(Received for publication, July 18, 1995; and in revised form, October 30, 1995)
From the
The effect of sphingomyelin (SPM) on the structure and function
of discoidal and spherical reconstituted high density lipoproteins
(rHDL) has been studied. Three preparations of discoidal rHDL with
1-palmitoyl-2-oleoyl phosphatidylcholine (POPC)/SPM/unesterified
cholesterol (UC)/apolipoprotein (apo)A-I molar ratios of
99.6/0.0/10.2/1.0, 86.0/13.6/10.8/1.0, and 72.5/26.3/11.4/1.0 were
prepared by cholate dialysis. SPM did not affect discoidal rHDL size or
surface charge. Esterification of cholesterol by lecithin:cholesterol
acyltransferase (LCAT) was inhibited in the SPM-containing discoidal
rHDL. When the discoidal rHDL of POPC/SPM/UC/apoA-I molar ratio
99.6/0.0/10.2/1.0 were incubated with low density lipoproteins (LDL)
and LCAT, SPM transferred spontaneously from the LDL to the rHDL (t = 0.8 h) and spherical particles with
a POPC/SPM/UC/CE/apoA-I molar ratio of 24.6/4.9/3.6/24.9/1.0 were
formed. Depleting the spherical rHDL of SPM head groups by incubation
with sphingomyelinase increased the negative charge on the surface, but
did not change their size. Cholesteryl ester transfer protein
(CETP)-mediated transfers of cholesteryl esters and triglyceride
between spherical rHDL and Intralipid were not affected by SPM head
group depletion. The effect of SPM on rHDL structure was assessed
spectroscopically. SPM increased POPC acyl chain and head group packing
in the discoidal rHDL. When the spherical rHDL were depleted of SPM
head groups, POPC acyl chain packing order decreased, but head group
packing order was not affected. SPM inhibited the lipid-water
interfacial hydration of discoidal rHDL. This parameter was not
affected when the spherical rHDL were depleted of SPM head groups. The
SPM molecule and the SPM head group, respectively, inhibited the
unfolding of apoA-I in discoidal and spherical rHDL. It is concluded
that (i) SPM influences the structure of discoidal and spherical rHDL,
(ii) SPM inhibits the LCAT reaction in discoidal rHDL, and (iii) the
SPM head group does not affect CETP-mediated lipid transfers into or
out of spherical rHDL.
Sphingomyelin (SPM) ()is a glycosphingolipid which is
present in cell membranes and plasma lipoproteins. For many years SPM
was thought only to maintain the structural integrity of membranes, but
recent studies have shown that it is also involved in a wide range of
metabolic events(1, 2) . The SPM molecule comprises a
phosphocholine head group and a ceramide backbone with a sphingosine
base and an amide-linked acyl chain. The ceramide backbone of SPM plays
a regulatory role in cell growth, differentiation, and
apoptosis(1, 2) . Ceramide also modulates protein
phosphorylation and has been implicated as a tumor-suppressor lipid (3) . The influence of SPM on lipoprotein metabolism is poorly
understood. It has been reported that the concentration of SPM in the
artery wall increases with aging and that it comprises 70-80% of
the phospholipids in atherosclerotic lesions(4) . These
observations suggest that SPM may be involved in the development of
atherosclerosis. The additional finding that the SPM in atherosclerotic
lesions is derived from plasma lipoproteins (4) emphasizes the
importance of understanding how this molecule influences lipoprotein
metabolism.
At present little is known about the origin of SPM in
lipoproteins. SPM reportedly transfers from cell membranes to
pre--migrating high density lipoproteins (HDL)(5) . SPM is
also present in discoidal, nascent HDL which are secreted from the rat
liver(6) . However, it is not known whether the SPM, which
enters the plasma compartment as a component of pre-
-migrating HDL
and nascent HDL, is subsequently incorporated into mature, spherical
HDL. Similarly, little is known of the origins of SPM in low density
lipoproteins (LDL) and very low density lipoproteins. It has been
reported that the lipoproteins in peripheral lymph are enriched in SPM
relative to their plasma counterparts(7) , suggesting that SPM
from cell membranes may be incorporated into lipoproteins before they
enter the plasma compartment.
Given that there are strong Van der
Waals interactions between SPM and unesterified cholesterol (UC) (8, 9) and that the concentrations of UC and SPM in
membranes and lipoproteins change in a coordinated manner (10) , it follows that SPM may participate in the regulation of
cholesterol transport and the maintenance of cell cholesterol
homeostasis. Evidence for this comes from studies which show that SPM
regulates the uptake and intracellular processing of
LDL(11, 12) . The additional finding that
SPM-containing lipid/apolipoprotein complexes are excellent acceptors
of cellular cholesterol (13) suggests that SPM may also be
involved in the initial step of the reverse cholesterol transport
process. Further support for the involvement of SPM in reverse
cholesterol transport comes from the observation that
[H]cholesterol efflux from fibroblasts to HDL
increases if the cells have been incubated with
sphingomyelinase(10) .
The aim of the present study is to better understand the influence of SPM on HDL metabolism. In order to overcome the problems of interpretation which may occur due to the heterogeneity of native HDL, the study has been carried out with well characterized preparations of discoidal and spherical reconstituted HDL (rHDL)(14, 15) . The results show that SPM influences both the structure and function of rHDL.
The rHDL were isolated from incubation mixtures by ultracentrifugation at 100,000 rpm in the 1.07 < d < 1.25 g/ml density range using a TLA-100.4 rotor or in the 1.063 < d < 1.25 g/ml density range using a TLA-100.2 rotor. Two 16-h spins at the lower density and one 16-h spin at the higher density were performed. These procedures were carried out at 4 °C in a Beckman TL-100 tabletop ultracentrifuge. The rHDL were dialyzed extensively against TBS before use.
Nondenaturing gradient gel electrophoresis on 3/35% gels (Gradipore, Sydney, Australia) was carried out as described previously (28) .
Packing of rHDL phospholipid head groups and acyl chains was monitored by labeling with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluene sulfonate (TMA-DPH) and 1,6-diphenyl-1,3,5-hexatriene (DPH), respectively(29, 30) . Polarity of rHDL lipid-water interfacial regions was assessed with 6-propionyl-2-(dimethylamino)-naphthalene (PRODAN)(31) . In all cases the molar ratio of phospholipid/probe was 500/1, and the phospholipid concentration was 0.5 mM. The labeling procedures and spectroscopic conditions are described in detail elsewhere(15) .
The unfolding of apoA-I was assessed from the wavelength of maximum fluorescence of samples following incubation at 25 °C for 0, 2, 5, 7, and 24 h with 0-8 M guanidine hydrochloride (GdnHCl). Data from the 24-h time points were used for the calculations described below. All calculations are based on the assumption that the unfolding of apoA-I is represented by a two-state process such that, at a given time, the only species present at significant concentrations are either completely folded or completely unfolded(32) . The central, linear regions of the unfolding curves were used for the calculations. For a given concentration of GdnHCl, the fraction of unfolded apoA-I was calculated as
where y, y
, and y represent the respective wavelengths of maximum fluorescence in
the folded, unfolded, and transition states.
The equilibrium constant (K) for unfolding was calculated as
and the free energy change was calculated as
where R is the gas constant (1.987 cal/degree/mol) and T is the absolute temperature (198.15 K). The concentration of
GdnHCl at the midpoint of the denaturation curve was calculated from
plots of G versus the concentration of GdnHCl using
values of
G between -1.5 and +1.5 kcal/mol.
G
where n is the difference in the number of binding
sites between the folded and unfolded states, k is the
equilibrium constant for binding at each site (0.6), and a,
the activity of GdnHCl, is calculated from the molarity (M) of
GdnHCl as follows
Figure 1: Influence of SPM on the size of discoidal and spherical rHDL. Discoidal and spherical rHDL with varying amounts of SPM were prepared as described under ``Experimental Procedures,'' electrophoresed on 3/35% polyacrylamide nondenaturing gradient gels, and stained with Coomassie Blue G-250. Laser densitometric scans of the stained gels are shown.
Figure 2:
Kinetics of the transfer of SPM from LDL
to rHDL. Discoidal rHDL (SPM/apoA-I molar ratio = 0/1; final
apoA-I concentration, 0.4 mg/ml) were incubated at 37 °C for 0, 1,
3, 6, 12, or 24 h with LDL (final apoB concentration, 1.5 mg/ml) and
LCAT (1.9 ml). The incubation mixtures also contained bovine serum
albumin (final concentration, 60 mg/ml) and -mercaptoethanol
(final concentration, 4.0 mM). The final volume of the
incubation mixtures was 5.0 ml. When the incubations were complete, the
rHDL were isolated by ultracentrifugation in the 1.07 < d < 1.25 g/ml density range. Concentrations of SPM and apoA-I
were determined as described under ``Experimental
Procedures.'' Molar ratios were calculated from means of
triplicate determinations which varied by less than 10%. The values in
the figure represent the mean of two separate
experiments.
Figure 3:
Influence of SPM on the structure of
discoidal and spherical rHDL. Discoidal rHDL with SPM/apoA-I molar
ratios of 0/1 (), 13.6/1 (
), and 26.3/1 (
) and
spherical rHDL with SPM/apoA-I molar ratios of 0/1 (
) and 4.9/1
(
) were labeled with DPH (Panel A), TMA-DPH (Panel
B), and PRODAN (Panel C). Steady state fluorescence
polarization of the DPH- and TMA-DPH-labeled samples and the wavelength
of maximum fluorescence of the PRODAN-labeled samples are shown. Values
represent the mean of at least three determinations. Experimental
errors for the polarization values are ±0.003 and ±1.0 nm
for the wavelength of maximum fluorescence.
When these discoidal rHDL
preparations were incubated with LDL and LCAT, the resulting spherical
rHDL all had SPM/apoA-I molar ratios of approximately 5/1 (result not
shown). This was consistent with SPM transferring spontaneously between
LDL and rHDL. The kinetics of the transfer of SPM from LDL to rHDL was
investigated by incubating discoidal rHDL with a POPC/SPM/UC/apoA-I
molar ratio of 99.6/0.0/10.2/1.0 in the presence of LDL and LCAT for
0-24 h. The rHDL were then isolated by ultracentrifugation and
the molar ratio of SPM/apoA-I was determined (Fig. 2). The
SPM/apoA-I molar ratio increased rapidly during the first hour of
incubation. Equilibrium was achieved between 6 and 12 h, with a t for the transfer of 0.8 h. After 24 h of
incubation, the POPC/SPM/UC/CE/apoA-I molar ratio of the spherical rHDL
was 24.6/4.9/3.6/24.9/1.0. In molar terms, SPM accounted for 17% of the
phospholipid in the spherical rHDL (Table 1).
When the spherical rHDL were depleted of SPM head groups by incubation with sphingomyelinase, the concentrations of the other constituents and the size of the particles did not change ( Fig. 1and Table 1). This was not the case for the SPM-containing discoidal rHDL, which were converted quantitatively to larger and smaller particles by incubation with sphingomyelinase (result not shown). These larger and smaller particles were not further characterized.
Various spectroscopic techniques were used to assess the effect of SPM on rHDL structure. The discoidal and spherical rHDL had comparable wavelengths of maximum fluorescence (Table 1). This is consistent with the environment of apoA-I Trp residues not being affected by (i) the shape of the rHDL, (ii) the presence of SPM in discoidal rHDL, or (iii) removal of SPM head groups from spherical rHDL by incubation with sphingomyelinase. The wavelengths of maximum fluorescence for the apoA-I in the rHDL were blue-shifted relative to lipid-free apoA-I. In other words, the apoA-I Trp residues in the rHDL are in a more hydrophobic environment than those in lipid-free apoA-I. This is in agreement with what has been reported elsewhere(23, 35) .
The local rotational motions of the rHDL apoA-I Trp residues were determined by steady state fluorescence polarization (Table 1). Although the discoidal rHDL polarization values decreased as the SPM content of the particles increased, the differences were not statistically significant. This is consistent with SPM having little effect on the local rotational motions of the apoA-I Trp residues in discoidal rHDL. The polarization of the spherical rHDL which contained SPM was comparable to that of the discoidal rHDL, suggesting that particle shape does not affect the rotation of apoA-I Trp residues. However, the polarization decreased when the spherical rHDL were depleted of SPM head groups (p < 0.01). This is consistent with the SPM head group restricting the rotation of apoA-I Trp residues in spherical rHDL. In all cases the polarization of lipid-associated apoA-I was significantly lower than that of lipid-free apoA-I (p < 0.001). In other words, lipid association enhances local rotational motions of apoA-I Trp residues. This confirms what has been reported previously(23, 35) .
The effect of SPM on rHDL surface charge was assessed by agarose gel electrophoresis. The electrophoretic mobilities of the discoidal rHDL were intermediate between lipid free apoA-I and native HDL and were not affected by SPM (Table 1). This demonstrates that the SPM molecule does not influence the surface charge of discoidal rHDL. The spherical rHDL with intact SPM migrated slightly slower than native HDL, but more rapidly than discoidal rHDL. After incubation with sphingomyelinase, their electrophoretic mobility increased and was indistinguishable from that of native HDL. This is consistent with the SPM head group decreasing the negative charge on the spherical rHDL surface.
The influence of SPM on phospholipid acyl chain packing order was assessed from the polarization of DPH-labeled spherical and discoidal rHDL (Fig. 3A). The SPM molecule increased discoidal rHDL acyl chain packing order as evidenced by the increase in polarization values with increasing SPM/apoA-I molar ratios. The polarization of the spherical rHDL which had been incubated with sphingomyelinase (open circles) was slightly lower than that of the spherical rHDL which had been incubated with TBS (closed circles). This is consistent with the SPM head group having a minor ordering effect on spherical rHDL phospholipid acyl chains. The additional finding that spherical rHDL have higher polarization values than discoidal rHDL demonstrates that phospholipid acyl chains are more ordered in spheres than in discs. This is in agreement with what has been reported by Jonas et al.(35) .
Phospholipid head group packing order was assessed from the polarization of TMA-DPH-labeled discoidal and spherical rHDL (Fig. 3B). The order of the discoidal rHDL phospholipid head groups increased as the SPM/apoA-I molar ratio increased from 0/1 (closed squares) to 13.6/1 (open squares) to 26.3/1 (diamonds). The values for the spherical rHDL with (closed circles) and without (open circles) SPM head groups were comparable. The additional finding that spherical rHDL have more ordered phospholipid head groups than discoidal rHDL confirms what has been reported elsewhere(35) .
The rHDL were also labeled with PRODAN, a polarity sensitive fluorescent probe (Fig. 3C). The wavelength of maximum fluorescence of the discoidal rHDL increased rapidly at temperatures above 25 °C. The increase was greatest for the discs without SPM (SPM/apoA-I molar ratio = 0/1) (closed squares), intermediate when the SPM/apoA-I molar ratio was 13.6/1 (open squares) and least when the SPM/apoA-I molar ratio was 26.3/1 (diamonds). In other words, SPM inhibits the hydration of discoidal rHDL lipid-water interfacial regions. The wavelength of maximum fluorescence of PRODAN in the spherical rHDL was comparable after incubation in the presence (open circles) and absence (closed circles) of sphingomyelinase. This demonstrates that SPM head groups do not influence the hydration of the spherical rHDL lipid-water interface. As the increase in the wavelength of maximum fluorescence of the spherical rHDL was small, it seems that the lipid-water interface of these particles is resistant to hydration.
Figure 4:
Influence of SPM on the GdnHCl-mediated
unfolding of apoA-I in discoidal and spherical rHDL. Discoidal and
spherical rHDL and lipid-free apoA-I were incubated with increasing
concentrations of GdnHCl for 0 (), 2 (
), and 24 (
) h
as described under ``Experimental Procedures.'' Results for
discoidal rHDL with SPM/apoA-I molar ratios of 0/1, 13.6/1, and 26.3/1
are shown in Panels A, B, and C,
respectively. Results for spherical rHDL with SPM/apoA-I molar ratios
of 0/1 and 4.9/1 are shown in Panels D and E,
respectively. The data in Panel F represents lipid-free
apoA-I. Each data point represents the mean of triplicate
determinations. Experimental errors for the wavelength of maximum
fluorescence are ± 1.0 nm.
Figure 5:
Influence of SPM on the kinetics of
unfolding of apoA-I in discoidal and spherical rHDL. Discoidal rHDL
with SPM/apoA-I molar ratios of 0/1 (), 13.6/1 (
), and
26.3/1 (
), spherical rHDL with SPM/apoA-I molar ratios of 0/1
(
) and 4.9/1 (
) and lipid-free apoA-I (
) were
incubated for 0-24 h with 2.5 M GdnHCl. Values for the
wavelength of maximum fluorescence represent the mean of triplicate
determinations. Experimental errors are ±1.0
nm.
Lipid-free apoA-I unfolded rapidly and completely, as evidenced by the comparable wavelengths of maximum fluorescence at 0, 2, and 24 h (Fig. 4F). The wavelengths of maximum fluorescence for the discoidal and spherical rHDL (Fig. 4, A-E) were blue-shifted at 0 and 2 h relative to 24 h, confirming that unfolding of apoA-I is inhibited by lipid association(23, 35) . Fig. 5shows the kinetics of the unfolding of apoA-I at 2.5 M GdnHCl. The apoA-I in the spherical rHDL which had been incubated with sphingomyelinase (closed circles) unfolded more rapidly than the apoA-I in the spherical rHDL which had been incubated with TBS (open circles). The rate of unfolding of apoA-I in discoidal rHDL decreased as the molar ratio of SPM/apoA-I increased from 0/1 (open squares) to 13.6/1 (closed diamonds) to 26.3/1 (open diamonds). Taken together, these results suggest that the SPM head group may be partly responsible for inhibiting the unfolding of apoA-I in discoidal rHDL. It should also be noted that, irrespective of the SPM content of the particles, the apoA-I in spherical rHDL unfolds more rapidly than the apoA-I in discoidal rHDL.
The influence of SPM on apoA-I stability was assessed from the
concentration of GdnHCl required for 50% unfolding of apoA-I
([GdnHCl]). Values for
[GdnHCl]
were determined directly from the 24-h
denaturation curves in Fig. 4and calculated as described under
``Experimental Procedures.'' The results in Table 2show good agreement between the two approaches.
[Gdn-HCl]
for lipid-free apoA-I was 1.0 M, confirming what has been reported elsewhere(36) .
[GdnHCl]
for the discoidal rHDL increased with
the molar ratio of SPM/apoA-I, suggesting that the stability of apoA-I
is enhanced by the SPM molecule. When the spherical rHDL were incubated
with sphingomyelinase, [GdnHCl]
was not
affected. In other words, SPM head groups do not influence the
stability of apoA-I in spherical rHDL. Although
[GdnHCl]
increased when the apoA-I was
associated with lipid, this change does not necessarily translate into
an increase in the stability of apoA-I. For example, although
[GdnHCl]
for apoA-I in spherical rHDL is
greater than that of lipid-free apoA-I, the two preparations have
similar conformational stabilities
(
G
Figure 6: Influence of SPM on the CETP-mediated transfer of core lipids between spherical rHDL and Intralipid. Spherical rHDL were incubated with sphingomyelinase or TBS and reisolated by ultracentrifugation as described under ``Experimental Procedures.'' Their composition is shown in Table 1. They were then incubated with TBS, TBS and Intralipid, or TBS, Intralipid, and CETP for 0, 1, 3, 6, 12, or 24 h. The final concentrations of rHDL CE and Intralipid TG (if present) in the incubation mixtures were 0.1 and 3.9 mmol/liter, respectively. The final activity of CETP (if present) was 2.6 units/ml. The final volume of the incubation mixtures was 2.0 ml. When the incubations were complete, the rHDL were isolated by ultracentrifugation in the 1.063 < d < 1.25 g/ml density range as described under ``Experimental Procedures.'' Concentrations of rHDL CE (Panel A), rHDL TG (Panel B), and rHDL CE + TG (Panel C) are shown. Values in Panels A and B represent the mean of triplicate determinations which varied by 10% or less.
Figure 7:
Influence of SPM on the LCAT reaction in
discoidal rHDL. Discoidal rHDL with POPC/SPM/UC/apoA-I molar ratios of
97.7/0.0/9.6/1.0 and 69.8/26.2/9.8/1.0 were prepared by cholate
dialysis. Both preparations were radiolabeled with
[H]UC as described under ``Experimental
Procedures.'' Aliquots of each preparation which contained 3.25
nmol UC were incubated at 37 °C for 5, 10, 15, 20, or 30 min with
purified LCAT. Esterification of cholesterol in the rHDL with (
)
and without (
) SPM is shown. The data points represent the mean
± S.D. of triplicate determinations. *p <
0.05,**p < 0.005
SPM, a glycosphingolipid consisting of a ceramide backbone and phosphocholine head group, is present in most cell membranes. SPM is transported in the plasma as a component of lipoproteins, but its impact on lipoprotein metabolism, and on HDL metabolism in particular, is poorly understood. This issue is addressed in the present study. Specifically we have determined how the SPM molecule and its head group influence the structure and function of discoidal and spherical rHDL.
To assess the influence of the SPM head group on rHDL metabolism, the rHDL were incubated with sphingomyelinase. Interpretation of these studies was dependent on sphingomyelinase affecting neither rHDL size nor the concentrations of other rHDL constituents. This was achieved for spherical rHDL ( Fig. 1and Table 1). The discoidal rHDL, by contrast, were converted into larger and smaller particles during incubation with sphingomyelinase (data not shown). This is not consistent with what has been reported by Subbaiah and Lui(26) , who found that the size of discoidal rHDL was not affected by incubation with sphingomyelinase. Given that the rHDL described by Subbaiah and Lui contained egg PC, as opposed to POPC in the present studies, it is possible that this discrepancy may be due to the structural differences between the two phospholipids. As egg PC has a higher proportion of unsaturated acyl chains than POPC(37) , it follows that egg PC-containing discoidal rHDL will have less ordered phospholipid acyl chains and more hydrated interfacial regions than POPC-containing discoidal rHDL(38) . Given that electrostatic repulsions between phospholipid head groups decrease as hydration of lipid bilayers increases(39) , it is possible that rHDL which contain egg PC may be more stable, and resistant to size changes, than rHDL which contain POPC. Regardless of the mechanism, the fact that sphingomyelinase altered the size of the POPC-containing discoidal rHDL precluded investigation of the influence of SPM head groups on their structure.
The problem of sphingomyelinase-mediated changes to the size of discoidal rHDL was circumvented by investigating the influence of the entire SPM molecule on their structure and function. To this end discoidal rHDL were prepared with a range of concentrations of SPM. The phospholipid/apoA-I molar ratio in these rHDL was maintained at approximately 100/1 by appropriate reductions in the concentration of POPC (Table 1). The rHDL size was not affected as the SPM/POPC molar ratio increased from 0/1 to 0.4/1 (Fig. 1). This differs from what has been reported by Subbaiah and Lui(26) , who found that the diameter of discoidal rHDL with egg PC increased from 10.0 to 16.8 nm when the SPM/egg PC molar ratio increased from 0/1 to 0.5/1. This discrepancy can be explained if the concentration of egg PC was not decreased when SPM was introduced into the rHDL. Under these circumstances the phospholipid/apoA-I molar ratio of the rHDL would increase and the particles would increase in size(40) . It is also possible that the increase in rHDL size was due to the somewhat disordered egg PC acyl chains being unable to accommodate the asymmetric SPM molecule. Under these circumstances the rHDL may undergo a structural reorganization to form larger, more stable particles.
The polarization results in Fig. 3show that SPM increases the packing order of phospholipid acyl chains and head groups in discoidal rHDL. This is probably because the SPM interfacial region contains a C4-C5 trans double bond which has been reported to increase acyl chain packing order(4) . The results in Fig. 3also show that the SPM head group has a slight ordering effect on the acyl chains in spherical rHDL. When these results are taken together it is tempting to speculate that the SPM head group is partly responsible for increasing the acyl chain packing order in discoidal rHDL. However, this is not necessarily the case as DPH partitions differently in discoidal and spherical rHDL. In spherical HDL, DPH is located at the interface of the phospholipid acyl chains and neutral lipid core(41) . In discoidal rHDL, DPH intercalates between phospholipid acyl chains(42) .
The results of the PRODAN studies (Fig. 3C) show that neither the SPM molecule nor its head group affect the hydration of rHDL lipid-water interfacial regions at temperatures less than 25 °C. This is in agreement with the report of Jonas et al.(35) . Above 25 °C, by contrast, there is a pronounced increase in discoidal rHDL lipid-water interfacial hydration which becomes less apparent as the concentration of SPM increases. This suggests that SPM limits the access of water to the discoidal rHDL lipid-water interface and is consistent with the observation that SPM-containing lipid bilayers have a low permeability to water(43) . The additional finding, that above 25 °C, spherical rHDL have less hydrated lipid-water interfaces than discoidal rHDL, suggests that access of water to the surface of spherical rHDL is restricted. This may occur if a proportion of the phospholipid head groups on the surface of spherical rHDL is masked by apoA-I. Finally, as incubation of spherical rHDL with sphingomyelinase does not affect the wavelength of maximum fluorescence of PRODAN, it follows that the SPM head group does not influence the interfacial hydration of these particles.
One of the most unexpected findings to emerge from the
present study is that SPM transfers rapidly and spontaneously between
LDL and rHDL with a t = 0.8 h. The t
for the spontaneous transfer of
phosphatidylcholine mass between discoidal rHDL and LDL, by contrast,
ranges from 5.8 to 6.9 h(44) . This marked difference in
half-times probably reflects structural differences between SPM and
phosphatidylcholine. The interfacial region of SPM is polar and
contains a trans double bond, a free hydroxyl group, and an
amide bond(4) . This region interacts strongly with water and
is probably responsible for the rapid transfer of SPM. The
corresponding region of phosphatidylcholine, by contrast, comprises a
glycerol backbone and possibly the carbonyl portion of the ester bonds.
These regions neither interact with water nor facilitate the
spontaneous transfer of phosphatidylcholines.
A primary aim of the present study was to determine how SPM influences the metabolism of HDL. The finding that SPM inhibits the LCAT-mediated esterification of UC in discoidal rHDL (Fig. 7) confirms what has been reported elsewhere(26) . This reduction in cholesterol esterification has been attributed to competition between SPM and phospholipids for binding to the active site of LCAT(26) . Our results also show that CETP-mediated transfers of CE and TG between spherical rHDL and Intralipid are not affected when the rHDL are depleted of SPM head groups (Fig. 6). As CETP reportedly binds to phospholipid head groups on the surface of HDL(45) , this result suggests that the concentration of phospholipids is not rate-limiting for CETP-mediated transfers of core lipids.
When spherical rHDL are subjected to agarose gel electrophoresis they migrate slower than native HDL (Table 1). This difference in mobility, which reflects the different surface charges of the preparations, is most likely due to variations in apolipoprotein and phospholipid composition. Native HDL contain several classes of apolipoproteins (46) , whereas the rHDL used in the present study contain only apoA-I. Davidson et al.(13) have also shown that phospholipid acyl chain composition affects HDL surface charge. As native HDL contain a range of phospholipids(47) , it is to be expected that their surface charge differs from that of rHDL which contain only POPC. When spherical rHDL are depleted of SPM head groups, their mobility is indistinguishable from that of native HDL. In other words, removing the SPM head group increases the negative charge on the surface of rHDL. One explanation for this observation is that removing the SPM head group exposes the polar, interfacial region of the molecule and that this region influences the surface charge of rHDL. Alternatively, it is possible that the conformation of apoA-I changes when spherical rHDL are depleted of SPM head groups. However, given that rHDL size is not affected by incubation with sphingomyelinase (Fig. 1), and the conformation of apoA-I is dependent on rHDL size(48) , this is not likely.
The finding that SPM decreases the GdnHCl-mediated unfolding of apoA-I in discoidal rHDL (Fig. 5) confirms what has been reported by Swaney (49) . This decrease may be due to the hydrogen bonds in the SPM interfacial region enhancing apoA-I-phospholipid interactions and stabilizing the particles. The additional finding that incubation with sphingomyelinase increases the unfolding of apoA-I in spherical rHDL suggests that SPM head groups also enhance phospholipid-apoA-I interactions. An alternative explanation for the increased unfolding of apoA-I in spherical rHDL is that removal of SPM head groups alters the orientation of phosphocholine head groups. Scherer and Seelig have shown that the orientation of these head groups is sensitive to surface charge(50) . As the negative charge on the surface of spherical rHDL increases after incubation with sphingomyelinase (Table 1), it follows that phosphocholine head group orientation may be altered such that phospholipid-apoA-I interactions decrease and the rHDL are destabilized.
In summary, this study provides an insight into the effect of SPM on the structure and function of discoidal and spherical rHDL. We have shown that the SPM molecule, and its head group, influence the structure and stability of both types of rHDL. When these results are considered, together with the observation that SPM inhibits cholesterol esterification in discoidal rHDL, it follows that factors which regulate the concentration of SPM in HDL may have a significant impact on plasma cholesterol transport.