn-84 → Gln or Asn-272 → Gln displayed a decreased ability to esterify cholesterol in either HDL or LDL. In contrast, the loss of a carbohydrate chain at position 384 was associated with an increase in enzyme activity for both HDL (1.5-fold) and LDL (2.5-fold) substrates. Kinetic analysis of these recombinant enzymes indicated that the apparent K m values for cholesterol in either HDL or LDL were not affected, but that the differences in activities were due to changes in the apparent V max. Heat inactivation studies were performed to assess the role of specific carbohydrate groups in enzyme stability. Loss of a carbohydrate chain at position 20, 272 or 384 decreased thermostability of LCAT whereas a mutation at position 84 did not affect thermostability. These results suggest that individual carbohydrate chains confer specific structural and functional properties to LCAT.

Recombinant lecithin:cholesterol acyltransferase containing a Thr123–>Ile mutation esterifies cholesterol in low density lipoprotein but not in high density lipoprotein.

Fish-eye disease is a rare genetic disorder of high density lipoprotein (HDL) metabolism that is characterized biochemically by a partial deficiency of the enzyme lecithin:cholesterol acyltransferase (LCAT). One of the mutations that is causative for fish-eye disease occurs at codon 123 of the LCAT gene. This mutation results in the exchange of a threonine residue for an isoleucine in the LCAT protein (Thr123-->Ile). In order to understand the functional significance of this exchange, we have used site-directed mutagenesis to reconstruct this mutation in an LCAT cDNA followed by expression of the mutant LCAT in COS-1 cells. The fish-eye disease mutation resulted in a 50% decrease in LCAT mass in the culture medium compared to wild type enzyme. The secreted mutant protein was incapable of esterifying cholesterol in HDL and HDL analogues. However, this protein retained the ability to esterify cholesterol in plasma and low density lipoprotein. These results support the hypothesis that this mutation is responsible for biochemical abnormalities of LCAT observed in fish-eye disease and the mutant LCAT protein has lost the potential to esterify cholesterol in the HDL pool but retains the ability to esterify cholesterol from other lipoproteins.