A simple and precise method to detect sterol esterification activity of lecithin/cholesterol acyltransferase by high-performance liquid chromatography.

The measurement of lecithin: cholesterol acyltransferase (LCAT, EC 2.3.1.43) activity is important in high-density lipoprotein (HDL) metabolism study and cardiovascular disease (CVD) risk assessment. However, current methods suffer from complex design and preparation of exogenous substrate, low reproducibility, and interference of cofactors. In this study, we developed a simple and precise high performance liquid chromatography (HPLC) method for the measurement of LCAT activity. By using 7-dehydrocholesterol (7-DHC) and 1,2-didecanoyl-sn-glycero-3-phosphocholine(10:0PC) as substrates, and an LCAT activating peptide (P642) as activator and emulsifier, the substrate reagent was easily made by vortex. The substrate reagent was mixed with serum samples (50:1, v/v) and incubated at 37 °C for 1 h. After incubation, the lipid was extracted with hexane and ethanol. With a conjugated double bond and ultraviolet absorption, 7-DHC and its esterification product could be separated and analyzed by a single HPLC run without calibration. LCAT activity was a linear function of the serum sample volume and the intra- and total assay coefficients of variation (CV) less than 2.5% were obtained under the standardized conditions. The substrate reagent was stable, and assay result accurately reflected LCAT activity. LCAT activities in 120 healthy subjects were positively correlated with triglyceride (P < 0.05), fractional esterification rate of HDL cholesterol (FERHDL) (P < 0.0001), and negatively correlated with apolipoprotein AI (apoAI) (P < 0.05) and HDL cholesterol (HDL-C) (P < 0.001). These results suggest that this method is sensitive, reproducible, and not greatly influenced by serum components and added substances, and will be a useful tool in the lipid metabolism study and the risk assessment of CVD.

BacMam production of active recombinant lecithin-cholesterol acyltransferase: Expression, purification and characterization.

Lecithin-cholesterol acyltransferase (LCAT) is a key enzyme in the esterification of cholesterol and its subsequent incorporation into the core of high density lipoprotein (HDL) particles. It is also involved in reverse cholesterol transport (RCT), the mechanism by which cholesterol is removed from peripheral cells and transported to the liver for excretion. These processes are involved in the development of atherosclerosis and coronary heart disease (CHD) and may have therapeutic implications. This work describes the use of baculovirus as a transducing vector to express LCAT in mammalian cells, expression of the recombinant protein as a high-mannose glycoform suitable for deglycosylation by Endo H and its purification to homogeneity and characterization. The importance of producing underglycosylated forms of secreted glycoproteins to obtain high-resolution crystal structures is discussed.

Binding of 8-anilino-1-naphthalenesulfonate to lecithin:cholesterol acyltransferase studied by fluorescence techniques.

The solvatochromic fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) has been used to study the hydrophobicity and conformational dynamics of lecithin:cholesterol acyltransferase (LCAT). The ANS to LCAT binding constant was estimated from titrations with ANS, keeping a constant concentration of LCAT (2 microM). Apparent binding constant was found to be dependent on the excitation. For the direct excitation of ANS at 375 nm the binding constant was 4.7 microM(-1) and for UV excitation at 295 nm was 3.2 microM(-1). In the later case, not only ANS but also tryptophan (Trp) residues of LCAT is being excited. Fluorescence spectra and intensity decays show an efficient energy transfer from tryptophan residues to ANS. The apparent distance from Trp donor to ANS acceptor, estimated from the changes in donor lifetime was about 3 nm and depends on the ANS concentration. Steady-state and time-resolved fluorescence emission and anisotropies have been characterized. The lifetime of ANS bound to LCAT was above 16 ns which is characteristic for it being in a hydrophobic environment. The ANS labeled LCAT fluorescence anisotropy decay revealed the correlation time of 42 ns with a weak residual motion of 2.8 ns. These characteristics of ANS labeled LCAT fluorescence show that ANS is an excellent probe to study conformational changes of LCAT protein and its interactions with other macromolecules.

A dual role for lecithin:cholesterol acyltransferase (EC 2.3.1.43) in lipoprotein oxidation.

Lecithin:cholesterol acyltransferase (LCAT) is a key enzyme involved in lipoprotein metabolism. It mediates the transesterification of free cholesterol to cholesteryl ester in an apoprotein A-I-dependent process. We have isolated purified LCAT from human plasma using anion-exchange chromatography and characterized the extracted LCAT in terms of its molecular weight, molar absorption coefficient, and enzymatic activity. The participation of LCAT in the oxidation of very low density lipoproteins (VLDL) and low-density lipoproteins (LDL) was examined by supplementing lipoproteins with exogenous LCAT over a range of protein concentrations. LCAT-depleted lipoproteins were also prepared and their oxidation kinetics examined. Our results provide evidence for a dual role for LCAT in lipoprotein oxidation, whereby it acts in a dose-responsive manner as a potent pro-oxidant during VLDL oxidation, but as an antioxidant during LDL oxidation. We believe this novel pro-oxidant effect may be attributable to the LCAT-mediated formation of oxidized cholesteryl ester in VLDL, whereas the antioxidant effect is similar to that of chain-breaking antioxidants. Thus, we have demonstrated that the high-density lipoprotein-associated enzyme LCAT may have a significant role to play in lipoprotein modification and hence atherogenesis.

Characterization of lecithin:cholesterol acyltransferase expressed in a human lung cell line.

Lecithin:cholesterol acyltransferase (LCAT) is a key enzyme for the transfer of mammalian cholesterol from peripheral tissues to the liver. In patients deficient in LCAT, serum cholesterol levels rise and can lead to corneal opacity, proteinuria, anemia, and kidney failure. As early as 1968, relatively low volume transfusion of normal plasma was shown to temporarily correct the abnormal lipoprotein profiles in LCAT-deficient patients. However, despite the cloning, study, and extensive expression of LCAT in mammalian cell lines, there is still no viable, clinical therapy for LCAT deficiency. The current study was initiated to provide a source of recombinant human LCAT for enzyme replacement therapy. Accordingly, human LCAT has been cloned and expressed for the first time in a human cell line. The recombinant LCAT secreted by these cells was purified by phenyl-Sepharose chromatography, analyzed to determine the nature of its glycosylation, and tested for its enzymatic properties. The activity and basic kinetic parameters for the enzyme were determined using both a fluorescent water-soluble substrate and a macromolecular (proteoliposome) substrate. The enzymatic properties and the carbohydrate components of the recombinant LCAT were all sufficiently similar to those of the circulating human plasma enzyme, suggesting that this source of LCAT may be appropriate for use in some form of enzyme replacement therapy.

Apolipoprotein A-II regulates HDL stability and affects hepatic lipase association and activity.

The effect of apolipoprotein A-II (apoA-II) on the structure and stability of HDL has been investigated in reconstituted HDL particles. Purified human apoA-II was incorporated into sonicated, spherical LpA-I particles containing apoA-I, phospholipids, and various amounts of triacylglycerol (TG), diacylglycerol (DG), and/or free cholesterol. Although the addition of PC to apoA-I reduces the thermodynamic stability (free energy of denaturation) of its alpha-helices, PC has the opposite effect on apoA-II and significantly increases its helical stability. Similarly, substitution of apoA-I with various amounts of apoA-II significantly increases the thermodynamic stability of the particle alpha-helical structure. ApoA-II also increases the size and net negative charge of the lipoprotein particles. ApoA-II directly affects apoA-I conformation and increases the immunoreactivity of epitopes in the N and C termini of apoA-I but decreases the exposure of central domains in the molecule (residues 98-186). ApoA-II appears to increase HL association with HDL and inhibits lipid hydrolysis. ApoA-II mildly inhibits PC hydrolysis in TG-enriched particles but significantly inhibits DG hydrolysis in DG-rich LpA-I. In addition, apoA-II enhances the ability of reconstituted LpA-I particles to inhibit VLDL-TG hydrolysis by HL. Therefore, apoA-II affects both the structure and the dynamic behavior of HDL particles and selectively modifies lipid metabolism.

Selective extraction of lecithin:cholesterol acyltransferase (EC 2.3.1.43) from human plasma.

A method for the rapid extraction of lecithin:cholesterol acyltransferase (LCAT) from human plasma or serum has been developed. The method is based on direct treatment of acidified plasma of fully conserved enzyme activity, with the strong ion exchanger Q-Sepharose, which under the experimental conditions bound all LCAT but only about 10% of the total protein content of the plasma, no albumin and essentially no lipoproteins. This corresponds to a 10-fold purification. Only traces of apolipoprotein A-I remained in the quantitatively desorbed LCAT preparation which, however, contained a residual fraction of apolipoprotein D and acidic plasma proteins. The present one-step procedure for extraction of LCAT in high yields from human plasma represents a simple and efficient alternative to the first step in previously described methods for preparation of the enzyme to homogeneity.

Detoxification of oxidized LDL by transferring its oxidation product(s) to lecithin:cholesterol acyltransferase.

In the present study, we isolated modified LCAT (m-LCAT) by hydroxyapatite column chromatography after incubation of crude LCAT (after DEAE SephadexA-50 column chromatography, penultimate step of LCAT purification) with oxidized LDL (oxLDL) at 37 degrees C for 1 h. The activity was found to be about 30% lower than that of native LCAT (n-LCAT). When activity was determined in the presence of oxLDL, m-LCAT was less inhibited than n-LCAT by oxLDL. Treatments of purified LCAT either at 56 degrees C for 30 min, at 100 degrees C for 10 min, or with 6 mM 5-5' -dithiobis-2-nitrobenzoic acid or 9 mM diisopropyl fluorophosphates (each at 37 degrees C for 30 min) resulted in the loss of its cholesterol-esterifying activity. When examined for their ability to detoxify oxLDL, native LCAT and LCAT treated at 56 degrees C for 30 min were found to detoxify oxLDL. These results indicate that oxidation product(s) of LDL is transferred and bound to LCAT in a way that does not depend on its cholesterol-esterifying activity, but rather on the availability of the sulfhydryl group of cysteine residue and the hydroxyl group of serine residue.

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.

Identification of domains in apoA-I susceptible to proteolysis by mast cell chymase. Implications for HDL function.

When stimulated, rat serosal mast cells degranulate and secrete a cytoplasmic neutral protease, chymase. We studied the fragmentation of apolipoprotein (apo) A-I during proteolysis of HDL(3) by chymase, and examined how chymase-dependent proteolysis interfered with the binding of eight murine monoclonal antibodies (Mabs) against functional domains of apoA-I. Size exclusion chromatography of HDL(3) revealed that proteolysis for up to 24 h did not alter the integrity of the alpha-migrating HDL, whereas a minor peak containing particles of smaller size with prebeta mobility disappeared after as little as 15 min of incubation. At the same time, generation of a large (26 kDa) polypeptide containing the N-terminus of apoA-I was detected. This large fragment and other medium-sized fragments of apoA-I produced after prolonged treatment with chymase were found to be associated with the alphaHDL; meanwhile, small lipid-free peptides were rapidly produced. Incubation of HDL(3) with chymase inhibited binding of Mab A-I-9 (specific for prebeta(1)HDL) most rapidly (within 15 min) of the eight studied Mabs. This rapid loss of binding was paralleled by a similar reduction in the ability of HDL(3) to induce high-affinity efflux of cholesterol from macrophage foam cells, indicating that proteolysis had destroyed an epitope that is critical for this function. In sharp contrast, prolonged degradation of HDL(3) by chymase failed to reduce the ability of HDL(3) to activate LCAT, even though it led to modification of three epitopes in the central region of apoA-I that are involved in lecithin cholesterol acyltransferase (LCAT) activation. This differential sensitivity of the two key functions of HDL(3) to the proteolytic action of mast cell chymase is compatible with the notion that, in reverse cholesterol transport, intactness of apoA-I is essential for prebeta(1)HDL to promote the high-affinity efflux of cellular cholesterol, but not for the alpha-migrating HDL particles to activate LCAT.

Characterization of C-terminal histidine-tagged human recombinant lecithin:cholesterol acyltransferase.

Lecithin:cholesterol acyltransferase (LCAT) is the plasma enzyme that catalyzes esterification of the sn-2 fatty acid of phospholipid to cholesterol. To facilitate the isolation of large quantities of LCAT and to assist in future structure;-function studies, LCAT containing a carboxy-terminal histidine-tag (H6) was expressed in Chinese hamster ovary cells (CHO). A high level of CHO-hLCATH6 expression ( approximately 15 mg L(-1)) was achieved over a 72-h period using 10 mm sodium butyrate to enhance transcription and PFX-CHO protein-free medium. The pure enzyme ( approximately 96%) was isolated by cobalt metal affinity chromatography with an activity yield of 82 +/- 26%. CHO-hLCATH6 and CHO-hLCAT species had identical specific activities (26 +/- 6 and 26 +/- 3 nmol CE formed microg(-1) h(-1), respectively). The enzymatic activity of CHO-hLCATH6 was stable at 4 degrees C in excess of 60 days. Substrate saturation studies, using rHDL composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol, and apolipoprotein A-I (80:5:1) indicated that the appK(m) for CHO-hLCATH6, CHO-hLCAT, and purified plasma LCAT were nearly identical at approximately 2 microm substrate cholesterol. We conclude that carboxy-terminal histidine-tagged LCAT is a suitable replacement for both plasma LCAT and CHO-hLCAT.

Biochemical and biophysical characterization of human recombinant lecithin: cholesterol acyltransferase.

We established a Chinese hamster ovary cell line that constitutively expresses up to 5 mg/L of human recombinant lecithin: cholesterol acyltransferase (rLCAT). We purified the rLCAT to > 96% purity, and characterized it along with plasma LCAT (pLCAT) biochemically and biophysically. The recombinant enzyme is more heavily glycosylated and more heterogeneous in its carbohydrate content than the plasma enzyme, as revealed by differences in molecular weight and pI isoforms, determined by mass spectrometry and isoelectric focusing. Recombinant LCAT is half as active enzymatically as pLCAT. The difference in activity is due to differences in the catalytic rates rather than in the apparent K(m) values, suggesting that the binding of the rLCAT to interfaces is not altered by its different glycosylation pattern. Despite these differences, rLCAT has essentially the same intrinsic tryptophan fluorescence emission spectrum and far-UV CD spectrum as pLCAT, indicating that the tertiary and secondary structures of both enzyme forms are very similar. Both enzyme forms have a propensity to self-associate, and their multimers appear resistant to dissociation by SDS and dilution. The free energies of unfolding (delta G(H2O)) of rLCAT and pLCAT are 3.4 +/- 0.2 and 3.2 +/- 0.2 kcal/mol, respectively, as determined by guanidine hydrochloride denaturation monitored by fluorescence. These relatively low delta G(H2O) values support the notion that LCAT is capable of undergoing major conformational changes upon interaction with interfacial substrates.

Purification of recombinant lecithin: cholesterol acyltransferase.

Production and purification of recombinant human lecithin:cholesterol acyltransferase (LCAT), secreted by baby hamster kidney (BHK) cells, has been improved by limiting the harvesting times for the conditioned medium and introducing an additional purification step. The recombinant BHK cells were grown until nearly confluent on multilayered flasks in a fetal-calf-serum-enriched medium. Subsequently, the cells were washed and supplied with serum free medium for 24-h periods. The conditioned medium, containing recombinant LCAT, was harvested at 24 and 48 h and subjected to chromatography on phenyl-Sepharose and ACA-44 agarose to isolate the recombinant enzyme. The second chromatography step revealed the presence of a low-molecular-weight contaminant that exhibited a carbohydrate/protein composition similar to proteoglycans. The major purified component contained LCAT activity and was homogeneous by acrylamide gel electrophoresis.

Structural and functional properties of full-length and truncated human proapolipoprotein AI expressed in escherichia coli.

Utilizing the Escherichia coli/pGex vector expression system incorporating a thrombin cleavage site, full-length (residues -6-243) and truncated forms of proapolipoprotein AI (proapoAI), terminating at amino acid residues 222, 210, 150, and 135, were purified to levels of at least 5 mg/L, after thrombin cleavage. Assessed by circular dichroism, the helical contents of L-alpha-dimyristoylphosphatidylcholine-associated forms of human plasma-derived apolipoprotein AI (apoAI) and recombinant proapoAI were comparable, being 69% and 65%, respectively. Circular dichroism measurements of the lipid-associated complexes of the truncated forms showed that between the sequence of residues 150-222 no additional helicity was gained until the carboxyl-terminal sequence was present in the molecule, indicating that the carboxyl terminus of the protein is required for the formation of helix within this central region. While tryptophan residues were more than 86% accessible, as assessed by iodide quenching, in the two truncated forms, proapoAI-6-135 and proapoAI-6-150, for both free and complexed protein, this figure fell to about 50% for full-length recombinant proapoAI, further indicating the influence of the carboxyl terminus on the structure of the whole protein. While cross-linking human plasma apoAI in solution with dithiobis-(succinimidyl propionate) revealed high molecular weight oligomers by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, recombinant proapoAI did not strongly form complexes larger than trimers. None of the truncated proapoAI molecules formed oligomers larger than trimers. The shortest form, proapoAI-6-135, only dimerized. Initial results from lecithin:cholesterol acyltransferase activation (apoAI peptide concentration 0.2 microM) indicated that truncation of the 21 carboxy-terminal amino acids resulted in a drop of approximately 53% in activation and 33 residues a drop of 67% relative to the full-length protein. Overall these results indicate the important influence of the carboxyl terminus on the structure of apoAI.

Glycosylation structure and enzyme activity of lecithin:cholesterol acyltransferase from human plasma, HepG2 cells, and baculoviral and Chinese hamster ovary cell expression systems.

The glycosylation state of lecithin:cholesterol acyltransferase (LCAT) may be important in determining its enzymatic activity. We compared glycosylation structure, enzyme kinetics, and phosphatidylcholine (PC) acyl specificity of human LCAT from four sources: human plasma (pLCAT), media from HepG2 cells (HepG2 LCAT), media from SF21 cells infected with a recombinant baculovirus (bLCAT) and media from stably transfected Chinese hamster ovary (CHO) cells (CHO LCAT). bLCAT was underglycosylated (molecular weight approximately 50 kDa) and resistant to digestion by N-glycanase F, endoglycosidase F, and neuraminidase. CHO and HepG2 LCAT were overglycosylated (approximately 68 kDa and approximately 70-75 kDa) compared to pLCAT (approximately 65 kDa). CHO LCAT, like pLCAT, was sensitive to N-glycanase F and neuraminidase but not to endoglycosidase F. HepG2 LCAT demonstrated resistance to N-glycanase F and endoglycosidase F. Apparent Km values for all four enzymes were similar (1.4-9.2 microM cholesterol) for recombinant high density lipoproteins (rHDL) containing sn-1 16:0, sn-2 18:1 PC (POPC). Apparent Vmax values (nmol cholesteryl ester formed/h per micrograms) were 52.6 for pLCAT, 48.6 for CHO LCAT, 15.3 for bLCAT, and 8.3 for HepG2 LCAT. Changes in PC acyl specificity in the presence and absence of cholesterol were characterized by comparing the ratio of LCAT activity on rHDL containing sn-1 16:0, sn-2 20:4 PC (PAPC) or POPC (PAPC/POPC activity ratio). The ratios for pLCAT, bLCAT, CHO LCAT, and HepG2 LCAT activity were 0.63, 0.49, 0.56, and 0.51 with cholesterol and 0.34, 0.29, 0.36, and 0.99 without cholesterol, respectively. We conclude that LCAT source influences glycosylation structure, which affects the apparent Vmax for cholesteryl ester formation with only minor changes in apparent Km or acyl substrate specificity.

Formation of pregnenolone- and dehydroepiandrosterone-fatty acid esters by lecithin-cholesterol acyltransferase in human plasma high density lipoproteins.

Pregnenolone- (PREG-), and dehydroepiandrosterone- (DHEA-) fatty acid esters (FA) are present in human plasma, where they are associated with lipoproteins. Because plasma has the ability to form PREG-FA and DHEA-FA in vitro from their unconjugated steroid counterparts, we postulated that the LCAT enzyme might be responsible for their formation. Here we show that lecithin-cholesterol acyltransferase (LCAT) has PREG and DHEA esterifying activities. First, VLDL, IDL, LDL, and HDL were isolated by the sequential ultracentrifugation micromethod from the plasma of fasting men and women and tested for their ability to form PREG-FA, DHEA-FA, and cholesteryl esters in vitro from their respective unconjugated counterparts. The results showed that the three steroids were esterified only in HDL subfractions. The rate of tritiated PREG esterification was clearly higher than that of tritiated cholesterol and DHEA, both in total plasma and isolated HDL, and no gender difference was observed. Second, human and guinea pig LCAT were purified and used in phosphatidylcholine-reconstituted vesicles containing human apoAI to show their ability to esterify tritiated cholesterol, PREG, and DHEA in the absence of unlabeled steroid. The amount of cholesteryl ester, PREG-FA, and DHEA-FA increased after incubation as a function of time and amount of purified LCAT, showing that PREG is preferentially acylated by LCAT compared to cholesterol and DHEA. The PREG and DHEA esterifying activities of LCAT were cofactor-dependent, as shown by the absence of acylation without apoAI. Finally, we determined by HPLC the fatty acid moiety of PREG-FA and DHEA-FA formed in human plasma and guinea pig and rat sera in vitro after incubation with unconjugated tritiated PREG and DHEA. We showed that the fatty acid moieties of newly formed tritiated PREG-FA and DHEA-FA were similar to that reported for cholesteryl esters in the plasma of the three species. We conclude that LCAT has a lecithin-steroid acyltransferase activity and that PREG is probably the preferential substrate of this enzyme. In addition, the fact that the differences in the fatty acid moieties of cholesteryl esters of human, guinea pig, and rat plasmas are also observed for PREG-FA and DHEA-FA suggests that the LCAT is the sole circulating enzyme that has PREG and DHEA esterifying activities.

The influence of sphingomyelin on the structure and function of reconstituted high density lipoproteins.

The effect of sphingomyelin (SPM) on the structure and function of discoidal and spherical reconstituted high density lipoproteins (rHDL) has been studied. Three preparations of discoidal rHDL with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC)/SPM/unesterified cholesterol (UC)/apolipoprotein (apo)A-I molar ratios of 99.6/0. 0/10.2/1.0, 86.0/13.6/10.8/1.0, and 72.5/26.3/11.4/1.0 were prepared by cholate dialysis. SPM did not affect discoidal rHDL size or surface charge. Esterification of cholesterol by lecithin:cholesterol acyltransferase (LCAT) was inhibited in the SPM-containing discoidal rHDL. When the discoidal rHDL of POPC/SPM/UC/apoA-I molar ratio 99.6/0.0/10.2/1.0 were incubated with low density lipoproteins (LDL) and LCAT, SPM transferred spontaneously from the LDL to the rHDL (t1/2 = 0.8 h) and spherical particles with a POPC/SPM/UC/CE/apoA-I molar ratio of 24.6/4.9/3. 6/24.9/1.0 were formed. Depleting the spherical rHDL of SPM head groups by incubation with sphingomyelinase increased the negative charge on the surface, but did not change their size. Cholesteryl ester transfer protein (CETP)-mediated transfers of cholesteryl esters and triglyceride between spherical rHDL and Intralipid were not affected by SPM head group depletion. The effect of SPM on rHDL structure was assessed spectroscopically. SPM increased POPC acyl chain and head group packing in the discoidal rHDL. When the spherical rHDL were depleted of SPM head groups, POPC acyl chain packing order decreased, but head group packing order was not affected. SPM inhibited the lipid-water interfacial hydration of discoidal rHDL. This parameter was not affected when the spherical rHDL were depleted of SPM head groups. The SPM molecule and the SPM head group, respectively, inhibited the unfolding of apoA-I in discoidal and spherical rHDL. It is concluded that (i) SPM influences the structure of discoidal and spherical rHDL, (ii) SPM inhibits the LCAT reaction in discoidal rHDL, and (iii) the SPM head group does not affect CETP-mediated lipid transfers into or out of spherical rHDL.

Reactivity of lecithin-cholesterol acyl transferase (LCAT) towards glycated high-density lipoproteins (HDL).

Hyperglycaemia in diabetic patients results in non-enzymatic glycation of plasma proteins, including lipoproteins such as high-density lipoproteins (HDL). We studied the effects of in vitro HDL glycation on the activity of lecithin-cholesterol acyl transferase (LCAT), a key enzyme in HDL plasma metabolism. LCAT was prepared from non-diabetic subjects and HDL by sequential density ultracentrifugation (in the density range of 1.063-1.21 g/ml) from both diabetic and non-diabetic patients. HDL from non-diabetic patients were glycated in vitro by incubating lipoproteins with 100 mmol/l glucose for various times at 37 degrees C with sodium cyanoborohydride as reducing agent. Glycation of HDL protein was quantified by measuring the percentage of derived amino acid residues using the TNBS assay. Kinetic parameters of LCAT were first determined using native HDL from non-diabetic patients and in vitro glycated HDL. With native HDL, Km and Vmax were 51.1 +/- 4.2 mumol/l (n = 8) and 12.9 +/- 2.4 nmol/ml/h (n = 8), respectively. Enzyme reactivity, calculated as the Vmax/Km ratio, was 0.25 +/- 0.04 h-1 (n = 8). In the case of moderate glycation (derived residues < 30%; n = 19) a significant increase in both Km (18.2 +/- 3.4%; mean +/- S.D.) and Vmax (9.3 +/- 2.4%) was observed. In contrast, with a high level of glycation (derived residues > 30%; n = 8), both parameters fell (Km, 25 +/- 6.3%; Vmax, 34.1 +/- 3.3%). In addition, whatever the level of glycation, enzyme reactivity was lower in the presence of in vitro glycated HDL. This decrease in LCAT reactivity was not due to a peroxidative process nor to an alteration of the protein and lipid composition of in vitro glycated HDL. It could, however, be explained by glycation of lysine residues in apolipoprotein A-I, which is the most potent activator of LCAT. In a second series of experiments, native diabetic HDL preparations were used as LCAT substrate. No alteration in Km values was observed, but there was a significant decrease in both Vmax (28%) and enzyme reactivity (32%). This difference in Km and Vmax alterations between native diabetic HDL and in vitro glycated HDL with low levels of glycation might be explained by the impact of physiological modifications, other than glycation, which could differently affect the chemicophysical properties of HDL in diabetic patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Hydrolysis and transesterification of platelet-activating factor by lecithin-cholesterol acyltransferase.

Purified lecithin-cholesterol acyltransferase (LCAT, EC 2.3.1.43) from human plasma was found to hydrolyze platelet-activating factor (PAF) to lyso-PAF and acetate. In addition, it catalyzed the transfer of the acetate group from PAF to lysophosphatidylcholine, forming lyso-PAF and a 1-acyl analog of PAF. In contrast to the cholesterol-esterification reaction carried out by the enzyme, the hydrolysis and transacetylation of PAF by LCAT did not require an apoprotein activator and were not inhibited by sulfhydryl inhibitors but were inhibited by serum albumin. When added to a proteoliposome substrate of LCAT or to whole plasma, PAF inhibited cholesterol esterification by LCAT competitively. PAF acetylhydrolase (EC 3.1.1.47), purified from human plasma, also catalyzed the transfer of acetate from PAF to lysophosphatidylcholine. However, the LCAT-catalyzed reactions of PAF were not due to contamination with PAF acetylhydrolase, since the ratio of acetyl transfer to acetyl hydrolysis was 3 times greater for LCAT, when compared with PAF acetylhydrolase under identical conditions. Furthermore, recombinant human LCAT secreted by baby hamster kidney cells also catalyzed the hydrolysis and transacetylation of PAF. These results demonstrate that LCAT can inactivate PAF in plasma by transacetylation and suggest that it may have a role in the metabolism of PAF, and possibly of oxidized phospholipids, in plasma.

Comparative study of phospholipid transfer activities mediated by cholesteryl ester transfer protein and phospholipid transfer protein.

In the present study, a sequential procedure was set up to separate simultaneously cholesteryl ester transfer protein (CETP), phospholipid transfer protein (PTP), and lecithin:cholesterol acyltransferase (LCAT) from human plasma. Subsequently, phospholipid transfer activities of purified lipid transfer proteins, deprived of LCAT activity, were compared and potential interactions between the two proteins were studied. Phospholipid transfer (PT) activity was determined by using three independent assays that measured the transfer of radiolabeled phosphatidylcholine ([14C]PC) either from phospholipid liposomes to high density lipoproteins-3 (PTliposome-->HDL3), from high density lipoproteins-3 to phospholipid liposomes (PTHDL3-->liposome), or from HDL3 to low density lipoproteins (PTHDL3-->LDL). Comparative study of CETP and PTP pointed out several differences in the ability of the two proteins to transfer phospholipids. i) Whereas both CETP and PTP were able to mediate phospholipid transfers from [14C]PC-HDL3 to LDL, only PTP facilitated phospholipid transfers from [14C]PC-liposomes to HDL3. ii) As PTP did not promote the transfer of phospholipids from [14C]PC-HDL3 to liposomes, it was concluded that it functions as a phospholipid transfer protein rather than a phospholipid exchange protein. This latter point was confirmed by the ability of purified PTP to induce the net mass transfer of phospholipids from PC-liposomes to HDL3. iii) While PTP presented no intrinsic cholesteryl ester transfer activity, it was able to significantly increase CETP-mediated cholesteryl ester transfers from HDL3 to LDL. iv) CETP did not influence the PTliposome-->HDL3 activity induced by PTP. v) Oleic acid was able to significantly increase the cholesteryl ester transfer activity of CETP, but not the PTliposome-->HDL3 activity of PTP. vi) PTHDL3-->LDL activity of purified CETP was explained, for a large part, by the copurification of nonesterified fatty acids. Taken together, data of the present report suggest that phospholipid transfer activity of CETP and PTP could occur through distinct processes. Since, in human plasma, PTP is not only responsible for the major part of phospholipid net mass transfer but is also able in vitro to modulate the CETP-mediated transfer of cholesteryl esters between various plasma lipoprotein fractions, it could play a determinant role in lipoprotein remodeling in vivo.