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PubMed1996

Sphingomyelin inhibits the lecithin-cholesterol acyltransferase reaction with reconstituted high density lipoproteins by decreasing enzyme binding.

Lecithin-cholesterol acyltransferase (LCAT) catalyzes the formation of cholesterol esters on high density lipoproteins (HDL) and plays a critical role in reverse cholesterol transport. Sphingomyelin, an important constituent of HDL, may regulate the activity of LCAT at any of the key steps of the enzymatic reaction: binding of LCAT to the interface, activation by apo A-I, or inhibition at the catalytic site. In order to clarify the role of sphingomyelin in the regulation of the LCAT reaction and its effects on the structure of apolipoprotein A-I, we prepared reconstituted HDL (rHDL) containing egg phosphatidylcholine, cholesterol, apolipoprotein A-I, and up to 22 mol % sphingomyelin. Because the interfacial properties of substrate particles can dramatically affect LCAT binding and kinetics, we also prepared and analyzed proteoliposome substrates having the same components as the rHDL, except for a 4-fold higher ratio of phospholipid to apolipoprotein A-I. The reaction kinetics of LCAT with the rHDL particles revealed no significant change in the apparent Vmax but showed a concentration-dependent increase in slope of the reciprocal plots and in the apparent Km values with sphingomyelin content. The dissociation constant (Kd) for LCAT with these particles increased linearly with sphingomyelin content up to 22 mol %, changing in parallel with the apparent Km values. No structural changes of apolipoprotein A-I were detected in the particles with increasing content of sphingomyelin, but fluorescence results with lipophilic probes revealed that significant changes in the acyl chain, backbone, and head group regions of the lipid bilayer of the particles are introduced by the addition of sphingomyelin. On the other hand, the proteoliposome substrates also had increased Kdvalues for LCAT at high sphingomyelin contents but compared with the rHDL particles had a 6-10-fold lower affinity for LCAT binding and exhibited kinetics consistent with competitive inhibition by sphingomyelin at the active site. These results show conclusively that the dominant mechanism for the inhibition of LCAT activity with rHDL particles by sphingomyelin is the impaired binding of the enzyme to the interface. The results also underscore the significant differences in the enzyme reaction kinetics with different substrate particles.

Apolipoprotein A-I↗
PubMed1996

Regulation of cellular cholesterol efflux by lecithin:cholesterol acyltransferase reaction through nonspecific lipid exchange.

Erythrocyte was found lacking in reactivity to lipid-free apolipoproteins to generate pre-beta-high density lipoprotein (HDL) with the cellular lipid and, therefore, was used to study cellular cholesterol efflux to plasma lipoproteins exclusively by a nonspecific exchange mechanism. Over the range of hematocrit from 1-20% (cellular cholesterol pool of 2.5 micrograms per 250 microliters), the fractional rate of cellular cholesterol efflux to lipoprotein was constant, and, therefore, absolute efflux rate was a linear function of the hematocrit of this range. In the absence of lecithin:cholesterol acyltransferase (LCAT), the cholesterol influx rate from lipoproteins was equal to the efflux rate from erythrocyte resulting in no net transfer of cholesterol, with either HDL or low density lipoprotein. In the presence of LCAT in the mixture of HDL and erythrocyte, cholesterol was esterified exclusively in HDL regardless of the origin. When the hematocrit was low and efflux of cellular cholesterol was slower than cholesterol esterification, the esterification of cell-originating cholesterol did not directly enhance the efflux. With high hematocrit that gives faster cholesterol efflux, the efflux was increased directly by the cholesterol esterification. On the other hand, the LCAT reaction significantly reduced HDL-cholesterol influx. The LCAT reaction thus induces substantial net cholesterol efflux from erythrocytes through a nonspecific cholesterol exchange mechanism.

Animals↗
PubMed1996

Structural studies of a peptide activator of human lecithin-cholesterol acyltransferase.

The synthetic lipid-associating peptide, LAP-20 (VSSLLSSLKEYWSSLKESFS), activates lecithin-cholesterol acyltransferase (LCAT) despite its lack of sequence homology to apolipoprotein A-I, the primary in vivo activator of LCAT. Using SDS and dodecylphosphocholine (DPC) to model the lipoprotein environment, the structural features responsible for LAP-20's ability to activate LCAT were studied by optical and two-dimensional 1H NMR spectroscopy. A large blue shift in the intrinsic fluorescence of LAP-20 with the addition of detergent suggested that the peptide formed a complex with the micelles. Analysis of the CD data shows that LAP-20 lacks well defined structure in aqueous solution but adopts helical, ordered conformations upon the addition of SDS or DPC. The helical nature of the peptides in the presence of both lipids was confirmed by upfield H alpha NMR secondary shifts relative to random coil values. Average structures for both peptides in aqueous solutions containing SDS and DPC were generated using distance geometry methods from 329 (SDS) and 309 (DPC) nuclear Overhauser effect-based distance restraints. The backbone (N, Calpha, C=O) RMSD from the average structure of an ensemble of 17 out of 20 calculated structures was 0.41 +/- 0.15 Angstrom for LAP-20 in SDS and 0.41 +/- 0.12 A for an ensemble of 20 out of 20 calculated structures for LAP-20 in DPC. In the presence of SDS, the distance geometry and simulated annealing calculations show that LAP-20 adopts a well defined class A amphipathic helix with distinct hydrophobic and hydrophilic faces. A similar structure was obtained for LAP-20 in the presence of DPC, suggesting that both detergents may be used interchangeably to model the lipoprotein environment. Conformational features of the calculated structures for LAP-20 are discussed relative to models for apolipoprotein A-I activation of LCAT.

Amino Acid Sequence↗
PubMed1996

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.

Animals↗
PubMed1997

Alteration in apolipoprotein A-I 22-mer repeat order results in a decrease in lecithin:cholesterol acyltransferase reactivity.

Apolipoprotein A-I contains eight 22-amino acid and two 11-amino acid tandem repeats that comprise 80% of the mature protein. These repeating units are believed to be the basic motif responsible for lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation. Computer analysis indicates that despite a fairly high degree of compositional similarity among the tandem repeats, significant differences in hydrophobic and amphipathic character exist. Our previous studies demonstrated that deletion of repeat 6 (143-164) or repeat 7 (165-186) resulted in a 98-99% reduction of LCAT activation as compared with wild-type apoA-I. To determine the effects of substituting one of these repeats with a more hydrophobic repeat we constructed a mutant apoA-I protein in which residues 143-164 (repeat 6) were replaced with repeat 10 (residues 220-241). The cloned mutant protein, 10F6 apoA-I, was expressed and purified from an Sf-9 cell baculoviral system and then analyzed using a number of biophysical and biochemical techniques. Recombinant complexes prepared at a 100:5:1 molar ratio of L-alpha-dimyristoylphosphatidylcholine:cholesterol:wild-type or 10F6 apoA-I showed a doublet corresponding to Stokes diameters of 114 and 108 A on nondenaturing 4-30% polyacrylamide gel electrophoresis. L-alpha-Dimyristoylphosphatidylcholine 10F6 apoA-I complexes had a 5-6-fold lower apparent Vmax/apparent Km as compared with wild-type apoA-I containing particles. As expected, monoclonal antibody epitope mapping of the lipid-free and lipid-bound 10F6 apoA-I confirmed that a domain expressed between residues 143 and 165 normally found in wild-type apoA-I was absent. The region between residues 119 and 144 in 10F6 apoA-I showed a marked reduction in monoclonal antibody binding capacity. Therefore, we speculate that the 5-6-fold lower LCAT reactivity in 10F6 compared with wild-type apoA-I recombinant particles results from increased stabilization within the 121-165 amino acid domain due to more stable apoprotein helix phospholipid interactions as well as from conformational alterations among adjacent amphipathic helix repeats.

Amino Acid Sequence↗
PubMed1997

Role of the Arg123-Tyr166 paired helix of apolipoprotein A-I in lecithin:cholesterol acyltransferase activation.

The Arg123-Tyr166 central and Ala190-Gln243 carboxyl-terminal pairs of helices of apoA-I were substituted with the pair of helices of apoA-II, resulting in the apoA-I(Delta(Arg123-Tyr166), nablaA-II(Ser12-Ala75)) and apoA-I(Delta(Ala190-Gln243), nablaA-II(Ser12-Gln77)) chimeras, respectively. The structures of these chimeras in aqueous solution and in reconstituted high density lipoproteins (rHDL) and the lecithin:cholesterol acyltransferase (LCAT) activation properties of the rHDL were studied. Recombinant human apoA-I and the chimeras were expressed in Escherichia coli and purified from the periplasmic space. Binding of the apolipoproteins with palmitoyloleoylphosphatidylcholine was associated with a similar shift of Trp fluorescence maxima from 337 to 332 nm, from 339 to 334 nm, and from 337 to 333 nm, respectively. All rHDL had a Stokes radius of 4.8 nm and contained 2 apolipoprotein molecules/particle. Circular dichroism measurements revealed eight alpha-helices per apoA-I and per chimera molecule. The catalytic efficiencies of LCAT activation were 1.5 +/- 0.33 (mean +/- S.D.; n = 3), 0.054 +/- 0.009 (p < 0.001 versus apoA-I), and 1.3 +/- 0.32 (p = not significant versus apoA-I) nmol of cholesteryl ester/h/microM, respectively. The lower LCAT activity of the central domain chimera was due to a 27-fold reduced Vmax with unaltered Km. Binding of radiolabeled LCAT to rHDL of apoA-I and apoA-I(Delta(Arg123-Tyr166), nablaA-II(Ser12-Ala75)) was very similar. In conclusion, although substitution of the Arg123-Tyr166 central or Ala190-Gln243 carboxyl-terminal pair of helices of apoA-I with the pair of helices of apoA-II yields chimeras with structure similar to that of native apoA-I, exchange of the central domain (but not the carboxyl-terminal domain) of apoA-I reduces the rate of LCAT activity that is independent of binding to rHDL.

Apolipoprotein A-I↗
PubMed1997

Novel function of lecithin-cholesterol acyltransferase. Hydrolysis of oxidized polar phospholipids generated during lipoprotein oxidation.

Although the major function of lecithin-cholesterol acyltransferase (LCAT) is cholesterol esterification, our previous studies showed that it can also hydrolyze platelet-activating factor (PAF). Because of the structural similarities between PAF and the truncated phosphatidylcholines (polar PCs) generated during lipoprotein oxidation, we investigated the possibility that LCAT may also hydrolyze polar PCs to lyso-PC during the oxidation of plasma. PAF acetylhydrolase (PAF-AH), which is known to hydrolyze polar PCs in human plasma, was completely inhibited by 0.2 mM p-aminoethyl benzenesulfonyl fluoride (Pefabloc), a new serine esterase inhibitor, which had no effect on LCAT at this concentration. On the other hand, 1 mM diisopropylfluorophosphate (DFP) completely inhibited LCAT but had no effect on PAF-AH. Polar PC accumulation during the oxidation of plasma increased by 44% in the presence of 0.2 mM Pefabloc and by 30% in the presence of 1 mM DFP. The formation of lyso-PC was concomitantly inhibited by both of the inhibitors. The combination of the two inhibitors resulted in the maximum accumulation of polar PCs, suggesting that both PAF-AH and LCAT are involved in their breakdown. Oxidation of chicken plasma, which has no PAF-AH activity, also resulted in the formation of lyso-PC from the hydrolysis of polar PC, which was inhibited by DFP. Polar PCs, either isolated from oxidized plasma or by oxidation of labeled synthetic PCs, were hydrolyzed by purified LCAT, which had no detectable PAF-AH activity. These results demonstrate a novel function for LCAT in the detoxification of polar PCs generated during lipoprotein oxidation, especially when the PAF-AH is absent or inactivated.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1998

The hydrophobic face orientation of apolipoprotein A-I amphipathic helix domain 143-164 regulates lecithin:cholesterol acyltransferase activation.

Apolipoprotein A-I (apoA-I) activates the plasma enzyme lecithin:cholesterol acyltransferase (LCAT), catalyzing the rapid conversion of lipoprotein cholesterol to cholesterol ester. Structural mutants of apoA-I have been used to study the details of apoA-I-LCAT-catalyzed cholesterol ester formation. Several studies have shown that the alpha-helical segments corresponding to amino acids 143-164 and 165-186 (repeats 6 and 7) are essential for LCAT activation. In the present studies, we examined how the orientation of the hydrophobic face, independent of an increase in overall hydrophobicity, affects LCAT activation. We designed, expressed, and characterized a mutant, reverse of 6 apoA-I (RO6 apoA-I), in which the primary amino acid sequence of repeat 6 (amino acids 143-164) was reversed from its normal orientation. This mutation rotates the hydrophobic face of repeat 6 approximately 80 degrees. Lipid-free RO6 apoA-I showed a marked stabilization when denatured by guanidine hydrochloride, but showed significant destabilization to guanidine hydrochloride denaturation in the lipid-bound state compared with wild-type apoA-I. Recombinant high density lipoprotein discs (rHDL) formed from RO6 apoA-I, sn-1-palmitoyl-sn-2-oleoyl phosphati-dylcholine, and cholesterol were approximately 12 A smaller than wild-type apoA-I rHDL. The reduced size suggests that one of the repeats did not effectively participate in phospholipid binding and organization. The sn-1-palmitoyl-sn-2-oleoyl phosphatidylcholine RO6 rHDL were a less effective substrate for LCAT. Mapping the entire lipid-free and lipid-bound RO6 apoA-I with a series of monoclonal antibodies revealed that both the lipid-free and lipid-bound RO6 apoA-I displayed altered or absent epitopes in domains within and adjacent to repeat 6. Together, these results suggest that the proper alignment and orientation of the hydrophobic face of repeat 6 is an important determinant for maintaining and stabilizing helix-bilayer and helix-helix interactions.

Apolipoprotein A-I↗
PubMed1998

Structural and functional properties of two mutants of lecithin-cholesterol acyltransferase (T123I and N228K).

Two naturally occurring mutants of human lecithin-cholesterol acyltransferase (LCAT), T123I and N228K, were expressed in COS-1 and Chinese hamster ovary cells, overproduced, and purified to homogeneity in order to study the structural and functional defects that lead to the LCAT deficiency phenotypes of these mutations. The mutants were expressed and secreted by transfected cells normally and had molecular weights and levels of glycosylation similar to wild type LCAT. The purified proteins (>98% purity) had almost indistinguishable structures and stabilities as determined by CD and fluorescence spectroscopy. Enzymatic activities and kinetic analysis of the pure enzyme forms showed that wild type LCAT and both mutants were reactive with the water-soluble substrate, p-nitrophenyl butyrate, indicating the presence of an intact core active site and catalytic triad. Both the T123I and N228K mutants had markedly depressed reactivity with reconstituted HDL (rHDL), but T123I retained activity with low density lipoprotein. To determine whether defective binding to rHDL was responsible for the low activity of both mutants with rHDL, the equilibrium binding constants were measured directly with isothermal titration calorimetry and surface plasmon resonance (SPR) methods. The results indicated that the affinities of the mutants for rHDL were only about 2-fold lower than the affinity of wild type LCAT (Kd = 2.3 x 10(-7) M). Together, the activity and equilibrium binding results suggest that the T123I mutant is defective in activation by apolipoprotein A-I, and the N228K mutant has impaired binding of lipid substrate to the active site. In addition, the kinetic binding rate constants determined by the SPR method indicate that normal LCAT dissociates from rHDL, on average, after one catalytic cycle.

Animals↗
PubMed1999

Cholesteryl ester transfer protein corrects dysfunctional high density lipoproteins and reduces aortic atherosclerosis in lecithin cholesterol acyltransferase transgenic mice.

Expression of human lecithin cholesterol acyltransferase (LCAT) in mice (LCAT-Tg) leads to increased high density lipoprotein (HDL) cholesterol levels but paradoxically, enhanced atherosclerosis. We have hypothesized that the absence of cholesteryl ester transfer protein (CETP) in LCAT-Tg mice facilitates the accumulation of dysfunctional HDL leading to impaired reverse cholesterol transport and the development of a pro-atherogenic state. To test this hypothesis we cross-bred LCAT-Tg with CETP-Tg mice. On both regular chow and high fat, high cholesterol diets, expression of CETP in LCAT-Tg mice reduced total cholesterol (-39% and -13%, respectively; p < 0.05), reflecting a decrease in HDL cholesterol levels. CETP normalized both the plasma clearance of [(3)H]cholesteryl esters ([(3)H]CE) from HDL (fractional catabolic rate in days(-1): LCAT-Tg = 3.7 +/- 0.34, LCATxCETP-Tg = 6.1 +/- 0.16, and controls = 6.4 +/- 0.16) as well as the liver uptake of [(3)H]CE from HDL (LCAT-Tg = 36%, LCATxCETP-Tg = 65%, and controls = 63%) in LCAT-Tg mice. On the pro-atherogenic diet the mean aortic lesion area was reduced by 41% in LCATxCETP-Tg (21.2 +/- 2.0 micrometer(2) x 10(3)) compared with LCAT-Tg mice (35.7 +/- 2.0 micrometer(2) x 10(3); p < 0.001). Adenovirus-mediated expression of scavenger receptor class B (SR-BI) failed to normalize the plasma clearance and liver uptake of [(3)H]CE from LCAT-Tg HDL. Thus, the ability of SR-BI to facilitate the selective uptake of CE from LCAT-Tg HDL is impaired, indicating a potential mechanism leading to impaired reverse cholesterol transport and atherosclerosis in these animals. We conclude that CETP expression reduces atherosclerosis in LCAT-Tg mice by restoring the functional properties of LCAT-Tg mouse HDL and promoting the hepatic uptake of HDL-CE. These findings provide definitive in vivo evidence supporting the proposed anti-atherogenic role of CETP in facilitating HDL-mediated reverse cholesterol transport and demonstrate that CETP expression is beneficial in pro-atherogenic states that result from impaired reverse cholesterol transport.

Animals↗
PubMed1999

Molecular and biochemical characterization of lecithin retinol acyltransferase.

The enzyme responsible for conversion of all-trans-retinol into retinyl esters, the lecithin retinol acyltransferase (LRAT) has been characterized at the molecular level. The cDNA coding for this protein was cloned and its amino acid sequence deduced. LRAT is composed of a polypeptide of 230 amino acid residues with a calculated mass of 25.3 kDa. Tissue distribution analysis by Northern blot showed expression of a 5.0-kilobase transcript in the human retinal pigment epithelium as well as in other tissues that are known for their high LRAT activity and vitamin A processing. Affinity labeling experiments using specific compounds with high affinity for LRAT and monospecific polyclonal antibodies raised in rabbits against two peptide sequences for LRAT confirmed the molecular mass of LRAT as a 25-kDa protein. High performance liquid chromatography analysis of the reaction product formed by HEK-293 cells transfected with LRAT cDNA confirmed the ability of the transfected cells to convert [3H]all-trans-retinol into authentic [3H]all-trans-retinyl palmitate as chemically determined.

Acyltransferases↗
PubMed2000

Formation of spherical, reconstituted high density lipoproteins containing both apolipoproteins A-I and A-II is mediated by lecithin:cholesterol acyltransferase.

Previous studies have provided detailed information on the formation of spherical high density lipoproteins (HDL) containing apolipoprotein (apo) A-I but no apoA-II (A-I HDL) by an lecithin:cholesterol acyltransferase (LCAT)-mediated process. In this study we have investigated the formation of spherical HDL containing both apoA-I and apoA-II (A-I/A-II HDL). Incubations were carried out containing discoidal A-I reconstituted HDL (rHDL), discoidal A-II rHDL, and low density lipoproteins in the absence or presence of LCAT. After the incubation, the rHDL were reisolated and subjected to immunoaffinity chromatography to determine whether A-I/A-II rHDL were formed. In the absence of LCAT, the majority of the rHDL remained as either A-I rHDL or A-II rHDL, with only a small amount of A-I/A-II rHDL present. By contrast, when LCAT was present, a substantial proportion of the reisolated rHDL were A-I/A-II rHDL. The identity of the particles was confirmed using apoA-I rocket electrophoresis. The formation of the A-I/A-II rHDL was influenced by the relative concentrations of the precursor discoidal A-I and A-II rHDL. The A-I/A-II rHDL included several populations of HDL-sized particles; the predominant population having a Stokes' diameter of 9.9 nm. The particles were spherical in shape and had an electrophoretic mobility slightly slower than that of the alpha-migrating HDL in human plasma. The apoA-I:apoA-II molar ratio of the A-I/A-II rHDL was 0.7:1. Their major lipid constituents were phospholipids, unesterified cholesterol, and cholesteryl esters. The results presented are consistent with LCAT promoting fusion of the A-I rHDL and A-II rHDL to form spherical A-I/A-II rHDL. We suggest that this process may be an important source of A-I/A-II HDL in human plasma.

Apolipoprotein A-I↗
PubMed2000

Distinct central amphipathic alpha-helices in apolipoprotein A-I contribute to the in vivo maturation of high density lipoprotein by either activating lecithin-cholesterol acyltransferase or binding lipids.

Recombinant adenoviruses with cDNAs for human apolipoprotein A-I (wild type (wt) apoA-I) and three mutants, referred to as Delta4-5A-I, Delta5-6A-I, and Delta6-7A-I, that have deletions removing regions coding for amino acids 100-143, 122-165, and 144-186, respectively, were created to study structure/function relationships of apoA-I in vivo. All mutants were expressed at lower concentrations than wt apoA-I in plasma of fasting apoA-I-deficient mice. The Delta5-6A-I mutant was found primarily in the lipid-poor high density lipoprotein (HDL) pool and at lower concentrations than Delta4-5A-I and Delta6-7A-I that formed more buoyant HDL(2/3) particles. At an elevated adenovirus dose and earlier blood sampling from fed mice, both Delta5-6A-I and Delta6-7A-I increased HDL-free cholesterol and phospholipid but not cholesteryl ester. In contrast, wt apoA-I and Delta4-5A-I produced significant increases in HDL cholesteryl ester. Further analysis showed that Delta6-7A-I and native apoA-I could bind similar amounts of phospholipid and cholesterol that were reduced slightly for Delta5-6A-I and greatly for Delta4-5A-I. We conclude from these findings that amino acids (aa) 100-143, specifically helix 4 (aa 100-121), contributes to the maturation of HDL through a role in lipid binding and that the downstream sequence (aa 144-186) centered around helix 6 (aa 144-165) is responsible for the activation of lecithin-cholesterol acyltransferase.

Animals↗
PubMed2000

A lecithin cholesterol acyltransferase-like gene mediates diacylglycerol esterification in yeast.

The terminal step in triglyceride biosynthesis is the esterification of diacylglycerol. To study this reaction in the model eukaryote, Saccharomyces cerevisiae, we investigated five candidate genes with sequence conservation to mammalian acyltransferases. Four of these genes are similar to the recently identified acyl-CoA diacylglycerol acyltransferase and, when deleted, resulted in little or no decrease in triglyceride synthesis as measured by incorporation of radiolabeled oleate or glycerol. By contrast, deletion of LRO1, a homolog of human lecithin cholesterol acyltransferase, resulted in a dramatic reduction in triglyceride synthesis, whereas overexpression of LRO1 yielded a significant increase in triglyceride production. In vitro microsomal assays determined that Lro1 mediated the esterification of diacylglycerol using phosphatidylcholine as the acyl donor. The residual triglyceride biosynthesis that persists in the LRO1 deletion strain is mainly acyl-CoA-dependent and mediated by a gene that is structurally distinct from the previously identified mammalian diacylglycerol acyltransferase. These mechanisms may also exist in mammalian cells.

Acyltransferases↗
PubMed2000

Identification of a sequence of apolipoprotein A-I associated with the activation of Lecithin:Cholesterol acyltransferase.

We aimed to distinguish between the effects of mutations in apoA-I on the requirements for the secondary structure and a specific amino acid sequence for lecithin:cholesterol acyltransferase (LCAT) activation. Several mutants were constructed targeting region 140-150: (i) two mutations affecting alpha-helical structure, deletion of amino acids 140-150 and substitution of Ala(143) for proline; (ii) two mutations not affecting alpha-helical structure, substitution of Val(149) for arginine and substitution of amino acids 63-73 for sequence 140-150; and (iii) a mutation in a similar region away from the target area, deletion of amino acids 63-73. All mutations affecting region 140-150 resulted in a 4-42-fold reduction in LCAT activation. Three mutations, apoA-I(Delta140-150), apoA-I(P143A), and apoA-I(140-150 --> 63-73), affected both the apparent V(max) and K(m), whereas the mutation apoA-I(R149V) affected only the V(max). The mutation apoA-I(Delta63-73) caused only a 5-fold increase in the K(m). All mutants, except apoA-I(P143A) and apoA-I(Delta63-73), were active in phospholipid binding assay. All mutants, except apoA-I(P143A), formed normal discoidal complexes with phospholipid. The mutation apoA-I(Delta63-73) caused a significant reduction in the stability of apoA-I.phospholipid complexes in denaturation experiments. Combined, our results strongly suggest that although the correct conformation and orientation of apoA-I in the complex with lipids are crucial for activation of LCAT, when these conditions are fulfilled, activation also strongly depends on the sequence that includes amino acids 140-150.

Amino Acid Sequence↗
PubMed2001

Analysis of glomerulosclerosis and atherosclerosis in lecithin cholesterol acyltransferase-deficient mice.

To evaluate the biochemical and molecular mechanisms leading to glomerulosclerosis and the variable development of atherosclerosis in patients with familial lecithin cholesterol acyl transferase (LCAT) deficiency, we generated LCAT knockout (KO) mice and cross-bred them with apolipoprotein (apo) E KO, low density lipoprotein receptor (LDLr) KO, and cholesteryl ester transfer protein transgenic mice. LCAT-KO mice had normochromic normocytic anemia with increased reticulocyte and target cell counts as well as decreased red blood cell osmotic fragility. A subset of LCAT-KO mice accumulated lipoprotein X and developed proteinuria and glomerulosclerosis characterized by mesangial cell proliferation, sclerosis, lipid accumulation, and deposition of electron dense material throughout the glomeruli. LCAT deficiency reduced the plasma high density lipoprotein (HDL) cholesterol (-70 to -94%) and non-HDL cholesterol (-48 to -85%) levels in control, apoE-KO, LDLr-KO, and cholesteryl ester transfer protein-Tg mice. Transcriptome and Western blot analysis demonstrated up-regulation of hepatic LDLr and apoE expression in LCAT-KO mice. Despite decreased HDL, aortic atherosclerosis was significantly reduced (-35% to -99%) in all mouse models with LCAT deficiency. Our studies indicate (i) that the plasma levels of apoB containing lipoproteins rather than HDL may determine the atherogenic risk of patients with hypoalphalipoproteinemia due to LCAT deficiency and (ii) a potential etiological role for lipoproteins X in the development of glomerulosclerosis in LCAT deficiency. The availability of LCAT-KO mice characterized by lipid, hematologic, and renal abnormalities similar to familial LCAT deficiency patients will permit future evaluation of LCAT gene transfer as a possible treatment for glomerulosclerosis in LCAT-deficient states.

Animals↗
PubMed2001

Interaction of lecithin:cholesterol acyltransferase (LCAT).alpha 2-macroglobulin complex with low density lipoprotein receptor-related protein (LRP). Evidence for an alpha 2-macroglobulin/LRP receptor-mediated system participating in LCAT clearance.

The reaction of lecithin:cholesterol acyltransferase (LCAT) with high density lipoproteins (HDL) is of critical importance in reverse cholesterol transport, but the structural and functional pathways involved in the regulation of LCAT have not been established. We present evidence for the direct binding of LCAT to alpha(2)-macroglobulin (alpha(2)M) in human plasma to form a complex 18.5 nm in diameter. Forty percent of plasma LCAT-HDL was associated with alpha(2)M; moreover, most of the LCAT in cerebrospinal fluid and in the medium of cultured human hepatoma cell line was associated with alpha(2)M. Purified recombinant human LCAT (rLCAT) labeled with (125)I bound to native and methylamine-activated alpha(2)M (alpha(2)M-MA) in vitro in a time- and concentration-dependent manner, and this binding did not depend on the presence of lipid. rLCAT bound to alpha(2)M-MA with greater affinity than to alpha(2)M. Furthermore, rLCAT did not activate alpha(2)M as phosphatidylcholine-specific phospholipase C does. Reconstituted HDL particles (LpA-I) inhibited the binding of rLCAT to alpha(2)M more efficiently than native HDL(3) did. LCAT associated with alpha(2)M was enzymatically inactive under both endogenous and exogenous assay conditions. Purified rLCAT alone did not bind to low density lipoprotein receptor-related protein (LRP) as lipoprotein lipase (LPL) does; however, when rLCAT was combined with alpha(2)M-MA to form a complex, binding, internalization, and degradation of rLCAT took place in LRP-expressing cells (LRP (+/+)) but not in cells deficient in LRP (LRP (-/-)). It is concluded that the binding of LCAT to alpha(2)M inhibits its enzymatic activity. Furthermore, the finding supports the possibility that the LRP receptor can act in vivo to mediate clearance of the LCAT-alpha(2)M complex and may significantly influence the bioavailability of LCAT.

Apolipoprotein E3↗
PubMed2001

Deletion of specific glycan chains affects differentially the stability, local structures, and activity of lecithin-cholesterol acyltransferase.

The enzymatic and interfacial binding activity of lecithin-cholesterol acyltransferase (LCAT) is affected differentially by the location and extent of its glycosylation. Two LCAT glycosylation-deficient mutants, N84Q and N384Q, were constructed, permanently expressed in Chinese hamster ovary cells, and purified to determine the effects of deleting individual glycan chains on its stability, structure, and function. These purified mutants were studied by spectroscopic structural methods and enzymatic and binding assays to develop a molecular rationale for the relationship between LCAT glycosylation and activity. The N84Q LCAT mutant did not possess measurable enzymatic activity or interfacial binding affinity for reconstituted high-density lipoproteins. In addition, in thermal and chemical denaturation studies, N84Q LCAT was found to be significantly less stable than wild-type LCAT. The N384Q variant was initially more enzymatically active than wild-type LCAT, but gradually lost activity within months; however, it retained full interfacial binding activity. Significant changes were detected over time by circular dichroism in the alpha-helical content of N384Q LCAT and in the beta-sheet content of N84Q LCAT, compared with wild-type LCAT. Fluorescence measurements with the probe 1-anilinonapthalene-8-sulfonate suggested an alteration of the active site cavity in both mutants. In conclusion, both mutants lost catalytic activity, N84Q shortly after purification and N384Q more gradually, and were destabilized, probably because the deletion of the glycan chains altered local structural elements near the active site cavity and/or the interfacial binding regions.

Amino Acid Sequence↗