Haptoglobin binds apolipoprotein E and influences cholesterol esterification in the cerebrospinal Fluid

Haptoglobin (Hpt) binds the apolipoprotein (Apo) A–I domain, which is involved in stimulating the enzyme lecithin-cholesterol acyltransferase (LCAT) for cholesterol esterification. This binding was shown to protect ApoA–I against hydroxyl radicals, thus preventing loss of ApoA–I function in enzyme stimulation. In this study, we report that Hpt is also able to bind ApoE. The Hpt binding site on the ApoE structure was mapped by using synthetic peptides, and found homologous to the Hpt binding site of ApoA–I. Hydroxyl radicals promoted in vitro the formation of ApoE-containing adducts which were detected by immunoblotting. Hpt impaired this oxidative modification whereas albumin did not. CSF from patients with multiple sclerosis or subjects without neurodegeneration contains oxidized forms of ApoE and ApoA–I similar to those observed in vitro . CSF was analyzed for its level of ApoA–I, ApoE, Hpt, cholesteryl esters, and unesterified cholesterol. The ratio of esterified with unesterified cholesterol, assumed to reflect the LCAT activity ex vivo , did not correlate with either analyzed protein, but conversely correlated with the ratio [Hpt]/([ApoE]+[ApoA–I]). The results suggest that Hpt might save the function of ApoA–I and ApoE for cholesterol esterification, a process contributing to cholesterol elimination from the brain.     

Haptoglobin inhibits lecithin-cholesterol acyltransferase in human ovarian follicular fluid

The activity of the enzyme lecithin-cholesterol acyltransferase (LCAT; E.C. 2.3.1.43) is involved in the removal of cholesterol excess from peripheral cells. This activity is stimulated by the HDL (high density lipoprotein) apolipoprotein A1 (ApoA1). Haptoglobin (Hpt) was previously found to be associated with ApoA1 in ovarian follicular fluid. LCAT activity was analyzed in follicular fluids, collected from an IVF program, containing different amounts of Hpt or Hpt/ApoA1 ratio. Addition of purified Hpt to follicular fluid caused a decrease in the enzyme activity, which was measured as the rate of synthesis of cholesteryl esters. In the fractions of fluid proteins, as obtained by gel filtration chromatography, Hpt and HDL were titrated by ELISA while the LCAT activity was assayed by using radioactive cholesterol and purified HDL. When isolated LCAT was incubated with fractions containing different Hpt/ApoA1 ratios, the enzyme activity was found negatively correlated with the Hpt/ApoA1 ratio (P < 0.01). LCAT kinetic parameters were measured in two fractions with the same amount of ApoA1 (5 microg/ml) but different amounts of Hpt (0.69 or 3.77 microg/ml): the V(max) did not change while the K(m) values were 24.1 or 78.6 microM in the presence of the low or high Hpt level, respectively. The analysis of fluids associated with cytoplasmically mature MII oocytes, in a cross-sectional study, confirmed that a negative correlation exists between the Hpt/ApoA1 ratio and the LCAT activity (P < 0.01). The results suggest that Hpt inhibits the reverse transport of cholesterol by preventing ApoA1 stimulation of the LCAT activity.     

Relevance of the amino acid conversions L144R (Zaragoza) and L159P (Zavalla) in the apolipoprotein A-I binding site for haptoglobin

The high-density lipoprotein apolipoprotein A-I (ApoA-I) stimulates the enzyme lecithin-cholesterol acyltransferase (LCAT) in the reverse cholesterol transport pathway. Two ApoA-I variants, Zaragoza (L144R) and Zavalla (L159P), are associated with low levels of HDL-cholesterol but normal LCAT activity. Haptoglobin interacts with ApoA-I, impairing LCAT stimulation. Synthetic peptides matching the haptoglobin-binding site of native or variant ApoA-I (native, P2a; variants, Zav-pep and Zar-pep) bound haptoglobin with different activity: Zar-pep>P2a>Zav-pep. They also differently rescued LCAT in vitro activity in the presence of haptoglobin (P2a=Zar-pep>Zav-pep). Therefore, both amino acid conversions affect haptoglobin binding and LCAT regulation. We highlight the role of haptoglobin in LCAT regulation in subjects with ApoA-I variants.     

Apolipoprotein A-I-dependent cholesterol esterification in patients with rheumatoid arthritis

Growing evidence suggests that atherogenesis is associated with inflammation or defective removal of cholesterol excess from peripheral cells. Apolipoprotein A-I [ApoA-I] activates the enzyme Lecithin-Cholesterol Acyl-Transferase to esterify cell cholesterol for transport to liver. Haptoglobin [Hpt] was previously found able to bind ApoA-I, and suggested to reduce the enzyme activation. The aim of this study was to demonstrate that enhanced levels of Hpt, as present during inflammation, are associated with low enzyme activity and increased thickness of the arterial wall. Enzyme activity and Hpt concentration were analysed in patients with rheumatoid arthritis having the same plasma levels of antioxidants (ascorbate, urate, alpha-tocopherol, retinol) or oxidation markers (nitrotyrosine, lipoperoxide) of healthy subjects. Cholesterol esterification, determined as ratio of cholesteryl esters with cholesterol in high-density lipoproteins, was lower in patients than in controls, and negatively correlated with the intima-media wall thickness of the common carotid. The ratio of Hpt with ApoA-I was negatively correlated with the enzyme activity, while positively correlated with intima-media wall thickness. The results suggest that high Hpt levels might severely impair the enzyme activity, thus contributing to cholesterol accumulation in vascular cells, and lesion formation in the endothelium.     

Effect of the human plasma apolipoproteins and phosphatidylcholine acyl donor on the activity of lecithin: cholesterol acyltransferase.

The human plasma apoproteins apoA-I and apoC-I enhanced the activity of partially purified lecithin: cholesterol acyltransferase five to tenfold with chemically defined phosphatidylcholine:cholesterol single bilayer vesicles as substrates. By contrast, apoproteins apoA-II, apoC-II, and apoC-III did not give any enhancement of enzyme activity. The activation by apoA-I and apoC-I differed, depending upon the nature of the hydrocarbon chains of phosphatidylcholine acyl donor. ApoA-I was most effective with a phosphatidylcholine containing an unsaturated fatty acyl chain. ApoC-I activated LCAT to the same extent with both saturated and unsaturated phosphatidylcholine substrates. Two of the four peptides obtained by cyanogen bromide cleavage of apoA-I retained some ability to activate LCAT. The efficacy of each of these peptides was approximately 25% that of the whole protein. Cyanogen bromide fragments of apoC-I were inactive. The apoproteins from HDL, HDL2, and HDL3, at low protein concentrations, were equally effective as activators of LCATand less effective than apoA-I. Higher concentrations of apoHDL, apoHDL2, and apoHDL3 inhibited LCAT activity. ApoC and apoA-II were both found to inhibit the activation of LCAT by apoA-I. The inhibition of LCAT by higher concentrations of apoHDL was not correlated with the aopA-II and apoC content.     

Apolipoprotein A-I: structure-function relationships

The inverse relationship between high density lipoprotein (HDL) plasma levels and coronary heart disease has been attributed to the role that HDL and its major constituent, apolipoprotein A-I (apoA-I), play in reverse cholesterol transport (RCT). The efficiency of RCT depends on the specific ability of apoA-I to promote cellular cholesterol efflux, bind lipids, activate lecithin:cholesterol acyltransferase (LCAT), and form mature HDL that interact with specific receptors and lipid transfer proteins. From the intensive analysis of apoA-I secondary structure has emerged our current understanding of its different classes of amphipathic alpha-helices, which control lipid-binding specificity. The main challenge now is to define apoA-I tertiary structure in its lipid-free and lipid-bound forms. Two models are considered for discoidal lipoproteins formed by association of two apoA-I with phospholipids. In the first or picket fence model, each apoA-I wraps around the disc with antiparallel adjacent alpha-helices and with little intermolecular interactions. In the second or belt model, two antiparallel apoA-I are paired by their C-terminal alpha-helices, wrap around the lipoprotein, and are stabilized by multiple intermolecular interactions. While recent evidence supports the belt model, other models, including hybrid models, cannot be excluded. ApoA-I alpha-helices control lipid binding and association with varying levels of lipids. The N-terminal helix 44-65 and the C-terminal helix 210-241 are recognized as important for the initial association with lipids. In the central domain, helix 100-121 and, to a lesser extent, helix 122-143, are also very important for lipid binding and the formation of mature HDL, whereas helices between residues 144 and 186 contribute little. The LCAT activation domain has now been clearly assigned to helix 144-165 with secondary contribution by helix 166-186. The lower lipid binding affinity of the region 144-186 may be important to the activation mechanism allowing displacement of these apoA-I helices by LCAT and presentation of the lipid substrates. No specific sequence has been found that affects diffusional efflux to lipid-bound apoA-I. In contrast, the C-terminal helices, known to be important for lipid binding and maintenance of HDL in circulation, are also involved in the interaction of lipid-free apoA-I with macrophages and specific lipid efflux. While much progress has been made, other aspects of apoA-I structure-function relationships still need to be studied, particularly its lipoprotein topology and its interaction with other enzymes, lipid transfer proteins and receptors important for HDL metabolism.     

Effect of lecithin:cholesterol acyltransferase on distribution of apolipoprotein A-IV among lipoproteins of human plasma

The effect of cholesterol esterification on the distribution of apoA-IV in human plasma was investigated. Human plasma was incubated in the presence or absence of the lecithin:cholesterol acyltransferase (LCAT) inhibitor 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) and immediately fractionated by 6% agarose column chromatography. Fractions were monitored for apoA-IV, apoE, and apoA-I by radioimmunoassay (RIA). Incubation resulted in an elevated plasma concentration of cholesteryl ester and in an altered distribution of apoA-IV. After incubation apoA-IV eluted in the ordinarily apoA-IV-poor fractions of plasma that contain small VLDL particles, LDL, and HDL2. Inclusion of DTNB during the incubation resulted in some enlargement of HDL; however, both cholesterol esterification and lipoprotein binding of apoA-IV were inhibited. Addition of DTNB to plasma after incubation and prior to gel filtration had no effect on the apoA-IV distribution when the lipoproteins were immediately fractionated. Fasting plasma apoE was distributed in two or three peaks; in some plasmas there was a small peak that eluted with the column void volume, and, in all plasmas, there were larger peaks that eluted with the VLDL-LDL region and HDL2. Incubation resulted in displacement of HDL apoE to larger lipoproteins and this effect was observed in the presence or absence of DTNB. ApoA-I was distributed in a single broad peak that eluted in the region of HDL and the gel-filtered distribution was unaffected by incubation either in the presence or absence of DTNB. Incubation of plasma that was previously heated to 56 degrees C to inactivate LCAT resulted in no additional movement of apoA-IV onto lipoproteins, unless purified LCAT was present during incubation. The addition of heat-inactivated LCAT to the incubation, had no effect on movement of apoA-IV. These data suggest that human apoA-IV redistribution from the lipoprotein-free fraction to lipoprotein particles appears to be dependent on LCAT action. The mechanism responsible for the increased binding of apoA-IV to the surface of lipoproteins when LCAT acts may involve the generation of "gaps" in the lipoprotein surface due to the consumption of substrate from the surface and additional enlargement of the core. ApoA-IV may bind to these "gaps," where the packing density of the phospholipid head groups is reduced.     

Preparation and incorporation of probe-labeled apoA-I for fluorescence resonance energy transfer studies of rHDL.

Apolipoprotein A-I (apoA-I), the major constituent of HDL, plays an essential role in regulating cholesterol metabolism, acting as the physiological activator of lecithin: cholesterol acyltransferase, which converts cholesterol to cholesterol ester. Thiol-reactive fluorescent probes attached to cysteine-containing apoA-I mutants are currently being used to investigate the "LCAT active" conformation of lipid-bound apoA-I. Herein, we report new methodologies allowing rapid expression, fluorescent labeling, and recombinant HDL (rHDL) preparation for use in apoA-I in fluorescence resonance energy transfer (FRET) studies. Cysteine-containing mutant forms of human apoA-I were cloned into the pTYB12 vector containing a T7 promoter, a modified self-splicing protein element (intein), and a small affinity tag [chitin binding domain (CBD)]. The fusion proteins were expressed in Escherichia coli, isolated from cell lysates, and bound to a chitin-affinity column. Release of mature human apoA-I was initiated by the addition of DTT, which induced self-cleavage at the COOH terminus of the intein - CBD fusion protein. ApoA-I was further purified by Q-sepharose and then used for fluorescent probe labeling. Discoidal rHDL were then prepared with donor and/or acceptor labeled apoA-I and characterized with respect to their size, composition and ability to activate LCAT.     

Multiple dysfunctions of two apolipoprotein A-I variants, apoA-I(R160L)Oslo and apoA-I(P165R), that are associated with hypoalphalipoproteinemia in heterozygous carriers

ApoA-I(R160L)Oslo and apoA-I(P165R) are naturally occurring apolipoprotein (apo) A-I variants that are associated with low HDL-cholesterol in heterozygous carriers. We characterized the capacity of these variants to bind lipid, to activate lecithin:cholesterol acyltransferase (LCAT), and to promote efflux of biosynthetic cholesterol from porcine aortic smooth muscle cells (SMCs) or exogenous cholesterol from lipid-loaded mouse peritoneal macrophages. During cholate dialysis, normal apoA-I and both variants associated completely with dipalmitoylphosphatidylcholine (DPPC) and formed rLpA-I of identical size. However, both apoA-I(P165R) and apoA-I(R160L)Oslo showed a reduced capacity to clear a turbid emulsion of dimyristoylphosphatidylcholine (DMPC). Compared to normal apoA-I, the LCAT-cofactor activity of apoA-I(P165R) and apoA-I(R160L)Oslo as defined by the ratio of Vmax to appKm was reduced significantly by 62% and 29%, respectively (here and throughout the text, the apparent Km is given as Michaelis-Menten kinetics do not take particle binding into account and therefore would result in errors with an interfacial enzyme such as LCAT; Vmax estimates are not affected by this error). ApoA-I/DPPC complexes induced biphasic cholesterol efflux from SMCs with a fast and a slow efflux component. Compared to rLpA-I reconstituted with wild type apoA-I, rLpA-I with apoA-I(P165R) or apoA-I(R160L)Oslo were significantly less effective in promoting cholesterol efflux from SMCs in incubations of 10 min duration but equally effective in incubations of 6 h duration. Lipid-free apoA-I did not induce efflux of biosynthetic cholesterol from SMCs but induced hydrolysis of cholesteryl esters and cholesterol efflux from acetyl-LDL-loaded mouse peritoneal macrophages. In the lipid-free form, both apoA-I variants promoted normal cholesterol efflux from murine peritoneal macrophages.We conclude that amino acid residues arginine 160 and proline 165 of apoA-I contribute to the formation of a domain that is very important for initial lipid binding and contributes to LCAT-activation and promotion of initial cholesterol efflux but not to the stabilization of preformed rLpA-I.     

Identification of plasma haptoglobin forms which loosely bind hemoglobin

Haptoglobin (Hpt) is known to capture circulating free hemoglobin (Hb) and bind apolipoprotein (Apo) A-I or E. Here, we report that Hb can be tightly bound by most of Hpt molecules (TB-Hpt, 80%), whereas loosely bound by a minor part of them (LB-Hpt, 20%). LB-Hpt amount was significantly increased (over 60%) in patients with acute coronary syndrome. LB-Hpt bound ApoA-I and ApoE less efficiently than TB-Hpt (8- and 4-fold less, respectively) and did not affect their activity of stimulating the enzyme lecithin-cholesterol acyltransferase. LB-Hpt and TB-Hpt displayed comparable levels of nitrotyrosine residues, but differences in glycan chains. Changes in LB-Hpt level might be associated with changes in Hpt functions.     

Disruption of the C-terminal helix by single amino acid deletion is directly responsible for impaired cholesterol efflux ability of apolipoprotein A-I Nichinan

Apolipoprotein A-I (apoA-I) Nichinan, a naturally occurring variant with ΔE235 in the C terminus, is associated with low plasma HDL levels. Here, we investigated the tertiary structure, lipid-binding properties, and ability to induce cellular cholesterol efflux of apoA-I Nichinan and its C-terminal peptide. Thermal and chemical denaturation experiments demonstrated that the ΔE235 mutation decreased the protein stability compared with wild type (WT). ApoA-I Nichinan exhibited capabilities to bind to or solubilize lipid vesicles that are intermediate to that of WT and a L230P/L233P/Y236P variant in which the C-terminal α-helix folding is completely disrupted and forms relatively larger and unstable discoidal complexes, indicating that perturbation of the C-terminal α-helical structure by the ΔE235 mutation leads to reduced lipid binding. Supporting this, apoA-I 209-241/ΔE235 peptide showed significantly decreased ability to form α-helix both in the lipid-free and lipid-bound states, and reduced efficiency to solubilize vesicles. In addition, both apoA-I Nichinan and its C-terminal peptide exhibited reduced activity in ABCA1-mediated cellular cholesterol efflux. Thus, the disruption of the ability of the C-terminal region to form α-helix caused by the E235 deletion appears to be the important determinant of impaired lipid binding and cholesterol efflux ability and, consequently, the low plasma HDL levels of apoA-I Nichinan probands.     

Characterization of functional residues in the interfacial recognition domain of lecithin cholesterol acyltransferase (LCAT)

Lecithin cholesterol acyltransferase (LCAT) is an interfacial enzyme active on both high-density (HDL) and low-density lipoproteins (LDL). Threading alignments of LCAT with lipases suggest that residues 50-74 form an interfacial recognition site and this hypothesis was tested by site-directed mutagenesis. The (delta56-68) deletion mutant had no activity on any substrate. Substitution of W61 with F, Y, L or G suggested that an aromatic residue is required for full enzymatic activity. The activity of the W61F and W61Y mutants was retained on HDL but decreased on LDL, possibly owing to impaired accessibility to the LDL lipid substrate. The decreased activity of the single R52A and K53A mutants on HDL and LDL and the severer effect of the double mutation suggested that these conserved residues contribute to the folding of the LCAT lid. The membrane-destabilizing properties of the LCAT 56-68 helical segment were demonstrated using the corresponding synthetic peptide. An M65N-N66M substitution decreased both the fusogenic properties of the peptide and the activity of the mutant enzyme on all substrates. These results suggest that the putative interfacial recognition domain of LCAT plays an important role in regulating the interaction of the enzyme with its organized lipoprotein substrates.     

Lipid-Sensing High-Throughput ApoA-I Assays

Apolipoprotein A-I (ApoA-I), a primary protein component of high-density lipoprotein (HDL), plays an important role in cholesterol metabolism mediating the formation of HDL and the efflux of cellular cholesterol from macrophage foam cells in arterial walls. Lipidation of ApoA-I is mediated by adenosine triphosphate (ATP) binding cassette A1 (ABCA1). Insufficient ABCA1 activity may lead to increased risk of atherosclerosis due to reduced HDL formation and cholesterol efflux. The standard radioactive assay for measuring cholesterol transport to ApoA-I has low throughput and poor dynamic range, and it fails to measure phospholipid transfer. We describe the development of two sensitive, nonradioactive high-throughput assays that report on the lipidation of ApoA-I: a homogeneous assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) and a discontinuous assay that uses the label-free Epic platform. The TR-FRET assay employs HiLyte Fluor 647-labeled ApoA-I with N-terminal biotin bound to streptavidin-terbium. When fluorescent ApoA-I was incorporated into HDL, TR-FRET decreased proportionally to the increase in the ratio of lipids to ApoA-I, demonstrating that the assay was sensitive to the amount of lipid bound to ApoA-I. In the Epic assay, biotinylated ApoA-I was captured on a streptavidin-coated biosensor. Measured resonant wavelength shift was proportional to the amount of lipids associated with ApoA-I, indicating that the assay senses ApoA-I lipidation.     

Deletion of N-terminal amino acids from human lecithin:cholesterol acyltransferase differentially affects enzyme activity toward alpha- and beta-substrate lipoproteins

Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for generation of the majority of the cholesteryl esters (CE) in human plasma. Although most plasma cholesterol esterification occurs on high-density lipoprotein (HDL), via alpha-LCAT activity, esterification also occurs on low-density lipoprotein (LDL) via the beta-activity of the enzyme. Computer threading techniques have provided a three-dimensional model for use in the structure-function analysis of the core and catalytic site of the LCAT protein, but the model does not extend to the N-terminal region of the enzyme, which may mediate LCAT interaction with lipoprotein substrates. In the present study, we have examined the functional consequences of deletion of the highly conserved hydrophobic N-terminal amino acids (residues 1-5) of human LCAT. Western blot analysis showed that the mutant proteins (Delta 1-Delta 5) were synthesized and secreted from transfected COS-7 cells at levels approximately equivalent to those of wild-type hLCAT. The secreted proteins had apparent molecular weights of 67 kDa, indicating that they were correctly processed and glycosylated during cellular transit. However, deletion of the first residue of the mature LCAT protein (Delta 1 mutant) resulted in a dramatic loss of alpha-LCAT activity (5% of wild type using reconstituted HDL substrate, rHDL), although this mutant retained full beta-LCAT activity (108% of wild-type using human LDL substrate). Removal of residues 1 and 2 (Delta 2 mutant) abolished alpha-LCAT activity and reduced beta-LCAT activity to 12% of wild type. Nevertheless, LCAT Delta 1 and Delta 2 mutants retained their ability to bind to rHDL and LDL lipoprotein substrates. The dramatic loss of enzyme activity suggests that the N-terminal residues of LCAT may be involved in maintaining the conformation of the lid domain and influence activation by the alpha-LCAT cofactor apoA-I (in Delta 1) and/or loss of enzyme activity (in Delta 1-Delta 5). Since the Delta 1 and Delta 2 mutants retain their ability to bind substrate, other factor(s), such as decreased access to the substrate binding pocket, may be responsible for the loss of enzyme activity.     

ApoA-I(MALLORCA) impairs LCAT activation and induces dominant familial hypoalphalipoproteinemia.

Apolipoprotein (apo)A-I is the major protein component of HDL and the cofactor for LCAT. We describe a large Spanish kindred, living in the Mediterranean Island of Mallorca, that presents a dominant form of hypoalphalipoproteinemia. The lipid profile of this family was studied because the proband, a 40-year-old male presenting signs of coronary atherosclerosis, showed severe HDL deficiency. However, none of the other family members had a known history of cardiovascular disease. Sequence analysis of the apoA-I gene in affected members identified a 33-base pair deletion, corresponding to residues 165-175 of the mature protein, eliminating the first 11 amino acids of the internal repeat 7. ApoA-I(MALLORCA) is associated with HDL-cholesterol deficiency (concentration ranging from 8-48% of the value in non-carriers), and a 2- to 3-fold decrease in plasma concentrations of apoA-I and apoA-II and endogenous LCAT activity, concomitant with a slight decrease in serum cholesterol efflux capability. Impairment of LCAT activity in HDL particles containing only mutated forms of apoA-I would not explain a pattern of dominant inheritance. HDL particles containing wild type apoA-I and at least one mutant apoA-I may also present impaired LCAT activity and/or other alterations leading to defective HDL maturation, a situation that would increase HDL lipid catabolism.We conclude that amino acids 165-175 of apoA-I are critical for normal HDL metabolism, at least in part because of their role in LCAT activation. However, apoA-I(MALLORCA) is not necessarily associated with clinical signs of atherosclerosis.     

Point mutations in apolipoprotein A-I mimic the phenotype observed in patients with classical lecithin:cholesterol acyltransferase deficiency

We have analyzed the effect of charged to neutral amino acid substitutions around the kinks flanking helices 4 and 6 of apoA-I and of the deletion of helix 6 on the in vivo activity of LCAT and the biogenesis of HDL. The LCAT activation capacity of apoA-I in vitro was nearly abolished by the helix 6 point (helix 6P-apoA-I[R160V/H162A]) and deletion {helix 6Delta-apoA-I[Delta(144-165)]} mutants, but was reduced to 50% in the helix 4 point mutant (helix 4P-apoA-I[D102A/D103A]). Following adenovirus-mediated gene transfer in apoA-I deficient mice, the level of plasma HDL cholesterol was greatly reduced in helix 6P and helix 6Delta mutants. Electron microscopy and two-dimensional gel electrophoresis showed that the helix 6P mutant formed predominantly high levels of apoA-I containing discoidal particles and had an increased prebeta1-HDL/alpha-HDL ratio. The helix 6Delta mutant formed few spherical particles and had an increased prebeta1-HDL/alpha-HDL ratio. Mice infected with adenovirus expressing the helix 4P mutant or wild-type apoA-I had normal HDL cholesterol and formed spherical alpha-HDL particles. Coinfection of mice with adenoviruses expressing human LCAT and the helix 6P mutant dramatically increased plasma HDL and apoA-I levels and converted the discoidal into spherical HDL, indicating that the LCAT activity was rate-limiting for the biogenesis of HDL. The LCAT treatment caused only a small increase in HDL cholesterol and apoA-I levels and in alpha-HDL particle numbers in the helix 6Delta mutant. The findings indicate a critical contribution of residue 160 of apoA-I to the in vivo activity of LCAT and the subsequent maturation of HDL and explain the low HDL levels in heterozygous subjects carrying this mutation.     

A proposed architecture for lecithin cholesterol acyl transferase (LCAT): identification of the catalytic triad and molecular modeling.

The enzyme cholesterol lecithin acyl transferase (LCAT) shares the Ser/Asp-Glu/His triad with lipases, esterases and proteases, but the low level of sequence homology between LCAT and these enzymes did not allow for the LCAT fold to be identified yet. We, therefore, relied upon structural homology calculations using threading methods based on alignment of the sequence against a library of solved three-dimensional protein structures, for prediction of the LCAT fold. We propose that LCAT, like lipases, belongs to the alpha/beta hydrolase fold family, and that the central domain of LCAT consists of seven conserved parallel beta-strands connected by four alpha-helices and separated by loops. We used the conserved features of this protein fold for the prediction of functional domains in LCAT, and carried out site-directed mutagenesis for the localization of the active site residues. The wild-type enzyme and mutants were expressed in Cos-1 cells. LCAT mass was measured by ELISA, and enzymatic activity was measured on recombinant HDL, on LDL and on a monomeric substrate. We identified D345 and H377 as the catalytic residues of LCAT, together with F103 and L182 as the oxyanion hole residues. In analogy with lipases, we further propose that a potential "lid" domain at residues 50-74 of LCAT might be involved in the enzyme-substrate interaction. Molecular modeling of human LCAT was carried out using human pancreatic and Candida antarctica lipases as templates. The three-dimensional model proposed here is compatible with the position of natural mutants for either LCAT deficiency or Fish-eye disease. It enables moreover prediction of the LCAT domains involved in the interaction with the phospholipid and cholesterol substrates.     

Cholesterol efflux by acute-phase high density lipoprotein: role of lecithin: cholesterol acyltransferase

HDL plays an initial role in reverse cholesterol transport by mediating cholesterol removal from cells. During infection and inflammation, several changes in HDL composition occur that may affect the function of HDL; therefore, we determined the ability of acute-phase HDL to promote cholesterol removal from cells. Acute-phase HDL was isolated from plasma of Syrian hamsters injected with lipopolysaccharide. Cholesterol removal from J 774 murine macrophages by acute-phase HDL was less efficient than that by control HDL because of both a decrease in cholesterol efflux and an increase in cholesterol influx. LCAT activity of acute-phase HDL was significantly lower than that of control HDL. When LCAT activity of control HDL was inactivated, cholesterol efflux decreased and cholesterol influx increased to the level observed in acute-phase HDL. Inactivation of LCAT had little effect on acute-phase HDL. In GM 3468A human fibroblasts, the ability of acute-phase HDL to remove cholesterol from cells was also lower than that of normal HDL. The impaired cholesterol removal, however, was primarily a result of an increase in cholesterol influx without changes in cholesterol efflux. When control HDL in which LCAT had been inactivated was incubated with fibroblasts, cholesterol influx increased to a level comparable to that of acute-phase HDL, without any change in cholesterol efflux. These results suggest that the ability of acute-phase HDL to mediate cholesterol removal was impaired compared with that of control HDL and the lower LCAT activity in acute-phase HDL may be responsible for this impairment. The decreased ability of acute-phase HDL to remove cholesterol from cells may be one of the mechanisms that account for the well-known relationship between infection/inflammation and atherosclerosis.     

The isolation and characterisation of a cDNA clone for human lecithin:cholesterol acyl transferase and its use to analyse the genes in patients with LCAT deficiency and fish eye disease

We have isolated cDNA clones coding for human lecithin:cholesterol acyl transferase (LCAT) from a liver-specific cDNA library by the use of two oligonucleotide probes based on the protein sequence. The clones span the sequence coding for the entire secreted LCAT, the 3' untranslated sequence and 12 amino acids of the signal peptide. The peptide sequence contains the conserved active site of serine lipases within a hydrophobic domain, flanked by a possible amphipatic alpha-helix. Only one gene for LCAT could be detected in genomic blots. We have used the cDNA as a probe to analyse the LCAT gene in patients suffering from LCAT deficiency and fish eye disease. No rearrangements or abnormal gene fragments were detected in these patients.     

Binding of partially reassembled high-density lipoprotein to isolated human small intestine epithelial cells. Effect of lipid composition

The binding of human high-density lipoprotein (HDL3), apolipoprotein A-I (apoA-I) and recombinants of apoA-I with cholesterol and/or dimyristoylphosphatidylcholine (DMPC) to the HDL receptor on isolated human small intestine epithelial cells was studied. ApoA-I competed for 125I-labelled HDL3 binding sites less effectively than HDL3, and a lower amount of 125I-labelled apoA-I than 125I-HDL3 was bound to cells. The apoA-I/DMPC recombinant competed for 125I-HDL3 binding sites nearly as well as HDL3, and 125I-apoA-I/DMPC recombinant bound to cells with at least the same efficiency as 125I-HDL3. The apoA-I/DMPC/cholesterol recombinant failed to compete for 125I-HDL3 binding sites, and the 125I-apoA-I/DMPC/cholesterol complex binding to cells was several-fold lower than that of other particles. All particles bound to cells with similar dissociation constants. Tetranitromethane-modified HDL3 failed to bind to high-affinity specific binding sites and compete with 125I-HDL3 for binding. The results obtained make it possible to assume that, while apoA-I may be a determinant of the HDL receptor, the lipid composition of the lipoprotein may affect its interaction with the receptor.