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J. Biol. Chem., Vol. 275, Issue 35, 26821-26827, September 1, 2000

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A Key Point Mutation (V156E) Affects the Structure and Functions of Human Apolipoprotein A-I^*

Kyung-Hyun Cho and Ana Jonas

From the Department of Biochemistry, University of Illinois College of Medicine at Urbana-Champaign, Urbana, Illinois 61801

Received for publication, April 3, 2000, and in revised form, May 31, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A naturally occurring point mutant of human apolipoprotein A-I (apoA-I), V156E, which is associated with extremely low plasma apoA-I and high density lipoprotein (HDL) levels, and coronary artery disease (Huang, W., Sasaki, J., Matsunaga, A., Nanimatsu, H., Moriyama, K., Han, H. Kugi, M., Koga, T., Yamaguchi, K., and Arakawa, K. (1998) Arterioscler. Throm. Vasc. Biol. 18, 389-396), was produced in an Escherichia coli expression system. The purified recombinant proapoA-I V156E mutant was examined in its structural and functional properties, both, in the lipid-free and lipid-bound states. In the lipid-free form the mutant protein exhibited small changes in conformation, but was more stable, and quite resistant to self-association, compared with control apoA-I. The V156E mutant was able to interact with phospholipid (PL) at high PL:protein ratios (95:1, mol/mol), but was inefficient in forming reconstituted HDL (rHDL) complexes at lower PL:protein ratios (40:1). In the lipid-bound, rHDL state, the mutant protein was somewhat more -helical and formed a larger complex (110 Å) than control apoA-I (97 Å). Furthermore, the rHDL particles containing the V156E mutant did not rearrange to smaller particles in the presence of low density lipoproteins, and had minimal reactivity with lecithin-cholesterol acyltransferase (LCAT), compared with rHDL particles made with control apoA-I. These results suggest a key role for Val-156, or the adjacent central region of apoA-I in the modulation of apoA-I conformation, stability, and self-association in solution, and in the formation of small HDL, the conformational adaptability of apoA-I leading to structural rearrangements of HDL, and the activation of LCAT.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Apolipoprotein A-I (apoA-I)¹ is the major protein component of high density lipoproteins (HDL). ApoA-I solubilizes phospholipids and cholesterol and transports them in blood from peripheral tissues to liver and steroidogenic tissues for excretion and metabolism. Thus apoA-I and HDL exhibit anti-atherogenic properties related to the process of reverse cholesterol transport (1). In addition to its major role in binding lipids and defining the structure of HDL, apoA-I is the main activator of lecithin-cholesterol acyltransferase (LCAT) in the esterification of cholesterol in plasma (2), and is a ligand for binding to cells and to the SR-BI receptor (3).

The functional properties of apoA-I have been correlated with its primary and secondary structure. The primary sequence of 243 amino acid residues contains repeated homologous segments of 22 amino acids separated by Pro residues (4). These 22-amino acid segments form amphipathic helices that constitute the phospholipid binding units of the apolipoprotein (5). While most of the apoA-I helices participate in lipid binding, various helical regions also have other functions: the helices spanned by residues 143-187 are critical for LCAT activation (6-8), and the helices from residue 192 to the COOH terminus are involved in interactions with cells and phospholipid bilayers (9, 10). In addition, amphipathic helices of apoA-I are thought to participate in structural rearrangements of HDL when their contents of lipids and apolipoproteins change during metabolism. A "hinge" or "mobile region" between residues 100 and 183 of apoA-I has been implicated in such rearrangements (11, 12). A well documented example of structural rearrangement is the conversion of 96-Å reconstituted HDL (rHDL) discs into 78-Å particles upon incubation with low density lipoproteins (LDL) (13, 14). The 78-Å products have reduced phospholipid content (lost to LDL) and altered apoA-I structure with a markedly decreased ability to activate LCAT (13, 15). The structural changes are a consequence of the conformational adaptability of apoA-I due to the presence of a hinge and the functional effect points to a role of the hinge in LCAT activation and perhaps in apoA-I interactions with other proteins.

As described above, various functions of apoA-I have been localized to specific helical regions; however, these regions are quite large and the roles of individual amino acids are unknown. Therefore, in this study we set out to analyze the structural and functional effects of an interesting point mutation, V156E, in apoA-I. This mutation occurs naturally and results in very low levels of apoA-I and HDL in a homozygous subject (16), furthermore, the mutation substitutes a nonpolar residue (Val) on the nonpolar face of helix 6 with a charged residue (Glu) which may lead to structural perturbation of apoA-I and of its hinge and LCAT-activating functions.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Radiolabeled [4-¹⁴C]cholesterol was obtained from NEN Life Science Products Inc.; 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), L--phosphatidylcholine from egg yolk (egg PC), and sodium cholate were purchased from Sigma. Bis-sulfosuccinimidyl suberate (BS₃) was obtained from Pierce (Rockford, IL). The quick change site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA). Enterokinase and the pET30a(+) expression vector were purchased from Roche (Germany) and Novagen (Madison, WI), respectively. Human apoA-I and LDL were prepared by routine methods used in our laboratory (14, 17) from blood plasma purchased from the Champaign County Blood Bank.

Construction of Expression Vector-- The expression system for wild-type (WT) proapoA-I cDNA was developed previously in our laboratory (18). The V156E mutant of the proapoA-I cDNA was generated using the polymerase chain reaction-based site-directed mutagenesis kit (Stratagene) employing oligonucleotides produced at the Biotechnology Center of the University of Illinois at Urbana-Champaign. The sequence of the mutagenic primer was as follows: 5'-CGCGACCGCGCGCGCGCCCACGAGGATGCACTGCGC-3'. The sequence of the construct was verified by appropriate endonuclease digestion and DNA sequencing on an Applied Biotechnology Systems DNA sequencer at the Biotechnology Center.

Expression and Purification of ProapoA-I and Mutant Protein-- The mutant construct and WT (proapoA-I) cDNA were subcloned into the pET30a(+) expression vector (Novagen) using NcoI and HindIII restriction sites. The plasmids were then transfected into BL21 (DE3) host cells (Novagen). Overexpression of the fusion protein, including a 6-kDa His tag, was carried out using a single colony picked from a freshly streaked plate in 1 liter of the Luria-Bertani (LB) medium supplemented with 30 µg/ml kanamycin, at 37 °C. Protein synthesis was induced when the cell density (A₆₀₀) reached 0.9-1.0 by the addition of isopropyl-1-thio--D-galactopyranoside (final concentration, 1 mM) in a New Brunswick C25 shaker rotating at 200 rpm. After the addition of isopropyl-1-thio--D-galactopyranoside, the induced cells were incubated 4 h longer under the same conditions.

After harvesting, the cells were resuspended in buffer, containing 20 mM Tris-HCl, 5 mM imidazole, 500 mM NaCl, pH 7.9, plus 1 mM phenylmethylsulfonyl fluoride and 0.02 mM 4-amidinophenylmethane-sulfonyl fluoride (this is the "binding buffer"). The cells were sonicated twice for 30 s or until they were no longer viscous, on ice using a Vibra Cell sonicator (Sonics and Materials, New Town, CT). The sonicated cells were fractionated by centrifugation at 10,000 rpm using a Beckman JA-17 rotor. The supernatant was directly applied to a His-affinity column that previously had been charged with NiSO₄ and equilibrated with the binding buffer. The pellet fraction was extracted with 6 M guanidine hydrochloride (GdnHCl) to solubilize precipitated protein and dialyzed against the binding buffer overnight, at 4 °C, prior to use. The dissolved pellet fraction was applied to the His-affinity column. The column was washed with the binding buffer followed by a buffer containing 60 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9.

After washing the column, the fusion protein was eluted with a high concentration of imidazole (1 M) in 20 mM Tris-HCl, 500 mM NaCl, pH 7.9. The eluted fusion protein was lyophilized after dialysis against 5 mM ammonium bicarbonate to remove the imidazole and buffer components.

Removal of the His Tag and Delipidation-- Egg PC-rHDL particles were synthesized with the fusion proteins (100/1, egg PC:protein, mol/mol) before enterokinase cleavage to avoid nonspecific degradation of the proteins. The enterokinase cleavage was carried out at an enzyme:protein ratio of 1/250 (w/w) for 16 h at room temperature with gentle shaking. The cleaved products were passed over the His-affinity column to remove the His tag. After the removal of the His tag, egg PC was removed by delipidation with cold ethanol:ether (3/2, v/v) and hexane:2-propanol (3/2, v/v), sequentially. Finally, delipidated and recovered proteins, mutant and WT, were dialyzed against ammonium bicarbonate and lyophilized.

Preparation and Characterization of the POPC-rHDL Particles-- The lyophilized proteins were solubilized in Tris-buffered saline, pH 8.0 (TBS), containing 3 M GdnHCl and then dialyzed against TBS before use. Discoidal reconstituted HDL (rHDL) particles were made by the sodium cholate dialysis method (19) described previously using initial molar ratios of POPC:FC:apoA-I of 95:5:1 and 40:0:1. The rHDL particles used as substrates in the LCAT reaction also contained trace amounts of ¹⁴C-cholesterol. The particles were used without further purification, and their sizes were determined from 8 to 25% native polyacrylamide gradient gel electrophoresis (PAGGE, Pharmacia Phast system) by comparison with standard globular proteins (Amersham Pharmacia Biotech). The gradient gels were stained with Coomassie Blue and scanned with an LKB Ultro Scan XL-laser densitometer. The number of apoA-I molecules per rHDL particle, as well as self-association properties of lipid-free proteins, were determined by cross-linking with BS₃ as described by Staros (20) and analyzing the products of the reaction by SDS-PAGE on 8-25% gradient gels.

POPC-rHDL Particle Size Conversion by Incubation with LDL-- In order to examine the conformational adaptability of WT proapoA-I and the mutant protein in POPC-rHDL particles, we observed particle size changes upon incubation with LDL and loss of phospholipid, as described previously by our research group (13, 14). To observe the changes in particle sizes, 100 µg (protein) of each POPC-rHDL (0.05 ml) were incubated with 120 µg (protein) of human LDL (0.05 ml) at 37 °C for a designated time interval up to 24 h (21). After the incubation, aliquots of the samples were analyzed directly by electrophoresis on the 8-25% native PAGGE, using the Pharmacia Phast system.

Circular Dichroism and Isothermal Denaturation Measurements-- The average -helical structure of lipid-free or lipid-bound apoA-I and the V156E mutant were determined by CD spectroscopy using a J-720 spectropolarimeter located in the Laboratory for Fluorescence Dynamics at the University of Illinois, Urbana-Champaign. The spectra were measured from 250 to 190 nm at 25 °C in a 0.1-cm path length cell using a 1.0 nm bandwidth, and 4-s response time. Four scans were collected and averaged. The protein concentration was adjusted to 0.07 or 0.1 mg/ml in TBS for lipid-free or lipid-bound protein, respectively. Percent -helix was calculated from the molar ellipticity at 222 nm as described by Chen et al. (22) using a mean residue weight for apoA-I of 115.3.

The effects of GdnHCl addition on the secondary structure of apoA-I and the mutant, in both lipid-free state and POPC-rHDL particles, were monitored by CD spectroscopy. The -helix contents of the proteins after 72-h incubations with increasing concentrations of GdnHCl at 4 °C were used to obtain their free energy of unfolding (G_D°) according to Aune and Tanford (23) and Sparks et al. (24). While denaturation of lipid-free apolipoprotein is instantaneous and reversible, denaturation of the lipid-bound proteins is very slow, requiring 72 h for equilibration. Furthermore, denaturation of the lipid-bound apolipoproteins is not a reversible process (18). Therefore, the G_D° for the lipid-bound proteins is only an apparent thermodynamic parameter.

Fluorescence Spectral Measurements-- Concentrations of all samples for fluorescence measurements were adjusted to 0.07-0.1 mg/ml (protein) in TBS. At these concentrations plasma apoA-I is monomeric in the lipid-free state. The wavelength of maximum fluorescence of the tryptophan residues in apoA-I, proapoA-I, and the V156E mutant were determined from uncorrected spectra using a Perkin-Elmer MPF-66 fluorescence spectrofluorometer with 4-nm excitation and emission band widths. The samples were excited at 295 nm to avoid tyrosine fluorescence, and the emission was scanned from 305 to 375 nm at room temperature.

Reaction of the POPC-rHDL with LCAT-- The LCAT assay was carried out as described in detail previously (25) using recombinant human LCAT (26). The reaction mixture contained the radiolabeled POPC-rHDL (with approximately 1.9 × 10^{4 14}C cpm/µg of free cholesterol, 4% defatted bovine serum albumin, and 4 mM -mercaptoethanol; the reaction was initiated by adding recombinant LCAT (0.163 µg, 50 µl) and was carried out at 37 °C for 30 min. The POPC-rHDL substrates were present in various concentrations in the range from 1.7 × 10⁷ to 1.4 × 10⁶ M apolipoprotein in the 0.5 ml of the reaction mixture. The reaction was performed in duplicate and the background values were determined by omitting only LCAT at each substrate concentrations from the reaction tubes. The initial reaction velocities at each substrate concentration were analyzed by Lineweaver-Burk plots, which gave the apparent K_m and V_max values by linear regression.

Miscellaneous Methods-- The protein concentrations were determined by using absorbance and the extinction coefficient for apoA-I and proapoA-I at 280 nm (1.13 and 1.28 ml/mg·cm, respectively) or the Lowry/Markwell protein assay (27) and a bovine serum albumin standard. The phospholipid and cholesterol contents were determined according to Chen et al. (28) and Heider and Boyett (29), respectively.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression and Purification of the V156E Mutant Protein-- A new expression system for proapoA-I and its V156E mutant was used in this study. The apolipoproteins were produced with a 6-kDa His tag attached to their amino terminus by a sequence susceptible to enterokinase cleavage (30). The fusion proteins were expressed in excellent yields (60-73 mg per liter of culture) and were readily purified by affinity chromatography on a Ni²⁺ column. However, enterokinase digestion of the fusion proteins gave unexpected, extensive cleavage of both proteins. To protect the proteins from nonspecific cleavage, the fusion proteins were reconstituted into rHDL particles with egg PC and were then digested with enterokinase. After another column step and delipidation, 27 and 32 mg of pure WT proapoA-I and its V156E mutant were recovered per liter of culture. The purity of the proteins, together with a plasma apoA-I control was checked by SDS-PAGE (Fig. 1A). From densitometric scanning of the gel the purity of the recombinant proteins is at least 95%.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Electrophoresis patterns of apolipoproteins in the lipid-free state. Panel A shows the purity of the apolipoproteins on 20% SDS-PAGE. At least 95% purity was estimated by laser densitometry. Lane M, molecular weight markers (low range, Bio-Rad); lane 1, plasma apoA-I; lane 2, V156E mutant; lane 3, propapoA-I (WT). Panel B shows the self-association behavior of the apolipoproteins at two concentrations, analyzed by BS3 cross-linking and 8-25% SDS-PAGGE. The cross-linking reactions were carried out in 20 mM phosphate buffer, pH 7.4, lane 1, plasma apoA-I (0.1 mg/ml); lane 2, plasma apoA-I (1.0 mg/ml); lane 3, V156E (0.1 mg/ml); lane 4, V156E (1.0 mg/ml); lane 5, proapoA-I (0.1 mg/ml); lane 6, proapoA-I (1.0 mg/ml).

Self-association of Lipid-free Proteins-- The self-association behavior of the proteins in the lipid-free state was examined at two different protein concentrations (0.1 and 1.0 mg/ml), by cross-linking with BS₃ in phosphate buffer. The cross-linking and SDS-PAGE analysis revealed that the V156E mutant has an impaired ability to self-associate as shown in panel B of Fig. 1. The control proteins (plasma apoA-I and WT propapoA-I) showed the typical oligomerization patterns with substantial formation (>60%) of dimers, trimers, and tetramers at 1.0 mg/ml concentrations, while the V156E mutant only formed small amounts of dimers (~10%). Apparently the V156E mutation is located in a region of apoA-I that is involved in protein monomer contacts, or alternatively, the mutation affects the protein structure preventing self-association. In all cases, the apolipoproteins do not self-associate at concentrations of 0.1 mg/ml.

Physicochemical Properties of Reconstituted POPC-rHDL Particles-- Two kinds of reconstituted rHDL particles were prepared using initial molar ratios of 95:5:1 and 40:0:1, POPC:FC:apoA-I. Those two preparations have been shown, with plasma apoA-I, to result primarily in 96-Å (95:1, POPC:protein) and 78-Å (40:1, POPC:protein) diameter particles containing two molecules of apoA-I per particle (15). The 40:1 preparations are quite heterogeneous and contain in addition to the 78-Å particles, larger 87- and 96-Å species, as well as small amounts of free apoA-I. The final compositions, particle sizes, and the content of apoA-I molecules per particle of the larger, essentially pure, rHDL particles are given in Table I. There was no significant difference between plasma apoA-I and WT proapoA-I in the composition and diameter (97 Å) of the rHDL. In contrast, the particles made with the V156E mutant had an increased PL to protein ratio and particle size of 110 Å, as shown in Fig. 2A. These particles were used in subsequent experiments without further purification because of their good homogeneity (Fig. 2A).

View larger version (67K): [in this window] [in a new window]   Fig. 2.   Electrophoresis patterns of apolipoproteins in POPC-rHDL complexes. The rHDL particles were prepared by the Na-cholate dialysis method using two molar ratios of PC:apolipoprotein. Panel A shows rHDL prepared with the larger molar ratio (95:5:1:150, POPC:FC:apoA-I:Na-cholate) on a 8-25% native gradient gel. Lane M, high molecular weight makers (Amersham Pharmacia Biotech); lane 1, plasma apoA-I rHDL; lane 2, V156E rHDL; lane 3, proapoA-I (WT) rHDL. Panel B shows the electrophoresis patterns of cross-linked POPC-rHDLs from panel A analyzed by 8-25% SDS-PAGGE. Panel C displays the electrophoresis patterns of rHDL prepared with the smaller molar ratios (40:0:1:150, POPC:FC:apoA-I:Na-cholate) on a 8-25% native gradient gel. Lane M, high molecular weight makers (Amersham Pharmacia Biotech); lane 1, plasma apoA-I rHDL; lane 2, V156E; lane 3, proapoA-I (WT) rHDL. Panel D shows the electrophoresis patterns of the cross-linked POPC-rHDLs from panel C analyzed by 8-25% SDS-PAGGE. For panels B and D: lane 1, lipid-free apoA-I; lane 2, plasma apoA-I; lane 3, V156E mutant; lane 4, proapoA-I (WT).

In addition, BS₃ cross-linking and SDS-PAGE analysis (Fig. 2B) revealed that the number of protein molecules per particle was two for all three rHDL, even though the V156E particle size was larger. While both plasma apoA-I and WT proapoA-I were completely associated with lipid in the POPC-rHDL, a small amount of lipid-free V156E protein was detected at the bottom of the native gel (lane 2, Fig. 2A).

In the rHDL preparations with 40:1 molar ratio of POPC:apoA-I, smaller particles (78 Å diameter) were produced together with larger particles and free protein when control proteins were used (see Fig. 2C). However, the V156E mutant, at the same 40:1 molar ratio, produced a slightly bigger 82-Å particle and a large proportion of lipid-free protein (lane 2, Fig. 2C). Cross-linking results (Fig. 2D) are also strikingly different for the control proteins and the V156E mutant particles. The particles formed with apoA-I and WT proapoA-I contain two protein molecules per particle, as expected, while the V156E mutant shows no cross-linking. The failure to cross-link the mutant protein in the 82-Å particles may be due to a changed apolipoprotein conformation, with inaccessible cross-linking sites, or to the presence of a single protein molecule per particle. The latter explanation appears unlikely because of the relatively large diameter of these particles. Furthermore, presence of free mutant protein in the 40:1 preparation contributes heavily to the monomer band on the SDS-PAGE.

Circular Dichroism and Fluorescence Measurements-- Far UV-circular dichroism spectra were measured to determine the average -helix structure in lipid-free and lipid-bound control apolipoproteins and the V156E mutant. The CD spectra of the V156E mutant exhibited two pronounced minima at 208 and 222 nm, characteristic of -helical proteins. The -helix contents expressed as the percentage of total amino acids are listed in Table II. For the control proteins the -helix contents are in reasonable agreement with published values (18), while the V156E mutant appears to be slightly less helical in the lipid-free state (52%) and somewhat more helical in the rHDL particles (74%) compared with the control proteins.

View this table: [in this window] [in a new window]   Table II -Helix content and denaturation of free (first three lines) and lipid-associated apoA-I, recombinant proapoA-I (WT), and mutant proapoA-I (V156E)

To determine the stability of the V156E mutant, we measured the standard free energy change (G_D°) by an isothermal denaturation method (23, 24) using increasing GdnHCl concentrations from 0 to 7 M and determining the change in -helical content, at equilibrium, for each GdnHCl concentration (Fig. 3). Interestingly, the lipid-free mutant protein has a mid-point of denaturation of 2.7 M GdnHCl, considerably higher than the control proteins (1.2 M), and a G_D° value of 3.0 kcal/mol, compared with 2.3-2.6 kcal/mol for apoA-I, indicating that the V156E mutant is significantly more stable in solution than apoA-I (see Table II). In the lipid-bound state, the midpoints of denaturation are similar for all three apolipoproteins (3.2-3.3 M GdnHCl) but the apparent G_D° value is larger for the mutant protein, suggesting a different denaturation behavior for the mutant compared with the control proteins in rHDL particles.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Denaturation of apolipoproteins in lipid-free state (A) and in lipid-bound POPC-rHDL particles (B). The fraction of folded apolipoprotein was obtained from CD ellipticity measurements at 222 nm, as a function of GdnHCl concentration after an equilibration of 72 h at 4 °C. Plasma apoA-I (), WT proapoA-I (), and V156E mutant ().

In addition to the increased stability, the mutant protein exhibits a slightly red-shifted tryptophan fluorescence, suggesting that structural changes in the V156 region in the COOH-terminal half of apoA-I, are transmitted to the NH₂-terminal half of the protein where the Trp residues are located. As indicated by the red-shifted fluorescence of the mutant protein, the Trp residues, on average, are exposed to a slightly more polar environment not only in the lipid-free, but also in the rHDL state.

Particle Size Rearrangements-- Native and rHDL particles are known to undergo structural and size rearrangements when their lipid contents are altered by spontaneous or facilitated lipid transfers (15, 31). In this study, we examined the ability of rHDL particles made with the control proteins and with the V156E mutant to rearrange into smaller particles when exposed to LDL. As reported in previous studies (13, 14), rHDL particles (97 Å) containing apoA-I lose phospholipids to LDL by spontaneous transfer and rearrange their protein conformations to produce some intermediate particles, and a 78-Å product which accumulates after 24 h (see Fig. 4, A and C). In contrast to the control rHDL particles, the rHDL containing the V156E mutant does not rearrange into smaller particles over the same time of incubation with LDL (Fig. 4B). The inability of the mutant rHDL to rearrange is most likely due to a structural change in the apolipoprotein that inhibits its ability to adapt to the altered lipid content. Several laboratories have suggested that apoA-I can change its conformation via a helical hinge domain when adapting to changing lipid contents in HDL (11, 12). Different reports have localized the hinge domain to the sequence of apoA-I between residues 100 and 183 (12, 32). Our present results suggest that either the helix containing V156E (helix 6) or adjacent helices are involved in the hinge and its structural rearrangements.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Size rearrangements of the rHDL particles in the presence of LDL. The rHDLs were prepared with a molar ratio of 95:5:1:150 (POPC:FC:apoA-I:Na-cholate). The reaction was initiated by adding 120 µg of human LDL (protein) to the 100 µg of rHDL (protein) at 37 °C. An aliquot of the incubation mixture was taken at time intervals and stored in 4 °C with gel loading buffer without SDS. The aliquoted samples were separated on the 8-25% native gels, and protein bands were visualized by Coomassie Blue staining. The rearrangement patterns of plasma apoA-I-rHDL, V156E-rHDL, and proapoA-I-POPC-rHDL are displayed in panels A, B, and C, respectively. Lane M, high molecular weight markers; lanes 1-5 correspond to 0, 4, 8, 12, and 24 h of incubation.

LCAT Activation-- It has now been firmly established that helices 6 and 7 of apoA-I (residues 143-186) are involved in LCAT activation (6-8, 33). However, the role of individual amino acid residues within this sequence is not yet understood. To determine the effect of a point mutation, V156E, in this region of apoA-I on LCAT activation, we measured the reaction kinetics of LCAT with the rHDL containing the V156E mutant in conjunction with the control rHDL particles. Fig. 5 shows plots of the initial velocity of the enzymatic reaction as a function of rHDL concentration, expressed as the apolipoprotein concentration. Clearly the reactivity of LCAT with the V156E rHDL is dramatically decreased compared with the control rHDL substrates. The apparent kinetic constants are listed in Table III. They are essentially the same for the apoA-I and WT proapoA-I rHDL and are comparable to previously published values (18). For the mutant rHDL the apparent V_max is 16-fold lower and the apparent K_m is 6-fold higher than for the control rHDL substrates. Thus the overall reactivity of the V156E rHDL with LCAT is only about 1% that of the normal apoA-I rHDL or WT proapoA-I rHDL. This minimal reactivity and the apparent kinetic constants are comparable to those of rHDL prepared with apoA-II, which is not considered an activator of LCAT (34, 35). Thus V156E or the local apoA-I conformation due to the point mutation are critically important in LCAT activation by apoA-I.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Kinetis of LCAT reaction with POPC-rHDL substrates. The rHDLs were prepared with molar ratios of 95:5:1:150 (POPC:FC:apoA-I:Na-cholate). The symbols correspond to initial velocities as a function of apolipoprotein concentrations for plasma apoA-I (), V156E (), and proapoA-I () rHDL complexes.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

This study has shown that the V156E mutant of apoA-I exhibits significant structural differences from WT proapoA-I and plasma apoA-I in both, the lipid-free and the lipid-bound state. In the lipid-free state the mutant was more stable and had slightly decreased -helix content, slightly increased polarity of its Trp residues, and markedly decreased self-association in solution, when compared with the control apolipoproteins. In functional tests the V156E mutant also had a strikingly different behavior from the control apolipoproteins. The mutant protein was less efficient in forming rHDL with POPC, and the particles formed had larger diameters and higher -helix contents than the corresponding apoA-I and WT proapoA-I rHDL. In tests of conformational adaptability and ability to activate LCAT, the V156E mutant in rHDL particles was essentially inactive compared with the normal apolipoproteins. The subsequent discussion will focus on the phenotype of the V156E mutant observed clinically (16), on the structure-function relationships of apoA-I, and on current structural models of discoidal rHDL, in light of the detailed in vitro results for the V156E mutant obtained in this study.

The phenotype reported by Huang et al. (16), in the original description of the natural V156E mutation in a homozygous subject, was a marked decrease in apoA-I and HDL concentrations, corneal opacities, and coronary artery disease. In addition, LCAT activity, cholesterol esterification rate and LCAT mass were about half of normal. Turnover studies of the mutant protein in rabbits showed rapid clearance, in contrast to normal human apoA-I. Our results provide an explanation for these clinical observations. We found that the V156E apoA-I mutant binds less efficiently to physiologic phospholipids (POPC), especially at low PC:apoA-I ratios. Therefore the formation of pre-₁ HDL particles may be impaired and their subsequent maturation to larger discoidal HDL and to spherical -HDL will be strongly inhibited by the inability of the mutant apoA-I to adapt to changing lipid contents and inability to activate LCAT. Indeed, Huang et al. (16) report that the variant apoA-I occurs predominantly in small HDL or in lipid-poor form. Such forms of apoA-I are expected to be cleared more rapidly from plasma than normal HDL leading to low apoA-I and HDL levels in the patient. The reduced activity of LCAT is related to its decreased mass, while the lower cholesterol esterification rate results from cholesterol esterification on LDL (36) and HDL particles containing apolipoproteins E and C-I, which are modest activators of LCAT (34, 37). The V156E variant apoA-I does not contribute to cholesterol esterification because it does not activate LCAT to any significant extent as found in this study.

The changes in the structure and function of the V156E apoA-I mutant, compared with the control proteins, also illuminate some properties of apoA-I structure and function. While there are numerous reports of natural and engineered point mutations of apoA-I, most of them have little effect on the properties of apoA-I (21, 38, 39). The V156E mutation is quite unusual in its dramatic structural and functional effects.

In the lipid-free state the mutant is more stable than normal apoA-I and has a markedly decreased tendency to self-associate. The substitution of a hydrophobic amino acid (Val) with a negatively charged amino acid (Glu) in position 156, on the hydrophobic face of helix 6 evidently interferes with the formation of protein-protein contacts that lead to self-association. Previous work by our group has shown that deletion of the COOH-terminal sequence of apoA-I also interferes with self-association of apoA-I (10). Therefore, we conclude that interprotein contacts involving the COOH terminus of one monomer with the central region (near residue 156) of another monomer, lead to dimerization and subsequent oligomerization of apoA-I. In agreement with this concept, elongation of oligomers by partial overlap of apoA-I monomers was predicted by early hydrodynamic studies of Barbeau et al. (40).

Stabilization of the mutant with respect to apoA-I suggests that a hydrophobic patch in this area of apoA-I, that participates in self-association, is exposed to water in the monomer form. Introduction of the Glu residue in place of Val-156 may result in favorable hydration and stabilization of the mutant monomer.

The lipid-binding properties of the V156E apoA-I mutant are significantly different from normal apoA-I. At relatively low POPC:apolipoprotein molar ratios (40:1) there is impaired formation of lipoprotein particles and large amounts of residual lipid-free V156E protein. Under the same conditions, apoA-I binds lipid quite efficiently. Presence of a charged amino acid residue (Glu) on the hydrophobic face of helix 6 may contribute to the decreased lipid binding by the mutant protein, but the inability to form small, stable 78-Å particles is probably the most important factor limiting the lipid binding by the mutant. At larger (95:1) molar ratios of POPC:apolipoprotein, the V156E mutant binds lipid and forms discoidal rHDL more efficiently (only about 10% of free protein remains). However, the diameter of the particles (110 Å) is atypical, and the conformation of the apolipoprotein appears distinct (increased -helix content and slightly red-shifted Trp fluorescence) compared with control apoA-I. In addition to the structural and conformational differences, the apparent G_D° is somewhat larger for the mutant compared with the control rHDL. This may explain, in part, the impaired rearrangement of the V156E rHDL in the presence of LDL, as discussed next.

Another very interesting finding in this study is the inability of the mutant rHDL to undergo structural and size rearrangements under conditions that readily convert control 97-Å rHDL particles into 78-Å rHDL products. This normal conversion is well documented and is attributed to the loss of phospholipid and resultant apoA-I conformational adaptation to a smaller particle diameter. It is possible that this conformational adaptation involves a hinge region of apoA-I, equivalent to 2 -helical segments, that swings away from the lipid interface and allows the particle to shrink, forming discrete, smaller particles (13, 15). The properties of these 78-Å rHDL are dramatically different from those of 97-Å rHDL. They have reduced -helix content, markedly decreased reactivity with LCAT (only 5% of normal), and decreased ability to bind to SR-BI receptors.² The V156E mutant rHDL does not undergo this structural rearrangement, strongly suggesting that the presence of the Glu residue at position 156, or a structural change resulting from this mutation in helix 6 itself, or in adjacent helices, is responsible for this behavior. At the amino acid level, the formation of a new salt bridge between Glu and a basic residue in an adjacent helix, or a local conformational distortion that shifts interprotein interactions, could contribute to the loss of conformational adaptability of the apoA-I mutant. In any case, a hinge region can be more precisely localized to helix 6 or nearby helices.

One of the most studied functions of apoA-I in rHDL is its activation of LCAT. The 97-Å rHDL, made with apoA-I, POPC and small amounts of cholesterol, is one of the most effective substrates for LCAT, exceeding the reactivity of natural HDL₃ substrates by a factor of 6-fold (36). In contrast, the mutant V156E rHDL particles are strikingly unreactive with LCAT, at least equaling apoA-II in its minimal reactivity with LCAT (34, 35). Most of the kinetic change is due to a dramatically decreased apparent V_max value which suggests that activation of LCAT is impaired; an increased apparent K_m (6-fold) suggests that the binding affinity of LCAT to the rHDL is also decreased. From recent work, using the surface plasmon resonance method, we have established that the association rate constant of LCAT with various lipoproteins and lipid vesicles is very similar, but that the dissociation rate is influenced, among other factors, by LCAT-apolipoprotein interactions (41). Thus the enzymatic kinetic constants point to a lack of interaction between LCAT and the V156E apoA-I mutant. To our knowledge, this is the first point mutant of apoA-I that has such a profound effect on LCAT activation. This suggests a direct involvement of V156E in LCAT activation and reinforces reports by Sorci-Thomas et al. (7, 33), Minnich et al. (6), and Holvoet et al. (8) implicating helix 6 of apoA-I in LCAT activation.

Finally, the relevance of the two current models of apoA-I organization on discoidal rHDL particles to the structure of the V156E mutant will be addressed. In one of the models of rHDL, the helical segments of each of two apoA-I molecules are organized as a "picket fence" on the periphery of the disc, while in the "belt model" two extended antiparallel molecules of apoA-I encircle the disc. In the first model (42), the Glu in the V156E mutant could form a salt bridge with a nearby Arg in helix 7, thus changing local stability and charge balance, which could affect conformational adaptability of the apolipoprotein. Furthermore, if there is indeed proximity of the NH₂-terminal and central regions of apoA-I in rHDL, as suggested by monoclonal antibody binding studies (12, 32) and our fluorescence results, then changes in the mutant apoA-I may release the NH₂ terminus which may result in the formation of the extra -helical structure and the increased rHDL diameter. The ability of apoA-I to form extra-helical structure (up to 85% -helix) has been demonstrated to occur under special conditions (e.g. in 30% 1-propanol) (17).

Considering the "belt" model of rHDL (43) the V156E mutation occurs in position 3 of the 6th 11/3 -helix. Position 3 is in the hydrophobic face of the helix, but is pointing away from the L protein-protein interface. Thus direct perturbation of protein-protein interactions by the Glu residue is not predicted; however, the mutation could locally influence the interaction with the charged groups of POPC. To explain the change in diameter of the rHDL, significant structural perturbations would have to be invoked, involving protein-protein salt bridge rearrangements of the most favorable LL5/5 rotamers. While neither model can fully explain the structural and functional changes observed with the V156E mutant, future refinements of the current models or new models will have to take into account the conformational flexibility of apoA-I, and the discrete diameter changes that apoA-I rHDL can undergo.

	ACKNOWLEDGEMENTS

We express our gratitude to Andrea Agree who developed the His tag expression and purification procedure for the preparation of proapoA-I mutants in our laboratory. We also thank Drs. S. Harvey and J. Segrest, University of Alabama, for comments regarding the belt model of rHDL and the implications of the V156E mutation to this model.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants HL 16059 and HL 29939.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.

To whom correspondence should be addressed: Dept. of Biochemistry, College of Medicine at Urbana-Champaign, University of Illinois, 506 South Mathews Ave., Urbana, IL 61801. Tel.: 217-333-0452; Fax: 217-333-8868; E-mail: a-jonas@uiuc.edu.

Published, JBC Papers in Press, June 16, 2000, DOI 10.1074/jbc.M002841200

² D. van der Westhuysen, D. Durbin, and A. Jonas, unpublished results.

	ABBREVIATIONS

The abbreviations used are: apoA-I, apolipoprotein A-I; HDL, high density lipoprotein; LCAT, lecithin-cholesterol acyltransferase; rHDL, reconstituted high density lipoprotein; POPC, 1-palmitoyl-2-oleoyl phosphatidylcholine; egg PC, egg phosphatidylcholine; BS₃, bis-sulfosuccinimidyl suberate; LDL, low density lipoprotein; WT proapoA-I, recombinant wild-type proapoA-I; TBS, Tris-buffered saline; GdnHCl, guanidine hydrochloride; FC, free cholesterol; PAGGE, polyacrylamide gradient gel electrophoresis; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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