(Received for publication, April 18, 1994; and in revised form, October 14, 1994)
From the
The effect of cholesteryl ester transfer protein (CETP) on the
size, composition, and structure of spherical, reconstituted HDL (rHDL)
which contain apolipoprotein (apo) A-I as their sole apolipoprotein has
been studied. Spherical rHDL were incubated with CETP and Intralipid
for up to 24 h. During this time CETP promoted transfers of cholesteryl
esters (CE) and triglyceride (TG) between rHDL and Intralipid. As a
result, the rHDL became depleted of CE and enriched in TG. However, as
the loss of CE from the rHDL was greater than the gain of TG, the
concentration of core lipids in the rHDL decreased. The decrease in the
concentration of rHDL core lipids, which was evident throughout the
incubation, was accompanied by a reduction in rHDL diameter from 9.2 to
8.0 nm, the dissociation of apoA-I from rHDL and a decrease in the
number of apoA-I molecules, from three/particle in the 9.2-nm rHDL, to
two/particle in the 8.0-nm rHDL. Spectroscopic studies showed that the
lipid-water interface and phospholipid packing of the 8.0-nm rHDL were,
respectively, more polar and less ordered than those of the 9.2-nm
rHDL. Quenching studies with KI revealed that the number of exposed
apoA-I Trp residues in the 9.2- and 8.0-nm rHDL was two and three,
respectively. Circular dichroism established that the 9.2- and 8.0-nm
rHDL had identical apoA-I -helical contents. The 9.2- and 8.0-nm
rHDL also had identical surface charges as determined by agarose gel
electrophoresis. Denaturation studies with guanidine hydrochloride
demonstrated that apoA-I is more stable in 8.0-nm rHDL than in 9.2-nm
rHDL. It is concluded that CETP converts rHDL to small, TG-enriched,
apoA-I-depleted particles with increased lipid-water interfacial
hydration and less ordered phospholipid packing. These changes are
associated with enhanced stability and minor changes to the
conformation of the apoA-I which remains associated with the rHDL.
Cholesteryl ester transfer protein (CETP) is a
multifunc-tional protein which promotes (i) homoexchanges of
cholesteryl esters (CE) between high density lipoproteins (HDL) and low
density lipoproteins (LDL)(1) ; (ii) heteroexchanges of CE and
triglyceride (TG) between HDL and very low density lipoproteins
(VLDL)(2) ; (iii) net mass transfers of CE from HDL to VLDL and
LDL(2, 3) ; and (iv) net mass transfers of TG from
VLDL to HDL(2) . CETP also changes the size of HDL during
incubation in vitro(4) .
The importance of understanding the effects of CETP on the structure and function of HDL has been highlighted by two recent observations. First, humans with a genetic deficiency of CETP have high concentrations of HDL which are larger than those found in normal subjects(5) . Such individuals appear to have a decreased incidence of coronary heart disease(6) . The second observation has been made in mice, a species which is normally deficient in CETP (7) and resistant to coronary heart disease(8) . When this species is made transgenic for CETP, their HDL decrease in size as well as concentration(9) . Such animals are also susceptible to diet-induced coronary heart disease (10) .
Although it is possible to study the effect of CETP on the structure and function of native HDL, the results of such experiments are difficult to interpret because native HDL consist of several subpopulations of particles which vary in size and composition. This problem has been overcome in the present report by using preparations of monodisperse, spherical reconstituted HDL (rHDL). We have shown recently that the interaction of rHDL with CETP is analogous to that of native HDL with CETP in that the rHDL acquire TG and lose CE during incubation with either VLDL or Intralipid(11) . We now report on the effects of these processes on the structure, size, and composition of rHDL.
In some experiments, the rHDL were isolated from the incubation mixtures by ultracentrifugation at 100,000 rpm in the 1.063 < d < 1.25 g/ml density range, with two 16-h spins at the lower density and a single 16-h spin at the higher density. These procedures were carried out at 4 °C using a Beckman TLA-100.2 rotor in a Beckman TL-100 Tabletop ultracentrifuge. As judged by gradient gel electrophoresis, the rHDL in the 1.063 < d < 1.25 g/ml density range were identical in size to the rHDL isolated as the fraction of d < 1.25 g/ml.
For the experiments with
VLDL, HDL, and CETP, the VLDL and HDL
were
isolated from the incubation mixtures as the supernatant and
infranatant, respectively, after 16 h of ultracentrifugation at 100,000
rpm at a density of 1.019 g/ml. A Beckman TLA-100.2 rotor and a Beckman
TL-100 Tabletop ultracentrifuge maintained at 4 °C were used for
this procedure. Completeness of the separation of VLDL and HDL
was established by the absence of apoB in the infranatant and the
absence of apoA-I in the supernatant.
Spectroscopic studies were carried out with a Perkin Elmer LS-50 luminescence spectrometer fitted with a thermostatted cell holder and polarizers. The cell holder was connected to a Lauda RM6T recirculating water bath (Lauda-Königshofen, Germany) and sample temperatures were monitored with a digital temperature probe (Baker Medical Research Institute, Melbourne, Australia). Uncorrected intrinsic fluorescence emission spectra were recorded from 300 to 380 nm using an excitation wavelength of 295 nm. The respective excitation and emission band passes were 10 and 5 nm. Intrinsic steady state fluorescence polarization values were determined using an excitation wavelength of 295 nm and excitation and emission band passes of 5 nm. These measurements were carried out at 25 °C.
Packing of rHDL phospholipid head groups was monitored with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH)(26) . Packing of phospholipid acyl chains at the interface of the rHDL core and surface monolayer was assessed with 1,6-diphenyl-1,3,5-hexatriene (DPH)(27) . Steady state fluorescence polarization of the TMA-DPH-labeled and DPH-labeled rHDL was measured at 5 °C intervals from 5 to 35 °C using an excitation wavelength of 366 nm. Polarity of rHDL lipid-water interfacial regions was determined with 6-propionyl-2-(dimethylamino)-naphthalene (PRODAN)(28) . The wavelength of maximum fluorescence of PRODAN-labeled rHDL was determined from 390 to 600 nm uncorrected emission spectra using an excitation wavelength of 366 nm and respective excitation and emission band passes of 5 and 6 nm. Labeling of the rHDL with DPH, TMA-DPH, and PRODAN was carried out exactly as described previously(12) . In all cases the molar ratio of probe/sample was 500:1 and the final concentration of phospholipid was 0.5 mM.
The stability of apoA-I in the rHDL was assessed by incubation with increasing concentrations of guanidine hydrochloride (GdnHCl). The rHDL were added to aliquots of 50 mM Tris-HCl (pH 8.0) containing 0-8 M GdnHCl. The final concentration of apoA-I in these mixtures was 20 µg/ml. Wavelengths of maximum fluorescence were determined from 300 to 380 nm emission scans using an excitation wavelength of 295 nm. The respective excitation and emission band passes were 10 and 5 nm. Initial readings (t = 0 h) were made at 25 °C, immediately after the rHDL had been added to the GdnHCl solutions. Measurements at t = 2, 4, 6, and 24 h were made after the samples had been incubated at 25 °C for the appropriate time.
To determine the fraction of exposed apoA-I Trp residues in the
rHDL, fluorescence quenching studies were carried out with KI. Varying
amounts of a 5 M KI stock solution were added to aliquots of
rHDL so that the final concentration of KI ranged from 0 to 0.33 M and the final concentration of apoA-I was 20 µg/ml. The KI
stock solution contained 1 mM NaS
O
to inhibit the formation
of I3-, which interferes with Trp
fluorescence(29) . To ensure the samples were of comparable
ionic strengths, appropriate amounts of 5 M NaCl were added
where necessary. The samples were incubated at 25 °C for 30 min to
allow for uptake of quencher. Fluorescence intensities of the samples
were determined from areas under the curves of 310-390 nm
uncorrected emission spectra using an excitation wavelength of 295 nm
and respective excitation and emission band passes of 10 and 5 nm.
Emission spectra of blank samples containing TBS and appropriate
concentrations of KI and NaCl were subtracted from the spectra of rHDL.
The results were analyzed according to the modified Stern-Volmer
equation(29) ,
where I is the emission intensity in the
absence of quencher, I is the emission intensity in the
presence of quencher, f
is the fraction of Trp
residues exposed to quencher and K
is the
Stern-Volmer quenching constant. Plots of (I
)/(I
- I) versus 1/([KI]) were linear, with r =
0.997 for lipid-free apoA-I, 0.897 for the 9.2 nm rHDL, and 0.964 for
the 8.0 nm rHDL.
The -helical content of rHDL apoA-I was
determined by circular dichroism. These studies were carried out by
Professor W. Sawyer, Department of Biochemistry, University of
Melbourne, Australia. The rHDL were adjusted to an apoA-I concentration
of 0.15 mg/ml with 0.02 M phosphate buffer (pH 7.4), and
dialyzed against the same buffer. Spectra were recorded from 190 to 250
nm at 24 °C on an AVIV circular dichroism spectrometer (AVIV
Associates Inc., Lakewood, NJ). Wavelength increments were 0.2 nm and
the averaging time for each point was 3 s. The %
-helix was
calculated from a fit of the entire spectrum using a mean residue
weight of 116.5 for apoA-I.
For immunoblot analysis, aliquots of incubation mixtures which had not been ultracentrifuged were subjected to nondenaturing gradient gel electrophoresis, transferred electrophoretically to nitrocellulose membranes, and immunoblotted with sheep anti-human apoA-I antiserum (Boehringer Mannheim)(32) .
Figure 1: Changes to the size of rHDL during incubation with CETP and Intralipid: influence of rHDL concentration. Varying amounts of rHDL were mixed with TBS, TBS and Intralipid, or TBS, Intralipid, and CETP. The mixtures of rHDL (final CE concentration 0.5 mmol/liter) and TBS were either maintained at 4 °C (Profile A) or incubated at 37 °C for 24 h (Profile B). Mixtures of rHDL (final CE concentration 0.5 mmol/liter) and Intralipid (final TG concentration 4.0 mmol/liter) were also maintained at 4 °C (Profile C) or incubated at 37 °C for 24 h (Profile D). The remaining incubations, which were carried out at 37 °C for 24 h, all contained the same concentrations of CETP (2.8 units/ml) and Intralipid (TG 4.0 mmol/liter) and varying concentrations of rHDL CE: 0.5 (Profile E), 0.25 (Profile F), 0.1 (Profile G), and 0.05 (Profile H) mmol/liter. In all cases the final volume of the incubation mixtures was 0.25 ml. When the incubations were complete the rHDL were isolated by ultracentrifugation as the fraction of d < 1.25 g/ml as described under ``Experimental Procedures.'' The isolated rHDL were electrophoresed on 3-35% nondenaturing polyacrylamide gradient gels and stained with Coomassie Blue G-250. The figure represents laser densitometric scans of the stained gels. The molar ratio of TG/CE is shown for individual incubations.
Figure 2: Time course of the changes in rHDL size during incubation with Intralipid and CETP. Spherical rHDL were incubated with TBS, TBS and Intralipid, or TBS, Intralipid, and CETP. Incubations containing rHDL (final CE concentration 0.1 mmol/liter) and TBS were either maintained at 4 °C (Profile A) or incubated at 37 °C for 24 h (Profile B). Mixtures containing rHDL (final CE concentration 0.1 mmol/liter) and Intralipid (final TG concentration 4.0 mmol/liter) were also maintained at 4 °C (Profile C) or incubated at 37 °C for 24 h (Profile D). Mixtures of rHDL (CE concentration 0.1 mmol/liter), Intralipid (TG concentration 4.0 mmol/liter), and CETP (2.7 units/ml) were incubated at 37 °C for 1 (Profile E), 3 (Profile F), 6 (Profile G), 12 (Profile H), or 24 h (Profile I). In all cases the final volume of the incubation mixture was 2.0 ml. When the incubations were complete an aliquot of each incubation mixture was ultracentrifuged at a density of 1.25 g/ml as described under ``Experimental Procedures.'' The rHDL in the fraction of d < 1.25 g/ml were subjected to gradient gel electrophoresis as described in the legend to Fig. 1. Laser densitometric scans of the stained gels are shown.
Figure 3:
Effect of CETP on concentration of core
lipids in rHDL and native HDL. Panels A-C show the
results of incubating spherical rHDL with Intralipid and CETP as
described in the legend to Fig. 2. When the incubations were
complete, the rHDL were isolated by ultracentrifugation in the density
range 1.063 < d < 1.25 g/ml as described under
``Experimental Procedures.'' Concentrations of rHDL CE (Panel A), rHDL TG (Panel B), and rHDL CE + TG (Panel C) are shown as a function of time. The values in Panels A and B represent the mean of duplicate
determinations which varied by 10% or less. Panels D-F show
incubations of native HDL
(final concentrations: CE and TG,
563 and 50 µmol/liter, respectively), VLDL (final concentrations:
CE and TG, 183 and 957 µmol/liter, respectively), and CETP (3.2
units/ml) at 37 °C for times ranging from 0 to 24 h. Concentrations
of HDL
CE (Panel D), HDL
TG (Panel
E), and HDL
CE + TG (Panel F) are shown
as a function of time. The values in Panels D and E represent the mean of duplicate
determinations.
To ensure
that the loss of rHDL core lipids in Fig. 3C was not
specific for the rHDL-CETP-Intralipid system, a further experiment was
carried out whereby native HDL were incubated with CETP and
VLDL for times ranging from 0 to 24 h. The results of this study showed
that the HDL
lost CE (Fig. 3D), gained TG (Fig. 3E), and sustained a net loss of core lipids
throughout the incubation (Fig. 3F).
The composition of the rHDL in the time course experiment is shown in Table 1as the number of moles of each constituent/mol of rHDL. Incubation with Intralipid in the absence of CETP had minimal effect on rHDL composition. When CETP as well as Intralipid were present in the incubation, CE was transferred from rHDL to Intralipid such that only a trace of CE remained in the rHDL by 6 h. During the first 3 h, the loss of CE from the rHDL was partially offset by the acquisition of TG from Intralipid. However, when the incubation was extended from 3 to 24 h there was a progressive loss of TG from the rHDL.
The moles of apoA-I/mol of rHDL decreased during the incubation (Table 1). This was confirmed by cross-linking studies, which showed that 9.2- and 8.0-nm rHDL contained three and two molecules of apoA-I/particle, respectively (Table 2). This decrease could be explained either by the dissociation of apoA-I from rHDL or by the formation of large, TG-rich rHDL of d < 1.063 g/ml. Given that (i) there was no loss of phospholipid from the 1.063 < d < 1.25 g/ml fraction (Table 1); (ii) there was no apoA-I in the d < 1.063 g/ml fraction (results not shown); and (iii) the rHDL in the 1.063 < d < 1.25 g/ml density range were identical in size to the rHDL in the d < 1.25 g/ml fraction, it is apparent that TG-rich rHDL of d < 1.063 g/ml were not formed during incubation. The decrease in the number of apoA-I molecules is therefore consistent with the dissociation of apoA-I from rHDL.
Further evidence of the dissociation of apoA-I from rHDL was obtained by immunoblot analysis. Aliquots of the incubation mixtures which had not been ultracentrifuged were subjected to nondenaturing gradient gel electrophoresis and immunoblotted for apoA-I. Lipid-free apoA-I was also applied to the gels. The lipid-free apoA-I migrated further than, and was distinct from, the apoA-I associated with rHDL. When the rHDL were incubated either alone or with Intralipid, apoA-I appeared as a single band in the size range of rHDL. This was also the case when the rHDL were incubated with CETP and Intralipid for 1 h. When the rHDL were incubated with CETP and Intralipid for 3, 6, 12, or 24 h, there were two bands of apoA-I, one of which co-migrated with rHDL while the other migrated to the same position as lipid-free apoA-I, exactly as described for incubations of native HDL(32) .
Figure 4:
Lipid packing of the 9.2-and 8.0-nm rHDL.
Aliquots of 9.2-nm (closed symbols) and 8.0-nm rHDL (open
symbols) were labeled with DPH (,
), TMA-DPH
(
,
), and PRODAN (
,
). Steady state
fluorescence polarization of rHDL labeled with DPH and TMA-DPH is shown
in Panel A. The wavelength of maximum fluorescence emission of
rHDL labeled with PRODAN is shown in Panel B. The values
represent the mean of at least three determinations. Experimental
errors for the polarization data are ±0.005 and ±1 nm for
the wavelength of maximum fluorescence.
The fraction of apoA-I Trp residues (f) in the 9.2- and 8.0-nm rHDL accessible to
quenching by KI is shown in Table 2. The f
for the 9.2-nm rHDL was 0.40 compared to 0.70 for the 8.0-nm
rHDL. As apoA-I contains 4 Trp residues(34) , this is
consistent with 2 residues being exposed to KI in the 9.2-nm
rHDL compared to three in the 8.0-nm rHDL. The Stern-Volmer quenching
constants (K
) for the 9.2-nm rHDL and lipid-free
apoA-I were comparable. The lower K
value for the
8.0-nm rHDL suggests that either the collisional rate constant for the
quenching process decreases with rHDL particle size or that the
lifetimes of the apoA-I Trp residues in 8.0-nm rHDL differ from those
in 9.2-nm rHDL and lipid-free apoA-I.
The packing order of the rHDL phospholipid acyl chains and head groups was assessed by labeling with DPH (Fig. 4A, squares) and TMA-DPH (Fig. 4A, diamonds), respectively. In all cases the polarization values were higher for the 9.2-nm rHDL (closed symbols) than for the 8.0-nm rHDL (open symbols). This is consistent with the phospholipid acyl chains and head groups in the 9.2-nm rHDL being more ordered than those in the 8.0-nm rHDL. The polarity of the rHDL lipid-water interfacial regions was assessed by labeling the 9.2-nm rHDL (closed symbols) and 8.0-nm rHDL (open symbols) with PRODAN (Fig. 4B). The higher values for the wavelength of maximum fluorescence of PRODAN in the 8.0-nm rHDL is consistent with their lipid-water interface being more hydrated than that of the 9.2-nm rHDL.
The unfolding of lipid-free apoA-I and apoA-I in rHDL was determined from the wavelength of maximum fluorescence of samples which had been incubated with increasing concentrations of GdnHCl for times ranging from 0 (closed symbols) to 24 h (open symbols) (Fig. 5). Lipid-free apoA-I unfolded rapidly, as evidenced by the similar values for the wavelengths of maximum fluorescence at 0 and 24 h (Fig. 5C). This was not the case for apoA-I in 9.2- (Fig. 5A) and 8.0-nm (Fig. 5B) rHDL, where the values for the wavelength of maximum fluorescence at t = 0 h were lower than the values at t = 2, 4, 6, or 24 h. For clarity the results for 0 and 24 h only are shown. The higher concentrations of GdnHCl required to achieve 50% denaturation of the apoA-I in rHDL relative to lipid-free apoA-I are consistent with lipid association enhancing the stability of apoA-I (Table 2). This is in agreement with what has been reported by others(35) . The additional finding that a higher concentration of GdnHCl is required to unfold apoA-I in 8.0-nm rHDL compared to 9.2-nm rHDL suggests that apoA-I is more stable in smaller particles.
Figure 5:
Denaturation of rHDL with guanidine
hydrochloride. Lipid-free apoA-I and rHDL were incubated at 25 °C
for 0 h () or 24 h (
) with varying concentrations of
GdnHCl as described under ``Experimental Procedures.'' Panels A, B, and C show the respective wavelengths of
maximum fluorescence of 9.2-nm rHDL, 8.0-nm rHDL, and lipid-free
apoA-I. The values represent the mean of triplicate determinations.
Experimental errors for the wavelength of maximum fluorescence are
±1 nm.
This report documents the influence of CETP on the size, structure, and composition of spherical rHDL. When rHDL of diameter 9.2 nm are incubated with Intralipid and CETP, the rHDL become depleted of core lipids and their diameter decreases to 8.0 nm. Formation of 8.0-nm particles is dependent on the concentration of rHDL in the incubation. Whereas 8.0-nm particles are not formed in incubations containing high concentrations of rHDL, quantitative conversion to 8.0-nm particles occurs when the concentration of rHDL is low. The simplest explanation for this observation is that when the concentration of rHDL in the incubation is high, there is increased transfer of core lipids between individual rHDL particles and reduced net mass transfer of core lipids from rHDL to Intralipid. Under these circumstances rHDL size does not decrease. Although these results can be explained in more complex terms, this simple interpretation is consistent with what has been reported for native lipoproteins. When high concentrations of native HDL are incubated with CETP and VLDL or LDL, transfers of core lipids within the HDL fraction increase and transfers of core lipids between HDL and either VLDL or LDL decrease(36, 37) .
The results of the present study show that CETP promotes transfers of CE and TG between rHDL and Intralipid. This confirms what we have reported previously for incubations of rHDL and either VLDL or Intralipid(11) . During the first 3 h of the incubation, CE was transferred from rHDL to Intralipid (Fig. 3A) and TG was transferred from Intralipid to rHDL (Fig. 3B) such that most of the CE was lost from the rHDL and their major core lipid was now TG. Therefore, beyond 3 h of incubation, TG, but not CE, was available for transfer from rHDL to Intralipid. As shown in Fig. 3B, when the incubation was extended from 3 to 24 h, CETP did, in fact, transfer TG from rHDL to Intralipid. Such a biphasic TG response has not been reported previously either for native HDL or for rHDL. One possible explanation of this observation is that CETP does not discriminate between CE and TG. In other words, the probability of CETP acquiring CE or TG from rHDL is proportional to their relative concentrations in the rHDL(38) . Furthermore, if, as has been observed for incubations of native HDL and VLDL(39) , transfers of core lipids out of rHDL exceeded transfers of core lipids into rHDL, we can now account for the progressive reduction in the concentration of rHDL core lipids in Fig. 3C. During the first 3 h of incubation, the imbalance in lipid transfers into and out of rHDL manifested as a transfer of CE out of rHDL into Intralipid (Fig. 3A) which was greater than the transfer of TG from Intralipid into rHDL (Fig. 3B). This is consistent with what has been reported previously for native HDL and VLDL(39) . When the incubation was extended beyond 3 h, at a time when the main core lipid in the rHDL was TG, the imbalance in transfers manifested as a decrease in the concentration of rHDL TG.
Although one would expect rHDL size
to decrease as core lipids are lost, the present results show that this
is not necessarily the case. After a 1-h incubation with CETP and
Intralipid, the concentration of rHDL core lipids decreased by 32%,
from 66 to 45 µmol/liter (Fig. 3C), but their size
did not change (Fig. 2). This observation suggested that the
reduction in core lipid volume was not as great as the decrease in the
concentration of core lipids. To determine if this was the case, rHDL
core volumes were calculated at 0 and 1 h. At 0 h the rHDL contained 84
molecules of CE/particle and at 1 h they contained 38 molecules of CE
and 19 molecules of TG/particle (Table 1). Given that the
respective molecular volumes of CE and TG are 1.13 nm and
1.61 nm
(Table 3), it follows that the rHDL core
volume was 95 nm
at 0 h compared to 73 nm
at 1
h. In other words, the rHDL core volume decreased by 22% whereas the
concentration of core lipids decreased by 32%. To confirm that there
was no reduction in particle size after 1 h of incubation, the rHDL
diameter was calculated as described in Table 3. The values
obtained by this approach for 1 h, as well as those for 12 and 24 h,
agreed closely with those obtained by gradient gel electrophoresis (Fig. 2). This result, which shows that rHDL can sustain a
reduction in core lipid volume without changing size, is consistent
with what has been reported previously(11) .
The results in Table 2show that the 9.2- and 8.0-nm rHDL contain two and three molecules of apoA-I/particle, respectively. This reduction in the number of apoA-I molecules can be explained in several ways. One possibility is that CETP mediates the fusion of two 9.2-nm rHDL to form a large, unstable particle with six molecules of apoA-I. Such a fusion product could rearrange into three smaller particles containing two molecules of apoA-I/particle. However, this mechanism does not account for the dissociation of apoA-I from rHDL. Furthermore, the calculated diameter of such a fusion product would be approximately 11.9 nm and there is no evidence of particles of this size in Fig. 2. However, as such fusion products are likely to be unstable and have short lifetimes, it may not be possible to detect them electrophoretically. It could also be argued that a fusion product may partially disintegrate during rearrangement and that one or two, rather than three, small particles are formed. If this occurred the rHDL would lose phospholipids as well as apoA-I. Although apoA-I was lost from rHDL, there was no evidence of a concommitant loss of phospholipids (Table 1). A more plausible explanation for the decrease in the number of apoA-I molecules/rHDL is that 9.2-nm rHDL which are depleted of core lipids contain an excess of surface constituents and are unstable. We suggest that: (i) once sufficient core lipids have been lost from the rHDL; and (ii) the core lipids remaining with the rHDL can be accommodated in a smaller volume, conversion to smaller, more stable particles occurs and a molecule of apoA-I dissociates from the rHDL.
A variety of spectroscopic techniques were used to determine
how rHDL structure was affected by the reduction in particle size and
dissociation of apoA-I. The results of the quenching studies in Table 2, which showed that 3 apoA-I Trp residues were accessible
to the quencher in the 8.0-nm rHDL compared to 2 residues in the 9.2-nm
rHDL, were consistent with the reduction in particle size affecting the
conformation of apoA-I. As circular dichroism studies did not reveal
any differences in the secondary structure of the 9.2- and 8.0-nm rHDL (Table 2), it is unlikely that the change in apoA-I conformation
occurred within an -helix. Trp
, in the random coil,
N-terminal region of the protein, and Trp
, which is
located in a
-turn, fulfill this requirement(40) .
Although Trp
is located in an
-helix(40) ,
the possibility that there was a change of conformation in this region
cannot be discounted. Indeed, it has been postulated that the
-helices in the vicinity of residue 100 form a hinged domain in
apoA-I which may, or may not, bind to the surface of HDL(41) .
The other results in Table 2suggest that the apoA-I
conformational change was minor. For example, the polarization and
wavelength of maximum fluorescence values in Table 2were
consistent with the apoA-I Trp residues in the 9.2- and 8.0-nm rHDL
having similar local rotational motions and average environments. In
addition, the reduction in particle size did not affect the apoA-I
-helical content or rHDL electrophoretic mobility (Table 2).
As electrophoretic mobility reflects rHDL surface charge(24) ,
and hence the charge of constituent apolipoproteins(42) , this
observation provides further evidence that changes in rHDL particle
size have little overall effect on the conformation of apoA-I.
The results in Fig. 4show that phospholipid packing and hydration of lipid-water interfacial regions is affected by rHDL particle size. The greater hydration of the 8.0-nm rHDL lipid-water interface relative to that of the 9.2-nm rHDL is consistent with the higher surface curvature of the small particles permitting increased access of water. The less ordered packing of phospholipid acyl chains in the 8.0-nm rHDL relative to 9.2-nm rHDL is in agreement with what has been observed by Ben-Yashar and Barenholz (27) for native HDL. These investigators also found that the packing order of phospholipid head groups was independent of HDL size. The results in Fig. 4A, by contrast, show that the phospholipid head groups in 9.2-nm rHDL are more ordered than in 8.0-nm rHDL. As native HDL contains several populations of particles with diameters ranging from 7.62 to 10.57 nm (43) , it is possible that differences in their phospholipid head group packing order may be obscured. The results of these spectroscopic studies also offer some insight as to why the limiting diameter of spherical rHDL is 8.0 nm. It is possible that the less ordered packing of the phospholipids and increased hydration of the surface of the 8.0-nm rHDL does not favor partitioning of CE and TG into the surface of particles, thus limiting the access of CETP to core lipids.
The results of the denaturation studies with GdnHCl (Fig. 5, Table 2) show that apoA-I is more stable in 8.0-nm rHDL than in 9.2-nm rHDL. In the case of the 9.2-nm rHDL, 50% denaturation of apoA-I was achieved with 3.35 M GdnHCl. This value differs from what has been reported by Leroy et al.(44) , who found that 4.8 M GdnHCl was required to achieve 50% denaturation of apoA-I in rHDL of comparable diameter. As the 9.2-nm rHDL in the current report are enriched in phospholipids and CE relative to those described by Leroy et al.(44) , this suggests that the stability of apoA-I in rHDL decreases as their lipid content increases. This is in contrast to what has been reported by Sparks et al.(45) , who found that apoA-I unfolded more readily as the lipid content of rHDL decreased. This discrepancy may relate to the fact that the rHDL described by Sparks et al. were prepared by sonication, whereas the rHDL of Leroy et al. were prepared in a more physiological manner, by incubating discoidal rHDL with LDL and LCAT. These different methodologies may generate rHDL which are structurally dissimilar. It has also been reported that the denaturation of apoA-I in spherical rHDL is multiphasic(35, 44) . There is, however, no evidence of stable intermediates in the present study (Fig. 5). Although the reason for this discrepancy is not clear, our findings are consistent with those of Sparks et al.(45) .
In conclusion, this study shows that CETP-mediated exchanges of core lipids between rHDL and Intralipid affect the size, composition, and structure of the rHDL. These results provide insight into the relationship between lipid transfers and rHDL structure and size which could not be achieved with native HDL. It is expected that future studies with the rHDL-CETP-Intralipid model system will provide further information about the relationship between rHDL structure and function and, as a result, enhance our understanding of the metabolism of HDL.