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Impaired function of lecithin:cholesterol acyltransferase in atherosclerosis-susceptible White Carneau pigeons: possible effects on metabolism of oxidized phospholipids.

Although White Carneau (WC) pigeons are known to be more susceptible to atherosclerosis than Show Racer (SR) pigeons, the reasons for this difference are not fully understood. While no major differences are known in the lipoprotein composition, a difference in the cholesteryl ester (CE) composition was reported. However, there is little information on the activity or specificity of lecithin:cholesterol acyltransferase (LCAT), the major source of plasma CE. In order to determine whether the esterification of cholesterol or other functions of LCAT are compromised in WC pigeons, we studied the various reactions catalyzed by LCAT in the two groups. The cholesterol esterification was found to be significantly lower in WC pigeons, whether assayed with exogenous or endogenous substrates. Furthermore, lyso phosphatidylcholine (PC) esterification and oxidized PC hydrolysis, two other reactions carried out by LCAT, were also lower in WC. We found evidence for the presence of an active lysophospholipase in pigeon plasma, and this activity was also lower in WC compared to SR. A significant increase in the FC/PC ratio, another reported atherogenic risk factor, was found in WC. plasma. Because of the absence of other hydrolytic enzymes in pigeon plasma, LCAT may play an important role in the metabolism of oxidized PC generated during lipoprotein oxidation, and therefore a decrease in its activity in White Carneau pigeons may contribute to increased risk of atherosclerosis.

Animals↗

Characterization of recombinant human plasma lecithin: cholesterol acyltransferase (LCAT): N-linked carbohydrate structures and catalytic properties.

The major N-linked carbohydrate structures were determined for recombinant human plasma lecithin:cholesterol acyltransferase (LCAT). The analysis of the structure of oligosaccharides by fast atom bombardment mass spectrometry (FAB-MS) and linkage analysis was preceded by reduction and carboxymethylation of the intact glycoproteins and digestion with trypsin and proline specific endopeptidase. The N-glycans were subsequently released from the glycopeptides by PNGase F digestion and the oligosaccharides were separated using a C18 Sep-pak cartridge. The data from the combination of FAB spectrometry and linkage analysis show that the N-linked glycans present on recombinant LCAT (rLCAT) were composed primarily of triantennary and tetraantennary structures with and without core fucosylation. A minor population of glycans (less than 5%) contained up to three repeats of N-acetyllactosamine in one or more antennae. The LCAT activities of both recombinant and circulating forms of plasma LCAT were determined using low molecular weight and lipoprotein substrates. The catalytic behavior of these two enzyme forms were found to be very similar if not identical. These findings validate the concept that the recombinant enzyme can serve as an appropriate model for structure/function studies of LCAT and provide the foundation for subsequent structural studies.

Apolipoprotein A-I↗

Binding and functional effects of transcription factors Sp1 and Sp3 on the proximal human lecithin:cholesterol acyltransferase promoter.

Human lecithin:cholesterol acyltransferase (LCAT) circulates in plasma bound to high density lipoproteins (HDL) and modulates the rate by which cholesteryl ester is transported to the liver. So far, little is known about the regulation of the expression of the LCAT gene. In this study we have defined the cis-elements, identified the trans-acting factors and demonstrated their functional effects and significance in determining transcriptional activity of the proximal LCAT promoter. Using deletion mutants having 5' variable ends (from nucleotides -72 to -27), we have identified the presence of two non-consensus GC-rich regions that stimulate transcription in HepG2 and HeLa cells. These regions designated sites A (-29 to -47) and B (-49 to -65) contain the CCTCC core sequence which in electromobility shift analysis is critical for the formation of two DNA-protein complexes designated I and II. Site-directed mutagenesis suggests that both sites are equally important in promoter activity, and that cooperative interactions between both sites are not required for activity. Electromobility shift and supershift experiments using oligonucleotides spanning sites A and B identified Sp1 and Sp3 as the transcription factors interacting at these sites. To determine the significance and functional effects that Sp1 and Sp3 have in regulating LCAT promoter activity, we performed transfection experiments in Drosophila SL-2 cells as they lack endogenous Sp1 and Sp3. Sp1 but not Sp3 activates the human LCAT promoter and when Sp1 is co-transfected along with Sp3, Sp3 functions as a dose-dependent repressor of Sp1-mediated activation. These findings indicate that Sp1 is capable of transactivating a reporter gene linked to the LCAT promoter containing Sp binding sites and suggests that the levels of Sp3 or the nuclear Sp1/Sp3 ratio may play an important role in determining the transcriptional activity of the LCAT promoter in vivo.

Base Sequence↗

Correction of hypoalphalipoproteinemia in LDL receptor-deficient rabbits by lecithin:cholesterol acyltransferase.

Familial hypercholesterolemia (FH), a disease caused by a variety of mutations in the low density lipoprotein receptor (LDLr) gene, leads not only to elevated LDL-cholesterol (C) concentrations but to reduced high density lipoprotein (HDL)-C and apolipoprotein (apo) A-I concentrations as well. The reductions in HDL-C and apoA-I are the consequence of the combined metabolic defects of increased apoA-I catabolism and decreased apoA-I synthesis. The present studies were designed to test the hypothesis that overexpression of human lecithin:cholesterol acyltransferase (hLCAT), a pivotal enzyme involved in HDL metabolism, in LDLr defective rabbits would increase HDL-C and apoA-I concentrations. Two groups of hLCAT transgenic rabbits were established: 1) hLCAT+/LDLr heterozygotes (LDLr+/-) and 2) hLCAT+/LDLr homozygotes (LDLr-/-). Data for hLCAT+ rabbits were compared to those of nontransgenic (hLCAT-) rabbits of the same LDLr status. In LDLr+/- rabbits, HDL-C and apoA-I concentrations (mg/dl), respectively, were significantly greater in hLCAT+ (62 +/- 8, 59 +/- 4) relative to hLCAT- rabbits (21 +/- 1, 26 +/- 2). This was, likewise, the case when hLCAT+/ LDLr-/- (27 +/- 2, 19 +/- 6) and hLCAT-/LDLr-/- (5 +/- 1, 6 +/- 2) rabbits were compared. Kinetic experiments demonstrated that the fractional catabolic rate (FCR, d(-1)) of apoA-I was substantially delayed in hLCAT+ (0.376 +/- 0.025) versus hLCAT- (0.588) LDLr+/- rabbits, as well as in hLCAT+ (0.666 +/- 0.033) versus hLCAT- (1.194 +/- 0.138) LDLr-/- rabbits. ApoA-I production rate (PR, mg x kg x d(-1)) was greater in both hLCAT+/LDLr+/- (10 +/- 2 vs. 6) and hLCAT+/LDLr-/- (9 +/- 1 vs. 4 +/- 1) rabbits. Significant correlations (P < 0.02) were observed between plasma LCAT activity and HDL-C (r = 0.857), apoA-I FCR (r = -0.774), and apoA-I PR (r = 0.771), while HDL-C correlated with both apoA-I FCR (-0.812) and PR (0.751). In summary, these data indicate that hLCAT overexpression in LDLr defective rabbits increases HDL-C and apoA-I concentrations by both decreasing apoA-I catabolism and increasing apoA-I synthesis, thus correcting the metabolic defects responsible for the hypoalphalipoproteinemia observed in LDLr deficiency.

Animals↗

[Case study of 14C-labelled lecithin placental transfer].

Because of the clinical and biochemical state of a 26 years old patient an erythroblastosis was expected. Continuous controls revealed that delivery in the 36th week of pregnancy was necessary. A therapeutic research with lecithine through the maternal circulation in this special case was carried out in order to prevent a respiratory distress syndrome of the premature infant.

Adult↗