Role of the Arg123-Tyr166 Paired Helix of Apolipoprotein A-I in Lecithin:Cholesterol Acyltransferase Activation*

(Received for publication, October 11, 1996, and in revised form, February 28, 1997)

Ann Dhoest Dagger §, Zhian Zhao Dagger , Bart De Geest Dagger , Els Deridder Dagger , Alain Sillen par , Yves Engelborghs par , Désiré Collen Dagger and Paul Holvoet Dagger **

From the Dagger  Center for Molecular and Vascular Biology and the par  Laboratory of Chemical and Biological Dynamics, University of Leuven, B-3000 Leuven, Belgium

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

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


INTRODUCTION

ApoA-I is synthesized as a prepropeptide, cotranslationally cleaved to pro-apoA-I, and, upon secretion, processed to mature 243-amino acid apoA-I (1). It is folded into amphipathic alpha -helices with hydrophilic and hydrophobic surfaces (2-4), as demonstrated with complexes of phospholipids with apoA-I or model peptides (5-11). ApoA-I, when associated with phospholipids in discoidal complexes (12-14), contains eight putative amphipathic alpha -helices oriented around the edge of the discs, parallel to the acyl chains of the phospholipids, with their hydrophobic surface toward the lipid core and their hydrophilic surface toward the aqueous phase. The first amino-terminal domain (residues 44-63) has the lowest helical structure probability, whereas the second alpha -helix (residues 69-85) is not involved in a pair (12-14). The six carboxyl-terminal alpha -helical structures most likely form pairs of antiparallel alpha -helices stabilized by protein-protein interactions. A minimum length of 17-20 amino acids (five to six helical turns) appears to be required for effective phospholipid binding and LCAT1 activation (12-14).

The structures in apoA-I involved in phospholipid binding and/or LCAT activation remain largely unidentified. Reported differences in LCAT activity of apoA-I and deletion mutants may result from altered folding and/or organization of these molecules in rHDL rather than from deletion of a functional domain (15, 16). Therefore, in this study, the apoA-I(Delta (Arg123-Tyr166),nabla A-II(Ser12-Ala75)) and apoA-I(Delta (Ala190-Gln243),nabla A-II(Ser12-Gln77)) chimeras were produced, in which the Arg123-Tyr166 central or Ala190-Gln243 carboxyl-terminal pair of alpha -helices of apoA-I was deleted (Delta ) and substituted (nabla ) with the pair of alpha -helices of apoA-II. The average structural properties in solution and in reconstituted high density lipoprotein particles of the two chimeras were found to be unaltered, but the central domain chimera had a markedly reduced LCAT activity.


EXPERIMENTAL PROCEDURES

Oligonucleotide-directed Mutagenesis and DNA Sequencing

All DNA manipulations were carried out essentially as described by Maniatis et al. (17). Oligonucleotide-directed mutagenesis was performed by the gapped-duplex method of Kramer et al. (18) using the pMa/c vector system of Stanssens et al. (19). This system employs phasmid (i.e. phage/plasmid hybrid) vectors, allowing cloning, site-directed mutagenesis, and sequencing using the same vector without recloning. Oligonucleotides were obtained by custom synthesis (Pharmacia, Brussels, Belgium). DNA sequences were determined using a primer walking strategy on an ALF DNA sequencer (Pharmacia, Uppsala). Template DNA was purified by alkaline hydrolysis followed by a polyethylene glycol precipitation step. The sequencing reactions were carried out using T7 DNA polymerase (Pharmacia) and a fast denaturation protocol as described (20). The reaction products were sized on 6% Hydrolink Long Ranger gels (AT Biochem, Malvern, PA) containing 1 × Tris borate/EDTA buffer and run with 0.6 × Tris borate/EDTA buffer. The sequence was assembled using the IntelliGenetics Suite 5.4 program.

Expression and Purification of ApoA-I and the ApoA-I/ApoA-II Chimeras

The pMc-5-apoA-I transfection vector, encoding wild-type recombinant apoA-I, for expression in Escherichia coli under control of the tac promoter and the PhoA signal peptide sequence was constructed starting from plasmid pA1-3 (21), the pUC-19 vector (22), and the pMa/c mutagenesis vector (19). Plasmid pA1-3, obtained by insertion of the apoA-I cDNA into the PstI site of pBR322 (21), was a kind gift of Dr. L. Chan (Baylor College of Medicine, Houston, TX).

Deletion mutagenesis with the 5'-deoxyoligonucleotide dCAAGCGCTGGCGCAGCTCGTCGCTTAAGGGCTCCACCTTCT was performed on the pMc-5-apoA-I transfection vector to delete the Arg123-Tyr166 coding sequence, resulting in the pMc-5-apoA-I(Delta (Arg123-Tyr166)) vector. By substitution of the CCG codon for Pro121 with the CCC codon for Pro and of the CTG codon for Leu122 with the TTA codon for Leu, an additional AflII restriction site was created. The Ser12-Ala75 segment of apoA-II was amplified by polymerase chain reaction using the 5'-deoxynucleotide dCATAAGCATGCTGTCTCAGTACTTCCAGACC primer, containing the apoA-II(Ser12-Thr17) sequence and the SphI restriction site, and the 3'-deoxynucleotide dATCAAATGCATGGCAGGCTGTGTTCCAAG primer, containing the apoA-II(Ile70-Ala75) sequence and the NsiI restriction site. Thirty cycles were performed, consisting of 1 min of denaturation at 94 °C, 2 min of annealing at 52 °C, and 1.5 min of extension at 72 °C. The reaction product was digested with NsiI and SphI, blunt-ended using T4 polymerase, and ligated in the AflII-treated and blunt-ended pMc-5-apoA-I(Delta (Arg123-Tyr166)) vector, resulting in the pMc-5-apoA-I(Delta (Arg123-Tyr166),nabla A-II(Ser12-Ala75)) vector for the expression of apoA-I(Delta (Arg123-Tyr166), nabla A-II(Ser12-Ala75)) in E. coli. The pMc-5-apoA-I(Delta (Ala190-Gln243),nabla A-II(Ser12-Gln77)) vector for the expression of apoA-I(Delta (Ala190-Gln243),nabla A-II(Ser12-Gln77)) in E. coli was constructed as described (23).

Apolipoproteins were expressed in the periplasmic space of E. coli WK6 host cells as described (16, 23). Standard apoA-I was isolated from normolipemic human plasma as described previously (1). The purity of proteins was established by SDS gel electrophoresis (24) and immunoblotting (25).

Preparation of Discoidal Apolipoprotein-POPC-Cholesterol Complexes

Complexes of the apolipoproteins with POPC (Sigma) and cholesterol, at an apolipoprotein/POPC/cholesterol ratio of 1:3:0.15 (w/w/w), were prepared using the cholate dialysis procedure (11, 26). The mixture was incubated at 43 °C for 16 h, and cholate was removed by extensive dialysis. Complexes were isolated by gel filtration on a Superdex 200 HR column equilibrated with 20 mM Tris-HCl, pH 8.1, containing 0.15 M NaCl and 0.02 mg/ml sodium azide in a Waters fast performance liquid chromatography system. One-ml fractions were collected. The sizes of these complexes were estimated by comparison of their migration positions on native 8-25% gradient polyacrylamide gels with those of standard proteins: thyroglobulin (Stokes radius of 8.5 nm), apoferritin (6.1 nm), catalase (5.2 nm), and lactate dehydrogenase (4.1 nm) (Pharmacia).

The number of apolipoprotein molecules/rHDL particle was determined following cross-linking of apolipoproteins with bis(sulfosuccinimidyl) suberate (final concentration of 1 mM) for 6 h, as described previously (27), and separation of cross-linked proteins by SDS-PAGE on 10-15% gradient gels. The extent of oligomer formation was estimated by comparison with plasma cross-linked apoA-I (27). The levels of phospholipid and cholesterol were determined using commercial enzymatic kits (Biomérieux for phospholipid and Boehringer Mannheim for cholesterol), and the concentrations of apoA-I were determined according to Bradford (28).

Circular Dichroism Spectrometry

Circular dichroism spectra of lipid-free apolipoproteins in solution and of apolipoproteins in discoidal apolipoprotein-POPC-cholesterol complexes were measured with a Jasco J600 spectropolarimeter (Japan Spectroscopy, Tokyo) at wavelengths between 200 and 250 nm, using 6 µM sample solutions and a 1-mm path length cuvette. Backgrounds were measured at 250 nm for 5 min, followed by measurements of the alpha -helical content at 222 nm for 5 min. The fraction of alpha -helices in the secondary structure of the apolipoproteins was estimated from the molar ellipticities at 222 nm ([phi ]222 = -30,300 fH - 2340, where fH is the fraction of alpha -helical structure) (29).

Fluorescence Analysis

Corrected steady-state fluorescence spectra and intensities were measured as described previously (16). Excitation was carried out at 295 nm, where the contribution of tyrosyl fluorescence to the total intensity is minimal. Fluorescence was measured with a 2-mm slit width in quartz cuvettes with optical path lengths equal to 1 and 0.4 cm and wavelength resolutions of 7.2 and 3.6 nm for the emission and excitation wavelengths, respectively. Excitation was vertically polarized, and fluorescence was detected with a polarizer at a 54° angle to reduce the influence of fluorescence depolarization and brownian motion on the detected intensity. The spectra were corrected for the wavelength dependence of the emission monochromator and the photomultiplier and for background intensities as described previously (30). In the fluorescence-quenching experiments, the spectra were recorded at increasing concentrations of KI. The results were analyzed by the Stern-Volmer equation as modified by Lehrer (31): F0/(F0 - F) = (1/fa + 1/faKSV) × (1/[KI]), where F0 and F are the fluorescence intensities observed in the absence and presence of a given concentration of KI, respectively, and fa is the fraction of quenchable fluorescence (32). The Stern-Volmer constant (KSV) is an index of the accessibility of tryptophan residues to the aqueous phase.

Generation of a Stable Cell Line Expressing Human LCAT

The pUC-LCAT.10 plasmid, covering the entire coding region of human LCAT, was obtained from Dr. J. Mc Lean (Department of Cell Biology, Genentech Inc., San Francisco, CA). The LCAT cDNA was cloned into the pcDNA3 expression vector (Invitrogen, San Diego, CA), which contains the enhancer-promoter sequences from the immediate-early region of human cytomegalovirus and a neomycin resistance gene. After trypsinization, 8 × 106 293 cells were resuspended in 600 µl of Dulbecco's modified Eagle's medium (Gibco, Gent, Belgium) supplemented with 10% fetal bovine serum (Gibco) and transfected with 75 µg of pcDNA3-LCAT, linearized with XbaI, by electroporation. After electroporation, cells were plated at densities of 5 × 106, 106, 5 × 105, and 105/10-cm dish. One day after transfection, cells were placed on Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 600 µg/ml Geneticin (Gibco). Cell colonies, appearing 14 days after transfection, were picked, replated, and screened for LCAT expression by spectrophotometric assessment of butyryl ester of p-nitrophenol (33). Transfected 293 cells were cultured in serum-free Dulbecco's modified Eagle's medium for 48 h, and conditioned medium was collected and concentrated on a Centricon-10 filter (Amicon, Inc., Beverly, MA).

Kinetics of LCAT Activation by Discoidal Apolipoprotein-POPC-Cholesterol Complexes

LCAT activation by discoidal apolipoprotein-POPC-cholesterol complexes was monitored by incubation of the equivalent of 5-40 µM unesterified cholesterol with a final concentration of 50 nM LCAT. The reaction was carried out in 10 mM Tris-HCl, pH 8.0, containing 5 mM EDTA, 0.15 M NaCl, 4-16 mg/ml delipidated bovine serum albumin, and 6 mM beta -mercaptoethanol (LCAT buffer). After preincubation for 20 min at 37 °C, LCAT was added. The mixture was incubated at 37 °C for 10-180 min, after which the reaction was arrested by extraction of cholesteryl esters with hexane/isopropyl alcohol (3:2, v/v) containing cholesteryl heptadecanoate as an internal standard. Under these reaction conditions, <20% of the cholesterol was esterified. Generated cholesteryl esters were quantified by isocratic high performance liquid chromatography on a reversed-phase ZORBAX ODS column, eluted with acetonitrile/isopropyl alcohol (50:50, v/v), as described previously (34). Initial activation rates were obtained from plots of the concentration of generated cholesteryl esters versus time, and the kinetic parameters Km (expressed in µM cholesterol) and Vmax (expressed in nmol of cholesteryl esters generated per h per µM of cholesterol (cf. Table III)) were determined by linear regression analysis from Lineweaver-Burk plots.

Table III. Apparent kinetic parameters for the reaction of LCAT with rHDL particles

Data represent mean ± S.D. for three independent measurements. Significance of differences as compared with wild-type apoA-I was determined by Student's t test. Vmax and Km were derived from linear (r >=  0.99) Lineweaver-Burk plots of 1/V0 versus 1/[C], with [C] ranging from 5 to 20 µM. LCAT concentration was 50 nM.

Apolipoprotein Apparent Vmax Apparent Km Apparent Vmax/Apparent Km

nmol CEa/h µM nmol CE/h/µM
ApoA-I 27  ± 3.8 18  ± 3.1 1.5  ± 0.33
ApoA-I(Delta (Arg123-Tyr166),nabla A-II(Ser12-Ala75)) 1.4  ± 0.21b 25  ± 5.0 0.054  ± 0.009b
ApoA-I(Delta (Ala190-Gln243),nabla A-II(Ser12-Gln77)) 21  ± 3.8 16  ± 2.4 1.3  ± 0.32

a CE, cholesteryl ester.
b p < 0.001.

Binding of 125I-Labeled LCAT to Discoidal Apolipoprotein-POPC-Cholesterol Complexes

Recombinant LCAT was labeled with 125I by the IODOGEN method. Following the reaction, radiolabeled LCAT was separated from free 125I by passage over a Sephadex G-25M PD-10 gel filtration column (Pharmacia) pre-equilibrated with LCAT buffer. Specific activity was typically 225 cpm/ng of protein. Radiolabeled LCAT (final concentration of 3 µg/ml) was incubated with rHDL (final apolipoprotein concentrations ranging between 4.4 and 140 µg/ml) for 20 min, and free LCAT was separated from bound LCAT by filtration on a Centricon-100 filter (Amicon, Inc.).


RESULTS

Fig. 1 is a schematic representation of the predicted amphipathic helical regions in apoA-I, apoA-II, apoA-I(Delta (Arg123-Tyr166),nabla A-II(Ser12-Ala75)), and apoA-I(Delta (Ala190-Gln243),nabla A-II(Ser12-Gln77)). The predicted number of amphipathic helices, according to Brasseur et al. (12), was eight for apoA-I and for the two chimeras. Wild-type apoA-I and the apoA-I/apoA-II chimeras were expressed in the periplasmic space of E. coli cells and purified to homogeneity as evidenced by their migration as single bands on 10-15% SDS-polyacrylamide gels (Fig. 2). The molecular masses of the apolipoproteins, calculated from a plot of the logarithm of the molecular masses of the standard proteins versus the migration distance, were 28.3 kDa for apoA-I and 30.4 and 29.8 kDa for the respective chimeras and thus were in agreement with those calculated on the basis of the respective amino acid compositions. Wild-type apoA-I migrated in the same position as plasma apoA-I. The identity of each band was confirmed by immunoblot analysis using polyclonal rabbit anti-human apoA-I antibodies (data not shown).


Fig. 1. Schematic representation of the predicted amphipathic helical regions in apoA-I (A), apoA-II (B), apoA-I(Delta (Arg123-Tyr166),nabla A-II(Ser12-Ala75)) (C), and apoA-I(Delta (Ala190-Gln243),nabla A-II(Ser12-Gln77)) (D). Hatched boxes, putative alpha -helices of apoA-I; dotted boxes, putative alpha -helices of apoA-II.
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Fig. 2. SDS-PAGE of purified apolipoproteins on 10-15% gradient gels. Proteins (5 µg/lane) were stained with Coomassie Brilliant Blue. Lane 1, protein calibration mixture consisting of phosphorylase b (94 kDa), albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), and trypsin inhibitor (20 kDa); lane 2, apoA-I; lane 3, apoA-I(Delta (Arg123-Tyr166),nabla A-II(Ser12-Ala75)); lane 4, apoA-I(Delta (Ala190- Gln243),nabla A-II(Ser12-Gln77)).
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The alpha -helical contents of the apolipoproteins in aqueous solution, as determined by circular dichroism scanning, were 48% for apoA-I and 43 and 41% for the respective chimeras. The recovery of apolipoproteins in discoidal apolipoprotein-POPC-cholesterol complexes after gel filtration was 90% for wild-type apoA-I and the chimeras. The Stokes radius of all rHDL was 4.8 nm (Fig. 3 and Table I). Cross-linking of the apolipoprotein molecules within all the discoidal apolipoprotein-POPC-cholesterol particles revealed 2 apolipoprotein molecules/particle (Fig. 4 and Table I). The phospholipid surface was calculated from the circumference of the disc using the measured diameter minus 3 nm, i.e. 2 × radius of an alpha -helix, multiplied by a disc height of 3.8 nm. The number of phospholipid molecules was calculated from the calculated surface divided by 0.45 nm2, the surface area/condensed phospholipid molecule. The calculated apolipoprotein/phospholipid molar ratios were 1:90, 1:83, and 1:89, respectively, and were in agreement with the ratios calculated on the basis of the composition of the rHDL.


Fig. 3. Determination of Stokes radii of rHDL by native polyacrylamide gel scanning. rHDL particles, isolated by gel filtration on a Superdex 200 HR column, were subjected to electrophoresis on 4-15% gradient polyacrylamide gels under nondenaturing conditions. A, apoA-I; B, apoA-I(Delta (Arg123-Tyr166),nabla A-II(Ser12-Ala75)); C, apoA-I(Delta (Ala190-Gln243),nabla A-II(Ser12-Gln77)); D, protein calibration mixture containing apoferritin (Stokes radius of 6.1 nm), catalase (5.2 nm), and lactate dehydrogenase (4.1 nm).
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Table I. Properties of discoidal apolipoprotein-POPC-cholesterol complexes


Properties Apolipoprotein
ApoA-I a ApoA-I(Delta (Arg123-Tyr166), nabla A-II(Ser12-Ala75)) ApoA-I(Delta (Ala190-Gln243), nabla A-II(Ser12-Gln17))

Stokes radius (nm)b 4.8 4.8 4.8
ApoA-I molecules/disc (n)c 2 2 2
 alpha -Helical content (%)d 74 75 72
Measured alpha -helices/apoA-I molecule (n)e 8 8 8
Estimated no. alpha -helices/apoA-I (n)f 8 8 8
Fraction of phospholipid surface covered with alpha -helicesg 0.77 0.77 0.77

a Values for plasma apoA-I and recombinant apoA-I were identical.
b Sizes of rHDL particles were determined by PAGE.
c The number of apolipoprotein molecules/HDL particle was determined by SDS-PAGE after cross-linking with bis(sulfosuccinimidyl) suberate.
d The alpha -helical content was determined by CD scanning.
e The number of alpha -helices/apolipoprotein molecule was calculated from the alpha -helical content and from the protein length assuming a length of 16 amino acids for the amphipathic helices and 6 amino acids for the adjacent beta -strands (2, 12).
f Values represent the estimated number according to the model of Brasseur et al. (12) (Fig. 1).
g The phospholipid surface was calculated from the circumference of the disc, using the measured diameter minus 3 nm, multiplied by a disc height of 3.8 nm. The area covered by an alpha -helix containing 16 amino acids was estimated to be 4 nm2.


Fig. 4. Determination of the number of apoA-I molecules/rHDL particle by SDS-polyacrylamide gel scanning. rHDL particles, in which apoA-I molecules were cross-linked with bis(sulfosuccinimidyl) suberate, were subjected to electrophoresis on 10-15% gradient SDS-polyacrylamide gels. A, apoA-I; B, apoA-I(Delta (Arg123-Tyr166),nabla A-II(Ser12-Ala75)); C, apoA-I(Delta (Ala190-Gln243),nabla A-II(Ser12-Gln77)); D, protein calibration mixture containing phosphorylase b (94 kDa), albumin (67 kDa), ovalbumin (43 kDa), and trypsin inhibitor (30 kDa).
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The alpha -helical contents of the apoA-I proteins in discoidal apolipoprotein-POPC-cholesterol complexes were 74% for apoA-I and 75 and 72% for the respective chimeras. The number of alpha -helices, calculated from the alpha -helical content determined by circular dichroism scanning and from the protein length assuming a length of 22 amino acids/alpha -helical repeat (2, 12), was eight per apoA-I or per chimera molecule (Table I) and was thus in agreement with the predicted values (12). The fraction of the phospholipid surface that was covered by the alpha -helix was calculated as the total phospholipid surface in nm2 divided by 4 nm2, the surface area of an alpha -helix, that contains 16 amino acids. The calculated fraction was 0.77 for apoA-I and the chimeras (Table I).

Binding of apoA-I and the chimeras to POPC was associated with a shift of Trp fluorescence maxima to lower wavelengths from 337 to 332 nm for apoA-I (both for plasma apoA-I and recombinant apoA-I), from 339 to 334 nm for the central domain chimera, and from 337 to 333 nm for the carboxyl-terminal domain chimera (Table II), suggesting that lipid binding is associated with a translocation of Trp residues to a more apolar environment. The quenching parameters KSV and fa are summarized in Table II. For totally exposed Trp residues, in the absence of electrostatic or viscosity effects, KSV = 12 M-1 and fa = 1, whereas for totally protected Trp residues, KSV = 0 and fa = 0. The quenchable fluorescence of all apolipoproteins in the respective rHDL represented, on average, 60% of the total fluorescence (Table II).

Table II. Fluorescence properties of apolipoproteins in rHDL particles

Data represent mean ± S.D. for three independent measurements. Data for apoA-I and the chimeras were not significantly different. Values for plasma apoA-I and recombinant apoA-I were identical.

Properties Apolipoprotein
ApoA-I ApoA-I(Delta (Arg123-Tyr166), nabla A-II(Ser12-Ala75)) ApoA-I(Delta (Ala190-Gln243), nabla A-II(Ser12-Gln77))

Wavelength of maximum fluorescence (nm) 332 334 333
KSV (M-1) for quenching of Trp fluorescence with I- 2.9  ± 0.7 3.4  ± 1.1 1.9  ± 0.06
Quenchable fraction of Trp fluorescence (fa) 0.61  ± 0.08 0.60  ± 0.05 0.58  ± 0.05

Recombinant LCAT was obtained in serum-free conditioned medium of transfected 293 cells. The homogeneity of the recombinant LCAT preparation is illustrated in Fig. 5. LCAT activation by the discoidal apolipoprotein-POPC-cholesterol complexes obeyed Michaelis-Menten kinetics, as shown by linear Lineweaver-Burk plots of the inverse of the initial activation rate (1/V0) versus the inverse of the cholesterol concentrations (1/[C]). The apparent kinetic parameters Vmax and Km and the Vmax/Km ratios for the different apolipoprotein-POPC-cholesterol complexes are summarized in Table III. Exchange of the Arg123-Tyr166 paired helix of apoA-I with the pair of helices of apoA-II reduced the LCAT activity of apoA-I 27-fold due to a reduction of Vmax and not of Km. In contrast, exchange of the Ala190-Gln243 carboxyl-terminal domain helices of apoA-I with the pair of helices of apoA-II had no effect on the LCAT activity of apoA-I.


Fig. 5. SDS-PAGE of recombinant LCAT on 10-15% gradient gels. Lane 1, protein calibration mixture consisting of phosphorylase b (94 kDa), albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), and trypsin inhibitor (20 kDa); lane 2, recombinant LCAT.
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Fig. 6 illustrates the binding of radiolabeled LCAT to rHDL of apoA-I and the apoA-I(Delta (Arg123-Tyr166),nabla A-II(Ser12-Ala75)) chimera. Fifty % of maximal binding was obtained with 34 µg/ml apoA-I and 27 µg/ml apoA-I(Delta (Arg123-Tyr166), nabla A-II(Ser12-Ala75)).


Fig. 6. Binding of radiolabeled LCAT to discoidal apolipoprotein-POPC-cholesterol complexes. bullet , apoA-I; black-square, apoA-I (Delta (Arg123-Tyr166),nabla A-II(Ser12-Ala75)).
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DISCUSSION

Reported differences in LCAT activity of apoA-I variants may be due to defective interaction with phospholipids, structural changes in rHDL, and/or deletion of functional domains. To further investigate the role of the central and carboxyl-terminal domains of apoA-I in LCAT activation, the apoA-I(Delta (Arg123-Tyr166),nabla A-II(Ser12-Ala75)) and apoA-I(Delta (Ala190-Gln243),nabla A-II(Ser12-Gln77)) chimeras were produced. The extent of in vitro phospholipid binding of these chimeras was similar to that of apoA-I, as demonstrated by comparable disc formation after mixing the apolipoproteins and phospholipids at equal weight ratios. This was evidenced by a shift of the maximum Trp fluorescence to a shorter wavelength and by a decreased accessibility of the Trp residues to I-. The sizes of rHDL reconstituted with apoA-I and the chimeras were identical: the respective rHDL contained 2 apolipoprotein molecules/particle, and circular dichroism scanning revealed eight alpha -helices per intact apoA-I molecule and per chimera molecule, in agreement with the predicted numbers according to Brasseur et al. (12). The calculated apolipoprotein/phospholipid molar ratios of the different rHDL particles were very similar. Thus, substitution of the Ala123-Tyr166 central or Ala190-Gln243 carboxyl-terminal domain helices of apoA-I with the pair of helices of apoA-II did not affect the size and the composition of rHDL, and the conformation and helical distribution in the different apolipoproteins in these particles were very similar. Substitution of the carboxyl-terminal domain of apoA-I with the helices of apoA-II did not reduce LCAT activity, but substitution of the central domain resulted in a 27-fold reduction of LCAT activity, suggesting that the Ala123-Tyr166 segment is critical for LCAT activation. Binding experiments revealed that the reduced LCAT activity of the apoA-I(Delta (Arg123-Tyr166),nabla A-II(Ser12-Ala75)) chimera was not due to reduced binding of LCAT to rHDL.

Based on data obtained with synthetic peptides, it was concluded that the LCAT-activating domain of apoA-I resides in a 22-mer tandem repeat located between residues 66 and 121 (35). This was further supported by the finding that monoclonal antibodies directed against an epitope that spanned residues 95-121 inhibited the LCAT activation with apoA-I (36). Binding of antibodies to an epitope in the amino-terminal domain of apoA-I may, however, induce conformational changes in the central domain of apoA-I that may be responsible for the reduction of LCAT activity (37). Deletion of the Leu44-Leu126 amino-terminal domain of apoA-I indeed did not reduce its LCAT activity (16), suggesting that the amino-terminal domain of apoA-I is not critical for LCAT activation.

Using deletion mutants of apoA-I, Minnich et al. (38) found that deletion of the Met148-Gly186 segment resulted in decreased LCAT activity, whereas Sorci-Thomas et al. (39) found that deletion of the Pro143-Ala164 segment reduced LCAT activity. In previous studies, the conformation of deletion mutants in their respective rHDL was not investigated. However, it has been demonstrated that the decreased LCAT activity of deletion mutants of apoA-I may be due to differences in folding (decreased alpha -helical content) and/or organization of the apolipoproteins in rHDL (3 or 4 molecules/particle as compared with 2 for intact apoA-I) rather than to the removal of specific domains for LCAT activity (16). Indeed, it has been demonstrated that rHDL discs containing wild-type apoA-I may have discrete sizes, compositions (with 2, 3, or 4 protein molecules/particle), and apoA-I conformations (with six, seven, or eight alpha -helices/apoA-I molecule in contact with lipid) and that differences in the apoA-I structure in these particles correlate with their ability to activate LCAT (40).

Chimeras in which alpha -helical segments of apoA-I are substituted with helical segments of apoA-II, which does not activate LCAT, in such a way that the average secondary structure of the apolipoprotein molecule as well as the organization of the apolipoprotein molecules in rHDL are not affected may be preferable reagents to address the function of a particular structural domain in LCAT activation. Indeed, rHDL containing apoA-II have a very low LCAT activity (41), and the addition of apoA-II together with apoA-I to liposomes reduces LCAT activity by 70% (42). Thus, substitution of sequences in apoA-I that are critical for the interaction with LCAT with sequences derived from apoA-II would result in decreased LCAT activity. Substitution of the carboxyl-terminal domain of apoA-I with helices of apoA-II was found not to affect LCAT activity, but substitution of the central domain resulted in a 25-fold reduction of LCAT activity, suggesting that the central domain (but not the carboxyl-terminal domain) is essential for LCAT activation.

The differences in LCAT activity were due to differences in apparent Vmax values, which reflect the activated enzyme concentration, and not to differences in apparent Km values, which reflect the affinity of LCAT for rHDL (43). Similar Km values are indeed in agreement with similar binding of LCAT to rHDL of apoA-I and the central domain chimera. Thus, the Ala123-Tyr166 segment of apoA-I appears to contain structures that are required for optimal phospholipid and cholesterol presentation to LCAT (44) that cannot be mimicked by the apoA-II segment. It is possible that substitution of the central domain affects the conformation of a hinged domain that is crucial for LCAT activation because antibodies that interfere with the mobility of a hinged domain in the central part of apoA-I inhibit LCAT activation (37). Previous data obtained with deletion mutants supported the existence of such a hinged domain overlapping either the Asn102-Lys140 or Ala124-His162 segment of apoA-I (16). Because conformational changes elsewhere in the molecule could not be excluded, these data were, however, not conclusive. The present study strongly suggests that this hinged domain most likely overlaps the Ala124-His162 domain of apoA-I.

In conclusion, substitution of the central or carboxyl-terminal pair of helices of apoA-I with the helices of apoA-II does not affect its average structure in rHDL. Substitution of the central domain (but not the carboxyl-terminal domain) results in a significant reduction of the rate of LCAT activation, although the binding of LCAT to rHDL is not reduced.


FOOTNOTES

*   This work was supported by Fonds voor Wetenschappelijk Onderzoek-Vlaanderen Project 3.0063.94.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Research assistant of the Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie.
   Research assistant of the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen.
**   To whom correspondence should be addressed: Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, O & N, Herestraat 49, B-3000 Leuven, Belgium. Tel.: 32-16-34-57-72; Fax: 32-16-34-59-90.
1   The abbreviations used are: LCAT, lecithin:cholesterol acyltransferase; rHDL, reconstituted high density lipoprotein(s); apoA-I(Delta (Arg123-Tyr166),nabla A-II(Ser12-Ala75)), chimera with the Arg123-Tyr166 segment of apoA-I substituted with the Ser12-Ala75 segment of apoA-II; apoA-I(Delta (Ala190-Gln243),nabla A-II(Ser12-Gln77)), chimera with the Ala190-Gln243 segment of apoA-I substituted with the Ser12-Gln77 segment of apoA-II; POPC, palmitoyloleoylphosphatidylcholine; PAGE, polyacrylamide gel electrophoresis.

ACKNOWLEDGEMENT

We thank G. Vanderheeren (Interdisciplinary Research Center, University of Leuven, Kortrijk, Belgium) for CD measurements.


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