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.     

Activation of lecithin cholesterol acyltransferase by human apolipoprotein E in discoidal complexes with lipids

In a continued investigation of lecithin cholesterol acyltransferase reaction with micellar discoidal complexes of phosphatidylcholine, cholesterol, and various water soluble apolipoproteins, we prepared complexes containing human apo-E by the cholate dialysis method. These complexes were systematically compared to apo-A-I complexes synthesized under the same reaction conditions. Apo-E complexes (134 A in diameter) were slightly larger than apo-A-I complexes (110 A) but were very similar in terms of their protein and lipid content (2.4:0.10:1.0, egg phosphatidylcholine/cholesterol/apolipoprotein, w/w) and in the percentage of apolipoprotein in alpha-helical structure (72-74%). Concentration and temperature-dependence experiments on the velocity of the lecithin cholesterol acyltransferase reaction revealed differences in apparent Km values and small differences in apparent Vmax but very similar activation energies (18-20 kcal/mol). These observations suggest that differences in lecithin cholesterol acyltransferase activation by apo-A-I and apo-E are primarily a result of different affinities of the enzyme for the particles but that the rate-limiting step of the reaction is comparable for both complexes. Apo-E was found to be 18% as effective as apo-A-I in activating purified human lecithin cholesterol acyltransferase. Addition of free apo-A-I to apo-E complexes resulted in the exchange of bound for free apolipoprotein causing a slight increase in the reactivity with the enzyme when the incubation mixture was assayed. When the unbound apolipoproteins were removed by ultracentrifugation reisolated complexes containing both apo-E and apo-A-I demonstrated an even greater increase in reactivity with the enzyme.     

Activation of lecithin: cholesterol acyltransferase by apolipoproteins E-2, E-3, and A-IV isolated from human plasma

Apolipoprotein A-IV, apolipoprotein E-2 and apolipoprotein E-3 were individually incorporated into defined phosphatidylcholine/cholesterol liposomes for study of lecithin:cholesterol acyltransferase activation. Enzyme activities obtained with these liposomes were compared with that from liposomes containing purified apolipoprotein A-I. Apolipoprotein A-IV, apolipoprotein E-2, and apolipoprotein E-3 all activated lecithin:cholesterol acyltransferase. With purified enzyme and with egg yolk phosphatidylcholine as the acyl donor, maximal activation was obtained at a concentration of approximately 0.5 nmol for apolipoprotein A-IV and 0.4 nmol for the apolipoprotein E isoforms. Apolipoprotein A-IV was approximately 25% as efficient as apolipoprotein A-I for the activation of purified enzyme; apolipoprotein E-2 was 40% as efficient, and apolipoprotein E-3, 30%. Similar activation results were obtained using plasma as the enzyme source. Analysis of the plasma of patients with absence of apolipoprotein A-I or with only trace amounts of apolipoprotein A-I exhibited a reduced rate of cholesterol esterification and lecithin:cholesterol acyltransferase activity that was proportional to the reduced level of the enzyme's mass. These results indicate that apolipoprotein A-IV and apolipoprotein E may serve as physiological cofactors for the enzyme reaction.     

Separation of free and apolipoprotein D-associated human plasma lecithin: cholesterol acyltransferase

A high performance gel filtration method for the rapid and reproducible separation of free and apolipoprotein D-associated lecithin: cholesterol acyltransferase (LCAT) originating from human plasma has been developed. Starting from step 3 of a previously invented covalent chromatography procedure, free LCAT was obtained as a well separated fraction in a yield of 55% of that injected into the column. The free LCAT had a specific activity of over 34,000 units/mg and did not contain apolipoprotein D or any other contaminant in the injected sample. Further 28% of LCAT with fully retained activity was recovered in a second fraction, demonstrating a 66,000 u LCAT associated with all apolipoprotein D occurring as a mean 33,000 u and a minor 66,000 u species and with at least two unidentified proteins with apparent molecular masses of 76,000 u and 43,000 u, respectively. Both free and apolipoprotein D-associated LCAT accepted the free cholesterol of heat-inactivated plasma selectively depleted of VLDL and LDL (alpha-LCAT activity) and of HDL (beta-LCAT activity) as substrate.     

Stimulation of lecithin:cholesterol acyltransferase activity by apolipoprotein A-II in the presence of apolipoprotein A-I

Various combinations of incorporation and addition of apolipoprotein A-I (apo A-I) and apolipoprotein A-II (apo A-II) individually or together to a defined lecithin-cholesterol (250/12.5 molar ratio) liposome prepared by the cholate dialysis procedure were used to study the effect of apo A-II on lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) activity of both purified enzyme preparations and plasma. When apo A-I (0.1-3.0 nmol/assay) alone was incorporated or added to the liposome, apo A-I effectively activated the enzyme. By contrast, when apo A-II (0.1-3.0 nmol/assay) alone was incorporated into or added to the liposome, apo A-II exhibited minimal activation of LCAT activity, approximately 1% of the activity obtained by an equal amount of apo A-I. Addition of apo A-II (0.1-3.0 nmol/assay) together with apo A-I (0.8 nmol/assay) to the liposome reduced the LCAT activity to approximately 30% of the level obtained with addition of apo A-I alone. On the other hand, addition of apo A-II (0.1-3.0 nmol/assay) or addition of lecithin-cholesterol liposome containing apo A-II (0.1-3.0 nmol/assay) to lecithin-cholesterol liposome containing apo A-I (0.8 nmol/assay) did not significantly alter apo A-I activation of LCAT activity. However, when the same amounts (0.1-3.0 nmol/assay) of apo A-II were incorporated together with apo A-I (0.8 nmol/assay) into the liposome, apo A-II significantly stimulated LCAT activity as compared to activity obtained with incorporation of apo A-I alone. The maximal stimulation was obtained with 0.4 nmol apo A-II/assay for both purified and plasma enzyme. At this apo A-II concentration, approximately 4-fold and 1.8-fold stimulation was observed for purified enzyme and plasma enzyme, respectively. These results indicated that apo A-II must be incorporated together with apo A-I into lecithin-cholesterol liposomes to exert its stimulatory effect on LCAT activity and that apo A-II in high-density lipoprotein may play an important role in the regulation of LCAT activity.     

Natural mutations of apolipoprotein A-I impairing activation of lecithin:cholesterol acyltransferase

Five natural mutations of apolipoprotein A-I (apoA-I), apoA-I(A95D), apoA-I(Y100H), apoA-I(E110K), apoA-I(V156E) and apoA-I(H162Q), were studied for their ability to activate lecithin:cholesterol acyltransferase (LCAT). Mutants apoA-I(E110K), apoA-I(V156E) and apoA-I(H162Q) had an impaired ability to activate LCAT. Combined with data on other apoA-I mutants this finding is consistent with the idea that the central region between amino acids 110 and 160 is likely to be the "active site" of apoA-I involved in the interaction with LCAT and that a specific sequence of apoA-I is required for activation of the enzyme.     

Purification of human plasma lecithin:cholesterol acyltransferase by covalent chromatography

Human plasma lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) has been purified more than 20,000 fold from plasma in 10% yield. This new procedure is composed of only four steps, including ultracentrifugation of plasma to yield a 1.21-1.25 kg/l density fraction, covalent binding of LCAT in this fraction to thiopropyl-Sepharose followed by adsorption of the enzyme to wheat-germ lectin-Sepharose for elimination of albumin and finally batch-wise treatment of the desorbed LCAT with hydroxyapatite to remove residual impurities. The purified enzyme was free of apolipoprotein A-I, A-II, B, C-I, C-II, C-III and E as checked by double immunodiffusion and SDS-electrophoresis, which latter method also demonstrated the absence of hitherto characterized lipid transfer proteins. Only traces of apolipoprotein D were present in the preparation as detected by immunoblotting. The purified enzyme retained alpha- and beta-LCAT activities. Non-denaturing and denaturing polyacrylamide gel electrophoresis yielded apparent molecular masses of 69 and 66 kDa, respectively, for the enzyme which on isoelectric focusing produced one major and one minor isoform with pI values of 4.20 and 4.25, respectively. Apolipoprotein A-I was required to transform artificial lecithin-cholesterol liposomes into substrates for the purified LCAT.     

Activation of phosphatidylcholine-sterol acyltransferase by human apolipoprotein E isoforms

The reaction catalysed by phosphatidylcholine-sterol acyltransferase (EC 2.3.1.43) is believed to be the major source of cholesteryl ester in human plasma; the enzyme requires a protein activator. Several human apolipoproteins were found to exhibit an activator function, the major one being apolipoprotein A-I. Human apolipoprotein E exists in the population mainly in three different genetic isoforms; apolipoprotein E-2, E-3 and E-4. These isopeptides were isolated from subjects homozygous for one of the isoforms, incorporated into phospholipid/cholesterol/[14C]cholesterol complexes by the cholate dialysis procedure and used to measure capacity to activate phosphatidylcholine-sterol acyltransferase in comparison to apolipoprotein A-I lipid substrate particles prepared by the same procedure. Acyltransferase activity was measured by the formation of [14C]cholesteryl ester from [14C]cholesterol using purified enzyme. With egg yolk phosphatidylcholine as acyl donor, apo E was 15-19% as efficient as apolipoprotein A-I for activation of the acyltransferase. Apo-E-stimulated cholesteryl ester formation by the enzyme was enhanced when 1-oleoyl-2-palmitoyl-glycerophosphocholine was used as a substrate phospholipid (45% of apo A-I/phosphatidylcholine control) and most pronounced with dimyristoylglycerophosphocholine (75% of apo A-I/phosphatidylcholine control). No significant difference in activation was found between apo E isoforms. It is concluded that apolipoprotein E activates phosphatidylcholine-sterol acyltransferase in vitro and that apolipoprotein E isoforms are similarly effective.     

Lipoprotein affinity of human apolipoprotein A-IV during cholesterol esterification

We have studied the lipoprotein distribution of human apo A-IV during cholesterol esterification by the action of endogenous lecithin-cholesterol acyltransferase. Using immunologic and radiotracer techniques at 4 degrees C, apo A-IV was found in two discrete monomeric and dimeric populations, unassociated with plasma lipoproteins. With incubation at 37 degrees C, apo A-IV initially associated with the high density lipoprotein-3 fraction, but thereafter dissociated from its surface, and reappeared as unbound protein and in association with a complex in the low density lipoprotein size range. Inclusion of LCAT inhibitors in the incubations abolished these changes. We conclude that the changes in lipoprotein distribution of human apo A-IV closely parallel the formation and exchange of plasma cholesteryl esters.     

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.     

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.     

Purification and characterization of human plasma lecithin:cholesterol acyltransferase.

A highly purified (approximately 12 000-fold) homogeneous preparation of human plasma lecithin:cholesterol acyltransferase (LCAT) with 16% yield was obtained by a combination of density ultracentrifugation, high density lipoprotein affinity column chromatography, hydroxylapatite chromatography, and finally chromatography on anti-apolipoprotein D immunoglobulin-Sepharose columns to remove apolipoprotein D. This enzyme preparation was homogeneous by the following criteria: a single band by polyacrylamide gel electrophoresis in 8 M urea; a single band on sodium dodecyl sulfate gel electrophoresis with an apparent molecular weight of 68 000 +/- 1600; a single protein peak with a molecular weight of 70 000 on a calibrated Sephadex G-100 column. Its amino acid composition was different from human serum albumin and all other apoproteins isolated from lipoprotein fractions.     

Three arginine residues in apolipoprotein A-I are critical for activation of lecithin:cholesterol acyltransferase

Previous studies have suggested that the helical repeat formed by residues 143;-164 of apolipoprotein A-I (apoA-I) contributes to lecithin:cholesterol acyltransferase (LCAT) activation. To identify specific polar residues involved in this process, we examined residue conservation and topology of apoA-I from all known species. We observed that the hydrophobic/hydrophilic interface of helix 143;-164 contains a cluster of three strictly conserved arginine residues (R149, R153, and R160), and that these residues create the only significant positive electrostatic potential around apoA-I. To test the importance of R149, R153, and R160 in LCAT activation, we generated a series of mutant proteins. These had fluorescence emission, secondary structure, and lipid-binding properties comparable to those of wild-type apoA-I. Mutation of conserved residues R149, R153, and R160 drastically decreased LCAT activity on lipid-protein complexes, whereas control mutations (E146Q, D150N, D157N, R171Q, and A175R) did not decrease LCAT activity by more than 55%. The markedly decreased activities of mutants R149, R153, and R160 resulted from a decrease in the maximal reaction velocity V(max) because the apparent Michaelis-Menten constant K(m) values were similar for the mutant and wild-type apoA-I proteins.These data suggest that R149, R153, and R160 participate in apoA-I-mediated activation of LCAT, and support the "belt" model for discoidal rHDL. In this model, residues R149, R153, and R160 do not form salt bridges with the antiparallel apoA-I monomer, but instead are pointing toward the surface of the disc, enabling interactions with LCAT. - Roosbeek, S., B. Vanloo, N. Duverger, H. Caster, J. Breyne, I. De Beun, H. Patel, J. Vandekerckhove, C. Shoulders, M. Rosseneu, and F. Peelman. Three arginine residues in apolipoportein A-I are critical for activation of lecithin:cholesterol acyltransferase J. Lipid Res. 2001. 42: 31;-40.