Synthetic substrates of lecithin: cholesterol acyltransferase

Investigation of the substrate specificity of lecithin: cholesterol acyltransferase has been greatly aided by the use of synthetic particles containing the molecular lipid substrates and the apolipoprotein activators of the enzyme. These synthetic particles, in vesicle or disc-like micelle form, are described in some detail noting their preparation, properties, advantages, and limitations as substrates for lecithin:cholesterol acyltransferase. The reactions of the enzyme with the synthetic particles are reviewed in terms of acyl donor and acceptor specificity, activation by apolipoproteins, effects of various inhibitors, and the kinetics of the reaction.     

Effects of detergents on the lecithin:cholesterol acyltransferase reaction

This paper describes the effect of an ionic (sodium dodecyl sulfate; SDS) and a nonionic detergent (Triton X-100) on the substrate and enzyme components of the lecithin: cholesterol acyltransferase (LCAT) reaction. When the enzyme sources (purified or partially purified) or the respective substrates [high-density lipoproteins (HDL) or proteoliposomes] were preincubated with detergents, a consistent trend in LCAT activity was only seen when partially purified LCAT was used as the enzyme source. This trend indicated an approximately 25% increase in enzyme activity over the control when 10(-4) M SDS and 2 X 10(-3)% Triton X-100 were present in the preincubation mixtures, respectively. Those observations suggested that, during the preincubations and subsequent assays, the enzyme (in the presence of detergents) was allowed to dissociate from the endogenous substrate and subsequently interact with the exogenous substrate molecules. Additional experiments utilizing molecular-sieve chromatography with whole plasma and partially purified enzyme also showed that dissociation of LCAT/lipoprotein complexes occurred in the presence of detergent. SDS was also shown to enhance the reaction of LCAT in whole plasma with anti-LCAT antibody in an enzyme-linked immunoassay system, indicating that the detergent treatment facilitated the exposure of additional antigenic sites, perhaps via dissociation of the enzyme from plasma lipoproteins.     

The interaction of apolipoproteins with lecithin:cholesterol acyltransferase

The rate of lecithin:cholesterole acyltransferase reaction was measured in a cholesterol-containing single bilayer lecithin vesicle system. ApolipoproteinA-I (apoA-I) activated the enzyme by itself; the other components of apolipoproteins of high density lipoproteins (HDL) (rho = 1.08--1.2 g/cm3), or rabbit serum gamma globulin inhibited the reaction. The reaction which was activated by pure apoA-I was strongly inhibited by anti-apoA-I antibody. Quantitative analysis of the results showed that the lecithin:cholesterol acyltransferase reaction was activated by the binding of apoA-I to the surface of lipid substrates. The rate of the lecithin:cholesterol acyltransferase-catalyzed reaction was strictly proportional to the surface density of apoA-I. The inhibition was due to the decrease of the amount of apoA-I on the lipid surface, either through competitive exclusion by apoA-II or by other proteins, or through specific extraction with antibody. The presence of components of apoHDL, other than apoA-I, prevented the inhibitory action of anti-apoA-I antibody.     

Isolation and properties of porcine lecithin:cholesterol acyltransferase

Lecithin: cholesterol acyltransferase (LCAT, phosphatidylcholine: sterol O-acyltransferase, EC 2.3.1.43) was purified approximately 20 000-fold from pig plasma by ultracentrifugation, phenyl-Sepharose and hydroxyapatite chromatography. Purified LCAT had an apparent relative molecular mass of 69 000 +/- 2000. By isoelectrofocusing it separated into five or six bands with pI values ranging from pH 4.9 to 5.2. The amino acid composition was similar to that of the human enzyme. An antibody against pig LCAT was prepared in goat. The antibody reacted against pig LCAT and gave a reaction of partial identity with human LCAT. Incubation of pig plasma or purified enzyme with the antibody virtually inhibited LCAT activity. The same amount of antibody inactivated only 62% of the LCAT activity in human serum. Pig and human LCAT were activated to the same extent by either human or pig apolipoprotein A-I (apo-A-I) using small liposomes as substrate. Human apoA-I, however, caused a higher esterification rate for both enzymes. Using apoA-I and small liposomes as a substrate, the addition of apoC-II up to 4 micrograms/ml had no effect on the LCAT reaction, but above this concentration LCAT was inhibited. Small liposomes with phosphatidylcholine/cholesterol molar ratios of 3:1 up to 8.4:1 did not show any significant differences in the LCAT reaction, when used as substrates in the presence of various amounts of apoA-I and albumin. In contrast, the LCAT activity was significantly reduced by liposomes with phosphatidylcholine/cholesterol molar ratios below 3:1.     

Continuous fluorescence assay for lecithin:cholesterol acyltransferase using a water-soluble phosphatidylcholine.

A water-soluble fluorescent phosphatidylcholine, 1,2-bis[4-(1-pyreno-butanoyl]-sn-glycero-3-phosphocholine (DPybPC) has been used to develop a sensitive, continuous assay for pure lecithin:cholesterol acyltransferase (LCAT) in solution. The monomeric substrate allowed us to examine the reaction of LCAT in the absence of a lipid/water interface in terms of the sensitivity of the enzymatic reaction to anions, ionic strength, apolipoproteins A-I and A-II, and a series of lysophosphatidylcholines and fatty acids. In contrast to the reaction of LCAT with aggregated phosphatidylcholines, the reaction of DPybPC with LCAT was not significantly affected by anions, ionic strength, nor apolipoproteins, indicating that these are only effectors of the interfacial reaction. Lysophosphatidylcholines and fatty acids inhibited LCAT in a chain-length-dependent manner below the critical micellar concentrations of these amphiphiles, indicating that the products of the LCAT reaction can bind to the enzyme and affect its kinetics even in the absence of an interface.     

Identification of the active-site serine in human lecithin: cholesterol acyltransferase

Purified human lecithin:cholesterol acyltransferase (LCAT) was covalently labeled by [3H]diisopropylflourophosphate with concomitant loss of enzymatic activity (M. Jauhiainen and P.J. Dolphin (1986) J. Biol. Chem. 261, 7023-7043). Some 60% of the enzyme was labeled in 1 h. Cyanogen bromide (CNBr) cleavage of the labeled, reduced, and carboxymethylated protein, followed by gel permeation chromatography yielded a 5- to 6-kDa peptide (LCAT CNBr-III) containing at least 60-70% of the incorporated label. Comparison of the amino acid composition of LCAT CNBr-III with that of the CNBr peptides predicted from the LCAT sequence (J. McLean et al. (1986) Proc. Natl. Acad. Sci. USA 83, 2335-2339) indicates that LCAT CNBr-III is peptide 168-220. In 22 cycles of automated Edman degradation of CNBr-III a radioactive derivative was only observed at cycle 14, and of the predicted CNBr fragments only peptide 168-220 contains a serine at position 14 from the amino terminus. Tryptic peptides predicted from the sequence should contain Ser181 at positions 22 and 23 from the N-terminus of fragments 160-199 and 159-199, respectively. On the other hand, Ser216 should be in position 15 from the N-terminus in fragment 202-238. Radiolabel sequencing of the tryptic digest of [3H]diisopropylphosphate-LCAT resulted in recovery of radioactivity in cycles 22 and 23, whereas cycle 15 yielded negligible radioactivity. These results establish that Ser181 is the major active site serine in human LCAT.     

Factors influencing the cholesterol esterification rate in lecithin cholesterol acyltransferase radioassay

Factors affecting the esterification rate of cholesterol by lecithin cholesterol acyltransferase (LCAT E.C. 2.3.1.43) in native cold labelled substrates (human, rabbit, rat serum, plasma, VLDL, LDL depleted serum, rabbit intraocular fluids) repaired by use of ready-made 14C-cholesterol discs (Cholesterol kinetics LCAT-test, UVVVR, Czechoslovakia) were investigated. EDTA added to the serum during the cold incubation (18 h, 0 degrees C-4 degrees C) increased the rate of esterification due to elimination of Ca2+ ions. The similar stimulating effect was found in the presence of mercaptoethanol (ME) in the serum, while in the plasma already stimulated by EDTA no additional effect by ME could be noticed. Freezing and thawing did not affect the fractional esterification rate (FER-per cent of total serum unesterified cholesterol esterified per hour) in normolipidaemic sera, whereas in hyperlipidaemic sera, particularly those with high levels of VLDL, FER was stimulated. Esterification partially proceeded during the cold incubation of serum or plasma with 14C-cholesterol ready-to-use discs, attaining the values of about 0.3%/h and 2-6%/h, respectively, in human sera and in rabbit and rat sera. The starting level of esterification did not affect the linearity of LCAT reaction during warm incubation (30 min at 37 degrees C), neither was the absolute value of FER changed as compared with cold labelled sera with those inhibited by DTNB and reactivated by ME. Substantial LCAT activity was also detected in extremely diluted substrates--such as intraocular fluid collected from rabbits with induced uveitis or after preceding paracentesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Familial lecithin:cholesterol acyltransferase deficiency. Biochemistry of the cornea

Opacification of the cornea from lipid accumulation is an early and characteristic feature of familial lecithin:cholesterol acyltransferase (LCAT) deficiency. Visual impairment in a female age 48 years led to keratoplasty and the first detailed analysis of cornea in this disorder. Multilaminar figures were present, and total lipid extracts were enriched with phospholipid and cholesterol; cholesteryl esters were reduced, and accounted for about 12% of the cholesterol. Linoleate C18:2 was the predominant residue in the cholesteryl ester fatty acid fraction, with a C18:1/18:2 ratio of 1:6.5. This ratio differs from that in normal cornea, and from that in plasma and in other tissue deposits in LCAT deficiency. Various disorders of the HDL/LCAT system in plasma can lead to corneal lipid accumulation and opacification. These disorders may share general defects of lipid clearance from the cornea, but this study of LCAT cornea indicates that the character of the accumulating lipid is significantly influenced by active local metabolism, irrespective of the defect in the HDL/LCAT system also present.     

Study of the components of reverse cholesterol transport in lecithin:cholesterol acyltransferase deficiency

Enzymatic and lipid transfer reactions involved in reverse cholesterol transport were studied in healthy and lecithin:cholesterol acyltransferase (LCAT), deficient subjects. Fasting plasma samples obtained from each individual were labeled with [3H]cholesterol and subsequently fractionated by gel chromatography. The radioactivity patterns obtained corresponded to the elution volumes of the three major ultracentrifugally isolated lipoprotein classes (very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL)). In healthy subjects, the LCAT activity was consistently found in association with the higher molecular weight portion of HDL. Similar observations were made when exogenous purified LCAT was added to the LCAT-deficient plasma prior to chromatography. Incubation of the plasma samples at 37 degrees C resulted in significant reduction of unesterified cholesterol (FC) and an increase in esterified cholesterol (CE). Comparison of the data of FC and CE mass measurements of the lipoprotein fractions from normal and LCAT-deficient plasma indicates that: (i) In normal plasma, most of the FC for the LCAT reaction originates from LDL even when large amounts of FC are available from VLDL. (ii) The LCAT reaction takes place on the surface of HDL. (iii) The product of the LCAT reaction (CE) may be transferred to either VLDL or LDL although VLDL appears to be the preferred acceptor when present in sufficient amounts. (iv) CE transfer from HDL to lower density lipoproteins is at least partially impaired in LCAT-deficient patients. Additional studies using triglyceride-rich lipoproteins indicated that neither the capacity to accept CE from HDL nor the lower CE transfer activity were responsible for the decreased amount of CE transferred to VLDL and chylomicrons in LCAT-deficient plasma.     

New substrate for determination of serum lecithin:cholesterol acyltransferase

Serum lecithin:cholesterol acyltransferase (LCAT) was estimated by enzymatically measuring the decrease in unesterified cholesterol after incubation of serum with liposomes. A high-performance liquid chromatography (HPLC) study showed the uptake of the lipids of liposomes by serum high density lipoprotein. Of all the examined liposomes prepared from cholesterol and various synthetic phosphatidylcholines, liposomes with dimyristoylphosphatidylcholine (DMPC) were found to be the most reactive in the LCAT reaction. When serum was used as an enzyme source, addition of purified apolipoprotein A-I, which is known to be an endogenous activator of LCAT, to the assay mixture resulted in a slight decrease in enzyme activity. Using DMPC-cholesterol liposomes as the substrate, the LCAT activities in 120 human sera showed a mean value of 485.4 +/- 64.6 nmol/hr per ml (mean +/- SD), which is 4.4- to 5.4-fold higher than the values obtained by self-substrate methods. LCAT activity was a linear function of the serum sample volume up to 670 nmol/hr per ml and coefficients of variation (CV) less than 4% were obtained under the standardized conditions. Moreover, when partially purified LCAT was added to various heat-inactivated sera, the activity was efficiently recovered. These results suggest that this method is sensitive, reproducible, and not greatly influenced by serum components.     

Reaction of lecithin cholesterol acyltransferase with water-soluble substrates

To separate the interfacial and catalytic reactions of lecithin cholesterol acyltransferase (LCAT), we carried out the first investigation of its reaction with water-soluble substrates. We used a continuous spectrophotometric assay for the hydrolysis of p-nitrophenyl esters of fatty acids to determine the chain length specificity of the enzyme and its modulation by anions and apolipoproteins in solution. By chemical modification of amino acid residues, we demonstrated that the active site serine and histidine residues participate in both the esterase and acyltransferase reactions but that cysteine residues are not involved in the esterase reaction. The kinetics of the LCAT reaction were measured for p-nitrophenyl esters of fatty acids having up to six (C-6) carbons in length. With increasing acyl chain lengths the optimal reaction rates occurred for the C-5 ester and Km and Vmax values decreased progressively, while the specificity constant, kcat/Km, increased. The same series of substrates and longer chain esters, up to C-16, were also reacted with LCAT in the presence of Triton X-100 in order to determine the general trends for the reaction rates as a function of chain length. The observed trends for the reaction rates and kinetic constants were attributed to an increasing binding affinity for the longer acyl chains in a large hydrophobic cavity, with a concomitant restriction in the motions of the substrates and a decreased probability for the correct positioning of the ester bond for hydrolysis, resulting in a decreased substrate turnover. Since the kinetics of the interfacial reactions of LCAT are very sensitive to the presence of anions and apolipoproteins, in particular apoA-I, we investigated the effects of these modulators on the reactions of LCAT in solution. Unlike the interfacial reactions, the hydrolysis of the p-nitrophenyl esters was not affected by 0.1 M concentrations of anions nor by water-soluble apolipoproteins (apoA-I, apoA-II, and apoCs). Thus the regulation of the activity of LCAT is mediated largely by the interfaces on which it acts.     

Activation of lecithin: cholesterol acyltransferase by human apolipoprotein A-IV

Human plasma apoproteins (apo) A-I and A-IV both activate the enzyme lecithin:cholesterol acyltransferase (EC 2.3.1.43). Lecithin:cholesterol acyltransferase activity was measured by the conversion of [4-14C] cholesterol to [4-14C]cholesteryl ester using artificial phospholipid/cholesterol/[4-14C]cholesterol/apoprotein substrates. The substrate was prepared by the addition of apoprotein to a sonicated aqueous dispersion of phospholipid/cholesterol/[4-14C]cholesterol. The activation of lecithin:cholesterol acyltransferase by apo-A-I and -A-IV differed, depending upon the nature of the hydrocarbon chains of the sn-L-alpha-phosphatidylcholine acyl donor. Apo-A-I was a more potent activator than apo-A-IV with egg yolk lecithin, L-alpha-dioleoylphosphatidylcholine, and L-alpha-phosphatidylcholine substituted with one saturated and one unsaturated fatty acid regardless of the substitution position. When L-alpha-phosphatidylcholine esterified with two saturated fatty acids was used as acyl donor, apo-A-IV was more active than apo-A-I in stimulating the lecithin:cholesterol acyltransferase reaction. Complexes of phosphatidylcholines substituted with two saturated fatty acids served as substrate for lecithin:cholesterol acyltransferase even in the absence of any activator protein. Essentially the same results were obtained when substrate complexes (phospholipid-cholesterol-[4-14C]cholesterol-apoprotein) were prepared by a detergent dialysis procedure. Apo-A-IV-L-alpha-dimyristoylphosphatidylcholine complexes thus prepared were shown to be homogeneous particles by column chromatography and density gradient ultracentrifugation. It is concluded that apo-A-IV is able to facilitate the lecithin:cholesterol acyltransferase reaction in vitro.     

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.     

Selectivity and contribution of lecithin: cholesterol acyltransferase to plasma cholesterol ester formation

Selectivity factors (Vm/Km) for human and rat lecithin: cholesterol acyltransferases (LCAT) for the transfer of various acyl groups from the 2-position of phosphatidylcholine were determined. By multiplying these values by the proportions of acyl groups at the 2-position of phosphatidylcholine, one can predict the proportions of molecular species of cholesterol ester which will be synthesized by LCAT. In human subjects fasted overnight, the molecular composition of plasma cholesterol ester was found to reflect the LCAT selectivity relatively accurately. This result supports the concepts that hepatic acyl-CoA:cholesterol acyltransferase (ACAT) does not contribute significantly to the synthesis of plasma cholesterol ester and that removal of cholesterol ester from plasma is not selective with respect to molecular species under these conditions. In contrast to the results with humans, the molecular composition of plasma cholesterol ester formed in spontaneously hypertensive rats fed a high-cholesterol diet and then fasted overnight differs from that which is predicted from LCAT selectivity and the proportion of various fatty acids at the 2-position of phosphatidylcholine: these results suggest that cholesterol ester is formed mainly via the ACAT reaction.     

A continuous fluorescence assay for lecithin cholesterol acyltransferase

A continuous fluorescence assay was adapted to the measurement of the phospholipase reaction of lecithin cholesterol acyltransferase (LCAT). The fluorescent phospholipid 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)aminocaproyl phosphatidylcholine (C6-NBD-PC) in micelle form reacted with LCAT to yield NBD-caproic acid, resulting in up to 5-fold increases in fluorescence in 30 min. The reaction rates were optimal in mixtures containing 0.1 M NaCl and 4 mM beta-mercaptoethanol at 37 degrees C. Apolipoprotein A-I did not activate the enzyme and bovine serum albumin bound monomeric substrate and interfered with the fluorescence assay. Under similar reaction conditions, bee venom phospholipase A2 was almost 100-fold more reactive than LCAT.