(Received for publication, October 23, 1996, and in revised form, December 26, 1996)
From the Departments of Apolipoprotein A-I contains eight
22-amino acid and two 11-amino acid tandem repeats that comprise 80%
of the mature protein. These repeating units are believed to be the
basic motif responsible for lipid binding and lecithin:cholesterol
acyltransferase (LCAT) activation. Computer analysis indicates that
despite a fairly high degree of compositional similarity among the
tandem repeats, significant differences in hydrophobic and amphipathic
character exist. Our previous studies demonstrated that deletion of
repeat 6 (143-164) or repeat 7 (165-186) resulted in a 98-99%
reduction of LCAT activation as compared with wild-type apoA-I. To
determine the effects of substituting one of these repeats with a more
hydrophobic repeat we constructed a mutant apoA-I protein in which
residues 143-164 (repeat 6) were replaced with repeat 10 (residues
220-241). The cloned mutant protein, 10F6 apoA-I, was expressed and
purified from an Sf-9 cell baculoviral system and then analyzed using a number of biophysical and biochemical techniques. Recombinant complexes
prepared at a 100:5:1 molar ratio of
L- There is a significant, negative correlation between the plasma
concentration of high density lipoproteins
(HDLs)1 and coronary artery disease in
human populations and in animal models of atherosclerosis (1-3).
Recent investigations using apolipoprotein A-I (apoA-I) transgenic and
knockout animals have clearly demonstrated the protective role of HDLs
in the prevention of atherosclerosis by "reverse cholesterol
transport" (4-8). The predominant protein constituent of HDL,
apoA-I, solubilizes and organizes phospholipid, cholesterol, and
cholesteryl ester in plasma and facilitates the distribution of
cholesteryl ester between hepatic and extrahepatic tissue. Conversion
of nascent HDLs or discoidal complexes into mature HDL particles is
mediated by the plasma enzyme lecithin:cholesterol acyltransferase
(LCAT). When activated by apoA-I, LCAT catalyzes the conversion of
HDL-cholesterol to HDL-cholesteryl esters (4, 5). However, the precise
mechanism of LCAT activation by HDL-bound apoA-I remains largely
unknown.
Investigations aimed at identifying how apoA-I organizes lipid and
activates LCAT have focused on the unique structural properties of this
protein (6-14). ApoA-I contains 10 tandem repeats, each having
substantial We have continued our investigation into the structure-function
relationships of apoA-I by constructing a 22-mer substitution mutant in
a region previously shown to be necessary for LCAT activation. Sequential deletion of any one of the 10 tandem repeats results in a
modest (30%) or dramatic (99%) reduction in LCAT reactivity compared
with wild-type apoA-I (25). However, several studies (26, 27) have
shown that the region corresponding to residues 143-186 (22-mer,
repeats 6 and 7) was absolutely essential for LCAT activation. In this
paper, we report the properties of a mutant apoA-I protein in which
repeat 6 (residues 143-164) was replaced with a copy of repeat 10 (residues 220-241). The cloned mutant protein, called 10F6 apoA-I, was
expressed and purified from an Sf-9 cell baculoviral system and used
for biophysical characterization, monoclonal antibody epitope mapping,
and LCAT reactivity measurements. In this report, we describe those
structural features of the residue 121-186 domain that are important
for optimal LCAT activation.
HPLC grade organic solvents were purchased from Fisher. All
other chemical reagents were purchased from Sigma
unless otherwise noted. [3H]Cholesterol was purchased
from DuPont NEN. Tissue culture reagents, restriction endonucleases,
and other DNA modifying enzymes were purchased from Life Technologies,
Inc.
Wild-type and 10F6 apoA-I cDNA:pBlueBac III
constructs were used in conjunction with the
Autographa californica nuclear polyhedrosis virus
linear viral DNA for co-transfection into Sf-9 cells, as described
previously (28). Recombinant wild-type and mutant baculoviral clones
were purified and used for the generation of high titer viral stocks as
described previously (28). To prevent degradation of the expressed
protein, pepstatin A and leupeptin (at a final concentration of 700 µg/liter and 500 µg/liter) were added to the culture medium 36 h postinfection (29).
Preparation and purification of recombinant wild-type and
10F6 apoA-I protein were carried out as previously reported (28). Briefly, at the time of harvest (50-72 h), Sf-9 medium was spun at
4 °C, 10,000 rpm for 10 min, and the supernatant was adjusted to
10% acetonitrile (v/v). The adjusted medium was loaded onto a C-18
reverse phase (50 × 5-cm) HPLC column and washed with 10% acetonitrile, 0.1% trifluoroacetic acid. ApoA-I was eluted from the
column in 1 h at a flow rate of 10 ml/min using a linear gradient of 10-95% acetonitrile, 0.1% trifluoroacetic acid at ambient
temperature. The partially purified apoA-I was adjusted to 5 mM Tris, pH 8.0, 8 M urea and then applied to a
DEAE-Fast-Sepharose anionic exchange column (16 × 3 cm). ApoA-I
eluted at approximately 9 mM Tris, pH 8.0, 8 M
urea using a linear gradient (200 ml each) that went from 5 mM Tris, pH 8.0, to 150 mM Tris, pH 8.0, 8 M urea at a flow rate of 144 ml/h. The final purified
protein was dissolved in 1 mM ammonium bicarbonate, pH 7.4, and its concentration was determined by the method of Lowry (30). The
molecular weight of the proteins was confirmed by electrospray mass
spectrometry on a Quattro II mass spectrometer.
A
molar ratio of 100:5:1 phospholipid to cholesterol to apoA-I protein
was used for making discoidal complexes as described previously (28).
Briefly, 2.1 mg of L- Discoidal
complex size was determined using nondenaturating gradient gel
electrophoresis. Briefly, gels were run for 3,000 V-h and then fixed in
10% sulfosalicylic acid (31). Following fixation, gels were stained in
Coomassie G-250 overnight and then destained in 7.5% acetic acid for
1-2 days. Gels were scanned using a Zeineh scanning densitometer model
SL-504-XL. Discoidal particle size was determined by comparison with
protein standards of known Stokes radii.
Circular dichroism spectra
were recorded with a Jasco J720 spectropolarimeter at 25 °C using a
0.1-cm path length cell. Ellipticity was measured at 222 nm. Five scans
were recorded and averaged, and the background was subtracted. Mean
molar residue ellipticity ( Competitive solid phase
immunoassays were used to assess the binding of monoclonal antibodies
to lipid-free apoA-I and recombinant DMPC phospholipid complexes
containing apoA-I. With two exceptions (antibodies AI-17 and AI-141.7),
each antibody used in this study has been described, and its epitopes
on wild-type apoA-I have been documented (32). The epitopes of
antibodies AI-17 and AI-147.7 were localized to residues 143-165 and
220-242, respectively, on the basis of their binding to numerous
apoA-I mutant proteins described
previously.2 The immunoassays were
performed as described (33). Briefly, isolated human plasma apoA-I or
plasma HDL (0.05 ml of 5 µg/ml) was immobilized onto 96-well Falcon
3911 Microtest III flexible assay plates. After coating the plates,
increasing amounts of purified apoA-I or recombinant A-I discs (0.025 ml) diluted in phosphate-buffered saline containing 3% normal goat
serum were added to wells in the presence of 0.025 ml of ascites fluid
containing a limiting amount of monoclonal antibody (typically
dilutions of 10 The LCAT reaction was monitored by
following the cholesterol to cholesteryl ester conversion using
recombinant discoidal complexes containing either wild-type or 10F6
apoA-I. The complexes were assayed in duplicate using 0-3.0 µg of
substrate cholesterol in a final concentration of 10 mM
Tris, pH 7.4, 140 mM NaCl, 0.25 mM EDTA, and
0.15 mM sodium azide, 0.6% fatty acid-free bovine serum
albumin, 2 mM The activation energy (Ea) for cholesterol ester
formation was derived from Arrhenius plots of the LCAT reactivity of
wild-type and 10F6 discoidal substrates. Reactions were performed at
37, 34, 31, 28, and 25 °C using 0.55 µg of discoidal cholesterol
substrate and 25 ng of purified human LCAT. Arrhenius plots were
constructed as the reciprocal of incubation temperature in Kelvin
versus the log of cholesteryl ester formed (nmol/h) (34).
Linear regression analysis was used to determine the activation energy
according to the formula, Ea(cal/mol) = Values are given as the mean ± S.D.
Statistical comparisons were made using Student's t
test.
To test whether substitution of a single 22-mer repeat located at
143-164 (repeat 6) would alter apoA-I's ability to bind lipid or to
activate LCAT, we replaced repeat 6 of wild-type apoA-I with repeat 10 (corresponding to amino acids 220-241). The boxed region in
Fig. 1 shows the primary amino acid sequence of
wild-type and 10F6 apoA-I within the residue 143-164 domain. The
mutant protein 10F6 apoA-I contains one copy of repeat 10 inserted in the region normally occupied by repeat 6 and another copy of repeat 10 in its native position, 220-241. Also shown in Fig. 1 is the calculated hydrophobic moment (<µH>) and the average
hydrophobicity (H) for the entire boxed 22-mer amphipathic
helix. The calculated values, <µH> and H, for
this region in wild type and 10F6 are <µH> = 0.37, H =
Recombinant HDL complexes were prepared using a molar ratio of 100:5:1,
DMPC:cholesterol:apoA-I, respectively. Fig. 2 shows a
Coomassie-stained 4-30% nondenaturing gradient gel of 10F6, wild-type, and plasma apoA-I containing recombinant discoidal complexes. The gel shows doublets corresponding to Stokes diameters of
114 and 108 Å for each of the recombinant apoA-I complexes prepared.
The final DMPC:cholesterol:apoA-I ratio of the three discoidal
preparations was 98.1:4.1:1, 90.7:4.8:1, and 104:4.0:1 for 10F6,
wild-type, and plasma apoA-I-containing particles, respectively.
The effect of the 10F6 apoA-I replacement mutation on the overall
secondary structure of wild-type apoA-I protein was first examined by
determining the percentage of
Percentage Comparative Medicine and
§ Biochemistry,
Departments of Immunology and Vascular Biology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
-dimyristoylphosphatidylcholine:cholesterol:wild-type or 10F6 apoA-I showed a doublet corresponding to Stokes diameters of
114 and 108 Å on nondenaturing 4-30% polyacrylamide gel
electrophoresis. L-
-Dimyristoylphosphatidylcholine 10F6
apoA-I complexes had a 5-6-fold lower apparent
Vmax/apparent Km as
compared with wild-type apoA-I containing particles. As expected,
monoclonal antibody epitope mapping of the lipid-free and lipid-bound
10F6 apoA-I confirmed that a domain expressed between residues 143 and
165 normally found in wild-type apoA-I was absent. The region between
residues 119 and 144 in 10F6 apoA-I showed a marked reduction in
monoclonal antibody binding capacity. Therefore, we speculate that the
5-6-fold lower LCAT reactivity in 10F6 compared with wild-type apoA-I
recombinant particles results from increased stabilization within the
121-165 amino acid domain due to more stable apoprotein helix
phospholipid interactions as well as from conformational alterations
among adjacent amphipathic helix repeats.
-helical character that typically begin with a proline
residue, a feature that is shared within the entire apolipoprotein
supergene family. These repeats comprise approximately 80% of the
total protein and are divided into two 11-mer units and eight 22-mer
units (15, 16), a feature that is highly conserved across species. When
displayed on an Edmunson wheel, the
-helices show a distinctive
amphipathic character with separate hydrophilic and hydrophobic faces.
The amphipathic nature of these repeating units is believed to be
responsible for the ability of apoA-I to organize lipid and to activate
LCAT (17). A number of different models have been suggested to explain
how apoA-I's amphipathic repeats associate with phospholipid and how
these repeats activate LCAT (6-14, 18-25). Progress toward obtaining crystals of either lipid-free or lipid-bound apoA-I has been slow, and
a three-dimensional crystal structure is not available to reconcile the
different models.
-dimyristoylphosphatidylcholine (DMPC) in chloroform (30 mg/ml) was added to 60 µg of cholesterol in
ethanol (10 mg/ml) and 10 µl of radiolabeled cholesterol
(1,2-3H) (50 Ci/mmol) in ethanol. Organic solvent was
removed under a stream of argon, and the tubes were placed under vacuum
for 30 min. 2.7 mg of sodium cholate was added, and the solution was vortexed and then incubated for 30 min at 39 °C. The mixture was briefly vortexed three times during the 30-min incubation. To this
mixture was added 0.9 mg of wild-type or mutant apoA-I, and the
incubation was continued for an additional 1 h at 39 °C. Sodium cholate was removed by dialysis against 10 mM Tris, pH 7.4, 140 mM NaCl, 0.25 mM EDTA, and 0.15 mM sodium azide at 39 °C. Recombinant discoidal
complexes were purified by passage through a Superose 12 (Pharmacia
Biotech Inc.) column (55 × 1.8 cm) at a flow rate of 1 ml/min.
Phospholipid, protein, and cholesterol assays were performed to
determine the final molar composition of the recombinant discoidal
complexes (28).
) is reported as
degrees·cm2·dmol
1 and calculated from the
equation, [
] =
obs·115/10·l·c,
where
obs is the observed ellipticity at 222 nm in
degrees, 115 is the mean residue molecular weight of the protein,
l is the optical path length in centimeters, and
c is the protein concentration in g/ml. The percentage of
-helix was calculated from the formula of Chen et al.
(32), [
]222 =
30,300fh
2,340. Stability of the discoidal
10F6 and wild-type DMPC complexes was determined by plotting the mean
residue ellipticity versus guanidine HCl concentration and
expressed as the concentration of guanidine HCl
(D1/2) that reduced the ellipticity by 50%.
4 to 10
6). Competitor
concentrations listed in the figures represent the final concentrations
(µg/ml) in the 0.05-ml reaction mixture. The plates were incubated
overnight at 4 °C. After washing the wells, mouse antibody binding
to the immobilized antigen was detected by a second 1-h incubation at
37 °C with 125I-labeled goat anti-mouse IgG. All data
were expressed as B/Bo, where B is the
cpm bound in the presence of competitor, and Bo is
the cpm bound in the absence of competitor. To compare the affinity of
the antibodies for DMPC discs containing wild-type or mutant apoA-I's
competitor, the slopes of logit-transformed B/Bo ratios were obtained by linear
regression and subjected to tests of equality.
-mercaptoethanol, and 25 ng of purified
human plasma LCAT (kindly provided by Dr. J. S. Parks). The reactions were carried out for 60 min at 37 °C, and the conversion of
[3H]cholesterol to [3H]cholesteryl ester
was determined by lipid extraction of the incubation mixture followed
by thin layer chromatography (28). The extent of cholesterol
esterification was kept below 15% to maintain first order kinetics.
Background values were determined by omitting LCAT from the reaction
tube. The fractional cholesterol esterification rate was multiplied by
the nmol of substrate cholesterol in the assay tube, corrected for the
background, and converted to nmol of cholesterol ester formed/h/ml of
LCAT. Apparent Vmax and Km
values were determined from Hanes-Woolf plots (34) of discoidal
cholesterol substrate concentration (µM) divided by the
cholesterol ester formation rate (nmol/h/ml of LCAT) versus discoidal cholesterol concentration (µM).
2.3R(slope), where R is the gas constant (1.987 cal/deg
1/mol
1).
0.30 and <µH> = 0.24, H = +0.24, respectively (35, 36). Replacing repeat 6 with repeat 10 increases the hydrophobicity of the 143-164 region in
10F6 apoA-I from
0.30 to +0.24, reflecting the greater proportion of
nonpolar and uncharged amino acids in this 22-mer.
Fig. 1.
Sequence comparison of the residue 143-164
domain of wild-type and 10F6 apoA-I. The boxed 22-mer
for wild-type apoA-I shows the amino acid sequence normally found in
repeat 6 (residues 143-165). The boxed 22-mer for 10F6
apoA-I shows the amino acid sequence that corresponds to repeat 10 or
residues 220-241. The calculated hydrophobic moment (35)
(<µH>) and the average hydrophobicity (36)
(H) for the entire boxed 22-amino acid
amphipathic helix are shown for wild-type and 10F6 apoA-I.
[View Larger Version of this Image (22K GIF file)]
Fig. 2.
Coomassie G-250-stained 4-30% nondenaturing
polyacrylamide gradient gel of DMPC cholesterol apoA-I discoidal
complexes. HMW lane, calibrating high molecular weight
standards and their corresponding Stokes radii: thyroglobin (8.5 nm),
ferritin (6.1 nm), catalase (4.6 nm), lactate dehydrogenase (4.1 nm).
10F6 lane, DMPC 10F6 apoA-I complexes. Wild-type
lane, DMPC wild-type apoA-I complexes. Plasma lane,
DMPC human plasma-derived apoA-I complexes. Each lane of recombinant
particles contained 6 µg of protein. All recombinant discoidal
preparations were prepared as described under "Experimental
Procedures."
[View Larger Version of this Image (53K GIF file)]
-helix content. Both lipid-free and
lipid-bound wild-type and 10F6 apoA-I were analyzed by circular
dichroism spectroscopy. As shown in Table I, a trend toward a higher
-helix content for both the lipid-free and
lipid-bound 10F6 apoA-I compared with wild-type apoA-I was noted.
However, these differences did not reach statistical significance at
the level of p < 0.05. When examined in the
lipid-bound state, the
-helix content for each of the apoA-Is
studied increased compared to its lipid-free state (Table I).
-helix content of wild-type and 10F6 apoA-I
Without DMPC
With DMPC
Wild-type
apoA-I
42.8 ± 7.1
67.2 ± 4.0
Plasma apoA-I
47.0 ± 7.8
65.3 ± 8.7
10F6
apoA-I
49.1 ± 4.8
72.5 ± 4.4
Although the 10F6 apoA-I substitution did not show a statistically
significant change in its -helix content compared with lipid-free or
lipid-bound wild-type apoA-I, the DMPC 10F6 apoA-I recombinant
complexes were found to be less susceptible to guanidine HCl
denaturation than DMPC wild-type apoA-I complexes. Guanidine HCl
denaturation of DMPC 10F6 apoA-I complexes gave a
D1/2 = 2.8 M, while DMPC wild-type
complexes gave a D1/2 = 2.1 M (data not shown). These results demonstrate that the lipid-protein interaction in
DMPC 10F6 complexes was more stable than that for DMPC wild-type apoA-I
complexes.
In the next set of experiments the effects of substituting apoA-I's repeat 6 with repeat 10 were evaluated with respect to the protein's ability to activate LCAT. Kinetic studies were conducted, and the Km and Vmax of the reactions are summarized in Table II. The apparent Km values are 1.2 and 6.0 µM and the apparent Vmax values are 51.0 and 48.0 nmol/ml·h for DMPC wild-type and 10F6 apoA-I complexes, respectively (Table II). The overall efficiency for the LCAT reaction, referred to as the apparent Vmax/apparent Km, was calculated to be 42.5 and 8.0 nmol/ml·h·/µM for DMPC wild-type and 10F6 apoA-I complexes, respectively. To probe the mechanistic basis for the lower LCAT efficiency observed with the 10F6 apoA-I-containing particles, the energy of activation was measured as a function of temperature. The Arrhenius plots shown in Fig. 3 produced activation energies of 16.0 and 25.3 kcal/mol for DMPC wild-type and 10F6 apoA-I complexes, respectively.
|
To further explore the conformational impact of the 22-mer replacement mutant on apoA-I secondary structure, epitope mapping studies were carried out using a panel of defined monoclonal antibodies (33). First, the binding capacity of each antibody for lipid-free wild-type or 10F6 apoA-I were compared in competitive immunoassays. The results are tabulated in Table III. Six antibodies that identify N-terminal epitopes between residues 1 and 126 and three antibodies that identify C-terminal epitopes between residues 178 and 242 bound well to both apoproteins. These antibodies gave binding ratios (10F6 to wild type) of 5 or less, indicating that these particular epitopes were expressed by both apoproteins and that there were only minimal differences in the extent of epitope expression between the two apoA-I proteins. In contrast, four antibodies that identify epitopes between residues 119 and 165 on wild-type apoA-I either did not bind to purified 10F6 apoA-I or bound very poorly, i.e. gave 10F6 to wild type binding ratios of 16 or greater (Table III).
|
To probe the conformational changes caused by association of apoA-I
with lipid, we studied the binding of the same 13 antibodies to the
recombinant DMPC discoidal complexes containing wild-type or 10F6
apoA-I. The six N-terminal antibodies, AI-16, AI-1.2, AI-19.2, AI-11,
AI-4, and AI-115.1, had comparable affinity for both apoprotein-lipid
complexes and had 10F6 to wild-type apoA-I binding ratios of 1.1 or
less. This indicates that each of these epitopes was fully expressed on
both the wild-type and 10F6 apoA-I when complexed with lipid.
Competitive binding curves for two of these six antibodies are shown in
Fig. 4. These antibodies bind epitopes that are linearly
adjacent to helix 6, the AI-4 epitope within residues 99-121 and the
AI-115.1 within residues 115-126. The superimposable curves confirm
that AI-4 has both comparable binding capacity and binding affinity for
the DMPC wild-type and 10F6 complexes. The displaced binding curves
indicates that 115.1 has a greater binding capacity for DMPC wild-type
apoA-I complexes. However, the similar slopes of the two AI-115.1
curves demonstrates that antibody AI-115.1 binds the two epitopes with comparable affinity (Fig. 4). The three C-terminal antibodies, AI-178.1, AI-187.1, and AI-144, also bound both lipid-associated apoproteins with ratios between 1 and 7, and this pattern of reactivity was consistent with the results obtained with the lipid-free
apoproteins (data not shown). Antibodies AI-137.1 and AI-17, which did
not bind the lipid-free 10F6 apoA-I, also did not bind to the DMPC 10F6
apoA-I (Fig. 5), although they bound the DMPC wild-type
apoA-I complexes. These data are in complete agreement with those
obtained with the lipid-free apoA-Is (Table III) and further confirm
the absence of these epitopes within the 137-165 region of 10F6 apoA-I protein. Competition curves for the two antibodies that bound residues
119-144, a region that both adjoins and slightly overlaps the deleted
repeat 6 (residues 143-164), are shown in Fig. 6. These
antibodies needed significantly higher concentrations of lipid-free
10F6 apoA-I to achieve 50% inhibition of binding (Fig. 6, top
panels), as compared with wild-type apoA-I. These results strongly
suggest that replacement of repeat 6 with repeat 10 changed the
conformation of epitopes in repeat 5 (residues 121-142) but had little
effect on the conformation of epitopes in repeat 7. However, these same
repeat 5 epitopes were more similar when the apoA-Is were complexed
with DMPC (Fig. 6, bottom panel). Taken together, these
results imply that repeats 5 and 6 of apoA-I interact with phospholipid
in a manner that normalizes and stabilizes a native conformation at the
lipid interface.
In this report we describe the biophysical and biochemical
properties of the structural mutant, 10F6 apoA-I. This mutant protein was designed to investigate the effect of a single 22-mer repeat substitution on apoA-I structure and function. In previous studies (25), we showed that deletion of repeat 6 (residues 143-164) or repeat
7 (165-186) reduces the overall LCAT reaction velocity by 98-99%
relative to wild-type apoA-I (25). From these observations and from
those of others (26, 27), we proposed that the reduced LCAT reactivity
observed with the 6 apoA-I 22-mer deletion mutants resulted from
disruption of interactions between adjacent helical repeats within
apoA-I. We also suggested that electrostatic and hydrophobic side chain
interactions were responsible for the proper alignment of the
phospholipid substrate for LCAT catalysis (25). In the present studies,
we have further refined our working model by characterizing apoprotein
helix-lipid interactions and the apoprotein helix-helix interactions
that are critical for optimal LCAT reactivity.
Recombinant DMPC complexes containing 10F6 apoA-I displayed a particle
size distribution that was similar to wild-type and human plasma
apoA-I-containing complexes. These data agree well with previous
reports describing the size, composition, and -helix content of DMPC
plasma apoA-I discoidal complexes (37). Despite the absence of a
significant difference in particle size or composition, DMPC complexes
containing 10F6 apoA-I showed a profound reduction in their ability to
co-activate LCAT. The reactivity of the DMPC 10F6 apoA-I complexes was
determined by measuring the initial velocity of the LCAT reaction as a
function of cholesterol concentration. The kinetic parameters are shown
in Table II and demonstrate that the overall catalytic efficiency
(apparent Vmax/apparent Km) of the LCAT reaction catalyzed by 10F6 apoA-I was reduced 5-6-fold compared with wild-type apoA-I. This difference was primarily due to a
5-6-fold increase in the apparent Km for 10F6 apoA-I complexes. These results suggest that the reduced binding affinity of LCAT to DMPC 10F6 apoA-I complexes was responsible for the
large decrease in catalytic efficiency (38). Thus, we conclude that
substitution of a single 22-mer repeat within the 143-165 domain
resulted in a substantial reduction in LCAT binding to the recombinant
substrate and ultimately in a lower catalytic efficiency. The decrease
in LCAT catalytic efficiency was accompanied by a 9 kcal/mol increase
in the activation energy for 10F6 compared with wild-type apoA-I
recombinant complexes (Fig. 3). This increase in activation energy for
catalysis of DMPC 10F6 complexes may reflect the interaction of
hydrophobic residues (residue 143-165 domain) with boundary
phospholipid, in addition to altering side chain interactions between
adjacent helices in this region (repeats 5, 6, and 7). This hypothesis
is strengthened by the guanidine denaturation studies in which DMPC
10F6 complexes were denatured at a higher molar concentration of
guanidine hydrochloride (2.87 ± 0.30 M) than DMPC
wild-type complexes (2.28 ± 0.10 M).
The carboxyl-terminal region or repeats 9 and 10 (residues 209-243) of apoA-I has been proposed to be important in cell binding (39), lipoprotein association (40, 41, 42), LCAT activation (25-27), and in vivo metabolism of HDL (40, 42). Recent studies using truncation mutants of the carboxyl-terminal region demonstrated that the domain corresponding to residues 227-243 was critical in modulating the association of apoA-I with lipoproteins as well as in the in vivo metabolism of apoA-I (40, 42). Other investigations show that substitution of the carboxyl-terminal region of apoA-I with a helical domain derived from apoA-II increases its hydrophobicity but does not restore the mutant protein's lipoprotein affinity or its ability to associate with HDL3 (42). It is possible that in vivo apoA-I's carboxyl-terminal domain interacts cooperatively with the amphipathic helices located in the middle of the protein, thus allowing a conformation that can conform to particles of varying size and composition (42). The mechanistic role of this region in LCAT activation appears to be secondary to its role in lipid-lipoprotein association. Several studies have shown that deletion of repeat 9 or 10 results in at least a 60-90% reduction in wild-type LCAT activation (25-27, 41), while in one study, truncation of the residue 193-243 domain resulted in a 245% increase in wild-type LCAT activation (41). Thus, although this region may play some role in LCAT activation, it appears that the contribution of the carboxyl-terminal region of apoA-I to LCAT activation is secondary to its intrinsic lipid binding affinity and particle formation properties.
Protein-lipid hydrophobic interactions are believed to play a critical
role in the activation of LCAT. Because LCAT is a surface active
protein that binds to the discoidal lipid-protein interface, it has
been suggested that apoA-I's amphipathic -helices serve to disrupt
the aqueous to lipid interface and expose phospholipid to LCAT (4, 5).
Recently, NMR studies using an apoA-I fragment, residues 166-185 (12),
as well as a synthetic peptide activator of LCAT, i.e.
LAP-20, have shown that hydrophobic interactions between the nonpolar
amino acids and the phospholipid acyl chains play an important role in
stabilizing lipid-protein complexes (43). Furthermore, the NMR studies
suggest that intermolecular salt bridges and "snorkeling" (44) of
basic amino acid side chains play a less important role compared with
hydrophobic interactions in stabilizing lipid-apoprotein interactions
(43). Recent studies using synthetic peptides corresponding to each of
the eight 22-mer repeats of apoA-I have shown that repeat 10 (220-241)
has the highest lipid binding affinity of all eight repeats (45) and the greatest calculated depth of penetration into phospholipid vesicles
(11). However, the relative lipid binding affinity of an amphipathic
helix does not directly relate to a peptides' ability to activate LCAT
(13).
Modeling studies have suggested that the mode of assembly of adjacent amphipathic helical repeats around the edge of a discoidal complex is determined by both the hydrophobic character of the residues and by the charge complementary along the edge of the helices (46). From crystallography studies, helix-helix interactions inside lipid bilayers have been shown to include interhelical salt bridges, hydrogen bonds, or precise packing interactions (47). Thus, these structural features may determine the overall stability and the relative orientation of the adjacent helices. Therefore, we suggest from our data that substitution of repeat 10 for repeat 6 increases the hydrophobic nature of the 121-165 region both with respect to apoprotein-phospholipid interaction and to adjacent helix-helix interaction. Taken together, these highly stabilized interactions most likely allow the helices in the 121-165 domain to penetrate more deeply into the phospholipid bilayer, which then restricts LCAT's access to the boundary phospholipid acyl chains and results in reduced overall catalytic efficiency. Our hypothesis is consistent with a proposed mechanism for LCAT activation in which the central helices of apoA-I are believed to be displaced from contact with lipid by an apoE-like segment within LCAT during the binding and activation process (5).
From the data presented in this report, it appears that the
substitution of repeat 10 within the residue 143-165 region had diverse effects on the expression of apoA-I antibody epitopes. First,
most epitopes N-terminal of the substituted repeat remained unchanged.
However, the substitution of repeat 6 altered the expression of at
least one repeat 5 epitope, the epitope between residues 115-126,
defined by antibody AI-115.1. The reduced (but not abolished) binding
capacity of this antibody for the DMPC 10F6 apoA-I complexes suggested
that at least a portion of the DMPC 10F6 apoA-I complexes did not
express an AI-115.1 epitope. Second, two epitopes between residues 119 and 144, the region of apoA-I that both adjoins and overlaps the
deleted repeat 6, had more comparable reactivity for both lipid-bound
apoproteins. Third, as expected, the repeat 6 epitopes defined by
antibodies AI-137.1 and AI-17 were lost. Finally, the three antibodies
that identified epitopes that were C-terminal to repeat 6 reacted
similarly with both lipid-free and lipid-bound wild-type and 10F6
apoA-I. Although the 10F6 apoA-I protein contains two copies per
molecule of the AI-141.7 epitope (residues 220-242), greater
reactivity of antibody AI-141.7 for this mutant apoA-I was not observed
with either lipid-free or lipid-bound 10F6 apoA-I. Overall, these
results suggest that substitution of repeat 6 for repeat 10 resulted in
alteration of native wild-type apoA-I epitopes in both repeats 5 and 6 (residues 121-165). The conformational alterations in repeats 5 and 6 were found to become less apparent when the apoprotein was complexed
with lipid, presumably as a result of lipid-apoprotein interactions
that dominated apoA-I's helix-helix interactions. In summary, these
studies have aided our understanding of the mechanism of apoA-I's
activation of LCAT by clarifying the nature of the interaction between
phospholipid and apoA-I's amphipathic -helices and the
intermolecular helix-helix interactions critical for optimal catalytic
efficiency.
We recognize the excellent technical assistance of Nell Nordin, Kathi Richards, Gregory Pate, Abraham Gebre, and Elizabeth Eagleson.